Targeted delivery aids in minimizing most of the drug-originated systemic toxic effects as well as improving the pharmacokinetic properties of anticancer therapeutics. Tumor targeting using hyaluronic acid (HA) as the targeting ligand has attracted a great deal of interest among a host of strategies developed to target the overexpressed tumor specific receptors. HA is an endogenous molecule that possesses a lot of biological functions in the human body. The role of HA synthases, HA degrading enzymes and the interaction of HA with its primary receptor CD44 in tumor metastasis and angiogenesis is really complex and controversial to date. However, overexpression of CD44receptors on tumor surface has been well studied, which have been utilized to direct tumor targeted drugs. Most of the HA based targeting systems were HA drug conjugates and surface modified colloidal carriers which required covalent modification. The lack of accurate structural characterization of these systems resulted in modification of HA binding sites that could affect the efficient cellular uptake. LbL technique is a simple and facile method to incorporate several materials into polyelectrolyte assemblies for drug delivery applications. HA being a negatively charged polysaccharide can be easily incorporated into such systems without any covalent modification. Although HA based polyelectrolyte multilayer films and microcapsules have been reported in combination with polycations like PAH, PLL and chitosan, their application as targeted drug delivery systems have not yet been explored. Herein, two LbL architectures with HA as the terminal layer have been investigated as targeted drug carriers, which can recognize overexpressed CD44 receptors in metastatic breast cancer cells. In the first part of the thesis, a novel polyelectrolyte nanocapsule system composed of biopolymers HA and protamine sulphate (PR) as the wall components was prepared and characterized. These pH and enzyme responsive nanocapsules were then utilized for efficient loading and release of anticancer drug doxorubicin (dox). Higher drug release was observed in simulated intracellular conditions like acidic pH and presence of hyaluronidase enzyme as compared to physiological pH. In the second part of the thesis, dox incorporated bovine serum albumin (BSA) nanoparticles modified with HA-Poly(l-Lysine) multilayers were developed and characterized. The drug release pattern of the dox loaded BSA nanoparticles was found to depend on the presence of a protease enzyme trypsin than pH variations. Both of these drug delivery systems were then evaluated for their cell targeting efficiency and cytotoxicity in CD44+ positive metastatic breast cancer cell line MDA MB 231. The final layer HA facilitated targeted delivery of these drug carriers via CD44 receptor mediated endocytosis. The enhanced cellular uptake followed by sustained delivery of dox by virtue of slow intracellular enzymatic degradation of the drug carriers resulted in their improved cytotoxicity as compared to free dox. Further in vitro biodistribution and tumor suppression efficiency of both the systems were studied in breast cancer xenograft models using BALB/c nude mice. Enhance accumulation of dox in the tumor tissue and significant tumor reduction were observed when treated with encapsulated dox using the HA based nanocarriers as opposed to free dox.

