Les sphéroïdes multicellulaires tumoraux (SMT) constituent un outil prometteur dans le domaine de l’étude biologique des tumeurs. Le but de la thèse était de développer une technique de la formation de SMT et de démontrer la disponibilité de ces sphéroïdes comme modèle in vitro 3D pour tester l’efficacité de principes actifs anticancéreux ainsi que celle de formulations de délivrance de médicaments. L'effet d’auto-assemblage de cellules induit par une addition des peptides RGD cycliques a été étudié pour 16 lignées cellulaires de différentes origines. Le peptide cyclique RGDfK et sa modification avec le cation triphenylphosphonium (TPP) ont permis de mettre en évidence l’induction de formation de sphéroïdes. Les sphéroïdes ont été employés comme modèles pour évaluer la cytotoxicité de principes actifs antitumoraux (doxorubicine, curcumine, temozolomide) et un certain nombre de formulations nano- et micrométriques (microréservoirs, nano-émulsions et micelles). / Multicellular tumor spheroids (MTS) are a promising tool in tumor biology. The aim of the Thesis was to develop a novel highly reproducible technique for MTS formation, and to demonstrate the availability of these spheroids as 3D in vitro model to test anticancer drugs and drug delivery vehicles. Cell self-assembly effect induced by an addition of cyclic RGD-peptides directly to monolayer cultures was studied for 16 cell lines of various origin. Cyclo-RGDfK peptide and its modification with triphenylphosphonium cation (TPP) were found to induce spheroid formation. The spheroids were used as a model to evaluate the cytotoxicity of antitumor drugs (doxorubicin, curcumin, temozolomide) and a number of nano- and micro- formulations (microcontainers, nano-emulsions and micelles).

Metal-nucleic acid interaction studies have been gaining attention due to their biological and chemical importance. Nucleic acids are negatively charged bio-polymers and neutralization of their negative charge is essential for the stability and function. In the cells, organic positive ions (positively charged amino acids and polyamines) and some of the metal ions (e.g. Na+, K+, Mg2+...etc) neutralize the charge of nucleic acids. Whereas, interactions of some metal ions (e.g. Cd2+, Hg2+…etc) with nucleic acids destabilize the structure. The stability and conformation of nucleic acids alter due to metal interactions. Further, metal interactions with nucleic acids can bring changes in conformation of ribose, H-bonding and π-π stacking interactions. To understand the metal interactions with nucleic acids, various spectroscopic techniques are being used. However, X-ray crystallographic technique is advantageous over all other spectroscopic techniques since it gives thorough detail of coordination mode and structure. However, crystallization of large molecules like nucleic acids with metals is associated with great difficulty. In order to simplify the problem, nucleic acid constituents and derivatives have been used as model systems for metal-nucleic acid interactions. Nucleic acid constituents and derivatives are multidentate ligands. Moreover, binding mode of metal with nucleic acid constituents and derivatives depends on various factors include pH, temperature, type of metal…etc. Further, understanding of metal nucleic acid interactions can aid to develop new anticancer drugs targeting nucleic acids. For example, cisplatin is a platinum based anticancer drug, which coordinates to N(7) of guanine in DNA brings cell death. There have been several reports in literature on the complexes of metal nucleic acid constituents. However, much more research is warranted for thorough understanding of metal-nucleic acid interactions. On the other hand, nucleic acid constituents and derivatives are used extensively in anticancer drug development. Some of nucleic acid constituent derivatives, 5-Fluro uracil and 6-Mercaptopurine, are currently in use for the treatment of colorectal cancer and leukemia, respectively. Moreover, cisplatin is a platinum based anticancer drug used in the treatment of various types of cancers. However, use of these drugs for long time poses severe side effects and drug resistance. Most of the side effects are due to non bio-compatibility of drugs. To overcome problems associated with present anticancer drugs, bio-compatible metal based anticancer drug development could be an attractive and alternative strategy. To address this, in this study, we report synthesis of a number of new metal complexes of nucleic acid constituents and their derivatives and characterization by various spectroscopic techniques. Also, the interactions of Ni, Cu and Zn ions with various nucleic acid constituents and their derivatives have been elucidated by single crystal X-ray crystallography. Interestingly, Ni, Cu and Zn ions showed various coordination modes to nucleic acid constituents and their derivatives. Further, anticancer studies were carried out for all these complexes in various cancer cell lines. Several complexes showed better cytotoxicity than the well-known drug cisplatin. My thesis work is divided into five parts based on the nature of molecules. I. Synthesis, X-ray crystallographic and anticancer studies on metal (Zn/Ni) complexes of guanine (G) based nucleic acid constituents In order to understand (Zn/Ni) interactions