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Osteoarthritis (OA) is a chronic and inflammatory disease with no effective regenerative treatments to date. The therapeutic potential of mesenchymal stem cells (MSCs) remains to be fully explored. Intra-articular injection of these cells promotes cartilage protection and regeneration by paracrine signaling and differentiation into chondrocytes. However, joints display a harsh avascular environment for these cells upon injection. This phenomenon prompted researchers to develop suitable injectable materials or systems for MSCs to enhance their function and survival. Among them, hydrogels can absorb a large amount of water and maintain their 3D structure but also allow incorporation of bioactive agents or small molecules in their matrix that maximize the action of MSCs. These materials possess advantageous cartilage-like features such as collagen or hyaluronic acid moieties that interact with MSC receptors, thereby promoting cell adhesion. This review provides an up-to-date overview of the progress and opportunities of MSCs entrapped into hydrogels, combined with bioactive/small molecules to improve the therapeutic effects in OA treatment.

Hyperthermia, an established adjuvant in cancer treatment, potentiates the effects of anticancer drugs and synergetic combination can be obtained improving the therapeutic outcome. Bone metastases may benefit from this combination when treated through in situ forming implants. Once loaded with magnetizable (nano-) particles and antineoplastic agents, these implants allow for the local application of magnetically-induced hyperthermia and the release of an anticancer drug. The implant can be heated applying an external magnetic field, sensitizing the surrounding tumoral tissues, while releasing the chemotherapeutic agent. Moreover, bone implants provide mechanical support to the weakened bone and consequently relieve pain. This review gives an overview of the current knowledge as well as the rationale of combining locally both magnetic hyperthermia and chemotherapy using in situ forming implants in the field of bone metastases treatment.

In this chapter we review both preformulation and formulation efforts relevant to magnetically-induced hyperthermia as a new and attractive modality for the treatment of cancer lesions eligible for a thermotherapy. Also addressed are the efforts to apply this method to de novo indications in specific clinical situations. Following a pharmaceutical approach, we first introduce the general biological rationale for the use of hyperthermia, considering the techniques available to generate hyperthermia. We then detail several different magnetically-induced heating modalities and review the literature on formulations in an attempt to compare their specificities, advantages and shortcomings. First, we consider the formulation of glass ceramics and cement biomaterials for magnetically mediated hyperthermia with respect to the biological specificities in the treatment of solid bone tumors. Secondly, formulations intended for magnetically mediated hyperthermia are considered for soft tissue solid tumors, emphasizing the potential for pharmacological modulation. In the final section, we consider magnetic liposome formulations that can be equally administrated in various types of tumors. We do not detail magnetic fluid hyperthermia that uses suspensions of magnetic nanoparticles stabilized by various coatings. Biological and immunological considerations revealed by liposomes are outlined. This chapter focuses on the importance of the formulation and on the highly complex interactions between formulation and hyperthermia that should be considered with respect to various medical applications.

Magnetic iron oxide nanoparticles with a narrow size distribution were synthesized by hydrothermally treating suspensions of iron oxide nanoparticles. Ricinoleic-acid-coated magnetic nanoparticles were co precipitated at room temperature from an aqueous solution of Fe2+/Fe3+ cations by the addition of a base. The presence of the ricinoleic acid on the nanoparticles' surfaces strongly suppressed their growth under the hydrothermal conditions. Because of the strong dependency of the rate of particle growth on their size, the size distribution significantly narrowed during the hydrothermal treatment. The size of the nanoparticles was successfully controlled by the temperature of the synthesis and the amount of ricinoleic acid present in the reaction mixture to between 9 and 30 nm. The presence of the ricinoleic acid on nanoparticles' surfaces enabled the preparation of colloidal suspensions in even moderately polar organic liquids. Measurements of the magnetic properties revealed that the nanoparticles smaller than 14 nm exhibited superparamagnetic behavior and nanoparticles larger than 15 nm displayed single domain ferrimagnetic behavior. The nanoparticles exhibited large values of saturation magnetization of up to 90 emu/g. The strong dependence of the nanoparticles' specific power losses when subjected to an alternating magnetic field on their average size and frequency was demonstrated. The ferrimagnetic nanoparticles showed much higher power losses than the superparamagnetic nanoparticles. (C) 2016 Elsevier B.V. All rights reserved.

