Janske Nel

Céline M Desmet

Benoit Driesschaert

Patrick Saulnier

Laurent Lemaire

Bernard Gallez

Florence Franconi

Nicolas Joudiou

Preparation and evaluation of trityl-loaded lipid nanocapsules as oxygen sensors for Electron Paramagnetic Resonance oximetry

Keywords: hypoxia, lipid nanocapsules, electron paramagnetic resonance, magnetic resonance imaging Les nanocapsules lipidiques en tant qu hypoxie, nanocapsules lipidiques, résonance paramagnétique électronique, imagerie par résonance magnétique Janske hydrogen proton, CA IX, carbonic anhydrase IX, p53, tumour suppressor gene Electron paramagnetic resonance, oximetry, hypoxia, lipid nanocapsules, trityl, nitroxide Chapter 3: Electron Paramagnetic Resonance and Lipid Nanocapsules Janske NEL | Lipid nanocapsules as a theranostic hypoxia, lipid nanocapsules, carbogen challenge, magnetic resonance imaging Lipid nanocapsules as a theranostic tool hypoxia, lipid nanocapsules, electron paramagnetic resonance, magnetic resonance imaging Les nanocapsules lipidiques en tant qu'outil théranostique hypoxie, nanocapsules lipidiques, résonance paramagnétique électronique, imagerie par résonance magnétique

This section includes the article Preparation and evaluation of trityl-loaded

Résumé: L'hypoxie est l'un des aspects les plus importants dans le microenvironnement tumoral. Ce phénomène, causé par une structure vasculaire anormale et un métabolisme perturbé, conduit à la formation de cellules hautement malignes résistantes aux thérapies cyto-et radio-toxiques. De ce fait, l'hypoxie est une préoccupation majeure et est à la base de notre nouvelle approche qui utilise des nanocapsules lipidiques (LNC) en tant que senseur d'oxygène. Il a été montré que les LNC sont d'excellents nanocarriers capables d'encapsuler des principes actifs à l'intérieur de leur coeur lipidiques et d'éviter le système immunitaire grâce au PEG de leur coque, permettant ainsi le traitement de tumeurs agressives. Nous avons émis l'hypothèse que le coeur lipidique des LNC pouvait être utilisé pour cartographier l'O 2 dans l'environnement tissulaire. En effet, la solubilité de l'O 2 étant supérieure dans les lipides que dans l'eau, le moindre changement d'oxygénation dans les tissus sera amplifié dans le coeur lipidique des LNC. Par conséquent, nous avons encapsulé une sonde paramagnétique lipophile, e.g. le tetrathiatriarylmethyl (TAM), et montré sa réponse in vitro à des variations en O 2 en utilisant la résonance paramagnétique électronique (RPE) montrant de ce fait la perméabilité des LNC à l'O 2.

Nous avons utilisé les TAM-LNC dans un modèle in vivo de tissu normal (muscle murin gastrocnémien) et pathologique (sarcome). Sans LNC, le TAM a rapidement été réduit et aucune mesure en O 2 n'a été possible toutefois, notre système TAM-LNC présentait une demi-vie supérieure à une heure et permettait la mesure en temps réel lorsque les animaux respiraient soit de l'air soit du carbogène (95 % O 2 , 5 % CO 2 ). Par ailleurs, la nature lipidique du coeur des LNC a été exploitée pour cartographier l'oxygénation des tissus grâce à l'imagerie par résonance magnétique (IRM) et plus précisément par l'utilisation de la séquence MOBILE qui permet d'imager la variation du temps de relaxation T 1 induite par l'O 2 dans les lipides. Avec une dose unique de LNC, nous avons été capables de représenter les changements de T 1 entre les différents modes de respiration et l'hétérogénéité de l'hypoxie dans des modèles de tissus murins sain et pathologique. En conclusion, nous avons apporté la preuve de faisabilité de l'utilisation des LNC en tant qu'outil diagnostique pour cartographier l'hypoxie dans des tissus sains et pathologiques.

Abstract: Hypoxia is one of the most challenging aspects of the tumour microenvironment. The phenomenon occurs due to abnormal vasculature and an exacerbated metabolism, and leads to highly malignant cells resistant to radio-and cyto-toxic therapy. As such, hypoxia is of major concern and prompted our novel approach in using lipid nanocapsules (LNCs) as an oxygen sensor. LNCs have been demonstrated as excellent core-shell nanocarriers, capable of encapsulating drugs within their lipidic core and avoiding the immune system due to their PEGylated shell, thus enabling treatment of highly aggressive tumours. We hypothesised that the lipidic-core of LNCs could also be used to assess the O 2 environment in tissue. Indeed, because O 2 solubility is greater in lipids than in water, any subtle changes in tissue O 2 will be heightened in the lipidic LNC core. Consequently, we encapsulated a lipophilic paramagnetic probe, e.g. tetrathiatriarylmethyl (TAM), and demonstrated its response to variations in O 2 in vitro, using Electron Paramagnetic Resonance (EPR), indicating the permeability of LNCs to O 2.

We applied the TAM-LNCs to an in vivo normal tissue model (gastrocnemius mouse muscle) and pathological model (sarcoma tumour). Herein, free TAM was rapidly reduced and no O 2 measurement was possible, however, our TAM-LNC system exhibited a half-life of over an hour and enabled realtime measurements whilst animals were breathing air and during a carbogen gas (95 % O 2 , 5 % CO 2 ) breathing challenge. Moreover, the lipidic-core nature of the LNCs was exploited to image tissue oxygenation using Magnetic Resonance Imaging (MRI), specifically the MOBILE sequence which enabled the mapping of O 2 -induced T 1 relaxation rate changes in lipids. Using a single dose of LNCs, we were able to portray the change in T 1 between air breathing and the carbogen challenge, and image the heterogeneous nature of hypoxia in murine normal tissue and pathological tumour models. In conclusion, we demonstrated the feasibility of using LNCs as a diagnostic tool for assessing hypoxia in both normal and pathological tissues.

List of Figures

Figure 2-1. Schematic of tumoural progression and the effects on angiogenesis, metabolism and apoptotic pathways. Adapted from the following citations 2,6,[START_REF] Harris | Hypoxia -A Key Regulatory Factor in Tumour Growth[END_REF][START_REF] Denko | Epigenetic Regulation of Gene Expression in Cervical Cancer Cells by the Tumor Microenvironment[END_REF][START_REF] Koong | Candidate Genes for the Hypoxic Tumor Phenotype[END_REF][START_REF] Wykoff | Hypoxia-Inducible Expression of Tumor-Associated Carbonic Anhydrases[END_REF][START_REF] Lal | Transcriptional Response to Hypoxia in Human Tumors[END_REF][START_REF] Semenza | The Metabolism of Tumours": 70 Years Later[END_REF][START_REF] Vaupel | Tumor Hypoxia: Causative Factors, Compensatory Mechanisms, and Cellular Response[END_REF] PET imaging of a high-grade glioma wherein the hypoxic regions (red) were assessed by [START_REF] Zhong | Overexpression of Hypoxia-Inducible Factor 1alpha in Common Human Cancers and Their Metastases[END_REF] F-FMISO (a) and the high glucose metabolism (red) were determined by [START_REF] Zhong | Overexpression of Hypoxia-Inducible Factor 1alpha in Common Human Cancers and Their Metastases[END_REF] F-FDG (b). Note the lack of concordance between the two images, suggesting that a complex relationship exists between the two metabolic processes. Image reproduced from Lee and Scott 279 . .................33

Statement of Original Authorship

The work contained in this thesis has not been previously submitted to meet requirements for an award at these or any other higher education institution. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made. Secondly, the lipidic-core nature of the LNCs was exploited using MRI, specifically using a sequence which enables the mapping of O 2 -induced spin-lattice (T 1 ) relaxation rate changes in lipids (MOBILE). This sequence, however, requires a sufficiently high intrinsic amount of lipids to acquire an oxygenation map, which unfortunately does not always occur spontaneously within tissues. Thus, LNCs were used to increase the lipidic signal-to-noise ratio within normal (gastrocnemius mouse muscle) and pathological tissues (neoplastic models including fibrosarcoma subcutaneous and intracerebral glioblastoma tumours) within murine models, and using the MOBILE sequence, oxygenation maps were produced of tissues in which this was not previously possible.

Signature: _________________________

Date: _________________________

AIMS AND OBJECTIVES

This thesis aims to investigate the feasibility and usefulness of the LNC therapeutic nanocarrier as a diagnostic O 2 sensor using EPR and MRI. The main objectives of the thesis are therefore: oxygenation map be produced of the area?

When applying

THESIS OUTLINE

In the following chapter (Chapter 2), the background of the thesis will be presented and will include an overview of; tumour hypoxia and its relevance to malignancy and treatment resistance in oncology, the current techniques for assessing and imaging tumour hypoxia with emphasis on their advantages and limitations, an introduction to the theoretical basis of MR techniques and the working principles of EPR and MRI on which special interest will be focused on the specific issue faced for each technique. These specific limitations will be addressed by the introduction and discussion of LNCs which will serve as the nanoparticle employed in this thesis.

In chapter 3, the ability of LNCs to encapsulate paramagnetic EPR spin probes is investigated, as well as their ability to respond to in vitro and in vivo oxygenation variation.

In chapter 4, the ability of LNCs to increase the lipidic content sufficiently to enable the mapping of O 2 using the MRI MOBILE sequence is investigated in both normal and pathological tissues.

Following this, in Chapter 5, the conclusions in the previous chapters are presented and their implications are discussed. Additionally, the future work which could stem from this thesis is considered.

INTRODUCTION TO CHAPTER 2

In this chapter, the literature relevant to the background of this thesis is reviewed. The aim is to provide a strong theoretical framework for the knowledge and context of this body of work, and identify previous work and their limitations pertaining to the assessment and imaging of tissue oxygenation.

In section 2.2, the general biology of the tumour microenvironment is introduced, and the relevance of hypoxia is described in regards to cancer progression and treatment. Several methods to assess and image tumour hypoxia in vivo have been previously developed and are described in section 2.3, with special attention given to their advantages and disadvantages.

This section will highlight the need for novel non-invasive methods for tumour hypoxia assessment and imaging such as EPR and MRI. Next, in section 2.4, the governing principles of EPR oximetry and its development in oncology will be explored. Similarly, in section 2.5, MRI will be discussed, including its usage in O 2 determination in oncology. Then, in section 2.6, the development of LNCs as a therapeutic nanomaterial will be reviewed and its use as an O 2 sensorand thus a potential theranostic toolusing EPR and MRI will be introduced.

Chapter 2: Background to the Research Area Janske NEL | Lipid nanocapsules as a theranostic tool 9

THE TUMOUR MICROENVIRONMENT AND HYPOXIA

O 2 plays a critical role in cellular metabolism and is involved in the pathogenesis of several maladies including carcinogenesis and its treatment regimes. Consequently, the ability to measure O 2 in tissue with sufficient sensitivity, accuracy and simplicity would be immensely beneficial as it would enhance our understanding and therapeutic strategies of solid tumours. When low levels of O 2 persist (referred to as hypoxia); changes in the gene and protein expressions of cells occur which causes an increase in vasculature growth (angiogenesis) to supply the proliferating cancer cells with O 2 and nutrients, a switch to anaerobic metabolism to combat the low O 2 environment leading to an increase in the pH of the microenvironment, and ultimately gives rise to cells which are highly malignant, metastatic, and resistant to therapy. This cascade is, of course, an oversimplification of hypoxia pathogenesis as tumours are complex systems consisting of a plethora of different cells and heterogeneous microenvironments which are constantly in flux and influenced by temporal and local changes 1 . Thus, the following sections will provide an overview of hypoxia and the challenges researchers face in elucidating the O 2 content of the tumour microenvironment.

What is hypoxia?

The status of 'hypoxia' is characterised by physiologically low levels of O 2 in which tissues can no longer function optimally. These low levels of O 2 tension often arise when the O 2 supply in limited but its consumption is increased, causing genetic and proteomic changes 1 . Several pathologies, beside cancer, are also associated with low levels of O 2 tension, including vascular and pulmonary diseases 2 . Clinical investigations over the last three decades have clearly demonstrated that 50 -60 % of locally advanced solid tumours 3 may exhibit hypoxic areas that are heterogeneously distributed within tumours larger than 1 mm 3 .

Such regions have been found in a wide range of malignancies including cancers of the breast, uterine, cervix, vulva, head and neck, prostate, rectum, pancreas, lung, brain tumours, soft tissue carcinomas, malignant melanomas, metastatic liver tumours and renal cell cancer [4][5][6] .

Hypoxia-induced proteome and genome changes in the tumour may promote tumour progression via mechanisms enabling cells to overcome nutritive deprivation, to escape from the hostile environment, and favour unrestricted growth. Sustained hypoxia in a growing tumour may also lead to cellular changes that can result in a more clinically aggressive phenotype 2 . It is therefore no surprise that hypoxia is an important physiological parameter, and the understanding of its pathogenesis requires consideration for tumour physiology and therapy.

The pathogenesis of tumour hypoxia

Cancer cells have a variety of mechanisms that enable them to adapt, evade and take advantage of their environment. The diversity of hypoxic stimuli within tumours is complexseveral intracellular signalling pathways are activated in tandem amongst a variety of shared genes making it difficult to generalise on its effect on tumour biology because the interplay between the O 2 tension, hypoxia-induced signalling, genomic and proteomic reorganisation, and the prevailing cellular damage, is labyrinthine. Ultimately, however, these mechanisms are primarily mediated by a family of transcriptional regulators; hypoxia-inducible factor (HIF) 7 . Numerous pathways involving factors such as cytokines, chemokines, and growth factors, are also stimulated in a cascade of both hypoxia-dependent and independent manners which all eventually lead to the activation of the HIF pathway and, more specifically, the stabilisation of HIF-alpha (HIF-α).

HIFs are heterodimers consisting of two subunits, alpha (α) and beta (β). Whilst the βsubunit is not O 2 sensitive, HIF-α rapidly responds to low O 2 tension at the post-translational level causing an increase in its quantity 8 . Among the HIF protein family members, the role of HIF-1 is well-established in cancer progression. Importantly, several studies have associated HIF-1α expression with human cancer progression and its prognostic impact has been the subject of numerous studies. Histological analyses have indicated that increased intracellular levels of HIF-1α is associated with poor prognosis and resistance to therapy in breast [9][10][11][START_REF] Trastour | HIF-1α and CA IX Staining in Invasive Breast Carcinomas: Prognosis and Treatment Outcome[END_REF] , head and neck [START_REF] Aebersold | Expression of Hypoxia-Inducible Factor-1alpha: A Novel Predictive and Prognostic Parameter in the Radiotherapy of Oropharyngeal Cancer[END_REF] , oesophagus [START_REF] Matsuyama | Expression of Hypoxia-Inducible Factor-1alpha in Esophageal Squamous Cell Carcinoma[END_REF] , stomach [START_REF] Griffiths | Hypoxia-Inducible Factor-1α Expression in the Gastric Carcinogenesis Sequence and Its Prognostic Role in Gastric and Gastro-Oesophageal Adenocarcinomas[END_REF] , lung cancers [START_REF] Swinson | Hypoxia-Inducible Factor-1? In Non Small Cell Lung Cancer: Relation to Growth Factor, Protease and Apoptosis Pathways[END_REF] , and many other types [START_REF] Talks | The Expression and Distribution of the Hypoxia-Inducible Factors HIF-1alpha and HIF-2alpha in Normal Human Tissues, Cancers, and Tumor-Associated Macrophages[END_REF][START_REF] Zhong | Overexpression of Hypoxia-Inducible Factor 1alpha in Common Human Cancers and Their Metastases[END_REF] . The full pathway, mechanism and impact of HIFs is a fascinating and immense topic and cannot be fully covered in this thesis thus for a full review the work of Harris [START_REF] Harris | Hypoxia -A Key Regulatory Factor in Tumour Growth[END_REF] is recommended.

It is important to note, however, that HIF-1 induces the expression of more than 30 known genes 2,6,[START_REF] Harris | Hypoxia -A Key Regulatory Factor in Tumour Growth[END_REF][START_REF] Denko | Epigenetic Regulation of Gene Expression in Cervical Cancer Cells by the Tumor Microenvironment[END_REF][START_REF] Koong | Candidate Genes for the Hypoxic Tumor Phenotype[END_REF][START_REF] Wykoff | Hypoxia-Inducible Expression of Tumor-Associated Carbonic Anhydrases[END_REF][START_REF] Lal | Transcriptional Response to Hypoxia in Human Tumors[END_REF][START_REF] Semenza | The Metabolism of Tumours": 70 Years Later[END_REF][START_REF] Vaupel | Tumor Hypoxia: Causative Factors, Compensatory Mechanisms, and Cellular Response[END_REF] ; at O 2 partial pressure (pO 2 ) levels between 10 -15 mmHg genes essential for angiogenesis are stimulated; at 10 mmHg or less decreased adenosine triphosphate (ATP) synthesis occurs leading to a change in energy metabolism; and at less than 1 mmHg hypoxia-induced apoptosis is stimulated (Fig. 2-1).

Angiogenesis

Arguably, the most fundamental characteristic of cancer cells involves their ability to sustain persistent proliferation. Whilst normal tissues carefully control the production and release of growth promoting signals for homeostatic progression through the cell growth-anddivision cycle, cancer cells dysregulate these signals. In cancer cells, signalling pathways are corrupted, negative-feedback loops are dampened, growth suppressors are evaded, and mechanisms of contact inhibition are suppressed; all of which enable the neoplastic cells to progress through the cell growth-and-division cycle and continuously increase in number and in size 1 . However, all cells require sustenance, even cancer cells. As such, tumour progression requires neovascularisation, which occurs either by the recruitment of circulating cells for the formation of blood vessels (vasculogenesis [START_REF] Brown | Vasculogenesis: A Crucial Player in the Resistance of Solid Tumours to Radiotherapy[END_REF] ) or by the sprouting of endothelial cells from nearby blood vessels (angiogenesis) in order to provide nutrients and O 2 and to remove catabolites from the expanding tumour mass. The 'angiogenic switch' is governed by a myriad of balanced, competing factors which either induce or oppose vascularisation, producing controlled and transient angiogenesis in normal tissue. For cancerous cells, however, this switch is tipped towards 'on' during the early development of the premalignant growth of the tumour mass and remains activated [START_REF] Raica | Angiogenesis in Pre-Malignant Conditions[END_REF][START_REF] Baeriswyl | The Angiogenic Switch in Carcinogenesis[END_REF][START_REF] Bergers | Tumorigenesis and the Angiogenic Switch[END_REF] . This activation is predominantly driven by vascular endothelial growth factor (VEGF), which is a well-known key angiogenic factor in normal tissues but with a markedly increased presence in several cancer types [START_REF] Goldmann | The Growth of Malignant Disease in Man and the Lower Animals, with Special Reference to the Vascular System[END_REF][START_REF] Hanahan | Patterns and Emerging Mechanisms of the Angiogenic Switch during Tumorigenesis[END_REF][START_REF] Toi | Vascular Endothelial Growth Factor: Its Prognostic, Predictive, and Therapeutic Implications[END_REF] .

Despite the enhanced angiogenesis within a tumour mass, angiogenesis cannot keep up with the cancerous proliferation, thus median O 2 levels are significantly lower than normoxia (normal physiological levels of O 2 within a given tissue) and can even reach 0 % O 2 [START_REF] Mckeown | Defining Normoxia, Physoxia and Hypoxia in Tumours-Implications for Treatment Response[END_REF][START_REF] Ballinger | Imaging Hypoxia in Tumors[END_REF] .

Furthermore, due to the haphazard growth of tumour cells and their associated vasculature, there is abundant evidence for the existence of substantial heterogeneity in the development and extent of tumour hypoxia due to pronounced intra-and inter-tumoural variability in vascularity, perfusion rates and regions of necrosis [START_REF] Vaupel | Hypoxia in Breast Cancer: Pathogenesis, Characterization and Biological/Therapeutic Implications[END_REF][START_REF] Vaupel | Blood Flow, Oxygen and Nutrient Supply, and Metabolic Microenvironment of Human Tumors: A Review[END_REF][START_REF] Dagogo-Jack | Tumour Heterogeneity and Resistance to Cancer Therapies[END_REF][START_REF] Allison | Heterogeneity and Cancer[END_REF][START_REF] Hendriksen | Angiogenesis, Hypoxia and VEGF Expression during Tumour Growth in a Human Xenograft Tumour Model[END_REF] , thus scattered gradients of mild and severe hypoxia appear within the tumour. These gradients occur because O 2 delivery is impaired by structural abnormalities within the neovasculature, including; blood vessels growing as dilated or narrowed single branches, non-hierarchical and chaotic vascular networks, disturbed capillary architecture, leakiness, and incomplete vascular walls [START_REF] Thomlinson | The Histological Structure of Some Human Lung Cancers and the Possible Implications for Radiotherapy[END_REF][START_REF] Less | Microvascular Architecture in a Mammary Carcinoma: Branching Patterns and Vessel Dimensions[END_REF][START_REF] Munn | Aberrant Vascular Architecture in Tumors and Its Importance in Drug-Based Therapies[END_REF][START_REF] Carmeliet | Angiogenesis in Cancer and Other Diseases. Nature[END_REF] .

These abnormalities cause numerous functional impairments such as increased vascular permeability making the blood prone to clotting and local tissue oedema [START_REF] Hashizume | Openings between Defective Endothelial Cells Explain Tumor Vessel Leakiness[END_REF][START_REF] Mcdonald | Significance of Blood Vessel Leakiness in Cancer[END_REF][START_REF] Dvorak | Vascular Permeability Factor/Vascular Endothelial Growth Factor and the Significance of Microvascular Hyperpermeability in Angiogenesis[END_REF] , interstitial hypertension [START_REF] Boucher | Tumor Angiogenesis and Interstitial Hypertension[END_REF] , and increased flow resistance. Furthermore, very often, tumour micro-vessels are perfused (at least transiently) by only plasma causing further stunting of O 2 delivery 48 .

Thus, O 2 gradients can arise due to an increase in diffusion distances causing cells far away (> 70 µm) from blood vessels to receive less O 2 than needed. This is referred to as diffusionlimited hypoxia or chronic hypoxia. Acute or transient hypoxia occurs due to perfusionlimited O 2 delivery wherein the collapse, reversal of blood flow, and subsequent re-perfusion of these imperfectly formed blood vessels cuts off the O 2 supply intermittently 4 . Both chronic and acute hypoxia have been implicated to cause an increase in reactive oxygen species (ROS) [START_REF] Hielscher | Hypoxia and Free Radicals: Role in Tumor Progression and the Use of Engineering-Based Platforms to Address These Relationships[END_REF] due to the interruption of the mitochondrial oxidation process at low O 2 tensionleading to tissue damage, the activation of stress-response genes HIF-1 [START_REF] Prabhakar | Invited Review: Oxygen Sensing during Intermittent Hypoxia: Cellular and Molecular Mechanisms[END_REF] , and the subsequent stabilisation of HIF-1α. In turn, HIF-1α induces the transcription of several growth factors such as transforming growth factor r-β and platelet-derived growth factors [START_REF] Denko | Epigenetic Regulation of Gene Expression in Cervical Cancer Cells by the Tumor Microenvironment[END_REF][START_REF] Koong | Candidate Genes for the Hypoxic Tumor Phenotype[END_REF][START_REF] Lal | Transcriptional Response to Hypoxia in Human Tumors[END_REF][START_REF] Wykoff | Identification of Novel Hypoxia Dependent and Independent Target Genes of the von Hippel-Lindau (VHL) Tumour Suppressor by MRNA Differential Expression Profiling[END_REF] , and of course, VEGF and its receptors [START_REF] Semenza | Regulation of Hypoxia-Induced Angiogenesis: A Chaperone Escorts VEGF to the Dance[END_REF] . But, notably, the expression of VEGF is not only influenced by hypoxia but also by glucose depletion [START_REF] Kotch | Defective Vascularization of HIF-1α-Null Embryos Is Not Associated with VEGF Deficiency but with Mesenchymal Cell Death[END_REF][START_REF] Marjon | Expression of the Pro-Angiogenic Factors Vascular Endothelial Growth Factor and Interleukin-8/CXCL8 by Bibliography Janske NEL | Lipid nanocapsules as a theranostic tool 121 Human Breast Carcinomas Is Responsive to Nutrient Deprivation and Endoplasmic Reticulum Stress[END_REF] and an acidic extracellular pH [START_REF] Xu | Acidic Extracellular PH Induces Vascular Endothelial Growth Factor (VEGF) in Human Glioblastoma Cells via ERK1/2 MAPK Signaling Pathway[END_REF] .