with guanine based nucleic acid constituents and their anticancer activity, several (Zn/Ni) complexes of 5′-GMP, 5′-IMP and hypoxanthine complexes were prepared. The synthesized complexes are (1) [Zn (5′-GMP)]n.11H2O, (2) [Ni (5′-GMP)2 Na2 (μ-OH2)3 (H2O)8].2H2O, (3) [Ni (5′-IMP)2Na2 (H2O)12]n.5H2O and (4) [Ni (hx)2 (H2O)4] Cl2 [Here 5′-GMP = 5′-Guanosine Mono Phosphate, 5′-IMP = 5′-Inosine Mono Phosphate and hx = Hypoxanthine). These complexes were characterized by various spectroscopic and X-ray crystallography techniques. Complex 1: The X-ray structure revealed that zinc is coordinated to 5′-GMP through N(7) position of purine and phosphate moieties, the uncoordinated water molecules are making interesting complicated network of hydrogen bonds in the unit cell. The geometry of zinc coordination centre is distorted tetrahedral. Fascinatingly, zinc exhibited two different coordination environments. In one case, all phosphate oxygens participated in coordination with zinc. In second case, N(7) position of purine and phosphate oxygens participated in coordination with zinc. Moreover, zinc formed a coordination polymer with 5′-GMP. The conformation of ribose changed upon zinc interaction with 5′-GMP from C(3′)-endo to C(2′)-endo, these results suggest that zinc interaction with nucleic acids may change their conformation. Complex 1 is stabilized in solid state by H-bonding and π-π stacking interactions. Complex 2: In complex 2, 5′-GMP is coordinated to nickel through N(7) position of purine but phosphate moiety did not take place in coordination. Two molecules of 5′-GMP and four water molecules coordinated to nickel and formed distorted octahedral geometry. The charge of complex 2 is balanced by sodium coordination to sugar hydroxyl groups and nickel coordinated water molecules. The geometry of sodium coordination centre is distorted octahedral. The conformation of 5′-GMP is altered due to nickel interaction. Moreover, complex 2 is stabilized in solid state by H-bonding and π-π stacking interactions. Complex 3: Nucleotide 5′-IMP also showed similar coordination modes like 5′-GMP towards nickel, where N(7) position of purine participated in coordination with nickel and phosphate moieties did not coordinate to nickel. Two molecules of 5′-IMP and four water molecules participated in coordination with nickel and formed distorted octahedral geometry. Interestingly, the charge of complex 3 is balanced by sodium coordination to sugar hydroxyl moieties. The geometry of sodium coordination centre is distorted octahedral. Moreover, nickel is forming coordination polymer with 5′-IMP. Further, nickel interactions with 5′-IMP brought changes in the conformation of ribose moiety. These results suggest that nickel interactions with nucleic acids may bring changes in their conformation. Interestingly, right hand helical structure formation is observed for complex 3 in crystal structure. Further, the chirality of complex 3 was confirmed by circular dichroism studies. Complex 3 is stabilized by both H-bonding and π-π stacking interactions in solid state. Complex 4: Surprisingly, nickel is coordinated to hypoxanthine through N(9) position of purine in acidic conditions and not through N(7) or N(3). The coordination mode of nickel with hypoxanthine is different from complexes 2 and 3. Two hypoxanthine moieties are coordinated to nickel in axial manner. The geometry of nickel coordination centre is distorted octahedral. Further, complex 4 is stabilized by H-bonding and π-π stacking interactions in solid state. Cytotoxicity studies of complexes 1-4 on various cancer cell lines revealed that complex 1 is better cytotoxic than complexes 2-4. Moreover, complex 1 exhibited comparable cytotoxicity with cisplatin on various cells lines and induced apoptotic cell death. II. Synthesis, structure elucidation and anticancer activity of copper-adeninyl complexes In order to understand copper-adenine interactions and anticancer activity, several copper complexes of adenine derivatives were prepared. Here, most of adenine derivatives used in complex preparation is known as cycline dependent kinase inhibitors. Prepared copper complexes are 1) [Cu (N6-benzyl adenineH)2Cl3 ].Cl.2H2O, 2) [Cu (2-amino-N6-benzyladenineH)2Cl3].(2-amino-N6-benzyl adenineH)2.3Cl.5H2O, 3) [Cu (α-(Purin-6-ylamino)-p-toluenesulfonamide H)2Cl4], 4) [Cu (kinetinH)2 Cl3].Cl.2H2O, 5) [Cu (N-1H-purine-6-yl-alanineH) (H2O) Cl3].H2O, 6) [(Cu (N-1H-purine-6-yl-alanineH)2Cl3).(Cu(N-1H-purine-6-yl-alanineH)Cl)2(μ-Cl)2].Cl.4H2O. All these complexes were characterized by X-ray crystallography and various spectroscopic techniques. Complex 1: Synthesis and X-ray structures of complex 1 were reported in literature. However, anticancer activity of complex 1 is not known. Therefore, it was prepared based on the reported lines to assess the anticancer activity. The anticancer activity of complex 1 was studied on various cell lines. Interestingly, complex 1 exhibited better cytotoxicity than cisplatin in MCF-7 and MDA-MB-231 cell lines. Complex 2: Ligand 2-amino-N6-benzyl adenine is coordinated to copper through N(9) of purine. In addition, two uncoordinated 2-amino-N6-benzyl adenine, three chloride and five water molecules are making it as a co-complex with uncoordinated ligands. The copper coordination centre adopted