The injectable formulation for treatment by hyperthermia comprises a liquid carrier and heat-generating superparamagnetic iron oxide nanoparticles having a mean diameter not greater than 20 nm. Said injectable formulation is able to form in-situ a hyperthermic solid or semi-solid implant upon contact with a body fluid or tissue. Said hyperthermic solid or semi-solid implant may be useful for treating a tumor or a degenerative disc disease by hyperthermia.

A method of producing a microencapsulated pharmaceutical formulation is disclosed comprising causing a dye to be attached to the surface of pharmaceutical particles or particle clusters and applying a source of radiant energy to the dye in the presence of a liquid polymeric or polymerisable material so as to cause the material to cross-link, producing a conformal layer of cross-linked polymer on the particulate surfaces. Preferably, the polymer provides an immuno-protective layer around the particles, while allowing therapeutic components to exit the microcapsules. Microencapsulated pharmaceutical formulations and their medical use are also disclosed, especially for the treatment of diabetes by encapsulating insulin secreting cells.

Les différentes études présentées dans cette thèse ont permis de faire face et ont tenté de répondre à un certain nombre de défis rencontrés dans la recherche de l'IA DDS pour le traitement de l'arthrose. Tout d'abord, le potentiel des molécules thérapeutiques pour l'administration locale. Les produits naturels sont de riches sources de molécules thérapeutiques ; les médicaments contre l'arthrose potentiellement modificateurs de la maladie (DMOAD) et les voies moléculaires méritent d'être explorées plus avant en appliquant des modèles précliniques précis de l'arthrose. Ensuite, le développement de systèmes d'administration de médicaments par des vecteurs polymériques et enfin, l'établissement de modèles in vitro précis d'arthrose pour l'évaluation des résultats de ce type de formulations. Les microparticules polymères avec des nanoparticules de médicament broyées par wet milling et des taux de chargement de médicament réglables semblent répondre à plus d'une demande de traitement de l'arthrose par IA. Pour améliorer la conception et le développement de ces DDS IA, il est nécessaire d'explorer des modèles in vitro d'arthrose améliorés et plus prévisibles, en accordant une attention particulière aux caractéristiques de ces formulations, comme la libération prolongée du médicament. Tous les angles explorés de la recherche sur les IA DDS méritent, et bénéficieront, d'améliorations supplémentaires dans le but ultime d'offrir de meilleures options de traitement de l'arthrose.

Injection of nanoparticles (NP) into the bloodstream leads to the formation of a so-called “nano–bio” interface where dynamic interactions between nanoparticle surfaces and blood components take place. A common consequence is the formation of the protein corona, that is, a network of adsorbed proteins that can strongly alter the surface properties of the nanoparticle. The protein corona and the resulting structural changes experienced by adsorbed proteins can lead to substantial deviations from the expected cellular uptake as well as biological responses such as NP aggregation and NP-induced protein fibrillation, NP interference with enzymatic activity, or the exposure of new antigenic epitopes. Achieving a detailed understanding of the nano–bio interface is still challenging due to the synergistic effects of several influencing factors like pH, ionic strength, and hydrophobic effects, to name just a few. Because of the multiscale complexity of the system, modeling approaches at a molecular level represent the ideal choice for a detailed understanding of the driving forces and, in particular, the early events at the nano–bio interface. This review aims at exploring and discussing the opportunities and perspectives offered by molecular modeling in this field through selected examples from literature.

The aim of this thesis was to explore the link between stability and immunogenicity of therapeutic proteins. Using various environmental stress conditions, protein aggregates were formed, biophysically characterized, and the relationship between the degree of aggregation and the impact on the protein bioactivity highlighted. Then, in view of simulating in vitro the phenomena of in situ protein aggregation once injected in the subcutaneous tissue, we reviewed the literature on 2D and 3D assays predicting the potential immunogenicity of (new candidate) therapeutic proteins. Based on that, an immunocompetent model of the human subcutaneous tissue was developed, combining hydrogels scaffolds into which antigen-presenting cells (MUTZ-3 dendritic cells) were embedded.