Metabolism

During normal cellular metabolism, energy is harnessed in the form of ATP from the anaerobic processing of glucose into lactate (i.e. anaerobic glycolysis) and through the oxidation of glucose into CO 2 by mitochondrial respiration (i.e. aerobic mitochondrial oxidation). Notably, a phenomenon named the Warburg effect (also known as aerobic glycolysis) can occur in both normal and tumoural cells, wherein the uptake of glucose dramatically surges and, even in the presence of sufficient O 2 and functional mitochondria, the anaerobic formation of glucose to lactate is preferred [START_REF] Warburg | The Metabolism of Carcinoma Cells[END_REF] (Fig. 2-2). The Warburg effect has been hypothesised to benefit tumoural cells immensely as it allows for the rapid synthesis of ATP, increased biosynthesis through the pentose-phosphate pathway, enhances the disruption of tissue architecture and immune cell evasion, and allows for signal transduction through ROS [START_REF] Liberti | The Warburg Effect: How Does It Benefit Cancer Cells?[END_REF][START_REF] Vander Heiden | Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation[END_REF] . This use of glycolysis and the pentose-phosphate metabolism seems counterintuitive; tumoural cells require large amounts of energy to keep proliferating whilst this metabolic switch provides considerably less energy. However, the glycolytic intermediate glucose-6-phosphate is used in the pentose-phosphate pathway to synthesise amino acids and nucleotides, which are essential for proliferation. Thus, not only is the use of the anaerobic metabolism an adaptive response for the tumour cells; it's an advantageous one.

At less than 10 mmHg, however, the O 2 content is no longer sufficient to supply ATP via mitochondrial respiration and the switch to anaerobic glycolysis is compulsory [START_REF] Rademakers | Molecular Aspects of Tumour Hypoxia[END_REF][START_REF] Dang | Oncogenic Alterations of Metabolism[END_REF][START_REF] Seagroves | Transcription Factor HIF-1 Is a Necessary Mediator of the Pasteur Effect in Mammalian Cells[END_REF][START_REF] Semenza | Defining the Role of Hypoxia-Inducible Factor 1 in Cancer Biology and Therapeutics[END_REF][START_REF] Lemaire | In Vitro Expansion of U87-MG Human Glioblastoma Cells under Hypoxic Conditions Affects Glucose Metabolism and Subsequent in Vivo Growth[END_REF] . As a result of the glycolytic metabolism pyruvate, lactate and hydrogen ions are excreted from the tumoural cells into its surroundings, leading to an acidic microenvironment and the activation of HIF-1α. It, in turn, regulates the expression of all enzymes in the glycolytic pathway, as well as the activation of the glucose receptors (GLUT-1 and GLUT-3) which mediate cellular glucose uptake [START_REF] Chen | Regulation of Glut1 MRNA by Hypoxia-Inducible Factor-1[END_REF] . The overexpression of GLUT-1 is correlated with poorer outcomes in breast [START_REF] Kang | Clinical Significance of Glucose Transporter 1 (GLUT1) Expression in Human Breast Carcinoma[END_REF] , head and neck 66 , oesophagus [START_REF] Tohma | Overexpression of Glucose Transporter 1 in Esophageal Squamous Cell Carcinomas: A Marker for Poor Prognosis[END_REF] , bladder [START_REF] Hoskin | GLUT1 and CAIX as Intrinsic Markers of Hypoxia in Bladder Cancer: Relationship with Vascularity and Proliferation as Predictors of Outcome of ARCON[END_REF] , stomach [START_REF] Kawamura | Expression of Glucose Transporter-1 in Human Gastric Carcinoma: Association with Tumor Aggressiveness, Metastasis, and Patient Survival[END_REF] , colorectal [START_REF] Furudoi | Clinical Significance of Human Erythrocyte Glucose Transporter 1 Expression at the Deepest Invasive Site of Advanced Colorectal Carcinoma[END_REF] , ovarian [START_REF] Cantuaria | GLUT-1 Expression in Ovarian Carcinoma: Association with Survival and Response to Chemotherapy[END_REF] , and lung cancer [START_REF] Younes | Overexpression of Glut1 and Glut3 in Stage I Nonsmall Cell Lung Carcinoma Is Associated with Poor Survival[END_REF] . Tumour cells have also been shown to increase their glycogen storage in response to acute hypoxia to maintain cell viability and proliferation [START_REF] Pescador | Hypoxia Promotes Glycogen Accumulation through Hypoxia Inducible Factor (HIF)-Bibliography Janske NEL | Lipid nanocapsules as a theranostic tool 123 Mediated Induction of Glycogen Synthase 1[END_REF][START_REF] Gatenby | Mathematical Models of Tumour Invasion Mediated by Transformation-Induced Alteration of Microenvironmental PH[END_REF] . This metabolic switch, and associated enhanced glycolytic rates, is directly associated with increased lactate production which, of course, affects the overall pH of tumours. Unfortunately, this acidic is the final electron acceptor to enable the complete oxidation of glucose, O 2 is essential for the process. However, when O 2 is limited, glycolysis can be used alternatively but results in fewer ATP than during oxidative phosphorylation. Conversely, in proliferating or tumoural tissue, although the mitochondria are functioning normally, the aerobic glycolysis pathway (Warburg effect) can be preferred. Image reproduced from Vander Heiden et al. [START_REF] Vander Heiden | Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation[END_REF] .

environment is no deterrent for tumoural cells, who have adapted to growing in a much lower pH environment than their counterpart normal cells.

This adaptation is made possible by, among others, the enzyme carbonic anhydrase IX (CA IX) which is able to counteract the intracellular acidification caused by glycolysis by reversibly converting CO 2 and water into carbonic acid, as well as increasing the protonextrusion capacity of cellular membranes, thus controlling the intracellular pH environment of the tumour cells. Several studies have correlated increased expression of CA IX with tumour cell survival [START_REF] Robertson | Role of Carbonic Anhydrase IX in Human Tumor Cell Growth, Survival, and Invasion[END_REF] , invasiveness [START_REF] Parkkila | Carbonic Anhydrase Inhibitor Suppresses Invasion of Renal Cancer Cells in Vitro[END_REF] , and poor outcomes in tumour therapy [START_REF] Hoskin | GLUT1 and CAIX as Intrinsic Markers of Hypoxia in Bladder Cancer: Relationship with Vascularity and Proliferation as Predictors of Outcome of ARCON[END_REF][START_REF] Koukourakis | Endogenous Markers of Hypoxia/Anaerobic Metabolism and Anemia in Primary Colorectal Cancer[END_REF][START_REF] Loncaster | Carbonic Anhydrase (CA IX) Expression, a Potential New Intrinsic Marker of Hypoxia: Correlations with Tumor Oxygen Measurements and Prognosis in Locally Advanced Carcinoma of the Cervix[END_REF][START_REF] Chia | Prognostic Significance of a Novel Hypoxia-Regulated Marker, Carbonic Anhydrase IX, in Invasive Breast Carcinoma[END_REF][START_REF] Kon-No | Carbonic Anhydrase IX Expression Is Associated with Tumor Progression and a Poor Prognosis of Lung Adenocarcinoma[END_REF][START_REF] Chen | Expression of Carbonic Anhydrase 9 at the Invasion Front of Gastric Cancers[END_REF] .

Notably, the full intricacies of the tumour metabolism is manifold and cannot be fully discussed in this section, thus the work of Eales [START_REF] Eales | Hypoxia and Metabolic Adaptation of Cancer Cells[END_REF] is highly recommended for a more complete understanding. But there is no doubt that a decreased pH is associated with hypoxia, and that CA IX is linked to the adaptive measures tumour cells employ to combat this acidification, as well as cell survival via apoptosis (a form of programmed cell death) avoidance [START_REF] Potter | Hypoxia Inducible Carbonic Anhydrase IX, Marker of Tumour Hypoxia, Survival Pathway and Therapy Target[END_REF][START_REF] Lagadic-Gossmann | Alterations of Intracellular PH Homeostasis in Apoptosis: Origins and Roles[END_REF] .

Apoptosis

As much as proliferation is a key characteristic of cancer cells, so too is the avoidance of death and the continuation of cell survival. Apoptosis is induced in a number of HIF-1 dependent and independent pathways, and tumour cells have developed many mechanisms to evade these pathways. Under hypoxia, HIF-1α activates the expression of pro-apoptotic proteins [START_REF] Seimiya | Hypoxia Up-Regulates Telomerase Activity via Mitogen-Activated Protein Kinase Signaling in Human Solid Tumor Cells[END_REF][START_REF] Vande Velde | BNIP3 and Genetic Control of Necrosis-like Cell Death through the Mitochondrial Permeability Transition Pore[END_REF][START_REF] Sowter | HIF-1-Dependent Regulation of Hypoxic Induction of the Cell Death Factors BNIP3 and NIX in Human Tumors[END_REF][START_REF] Suzuki | Dephosphorylated Hypoxia-Inducible Factor 1α as a Mediator of P53-Dependent Apoptosis during Hypoxia[END_REF] , leading to the activation of the p53 gene; a tumour-suppressor gene which, once activated, causes a cascade of pathways leading to the apoptosis of the tumoural cell.

However, the relationship between p53, apoptosis and HIF-1 is not straightforward; hypoxia has been shown to induce p53 activation in some conditions but not in others, and the mechanisms by which this is controlled is not well understood [START_REF] Schmid | HIF-1 and P53: Communication of Transcription Factors under Hypoxia[END_REF][START_REF] Hammond | The Role of P53 in Hypoxia-Induced Apoptosis[END_REF] . What is known is that the severity and duration of the hypoxic condition plays a role. The endurance of chronic hypoxia by tumour cells, and the associated tissue damage via ROS, is correlated to a high frequency of DNA breaks and accumulation of DNA replication errors leading to genetic instability and mutagenesis in a variety of genes including p53 [START_REF] Chan | Chronic Hypoxia Decreases Synthesis of Homologous Recombination Proteins to Offset Chemoresistance and Radioresistance[END_REF][START_REF] Luoto | Tumor Hypoxia as a Driving Force in Genetic Instability[END_REF][START_REF] Kondo | Hypoxia-Induced Enrichment and Mutagenesis of Cells That Have Lost DNA Mismatch Repair[END_REF] . This enables the tumour cells to avoid apoptosis via the mutated p53 gene not activating the apoptosis pathways.

Hypoxia also affects chromosomes to promote transformation via telomerases. Telomerases are ribonucleic enzymes that function to maintain telomere lengthwherein increased telomere length means increased cell survivaland their activity is suppressed in normal somatic cells, thus enforcing normal cell death following chromosome degradation. However, this safety featureto self-destruct cells with damaged DNAmalfunctions in cancer cells.

Instead, telomerase activity is increased and thus promotes cellular immortalisation [START_REF] Seimiya | Hypoxia Up-Regulates Telomerase Activity via Mitogen-Activated Protein Kinase Signaling in Human Solid Tumor Cells[END_REF] .

Conversely, acute hypoxia leads to genomic instability due to a delay in the DNA damage response and p53-dependent apoptosis [START_REF] Pires | Effects of Acute versus Chronic Hypoxia on DNA Damage Responses and Genomic Instability[END_REF] . This encourages the cancer cells to enter quiescence; a state of cell-cycle arrest causing reduced cell proliferation and conferring a stem-cell like quality [START_REF] Terzi | The Cell Fate: Senescence or Quiescence[END_REF] . This would appear a paradox in the understanding of cancer cells seeing as their defining characteristic is uncontrolled proliferation; however, this reduced hibernation-like state is the perfect defence. By entering quiescence, cancer cells are able to conserve ATP supplies, avoid apoptosis (and senescence) and be protected from external stresses such as nutrition/O 2 deprivation and cancer therapies [START_REF] Das | Hypoxia Enhances Tumor Stemness by Increasing the Invasive and Tumorigenic Side Population Fraction[END_REF][START_REF] Vaupel | Oxygen Status of Malignant Tumors: Pathogenesis of Hypoxia and Significance for Tumor Therapy[END_REF][START_REF] Collado | Senescence in Tumours: Evidence from Mice and Humans[END_REF][START_REF] Evan | Cellular Senescence: Hot or What?[END_REF] .

This interplay between HIF, p53 and hypoxia is complex and a more detailed description has been provided by Eales [START_REF] Eales | Hypoxia and Metabolic Adaptation of Cancer Cells[END_REF] . Hypoxia and its associated severities drive a physiological selective pressure in tumours for the expansion of cells that have lost their apoptotic potential and, in particular, for cells with p53 mutations, wherein cells lacking functional p53 are more susceptible to genomic instability and potential malignancy 100-102 .

Other hypoxia-response factors

A number of other pathways are also stimulated during hypoxia including; the cell cycle pathway relating to quiescence and proliferation referred to as PI3K/AKT/mTOR 103,104 , the receptor pathways relating to the communication between cell surface receptors and DNA referred to as MAPK/ERK [105][106][107] , and a stress response pathway relating to cytokine production and cell survival controlled by nuclear factor-ĸB (NF-ĸB) 108 . These pathways, and many others, are activated and induces the activity of; metal transcription factor-1 (MTF-1; for increased iron metabolism), erythropoietin (EPO; for increased production of haemoglobin), human lactate dehydrogenase isoenzyme-5 (HLDH5; for lactate metabolism), Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3; for apoptosis avoidance), matrix metalloproteinases (MMPs; for metastatic progression), urokinase plasminogen activator receptor (uPA-R; for metastatic progression), lysyl oxidase (LOX; for metastatic progression), osteopontin (OPN; for metastatic progression), and many more 1,[109][110][111][112] .

Hypoxia also regulates inflammatory mediators and growth factors to stimulate platelet, EMT, epithelial-to-mesenchymal transition.

leukocyte, and smooth muscle cell activity for immune-resistance and immune-suppression, helping the tumour cells escape surveillance and destruction 113,114 . Combined, all of these pathways form the critical steps involved in helping tumour cells persist and invade the body;

the consequences of which include metastasis, increased malignancy and resistance to therapeutic interventions.

The consequences of tumour hypoxia

The increased proliferation of tumoural cells, and their associated chaotic and malfunctioning blood vessels, gives rise to the heterogeneous, hypoxic microenvironment associated with neoplasia. In turn, hypoxia induces a reduced stem-cell like proliferation, apoptosis avoidance, and cell-cycle arrest; all factors which contribute to the resistance of tumour cells to therapy (Fig. 23). These therapeutic problems apply to radio-, chemo-, photodynamic-and immuno-therapy due to the development of resistance in the tumoural cells 2,5,[115][116][117][118] .

This resistance is often the circumstance of the low O 2 tension itself. In the case of radio-therapy, sensitivity to the therapeutic regime is drastically reduced when the pO 2 in tissue is below 10 mmHg [119][120][121][122][123][124][125] . This is because O 2 is a potent radiosensitiser which, upon being bombarded with ionising radiation forms ROS (such as hydroxyl), and can produce single-and double-stranded DNA breaks within cells which facilitates cell death 126 .

However, when O 2 levels are too low this mechanism cannot occur or occurs less effectively.

This phenomenon is known as the 'O 2 enhancement effect'; wherein the radiation dose required to achieve the same biological effect is approximately three times higher in the absence of O 2 than in the presence of normal levels of O 2 126,127 . Resistance is also compounded by the tortuous and chaotic vasculature network of the tumoural mass. Cells located distant from a functional blood supply can be resistant to drug therapy because of the limited penetration of the anti-cancer agents and thus its lower efficacy due to leaky vasculature and dead-ends associated with tumoural angiogenesis 128 ; and the reduced rate of cell proliferation (i.e. quiescence) entered by cells further away from vasculature offers protection from the effects of chemotherapeutic agents whose activity is selective for rapidly dividing cell populations 128,129 .

These resistance factors are prominently showcased in the chemo-and radiotherapeutic challenges faced in treating primary brain tumours (referred to as gliomas or glioblastomas). These neoplasms are extremely difficult to treat due to their increased proliferation, invasive nature, propensity for recurrence, and treatment resistance 130,131 . The standard treatment regime for newly diagnosed malignant gliomas involves surgical resection, and radio-and chemo-therapy [132][133][134][135][136][137] , unfortunately however patient prognosis remains poor. The incurability of glioblastomas is underscored by the invasive, diffuse, and ill-defined borders of these tumours. Notably, image-guidance by MRI is routinely used as a guide for planning therapy strategiesincluding intensity-modulated radiation therapy (IMRT)to help identify tumour location, oedema and necrosis, along with the tissue microstructural changes and regions displaying dysfunction of the blood-brain barrier [138][139][140] .

Unfortunately, however, commonly used MRI sequences are not able to provide images that clearly delineate tumour boundaries and thus do not always correspond with sites of tumour infiltration or other molecular events, and provide only indirect information about tumour pathophysiology. This pathophysiology includes key factors that contribute to treatment failure such as tumour hypoxia. Current research strongly emphasises the need to study and determine the distribution of hypoxia in tumours for radiation treatment planning purposes. Ling et al. 141 proposed the concept of using functional imagingbased on known factors of resistance (such as hypoxia)to determine a heterogeneous multidimensional dose distribution of radiotherapeutics within tumours; this approach, closely related to IMRT, was named 'dose painting'. Using this method it is possible to apply a strategy of IMRT wherein higher doses of radiation are focused on areas of increased treatment resistance determined by functional imaging. To employ effective 'dose painting' strategies, the visualisation of tumour hypoxia in vivo would have a significant impact on the treatment strategy for tumours such as glioblastoma.

Notably, numerous studies have demonstrated that tumour hypoxia, in addition to diminishing therapeutic efficacy, plays a major role in metastasis 142 and malignant progression 143 . Hypoxia promotes these consequences by inducing the expression of HIF-1α, which in turn activates a metastatic cascade which provides a selection pressure for a more aggressive phenotype. This metastatic cascade and selective pressure involves the creation of favourable metastatic niches for tumoural cells and the trafficking of cells towards those locations. This is achieved by the expression of VEGF (essential for the dissemination of tumoural cells from the primary tumour cells) 144 , the transition of epithelial cells to mesenchymal (EMT) (essential for invasive and migratory behaviour), and a transdifferentiation of cells to acquire plasticity (essential for mobility) 1,6,145 . Furthermore, hypoxic stress proteins and the loss of apoptotic potential can impart resistance to certain chemotherapeutic drugs [146][147][148] . Finally, the resistance of solid tumours to chemo-therapy may also be the result of another largely ignored perspective: that of fluctuations in blood flow such as seen in acute hypoxia. Acute hypoxia has significant implications for delivery of chemotherapeutic agents, cellular responsiveness to those agents, and the regrowth potential of the surviving tumour cells. Moreover, regions actively proliferating at one point in time may not have cells actively synthesising DNA at a different time 149,150 . This serves as a possible selection mechanism, wherein cells which survive this high-stress hypoxic environment, are likely to be a source of clonogenes which repopulates tumours with more malignant cells following radio-therapy 100,151,152 . Furthermore, tumour cells and their associated local microenvironment also change in response to anti-cancer treatments such as the transient changes in the oxygenation status of tumours induced by radiation 153 . It is important to emphasise that heterogeneities in tumour oxygenation vary throughout the tumoural microenvironment, thus there is a change in hypoxia both spatially and temporally.

Furthermore, these changes differ between tumour types, patient, and from tumour to tumour.

Electrode based methods

Assessment of the tumour oxygenation status by invasive and non-invasive procedures has been studied vigorously over the years. The most direct method, so far, for measuring hypoxia in solid tumours is the polarographic electrode measurement method. Polarographic electrodes (also known as Clark electrodes), which were introduced in the 1950s, were the first technology to allow for the accurate measurement of tissue oxygen levels. These electrodes measure the electric current formed at a cathode upon reduction of O 2 , with the generated current being directly proportional to the pO 2 within the tissue 167 . The design generally includes a needle-like electrode, the tip of which is enclosed by a gas-permeable membrane. The pO 2 measurements made by the electrode are highly accurate, and was used extensively by Vaupel and colleagues to analyse tumour hypoxia in a range of models 118,168 .

The technique offers high spatial resolution, and a direct method of assessing intratumoural pO 2 values (Fig. 234) 169 .

(a) (b) Unfortunately, however, the use of the polarographic electrode technique includes several limitations and drawbacks; only tumours which are readily accessible can be measured, areas of necrosis cannot be identified, the technique is invasive and the insertion of the needle-like probes is not well-tolerated in human patients, the electrodes require careful calibration for each environment as many factors (e.g. temperature and ionic strength) affect performance, O 2 readings are highly location-dependent as the operator places the probe, the technique is destructive as it induces damage to capillaries and small blood vessels which further complicates matters of hypoxia and the microenvironment, and lastly, the electrode consumes O 2 during measurement 170 .

To address some of these issues, the Eppendorf electrode system was developed, in which a computerised driver moves the electrodes to precise locations and depths 170 .

Furthermore, recessed-tip microelectrodes were developed to reduce tissue damage and microcirculatory disturbance, providing faster responses and finer spatial resolution 171 . But, neither of these advances can eliminate the consumption of the O 2 within the tumour and the invasiveness of the technique. Recently, however, the concept of wearable, bio-conformable electronics was introduced to overcome this limitation 172 . Planar electrodes have been designed which enable the measurement and mapping of transcutaneous pO 2 by attachment of the probes to the skin surface 173 . However, the technique requires the elevation of the skin to 44 -48 °C which perturbs the examined tissue. The Eppendorf polarographic measurement of tumoural pO 2 has been touted as the "gold standard" but is disputed. Often the pO 2 measurements are followed by a core biopsy of the tumour area (where the pO 2 determination occurred) to be processed with immunohistochemistry.

Immunohistochemistry (IHC) based methods

Regarding the development of suitable probes for tumour hypoxia, the nitroimidazoles have received particular attention. Originally, 2-nitroimidazole was developed as an antibiotic against anaerobic bacteria and protozoa in 1953 174 , but its unique behaviour in hypoxic environments propelled its popularity. In hypoxic conditions, nitro-aromatics are selectively reduced by nitro-reductase enzymes to form hydroxylamine intermediates that can bind irreversibly to cellular nucleophilic groups present in proteins and DNA 166 . This irreversible retention in cells is exploited to provide researchers with a microscopy based technique for visualising tumour hypoxia named immunohistochemistry (IHC) (Fig. 2345) 175 .

The detection of these exogenous hypoxia markers are based on antigen-antibody interactions which includes the injection of 2-nitroimidazole derivatives, such as pimonidazole or EF5 (pentafluoropropylacetamide), into the patient before biopsy of the tumour tissue and the detection of the reductants using antibodies conjugated with a counterstain for detection. Importantly, due to the involvement of enzymes during the nitroimidazole reductions, the method only accounts for viable cells. Attempts to design optimised nitroimidazole molecules has been successful, enabling improved; stability, ability to attach moieties (such as isotopes) which enables multimodal imaging, solubility, tissue penetration and biodistribution 162,176 . Further improvements have enabled the vascular parameters of the hypoxic tissue to be related to O 2 concentration both in vitro 177,178 and in vivo 179,180 . It should be noted that nitroimidazole-based markers target highly hypoxic cells, such as cells with pO 2 below 1 mmHg, leading to cells with higher O 2 concentrations, but still deemed hypoxic, to be excluded 162 . Studies supporting the diagnostic significance of IHC have been published for soft tissue sarcoma 181 , brain tumours 182 , and head and neck cancers 183 , however conflicting results have arisen, such as for cervical cancer 184 .

IHC can also involve the use of surrogate markers of hypoxia, such as the endogenous makers for HIF-1, CAIX, VEGF and GLUT. Here, both exogenous and endogenous markers are used to provide information regarding the intercapillary distance, vascular density, and distance from tumour cells to the vessels, within the tumour microenvironment at a pO 2 of < 10 mmHg [185][186][187] . Several methods have been developed to directly measure HIF-1 activity using the introduction of transgenes with hypoxic response elements as promoter sequences coupled to reporter genes, such as luciferase 188,189 or green fluorescence protein (GFP) 190 .

However, the heterogeneity of the tumour gene response makes HIF-1 a complex target; many studies have shown weak correlation between HIF-1α expression and the polarographic electrode technique 191,192 . Attempts to predict and quantify the re-oxygenation of acute hypoxia have been made using a combination of exogenous and endogenous probes 193 .

However, although combination CA IX and proliferation markers can identify cells which are proliferating under hypoxic conditions, there is still no correlation between the amount of CA IX and direct O 2 measurements as compared to the polarographic electrode 194,195 . Markers such as VEGF and GLUTs have also been explored [196][197][198] , and imaging strategies have been developed to reflect tumour vasculature and proliferation, however a direct relationship between pO 2 measurements and protein expression has not been established 199,200 .

Thus, whilst IHC can provide important information regarding the tumour microenvironment, with only viable cells providing information at high spatial resolution and following a highly standardised technique, limitations include; invasiveness and limited sampling size, limited quantification of averaged hypoxic areas over sampled size, and a difficulty in performing repetitive measurements to monitor changes in oxygenation due to the necessity for biopsy or surgical removal of the tumour tissue for evaluation thus hindering the ability to measure the heterogeneity of tumours. Furthermore, 2-nitroimidazole binds to keratinised tissue which could be partially responsible for unspecified staining thus explaining the disparities with other methods 201 . Although attempts have been made to use IHC techniques for chronic and acute hypoxia determination, overall the technique still provides only a snapshot in time of tumour dynamics [202][203][204][205][206][207] .