distorted trigonal bipyramidal geometry [3+2] with τ = 0.671 (α-β/60, where α, β are two greatest valence angles of coordination centre). Further, complex 2 is stabilized in solid state by both H-bonding and π-π stacking interactions. H-bonding is observed between N-H···Cl. Uncoordinated water molecules formed six-member rings with H-bonding network. The π-π stacking interactions are observed between phenyl and purine moieties. Complex 2 exhibited better cytotoxicity than 2-amino-N6-benzyl adenine and copper salt. Complex 3: Ligand α-(2-Amino purin-6-ylamino)-p-toluene sulfonamide is coordinated to copper through N(9) position and protonation is observed at N(3) position. Two molecules of α-(2-Amino purin-6-ylamino)-p-toluene sulfonamide and four chloride ions are forming a distorted octahedral geometry with copper. Complex 3 is stabilized by N-H···Cl and N-H···O H-bonding. Further, complex 3 exhibited better cytotoxicity than cisplatin in U251 cells. Complex 4: Kinetin is coordinated to copper through N(9) position of purine. Protonation is observed on N(3) position and balanced the charge of complex 4. Two molecules of kinetin and three chloride moieties are coordinated to copper and forming distorted trigonal bipyramidal geometry [3+2] with τ = 0.431. Moreover, complex 4 is stabilized by both H-bonding interactions and π-π stacking interactions. The H-bonding of complex 4 is observed between N-H···Cl and C-H···Cl. The π-π stacking interactions are observed between furanyl aromatic ring and imidazole ring of purine. Complex 4 exhibited better cytotoxicity than kinetin and copper salt. Complex 5: The N-1H-purine-6-yl-alanine is coordinated to copper through N(9) position of purine. Complex 5 crystallizes in the monoclinic space group P21 with Z=4. One molecule of N-1H-purine-6-yl-alanine, two chloride ions and one water molecule coordinated to copper. The geometry of copper coordination centre is distorted trigonal bipyramidal [3+2] with Cu(1) τ1 = 0.613 and Cu(2) τ2= 0.671. Protonation is observed on N(3) position. Complex 5 is stabilized by both H-bonding and π-π stacking interactions. The H-bonding of complex 5 is observed between N-H···Cl and C-H···Cl. The π-π stacking interactions are observed between imidazole moieties. Moreover, complex 5 exhibited better cytotoxicity than N-1H-purine-6-yl-alanine and copper salt. Complex 6: Complex 6 is a co-complex, where two different complexes are co-crystallized. The crystal structure of complex 6 indicate that geometry of Cu(1) and Cu(2) coordination centre are distorted trigonal bipyramidal [3+2] with τ1 = 0.3261 and τ2 = 0.8, respectively. Two molecules of N-1H-purine-6-yl-alanineH are coordinated to Cu(2) through N(9) position of purine. The N-1H-purine-6-yl-alanineH ligands are arranged in geometry in trans manner with respect to axis passing through the N(9) atom and copper. Whereas, in second co-complex two N-1H-purine-6-yl-alanineH are coordinated to Cu(1) through N(9) and N(3) position of purine. Both Cl(1) and Cl(3) coordinated to copper are forming a bridge between copper. In addition, one uncoordinated chloride and two water molecules are present in the unit cell. Complex 6 is stabilized in crystalline state by both H-bonding and π-π stacking interactions. Complex 6 exhibited better cytotoxicity than complex 5, N-1H-purine-6-yl-alanine and copper salt on various cell lines. III. Synthesis, structure and anticancer activity of zinc complexes of adenine derivatives In order to understand zinc interaction with adenine and their anticancer activity, several zinc complexes of adenine derivatives were prepared. The prepared complexes are (1) [Zn (N6-benzyladenineH).Cl3].2H2O, (2) [Zn2 (μ -N6-benzyladenine)2( μ-H2O)2(H2O)4].(OTf)4.H2O, (3) (N6-benzyl adenineH2) [ZnCl4].2H2O, (4) [Zn (2-Amino-N6-Benzylpurine)Cl3).2-Amino-N6-BenzylpurineH).EtOH, (5) (2-Amino-N6-(3-picoyl)purineH2)[ZnCl4].H2O, (6)(2-Amino-N6-(3-picoyl)purineH2)[ZnCl4].HCl, (7) (2-Chloro-N6-(3-picoyl) purineH2) [ZnCl4].H2O, (8) ((α-Purine-6-ylamino)-p-toluene sulfonamide H)2[ZnCl4].2HCl.2H2O. Complex 1: The N6-benzyl adenine is coordinated to zinc through nitrogen atom N(7) of purine. One molecule of N6-benzyl adenine and three chloride ions are coordinated to zinc and forming distorted tetrahedral geometry. Interestingly, the nitrogen atom N(1) of purine is protonated. Complex 1 exhibited strong H-bonding interactions between N-H···O, N-H···Cl and N-H···N. The complex 1 showed better cytotoxicity than N6-benzyl adenine and ZnCl2. Complex 2: The N6-benzyl adenine formed a dimeric complex with zinc at neutral pH. Complex 2 crystallizes in the triclinic space group P-1with Z=1. Two Zn metal centres are bridged by two molecules of N6-benzyl adenine through nitrogen atoms N(3) and N(9) of purine forming a di-nuclear complex, further two zinc centres is bridged by two water molecules and other two water molecules on the other side completing the octahedral coordination for the Zn. Complex 2 is stabilized in crystalline state by H-bonding interactions. The H-bonding of complex 2 is observed between O-H···O and N-H···O. Complex 2 exhibited better cytotoxicity than N6-benzyl adenine and ZnCl2 on various cell lines. Complex 3: The N6-benzyladenine is not coordinated to the Zn metal at acidic pH and forms an ion-pair complex. Ion-pair