The aim of this thesis was to explore iron oxide nanoparticles (IONPs) for ultimate detection and co-treatment of early prostate cancer (PC) lymph node metastases. IONPs previously optimized as a theranostic tool for the combined use in MRI and magnetic hyperthermia were functionalized with an RNA aptamer or a small molecule, both actively targeting the prostate-specific membrane antigen (PSMA). Functionalized IONPs were developed to meet the requirements for lymphatic targeting and specific binding to PSMA-expressing PC cells while preserving high MRI relaxivity and heating properties. In a second project, we designed and tested an in situ forming implant entrapping IONPs as a heating source for magnetic hyperthermia with intended application in localized PC. Besides appropriate syringeability and implant formation upon injection, we confirmed homogeneous intrinsic radiopacity in micro-CT in vivo. When applying magnetic field strengths and frequencies eligible for use in humans, the implant reached the hyperthermia target temperature of 48 °C.

Osteoarthritis (OA) is the most common type of arthritis affecting 80 % of the world's population over 75 years. Despite this rising trend, there is as yet no cure for OA. This thesis aimed to design two efficient biodegradable and biocompatible extended release formulations for intra-articular (IA) administration to treat the disease and improve the quality of life of patients. Firstly, Nanocrystal-Polymer Particles (NPPs), with a very high drug loading of PH-797804 or Kartogenin, were formulated by a wet-milling/spray drying process. Secondly, a thermosensitive hyaluronic acid able to form nanoparticles in situ was designed and developed as a new way of lubricating joints. In vivo, both of these technologies showed a positive effect based on histological scoring, epiphyseal thickness, OA biomarkers and prolonged IA persistence in a murine OA model. This thesis provides proofs-of-concept of novel and innovative extended drug delivery system/viscosupplement with the potential to treat human OA.

We explored strategies to modulate epithelial tight junctions for barrier protection and for drug delivery. Our findings indicate that toll-like receptor 2 (TLR2) stimulation in human bronchial epithelial cells increased tight junction barrier function by increasing the expression of claudin-1, and through modulation of PKCζ activity. We designed and characterized cell permeable short peptides that can transiently open tight junctions. The peptides induced redistribution of tight junction proteins claudin-1 and ZO-1 into the intracellular compartment thereby inducing a transient increase in paracellular permeability. We investigated the lead short peptide (SG-01) for its ability to act as an anti-tumor adjuvant and mucosal vaccine adjuvant. The peptide and its combination with a cytotoxicity drug gefitinib showed a significant improvement in the reduction of tumor size in a 3D tumor model. A preclinical study in mice showed that SG-01 significantly improved immunogenicity of ovalbumin probably by improving its permeation across nasal mucosa.

In this thesis, we explore a novel anti-angiogenic strategy for the treatment of intermediate hepatocellular carcinoma. Patients suffering from this stage of the disease are usually treated by transarterial chemoembolization, which has proven clinical benefit. Nevertheless, ischemia created by the embolization leads to post-interventional neoangiogenesis, and may eventually result in tumor rebound. Therefore, the idea evolved to combine marketed drug-capable embolic beads, which are successfully established in this technique, with an anti-angiogenic drug. An additional advantage of the beads is the possibility to deliver the drug locally and in a controlled manner, while possibly reducing undesired drug-related systemic toxicity.------- L'objectif de cette thèse est l'exploration d'une stratégie anti-angiogénique pour le traitement du carcinome hépatocellulaire. Les patients au stade intermédiaire de la maladie sont généralement traités par la chimioembolisation transartérielle, qui a fait preuve d‘un bénéfice clinique. Cependant, l'ischémie créée par l'embolisation induit une néoangiogénèse post-interventionnelle pouvant aboutir à la récurrence de la tumeur. Pour cette raison il a été proposé de combiner des sphères d'embolisation déjà commercialisées et établies cliniquement, avec un principe actif anti-angiogénique. Un avantage supplémentaire de ces « chimiosphères » est la possibilité de libérer le médicament de manière locale et contrôlée, tout en réduisant la toxicité systémique indésirable liée au principe actif.