Multipotent mesenchymal stromal cells (MSC) were first discovered in bone marrow and can be isolated from “virtually all organs”. They could participate in tissue maintenance and self-renewing process. They are able to adhere to plastic surfaces and acquire a fibroblastic shape when isolated. They are characterized by a particular phenotype and are able to differentiate into several cell types if cultivated in a specific induction medium. These characteristics were defined on MSC in culture and do not represent how they may be in situ.MSC present particular properties. They can secrete growth factors and several cytokines that give them a trophic activity on one hand and the ability to modulate the immune system on the other hand. They are also able to differentiate. These different properties make them an attractive candidate for cell therapy.MSC are already the focus of several pre-clinical and clinical studies. Nevertheless, the results of these studies are difficult to interpret due to limited understanding of their basic biology. MSC are poorly defined in situ and are heterogeneous. Their heterogeneity is dictated by their tissue of origin and cell preparation. To date, there is no standard protocol for MSC isolation and culture. This leads to numerous questions regarding patient safety, and these questions require answers.The first part of the work deals with the methods used to optimize the extraction of MSC and purification from adipose tissue, one of the main sources of autologous MSC with bone marrow. Classical methods require an enzymatic digestion step. The enzyme used and the duration of adipose tissue digestion time can induce cellular alterations and modify cell functions. Moreover, the addition of a xenobiotic increases the risk of contamination and complicates the monitoring of good manufacturing practices (GMP). We propose a method that does not require this enzymatic digestion step while being easier, safer, faster, gentler and less expensive. Compared to the classical enzymatic method, our method yields an equivalent number of MSC from adipose tissue while preserving their properties.The second part of this work focuses on the characterization of the MSC subpopulations from adipose tissue and compares them to those from bone marrow, which are the historical gold standard. The study made it possible to deepen the knowledge of MSC surface markers in situ from these 2 sources. It also evaluated the various properties of the isolated subpopulations thanks to the cell surface markers CD271, SUSD2, MSCA-1, CD44 and CD34. We showed that MSC from bone marrow express MSCA-1, CD271 and SUSD2 markers in situ. We also found that a population clearly positive for the CD34 does exist in situ with different properties compared to those of the unselected populations or the negative counterpart. 2 populations that are negative and positive for CD44 also exist with similar properties.In contrast to bone marrow MSC, only one selection was able to effectively isolate MSC from adipose tissue by a positive selection based on the expression of CD34. We also isolated a CD271+ population but only from lipoaspirate samples and not from abdominoplasty samples. Collectively, our results suggest that MSCA-1 seems to be the best marker through which to isolate MSC from bone marrow and that CD34 is the only marker able to positively isolate cells from adipose tissue. Thus, we show that the MSC from the different sources share similar properties although they have specific characteristics. The choice of the source and of the marker with which to isolate a particular subpopulation is important depending on their intended clinical use. / Les cellules stromales mésenchymateuses multipotentes (CSM) ont été mises en évidence dans la moelle osseuse et peuvent être isolées de « virtuellement tous les organes ». Elles participeraient à la maintenance et au renouvellement des tissus. Une fois isolées, elles sont capables d’adhérer à des surfaces en plastique en prenant une forme fibroblastique. Elles sont caractérisées par un phénotype particulier et peuvent se différencier en divers types cellulaires lorsque cultivées dans un milieu d’induction spécifique. Ces caractéristiques ont été définies sur les CSM en culture et ne reflètent pas forcément ce qui se passe in situ.Les CSM présentent des propriétés particulières. Elles peuvent sécréter des facteurs de croissance ainsi que de nombreuses cytokines qui leur permettent d’une part d’avoir une activité