complex 3 crystallizes in the monoclinic space group Cc with Z=4. The protonation is observed at N(1) and N(9) atoms of N6-benzyl adenine. The positive charges on N6-benzyl adenine is neutralized by the presence of two chloride ions in [ZnCl4]2-. Alternative arrangement of cation and anion arrangement is observed in complex 3. Water channel formation is observed between cation and anion arrangement. Moreover, complex 3 is stabilized by H-bonding and π-π stacking interactions. H-bonding is observed in complex 3 between N-H···Cl, O-H···Cl and N-H···O. The π-π stacking interactions in complex 3 are observed between benzyl six-membered aromatic ring and purine six-membered rings. Complex 3 exhibited better cytotoxicity than N6-benzyl adenine and ZnCl2 in various cell lines. Complex 4: Ligand 2-amino-N6-benzyl adenine resulted in a different structure from N6-benzyl adenine with zinc. One molecule of 2-amino-N6-benzyl purine is coordinated to zinc through nitrogen atom N(7) of purine. Surprisingly, one uncoordinated positively charged 2-amino-N6-benzyl purineH is present in the asymmetric unit, which is balancing the charge of zinc complex 4. Protonation is observed on N(3A) atom. Interestingly, tautomeric proton is located on coordinated purine of N(9) atom and uncoordinated purine of N(7A) atom. Geometry of ‘Zn coordination centre’ is distorted tetrahedral. Complex 4 is stabilized by H-bonding and π-π stacking interactions. The H-bonding interaction in complex 4 is observed between N-H···O and N-H···Cl. The π-π stacking interactions are observed between five-member aromatic rings and six-membered aromatic rings. Complex 4 exhibited better cytotoxicity than 2-amino-N6-benzyl purine and ZnCl2 in various cell lines. Complex 5: 2-Amino-N6-(3-picoyl) purine forms an ion-paired complex with zinc at acidic pH. The protonation in 2-Amino-N6-(3-picoyl) purine is observed at N(3) of the purine and picolyl N(14). The positive charge of 2-Amino-N6-(3-picoyl) purine is neutralized by the presence of two chloride ions in [ZnCl4]2-. Moreover, complex 5 exhibited both H-bonding interactions and π-π stacking interactions. The H-bonding interactions are observed between N-H···Cl, N-H···N, O-H···Cl, N-H···O and C-H···N. One uncoordinated water molecule is present in unit cell, which is involved in H-bonding with both ions. The π-π stacking interactions are observed between purine five-membered rings and purine six-membered ring. Complex 5 exhibited better cytotoxicity than cisplatin in HeLa and MDA-MD-231 cells. Complex 6: 2-Amino-N6-(3-picoyl) purine formed similar structure of complex 5 in strong acidic conditions. Complex 6 exhibited both H-bonding and π-π stacking interactions. The H-bonding in complex 6 is observed between N-H···Cl and N-H···N. In complex 6, the π-π stacking interactions are observed between pyridyl six-membered rings and purine six-membered rings. Purine-Purine stacking interactions are observed between purine six-membered ring and five-membered rings. Complex 6 exhibited better cytotoxicity than cisplatin in HeLa, MCF-7, MDA-MB-231 and HeLa-Dox cells. Interestingly, complex 6 arrested (G2/M phase) cell cycle in HeLa and MCF-7 at higher concentration and induced apoptosis. Complex 7: 2-chloro-N6-(3-picoyl) purine formed ion-pair complex with zinc. The protonation in 2-chloro-N6-(3-picoyl) purine is observed on N(9) of purine and N(14) of picolyl atoms. The positive charge of 2-chloro-N6-(3-picoyl) purine is neutralized by the presence of two chloride ions in [ZnCl4]2-. Complex 7 is stabilized by both H-bonding and π-π stacking interactions. The H-bonding is observed between N-H···Cl, O-H···Cl and N-H···O in complex 7. The π-π stacking interactions are observed between pyridyl six-membered ring and six-membered ring of purine. Complex 7 exhibited better cytotoxicity than cisplatin in HeLa, MCF-7, U251 and HeLa-Dox cells. Complex 8: (α-Purine-6-ylamino)-p-toluene sulphonamide formed ion-pair complex with zinc. Ion-pair complex 8, crystallizes in the triclinic space group P-1 with Z=4. The protonation on (α-Purine-6-ylamino)-p-toluene sulfonamide is observed at N(9) and N(1) atoms of purine. The positive charge of the ligand is neutralized by two chloride ions present in [ZnCl4]2 -. The H-bonding is observed between N-H···Cl, O-H···N, N-H···O and O-H···Cl. The π-π stacking interactions are observed between benzyl rings of benzene sulfonamide moieties. Complex 8 exhibited better cytotoxicity than cisplatin in HeLa, MCF-7 and HeLa-Dox cells. Moreover, these complexes induced apoptotic cell death as revealed by Annexin V/PI assay, FACS and microscopy analysis. IV. Synthesis, structure and cytotoxicity studies of zinc complexes of uracil-1-acetic acid and N6-adeninebutyric acid To understand the zinc interactions with nucleic acid constituent derivatives and their anticancer activity, zinc complexes of uracil-1-acetic acid and N6-adeninebutyric acids were prepared. (1) [Zn (uracil-1-acetato)2 (H2O)4] and complex (2) [Zn (N6-adeninebutyric acid)2 (H2O)2]) were characterized by X-ray crystallography and various spectroscopic techniques. The X-ray structures showed acetate moiety coordination to zinc rather than purine and pyrinidine moities. The geometry of zinc coordination centre is distorted octahedral. Complexes 1 and 2 are stabilized by