Layer-by-Layer technology (LbL), a technique based on the deposition of oppositely charged polyelectrolytes layer-wise on the surface of interest, has gained increasing interest in the field of drug delivery. The presented work in this thesis explores new applications for Layer-by-Layer (LbL) technology in the field of controlled protein delivery. The basic motives for these studies were the protein-friendly mild formulation conditions where only aqueous solutions are used, in contrast to organic solvents typically employed in the fabrication of many other protein formulations, thus correct protein folding and activity are preserved. Several formulations were designed for the loading and slow release of model and therapeutic biologicals from different carriers. Special attention was paid to testing the activity of released fractions. Moreover, double chambered nanoparticles were developed to host and co-deliver a protein and a small molecule drug to the same cells.

For a long time, treatment of osteoarthritis (OA) has been limited to oral administration of analgesic and anti-inflammatory drugs, called symptoms-modifying OA drugs. The aim of this thesis was to investigate a new way of treating arthritic diseases by intra-articular injection. A disease-modifying OA drug, VX-745 was successfully encapsulated in poly(lactic) and poly(lactic-co-glycolic) acid particles. The in vivo bioactivity of the drug was demonstrated for the first time in this thesis following intra-articular administration. The retention of the drug delivery systems in the joint was observed for 6 weeks and the drug content was released over extended periods of time. The possibility to adjust the drug loading and to modulate the release kinetics render polymeric particles interesting for the delivery of poorly water soluble drugs for the treatment of chronic diseases.

La chimioembolisation transarterielle est une technique couramment utilisée dans la thérapeutique pour les tumeur hépatiques non résecables. Elle consiste en la combinaison d'un agent antitumoral et l'embolisation.

Les maladies cardiovasculaires sont une cause majeure d'incapacité et de décès prématuré dans le monde [1]. On dénombre à 16,7 millions le nombre de décès lié aux maladies cardiovasculaires, plus du tiers de ces décès concernent des adultes d'âge mur [2]. La pathologie sous-jacente est l'athérosclérose se développant sur de longue années [1]. Les sténoses artérielles résultent le plus souvent de plaque d'athérosclérose obstruant progressivement la lumière artérielle . Lorsque l'obstruction atteint 50% à 70% du diamètre artériel, les premiers symptômes apparaissent reflétant un stade avancé de la pathologie [3]. Hemodynamiquement, la sténose artérielle va entrainer une augmentation des forces de cisaillement locales, contribuant à favoriser l'activation plaquettaire au niveau de la plaque athéromateuse [3]. La plaque d'athérosclérose subit alors des remaniements et peut venir à se fissurer, la surface endothéliale peut alors être le site d'un thrombus local [3]. Chez certain patient souffrant d'une obstruction de la circulation coronarienne une intervention chirurgicale est nécessaire, le pontage coronarien [4]. Le pontage dérive la circulation sanguine, permettant une augmentation distale du flux par rapport à l'obstruction [4]. En vue d'une revascularisation la déviation est généralement effectuée à partir de prélèvement de la veine saphène ou d' une artère thoracique [4]. Cependant l'intervention chirurgicale est associée à des séquelles clinique

La colonne vertébrale est le site métastatique le plus fréquent dans le cancer de la prostate, ce qui réduit considérablement la qualité de vie des patients atteints. Dans le cadre du traitement des métastases osseuses, ce projet a été consacré à la formulation et à la caractérisation dʼun implant se formant in situ destiné à agir localement par hyperthermie et chimiothérapie combinées. Le but de cette étude était dʼétablir une formulation dʼun ciment acrylique ayant pour base le polyméthacrylate de méthyle (PMMA) et incluant un agent antiblastique (doxorubicine, DOX) et des SPIONs (Superparamagnetic iron oxide nanoparticles) incorporés dans une matrice de PMMA (PMMA/SPIONs) qui, une fois exposés à un champ magnétique alternatif, produisent localement des températures dans la gamme de lʼhyperthermie modérée (42-45°C) tout en obtenant une libération locale de principe actif. La première étape a consisté en la détermination dʼun taux adéquat de SPIONs au sein de la formulation du ciment permettant dʼatteindre ces températures, avant que, dans un second temps, la DOX nʼy soit incorporée à raison de 25 mg/g de ciment.