Optical based methods

Fluorescence and near-infrared (NIR) spectroscopy

Both fluorescence and NIR spectroscopy enables the determination of haemoglobin saturation via the absorption and scattering of incident wavelengths in tissue. These techniques are non-invasive, and includes the used of near-infrared spectroscopy (NIRS), optical coherence tomography (OCT), and diffuse optical spectroscopy and tomography (DOS/T). These techniques enable the determination of haemoglobin saturation via the absorption and scattering of near-infrared wavelengths (400 -1000 nm) which readily cross through biological tissues. This is achieved by the illumination of tissue, and the measurement of backscattered light as a function of wavelength, specifically when the haeme groups of haemoglobin bind O 2 , their absorption spectrum changes and enables the differentiation between oxygenated (HbO 2 ) and deoxygenated (Hb) haemoglobin 208 . This differentiation can be expressed as the haemoglobin saturation fraction and can be related to the tissue pO 2 by using the haemoglobin dissociation curve 209,210 . For clinical studies, this method can be used under normal conditions without a major restriction of motion. Its main advantage is the ability to perform real-time measurements repeatedly 211 .

For haemoglobin saturation measurements, four main applications of optical spectroscopy principles are used, namely; diffuse reflectance, hyperspectral imaging, and diffuse optic topography and tomography, all of which enables the quantification of light absorption and scattering to determine haemoglobin saturation in tissues. Bulk tissue oxygenation and the non-invasive monitoring of human intra-tumour chemotherapy response during treatment regimes can be determined via diffuse reflectance spectroscopy [212][213][214] . Window chamber models for high-resolution imaging of tumour microvasculature and haemoglobin saturation measurements have been achieved in vivo via hyperspectral imaging [215][216][217] . DOS/T involve acquiring oxygenation measurements through the use of an array of NIR sources/detectors thus allowing for a relatively deep (tens of mm) measurement in tissue [218][219][220] (Fig. 23456).

Unfortunately, although fluorescence and NIR spectroscopy techniques are simple, low cost, and relatively non-invasive, they are fundamentally indirect methods for determining tumour pO 2 which depends on the tumours under investigation to have well-perfused vasculature. Direct spectroscopy methods are suitable to measure pO 2 from 20 to 100 mmHg, but suffer from a long response time (approximately 3 min) with relatively poor resolution (tissue scatters light strongly causing low absorption). To add to the complications, it should be noted that the useful lifetime of spectroscopy is short because the response to pO 2 is nonlinear and not readily described mathematically, and pH and other gases, such as CO 2 , can skew calculations. Regardless of this, a significant advantage of these optic techniques is that they are able to provide functional information regarding the tumour tissue [221][222][223][224][225][226][227] .

Photoacoustic tomography (PAT)

The absorption of light by haemoglobin can also be directly detected by taking advantage of photo-thermal effects. For the use of the photoacoustic tomography (PAT)

technique, laser energy is directed at a tissue sample, wherein the molecules absorb the light and converts it into heat, leading to a rapid but transient localised thermos-elastic expansion, generating an ultrasonic emission that can be detected. In this way, PAT enables the threedimensional visualisation of the position of molecules within a tissue by absorption contrast.

This contrast is largely determined by the wavelength-dependant changes in optical PAT can also obtain information on haemoglobin oxygen saturation at high resolution and contrast without the use of exogenous contrast agents 230 . Another advantage of PAT is its compatibility with widely used ultrasound imaging (USI) techniques 231 ; when combined, PAT and USI can simultaneously provide anatomical and functional information on tumours 232,233 , enabling simultaneous imaging of cross-sections of blood vessels, the concentration and oxygenation of haemoglobin, as well as blood flow in vivo 232,234 (Fig. 234567). These Although PAT excels at providing high resolution, millimetre-deep imaging of the vasculature of tissue 232 , this approach is considered an indirect method of pO 2 determination, and relies on the existence of blood and its perfusion, and does not reveal information regarding the concentration of O 2 within the tissues and cells themselves. Furthermore, a penetration depth issue is inherent in this technique. This limitation can be circumvented by using longer wavelengths but endogenous tissue chromophores have less optical absorption at these wavelengths thus creating a trade-off between imaging depth and resolution.

Furthermore, absolute quantification for oxygenation in deep tissue remains a challenge, mainly due to unknown sources of optical influence.

Luminescence quenching probes

Optical-imaging approaches based on luminescence quenching enable the direct measurement, quantification and imaging of O 2 concentration within tissues, even in the absence of blood. Optical O 2 sensors based on luminescence quenching operate through energy exchange; excited state luminophores transfer energy to surrounding O 2 and thereby no longer emit. The quenching effect on the luminescence intensity and lifetime of a sensor is described by the Stern-Volmer equation. Many hundreds of different optical probes capable of sensing a myriad of different products have been designed, developed and applied in several applications [241][242][243][244] .

Luminescence-intensity based approaches are advantageous in their simplicity and adaptability to assist existing imaging systems used to capture the distribution of phosphorescence intensity and quantify O 2 across two-dimensional surfaces 245,246 .

Limitations such as inhomogeneous illumination and non-uniform distribution of the probes have been overcome by introducing O 2 -independent references dyes that co-localise with the phosphorescent sensor. In contrast, lifetime-based approaches are advantageous because they are independent of excitation energy, detector sensitivity, and probe concentrations. These methods have enabled a host of studies involving measurements of intravascular pO 2 values in various tissue types, including tumours, the brain, retina, and heart [247][248][249][250] . The most common, and oft used, example of O 2 dependent phosphorescent lifetime measurements is the OxyLite® probe. Since 1996, the assessment of tumoural oxygenation has been possible using fiber-optic O 2 sensors named OxyLite® 251 . Whereas the polarographic microelectrode provides static information of tissue oxygenation status, the fiber-optic technique used with the OxyLite® system, provides continuous pO 2 measurements. In this technique, the microelectrode probe is inserted within the concerned tissue for a prolonged period of time producing several repeated measurements in the same position and can directly image cell pO 2 within the range of 0 -15 mmHg, as well as accurately map O 2 distribution [251][252][253] .

Luminescent materials can be detected using a variety of optical imaging modalities, enabling a wide range of multi-modality capabilities. With regard to pre-clinical in vivo applications used to study tissue hypoxia, phosphorescence lifetime imaging has become increasingly important due to the capacity for successful three-dimensional spatial registration using confocal imaging 254 and diffuse tomography 255 . For example, phosphorescence lifetime imaging via OxyphorG2 was used to measure fluctuations in vascular pO 2 in fibrosarcomas, gliomas, and mammary adenocarcinomas grown in dorsal skin fold window chambers 256 .

Aside from these two commonly used intensity-and lifetime-based O 2 determination systems, many other approaches, such as fluorescence anisotropy measurements, Fӧrster resonance energy transfer (FRET), and two-photon excitation techniques have also been explored 249,257,258 . Various O 2 probes have been explored for decades and includes; transition metal Ru 2+ -, Os 2+ -, Ir 3+ -complexes [259][260][261][262] , organic dyes such as cyanine 263 , rhodamine 264,265 , fluorescein (including fluorescein isothiocyanate [FITC] and erythrosin B) [266][267][268] , oxazine 269,270 , and phosphorescent metalloporphyrins (Pt 2+ , Pd 2+ , and Zn 2+ ) 271-273 (Fig. 2345678).

These O 2 probes have been designed as complexes, films and foils, and encapsulated within nanoparticles, with each system bringing its own advantages and drawbacks. Due to the low cost, simple preparation and implementation, and their rapid and non-destructive characteristics, the use of optical O 2 probes is by far the largest sector of development as hypoxia sensors. However, the use of these probes come with some intrinsic disadvantages, including their poor chemical stability, poor photostability, cytotoxicity, perturbation of the host system, and poor spatial resolution due to high background noise 274 . The most popular hypoxia imaging agent for PET is 2-nitroimidazole, which is labelled with a radioisotope (e.g. [ 18 F]Fluoromisonidazole thus 18 F-FMISO) 166,275,278 and, is taken up by viable cells with <10 mmHg O 2 279 in gradients (indicating the degree of adduct accumulation in the area). [START_REF] Zhong | Overexpression of Hypoxia-Inducible Factor 1alpha in Common Human Cancers and Their Metastases[END_REF] F-FMISO has been used to demonstrate hypoxia in glioma, head and neck cancer, renal tumours, soft-tissue sarcomas, and non-small cell lung cancer [279][280][281] but it is not suitable for all cancers 279,282 . Notably, [START_REF] Zhong | Overexpression of Hypoxia-Inducible Factor 1alpha in Common Human Cancers and Their Metastases[END_REF] F-FMISO remains one of the most widely used radiotracer, specifically for glioblastomas, as it provides balance between sensitivity, specificity, tumour accessibility, imaging time, and invasiveness 283,284 . Second generation nitroimidazoles for PET have been developed including [START_REF] Zhong | Overexpression of Hypoxia-Inducible Factor 1alpha in Common Human Cancers and Their Metastases[END_REF] that delayed imaging provides no advantages as compared to the short half-life of [START_REF] Zhong | Overexpression of Hypoxia-Inducible Factor 1alpha in Common Human Cancers and Their Metastases[END_REF] F, and rather demonstrated poor image resolution, higher doses of radiation, poor biodistribution, and reversible binding over time as compared to [START_REF] Zhong | Overexpression of Hypoxia-Inducible Factor 1alpha in Common Human Cancers and Their Metastases[END_REF] F-compounds 293,294 . Other organic makers include the copper complexed dithiosemicarbazones (Cu-ATSM) for which the mechanism of hypoxia specificity is debated 295,296 . Regardless, Cu-ATSM can be an effective tracer for global hypoxia and is especially useful in situations where blood flow may be limited 297 .

When compared to [START_REF] Zhong | Overexpression of Hypoxia-Inducible Factor 1alpha in Common Human Cancers and Their Metastases[END_REF] F-FMISO, Cu-ATSM uptake is significantly higher in the target tissue than in non-hypoxic areas and occurs at a faster rate in animal models (10 -15 min as compared to 2 -4 h) 298,299 . Although Cu-ATSM has been successfully applied to several tumoural types for the detection of hypoxia [300][301][302][303] , some preclinical data suggests that it may not be suitable for all tumour types 295,304,305 . Furthermore, opposite findings have observed between studies applying the same cell line to different animal species 306,307 .

PET organic markers can also indicate glucose metabolism (e.g. [START_REF] Zhong | Overexpression of Hypoxia-Inducible Factor 1alpha in Common Human Cancers and Their Metastases[END_REF] Ffluorodeoxyglucose [FDG]) (Fig. 23456789), or blood perfusion ( 15 O-labelled water). FDG is the most commonly used PET marker in oncology and, as previously discussed, under hypoxic conditions mitochondrial ATP synthesis is abandoned and glycolysis is used in compensation thus making the use of FDG a surrogate marker for tumour hypoxia 308,309 . However, as most cancer cells depend heavily on glycolytic ATP production even under well-oxygenated conditions, weak correlations between tumour hypoxia and FDG retention has been observed 308,309 , thus the potential of the marker is limited. On the other hand, perfusion can be estimated with PET by using [START_REF] Griffiths | Hypoxia-Inducible Factor-1α Expression in the Gastric Carcinogenesis Sequence and Its Prognostic Role in Gastric and Gastro-Oesophageal Adenocarcinomas[END_REF] O-labelled water. O 2 delivery to tumours occurs via the vascular supply, indicating that blood perfusion and hypoxia are related; poor perfusion will increase the degree of chronic hypoxia, and changes in perfusion are responsible for acute hypoxia 310 . However, clinical studies using this approach have produced conflicting results 311 ; high blood perfusion has been associated with metastasis and poor survival after radiotherapy 277 , rather than the low perfusion one would expect to be indicative of hypoxia.

Single-photon emission computed tomography (SPECT)

Whereas PET detects the emission of gamma ray pairs, the single-photon emission computed tomography (SPECT) technique simply relies on individually detected gamma ray photons. 123 I, iodinated azomycin arabinosides, was the isotope initially used for this purpose retention and hypoxia, as measured by the polarographic electrode 318 . Thus, SPECT provides the advantage of being much simpler, less costly to acquire, and the ability to use a wide range of available gamma-emitting isotopes, however, its intrinsic spatial resolution is lower than that of PET.

Overall, PET and SPECT provide highly sensitive, relatively non-invasive information on viable hypoxic cells and can demonstrate O 2 gradients with high sensitivity. However, the use of radioactive compounds, with short half-lives, severely impedes the usage of the technique, as well as the variable uptake of the compounds in patients and tumours.

Furthermore, one of the issues with PET markers is that the cells must be hypoxic for a significant period of time to be detected, which means that such markers are more likely to identify chronic rather than acute hypoxia. The major problem facing the application of PET and SPECT hypoxia markers however is one of resolution; often, the voxel size that would be identified in a PET scan is much larger than most of the hypoxic structures, thus the actual PET image does not accurately reflect the true hypoxia heterogeneity at the micro-regional level.

Magnetic resonance (MR) based methods

Having explored the developments, applications, advantages and limitations of electrode, immunohistochemistry, optical and nuclear based techniques for hypoxia analyses and imaging, the following sections will address the final technique: magnetic resonance (MR) based techniques as a major non-invasive technique capable of monitoring anatomy and tumour physiology. The phenomena of nuclear magnetic resonance (NMR) and EPR were discovered in the early 1940s, and was based on similar physical principles of interactions between protons or electrons with electromagnetic fields [319][320][321] . Notably, EPR spectroscopy utilises paramagnetic species containing unpaired electrons, such as free radicals and transition metals, whereas NMR spectroscopy detects the nuclei of non-zero nuclear spins such as 1 H, 31 P and [START_REF] Aebersold | Expression of Hypoxia-Inducible Factor-1alpha: A Novel Predictive and Prognostic Parameter in the Radiotherapy of Oropharyngeal Cancer[END_REF] C. The interactions of these electron or non-zero nuclear species with an applied magnetic field give rise to the characteristic longitudinal (T 1 ) and transverse (T 2 ) relaxation times associated with MR based methods.

ELECTRON PARAMAGNETIC RESONANCE (EPR)

Development of EPR probes for oximetry

EPR is a non-invasive MR based technique, capable of making repeated measurements and provides direct O 2 quantification. EPR is the resonant absorption of radiation by paramagnetic systems in the presence of an applied magnetic field, which causes a separation in the energy fields of the unpaired electrons (i.e. Zeeman splitting 322 ). Resonance absorption of electromagnetic radiation occurs when the applied energy exactly matches the energy level separation, and the degree of absorption is proportional to the number of free radicals in the material.

Notably, there are some differences between NMR and EPR. Firstly, as the magnetic moment of an electron is approximately 2000 times larger than that of protons, the extent of the external magnetic field required to separate the energies of the spin state to the same extent as that of protons, is correspondingly 2000 times lower in amplitude. Secondly, the spectral responses of the two spin systems also show some interesting differences. When perturbed from equilibrium by a pulse of radiation (at specific fields from microwave to RF), the spin system of protons returns to equilibrium with characteristic time constants, called T 1 and T 2 . For protons, T 1 and T 2 are in the range of 1.0 to 0.1 seconds, and in vivo the T 1 and T 2 of protons vary depending on the tissue. In the case of electrons, T 1 and T 2 are occurring very rapidly in the range of 5 microseconds to 10 nanoseconds.

EPR oximetry is based on the paramagnetic characteristics of molecular O 2 which, in its ground state, possesses two unpaired electrons. As mentioned in the working principles of MR, macroscopic magnetisation occur when there is a net absorption of energy upon application of an external magnetic field; this absorption by the electron species is what is monitored by the EPR instrumentation (Fig. 2345678910) and is converted into an EPR spectrum (Fig. 234567891011). This spectrum indicates the simulated absorption for a system of electrons in a varying magnetic field. Furthermore, it should be noted, that electrons are not solitary in systems but instead are associated with one or more atoms. This causes several consequences, the most important of which is hyperfine coupling. This phenomenon occurs when the magnetic moment of a nucleus with a non-zero spin affects any unpaired electrons associated with that atom leading to the splitting of the EPR resonance signal into doublets, triplets, etc.

This causes spectral lines to be either singular, or with several peaks and troughs.

Importantly, however, EPR cannot measure O 2 itself. In the gas phase, at very low pO 2 (< 1 mmHg) it is possible to record an EPR spectrum of molecular O 2 with a conventional EPR spectrometer 323,324 . However, the T 1 for molecular O 2 dissolved in fluids was found to be 7.5 ps 325 , making it much too short for measurement and making the direct measurement of O 2 impossible on current EPR spectrometers. Furthermore, in biological systems, direct EPR measurements cannot be achieved as the spectral lines are so broadened as to be undetectable. Hence the need for paramagnetic spin probes arose, whose measurements depend on T 1 and T 2 , and can provide a direct indication of the concentration of O 2 326,327,336,[328][329][330][331][332][333][334][335] . Particulate probes include lithium phatalocyanine (LiPc) crystals and carbon-based products. LiPc aggregates have a single sharp EPR line, the width of which is highly sensitive to pO 2 . LiPc crystals are implanted into the tissue of interest and the EPR spectra reflect the average pO 2 on the surface of each LiPc aggregate and enables the measurements of tumour pO 2 using only a few crystals 322,[339][340][341] . Carbon-based materials include coals (such as fusinite 342,343 and gloxy 344 ), chars 345,346 , and carbon black 347 (India ink). These materials have been extensively used for in vivo EPR oximetry, with India ink thus far being the only spin probe approved for in vivo clinical use [347][348][349][350] . It should be noted that all particulate EPR probes require implantation into the tissue of interest several days before measurements can occur to allow the tissue damage caused by its insertion to be minimised, and can only measure pO 2 content at their site of implantation 339,351,352 .

In contrast, soluble spin probes (e.g. nitroxides and triarylmethyl radicals) are chemically well-defined and can distribute evenly in tissues. Among soluble paramagnetic materials, two types of structures are particularly interesting: the nitroxides and the triarylmethyl radicals. Nitroxides are free radicals where the unpaired electron is delocalised between nitrogen and O 2 and include 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) and 4hydroy-TEMPO benzoate (TB) (Fig. 23456789101112). A number of different properties of nitroxides can be manipulated. For example, a charged nitroxide will not cross the plasma membrane and thus can be used to measure the concentration of O 2 in the extracellular compartment; a neutral nitroxide will be distributed throughout the intracellular and extracellular environments 353 . Moreover, nitroxides can be linked to carrier molecules to achieve tissue or organ selectivity 354,355 . The effect of O 2 on the LWs of these compounds is modest but sufficient for several applications. For most nitroxides, the LW in the absence of O 2 is approximately 1 Gauss. To increase the sensitivity to O 2 , it is convenient to use perdeuterated nitroxides 326,335 for which the LW is lower than 200 mG. It is also possible to use nitroxides with a resolved super-hyperfine structure to increase sensitivity to O 2 356 . This method is only valid at low O 2 because, at high a concentration of O 2 , the super-hyperfine structure is not resolvable. Furthermore, nitroxides have the limitation of being metabolically converted to diamagnetic hydroxylamines thus rendering it unable to produce an EPR signal [357][358][359][360] . This limitation is compounded in strongly reducing systems, such as the low pH microenvironment of hypoxic tumours. A possible solution for these shortcomings is to encapsulate nitroxides in lipophilic environments. As O 2 is more soluble in organic solvents than in water, an increase in sensitivity has been demonstrated using these systems 337 .

(a) (b)

The second interesting group of soluble spin probes are the triarylmethyl (trityl or TAM) radicals. These radicals were first demonstrated by Gomberg in 1900 361 , and were further developed by Nycomed Innovations in the 1990s 362 by eliminating hyperfine coupling with hydrogen nuclei to achieve a single sharp EPR line, and thus became known as tetrathia-TAM radicals (Fig. 2345678910111213). Salts of these trityl radicals have good water solubility as well as stability in the presence of reducing agents such as ascorbate and glutathione 363,364 .

The EPR spectrum of these compounds displays one single line 365 . An important characteristic of triarylmethyl radicals is the narrowness of their EPR LW (typically less than 100 mG). As the signal intensity is inversely proportional to the square of the EPR LW, the sensitivity of detection is particularly convenient for in vivo applications using these compounds. Recent development of triarylmethyl radicals has significantly expanded their potential as O 2 -sensitive free radicals in EPR [365][366][367][368][369][370][371] . In human blood, the stability of triarylmethyl radicals varies from a half-life of a few hours to one of more than 24 h depending on the particular structure of the compound. However, their oxygen sensitivity is affected by changes in viscosity and pH 364,371 . Additionally, when EPR measurements are performed in biological systems, the spin probes may lose their signal intensityand thus their oxygen responsivenessdue either to; the removal of the probes from the site of introduction due to the vasculature; or from the interactions with tissues and cells; or by reduction of the unpaired electrons by chemical reactions or protein binding 372,373 . Notably, the presence of several fluorine atoms within the TAM (F15T-03 374 ) radical enable it to be easily emulsified or encapsulated within lipophilic environments which confers an excellent environment for both O 2 solubility and protection from the biological milieu 366,368,375 .

EPR oximetry in oncology

EPR oximetry is a technique that can provide repeated assessments of average tumour pO 2 with minimal perturbation to the microenvironment 376 , and it has been extensively used for pO 2 measurements in animal models [377][378][379] . Importantly, EPR oximetry has been used to examine tumour re-oxygenation after radiation treatment in murine tumours 380,381 to determine tumour cure dependency on hypoxic fraction 333,382 and to examine changes in tumour oxygenation in response to vascular changes 383,384 . One of early applications of in vitro EPR oximetry was to measure the O 2 consumption rate in tumour cell suspensions 385 and to study O 2 transportation both across and within a lipid bilayer model membrane 338 . In vivo EPR applications, however, generally include the study of oxygenation of several tissues and organs (including the brain [386][387][388][389][390] , heart 329,[391][392][393][394][395][396][397] , liver 351,[398][399][400][401][402][403] , kidney 344,404 , gastrointestinal tract 405 , skeleton muscles 337,339,342,344,[406][407][408][409][410] , and skin 347,411,412 ) under various pathophysiological conditions (diabetes and wound healing 334,413,414 ) of which, by far, the largest application is tumoural oximetry.

In several studies, measurements of tumour oxygenation using EPR have been designed

as to show that a specific method or a newly developed paramagnetic material could be applied to tumour oximetry 326,351,408,[415][416][417] . Consequently, EPR oximetry has been compared

to other methods that provide direct or indirect measurements of tumour oxygenation, such as; polarographic electrodes 331,418 , the distribution of nitroimidazoles 419 , perfusion-based MRI techniques 389,420,421 , and pO 2 recordings using OxyLite® 384,422,423 . However, since oxygenation is of crucial importance in tumours to predict the outcome of a treatment, and since there is hope for manipulating local oxygenation to improve the efficacy of anti-cancer therapies; many efforts have been developed to apply EPR oximetry for the determination of tumour oxygenation in relation to a treatment regime.

For tumours, the ability to monitor changes in O 2 before and after treatment could have profound implications for the planning of effective treatment strategies 424 . In particular radiotherapy could benefit from modulated treatment based on regional variations in O 2 . One way to increase the tumour pO 2 is the breathing of O 2 enriched gas. Generally, carbogen, a gas containing 95% O 2 and 5% CO 2 , is preferred to pure O 2 to avoid the vasoconstrictive effects of O 2 . Breathing carbogen (possibly combined with nicotinamide administration) is now applied successfully in the clinic together with irradiation 425 . Currently, raising pO 2 by inhalation of hyperoxic gases is the most effective way to decrease diffusionlimited hypoxia. The increase in pO 2 in tumours induced by breathing carbogen (and often referred to as the carbogen challenge) was shown in several tumour lines using EPR spectroscopy 389,408,426,427 . This effect of carbogen on tumour pO 2 and on tumour regrowth delay is particularly interesting as a positive control for comparing the efficacy of other types of treatments 384,422,423 . Carbogen is also effective in human tumour models and patients as

shown by the use of various different measures of tissue oxygenation 252,[428][429][430][431][432][433][434] . Most studies found a significant decrease in hypoxia irrespective of the tumour type or method used.

However, a few studies have observed no effect or even a worsening of the oxygenation status 435,436 . These studies of tumour oxygenation and re-oxygenation using EPR oximetry have been designed using both particulate and soluble spin probes. However, as mentioned previously, both categories have their advantages and disadvantages. In the case of tumour oximetry, a need for new spin probes which can meet the main requirements; excellent O 2 sensitivity in vivo with a narrow single EPR spectrum line which linearly corresponds to O 2 concentration, preparations which are stable and consistently produce similar products, good stability in biological media thus presenting no pharmacotoxicity issues, and capable of performing immediate measurements throughout the tumour mass; is of great need.