non-covalent interactions. Anticancer studies of these complexes showed better cytotoxicity than cisplatin in MDA-MB-231cells. V. Copper (II) complexes of 6-mercaptopurine, hypoxanthine and uracil-1-acetic acid: Synthesis, structures, antioxidant and potent anticancer activity To delineate copper interactions with purine and pyrimidine derivatives and anticancer activity, several copper complexes of 6-mercaptopurine, hypoxanthine and uracil-1-acetic acid were prepared. The prepared complexes are (1) [Cu (6-MP) (bpy) Cl2], (2) [Cu (hx) (phen) Cl2].H2O and (3) [Cu (bpy)2 (uracil-1-acetato)].6H2O)] (bpy = 2, 2′-bipyridine, phen = 1, 10-phenanthroline, 6-MP = 6-Mercapto Purine and hx = hypoxanthine). All these complexes were chracterized by various spectroscopic and X-ray diffraction techniques. Complexes 1 and 2 crystallize in the monoclinic space groups Cc and C2/c, respectively with eight molecules in the unit cell. All the complexes 1-3 adopt distorted trigonal bipyramidal geometry. Surprisingly, most potent coordination sites of sulfur in 6-MP and acetato in uracil-1-acetato did not participate in coordination with copper. In complexes 1 and 2, the N(7) position of purine and the N(3) position of pyrimidine in complex 3 are coordinated with copper. All these complexes 1-3 are stabilized by non-covalent interactions in solidstate. Anticancer studies showed better cytotoxicity for copper complexes than cisplatin, 6-meracptopurine and temozolomide in various cell lines. Interestingly, copper complexes of 6-MP and hypoxanthine showed antioxidant activity and reduced ROS level in cells. In contrast, copper complex of uracil-1-acetic acid produced ROS in cells. In contrast, copper hypoxanthine showed better cytotoxicity than cisplatin in HeLa-Dox cells. All these complexes induced apoptotic cell death. In summary, we studied the interaction of metal-nucleic acid constituents and derivatives by X-ray crystallography. We found new coordination modes for Ni, Cu and Zn towards various nucleic acid constituents and derivatives. Some of these complexes showed better cytotoxicity than well known anticncer drugs cisplatin, 6-meracptopurine and temozolomide. Complex [Cu (hx) (phen) Cl2].H2O showed better cytotoxicity than cisplatin in doxorubicin resistant (HeLa-Dox) cells. These complexes induced apoptotic cell death in various cancer cells. All in all, the results of present studies/findings could form a potential lead for the development of newer anticancer therapeutics.

L’objectif de ce travail a été la préparation de nanoparticules pour l’administration intratumorale. Ladouble functionalisation de ces nanoparticules est rapidement approuve comme extrement intéressant, car ellespeuvent être utilisé pour le diagnostic in vivo et la thérapie (théranostics). Pour réaliser ce type desnanoparticules, l’oxyde de fer a été choisi comme agent de contraste pour une utilisation en imagerie parrésonance magnétique (IRM) pour le diagnostic de cellules cancéreuse. Les nanoparticules d’oxyde de fer ont étépréparées dans des milieux aqueux et organique. La méthode d’émulsification multiple suivie de l’évaporationde solvant a été utilisée pour l’encapsulation concomitante d’un principe actif et des nanoparticules de l’oxydede fer. Tous les paramètres affectant la taille des nanoparticules pendant le procédé ont été étudiés en utilisantune molécule active hydrophile modèle (une dérivative de la Stilbene) et le protocole standard a ensuite étéévalué. Dans une seconde étape, les particules d’oxyde de fer ont été encapsulées par la même méthoded’émulsion évaporation et entièrement caractérisées en termes de morphologie, taille, magnétisation, etcomposition chimique. La visualisation in vitro des particules modèles a été réalisée par IRM et comparée a unproduit commercial à base de Gadolinium (Gd). D’autre part, la quantité nécessaire d’oxyde de fer permettantune bonne visualisation par IRM a été déterminée par une étude in vivo menée sur des souris. Enfin la doubleencapsulation d’oxyde de fer avec une molécule anticancéreuse a été effectuée par la méthode développée, laformulation obtenue a été entièrement caractérisée. / The objective of this work was the preparation of nanoparticles for intratumoral administration.Important characteristic was dual functionality of these nanoparticles that they can be used for diagnosis andtherapy so the resulting nanoparticles will serve as theranostic agents. For this purpose iron oxide was chosen asa contrast agent to be used in Magnetic resonance imaging (MRI) for diagnosis of cancerous cells. Iron oxidenanoparticles (IONPs) were prepared in aqueous and organic medium. A multiple emulsion evaporation methodwas designed for the encapsulation of active ingredient (hydrophilic drug i-e Stilbene) and the IONPs. All theparameters affecting the colloidal properties final hybrid particles were studied and characterization was done forfinal particles. Then prepared particles were evaluated for in vitro MRI and also compared with commerciallyavailable products such as gadolinium (Gd). At same time, the minimum detectable quantity of iron oxide in vivowas determined using a commercialized iron oxide emulsion on rat models. Finally an anticancer agent wasencapsulated with IONPs using same multiple emulsion method and characterization was done.