trophique et d’autre part de moduler le système immunitaire. Elles sont aussi capables de se différencier. Ces différentes propriétés les rendent particulièrement attractives pour la thérapie cellulaire.Les CSM font déjà l’objet de nombreuses études pré-cliniques et cliniques dont les résultats sont difficilement interprétables car nous n’avons à l’heure actuelle qu’une compréhension limitée de leur biologie de base. Les CSM sont encore mal définies in situ et sont hétérogènes. Cette hétérogénéité provient de leur différence d’origine et de leur préparation cellulaire :il n’existe aucune standardisation des protocoles d’isolation et de culture. Cette hétérogénéité entraine de nombreuses questions relatives à la sécurité du patient qui doivent être élucidées.La première partie de ce travail cherche à optimiser les méthodes d’extraction et de purification des CSM du tissu adipeux humain, la principale source de CSM autologues avec la moelle osseuse. Les méthodes classiques requièrent une étape de digestion enzymatique dont l’enzyme utilisée et le temps de digestion du tissu adipeux peuvent induire des altérations cellulaires et modifier leurs fonctions. De plus, l’adjonction de xénobiotiques augmente le risque de contamination et complique le suivi des bonnes pratiques de fabrication (BPF). Nous proposons une méthode qui s’affranchit de cette étape de digestion enzymatique tout en étant plus facile, plus sûre, plus rapide, moins chère et moins traumatisante pour les cellules. Elle permet d’obtenir un nombre tout aussi important de CSM du tissu adipeux que la méthode enzymatique classique en préservant leurs propriétés.La deuxième partie de ce travail vise à caractériser les sous populations de CSM du tissu adipeux humain en les comparant à celles de la moelle osseuse, source de référence historique. Cette étude a permis d’approfondir la connaissance des marqueurs de surface des CSM de ces 2 sources in situ, tout en évaluant les différentes propriétés des sous-populations isolées grâce aux marqueurs de surface CD271, SUSD2, MSCA-1, CD44 et CD34. Nous avons montré que les CSM de la moelle osseuse expriment les marqueurs MSCA-1, CD271 et SUSD2 in situ et qu’il existait une sous-population clairement positive pour le CD34 avec des propriétés différentes de celles de la population non sélectionnée ou négative pour ce marqueur. Il existe aussi 2 sous-populations positive et négative pour le CD44 avec des propriétés similaires.Contrairement aux CSM de la moelle osseuse, une seule sélection a permis d’isoler efficacement les CSM du tissu adipeux par une sélection positive sur base de l’expression du CD34. Nous avons pu aussi isoler une population CD271+ mais seulement des prélèvements de lipoaspirations et non des abdominoplasties.Au vu de nos résultats, MSCA-1 semble le meilleur marqueur pour isoler les CSM de la moelle osseuse tandis que le CD34 est le seul marqueur capable d’isoler positivement celles du tissu adipeux. Ainsi, nous montrons que les CSM issues de différentes sources partagent des propriétés similaires avec cependant des caractéristiques propres. Le choix de la source et du marqueur pour isoler une sous-population sont donc importants en fonction de leur utilité clinique envisagée. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Abstract Oral lichen planus (OLP) is a chronic immune-mediated mucosal disease with unknown etiology. According to the current view, the pathogenesis of OLP involves activation of T-cell mediated immunity against the epithelial keratinocytes. A proportion of OLP patients are affected by painful symptoms, and the risk of oral cancer is increased in OLP. There is no curative treatment for OLP. Topical corticosteroids are used most commonly in the management of OLP. However, the evidence base for the effectiveness of any therapy is weak. The objective of this thesis was to study novel aspects of OLP etiopathogenesis and management. An epidemiologic, retrospective case-control study was conducted to determine whether systemic diseases, in particular thyroid diseases, are associated with OLP. In addition, a randomized controlled trial comparing the effectiveness of topical tacrolimus, triamcinolone acetonide and placebo in symptomatic OLP was carried out. Furthermore, immunohistochemical expression of toll-like receptors 4 and 9, hyaluronan and its principal receptor CD44 antigen, hyaluronan synthases 1-3, hyaluronidases 1-2 and cathepsin K was studied in OLP tissue samples and in healthy oral mucosa. The effect