Unfortunately, the particulate spin probes do not meet all these requirements but the soluble spin probes meet several. Both nitroxides and triarylmethyl radicals are chemically welldefined with well-established synthesis protocols, and benefit from being encapsulated as this confers protection to the radical (i.e. avoidance of metabolic conversion) as well as eliminating any possible toxicity the probe might confer to the tissue. Furthermore, nitroxides, such as TEMPO and TB, and triarylmethyl radicals, such as TAM (F15T-03), are capable of being encapsulated within a lipophilic environment (determined by their partition coefficient [log P] value) which is highly advantageous as this increases the sensitivity of the EPR radical because O 2 is highly soluble in lipids. A suitable encapsulation device is thus a priority. Several studies have involved the encapsulation of nitroxides within various carriers including microspheres, micelles, liposomes, and dendrimers [437][438][439][440][441][442][443][444] , as well as the emulsification and encapsulation of triarylmethyl radicals in various organic materials including perfluorocarbons, liposomes, and even hydrogels 439,[445][446][447][448][449] . These studies all indicate that the EPR radicals benefit from encapsulation. However, not all these encapsulation and emulsification methods can be applied in vivo as some concerns of biosafety 450 and sensitivity have been noted.

It is thus, to this end, that this body of work introduces the use of the biocompatible nanocarrier lipid nanocapsules (LNCs) as an encapsulation tool in section 2.6.

MAGNETIC RESONANCE IMAGING (MRI)

Development of MRI for determining oxygenation

MRI is based on the ability of hydrogen atoms to align and spin around an applied magnetic field, . Upon application of a transverse RF pulse ( ), these protons are perturbed from . The subsequent process through which these protons return to their original state is referred to as relaxation. Two independent processes, T 1 relaxation and T 2 relaxation, can be monitored and converted to generate an MR image. Notably, local variation in relaxation is due to the chemical and physical nature of the sample. In the case of T 1 relaxationalso termed spin-lattice or longitudinal relaxationthese perturbations are thermal based, in that they involve the transfer of energy from the nuclear spins to the surrounding molecular environment, which results in the recovery of the longitudinal magnetisation to its equilibrium. This energy transfer occurs because there are fluctuating fields associated with the 'lattice' or surrounding molecules. Conversely, T 2 relaxationoften called spin-spin relaxationoccurs due to internuclear and electron-nuclear interactions which result in the exponential decay of the transverse magnetisation. In a system of identical non-interacting nuclei such decay would not occur but, in reality, different nuclei precess at slightly different frequencies as a result of interactions between neighbouring nuclei, thus leading to a loss of overall transverse magnetisation. Notably, this decrease in signal occurs rapidly because the local magnetic field is always heterogeneous; either because the applied field is non-uniform or because of spatial variations in magnetic susceptibly. This is taken into account by another relaxation time, T 2 *, and its relaxation is governed by the free induction decay (FID) resulting from the inhomogeneities of the magnetic field.

MR procedures for measurement of the oxygenation status of tumours have the advantage of allowing the assessment of hypoxia in deep-seated tumours in a non-invasive manner which does not disturb vasculature, and enables repeated measurements. MR techniques for hypoxia determination include perfusion and probe-based (both exogenous and endogenous) imaging and spectroscopy.

Perfusion-based MR methods

BOLD, or blood-oxygen level dependent, contrast is a functional MRI method able to measure blood oxygenation using gradient echo sequences sensitive to changes in transverse decay (also known as the effective T 2 or T 2 *) 451 . Originally, the BOLD approach was developed to investigate cerebral activity but, recently, its use has been extended to the assessment of tumour oxygenation in response to hyperoxic gas challenges (e.g. carbogen) 296,452 . BOLD-MRI relies on red blood cells to be delivered to the tissue of interest and provide information regarding tissue oxygenation 453 and is based on the paramagnetic nature of deoxyhaemoglobin; as the O 2 saturation of the haemoglobin increases, the iron within the haeme subunit changes from a paramagnetic high spin state, under low pO 2 , to a diamagnetic low spin state under high pO 2 . Therefore, changes in deoxyhaemoglobin content in blood will influence water relaxation (particularly T 2 *), enabling blood oxygenation levels to be measured by MRI. Thus, BOLD-MRI is sensitive to oxygenation variation in the vascular compartment 454 , as well as having high spatial and temporal resolution 455 . The major drawback, of course, is that BOLD does not provide actual pO 2 data but rather the change of O 2 tension in the vasculature, which is not directly correlated to blood oxygenation in tissue with poor blood perfusion such as in solid tumours and implantations 456 . Furthermore, a quantitative relationship between the BOLD effect and pO 2 has not been found, due to the complex relationship between these two parameters 457 . Indeed, in addition to oxygenation, the BOLD effect is influenced by other factors, including blood flow, haemoglobin levels, haematocrit levels, vasculature, temperature, and pH 454,455,[457][458][459] . These circumstances radiotherapy outcome for patients with cervical tumours, concluding that patients with hypoxic tumours has a worse response to radiotherapy [468][469][470] .

DCE-MRI is a rapid, minimally invasive technique with which repeated whole body imaging can be performed enabling the tracking of tumour heterogeneity with sub millimetre sized voxels for easily tracking chronic and acute hypoxia. However, although this technique has been used both pre-clinically and clinically, the technique provides only descriptive data pertaining to blood flow within the vascular compartment but not pO 2 measurementsmuch like BOLD. Furthermore, as with all the perfusion techniques, a complicated relationship between oxygenation, blood flow and blood supply exists in solid tumours, which must be accounted for using pharmacokinetic modelling to correctly interpret results. . The same paramagnetic effect has been reported in other molecules, including water [472][473][474] . However, in the latter case, many other factors, including metal ions, pH and temperature, also induce changes in water relaxivity in vivo, making the results observed more difficult to interpret exclusively in terms of pO 2 variations. In contrast, T 1 of PFCs is not usually affected by hydrophilic charged species 475 as these hydrophobic molecules do not mix with the surrounding aqueous phases, instead forming droplets that inhibit ions from entering the hydrophobic core 476 . Notably, O 2 solubility is greater in PFCs than in water, meaning these compounds could be considered as molecular amplifiers of O 2 tension but the differences in O 2 solubility between PFCs and the surrounding medium, pO 2 between both phases will become the same after a short (usually seconds) equilibration interval 473 . As a consequence, PFCs exhibit high sensitivity to O 2 changes and their detection and have been investigated for the measurement of oxygenation in vivo using O 2 maps 473,477,478 . Standardisation of PFC probes for oxygenation determination in tumours have been attempted with microelectrodes but discrepancies have been found [479][480][481] .

Probe-based MR methods

Probe

Several PFCs have been developed, including the cyclic hexafluorobenzene (HFB) 482 and perfluoro-15-crown-5-ether (PFCE) [483][484][485] (Fig. SR-4554 has shown good results at detecting hypoxia in a Phase I clinical study by Lee et al. 495 , and is being further developed for the detection of tumour hypoxia in some patients.

However, the toxicity profile of nitroimidazole drugs is the major drawback; misonidazole showed neuropathy and acute central nervous system toxicity 496 . To circumvent some of the issues presented by PFCs, Kodibagkar et al. 474 developed a proton analogue to PFCs, hexamethyldisiloxane (HMDSO), as a novel 1 H MRS oximetry probe. HMDSO shares some important properties with PFCs: it is highly hydrophobic and non-toxic, shows high O 2 solubility, is biochemically inert in vivo, and is readily available and relatively inexpensive 497 . However, HMDSO also presents some disadvantages when compared with PFCs, mainly because of the strong background signal in 1 H NMR, a circumstance not occurring in [START_REF] Harris | Hypoxia -A Key Regulatory Factor in Tumour Growth[END_REF] F NMR. To this end, proton imaging of siloxanes to map tissue oxygenation levels (PISTOL) was developed 498 but was hindered by poor spatial resolution.

Overall, PFCs enable the dynamic imaging and mapping of pO 2 in tumours but the utilisation of [START_REF] Harris | Hypoxia -A Key Regulatory Factor in Tumour Growth[END_REF] F oximetry requires that clinical scanners are equipped with a specific [START_REF] Harris | Hypoxia -A Key Regulatory Factor in Tumour Growth[END_REF] Ftuned probe. Furthermore, PFCs are subject to flow artefacts and the oxygen sensitivity of some [START_REF] Harris | Hypoxia -A Key Regulatory Factor in Tumour Growth[END_REF] F MR imaging compounds are easily affected by temperature, dilution, pH, common proteins, and blood 499 . After intravenous injection, the majority of the PFC contrast agent is extensively ingested by the reticuloendothelial system (RES), and the slow clearance can have adverse effects. Intratumoural injection can also be risky as PFC emulsions could accidentally be injected into tumoral vein, leading to embolism 486 . Furthermore, although the technique allows for high anatomical resolution it cannot distinguish between necrotic areas and offers low sensitivity.

To circumvent the need for specially equipped scanners and the introduction of an exogenous probe for oximetry imaging, endogenous nuclei have been explored including the change in water T 1 relaxation rates in tissues and CH 2 in the form of lipids. This technique includes the use of oxygen-enhanced MRI (OE-MRI). OE-MRI relies on the quantification of variation in water T 1 relaxation rate by the paramagnetic change in O 2 dissolved in blood plasma, interstitial tissue fluid and intracellular water under 100 % O 2 breathing. This technique can identify spatial heterogeneity in tumour hypoxia for renal 500 as well as murine glioma and human glioblastoma multiforme carcinomas 501 . OE-MRI enables repeated measurements with assessment of chronic and acute hypoxia. However, although the anatomical spatial resolution is well-defined and is not influenced by perfusion changes in the vascular compartment or haematocrit (such as BOLD); the technique is unable to identify necrotic regions, and relies on the use of hyperoxic gases which can be vasoactive and lead to difficulty in interpreting results. Furthermore, this technique is severely hindered by the heterogeneity of tumours; regions of tumours do not respond to gas challenges as the vasculature has collapsed making the gases inaccessible to the tumour 502 .

It was to address this gap in tumour oxygenation determination that the use of CH 2 in the form of endogenous lipids were used to map oxygenation by T 1 relaxation rates (MOBILE; mapping of O 2 by imaging lipids relaxation enhancement) was developed.

Mapping of O 2 by imaging lipids relaxation enhancement (MOBILE)

While T 2 * is sensitive to the relative changes in Hb/HbO 2 in vessels (exploited in BOLD 162,503 ), it cannot provide a quantitative relationship between signal intensity and true changes in tissue pO 2 . Likewise, although the OE-MRI method provides an alternative to the BOLD method as a non-invasive method for measuring O 2 level variations, unfortunately this Here, the Look-Locker correction 510 is applied to calculate T 1 from the effective T 1 of the tissue, T 1 * with T 1 * < T 1 . This correction is implemented to compensate for the T 1 shortening induced by imperfect inversion pulses. The T 1 is then calculated for each pixel using the following equation:

T 1 = T 1 *(B/A -1).
In vivo assessment was achieved using several models of pathogeneses including severely hypoxic tumours, peripheral ischemia, cerebral ischemic stroke, and liver steatosis.

The group also used normal human volunteers to demonstrate oxygenation mapping of adult brains before and after a hyperoxic carbogen challenge. Using MOBILE, the researchers were able to show the much greater sensitivity of the R 1 of lipids than water to O 2 variation, and succeeded in determining pO 2 values, comparable to OxyLite® and EPR oximetry methods, for all in vivo models (Fig. 2345678910111213141516).

In fact, for the tumour oxygenation experiments, the R 1 of lipids was more sensitive to O 2 variation by a factor of 4.8 as compared to the R 1 of water. Notably, the correlation within the same tumoural experiments between the R 1 of H 2 O and pO 2 was not significant, whilst a positive linear significant relationship was found for the R 1 of lipids and pO 2 . Unfortunately, however, the MOBILE technique can only be applied to tissues which contains lipids at a sufficiently high enough intrinsic content. In the case of the aforementioned study, the mean value of total lipid content for human embryonal carcinoma-induced tumours at 6 mm in length was 106.1 ±20.4 µg/mg, and the normal adult human brain was 93.6 ±11.8 µg/mg.

Thus, a signal-to-noise ratio (SNR) limitation is apparent; a sufficiently large enough amount of lipids is required in the tissue of interest otherwise the signal acquired by the MOBILE sequence would not be able to discern the lipid R 1 on a pixel-to-pixel basis as voxel size in MRI are limited. Thus, if no lipids, or insufficient amounts of lipids, occur in the tissue then

LIPID NANOCAPSULES (LNCS)

Formation and development of LNCs

Nanotechnology has emerged as an outstanding tool for the development of technologies for diagnosis, treatment and prevention of cancer 511 . Their applications in cancer treatment include molecular imaging, early detection, diagnosis, targeted therapy, overcoming drug resistance, and cancer bioinformatics 512-520 . Several nanomaterials have been developed as bioanalytical platforms in cancer for the detection, measuring and/or imaging of pH 521 , apoptosis 522,523 , ROS [524][525][526] , and oxygen. Bioanalytical sensors specifically designed for oxygen determination include inorganic (e.g. quantum dots) and organic (e.g. polymer-based) nanoparticles which can be conjugated with phosphorescent metalloporphyrins 262,527-530 or near-infrared oxygen-sensitive dyes 531,532 , wherein the nanoparticles can be embedded in hydrophobic matrices (such as organically modified silica) [533][534][535][536] or encapsulated into liposomes 537 or micelles 538 . An interesting application of nanotechnology is the preparation of dual-purpose nanomaterials used for simultaneous diagnosis and therapy. This dual-purpose targeted research has given rise to a new term:

theranostics [539][540][541][542] . It is assumed that a combined technique will result in the acceleration of drug development, improved disease management, reduced risks and reduced cost.

Nanocarriers for drug delivery generally consist of 2 main components: the nanoparticle itself, which is used as the carrier agent, and the drug 543 . The drug can either be adsorbed, dissolved, or dispersed throughout the nanoparticle complex or, alternatively, it can be covalently attached to the surface. Studies using paclitaxel have shown that when compared with the conventional formulation, the nanoparticle formulation of the drug increases both its cytotoxicity profile in cell culture and its therapeutic efficiency in a living animal model 544 . This has been attributed to the nanoparticle formulation having greater bioavailability and a longer sustainable therapeutic time, which allows the drug concentration to remain above the minimum effective value for an extended period of time. There are several types of nanoparticle systems that have been used as carriers including liposomal 545,546 , solid lipid 547-549 , polymeric [550][551][552] , mesoporous silica [553][554][555] , and inorganic nanoparticles 556,557 . Among these carriers, Heurtault and colleagues 558-560 developed a nanoparticle characterised as a hybrid between polymer nanocapsules and liposomes called lipid nanocapsules (LNCs). These nanoparticles are classified as spherical, nanometre-sized biomimetic colloidal lipid emulsions, the core of which is composed of an oil surrounded by a thin amorphous shell (Fig. 234567891011121314151617). These nanoparticles can be prepared in various sizes using the low-energy phase inversion temperature (PIT) method which employs no organic solvents.

LNCs are composed of a medium chain triglyceride core (i.e. lipids) known under the commercial name of Labrafac®, surrounded by a thin capsule consisting of non-ionic and amphiphile surfactants. This lipidic core is responsible for the size of the LNCs, as well as providing the oily reservoir in which lipophilic drugs are encapsulated. The surfactants of the system, both the non-ionic Kolliphor® and the lipophilic Lipoïd®, are the major influencers of LNC formation and stability 561,562 . Additionally, Kolliphor® provides a shell separating the loaded agent from the outside environment by a coating of polyethylene glycol (PEG) 561,563 . PEG is widely used material, as its addition lends increased stability to nanoparticles in biological fluids by creating a dynamic cloud of hydrophilic and neutral chains at the surface that reduces protein opsonisation, thereby preventing rapid clearance from the circulation system by mononuclear phagocytic cells from the RES 564,565 . A major drawback often arising in intravenous injection of drug carriers is the RES recognition of the nanoparticles and their subsequent removal from the vasculature system and accumulation in the liver and spleen.

The PEGylation of a nanoparticle's shell thus serves to minimise the interaction of the nanoparticles with the RES by repelling plasma proteins 566 , achieving the so-called Stealth® nanosystems [567][568][569][570][571] . This enables LNCs to have an increased half-life within the blood and cross certain biological barriers 572 . Notably, all the constituents of LNC synthesis are biocompatible and FDA approved.

During the phase inversion temperature (PIT) process (Fig. 2 to the study) is heated under magnetic stirring from room temperature to 95 °C. This temperature is above the PIT and thus a water in oil (w/o) emulsion is obtained. This is followed by cooling to 50 °C (thus below the PIT) leading to the formation of an o/w emulsion. This temperature cycling is repeated several times, crossing the phase-inversion zone (PIZ) between high temperature and cooling repeatedly. During the final cooling phase, an irreversible cold dilution (4 °C) is added to the mixture during the PIZ (i.e. approximately 72 °C). This is done to break the microemulsion system obtained in the PIZ, and leads to the formation of stable nanocapsules 573 . Therefore, the formulation process is a function of the physiochemical properties of the components since the cycling of the temperature during the PIT method increases the quality of the nanoemulsions in terms of size and PDI by increasing the amount of surfactant at the water/oil interface 574 . Furthermore, it was necessary for the LNC formulation to be osmotically adjusted for in vivo administration in this thesis 562,575 .

This involved the dialysis of the LNC formulation (MWCO = 100 kDa) with magnetic stirring, and occurred over several hours with continual water changes to allow for the maximal escape of the free components. Then, to reobtain the original concentration of LNCs, evaporation with N 2 was performed (Fig. 2 Due to their very small size, in the range of 10 -100 nm, and their capacity to encapsulate lipophilic agents, LNCs have undergone extensive investigation as drug carriers for improving therapeutic agent delivery 558,573,[576][577][578][579][580] . LNCs have been applied to treatment of tumours by delivering known anti-cancer agents, such as docetaxel, paclitaxel, etc., loaded within the nanoparticles to malignancies including breast cancer, melanoma, small cell lung cancers, metastatic cancers, and most notably, glioblastomas 568,[581][582][583][584][585][586][587][588] . Glioblastomas are highly malignant, aggressive and drug-resistant malignancies of the central nervous system which are often associated with high morbidity and mortality, and dismal prognosis [589][590][591] .

Numerous studies involving LNCs loaded with various anti-cancer drugs specifically for glioblastoma treatment have been published [583][584][585][592][593][594][595][596][597][598] . These treatments have explored various strategies, including; passive 594,599-602 and active 586,592,[603][604][605] targeting, direct introduction of the drug-loaded LNCs into the glioblastoma tumour using a convectionenhanced delivery (CED) protocol 585,606 , the use of hydrogels imbedded with various drugloaded LNCs specifically to address the recurrence issue of glioblastomas 596,597,607,608 , as well as the introduction of drug-loaded LNCs into stem cells used for migration through the bloodbrain-barrier for targeting glioblastoma 593,609 . LNCs have also been employed as carriers; to enhance the effect of radiopharmaceuticals for cancer treatments 594,[610][611][612] ; to test novel anticancer and cancer associated-symptom drugs including curcumin, artesunate, etc. 560,595,598,608,[613][614][615][616] ; as an oral 617,618 and aerosol 619 delivery nanomaterial; and as a diagnostic tool for monitoring hypoxia in glioblastomas 485 . All of these advances have been due to several factors of LNCs. Firstly, LNCs are synthesised with an organic solvent-free formation process using biocompatible constituents which are all FDA approved for oral, topical and parenteral in vivo administration in both animal models and human patients 620,621 . Also, LNCs exhibit adjuvant effects, such as the inhibition of the plasma membrane multi-drug resistance pump (such as glycoprotein P; P-gp), and generally have a slightly negatively charged surface, due to the phospholipid molecules and the presence of PEG dipoles in their shell. This enables the LNCs to cross membranes, become internalised within cells, and enables endo-lysosomal escape (ensuring the nanoparticles are not destroyed by the cells) thus offering increased bioavailability and protection of the LNCs' payload, as well as enabling the nanoparticles to avoid the immune system. LNCs also have an excellent drug loading capacity, as their oily core allows for the encapsulation of lipophilic and amphiphilic drugs, agents or compounds. Lastly, and most importantly for this thesis, LNCs have been shown to be permeable to O 2

INTRODUCTION TO CHAPTER 3

From reviewing the literature in Chapter 2, it is apparent that EPR is an excellent tool for investigating tumour hypoxia as it is capable of providing non-invasive and repeatable oxygen measurements 341,622 . The application of EPR oximetry is accomplished through the use of paramagnetic spin probes. As described, two classes of oxygen-sensitive EPR spin probes have been developed both of which have certain advantages and drawbacks. In particular, soluble spin probes (e.g. nitroxides and trityl) have been chemically well-defined and demonstrated to distribute well within samples 322 but suffer when used in biological media due to signal intensity loss (from metabolic conversion and/or washout from the tissues) 373,623 .

In this chapter, these limitations were addressed by the encapsulation of spin probes within LNCs. Encapsulation would provide several advantages including; improving the oxygen sensitivity 337,447,624,625 , creating a defined microenvironment for the spin probe ensuring specificity, and providing a shell which might protect from bio-reductants 337,363,626 as well as prevent biocompatibility issues. In this regard, LNCs was good a candidate as not only could they provide the aforementioned advantages, but they are prepared using FDA approved constituents and have previously been employed in several studies of tumour therapeutics as an encapsulation agent (see Chapter 2). Thus, the incorporation of EPR spin probes within the oily-core of the LNCs were hypothesised to be able shield the spin probe from the tissue environment and provide an oxygen permeable shell, thus enabling the oxygen sensing mechanism of the EPR probe loaded-LNCs.

In this chapter, published data regarding the synthesis, characterisation and development of trityl-loaded LNCs as an EPR oxygen nanosensor is presented in section 3.2.

The trityl-loaded LNCs (referred to as TAM-LNCs) were evaluated in vitro and in vivo to determine its O 2 sensing capabilities, and included the use of two animal models representing normal-functioning tissue and pathological tissue, i.e. subcutaneous fibrosarcoma. This tumoural model has well-characterised vasculature, a known response to the carbogen gas challenge, and established pO 2 values previously measured by EPR 384,422,627 . In section 3.3, unpublished data regarding the findings from the article in section 3.2 are presented. Herein, the kinetics of the TAM-LNCs within in vivo models is investigated regarding the sensor's pO 2 sensing reversibility and the possibility of an O 2 reservoir effect occurring in vivo. 337,372,375,406,447,635,636 .

The aim of the present study was to explore the possible use of lipid

MATERIALS AND METHODS

Materials

Labrafac® WL 1349 (caprylic-capric acid triglycerides) was purchased from Gattefossé S.A. (Saint-Priest, France).

Preparation of EPR probe encapsulated lipid nanocapsules

The preparation of nitroxide TB-and TAM-loaded LNCs (henceforth referred to as TB-and TAM-LNCs) was based on a phase inversion method 559 . In brief, the EPR probe was weighed, and dissolved in Labrafac® (oil phase) to reach a final concentration of 10 mM which produced a sufficient signal-to-noise ratio but did not exceed the self-induced line broadening threshold 367,639 . 1.028 g of Labrafac, containing the respective probe, was

Size characterisation

The average hydrodynamic size and polydispersity index (PDI) of the probe-

Osmolarity adjustment of EPR probe loaded LNCs

Dialysis of the synthesised probe loaded LNCs (4 mL) was performed using dialysis tubing (Spectra Biotech Cellulose Ester Dialysis Membrane, MWCO = 100 kDa) for 24 h against pure water (obtained through a Milli-Q apparatus), changing the water hourly for the first 6 h. Then, to reobtain the original volume of LNCs ( 1mL), the dilution factor of the dialysed solution was calculated and evaporation, using N 2 , was applied under magnetic stirring. Once the initial volume was obtained, evaporation was ceased and 30 µL of a salt solution (320 mg/mL) was added to adjust the osmolarity of the formulation. Osmolarity measurements were performed using a 5520 Vapro vapour osmometer from Wescor (Logan, Utah, USA).

2.3

In vitro EPR measurements

In vitro EPR spectra acquisition

EPR spectra were acquired with a Bruker

Ascorbic acid assay

The protective properties of LNCs were

In vitro oxygen calibration curve

A calibration curve (LW as a function of

2.4

In vivo experiments

Animal models

The syngenic FSaII fibrosarcoma murine challenge.

Statistical analysis

For the in vitro characterisation of probe loaded-LNCs, the data are presented as mean ±SD from triplicate experiments.

For in vivo pO 2 measurements with TAM-LNCs, the paired Student's t-test was implemented, wherein a p value of < 0.05 was considered significant.