Nos travaux de doctorat s’inscrivent dans la démarche de la valorisation de la biodiversité végétale cambodgienne. Les objectifs portaient sur les travaux phytochimique et pharmacologique de deux plantes cambodgiennes : Vernonia cinerea et Vernonia elliptica. Une méthode de dosage par CLHP du composé 8α-tigloyloxyhirsutinolide 13-O-acetate (H1) appartenant à la classe des lactones sesquiterpènes de V. cinerea a été mise au point et validée dans le but de déterminer la teneur de ce composé. La teneur en H1 varie entre 0,08 % à 0,17 % pour les échantillons étudiés provenant de 4 régions du Cambodge. L’évaluation de l’activité antiproliférative in vitro des extraits de V. cinerea, et du composé H1 a été réalisée vis-à-vis de deux lignées cellulaires cancéreuses humaines HT29 et HepG2. Le composé H1 présente une CI50 similaire à celle du contrôle positif 5FU, 4,3 ± 0,2 versus 5,4 ± 0,9 µM vis-à-vis de la lignée HepG2. Les résultats intéressants obtenus sur V. cinerea nous ont incités à travailler sur d’autres espèces du genre Vernonia qui poussent au Cambodge, Vernonia elliptica. L’étude phytochimique menée sur cette plante a permis d’isoler un lignane [(+)-Syringaresinol] et deux lactones sesquiterpènes [(+)-8,13-diacetyl-piptocarphol et Glaucolide B]. Ces composés sont décrits dans cette plante pour la première fois. L’étude in vitro de l’activité antiproliférative des extraits de V. elliptica montre que l’extrait dichlorométhane obtenu à partir de l’extrait aqueux est très actif. / Our PhD work is in the process of valorization of the Cambodian plant biodiversity. The objectives focused on phytochemical and pharmacological studies of two Cambodian plants of the Vernonia genus: Vernonia cinerea and Vernonia elliptica.An analytical HPLC method was developed and validated to quantify the major compound 8α-tigloyloxyhirsutinolide 13-O-acetate (H1) of Vernonia cinerea. We found that the compound H1 content ranges from 0.08% to 0.17% in samples collected from cambodian regions. We thus evaluated the anti-proliferative activity in vitro of extracts of V. cinerea and H1 compound against two human cancer cell lines HT29 and HepG2. For HepG2 cell line, the compound H1 has a similar IC50 to the positive control 5FU, 4.3 ± 0.2 versus 5.4 ± 0.9 µM. The interesting results on V. cinerea encouraged us to study other species of Vernonia genus growing in Cambodia, Vernonia elliptica. The phytochemical study conducted on this plant led to isolate a lignan [(+)-syringaresinol] and two sesquiterpene lactones [(+)-8,13-diacetyl-piptocarphol and Glaucolide B]. These compounds were described in this plant for the first time. The in vitro study of the anti-proliferative activity of the extracts of V. elliptica shows that the dichloromethane extract obtained from aqueous extract is very active.

Introdução: O carcinoma de células renais metastático (CCRm) é um tumor altamente agressivo e resistente. Seu tratamento é baseado em terapia alvo molecular e citocinas. Avaliamos os efeitos antineoplásicos do Amblyomin-X, sobre CCR. Métodos: Avaliadas culturas de CCR RENCA e fibroblastos normais NIH/3T3 tratadas ou não com Amblyomin-X. Realizados ensaios de viabilidade celular por MTT e determinação, por citometria de fluxo, da proporção de células em apoptose/necrose; expressão da P-gp; Bad; Bax; Bcl-2; ciclina D1; caspase 3; Ki-67; p53; VEGFR1; citocromo c; análise das fase do ciclo celular; e atividade do proteassoma. Analisamos as populações celulares por microscopia eletrônica de varredura. Empregados testes T e One-way ANOVA para análise estatística. Resultados: O Amblyomin-X demonstrou citotoxicidade em células RENCA por indução de apoptose, diminuição de proliferação celular, inibição do proteassoma e modulação do ciclo celular em G0/G1. Em fibroblastos normais não houve citotoxicidade Conclusão: O Amblyomin-X apresentou efeito antineoplásico em CCR e não exerceu efeito citotóxico em células normais, demonstrando um possível potencial terapêutico no tratamento do CCRm. Estudos futuros deverão confirmar estes resultados. / Introduction: Metastatic renal cell carcinoma (mRCC) is a highly agressive and resistant tumour. Its treatment is based on targeted therapies and cytokines. We have evaluated the antineoplasic effects of Amblyomin-X on RCC. Methods: RCC (RENCA) and fibroblasts (NIH/3T3) cell cultures treated or not with Amblyomin-X were evaluated. MTT assay was performed to determine cell viability. Apoptosis/necrosis ratio; expression of P-gp; Bad; Bax; Bcl-2; cyclin D1; caspase-3; Ki-67; p53; VEGFR1; cytochrome c; cell cycle analysis and proteasome activity were obtained by flow cytometry. Cellular populations were analised by Scanning Electron Microscopy. Statistical analyses was performed using T-Tests and One-way ANOVA. Results: Amblyomin-X showed cytotoxic activity on RENCA tumor cells. It has induced apoptosis, decreased tumor cell proliferation, targeted the ubiquitinproteasome system and modulated genes related to cell cycle in G0/G1. There was no toxicity on fibroblasts. Conclusion: Amblyomin-X showed antineoplasic effects on RCC cells preserved normal fibroblast cells. There is a potential role of its therapeutic use in mRCC treatment. Future studies should confirm our initial results.