of topical tacrolimus on the expression of these molecules in OLP was also studied. The results of the present study showed that a history of hypothyroidism was associated with an approximately twofold risk of having OLP. Furthermore, both tacrolimus and triamcinolone acetonide were more efficient than placebo in reducing the signs and symptoms of OLP. No statistically significant differences were noted in the efficacy between tacrolimus and triamcinolone acetonide. In addition, the expression of the studied molecules was altered in the epithelium or stroma in OLP compared to healthy oral mucosa. Tacrolimus treatment decreased the expression of CD44 antigen in the stroma and the expression of cathepsin K in the epithelium in OLP. In conclusion, the present study extends our knowledge about systemic associated factors and management of OLP. In addition, the results improve our understanding of molecular level changes that occur in OLP. / Tiivistelmä Suun punajäkälä on krooninen immuunivälitteinen limakalvotauti, jonka etiologia on tuntematon. Taudin syntymekanismiin liittyy tämän hetkisen näkemyksen mukaan T-soluvälitteisen immuniteetin aktivoituminen epiteelin keratinosyyttejä vastaan. Suun punajäkälä aiheuttaa osalle potilaista kivuliaita oireita ja lisää suusyövän riskiä. Parantavaa hoitoa tautiin ei ole. Yleisimmin suun punajäkälän oireiden hoidossa käytetään paikallisia kortikosteroidivalmisteita. Kuitenkin eri hoitomuotojen tehosta on vain heikkoa näyttöä. Tämän väitöskirjatyön tarkoituksena oli tutkia uusia näkökohtia liittyen suun punajäkälän etiopatogeneesiin ja hoitoon. Epidemiologisessa tapaus-verrokkitutkimuksessa selvitettiin, liittyvätkö yleissairaudet, erityisesti kilpirauhassairaudet, suun punajäkälään. Lisäksi satunnaistetussa kontrolloidussa tutkimuksessa verrattiin paikallisen takrolimuusin, triamsinoloniasetonidin ja lumelääkkeen tehoa oireisesta suun punajäkälästä kärsivillä potilailla. Tutkimuksessa selvitettiin myös tollin kaltaisten reseptorien 4 ja 9, hyaluronaanin ja sen pääasiallisen reseptorin CD44-antigeenin, hyaluronaanisyntaasien 1–3, hyaluronidaasien 1–2 sekä katepsiini K:n immunohistokemiallista ilmentymistä suun punajäkälänäytteissä ja terveessä suun limakalvossa. Lisäksi tutkittiin takrolimuusihoidon vaikutusta näiden molekyylien ilmentymiseen suun punajäkälässä. Tämän tutkimuksen tulokset osoittivat, että kilpirauhasen vajaatoimintaan liittyi noin kaksinkertainen riski sairastaa suun punajäkälää. Lisäksi havaittiin, että suun punajäkälässä sekä takrolimuusi että triamsinoloniasetonidi ovat tehokkaampia kuin lumelääke oireiden ja kliinisen taudinkuvan lievittämisessä. Takrolimuusin ja triamsinoloniasetonidin tehossa ei todettu tilastollisesti merkitseviä eroja. Lisäksi suun punajäkälänäytteissä tutkittujen molekyylien ilmentyminen oli muuttunut joko epiteelissä tai stroomassa verrattuna terveeseen limakalvoon. Takrolimuusihoito vähensi CD44-antigeenin ilmentymistä stroomassa ja katepsiini K:n ilmentymistä epiteelissä suun punajäkälässä. Yhteenvetona voidaan todeta, että tämä tutkimus lisää tietoa suun punajäkälään liittyvistä systeemisistä tekijöistä ja suun punajäkälän hoidosta. Lisäksi löydökset lisäävät ymmärtämystä suun punajäkälässä tapahtuvista molekyylitason muutoksista.

Metastatic breast cancer treatment has seen few advances in recent years, yet treatment resistance continues to rise, causing disease recurrence. A pilot study was performed to determine the efficacy of ex vivo expansion and reprogramming of tumor-reactive immune cells from experimental metastatic tumor-sensitized mice. Also, phenotypic changes in tumors due to metastasis or tumor microenvironment influences were characterized. Metastatic neu+ mouse mammary carcinoma (mMMC) and its distant relapsing neu-antigen-negative variant (mANV) were investigated in FVBN202 mice. Tumor-reactive central memory CD8+ T cells and activated NK/NKT cells were successfully reprogrammed and expanded during 6-day expansion from mMMC- and/or mANV-sensitized mice, resulting in tumor-specific cytotoxicity. mMMC exhibited a flexible neu-expression pattern and acquired stem-like, tumorigenic phenotype following metastasis while mANV remained stable except decreased tumorigenicity. Myeloid-derived suppressor cell (MDSC) levels were not increased. Adoptive cellular therapy (ACT) with reprogrammed tumor-reactive immune cells may prove effective prophylaxis against metastatic or recurrent breast cancer.

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