RESULTS AND DISCUSSION

Probe loaded-LNCs characterisation

The phase inversion process was exploited to obtain lipophilic EPR probes encapsulated within the lipidic core of the LNCs. The physicochemical properties of different types of the LNC preparations are presented in Table 3- This is consistent with an encapsulation of TB into the lipidic core of the LNCs, with a slow exchange of a small fraction at the interfacial surface leading to a slow reduction of the nitroxide. TAM-LNCs were not tested using this protocol as trityls are not sensitive to the presence of ascorbic acid.

In vitro EPR oximetry with TBand TAM-LNCs

The EPR LW (peak-to-peak) of TB-and TAM-LNCs increased linearly with pO 2 .

From nitrogen to air, the TB-LNCs LW

In vivo measurements using TAM-LNCs

KINETICS OF TAM-LNCS IN VIVO

Introduction

In this section, the unpublished work pertaining to the above article in section 3.2 will be discussed. Herein, the in vivo studies using normal and tumoural tissues were expanded upon to elucidate the ability of TAM-LNCs (Fig. 345678) to reversibly respond to O 2 variation in tissue by using the carbogen challenge 323,646,647 and reversible occlusion of the femoral artery within the mouse muscle. Additionally, the possibility of LNCs introducing auxiliary O 2 within the tissue and thus acting as an "oxygen reservoir" was explored.

Materials and Methods

Synthesis and characterisation of TAM-LNCs

Osmolarity adjusted LNCs loaded with TAM (herein referred to as TAM-LNCs) were prepared using the phase-inversion temperature (PIT) process 559 and the osmolarity adjustment protocol described in section 3.2. The average hydrodynamic size and polydispersity index (PDI) of the LNCs were determined by dynamic light scattering using a Malvern Zetasizer® (Nano Series DTS 1060, Malvern Instruments S.A., Worcestershire, UK).

In vitro EPR spectra acquisition

EPR spectra were acquired with a Bruker EMX-Plus spectrometer (Bruker, Rheinstetten, Germany), operating in X-band (9.85 GHz) and equipped with a PremiumX ultra low noise microwave bridge and a SHQ high sensitivity resonator. General settings were as previously described in section 3.2 for the TAM EPR probe.

In vivo EPR measurements

Animal models

For in vivo studies, two groups of male C3H mice (Janvier, Le Genest Saint Isle, France) were used; one group for intramuscular experiments (n = 13) and another for intratumoural (n = 12).

Intramuscular and intratumoural EPR measurements with TAM-LNCs

A bolus injection of 50 µL of TAM-LNCs were introduced intramuscularly (n = 10)

or intratumourally (n = 9) into animals as previously described in section 3.2, and EPR spectra were recorded as per the following protocol (Fig. 3456789); firstly, during air breathing;

then after 20 min of carbogen breathing; then, following a return to air breathing for 10 min, an occlusion of the ipsilateral femoral artery of the gastrocnemius muscle was applied for 5 min using a rubber band tied tightly above the knee of the animal. Total time taken for the full experiment was approximately 40 min.

EPR spectra were acquired in vivo with a low-frequency microwave bridge operating at 1.2 GHz L-band EPR spectrometer (Magnettech, Berlin, Germany) with an extended loop resonator possessing an inner diameter of 12.5 mm, and a thickness of 2 mm. General settings were as follows: incident microwave power of 2.8 mW; modulation amplitude of 0.019 mT; modulation frequency: 100 kHz; sweep width: 1.0 mT; scan time: 30 s.

Evolution of LW over time

A 50 µL bolus injection of TAM-LNCs was injected intramuscularly (n = 3) or intratumourally (n = 3) in anaesthetised animals. The animal was placed as described previously in section 3.2 within the L-band EPR spectrometer.

Results

Intramuscular and intratumoural EPR measurements with TAM-LNCs

To further explore TAM-LNCs as an oxygen sensor, in vivo experiments were applied (Fig. 345678910). Similarly, the LWs were measured for subcutaneous tumours, 0.745 ±0.035 G during air breathing, 0.912 ±0.196 G after the carbogen challenge, and 0.649 ±0.054 G after occlusion (Fig. 345678910). For both muscles and tumours a significant difference between the means was determined using the one-way ANOVA, p < 0.0001 and p < 0.0014, respectively). Between the air and carbogen breathing a difference was noted (p < 0.0001 and p < 0.05, respectively) and between the carbogen breathing and occlusion (p < 0.0001 and p < 0.01, respectively). However, no significant difference (p > 0.

Evolution of LW over time study

To address the issue of the 'reservoir effect', wherein O 2 is hypothesised to accumulate within the lipidic core of the LNCs (during formulation and handling) and be subsequently deposited in vivo during the LNC deposit; a kinetics study was done to note the evolution of the LW over time (Fig. 34567891011).

Indeed, if the 'reservoir effect' was occurring, a decrease and eventual plateau in the change in LW would be apparent over time thus indicating the diffusion of O 2 from the LNCs to the surrounding tissue. As shown in Figure 3-11, the LW did not decrease with time thus excluding the 'reservoir effect'.

Discussion and conclusions

A significant difference was observed in LWs measured during air and carbogen breathing as previously noted in the published article. Notable in this study, however, was the application of an ipsilateral occlusion of the femoral artery promoting the decrease in arterial blood flowand thus the pO 2near the TAM-LNCs. Here, it was demonstrated that the TAM-LNCs could respond significantly and reversibly to a change in its O 2 environment in both a normal and tumoural tissue environment (Fig. 345678910). Unfortunately, no significant difference was noted between the air breathing and the occlusion LWs in of the in vivo model group. This is surprising, as lower LWs were expected during occlusion due to the halt of arterial blood flow, and thus oxygen, towards the injected TAM-LNCs. Furthermore, a potential limitation for the use of LNCs could be the 'reservoir effect' where an import of O 2 is dissolved in the lipid core which influences the pO 2 measurement during the experimental studies. If the 'reservoir effect' was occurring, a decrease in the LW would be apparent over time due to the distribution of O 2 from the LNCs into the tissue as the system equilibrates. However, this was not observed (Fig. 34567891011). Instead a relatively stable difference throughout the experiment was noted, indicating this was not necessarily a factor attributing to the TAM-LNCs overestimation of pO 2 . Another aspect contributing to the overestimated pO 2 values could be the system itself. Indeed, unlike for solid EPR probes, e.g. LiPc, the liquid probes are susceptible to viscosity; such as the difference between tissue and an aqueous environment 367 , and the contribution from the choice of oil used (i.e. Labrafac®).

Labrafac® was used as the oily-core constituent of LNCs, as described by Heurtault et al. 558 , and is considered biologically inert and medically safe for use 620,621 but has a relatively high viscosity (25 -33 mPa.s, 20 °C) 648 which could have a broadening effect on the EPR LW, thus effecting resolution and the conversion between LW and pO 2 .

In conclusion, LNCs were capable of encapsulating lipophilic EPR probes, including both nitroxides and trityl. In vitro experiments, however, indicated that only TAM-LNCs could be used with in vivo animal models as the nitroxides suffered from EPR signal loss and incomplete incorporation of the EPR molecule within the LNCs. TAM-LNCs performed well as an oxygen sensor in both a normal tissue environment and in a pathological environment, with LNCs providing protection and in vivo biocompatibility for the EPR probe, and an O 2 permeable environment thus enabling the demonstration of its ability to respond to variations in pO 2 . As a matter of fact, the LNCs were responsive enough to enable a reversible measurement of pO 2 within tissues. But an over estimation of pO 2 was apparent, thus only relative change in the level of oxygenation could be noted. These developments and results with TAM-LNCs warrant further investigation of the nanoplatform as an oxygen sensor.

INTRODUCTION TO CHAPTER 4

MRI is a non-invasive technique used extensively in oncology, as noted in Chapter 2, for imaging anatomical features and monitoring the growth of tumours. In the case of perfusion-based methods, however, this technique does not measure pO 2 directly. Furthermore, regarding probe-based methods such as PFCs, these techniques rely on specialised MR equipment. Thus, a different strategy for monitoring tumoural hypoxia via the mapping of oxygen by imaging lipid relaxation enhancement (MOBILE) was developed. This sequence enables the imaging of pO 2 variations within various in vivo models (see Chapter 2, section 2.5.2). However, due to signal-to-noise issues which require the intrinsic lipid content to be sufficiently high enough for the MOBILE sequence to produce a map of the area, the MOBILE sequence cannot be applied to all tissue. Thus, in this chapter, the limitation of the MOBILE sequence is addressed by the use of LNCs. LNCs consists of a thin polymer coating surrounding a lipidic core, produced from entirely biocompatible components approved by the FDA for in vivo usage, and have been employed in treatment strategies of several cancer types, most notably the highly malignant and treatment resistant glioblastoma. The lipidic core of LNCs makes them an excellent tool for assessing and imaging O 2 in tissue, in regard to the MOBILE sequence, as the solubility of O 2 is much greater in lipids as compared to water 506 . Thus, the use of LNCs to increase the SNR will enable oxygenation mapping within tissues regardless of their intrinsic lipidic contents and will provide the basis of its usage as a theranostic tool.

In this chapter, data submitted for publication regarding the development of LNCs as an MRI O 2 sensor using the MOBILE sequence is presented in section 4.2. Herein, LNCs were evaluated for their ability to improve the SNR within both normal and pathological tissues, and produce pixel-by-pixel analysis of oxygenation maps. Importantly, the effect of using different techniques (i.e. bolus injection vs. convection-enhanced delivery [CED]) for introducing the LNCs into the tissues was examined. Of note was the choice of pathological tissue used; the subcutaneous fibrosarcoma and the intracerebral glioblastoma models. As previously described, the subcutaneous fibrosarcoma model is a well-known model in regards to its pO 2 values and response to the carbogen gas challenge 384,422,627 . Likewise, the intracerebral glioblastoma model is known to be highly vascularised yet hypoxic 649,650 . In the last sections, unpublished data regarding the in vivo fate of LNCs was investigated (section 4.3).

INTRODUCTION

Hypoxia is a key feature in solid tumours caused by abnormal and torturous vasculature which are unable to meet the O 2 demand of the tumour mass. This oxygen deprivation causes; a change in gene expression; leading to more aggressive tumours; and results in metastatic progression and resistance to radiation and chemotherapy [START_REF] Vaupel | Tumor Hypoxia: Causative Factors, Compensatory Mechanisms, and Cellular Response[END_REF]651 . Thus The drawback of this method however, is that there is not always a sufficient amount of intrinsic lipids in the tissue of interest to enable the use of the MOBILE method. This limitation can be overcome by the use of lipid nanocapsules (LNCs) 559 : a nanocarrier with an oil-filled core surrounded by a protective polymer shell, capable of encapsulating drugs, with proven treatment efficiency in preclinical glioblastoma cancer models 597,602,656 . Thus, while delivering therapy, an increase in lipid level can be reached allowing MR oxygenation mapping using the described MOBILE method. Notably, LNCs are also of particular interest due to their O 2 permeability 485 , solvent free and FDAapproved constituents, and their ability to avoid the immune system 657 . Using the MOBILE MR sequence, LNCs were investigated for their ability to respond to changes in O 2 concentrations in vitro. For in vivo experiments, a muscle model and two tumour models were employed to assess the ability of the LNCs to act as an oxygen sensor during carbogen respiratory challenges.

MATERIALS AND METHODS

Reagents

The lipophilic Labrafac® WL 1349

Normal gastrocnemius muscle animal model

Five 7-week old male C3H/HeJ mice (Janvier, Le Genest Saint Isle, France)

were used for the intramuscular tissue study. The difference between R 1 (= 1/T 1 ) during carbogen and air breathing for each pixel was calculated and represented as oxygenation maps.

Cell

Statistical analysis

For the in vivo pO 2 measurements, onetailed Wilcoxon tests were used to compare the mean changes between groups (air vs. carbogen breathing within the intramuscular, fibrosarcoma and glioblastoma groups). Histogram distributions, linear fits and Wilcoxon rank tests (for pixel-to-pixel analyses) with p values < 0.05 (*) were considered significant, and were calculated using Prism 5 software.

RESULTS

LNC preparation and characterisation

The phase inversion process was exploited to obtain LNCs consisting of a lipidic-core (i.e. Labrafac) enclosed within a polymerbased (i.e. PEG) shell. After osmolarity adjustment, the LNCs had an average size and PDI of 60.7 ±0.9 nm and 0.152 ±0.016, respectively. These physicochemical properties are comparable to previously studied LNCs 574,641,642 .

In vitro validation of LNCs sensitivity to oxygen

To assess the ability and sensitivity of the (median change of 0.3187 s -1 for all rats). Thus LNCs, which are already used as a therapeutic nanocarrier 573,593,596,597,661 , viscosity and iron content for example 667- 670 , all of which are variables that cannot be easily mimicked in vitro.

could

CONCLUSION

Herein, our aim was to demonstrate that LNCs, besides its proved therapeutic nanocarrier efficiency, could be used as a diagnostic tool in the form of an oxygen sensor for normal and tumoural tissue using the MOBILE MR sequence. LNCs that LNCs could be used as both a therapeutic and diagnostic tool for cancer.

IN VIVO FATE OF LNCS AFTER DIRECT DEPOSIT IN TISSUE

Introduction

In this section the unpublished work pertaining to the above article in section 4.2, will be presented and discussed. Herein, the in vivo fate of the LNC formulation was explored as, according to literature and with respect to their therapeutical capabilities, they are broken apart or metabolised by cells and tissues over time. The objective of the experiments presented here was to address this issue.

Materials and methods

Synthesis of LNCs

Chemicals and formulation process for LNCs were as described in section 4.2.

Animal models

Three murine animal models were used in this in vivo study; two normal tissue models, (Harvard Apparatus, Les Ulis, France) at 0.5 µL/min rate for 80 min. Afterward, the needle was held in place for 5 additional minutes to avoid the expulsion of the LNC formulation from the brain during removal of the needle, which was withdrawn slowly (0.5 mm per 30 seconds). The incision was stitched and the animal was prepared for the MR experiments.

Evaluation of the LNC formulations fate was assessed using point resolved spectroscopy (PRESS).

PRESS measurements

The PRESS sequence was used to determine the lipid content (CH 2 associated with the backbone of Labrafac®) of the injected LNCs. A single voxel of ~4 x 4 x 4 mm 3 was used for all the animal models. For each individual, the voxel was placed over the LNC area, avoiding vascular structures and subcutaneous fat tissue, and the spectra was recorded using the PRESS pulse sequence (TR = 3000 msec, TE = 10 msec) after water suppression using the variable power and optimised relaxation delays (VAPOR) pulse train 671 . The methylene signal, which represents CH 2 backbone of the Labrafac® within the LNCs, was measured at 1.2 ppm. This peak was measured in comparison to the water peak (4.7 ppm).

Results

Dispersion of LNC lipids over time

After the introduction of the LNC formulation, the PRESS MR sequence was used to acquire the spectra of the compounds present in the voxel within the animal models. An A semi-quantitative analysis, using the ratio between the unsuppressed water and CH 2 , was performed for all the animal models (Fig. 456). Here, the signal corresponding to the LNC, especially the CH 2 used for pO 2 estimation, decreases after one hour to about 80 % of the value measured just after injection for all the animal models explored. Two hours later, a further 25 % decrease is observed, and for two animalsone normal brain and one brain tumourexplored at 24 h after the initial injection, the CH 2 peak returns to pre-injection values.

Of note, and in complement to the main results included in the publication, the aforementioned in vivo LNC fate experiment enabled the response of the normal brain to the carbogen challenge to be measured. Interestingly, on both the RARE and MOBILE images it is apparent that a portion of the LNC formulation settled in the corpus callosum of the rat brain despite an injection into the striatum, thus showing a reflux of the LNC formulation from the well-organised normal brain. This phenomenon was noted in 5 of 6 animals.

However, this did not impair the calculation of R 1 in the striatum before and after the carbogen challenge following the application of the MOBILE MR sequence, and as shown in

Discussion and conclusions

Assessing the fate of LNC after injection was an important factor to assess as this information has a direct impact in the time frame in which LNCs can be used as an O 2 sensor within the tissue. Among the soluble paramagnetic materials, two types of spin probes are particularly interesting: the nitroxides and the triarylmethyl radicals. The nitroxide spin probes have a rich history in EPR; the compounds have been used to measure numerous in vivo parameters, including pO 2 , with accuracy and sensitivity 674 . However, although these compounds have been extensively modified to modulate their intracellular accumulation and tissue sensitivity 354,355 for improved in vivo use, upon contact with certain metabolic enzymes they are converted to the EPR-silent hydroxylamines which can hinder their usefulness. Alternatively, the triarylmethyl radicalsincluding the spin probe TAMare considered to be less sensitive to metabolic enzymes with possible higher stability, although this has been reevaluated in the literature 675 . TAM is also characterised by a narrower LW than nitroxides, such as TEMPO and TB (Fig. water, an increase in sensitivity can be achieved using these systems. Thus, the encapsulation of nitroxide probes -TEMPO and TBand a triarylmethyl radical -TAMwithin the lipophilic core of LNCs was proposed. The encapsulation was hypothesised to improve O 2 sensitivity, create a defined microenvironment for the spin probe which ensures specificity, and prevent biocompatibility issues in vivo.

In this regard, LNCs were an excellent candidate as not only could they provide the aforementioned advantages, but they are also prepared using FDA approved constituents and have previously been employed in several studies of tumour therapeutics as an encapsulation agent (see Chapter 2, section 2.6). In this body of work, it was demonstrated that LNCs could incorporate either the TB or TAM EPR spin probes within its lipophilic core and provide adequate EPR signal after formulation. The TAM-LNCs were found to be responsive to O 2 variation in vitro and demonstrated by a direct relationship between the LW and O 2 variation (Fig. 34), thus indicating the permeability of LNCs to O 2 . Notably, TB-LNCs were also found to be sensitive to O 2 variation but at a lower extent than the TAM-LNCs. TAM-LNCs were also applied to in vivo systems, both in normal muscle and subcutaneous fibrosarcoma tumour models, wherein they were noted to be responsive to an increase in the O 2 concentration within the tissues (Fig. 3-6 and 3-7) (following the carbogen challenge), as well as a decrease in the O 2 environment (Fig. 3456789101112) (following the temporary occlusion of an artery). This indicated that the TAM-LNCs were reversibly responsive to O 2 changes in vivo.

The LNCs were also noted to protect the TB EPR spin probe it encapsulated from metabolic conversion (Fig. 3-3). Interestingly, although the TAM-LNCs were protected within the LNCs (as demonstrated by the sustained TAM signal over time in vivo, Fig. 3 This limitation was overcome by the use of LNCswhich possess an oily-corewhich could thus serve to increase the SNR by strategically increasing the lipidic content within tissue.

Here, LNCs were assessed for their ability to respond to O 2 variations both in vitro and in vivo using the MOBILE MR sequence, and a direct relationship between the R 1 and O 2 % was observed (Fig. 4-1). This coincided with the EPR results wherein the TAM within the It was also possible to produce an oxygenation map wherein the R 1 during air and carbogen breathing could be visualised (Fig. 4-2 and 4-3, b insert). This was possible due to the increase in the SNR of lipids which enabled the pixel-to-pixel analysis of an oxygenation map. Interestingly, but not entirely unexpectedly, although an oxygenation map of the injected LNCs could be produced, the bolus injection -and thus the 'drop' of lipidsallowed for a limited area within the tissue to be mapped. Thus, a second method of introducing the LNCs within tumoural tissue was explored; local diffusion using a CED protocol. It should be noted, that LNCs have previously been introduced as a therapeutic agent nanocarrier into several cancer typesincluding the highly malignant glioblastoma (see section Chapter 2)using the CED protocol. This is because CED allows for a direct, local delivery into the tissue of interest and enables an efficient distribution of the introduced LNC formulation. Hence, an intracerebral glioblastoma model was used for the CED introduction of the LNC formulation, and the MOBILE MR sequence was able to measure the R 1 change between air and carbogen breathing (Fig. 4-4). Furthermore, due to the large distribution of the LNCs, a map representing the oxygenation over the entire tumour was possible and the pixel-to-pixel analysis highlighted the heterogeneity of the glioblastoma tissue to the hyperoxic challenge (Fig. 4-4, b insert). However, the signal was only measurable for a few hours (2 -3 h) after the LNC injection as the lipid signal decreased over time. To demonstrate this observation, the fate of the LNCs in vivo was explored using MR spectroscopy (PRESS) in the context of CED delivery in both normal brain and tumoural brain tissue. This semi-quantitative analysis indicated a decrease of approximately 50 -60 % of the signal arising from the LNC formulation 3 h after its introduction in the tissue, and a return to base quantities after 24 h (Fig. 456). This decline could be associated with the metabolic degradation of the LNCs within the tissue or the movement of the LNCs out of the area being studied.

In summation, this body of work showed that LNCs could serve as an O 2 sensor within tissues using both EPR and MRI. For the EPR spin probes, it served as an effective encapsulation device capable of protecting the TAM spin probes which proved to be O 2 permeable, allowing for pO 2 measurements to be determined for both normal and tumours tissues. Furthermore, the lipids within LNCs were not only detectable by the MOBILE MR sequence, but also increased the SNR sufficiently to allow for the pixel-to-pixel analysis of oxygenation maps within hypoxic tumoural tissue.

LIMITATIONS AND PERSPECTIVES

In this thesis, the feasibility and usefulness of LNCs as a diagnostic O 2 sensor using EPR and MRI within in vitro studies and in vivo models was demonstrated, showing that this system is a promising nanoplatform for combining cancer therapeutics and diagnostics. The

In vitro studies: Sensitivity curve

As noted for both the EPR and MRI studies, higher-than-expected pO 2 values were estimated when LW or T 1 were converted from the sensitivity curves. In Chapter 3, regarding the TAM-LNCs, the presented article noted a discrepancy between the in vitro pO 2 sensitivity curve and the pO 2 measured in vivo. A potential explanation for this observation, as shown by Frank et al. 375 , could arise from local viscosity changes which was demonstrated to have an impact on LW and thus on the conversion to pO 2 . Indeed in our study, the sensitivity curve was performed at 37 °C, and the in vivo measured LWs were converted to pO 2 on this basis.

However, this conversion implies that the temperature of the tumour tissue was also 37 °C which, according to is not always the case as it is significantly dependent on the size of the tumour, blood flow, as well as the type and location of the tumour [679][680][681] . At reduced tumoural temperature, the viscosity of the formulation would be affectedwhich was not represented in the sensitivity curvewhich could lead to the overestimation of pO 2

we encountered. This brought into question the nature of the medium in which the sensitivity curve was performedfor both the EPR and MRI experimentsand its relevance when converting T 1 and LW values into pO 2 .

In regards to the MRI experiments, the in vitro sensitivity curve between the T 1 relaxation rate from the lipidic core of LNCs and O 2 variation demonstrated a linear relationship but, when R 1 was transposed to pO 2 , a higher estimation of pO 2 was also noted within the in vivo models, and could be attributed to the environment surrounding the LNCs.

Apart from O 2 , various factors influence T 1 relaxation and many of which can be foundand must be taken into accountin vivo. Such factors include but are not limited to; temperature, viscosity, and iron content. Indeed, as for the EPR experiments, temperature was assumed to be 37 °C. However, as reported in literature 485 , a decrease in temperature results in a an increase in R 1 and a proportional increase in pO 2 . Thus, if the tumour temperature is lower than the temperature used for the sensitivity curve, converting the R 1 measured in vivo on this basis would lead to an overestimation. It could thus have been advantageous, and more representative, to measure the response of T 1 to O 2 and temperature variation in a step-wise fashion.

Another factor which influences T 1 relaxation is viscosity. The use of plasma as a mediumwhich has a higher viscosity than water at approximately 1.10 -1.30 mPa.s 682could have been used to overcome this issue, however, it would not account for the complexity encountered in tissue wherein iron could be present or the general organisation of the tissue with a multitude of cells, proteins, and macromolecules interacting with the LNCs which also affects the T 1 relaxation time.

Thus, the development of a more pertinent ex vivo model could be of interest.

Unfortunately, however, this system presents its own issues as ensuring a homogenous perfusion of the gas throughout the tissue massto ensure good homogeneity for the sensitivity curveand ensuring the LNCs interaction with the gas within the mass would be difficult with the system available during the MRI experiments. Thus, although the use of an aqueous system of LNCs was not the ideal experimental conditions it was used to demonstrate the feasibility of the LNCs system as an O 2 probe which responds well to O 2 variation both in vitro and in vivo.

In vitro studies: LNC formulation

Notably, for the encapsulation and retention of soluble EPR spin probes within LNCs, the use of compounds with a log P equal or less than 1.30 (i.e. TEMPO) is not recommended for oximetry use as presented in this thesis. In the case of the encapsulation of TEMPO, the molecule proved insufficiently lipophilic and separated from the LNCs during formulation.