Le cancer du foie se place, au niveau mondial, en cinquième position des cancers les plus fréquents. 350'000 à un million de nouveaux cas de cancer du foie sont détectés chaque année dans le monde. En outre, il est le troisième cancer le plus mortel au monde [1]. En effet, après le diagnostic de la pathologie, la médiane de survie est de seulement 4 mois. Ce cancer est plus fréquent en Afrique et en Asie et se fait plus rare en Europe [2]. L'abus d'alcool, l'hépatite B et C sont généralement les causes de cette pathologie [3]. Des métastases hépatiques peuvent survenir dans 80% des cas suite à un carcinome colorectal, un cancer de l'estomac ou du pancréas [2]. Le carcinome hépatocellulaire est le cancer du foie visé dans ce travail. L'incidence de cette pathologie est en progression avec les années. Cette observation peut être corrélée à l'augmentation d'hépatite B. En Suisse, 100 à 150 nouveaux cas sont décelés chaque année

L'objectif du travail présenté dans cette thèse était de développer des nanoparticules fonctionnelles ayant la capacité d'induire l'adhésion et la migration de fibroblasts et kératinocytes humains normaux. L'utilisation de systèmes particulaires dans la cicatrisation constitue une nouvelle approche de l'ingéniérie tissulaire. Dans cette optique, un dérivé hydrosoluble du chitosan rendu fonctionnel par l'ajout de peptides RGD a été développé et évalué. Les nanoparticules furent développées par coacervation complexe et la capacité du système particulaire à induire un changement cellulaire phénotypique a été évaluée in vitro. In vitro, aucune conclusion n'a pu être tirée des études d l'interactions entre le polymère et les kératinocytes. Cependant, aussi bien les polymères que les nanoparticules ont induit l'adhésion et l'étalement de fibroblastes dermiques humains, confirmant le concept de nanoparticules bio-actives. Pour résumer, un système particulaire bio-actif a été développé, mais pour induire la migration des kératinocytes, le choix des peptides doit être réévalué pour la réalisation d'études ultérieures., The aim of the work presented in this thesis, was to develop functionalized nanoparticles with the ability to induce adhesion and migration in normal human keratinocytes and fibroblasts. Using particulate systems in dermal restoration is a novel approach of tissue engineering. In this view, a water-soluble chitosan derivative functionalized with RGD peptides was developed and evaluated. Nanoparticles were formed through complex coacervation and the particulate system was evaluated in vitro for its ability to change phenotype in cells. In vitro, no clear conclusions could be drawn from assays looking at the interaction between keratinocytes and the chitosan derivative. However, both the polymer and the particles formed thereof induced cell adhesion and spreading in human dermal fibroblasts, proving the concept of bioactive nanoparticles. To summarize, a bioactive particulate system was developed, but the choice of peptides to induce migration in keratinocytes needs to be re-evaluated for further investigations.

Dans le cadre du traitement du cancer, l'hyperthermie locale est une approche qui consiste en un chauffage local du site tumoral aboutissant à la destruction de la tumeur. En effet, à l'aide de nanoparticules superparamagnétiques d'oxyde de fer (SPIONs) et d'un champ magnétique alternatif, il est possible de chauffer localement une tumeur. En combinant hyperthermie et chimiothérapie locales, il devient possible de cibler la tumeur tout en minimisant les effets secondaires et systémiques liés à la chimiothérapie conventionnelle.