Ce travail de recherche rapporte l'utilisation de techniques biophotoniques pour investiguer des questions biomédicales, de la recherche fondamentale (interaction champs électriques impulsionnels / cellules) aux études cliniques (toxicité cutanée induite chez les patients traités par des thérapies ciblées).La microspectroscopie confocale Raman et de la microscopie terahertz ont été utilisées pour étudier le processus d’électroperméabilisation cellulaire d'un point de vue moléculaire. Nos résultats démontrent l'implication des protéines. De plus, nous avons montré que la signature Raman des cellules peut être utilisée comme un biomarqueur précis des différents états des cellules exposées aux chocs électriques, correspondant à une électroperméabilisation non détectable, électroperméabilisation et irréversibleEn tant que projet parallèle de ce doctorat, une recherche clinique a été réalisée afin d'étudier la toxicité cutanée induite chez les patients traités par des thérapies anticancéreuses ciblées. Bien que l'efficacité de ces thérapies ne soit pas discutée, de nombreux effets cutanées secondaires graves sont associés. Dans cette étude, nous avons étudié l'opportunité de prédire l’apparition de la toxicité cutanée au moyen de la microspectroscopie Raman confocale réalisée sur la peau des patients. Nous avons réussi à déterminer un nouveau biomarqueur pharmacodynamique spécifique de la toxicité cutanée grâce aux signatures Raman de la peau des patients; alors que l'évaluation dermatologique ou histologique n'a détecté aucune modification. / This research work reports the use of various biophotonics techniques to investigate biomedical questions, from basic research (interaction between pulsed electric fields and cells) to clinical studies (skin toxicity induced in patients treated with targeted anticancer therapies).Confocal Raman microspectroscopy and terahertz microscopy have been used to investigate cell electropermeabilization process from a molecular point of view. Our results demonstrate the involvement of the proteins in cell electropermeabilization. Moreover, we have shown that the Raman signatures of the cells could be used as an accurate biomarker of the different states of the cells exposed to pulsed electric fields, corresponding to no detectable electropermeabilization, reversible and irreversible electropermeabilization.Finally, this doctorate research demonstrates the opportunity to predict skin toxicity induced by targeted anticancer therapies by means of confocal Raman microspectroscopy. We succedded to determine a novel and specific pharmacodynamic biomarker for skin toxicity based on the Raman signatures of the patient’s skin, whereas dermatological or histologicalevaluation did not detect any modifications.

La metformine, un médicament couramment utilisé pour le traitement du diabète de type II, fut récemment identifiée comme un composé ayant des propriétés anticancéreuses très intéressantes, notamment pour le cancer du pancréas. Toutefois, malgré les nombreuses expériences in vitro et sur des modèles murins qui ont confirmé cet effet, les essais cliniques sur les humains sont restés infructueux. Un des facteurs mis en cause pour expliquer ces résultats est la grande hydrophilie de la metformine, qui diminue sa biodisponibilité et limite son transport au travers des membranes cellulaires. Nous nous sommes donc intéressés à la synthèse de sels de biguanidium amphiphiles inspirés de la metformine qui se partitionnent plus facilement dans les bicouches phospholipidiques et qui possèdent de meilleures activités anticancéreuses. Pour ce faire, nous avons tout d’abord étudié la perturbation des membranes par de simples alkylbiguanidium. Nous avons démontré que ces composés peuvent transporter des ions H+/OH- et dépolariser les membranes bactériennes, ce qui leur confère des propriétés antibactériennes et antifongiques. Afin de limiter l’hémolyse associée à ces composés, des sels de biguanidium substitués par le groupement phényléthynylbenzyle ont par la suite été synthétisés. La structure de ceux-ci leur permet de mieux se partitionner dans les membranes et s’accumuler dans les mitochondries, tout en diminuant la toxicité associée à une perturbation membranaire trop forte. Leur activité sur les cellules cancéreuses du pancréas est ainsi beaucoup plus importante que celle de la metformine, de même que leur capacité à inhiber la croissance de xénogreffes chez les souris. Ces résultats nous ont ensuite amené vers la synthèse d’une petite librairie de sels de biguanidium et l’étude de leurs activités anticancéreuses et antibactériennes. Les modifications structurales et les changements de contre-ion apportés à cette librairie ont permis d’obtenir des composés encore plus efficaces et surtout beaucoup plus sélectifs envers les cellules saines, ouvrant ainsi la porte à une nouvelle classe de médicaments anticancéreux à base de sels de biguanidium. / Metformin, a common drug used for the treatment of type II diabetes, has recently been linked to interesting anticancer properties, notably on pancreatic cancer. Although there have been many experiments in vitro and on murine models that have confirmed this effect, human clinical trials featuring metformin have been unsuccessful. One of the reasons brought forward to explain these results is the high hydrophilicity of metformin, which limits its bioavailability and transport through cellular membranes. For this reason, we have been interested in the synthesis of amphiphilic biguanidium salts inspired from metformin that can partition more easily in phospholipid membranes and thus have better anticancer properties. We first studied the membrane perturbation properties of simple alkylbiguanidium salts and showed that these compounds can transport H+/OH- ions and depolarize bacterial membranes, which in turn gives them antibacterial and antifungal properties. To limit the hemolytic activity associated with these compounds, biguanidium salts substituted by the phenylethynylbenzyl moiety were then synthesised. Their structure allows them to partition more easily in membranes and accumulate in mitochondria, while lowering the toxicity associated with high membrane perturbation. For those reasons, their activity on pancreatic cancer cells is much higher than metformin, as is their inhibition of xenograft growth in mice. These results encouraged us to synthesise a small library of biguanidium salts and study their anticancer activity. The structural modifications and counter-anion variations brought to this library have improved the efficiency of these compounds as well as their selectivity towards healthy cells, thus opening the door to a new class of anticancer drugs based on biguanidium salts.