After the encapsulation of TB (log P = 3.10) within LNCs, an EPR spectrum with reduced height in the third line was noted and is indicative of an EPR spin probe whose motion is restricted with possible interaction with the LNC shell. Moreover, when the TB-LNCs were exposed to a reductive environment, the TB signal decreased with time, which is consistent if the TB probe was exposed from the LNC shell. Despite this being non-advantageous for long-term oxygenation measurements, the characteristics of the system could be further analysed using interface analysis techniques such as the pendant drop method. Our conclusion for this work was that a highly lipophilic molecule was required to remain within the O 2 soluble lipidic core of the LNCs whilst being protected by the PEGylated shell.

However, for other work such as free radical scavenging within tumoural cells the exposure of the nitroxide to the biological milieu could be useful. As an example, nitroxide derivatives such as piperidine, pyrroline and pyrrolidine mimic the superoxide dismutase which scavenges reactive free radicals and is useful as an antioxidant against the oxidative stress induced by anti-cancer drugs as well as inhibiting tumoural proliferation 678 . If combined with LNCs, the system could provide a targeted delivery to tumoural cells through the use of a functionalised shell 683 . But, for this thesis, only the use of lipophilic EPR spin probes, such as TAM which remained within the lipidic core of the LNCs, was useful.

As noted previously, the LNCs used in this thesis had comparable physio-chemical properties as previously used formulations (in terms of size, PDI and zeta potential, see

Chapter 2) both before and after the osmolarity adjustment process. A notable exception, however, is sterility. Aqueous solutions of LNCs are commonly sterilised using mechanical filtration with a pore size of 0.22 µm 573 . This route of sterilisation removes any detectable bacteria, fungi and yeast and does not causes the nanocapsules to agglomerate, nor does it cause changes to their size or PDI (slight changes to zeta potential have been observed) 684 .

However, although this method is effective, the use of highly concentrated LNC formulations such as in this thesisdid not allow for such a sterilisation process and may require the use of a sterilised room with sterilised constituents. Furthermore, the use of such a highly concentrated LNC formulation injected in vivo raises concerns of toxicity. Although the LNCs were formulated using regulatory-approved components with similar characteristics (i.e. 50 nm-sized) as previous formulations which were well-tolerated in vivo in the past (see Chapter 2, section 2.6), an investigation at the cellular level to elucidate the effect of the highly concentrated formulation would be beneficial.

In vivo studies: Animal models

Notably, as a proof of concept for the sensitivity of LNCs to pO 2 an in vivo model of C6-induced intracerebral glioblastoma animal model was used. This model, although useful, highlighted a limitation for the use of the LNC formulation; if foci of necrosis of haemorrhaging were present, the lipid T 1 relaxation change following a carbogen challenge could not be measured and a R 1 map could not be produced in areas without blood perfusion or limited O 2 accessibility. This, however, could be an advantage for future work.

The use of the a R 1 map produced from the LNC formulation following a carbogen challenge could be used as an indicator for poor O 2 accessibility and could be used as a tool for dose painting during IMRT [685][686][687] . It is also important to note, that although our work indicated a response within the tissue to carbogen challengereflected as changes in LW and T 1 relaxation timeno associated proof of hypoxia was presented. Thus, the use of immunohistochemical staining of tissue samples, such as a 2-nitroimidazole derivative, for hypoxia confirmation could be useful.

Lastly, in regard to the animal models, we demonstrated that TAM-LNCs could respond reversibly to pO 2 changes in vivo by using the carbogen challenge (to increase the pO 2 ) and the occlusion of the femoral artery performed by means of a rubber band tied tightly around the knee (to decrease the pO 2 ). Here, the LW values seemed to indicate that there was no difference between the two experimental conditions. This could indicate that the TAM-LNCs were not sensitive enough to discern the difference at such a low O 2 tension, however, the sensitivity curve demonstrates that the TAM-LNCs are capable of distinguishing between 0 -5 % O 2 concentrations. Thus, the use of a different model to induce transient and severe hypoxia could be beneficial, such as the use of surgical severance of the femoral artery or for the animal to be sacrificed. In this way, the reversible response of the TAM-LNCs could be verified.

In vivo studies: LNCs as a theranostic tool

A concern, regarding the use of LNCs as an O 2 sensor in tissue, could be the possibility of the LNCs harbouring auxiliary O 2 . In other words, during the formulation and handling process of the LNCs, O 2 accumulates in the lipidic core of the nanocapsules (due to the higher solubility of O 2 in lipids than in water) but once introduced in vivo the O 2 diffuses from the lipidic core into the tissue causing an initial large release of O 2 which equilibrates.

Or, conversely, the O 2 from the tissue could accumulate within the LNC's lipidic core and reduce the O 2 available to the tissue. Indeed, Zhou et al. 688 recently demonstrated that PFC nanoparticles could be loaded with O 2 for release within tumours to induce an enhanced radio-therapeutic effect. In our studies, we did not evaluate the latter but did demonstrate the lack of the former, i.e. 'reservoir effect', occurring by means of measuring the TAM-LNCs LWs over time.

In Chapter 4, section 4.2, two routes of delivery for the LNC formulation was described, namely an intramuscular injection and the intracerebral CED protocol. The use of the intracerebral CED protocol has been used in clinic for glioblastoma multiforme drug delivery as a means to bypass the blood-brain barrier, reduce systemic toxicity, and to overcome the limitations of diffusion-mediated delivery of compounds with a larger molecular weight 660,689,690 . However, although this method is an effective strategy for drug delivery, it is still under development; a balance must be struck between the quantity of the therapeutic product delivered to the target site and the potential harmful side-effect. In this regard, several new methods of drug delivery are being developed to both improve the current methods (e.g. multiport catheter designs approaches for CED protocols 691,692 ) as well as develop new methods (e.g. miniaturised implantable systems 693,694 ).

Lastly, it is also notable, that although LW or T 1 relaxation measurements could be performed for 1 -3 h, respectively, a signal loss related to LNCs biodegradation and/or the dispersion was evident over time. This observation raises the question of the 'dual function'

that is expected from a theranostic tool; i.e. the ability to treat and diagnose, and in our case, follow the change induced by therapy. Using the MR PRESS sequence, an increase in the CH 2 lipid peak (1.2 ppm) and the PEG peak (3.7 ppm) was notable after LNC introduction in all models, and was associated with an increase in SNR enabling the production of lipid images by the MOBILE sequence. However, after 24 h this characteristic signal of LNCs (1.2 ppm lipid peak and 3.7 ppm PEG peak) could no longer be observed. Previous studies regarding the in vivo application of standard LNCs (with 15 units of HS-PEG within the shell) have determined that the nanocapsules are stable in the blood for up to 24 h (whereupon their hydrodynamic size increased dramatically) 695 , however this study gave no quantitative indication. An attempt to discern the integrity of LNCs in vivo using two FRET embedded dyes within the nanocapsule has been studied, and demonstrated that LNCs can still be intact several days after introduction. Unfortunately, however, no information regarding the percentage of intact LNCs as compared to the introduced LNCs was determined 596 . As a consequence, if the use of LNCs in a diagnostic capacity is to be pursued, the issue of their integrity over time would have to be addressed. This could be achieved by avoiding their potential biodegradation (a different shell which is still O 2 permeable) or internalisation in cells (using different particle sizes).

Nevertheless, and considering these limitations, although the feasibility of the LNCs as an O 2 sensor within tissues have been demonstrated, their usefulness as a theranostic tool in long-term therapeutic regimes appears unlikely unless a mixture of LNC formulations is developed which possesses both diagnostic and therapeutic LNC formulations (assuming that the behaviour of the latter can be monitored by the former). Interestingly, measurements wherein the ability of the tissues to be oxygenated, i.e. an 'oxygen-ability map', is required could benefit from our system demonstrated in this thesis. Such applications include dose painting during IMRT. Here, the use of an O 2 sensor could be useful and act as a vehicle for radio-enhancers, such as oxygen-loaded PFCs or gold nanoparticles. Abstract: Hypoxia is one of the most challenging aspects of the tumour microenvironment. The phenomenon occurs due to abnormal vasculature and an exacerbated metabolism, and leads to highly malignant cells resistant to radio-and cyto-toxic therapy. As such, hypoxia is of major concern and prompted our novel approach in using lipid nanocapsules (LNCs) as an oxygen sensor. LNCs have been demonstrated as excellent core-shell nanocarriers, capable of encapsulating drugs within their lipidic core and avoiding the immune system due to their PEGylated shell, thus enabling treatment of highly aggressive tumours. We hypothesised that the lipidic-core of LNCs could also be used to assess the O 2 environment in tissue. Indeed, because O 2 solubility is greater in lipids than in water, any subtle changes in tissue O 2 will be heightened in the lipidic LNC core. Consequently, we encapsulated a lipophilic paramagnetic probe, e.g. tetrathiatriarylmethyl (TAM), and demonstrated its response to variations in O 2 in vitro, using Electron Paramagnetic Resonance (EPR), indicating the permeability of LNCs to O 2.

We applied the TAM-LNCs to an in vivo normal tissue model (gastrocnemius mouse muscle) and pathological model (sarcoma tumour). Herein, free TAM was rapidly reduced and no O 2 measurement was possible, however, our TAM-LNC system exhibited a half-life of over an hour and enabled realtime measurements whilst animals were breathing air and during a carbogen (95 % O 2 , 5 % CO 2 ) breathing challenge. Moreover, the lipidic-core nature of the LNCs was exploited to image tissue oxygenation using Magnetic Resonance Imaging (MRI), specifically the MOBILE sequence which enabled the mapping of O 2 -induced T 1 relaxation rate changes in lipids. Using a single dose of LNCs, we were able to portray the change in T 1 between air breathing and the carbogen challenge, and image the heterogeneous nature of hypoxia in murine normal tissue and pathological tumour models. In conclusion, we demonstrated the feasibility of using LNCs as a diagnostic tool for assessing hypoxia in both normal and pathological tissues.

Figure 2 - 4 . 22 Figure 2 - 5 . 24 Figure 2 - 6 . 27 Figure 2 - 7 .Figure 2 - 8 . 31 Figure 2 - 9 .

 24222524262727283129 Figure 2-4. Determination of murine skin oxygen tension using the polarographic microelectrode technique. Note the diameter of the microelectrode tip (100 µm) shown in relation to the mouse footpad (a). Figure (b) shows the time frame of measurements, performed over seconds, wherein (I) -(III) indicates the time wherein the tip is brought into contact and penetrates the skin. Time (IV) indicates the measurement of the oxygen tension. Note the decrease of pO 2 overtime due to the consumption of oxygen by the electrode. Image reproduced from Hofmann et al. 169 . .............................................................22 Figure 2-5. Example of immunohistochemical staining of HIF-1α and VEGF in glioblastoma tissue samples (a) and (b) and normal brain samples (c) and (d). Arrow indicates area of necrosis within the sample. Image reproduced from Irshad et al. 175 . ..............................................................................................24 Figure 2-6. Example of diffuse optical tomography (DOT) imaging wherein an invasive ductal carcinoma is described in terms of relative deoxyhaemoglobin (rHb), oxygenated haemoglobin (rHbO 2 ), and relative tissue oxygen saturation (rStO 2 ). The black solid line indicates the region identified as the tumour. Notably, these parameters do not directly measure the tumoural pO 2 values. Image reproduced from Chung et al. 220 . ......................................................27 Figure 2-7. Combined ultrasound and photoacoustic images of subcutaneous tumours induced in nude mice before and after photodynamic therapy (PDT). Grayscale ultrasound images, presented in the top panel, are overlaid with oxygen saturation (StO 2 ) map where blue and red represent hypoxic and oxygenated regions. Similarly, the bottom panel is overlaid with a scale to represent the total haemoglobin (HbT). Image reproduced from Mallidi et al. 234 . ...........28

Figure 2 - 10 . 36 Figure 2 - 11 . 36 Figure 2 - 12 . 38 Figure 2 - 13 . 39 Figure 2 - 14 . 45 Figure 2 - 15 . 48 Figure 2 - 16 . 50 Figure 2 - 17 . 52 Figure 2 - 18 . 53 Figure 3 - 1 . 60 Figure 3 - 2 . 64 Figure 3 - 3 . 65 Figure 3 - 4 . 66 Figure 3 - 5 . 67 Figure 3 - 6 . 68 Figure 3 - 7 . 68 Figure 3 - 8 . 70 Figure 3 - 9 . 72 Figure 3 - 10 . 73 Figure 3 - 11 .

 21036211362123821339214452154821650217522185331603264336534663567366837683870397231073311 Figure 2-10. Examples of EPR instrumentation used in oximetry. Left: EPR X-band used for in vitro samples and, right: EPR clinical L-band used for in vivo work. ......................................................................................................................36 Figure 2-11. Example of EPR spectrum wherein the effect of O 2 on a paramagnetic species is demonstrated. The broad LW spectrum indicates measurement during 21 % O 2 and the thin LW spectrum indicates measurement during 100 % N 2 . Image reproduced from Gallez et al. 341 . ............................................36Figure 2-12. Chemical structures of two nitroxides; TEMPO (a) and 4-hydroy-TEMPO benzoate (b), and the EPR spectrum of TEMPO under 21 % O 2 showing the characteristic 3 spectral lines of nitroxides (c). ............................................38 Figure 2-13. Chemical structure of a triarylmethyl EPR radical, namely TAM (F15T-03) (a), and its associated EPR spectra (b) in 21 % O 2 showing the characteristic 1 spectral line of a triarylmethyl radical. ................................39 Figure 2-14. Measurement of tumour hypoxia (in mmHg) within a mouse brain after a single injection of a perfluorocarbon (perfluoro-15-crown-5-ether; PFCE) loaded within lipid nanocapsules (LNCs).Image reproduced from Lemaire et al. 485 . ............................................................................................................45 Figure 2-15. Relationship between changes in R 1 (=1/T 1 ) as compared to changes in pO 2 in vitro in (a) water and (b & c) pure oil measured at different chemical shifts (i.e. [b] 1.2 ppm and [c] 4.0 ppm). Image reproduced from Jordan et al. 508 . ................................................................................................................48 Figure 2-16. The sensitivity of the MOBILE sequence to variations in tumour oxygenation. Notably, (a) illustrates the anatomical transverse MR image of a mouse with a tumour (shown by arrow), wherein the dark spot within the tumour shows where the tip of the OxyLite® probe is situated. Following the carbogen gas challenge, maps of changes in relaxation times (ΔR 1 = R 1 carbogen -R 1 air ) in response to the challenge can be produced using the MOBILE sequence (b & c). Colour scales highlight the higher sensitivity of the MOBILE technique wherein a higher proportion of the pixels are in the red zone indicating a greater change in R 1 . ........................................................50 Figure 2-17. Schematic of the nanoparticle named LNCs including its constituent components. ..................................................................................................52 Figure 2-18. The use of the phase inversion temperature (PIT) method to formulation lipid nanocapsules (LNCs) (a), and their osmotic adjustment (b) for in vivo use. ................................................................................................................53 Figure 3-1. Chemical structure illustrations of (a) TEMPO benzoate and (b) F15T-03, and (c) a schematic of lipid nanocapsules (LNCs). ......................................60

Figure 4 - 1 . 86 Figure 4 - 2 . 88 Figure 4 - 3 . 89 Figure 4 - 4 . 90 Figure 4 - 5 . 97 Figure 4 - 6 . 97 Figure 4 - 7 . 98 Figure 0 - 1 .Figure 0 - 2 .Figure 0 - 3 .Figure 0 - 4 .

 418642884389449045974697479801020304 Figure 4-1. In vitro sensitivity of the lipid relaxation rate (R 1 ) arising from lipid nanocapsules (LNCs) to oxygenation as measured by using the MOBILE MR sequence (R 2 = 0.9948). ................................................................................86 Figure 4-2. Application of lipid nanocapsules (LNCs) to assess variations of oxygenation in the intramuscular tissue of mice using lipid relaxation rate measured with the MOBILE MR sequence, wherein (a) shows the transversal anatomical image of a mouse gastrocnemius muscle and the injected LNCs

Firstly, I would

  like to express my deep gratitude to Dr Laurent LEMAIRE and Prof Bernard GALLEZ, my research supervisors, for the opportunity to work on this project. But most importantly, for their guidance, encouragement and patience in teaching me the skills needed to become a researcher. I would also like to thank Dr Florence FRANCONI and Dr Nicolas JOUDIOU for their invaluable knowledge and technical support during the process of creating this thesis. Similarly, I am also grateful to Prof Frank BOURY, the coordinator of the European Doctorate in Nanomedicine and Pharmaceutical Innovation (NanoFar), Marion TOUCHETEAU, Sandrine WOLLANDERS and Ourega KOFFI, for their dedication to the NanoFar program and its students. Through their hard work they enabled many students, including myself, to realise the dream of pursuing a PhD, and I will be forever thankful. I would like to thank the members of my dissertation committee; Prof. Anne DES RIEUX, Dr Myriam BERNAUDIN, Dr Yves Michel FRAPART, and Dr Fanny NOURY; for generously offering their time, expertise and guidance in review of my thesis. My thanks to my colleagues in Belgium and France; it was a pleasure to work with them for three years and I am sincerely grateful for their professionalism and technical support. Specifically, I would like to thank Dr Céline M. DESMET and the staff at the Service Commun d'Animalerie Hospitalo-Universitaire (SCAHU, Angers, France) for their unfailing assistance and guidance. Many thanks to my friends and colleagues in the NanoFar program, especially Valentina, Kita, Chiara, Bathabile, Maruthi, Surasa, Pauline, Natalija, Vincent, Audrey, Raneem and Mathie. Thank you for the coffee, delicious meals, and camaraderie that made my PhD so incredibly memorable. I owe a great deal to my parents, Suzette and Marius NEL, as well as my sister, Sumari NEL, who have always encouraged and supported me no matter where in the world I am. Finally, to Baptistethank you. An acknowledgement here cannot express how grateful I am to you for helping me attain something I thought was impossible. characteristic feature of tumours. The phenomenon results from tissues increasingly consuming oxygen (O 2 ) whilst receiving a limited supply. Mounting evidence indicates that hypoxia plays a vital role in tumour development, angiogenesis, metabolism, and resistance to treatment. Alterations in the gene and protein expressions of tumours induced by hypoxia lead to more aggressive survival patterns and result in resistance to several forms of therapy, including radio-, immuno-photodynamic, and cytotoxic chemotherapy. Thus, techniques to assess and image tumour hypoxia has become of crucial importance for the prediction of therapeutic outcomes.Fortunately, the chemical and physical properties of O 2 facilitate a wide variety of methods for monitoring and imaging hypoxia. These methods involve electrochemical reduction, optical-based methods including spectroscopy and luminescence, nuclear-based methods including positron emission tomography, and magnetic resonance (MR) based methods including electron paramagnetic resonance (EPR) and magnetic resonance imaging (MRI). Unfortunately, no 'gold standard' amongst the methods exists as, although each method provides unique advantages, several limitations regarding the tumour microenvironment itselfsuch as tumour heterogeneity and necrosisand the techniquesincluding invasiveness, use of ionising radiation, and poor spatial resolutionhave stunted progress. Recent advances, however, have provided modern medicine with a new aid in the form of nanotechnology, which has emerged as an outstanding tool for the development of technologies for diagnosis, treatment and prevention of cancer. To this end, this thesis aims to demonstrate that lipid nanocapsules (LNCs), in combination with EPR and MRI techniques, can be used as an O 2 sensor to address these limitations. LNCs have been exemplified as excellent core-shell nanocarriers capable of encapsulating drugs within their lipidic core and avoiding the immune system due to their 'stealth' shell, thus enabling the treatment of highly aggressive tumours.Firstly, in this thesis, LNCs will be used in combination with EPR oximetry. EPR oximetry enables repeated and direct O 2 quantification within tumours from the interactions between the unpaired electrons of molecular O 2 and a paramagnetic probe. These interactions lead to a decrease in spin-spin (T 2 ) relaxation rates and can be evaluated by the EPR signal line width (LW) which is directly proportional to O 2 tension. Unfortunately, paramagnetic probes can often be rapidly bioreduced and thus require encapsulation before their introduction into an in vivo system. Thus, in this thesis, LNCs were used to encapsulate and Chapter 1: Introduction Janske NEL | Lipid nanocapsules as a theranostic tool 3 protect paramagnetic probes and to demonstrate its response to variations in O 2 in vitro thus indicating the permeability of LNCs to O 2 . We applied the successfully encapsulated LNC paramagnetic probes to an in vivo normal tissue model (gastrocnemius mouse muscle) and a pathological model (fibrosarcoma subcutaneous tumour).

  LNCs to the EPR technique: a) Can LNCs encapsulate paramagnetic EPR probes? b) If so, can the LNCs protect the encapsulated probe from the biological milieu? c) Once encapsulated, are the probe-loaded LNCs sensitive to O 2 variation in vitro? d) Furthermore, can the probe-loaded LNCs serve as O 2 sensors by responding to O 2 variation achieved by a hyperoxic gas (95 % O 2 and 5 % carbon dioxide (CO 2 ); carbogen) breathing challenge within normal murine tissue? e) Lastly, can the probe-loaded LNCs serve as an O 2 sensor within murine tumours? When applying LNCs to MRI using the MOBILE sequence; a) Are the lipids within the LNCs detectable by MRI using the MOBILE sequence? b) If so, do the lipids within the LNCs respond to O 2 variation in vitro? c) Can the LNCs serve as O 2 sensors by responding to O 2 variation, achieved by a hyperoxic gas breathing challenge, within normal murine tissue using the MOBILE sequence? If so, can an oxygenation map be produced of the area? d) Similarly, can the LNCs serve as O 2 sensors by responding to O 2 variation, achieved by a hyperoxic gas breathing challenge, within tumoural tissues; such as intracerebral glioblastoma and fibrosarcoma subcutaneous murine tumours? If so, can an

Figure 2 - 1 .

 21 Figure 2-1. Schematic of tumoural progression and the effects on angiogenesis, metabolism and apoptotic pathways. Adapted from the following citations2,6,19-25 .

Figure 2 - 2 .

 22 Figure 2-2. Schematic illustrating the difference between oxidative phosphorylation, anaerobic glycolysis, and aerobic glycolysis (Warburg effect). In the presence of O 2 , non-proliferating cells (differentiated) preferentially metabolise glucose through the oxidative phosphorylation pathway, via the mitochondria. In this instance, because O 2

Figure 2 - 3 .

 23 Figure 2-3. Schematic illustrating the role of hypoxia in cancer progression, angiogenesis, metastasis and, inevitably, resistance to therapy. Image reproduced from Muz et al. 6 .

Figure 2 - 4 .

 24 Figure 2-4. Determination of murine skin oxygen tension using the polarographic microelectrode technique. Note the diameter of the microelectrode tip (100 µm) shown in relation to the mouse footpad (a). Figure (b) shows the time frame of measurements, performed over seconds, wherein (I) -(III) indicates the time wherein the tip is brought into contact and penetrates the skin. Time (IV) indicates the measurement of the oxygen tension. Note the decrease of pO 2 overtime due to the consumption of oxygen by the electrode. Image reproduced from Hofmann et al. 169 .

Figure 2 - 5 .

 25 Figure 2-5. Example of immunohistochemical staining of HIF-1α and VEGF in glioblastoma tissue samples (a) and (b) and normal brain samples (c) and (d). Arrow indicates area of necrosis within the sample. Image reproduced from Irshad et al. 175 .

Figure 2 - 6 .

 26 Figure 2-6. Example of diffuse optical tomography (DOT) imaging wherein an invasive ductal carcinoma is described in terms of relative deoxyhaemoglobin (rHb), oxygenated haemoglobin (rHbO 2 ), and relative tissue oxygen saturation (rStO 2 ). The black solid line indicates the region identified as the tumour. Notably, these parameters do not directly measure the tumoural pO 2 values. Image reproduced from Chung et al. 220 .

  parameters can be used to quantify O 2 metabolism at a high spatial resolution without the requirement of an exogenous contrast agent. Photoacoustic imaging overcomes the diffusion limit of traditional optical methods by detecting ultrasound generated from light absorption 235 , and can thus overcome the tissue scattering associated with most spectroscopy techniques 236 .

Figure 2 - 7 .

 27 Figure 2-7. Combined ultrasound and photoacoustic images of subcutaneous tumours induced in nude mice before and after photodynamic therapy (PDT). Grayscale ultrasound images, presented in the top panel, are overlaid with oxygen saturation (StO 2 ) map where blue and red represent hypoxic and oxygenated regions. Similarly, the bottom panel is overlaid with a scale to represent the total haemoglobin (HbT). Image reproduced from Mallidi et al. 234 .

  One of the most widely used, and clinically applied, nuclear based techniques for hypoxia analyses and imaging in tumours is positron emission tomography (PET). PET, as the name suggests, is based on the positrons emitted as radioisotopes decay which, upon deceleration, encounters an electron causing the annihilation of both the electron and positron and the emission of a pair of gamma (annihilation) photons which are detectable by the PET equipment. The most common positron-emitting radioisotopes are 18 F, 124 I and60/64 Cu. These radioisotopes are attached to organic markers which includes; 2-nitroimidazoles, such as fluoromisonidazole (FMISO), 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-penta-fluoropropyl)acetamide (EF5), and fluoroetanidazole (FETA); nucleoside conjugates, such as iodoazomycin arabinoside (IAZA); and copper compounds, such as Cu(II)-diacetyl-bis(N4methylthiosemicarbozone) (Cu-ATSM)[275][276][277] . These markers specifically bind to viable hypoxic cells and form stable adducts, wherein the radioisotopes accumulate and are detected by a PET scanner.