In this thesis work, injectable formulations forming in-situ an implant that entraps superparamagnetic microparticles are developed in view of treating solid tumors by inducing magnetically a local moderate hyperthermia. We first review physical, biological and clinical aspects of induced hyperthermia for cancer treatment, emphasizing electro-magnetic modalities. The in vitro and in vivo investigations of materials and their formulation is then presented. In the next chapter, we characterize the physico-chemical, magnetic and heating properties under an alternating magnetic field (115 kHz, 9 - 12 mT) of silica microparticles embedding superparamagnetic iron oxide nanoparticles (SPIONs) and of selected formulations thereof: an alginate hydrogel and an organogel oof poly(ethylene-co-vinyl alcohol) in dimethysulfoxide). Finally, we demonstrate the therapeutic potential of the superparamagnetic injectable organogel to treat subcutaneous necrotizing solid tumors of human colocarcinoma in a murine model by 20-min magnetic induction of local hyperthemia., Cette thèse présente les travaux de développement de formulations injectables capables de se solidifier in situ, formant ainsi un implant piégeant des microparticules magnétiques en vue du traitement de tumeurs par induction magnétique d'une hyperthermie locale modérée. Nous exposons tout d'abord le contexte physique, biologique et clinique de l'hyperthermie comme traitement anticancéreux, particulièrement des modalités électromagnétiques. Les performances in vitro et in vivo des matériaux et formulations sont alors présentées. L'objet du chapitre suivant est la caractérisation des propriétés physicochimiques, magnétiques, et chauffantes, dans un champ magnétique alternatif (115 kHz, 9 - 12 mT), des microparticules de silice renfermant des nanoparticules d'oxyde de fer superparamagnétiques (SPIONs) et de deux de leurs formulations: un hydrogel d'alginate de sodium et un organogel de poly(éthylène-co-alcool vinylique) dans le diméthylsulfoxide. Finalement, nous présentons le potentiel thérapeutique de 20 minutes d'hyperthermie locale induite après injection de l'organogel superparamagnétique dans un modèle murin sous-cutané de tumeurs nécrosantes de colocarcinome humain.

In vivo, one of the key signals to induce cell migration is interaction between receptors on the cell surface and proteins in the surrounding extracellular matrix. In 1984 Pierschbacher and Ruoslathi discovered that the small peptide sequences interacting with the receptor also was able to induce a similar response as the whole protein. Cell migration is an important step in the process of wound healing in skin. The aim of this project is to prepare and characterise polymeric vectors for small peptides favouring cell migration. To develop vectors for this purpose, two oppositely charged biopolymers will be used, one catioinc, e.g. chitosan or its derivatives, and one anionic, e.g. a glycosaminoglycan. When mixing two oppositely charged polymers, polyelectrolytic complexes (PECs) are formed. The influence of different physico-chemical parameters, such as charge ratio (n+/n-), the presence of different salts along with other parameters will be investigated, as well as the size, form and stability of the complexes. Different approaches of incorporation of the peptide will be considered. Once the formulations are optimised the chosen particles will be tested in vitro for cytotoxicity on immortalised keratinocytes, which are often used for testing skin formulations.

Bone regeneration may benefit from the local delivery of bone-promoting growth factors. In this view, we developed, optimized and evaluated in vivo a novel syringeable hydrogel based on chitosan, intended as a drug carrier. The effect of the carrier pH and nature on recombinant human Bone morphogenetic protein-2 (rhBMP-2) bioactivity was demonstrated. As for carrier pH, rhBMP-2 bone-forming ability decreased when shifting hydrogel pH from acidic to neutral conditions. Observed aggregation and conformational changes of rhBMP-2 in solution might explain the decrease of growth factor bioactivity. Although combining rhBMP-2 with a cross-linked hyaluronan hydrogel induced formation of higher amounts of bone, a more mature bone was observed with the chitosan hydrogel carrier. The addition of β-tricalcium phosphate to chitosan hydrogel increased rhBMP-2-induced bone formation. Finally, the sustained release of a platelet growth factor from chitosan hydrogel formulations demonstrated the potential of this approach as an alternative to exogenous growth factors.

In this work, we have produced and characterised biodegradable microparticles for the local tratment of arthritis. Two active infredients were co-encapsulated: dexamethasone acetate, with anti-inflammatory effect, and superparamagnetic nanoparticles (SPION) to keep the whole system in place with an external magnetic field. The formulation has been optimised by an experimental design. Microparticles were physicochemically characterized and their interaction with synovial fibroblasts was studied, revealing that neither the microparticles nor their individual components were toxic for synoviocytes. Finally, the biological action of the microparticles was studied in a antigen-induced arthritis model in mice, with and without a subcutaneously implanted magnet near the mouse knee. Following intra-articular injection of dexamenthasone and SPION-containing microparticles in arthritic joints, a diminutaion int the synovial inflammation was observed 4 days afer the injection. This versatile type of microparticles could be used for the joint delivery of other drugs, such as p38 MAPK or IL-1 inhibitors.