Yaku’amide A (YA) is a linear anticancer peptide that is rich in bulky dehydroamino acids (ΔAAs) and β-hydroxyamino acids (β-OHAAs). In our recent total synthesis of YA, we featured a one-pot anti dehydration–azide reduction–O→N acyl transfer process for the stereospecific construction of Z- and E- ∆Ile residues. Despite previous total syntheses and our efforts, the synthesis of YA remains lengthy. Via computational studies, we identified two analogue peptides that closely resemble the conformation of YA. The use of simpler and symmetrical bulky ΔAAs such as dehydrovaline (ΔVal) and dehydroethylnorvaline (ΔEnv) as surrogates of ∆Ile, along with azlactone chemistry for their incorporation, significantly decreased the overall number of synthetic steps. Biological studies revealed that our analogues exhibited very similar activity to that of the natural product YA, demonstrating their suitability as mimics and consistency with our computational model. Despite its utility in the construction of YA analogues, azlactone chemistry is sluggish and moderate to low yielding. For this reason, we have explored strategies to streamline the synthesis of peptides containing Z-dehydroaminobutyric acid (∆Abu), ∆Val, and Z-dehydrophenylalanine (∆Phe). The key process is to form the alkene moiety via elimination of a β-sulfonium or β-OHAA embedded within a peptide, avoiding the need to form the alkene moiety via azlactone-dipeptide dehydration and bypassing sluggish amidation/ring opening steps. β-sheet disruption of Tau-model hexapeptides is a key type of inhibition for modulating Alzheimer’s disease progression. Previous studies replaced key residues with proline, due to its rigidity and lack of amide proton, to inhibit β-sheet formation. Similar to proline, ∆AAs are also known for their rigidity and ability to favor other conformations (e.g. β-hairpin, 310-helix) along with increasing peptide half-life. We have incorporated ∆Abu, ∆Val and dehydrocyclohexylglycine (∆Chg) in a highly aggregative hexapeptide sequence, using previously studied methods, to assess their capabilities as putative β-sheet breakers and to stabilize against proteolysis. Studies are continuing.

Dans la majorité des cas, les mitochondries sont nécessaires à la tumorigenèse et à la réponse des cellules cancéreuses aux signaux générés par les facteurs micro-environnementaux (exemples : privation de nutriments, hypoxie) ou par les traitements anticancéreux (exemples : chimiothérapie, radiothérapie). Presque toutes les protéines mitochondriales sont codées par le génome nucléaire et importées dans l'organelle. Des machineries d'import ont donc évolué afin de répondre aux besoins d'import protéique. Dans ce contexte, la machinerie régulée par CHCHD4/Mia40 fonctionne dans l’espace intermembranaire et contrôle l’import d’un groupe de protéines (substrats) qui joue des rôles importants dans la survie et la réponse au stress. Les substrats de CHCHD4/Mia40 sont impliqués dans un vaste panel d’activités mitochondriales qui inclut la biogenèse des complexes de la chaîne respiratoire, l’homéostasie lipidique, le stockage du calcium, ainsi que l'ultrastructure et la dynamique mitochondriale. Ce programme de thèse a été dédié à l’étude de la voie d’import CHCHD4/Mia40 dans les cellules cancéreuses et a porté un intérêt tout particulier à l'un des substrats CHCHD4/Mia40 qui façonne l'ultrastructure mitochondriale. En utilisant des techniques d’ARN interférence et de sur-expression de protéines recombinantes, dans un modèle de cancer du côlon, nous avons montré que l’expression du substrat étudié a un effet crucial sur la prolifération et la croissance tumorale. Nos données ont également impliqué cette protéine dans la réponse aux traitements anticancéreux. Dans l'ensemble, ces travaux ouvrent un nouveau champ d'investigations qui non seulement permettra de mieux comprendre la plasticité métabolique des cellules cancéreuses, mais aidera également à identifier de nouveaux biomarqueurs métaboliques. / In the vast majority of cases, mitochondria are required for tumorigenesis and also for the tumoral response to signals generated by the microenvironmental factors (e.g. nutrient deprivation, hypoxia) or to the effects of anti-cancer treatments (e.g. chemotherapy, radiotherapy). As almost all mitochondrial proteins are nuclear-encoded and imported into the organelle, specialized import machineries have evolved in order to meet the need for protein import. Among these machineries, the one that operates in the intermembrane space and is controlled by CHCHD4/Mia40, regulates the import of a group of proteins (substrates) that play important roles in survival and stress response. Substrates of CHCHD4/Mia40 are involved in a broad panel of mitochondrial activities that includes the biogenesis of respiratory chain complexes, lipid homeostasis, calcium storage, as well as ultrastructure and mitochondrial dynamics. This thesis program was dedicated to the study of the CHCHD4/Mia40 import pathway in cancer cells, with a particular interest for one of the CHCHD4/Mia40 substrates that shapes mitochondrial ultrastructure. Using RNA interference approach and recombinant protein overexpression technique, in a colon cancer model, we showed that the expression of this substrate had a crucial effect on proliferation and tumor growth. Our data also involved this protein in the response to anti-cancer treatments. All together, this work opens a new field of investigations that will not only shed new lights on the metabolic plasticity of cancer cells but also help to identify new metabolic biomarkers.

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