Figure 2 - 8 .

 28 Figure 2-8. Example of a phosphorescence lifetime (PLI) image (using a Pd 2+ porphyrin probe) and a pixel-pO 2 histogram of an in vivo tumour. Image reproduced from Erickson et al.

  F;fluoroerythronitroimidazole ([ 18 F]FETNIM)285,286 , -fluoroetanidazole ([ 18 F]FETA) 287 ,fluoroazomycin arabinoside ([ 18 F]FAZA) 288 , and EF1/5[289][290][291] . These markers have a relatively high lipophilicity, which ensured easy penetration of cell membranes and diffusion into tumour tissue, but limited the clearance of unbound tracer which leads to relatively low tumour-to-reference tissue ratios 292 . The iodinated analogues include iodoazomycin galactopyranoside (IAZGP) and IAZA, both of which are labelled with the long-lived radioisotope 124 I, thus allowing for delayed imaging. However, numerous studies have shown

Figure 2 - 9 .Chapter 2 :

 292 Figure 2-9. PET imaging of a high-grade glioma wherein the hypoxic regions (red) were assessed by 18 F-FMISO (a) and the high glucose metabolism (red) were determined by 18 F-FDG (b). Note the lack of concordance between the two images, suggesting that a complex relationship exists between the two metabolic processes. Image reproduced from Lee and Scott 279 .

Figure 2 - 10 .Figure 2 - 11 .

 210211 Figure 2-10. Examples of EPR instrumentation used in oximetry. Left: EPR X-band used for in vitro samples and, right: EPR clinical L-band used for in vivo work.

Figure 2 - 12 .

 212 Figure 2-12. Chemical structures of two nitroxides; TEMPO (a) and 4-hydroy-TEMPO benzoate (b), and the EPR spectrum of TEMPO under 21 % O 2 showing the characteristic 3 spectral lines of nitroxides (c).

Figure 2 - 13 .

 213 Figure 2-13. Chemical structure of a triarylmethyl EPR radical, namely TAM (F15T-03) (a), and its associated EPR spectra (b) in 21 % O 2 showing the characteristic 1 spectral line of a triarylmethyl radical.

  prompted the use of FLOOD (flow and oxygenation dependent) to refer to the BOLD experiment when tissue oxygenation is measured and applied to the study of oxygenation in tumours454 .Dynamic contrast-enhanced imaging techniques (DCE-MRI) characterises the physiological microenvironment of tumours via the uptake of contrast agent into tumour tissue. This imaging technique relies on repeated image acquisition before, during, and after the intravenous administration of a low-molecular-weight contrast agent, such as gadoliniumbased (e.g. Gd-DOTA)460 . DCE can be combined with computer tomography (CT) which enables the monitoring of temporal changes in the contrast enhancement. These changes can provide information regarding the perfusion, relative blood volume, and the microvasculature of the tumour 461 . DCE-MRI parameters reflecting heterogeneous tumour perfusion and subtle tumour volume change early during treatment were better predictors of tumour recurrence and death than clinically accepted prognostic factors (e.g., stage, lymph node status, and histology)462 . From a clinical standpoint, several trials have investigated the relationship between various MRI parameters and more-specific markers of hypoxia453,463-

  -based MR techniques include the use of exogenous probes (e.g. perfluorinated compounds [PFCs]) and endogenous probes (e.g. metabolic markers and lipids). PFCs are organic molecules, in which all the hydrogen atoms have been replaced by fluorine atoms, hence providing strong 19 F MR signal. PFCs possess a series of properties making them very suitable as an MR contrast agent 162 , in particular they are; biologically inert, exhibit minimal toxicity, and are commercially available. Parhami and colleagues were the first to demonstrate that at a given magnetic field and temperature a linear relationship exists between 19 F MR T 1 relaxation of PFCs and O 2 tension, a circumstance attributed to the paramagnetic nature of O 2 471

2 - 14 ).Figure 2 - 14 .

 214214 Figure 2-14. Measurement of tumour hypoxia (in mmHg) within a mouse brain after a single injection of a perfluorocarbon (perfluoro-15-crown-5-ether; PFCE) loaded within lipid nanocapsules (LNCs).Image reproduced from Lemaire et al. 485 .

Figure 2 - 15 .

 215 Figure 2-15. Relationship between changes in R 1 (=1/T 1 ) as compared to changes in pO 2 in vitro in (a) water and (b & c) pure oil measured at different chemical shifts (i.e. [b] 1.2 ppm and [c] 4.0 ppm). Image reproduced from Jordan et al. 508 .

Chapter 2 :

 2 Background to the Research Area Janske NEL | Lipid nanocapsules as a theranostic tool 50no oxygenation mapping could be determined or a very poor resolution of the map would be produced.Thus, considering the potential use of MOBILE for mapping oxygenation in vivo, this body of work introduces the use of the highly lipidic nanoparticle, lipid nanocapsules (LNCs).

Figure 2 - 16 .

 216 Figure 2-16. The sensitivity of the MOBILE sequence to variations in tumour oxygenation. Notably, (a) illustrates the anatomical transverse MR image of a mouse with a tumour (shown by arrow), wherein the dark spot within the tumour shows where the tip of the OxyLite® probe is situated. Following the carbogen gas challenge, maps of changes in relaxation times (ΔR 1 = R 1 carbogen -R 1 air ) in response to the challenge can be produced using the MOBILE sequence (b & c). Colour scales highlight the higher sensitivity of the MOBILE technique wherein a higher proportion of the pixels are in the red zone indicating a greater change in R 1 .

Figure 2 - 17 .

 217 Figure 2-17. Schematic of the nanoparticle named LNCs including its constituent components.

Figure 2 - 18 .

 218 Figure 2-18. The use of the phase inversion temperature (PIT) method to formulation lipid nanocapsules (LNCs) (a), and their osmotic adjustment (b) for in vivo use.

  greatly increased by the high solubility of oxygen in lipids.Because nitroxides and TAM radicals could be used in in vivo EPR oximetry,

Lipoïd

  F15T-03) radical was synthesised as previously described374 . NaCl was purchased from Prolabo (Fontenay-sous-Bois, France). Water was obtained from a Milli-Q (Millipore) ultrafiltration system. No metal contaminates such as iron were observed in the EPR spectra.

Figure 3 - 1 .

 31 Figure 3-1. Chemical structure illustrations of (a) TEMPO benzoate and (b) F15T-03, and (c) a schematic of lipid nanocapsules (LNCs).

  with a 633 nm laser beam (helium-neon laser, 4 mW) at a fixed scattered angle of 173°. The formulations were diluted with pure water (dilution factor: 60) before measurement. Values are expressed as mean of three measurements ±standard deviation (SD).

  settings were as follows: incident microwave power of 2.5 mW for TB and 0.3 mW for TAM; modulation frequency: 100 kHz; sweep width: 50 G for the nitroxide and 10 G for TAM; modulation amplitude: 0.6 G for TB and 0.2 G for TAM; scan time: 10 s.

  investigated with an ascorbic acid reduction assay 640 . This was achieved by incubating the TB-LNCs in ascorbic acid, a compound known to reduce the paramagnetic nitroxide into diamagnetic hydroxylamine. Nitroxide-loaded LNC formulations were combined 1:1 with a 2.5 mM ascorbic acid solution, and an aliquot of 100 µL was placed in an open quartz capillary tube (0.5 mm diameter). The non-encapsulated TB (dissolved in dimethyl sulfoxide (DMSO)) was used for comparison by mixing 1:1 with a 2.5 mM ascorbic acid solution diluted with Milli-Q water. EPR spectra were repeated over time to measure the decrease in signal intensity. All measurements were done in triplicate.

pO 2 )

 2 was obtained for both probe encapsulated-LNCs. LWs were obtained at various oxygen levels to determine the sensitivity of the LW to pO 2 . Measurements were performed using the same spectrometer as mentioned in 2.3.1, equipped with a variable temperature controller (ER 4131 VT) equilibrated at 310 K. Samples were placed in a gaspermeable Teflon tube (0.625 mm inner diameter, 0.05 mm wall), which was folded and placed in a quartz open ended EPR tube. The oxygen content was varied between 0 % and 21 % O 2 by using an Aalborg gas mixer. The oxygen content in the mixed gas was measured by a Servomex MiniMP 5200 oxygen analyser (the precision of which is 0.1 % with oxygen content).

  Abbot, Queensborough, UK): 3 % inhalation mixed with continuous air flow (2 L/min) for induction, and 1.5 % isoflurane for anaesthesia maintenance. A circulating water system was used for mouse-body temperature regulation. 50 µL injection of either TAM-LNCs or TAM dissolved in DMSO (10 mM) was injected intramuscularly or intratumourally (for intramuscularly; n = 5 encapsulated TAM-LNCs and n = 2 of unencapsulated TAM dissolved in DMSO, for intratumourally; n = 3 encapsulated TAM-LNCs and n = 2 of unencapsulated TAM dissolved in DMSO) in anaesthetised animals. Prior to the injection of TAM dissolved in DMSO, an occlusion of the femoral artery using a rubber band was performed to enable the measurement of the signal. The animal was placed in a side-way position to enable the access of the experimental gastrocnemius muscle, and the loop resonator was placed over the area of injection. The first EPR spectrum was acquired using the low-frequency EPR spectrometer 2 min after the injection, and this signal intensity was defined as the 100 % signal value. All other values were calculated relatively.

1 .

 1 The size of TB-LNCs and TAM-LNCs were 60 nm and 57 nm, respectively with a PDI under 0.2. The size of the probe loaded-LNCs was comparable to previously studied standard blank LNCs which showed good stability stored at 4 °C from 12-18 months 641,642 . Both probe loaded-LNCs types exhibited an EPR signal demonstrating the effective inclusion of TB and TAM in the LNCs (Fig. 3-2). Of note, our attempt to prepare TEMPO loaded-LNCs was not successful as no EPR signal was recorded after the preparation. This could be due to the lower lipophilicity of TEMPO (log P = 1.40) compared to TB (log P = 3.10) 643 . Interestingly, the intensity of the three EPR lines in the EPR spectrum of TB-LNCs was not equivalent, with a reduced height of the third line (Fig. 3-2), indicative of motion restriction of TB within the LNCs. As expected the LW of the TAM-LNCs (equilibrated in air at room temperature) was narrow (0.71 G, under air) compared to the LW of the TB-LNCs (1.92 G, measured on the central peak of the nitroxide). To further ascertain the entrapment of the probes inside the LNCs, we used an ascorbic acid assay on TB-LNCs. It is well established that ascorbic acid rapidly reduces nitroxides in solution, and similarly it was found

Figure 3 - 2 . 3 - 3 )

 3233 Figure 3-2. EPR spectra acquired in vitro (under air) of TB-LNCs (top) and TAM-LNCs (bottom).

(

  mean ±SD) increased from 1.621 ±0.001 to 1.846 ±0.011 G (presenting only a 14 % increase in LW) whilst TAM-LNCs increased from 0.511 ± 0.003 to 0.711 ±0.002 G (presenting a 39 % increase in LW) (Fig. 3-4), indicating that TAM-LNCs were more sensitive to change in O 2 . The slope of the TAM-LNCs calibration curve (1) 447 . Concerns about the biocompatibility of hexafluorobenzene 450 led us to consider other excipients such as

Figure 3 - 3 .

 33 Figure 3-3. Evolution of EPR signal intensity over time of unencapsulated TB (squares) or TB-LNCs (circles) in the presence of ascorbic acid. Results are expressed as relative to the initial signal intensity (mean ±SD) (n = 3) wherein the dotted line indicates the trend

  experiments as EPR spectra can be recorded in tissues at a depth of approximately 1 cm. After intramuscular or intratumoural administration of unencapsulated TAM, the EPR signal

Figure 3 - 4 .Figure 3 - 5 .

 3435 Figure 3-4. Calibration of the EPR LWs recorded with TB-LNCs (top, left) and TAM-LNCs (top, right) as a function of LW (G) over % O 2 . Relative sensitivity to O 2 as % change in LW as a function of O 2 concentration (bottom) shown with both TB-LNCs (cross) and TAM-LNCs (triangle). Curves show an R 2 of 0.987 and 0.994, respectively.

Figure 3 - 7 .

 37 Figure 3-7. In vivo EPR LW from TAM-LNCs injected in the muscle (n = 10) when mice were breathing air or carbogen. P value of < 0.0001 indicated with *** as determined by the paired Student's t-test.

Figure 3 - 6 .

 36 Figure 3-6. In vivo EPR LW from TAM-LNCs injected in the FSaII fibrosarcoma (n = 9) when mice were breathing air or carbogen. P value of 0.03 indicated with * as determined by the paired Student's t-test.

Figure 3 - 8 .

 38 Figure 3-8. Chemical structure of the EPR probe (a) TAM, and a schematic of the EPR probe-loaded LNCs (b) representing the inclusion of the EPR probe within the LNC lipidic core.

  performed including normal tissue (gastrocnemius muscle) and pathological tissue (FSaII fibrosarcomas-induced subcutaneous tumour) animal models. After administration of TAM-LNCs in the muscle or subcutaneous tumours in the form of a bolus injection, three EPR spectra were acquired; during air breathing, carbogen breathing, and the occlusion. This experiment was applied to both the muscle (n = 10) and subcutaneous tumour (n = 9) models (Fig.3-10 (a) and (b), respectively).The LWs (mean ±SD) measured in the muscles were 0.748 ±0.039 G in mice breathing air and 0.943 ±0.095 G in mice breathing carbogen, and 0.687 ±0.054 G after occlusion was

Figure 3 - 9 .

 39 Figure 3-9. Experimental protocol for EPR spectra acquisition during in vivo experimentation with TAM-LNCs.

Figure 3 - 10 .

 310 Figure 3-10. In vivo EPR LW from TAM-LNCs injected in the (a) muscle (n = 10) or (b) FSaII fibrosarcoma subcutaneous tumour (n = 9) when mice were breathing air or carbogen or underwent occlusion. For statistical analysis, the one-way analysis of variance (ANOVA) test, with the Tukey post-hoc test applied, was used, wherein a p value of < 0.05 indicated with *, p value of < 0.01 indicated with **, and p value < 0.001 indicated with ***. Notably, a nonsignificant result occurs when the p value is > 0.05.

Figure 3 - 11 .

 311 Figure 3-11. The change in EPR LW (mean ±SD) over time for muscle (n = 3, closed circle) and FSaII fibrosarcoma subcutaneous tumour (n = 3, closed square).

  , a need has risen to combat the poor outcome of treatment interventions by developing techniques to image and assess the hypoxic tumour environment. Ideally, the imaging of hypoxia should be noninvasive, sensitive, robust, quantitative, widely available and able to reflect tumour heterogeneity. To this end, techniques such as electron paramagnetic resonance and positron emission tomography imaging have been developed, but magnetic resonance imaging (MRI) techniques are more appealing due to its ability to provide spatial information and its use of nonionising probes. The development of perfluorinated compounds began the use of 19 F-based MR oximetry and introduced the use of exogenous probes 471,652,653 , however their in vivo application was limited by the need for specialist MR equipment regarding the 19 F-tuned probe. Endogenous probes, such as water and lipids, had no such limitation. Regardless the origin of the sensor, oxygen measurement is based on the change in longitudinal relaxation time (T 1 ). Indeed, molecular oxygen (O 2 ) possesses two unpaired electrons which enable its paramagnetic ability to act as a T 1shortening agent in tissues 654 , and therefore as a signal intensity enhancer on MR T 1 -weighted sequences 323 . Therefore, the change of oxygenation is also directly correlated with the longitudinal relation rate (1/T 1 = R 1 ) 471,655 . Despite being attractive for oxygenation measurements, with respect to the high water content of biological tissues, water T 1 change with oxygen concentration is limited, and lower than the change observed with lipids, due the six times higher solubility of oxygen in lipids than in water 506 . This property was exploited by Jordan and colleagues who developed an MR sequence to map oxygen based on imaging of lipid relaxation rates (MOBILE; acronym for mapping of oxygen my imaging of lipid relaxation enhancement) 508 .

2 . 3

 23 Kolliphor® HS 15 (0.846 g), NaCl (0.148 g) and water (2.962 g) were mixed and heated to 95 °C with magnetic stirring.Thereafter the mixture underwent three cycles of cooling and heating between 50 °C and 95 °C. After the final heating phase, the solution was cooled to 72 °C and subjected to an irreversible shock induced by adding 1 mL of 4°C water. This fast cooling allowed for the formation of stable, core-shell LNCs. Thereafter, the osmolarity of the formulation was adjusted to be physiologically safe for in vivo administration. To achieve this, 1 mL of the LNC formulation was dialysed using dialysis tubing (Spectra Biotech Cellulose Ester Dialysis Membrane, MWCO = 100 kDa) for 24 h against pure water (Milli-Q plus® system, Millipore), changing the water hourly for 6 h. Then, to obtain the original volume of LNCs, the dilution factor of the dialysed solution was calculated and evaporation, using N 2 , with magnetic stirring was done. Once the initial volume was obtained, evaporation was ceased and 30 µL of a salt solution (320 mg/mL) was added. Osmolarity measurements were performed using a 5520 Vapro vapour osmometer from Wescor (Logan, Utah, USA). Osmolarity adjusted LNCs were used for all in vivo experiments. The average hydrodynamic size and polydispersity index (PDI) of the LNCs were determined by dynamic light scattering using a Malvern Zetasizer® (Nano Series DTS 1060, Malvern Instruments S.A., Worcestershire, UK) fitted with a 633 nm laser beam (heliumneon laser, 4 mW) at a fixed scattered angle of 173°. To ensure accuracy, LNCs were diluted 1:60 (v/v) in deionised water to produce scattering intensity values that were in the mid-range of the instrument's detector. Values are expressed as mean of three measurements ±standard deviation (SD) for five formulations. In vitro sensitivity of LNCs to pO 2 To evaluate the response of LNCs to variations in O 2 , the MOBILE sequence was applied wherein the T 1 of the methylene group (~1.2 ppm) of the lipid core of the LNCs was measured at 37 °C in sealed tubes bubbled with nitrogen (0 % O 2 ), air (21 % O 2 ) or pure oxygen (100 % O 2 ) for 30 min with magnetic stirring. Temperature of the samples was maintained using a warm water circulation system. MOBILE sequence acquisition parameters were as follows; TR: 1520 ms; mean echo time (TE): 0.8 ms, flip angle: 5°; bandwidth (BW): 200 kHz at 30 % echo position; matrix: 32 x 32, field of view (FOV): 5 x 5 cm; 16 segments, a series of 50 images spaced at TR 29.80 ms with a slice thickness of 4 mm, and a total acquisition time of approximately 35 min. Determination and acquisition of the lipid signal was performed as described by Jordan et al 508 . Briefly, the difference in frequency between the water and lipid peaks (i.e. 1.2 ppm for the methylene group of the lipid core of LNCs) in a 1 H proton spectrum was determined and used as the offset for the imaging frequency for the MOBILE sequence. A Gaussian saturation pulse of 2.74 ms with a 1000 Hz frequency range to spoil the water signal was applied.

5 %

 5 array coil positioned over the rat head for C6-glioma experiment and or a 2 cm diameter loop coil positioned under the mouse leg or tumour. For the duration of all MR experiments, animal temperature was regulated by a heated watercirculating system. For anatomical images, T 2 -weighted rapid acquisition with refocused echoes (RARE) set of images was acquired to follow the volume of tumours (TR = 3200 ms; TE = 21.3 ms; RARE factor = 4; FOV = 4 x 4; matrix = 256 x 256; nine contiguous slices of 0.5 mm, Nex = 1). Thus, once tumours were of proper size, the LNC suspension was injected into the tissue. Animals were anaesthetised, and a bolus injection of 40 µL was injected intramuscularly in the normal tissue model, or intratumourally for the subcutaneous fibrosarcoma tumour model. For LNC delivery into the intracerebral glioblastoma tumour model, a convection-enhanced delivery (CED) protocol 606,660 was implemented to provide a well-diffused distribution of the 40 µL LNCs in the brain tumour. Once the LNC suspension was introduced into the animal model, T 1 was measured using the MOBILE MR sequence (with acquisition parameters described in section 2.3, with geometry changes for in vivo experiments adjusted as follows; SL of 3 mm, and a 1 x 1 cm FOV for mice and 3 x 3 cm for rats). LNC T 1 sensitivity to oxygen variation in the different animal models was assessed using a 20 min carbogen gas (95 % O 2 , Jordan et al. 508 as a segmented inversionrecovery fast imaging with steady state precession (IR FISP), was used to acquire images in order to calculate T 1 relaxation time maps whilst the animal was breathing air and carbogen. After the acquisition of the T 1 -weighted MOBILE MR image set, the T 1 was calculated using a non-linear fit with a home-made program written in Matlab (The MathWorks, Inc., Natick, MA) for a global value of a manually drawn region of interest (ROI), and for each pixel within the same ROI, wherein the magnetisation M z is a function of the inversion time (TI), according to: according to: M z (TI) = A -Bexp(-TI/C); with A = M 0 (T 1 */T 1 ); B = M 0 (1 + T 1 */T 1 ); and C = T 1 *, and then T 1 = (B/A -1)T 1 *.

  of LNCs, a set of T 2 -weighted RARE images was acquired to produce an anatomical image (Fig. 4-2, a), which was used to define the location wherein the LNCs were deposited.

Figure 4 - 2

 42 (b) presents the last image of the MOBILE set, and shows the lipid deposit. It should be noted that the bright areas of the MOBILE image in Fig. 4-2 (b) corresponds to the lipid deposit as well as to the subcutaneous fat surrounding the muscle tissue, while the dark areas within the muscle are where no LNCs were introduced. The absence of signal in the muscle highlights that basal muscular lipid content is too low to give rise to significant lipid signal. A ROI was drawn around the lipid hypersignal arising from the LNCs, and was used to calculate the global R 1 for each animal during air or carbogen breathing. Herein, the carbogen challenge was used to induce a transient increase in oxygenation within the normal and tumoural tissue, as previously demonstrated 323,646,647,649 . In the present study, an increase in oxygenation was observed through the increase in the global R 1 over the LNC area for all explored mice (Fig. 4-2, c: p value = 0.0313, n = 5) with an overall 6 % mean increase. Moreover, a pixel-to-pixel analysis of the LNC area was also performed. The insert in Figure 4-2 (b) shows the R 1 change in the lipid deposit area induced by the carbogen challenge on a pixel-to-pixel basis with the corresponding ΔR 1 distribution histogram shown in Figure 4-2 (d). For all mice, a median of 61 % of pixels responded to the carbogen challenge and showed an increase in R 1 . For the mouse presented in

Figure 4

 4 Figure 4-2 (d), the median change in R 1 after the carbogen challenge was calculated at 0.1509 s -1 with 66 % of the pixels responding (for all mice = median change of 0.3450 s -1 ). For data regarding all individuals in the intramuscular study, see the supplementary documentation (Appendix A, Fig. 0-1).

Figure 4 - 1 .Figure 4 - 2 .

 4142 Figure 4-1. In vitro sensitivity of the lipid relaxation rate (R 1 ) arising from lipid nanocapsules (LNCs) to oxygenation as measured by using the MOBILE MR sequence (R 2 = 0.9948).

Figure 4 - 3 .Figure 4 - 4 .

 4344 Figure 4-3. Application of lipid nanocapsules (LNCs) to assess variations of oxygenation in a subcutaneous FSaII fibrosarcoma tumour mouse model using lipid relaxation rate measured with the MOBILE MR sequence, wherein (a) shows the transversal anatomical image of a fibrosarcoma tumour and the injected LNCs (indicated by arrow) and (b) shows the lipid image issued from the set of images acquired with the MOBILE MR sequence wherein the lipids from the LNCs can be observed. The global R 1 measured over the entire lipid deposit signal for all mice (n = 7) is shown in (c) (p value = 0.0452 indicated by *). The insert in (b) indicates the change in R 1 (ΔR 1 = R 1 carbogen -R 1 air ) for each pixel of lipid signal in the MOBILE image and a colour scale highlights the more responsive pixels in red (s -1 ). ΔR 1 distribution of all encompassing pixels in the lipid deposit region of interest is represented as a histogram (d), wherein the median change of R 1 is indicated with a dotted red line.

  level variation accounts for a substantial change in R 1 and a linear relationship could be established (Fig. 4-1); as oxygenation increased, R 1 increased correspondingly. This relationship between the R 1 of lipids found in the LNCs and oxygen content could be reliably demonstrated and led to in vivo experimentation. Thus, to further test the ability of the LNCs to probe oxygenation variation in vivo, a carbogen challenge was performed using three animal tissue models, namely a normal gastrocnemius muscle tissue model and two tumoural tissue models 428,650,662,663 . A bolus injection was used to introduce the LNC formulation into the muscle tissue, and the MOBILE MR image after the injection showed that the solution remained as a conglomeration, i.e. as a 'drop' (Fig. 4-2 & 4-3, b). This 'drop' of injected LNCs remained as is for the duration of the experiments and did not diffuse throughout the tissue. This can be attributed to the direct injection into the tissue; since the LNCs circumvent the washout and biodistribution associated with an intravenous injection 568,664-666 this allows for the formulation's sustained presence within the tissue. Thus, the persistence of a 'drop' of injected LNCs within the tissue is to be expected, as the normal gastrocnemius muscle is a highly hierarchized tissue and the bolus injection takes place over a short period of time, thus not allowing the liquid to disperse throughout the tissue. Regardless of the injected LNCs in the tissue, the lipids in the LNCs responded to the carbogen challenge and a map portraying the variation of the T 1 relaxation rate induced by percentage O 2 inhaled could be produced. This map enabled the visualisation of tissue pO 2 of the normal gastrocnemius muscle. Of note, the intrinsic muscular lipidic signal is too low to portray the effect of the challenge, thus making the LNC injection compulsory as no lipid signal can be observed outside the lipid drop (Fig. 4-2, b). This observation was also noted in the tumoural models used in this study (Fig. This muted response could be attributed to the poor blood perfusion prevalent in this tumour model 384,422,627 , which limits the blood's transportation of the carbogen gas to the tissue environment near the drop of LNCs. Similarly, this could explain the instance in which the R 1 decreased after the carbogen challenge in an individual animal. On the contrary, the intracerebral glioblastoma model, is associated with a heterogeneous tumour microenvironment which is highly vascularised 649 . This readily explains the increased response of this tumour model, comparative to the normal gastrocnemius muscle tissue, to the carbogen challenge. Furthermore, the methods of LNC introduction between the two tumour models should be noted. The CED protocol allowed for a more even distribution of the LNCs throughout the brain tumour tissue, whereas the bolus injection within the muscle and subcutaneous tumour model only allowed for the mapping of the LNCs within the drop. Oxygenation mapping within the normal tissue and tumoural models showed that LNCs were able to improve the lipid signal-to-noise ratio to such an extent that pixel-to-pixel analysis of the area was achievable. Therefore, it was possible to image and analyse the response of each pixel to the carbogen challenge within all the animal models without using ionising probes or specialised 19 F-based MR equipment 294,485 , thus making our system more widely applicable. Furthermore, in our study, the bolus injection allowed for a very specific site within the muscle and subcutaneous fibrosarcoma tumour to be analysed, whereas the CED diffusion of the LNCs throughout the intracerebral glioblastoma model allowed for the mapping and analysis of the entire area of the injected LNCs. Using the pixel-topixel analysis, it was possible to map the heterogeneity of the response from air to carbogen breathing for each animal (Fig. Although mapping of pO 2 change in tissue was achievable and successful, the conversion of R 1 to pO 2 proved difficult in vivo. Direct conversion, on the basis of data presented in Figure 4-1, of R 1 into absolute pO 2 values cannot be done because although R 1 has been shown to be dependent on oxygenation it is also dependant on the heterogeneity of tissues,

  were synthesised using the phase-inversion process, and the osmolarity of the formulation was adjusted for safe in vivo use. In vitro validation showed a linear relationship between oxygen variation and R 1 , thus demonstrating the permeability and responsiveness of the LNCs to oxygen. For in vivo experimentation, induce transient oxygenation changes within the tissue. From a single bolus injection of the LNCs, both a global R 1 value, as well as a pixel-to-pixel analysis was possible, due to the improved signal-to-noise provided by the lipid-core of the LNCs. However, because the bolus injection occurred over a short period of time, a limited area of tissue could be mapped. Thus, a CED injection was used to diffuse the LNCs throughout the intracerebral glioblastoma tumours to provide a more detailed oxygenation and heterogeneity map. Unfortunately, only relative pO 2 values were achievable from the LNC system as a myriad of parameters influence T 1 relaxation in vivo. Regardless, these developments and results indicate

  i.e. a normal gastrocnemius muscle model in C3H/HeJ mice (Janvier, Le Genest Saint Isle, France) (n = 5), a normal brain model in female Sprague Dawley rats (SCAHU-Angers, France) (n = 6), and one tumoural tissue model, i.e. the C6 intracerebral glioblastoma tumour model in female Sprague Dawley rats (SCAHU-Angers, France) (n = 5) as described.4.3.2.3 MR experimentsMR imaging was performed using a 7T scanner (Biospec 70/20 Avance III, Bruker Wissembourg, France) equipped with BGA12S gradient system (675 mT/m) as described in section 4.2. Lipid signal was attained from the described MOBILE sequence (acquisition parameters described in section 4.2). LNC fate was evaluated after a 40 µL injection of the formulation using either a bolus injection in the normal gastrocnemius muscle or the CED protocol for the normal brain and the C6 intracerebral glioblastoma model. The CED protocol involved the injection of 40 µL of the LNC formulation at the coordinates of the tumour cells (or, in the case of the normal brain animals, in the same coordinates in which the cells would have been injected). Infusions were performed at the depth of 5 mm from the brain surface using a 40 µL Hamilton® syringe with a 22G needle. CED was performed with an osmotic pump PHD 2000 Infusion Chapter 4: Magnetic Resonance Imaging and Lipid Nanocapsules Janske NEL | Lipid nanocapsules as a theranostic tool 96

4 - 5 .

 45 example of the constituents associated with the LNC formulation within the intracerebral brain tumour model is shown in Figure In this example, the resonance associated with the water (chemical shift = 4.7 ppm) is unsuppressed and was used to determine the ratio between the water and CH 2 resonance (chemical shift = 1.2 ppm) associated with the LNC constituent Labrafac® over time. In the insert of Figure 4-5, the metabolitescreatine (chemical shift = 3.0) and choline (chemical shift = 3.2)commonly associated with a tumour induced with C6 glioblastoma cells can be observed for both the spectra 672,673 . In both the water unsuppressed and suppressed spectra, a decrease in the resonances of PEG and CH 2 can be noted over time, with no PEG being measured after 24 h.

Figure 4 - 5 .Figure 4 - 6 .

 4546 Figure 4-5. Percentage (%) of CH 2 , associated with the lipids from LNCs, within the normal brain (NB, blue circle) model, the brain tumour (BT, red square) model and the normal muscle (MSCL, green triangle) model over time as compared to water.

Figure 4 - 7 ,Figure 4 - 7 .

 4747 Figure 4-7, an average increase of 9 % was observed (p = 0.0469, n = 6). For the data regarding all individuals in the normal brain tissue study, see the supplementary documentation (Appendix B, Fig. 0-4).

  LNCs could respond to O 2 changes, thus further demonstrating the permeability of the LNCs to O 2 . To demonstrate the ability of the LNCs to respond in vivo, several animal models including both normal tissue and hypoxic tumoural tissues were used. LNCs were evaluated as a tool to assess and image the heterogeneity of hypoxia within tumours. Here, a bolus injection was usedsimilar to the EPR experiments -which deposited a lipid 'droplet' within normal muscle and subcutaneous fibrosarcoma tumour models. Using the MOBILE MR sequence it was possible to image the introduced lipidsin the form of LNCsas well as measure their response to O 2 variation (Fig.4-2 and 4-3) following a carbogen challenge.

  LNC formulation is simple to synthesise and has been well-characterised, its constituents are FDA approved for in vivo use, and the therapeutic uses of drug-loaded LNCs have been demonstrated in several pre-clinical studies. Similarly, the magnetic resonance techniques used in this thesis, EPR and MRI, are frequently used for determining tumour physiology and oxygenation. These MR methods have well-understood limitations with respect to oxygen assessmentsuch as the degradation of the soluble EPR spin probes with time and the intrinsically low signal-to-noise ratio of the lipids assessed with the MOBILE MR sequence in MRIwhich were addressed in this thesis by the use of the LNC formulations. However, there are still several limitations that should be addressed in regards to the application of LNCs as an O 2 sensor in tumours. Some of these limitations, and their associated perspectives, are evaluated and discussed in this section wherein Tables5-1 and 5-2 consider the in vitro parameters and Tables 5-3 and 5-4 the in vivo parameters using a Strengths, Weakness, Opportunities and Threats (SWOT) analysis.

Chapter 5 :

 5 Conclusions, Limitations and PerspectivesJanske NEL | Lipid nanocapsules as a theranostic tool 110

( 158 )Figure 0 - 1 .Figure 0 - 2 .Figure 0 - 3 .

 158010203 Figure 0-1. Supplementary data regarding the intramuscular normal muscle model experiments. Application of LNCs to assess variations of oxygenation in the intramuscular tissue of mice using lipid relaxation rate measured with the MOBILE MR sequence, wherein (a), (d), (g) and (j) show the transversal anatomical images of mice gastrocnemius muscles and the injected LNCs (indicated by an arrow). Images (b), (e), (h) and (k) show the lipid image issued from the set of images acquired with the MOBILE MR sequence wherein the lipids from the LNCs can be observed. The insert indicates the change in R 1 (ΔR 1 = R carbogen -R air ) for each pixel of lipid signal in the MOBILE image and a colour scale highlights the more responsive pixels in red (s -1 ). ΔR 1 distribution of all encompassing pixels in the lipid deposit region of interest is represented as a histogram in (c), (f), (i) and (l), wherein the median change of R 1 is indicated with a dotted red line.

  . ...........................................................................................................11 Schematic illustrating the difference between oxidative phosphorylation, anaerobic glycolysis, and aerobic glycolysis(Warburg effect). In the presence of O 2 , non-proliferating cells (differentiated) preferentially metabolise glucose through the oxidative phosphorylation pathway, via the mitochondria. In this instance, because O 2 is the final electron acceptor to enable the complete oxidation of glucose, O 2 is essential for the process. However, when O 2 is limited, glycolysis can be used alternatively but results in fewer ATP than during oxidative phosphorylation. Conversely, in proliferating or tumoural tissue, although the mitochondria are functioning normally, the aerobic glycolysis pathway (Warburg effect) can be preferred. Image reproduced from Vander Heiden et al.

	Figure 2-2.

58 . .................................................................................14 Figure 2-3. Schematic illustrating the role of hypoxia in cancer progression, angiogenesis, metastasis and, inevitably, resistance to therapy. Image reproduced from Muz et al. 6 . .......................................................................17

Table 2 - 1 .

 21 Main characteristics of methods used to assess in vivo hypoxia. .............21

Table 3 - 1 .

 31 Characteristics of EPR probe-loaded LNC preparations ..........................64

Table 5 - 1 .

 51 SWOT analysis of the in vitro studies regarding the sensitivity curve. ..106

Table 5 - 2 .

 52 SWOT analysis of in vitro studies regarding the LNC formulation. ......106

Table 5 - 3 .

 53 SWOT analysis of the in vivo studies regarding the animal models. .....107

Table 5 - 4 .

 54 SWOT analysis of the in vivo studies regarding the applicability of LNCs as a theranostic tool .........................................................................................108

.3 EXISTING METHODS TO MEASURE HYPOXIA

	Chapter 2: Background to the Research Area
	2Considering the severe consequences of hypoxia, it is no surprise that the measurement
	of O 2 tension in cancer represents a long-standing endeavour of medicine and biology.
	Because low O 2 tension is linked to such dire therapeutic outcomes, a collection of adjuvant
	therapeutic interventions involving increased O 2 administration have been proposed over the
	last several decades to improve the therapeutic outcomes of cancers. These include hyperoxic
	gas breathing -including the use of 100 % O 2 , 95 % O 2 /5 % CO 2 (carbogen gas) breathing
	and accelerated radiotherapy combined with carbogen gas breathing and nicotinamide
	(ARCON) -hypoxic cell-selective cyto-toxins, and IMRT 154-160 . However, despite the
	promising perspectives these therapeutic approaches have gained moderate success at the
	clinical level. This can be attributed to several factors, including the heterogeneity of hypoxia
	and a lack of accurate methods to characterise tissue O 2 levels in vivo. On these grounds, a
	more precise and robust measurements of the O 2 levels in vivo would allow a better selection
	of patients responding positively to hypoxia-directed treatments. The ideal properties of a
	hypoxia measurement technique should include; non-invasiveness, adequate spatial and
	temporal resolution, quantitative measurement, no O 2 consumption, adequate range of O 2
	tension values measured, and if an exogenous agent is required, exhaustive knowledge of its
	toxicology and pharmacodynamics 161 . Unfortunately, there is currently no in vivo method
	that fulfils satisfactorily all these requirements. Table 2-1 summarises the most relevant
	methodologies, together with their mechanisms, characteristics, advantages and limitations.
	It is clear, therefore, that the challenges faced by researchers to develop methods to determine
	tumoural O 2 tensions are immense and of crucial importance.
	Janske NEL | Lipid nanocapsules as a theranostic tool	20

Table 2 -1. Main

 2 characteristics of methods used to assess in vivo hypoxia.

	O 2 electrodes	IHC

IHC, immunohistochemistry; PAT, photoacoustic tomography; PET, positron emission tomography; SPECT, single-photon emission tomography; Hb/HbO2, deoxygenated and oxygenated haemoglobin ratio. Adapted from citations

[162][163][164][165][166]

.

  312,313 but has shown inconsistent uptake into various tumours, thus efforts have been made

	towards its modification 314 . Newer agents include iodoazomycin pyranoside (IAZP), beta-D-
	iodinated azomycin galactopyranoside (IAZGP), and iodoazomycin xylopyranoside (IAZXP)
	based on the azomycin-nucleoside structure 315 . 99m Tc is an isotope gaining popularity, with
	nitroimidazoles and non-nitroimidazoles labelled with 99m Tc increasingly being used to study
	tumour hypoxia, such as; the nitroimidazole compounds BMS-181,321 and BRU 59-21; and

complex ligands without the nitroimidazole group, such as N2IPA, and 99m Tc-HL91 315 .

Unfortunately, BMS-181,321 suffer from instability, low clearance from the blood, and high background levels in normal tissues 316 . BRU 59-21 has greater stability in vitro and more

  Another factor influencing EPR spectra is the interactions of unpaired electrons with its environment thus influencing line shape. Biomolecular collisions between O 2 and free radicals alter the resonance characteristics of the radical and consequently the EPR spectrum.Notably, molecular O 2 is an efficient relaxer (i.e. capable of shortening relaxation times) of other paramagnetic species. The enhancement of relaxation times scales linearly with the concentration of O 2 , thus measurements that depend on T 1 and T 2 of EPR spin probes can provide a direct indication of the concentration of O 2 . The most common method is to use the broadening of the principal hyperfine line (related to T 2 ). This line broadening occurs by direct biomolecular collisions between two paramagnetic species (with the rate of collision given by the Smoluchowski equation 337 ) and by Heisenberg spin exchange 338 .

Unfortunately, there is a lack of sufficient amounts of endogenous naturally occurring paramagnetic metal ions and free radicals to be detected directly by EPR in vivo. This deficiency makes it necessary to use exogenous paramagnetic materials, but not all paramagnetic substances are convenient for oximetry purposes. The main requirements for a suitable exogenous spin probe for oximetry applications are; a few narrow EPR lines in the EPR spectrum, good stability in biological media, and no pharmacotoxicity issues. Two types of probes are used; soluble probes that report the concentration of dissolved O 2 , and particulate probes that measure pO 2 in tissue. Both have certain advantages and drawbacks.

  INTRODUCTION

					providing estimates of pO 2 values.
	Tissue oxygenation is a key parameter in	However, they are also prone to signal
	physiology	and	pathophysiological	decrease due to rapid washout and/or
	conditions, including wound healing,	metabolic	biotransformation.	The
	ischemia, peripheral vascular disease	encapsulation of EPR spin probes could
	and cancer. In oncology, tumour hypoxia	potentially	circumvent	these
	is known to decrease the response to	disadvantages
	radiation therapy 127,628 , decrease the
	efficacy of cytotoxic drugs 129,629 , and
	play a pivotal role in malignant
	progression	including	metastasis
	(reviewed in 4,101,630-633 ). Therefore,
	appropriate knowledge of tumour
	oxygenation may help in guiding anti-
	cancer treatments. An assortment of
	different techniques has been developed
	for measuring tumour oxygenation,
	including	electron	paramagnetic
	resonance	(EPR)	oximetry.	EPR
	oximetry is capable of producing real-
	time, repeated measurements of partial
	pressure of oxygen (pO 2 ) values in a
	non-invasive manner without consuming
	O 2 . This technique uses paramagnetic
	spin probes which can be either in
	solutions or in particulate forms.
	Interactions between the unpaired
	electron spin centres 164,634 of the probes
	and oxygen molecules cause a
	shortening	of	relaxation	times,
	producing a broadening effect on the
	EPR line width (LW) of the spin probes.
	Soluble probes have the advantage of
	diffusing throughout a tissue and

Table 3 - 1 .

 31 Characteristics of EPR probe-loaded LNC preparations

1 mean ±SD (n = 3) 2 measured on the central peak of the nitroxide

  The semi-quantitative PRESS analysis revealed that the lipid signal arising from the LNCs decreased by 50 -60 % for all the animal models tested. Most notable, however, was the decline in observable lipid (1.2 ppm) and PEG (3.7 ppm) resonances associated with the LNCs after 24 h. These results indicate that the LNCs cannot be used in the context of long-term (e.g. several days) oximetry measurements in vivo. Such a decline in LNCs could be associated with the metabolic degradation of the nanocapsules within the tissue, or the displacement of the LNCs from the measured area (i.e. the injected area).

	5.1 CONCLUSIONS
	This thesis was focused on developing LNCs -which have already been established as
	a nanocarrier for cancer therapeutics -as an O 2 nanosensor capable of measuring and
	imaging pO 2 in hypoxic tissues, including tumours, using EPR and MRI methods.
	EPR oximetry is potentially capable of producing real-time, repeated measurements of
	pO 2 values within tissues in a non-invasive manner without consuming O 2 . The technique
	uses paramagnetic spin probes which can be either in solutions or in particulate forms.
	Chapter 5:
	Conclusions, Limitations and Perspectives

2-12 and 2-13), at

  21 % O 2 thus indicating its increased O 2 sensitivity 676 . Altogether, the soluble spin probes have the advantage of diffusing throughout a tissueas opposed to the particulate EPR spin probes which are implanted in an areaand providing measurements of O 2 values. Unfortunately, the soluble spin probes are prone to signal decrease due to rapid washout and metabolic biotransformation. Furthermore, soluble EPR probes are less sensitive to O 2 changes compared to particulate EPR probes which can be problematic for evaluating subtle changes in tissue oxygenation. To overcome this limitation, encapsulation within a lipophilic environment has been explored 442 . Consistent with the Smoluchowski equation, as O 2 is more soluble in lipophilic environment than in

  Notably, the water T 1 change with O 2 concentration is limited and lower than the change observed in lipids due to the six times higher solubility of O 2 in lipids than in water 506 . Thus, Jordan and colleagues developed the MOBILE MR sequence 508 which exploited this phenomenon to map O 2 in tissues by imaging lipids relaxation enhancement. The drawback of this method, however, is that it requires a sufficient amount of lipids in the tissue of interest to enable the MOBILE sequence to produce a lipid image.

	MRI-based techniques are ideally suited for the assessment and imaging of tumour
	physiology and hypoxia as it is non-invasive, quantitative, widely available and able to reflect
	tumour heterogeneity. MRI techniques for hypoxia determination include perfusion-and
	probe-based (both exogenous and endogenous) imaging. Notably, however, perfusion-based
	techniques -such as BOLD, FLOOD or OE-MRI -do not provide direct pO 2 values but
	instead rely on relative changes in blood flow and Hb/HbO 2 ratio, furthermore these methods
	suffer from poor sensitivity, and changes to relaxation times not related to O 2 level 504 .
	Alternatively, probe-based methods rely on either exogenous or endogenous probes for pO 2
	measurements. Notable exogenous probes include 19 F which, although sensitive to tumoural
	levels of O 2 , requires specialised MR equipment. Of course, endogenous probes -such as
	water and lipids -have no such limitation. However, although water is an attractive option
	for monitoring tumoural pO 2 -due to its abundance in biological tissue -methods which
	monitor its relaxation rate could be considered as not sensitive enough for measuring hypoxia
	in tumours 323,504,505,677 .

-5), a decrease in the signal intensity is notable over time which was similar to the decrease of measurable signal over time noted for the LNCs in the MRI experiments.

Table 5 - 1 .

 51 SWOT analysis of the in vitro studies regarding the sensitivity curve.

	Strengths	Weaknesses	Opportunities	Threats
		In

vitro studies: Sensitivity curve TAM-LNCs are sensitive to O 2 variation as assessed by EPR (see Fig. 3-2 and 3-4) T 1 of lipid CH 2

			Develop an improved ex
		pO 2 values are	vivo model which	Cannot directly convert
	backbone (Labrafac®)	overestimated	mimics the in vivo	LW or T 1 to pO 2
	is detectable by the		situation
	MOBILE sequence,
	demonstrated linear
	sensitivity to O 2
	variation
	(see Fig. 4-1)

Table 5 - 2 .

 52 SWOT analysis of in vitro studies regarding the LNC formulation.

	Strengths	Weaknesses	Opportunities	Threats
		In

vitro studies: LNC formulation Soluble EPR spin probes (nitroxides and trityl) could be encapsulated in LNCs (see Table 3-1, Fig. 3- 3 and Fig. 3-5)

			TB-LNC system could
		Probes with low log P	be used as a new tool	Bio-reduction of nitroxides;
		did not remain within	for free radical	not suitable for oximetry
		lipidic core of LNCs	scavenging in cells	studies
			678
	Osmolarity adjusted LNCs comparable to previously published formulations	Dialysis to eliminate consuming) salt, followed by re-concentration for in vivo use (time	Use of low energy process	Sterilisation of LNC formulation will require advanced processes

Table 5 - 3 .

 53 SWOT analysis of the in vivo studies regarding the animal models.

	Strengths	Weaknesses	Opportunities	Threats
		In vivo studies: Animal models
	No tissue damage
	(visible by MRI)
	when LNCs
	introduced into tissues (see Fig. 4-2, 4-3, 4-4 and 4-7)		Use of immunohistochemistry techniques for hypoxia staining
	pO 2 changes induced techniques EPR and MRI be assessed using challenge tissue could by the carbogen	No associated proof of tissue hypoxia	radiation therapy in dose painting for carbogen challenge; use where O 2 diffuses during ΔpO 2 map indicating	LNC signal cannot be visualised in necrotic regions
	(see Fig. 3-6, 3-7, 4-2,
	4-3, 4-4
	and 4-7)

Table 5 - 4 .

 54 SWOT analysis of the in vivo studies regarding the applicability of LNCs as a theranostic tool

	Strengths	Weaknesses	Opportunities	Threats
		In

vivo studies: LNCs as a theranostic tool No 'reservoir effect' was observed for the LNCs (see Fig. 3-11)

	Did not evaluate ability
	of LNCs to accumulate O 2 during carbogen challenge	Design of nanocarrier to sequester O 2	Synthesis of formulation capable of 'reservoir effect'
	Increase in lipids and
	PEG was detectable
	in vivo after LNC
	introduction with
	MRI, and showed
	good SNR
	(

see Fig. 4-9, insert for an example) TAM-LNC signal measurable in vivo for at least 60 min (see Fig. 3-5) LNCs produce sufficient signal for MOBILE lipid measurements for 2 - 3 h in vivo (see Fig. 4-2, 4-3, 4-4, 4-9 and 4-7)

		Determination of the
		origin of the signal
		decreases: LNC
	Decrease of signal over time	biodegradation and/or formulation dispersion	Stabilisation of LNCs to keep the diagnostic valence may impair the therapeutic one
		Modification of LNC
		formulation to limit
		biodegradation

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Janske NEL | Lipid nanocapsules as a theranostic tool

Acknowledgements

I would like to extend my thanks to the many people, in many countries, who so graciously contributed to the work presented in this thesis.

ACKNOWLEDGEMENTS

The authors would like to thank the NanoFar Erasmus Mundus program, the "Fonds du Patrimoine" (Secteur des Sciences de la Santé, UCLouvain) and comité départemental de Maine and Loire de la Ligue Contre le Cancer (France) for providing the funding for this project. ACKNOWLEDGEMENTS The authors would like to thank the NanoFar Erasmus Mundus program, le comité départemental de Maine et Loire de la Ligue Contre le Cancer (France), and la Chapter 4: Magnetic Resonance Imaging and Lipid Nanocapsules Janske NEL | Lipid nanocapsules as a theranostic tool 94 Commission du Patrimoine (Université catholique de Louvain) for providing the funding for this project.