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Transition metal bis(thiosemicarbazone) complexes have been of great interest in the last five decades. One of the most striking features of these complexes is that they possess a wide range of biological properties including antimalarial, antibacterial and anticancer activity. Zinc and copper bis(thiosemicarbazone) complexes have recently attracted attention due to their intracellular fluorescence and anticancer activity, respectively. The present work “Targeting Cancer Cells and Live Cell Imaging Using Bis(thiosemicarbazone) Complexes of Copper and Zinc” is an effort to target cancer cells using folic acid or biotin linked anticancer active copper bis(thiosemicarbazone) complexes. Interestingly, bis(thiosemicarbazone) ligands form zinc complexes that could be used to image cancer cells and one of the ligands could be used for imaging zinc in the cells. Chapter 1, provides a brief introduction to metal complexes in medicine. Different classes of metallodrugs and their mechanism of action are listed. A short discussion on different types of diagnostic drugs and transition metal complexes possessing anticancer activity is presented. An overview of the strategies available to target cancer cells is included. Furthermore, the use of thiosemicarbazone compounds for anticancer activity is reviewed in detail. Recent examples of bis(thiosemicarbazone) compounds in medicinal studies is briefly mentioned. This section ends with the scope of the present work which involves bis(thiosemicarbazone) complexes of zinc and copper. Chapter 2, “Zinc bis(thiosemicarbazone) complexes for live cell imaging and anticancer activity” deals with the synthesis and characterization of a series of mononuclear and binuclear zinc bis(thiosemicarbazone) complexes by varying substituents at the diketone moiety or at the thiosemicarbazide fragment of the ligand. The crystal structures of mononuclear ligand benzil-bis(4-pyrrolidine-3-thiosemicarbazone) (BTSCH2), zinc glyoxal-bis(4-methyl-4-phenyl-3-thiosemicarbazone) [Zn(GTSC)]3 and [Zn(BTSC)(DMSO)] complexes were determined using single-crystal X-ray crystallography. Here, the mononuclear zinc complexes were utilized as live cell imaging agents whereas binuclear zinc complexes proved to be anticancer agents. Among the many mononuclear complexes prepared, the trimeric zinc complex derived from glyoxal- bis(4-methyl-4-phenyl-3¬thiosemicarbazone) was found to be the most fluorescent complex owing to its unique structure. This permitted live cell imaging in a number of cancer cell lines. In comparison with the well studied zinc biacetyl-bis(4-methyl-3-thiosemicarbazone) Zn(ATSM) complex, which was used as a reference, [Zn(GTSC)]3 had a 2.5 fold higher fluorescence quantum yield in DMSO. The cellular fluorescence was measured in collaboration with Prof. K.Somasundaram’s laboratory at MCBL using flow cytometry. It was observed that [Zn(GTSC)]3 had 3 to 12 fold higher fluorescence than Zn(ATSM) in various cell lines (n = 9) of different tissue origin. Confocal fluorescence microscopy studies established that [Zn(GTSC)]3 localizes in the nucleus of human breast cancer MCF-7 and MDA-MB-231 cells within 30 minutes of addition. Moreover, [Zn(GTSC)]3 showed relatively less cytotoxicity compared to the Zn(ATSM) complex in all the cancer cell lines tested. DNA interaction studies such as binding and cleavage showed that [Zn(GTSC)]3 was less harmful to DNA as well. All these features make [Zn(GTSC)]3 a good fluorescent imaging agent for live cells. Binuclear zinc bis(thiosemicarbazone) complexes were also synthesized and their cytotoxicity was evaluated in different cancer cells. One of the ligands, 1,3-bis{biacetyl-2′-"-N-pyrrolidinethiosemicarbazide)-3′-(4"-N-thiosemicarbazide)} propane (ProBATpyrH4), and its zinc complex were found to show excellent anticancer activity against human hepatocellular cancer (HepG2) cell line. However, the cellular uptake studies as followed by flow cytometry revealed that these compounds do not fluoresce inside the cells. However, the DNA interaction studies using ethidium bromide displacement assay revealed that these complexes have better binding ability to DNA than mononuclear zinc complexes and the viscometric titrations suggested the binding mode to DNA is through partial intercalation. Apparently, these complexes do not induce DNA cleavage as evident from the cleavage experiments performed on pBR322 DNA. It is likely that their anticancer activity is due to unique DNA binding properties. Imaging zinc is important in the field of metallomics as alteration of zinc concentration in cells is associated with, or attributed to various diseases. In this regard, bis(thiosemicarbazone) ligands are useful. Chapter 3, “Imaging intracellular zinc using glyoxal-bis(4-methyl-4-phenyl-3-thiosemicarbazone) ligand” deals with imaging zinc in live cells using the bis(thiosemicarbazone) ligand, GTSCH2. Since the trimeric zinc complex is fluorescent, the corresponding ligand, GTSCH2, was utilized to visualize the zinc present within cells. The ligand GTSCH2 is found to be a selective fluorescence “turn-on” sensor for zinc. This sensor exhibited excellent sensitivity and selectivity towards zinc over other physiologically relevant cations. The binding affinity of GTSCH2 to zinc was estimated to be 0.59 nM in an aqueous MOPS (50 mM, NaCl; 100 mM; pH 7.3) buffer containing 30% DMSO, from competitive binding experiments carried out with ethylene glycol tetraacetic acid (EGTA). The sensor displayed maximal fluorescence response to zinc ion when present in the ratio of 1:1 and displayed stable fluoresence in the pH range 5.0 to 7.8, which suggests that the probe may be suitable for imaging zinc in both normal and cancer cells. The potential of GTSCH2 to image zinc inside the cell has been demonstrated in two human breast cancer cell lines using confocal fluorescence microscopy. Unlike mononuclear zinc complexes, the mononuclear copper bis(thiosemicarbazone) complexes are cytotoxic. Chapter 4, “Anticancer activity of copper bis(thiosemicarbazone) complexes” deals with the synthesis, characterization and anticancer activity of mononuclear copper bis(thiosemicarbazone) complexes. All of them were characterized by spectroscopic methods and in three cases by single crystal X-ray diffraction. The redox properties, studied by cyclic voltammetry, showed reversible one electron- reduction process that varied from –0.53 V to –0.18 V vsSCE. Anticancer activity for the synthesized complexes and their ligands were tested against many human cancer cell lines where the complexes Cu(GTSC) and Cu(GTSCHCl) derived from glyoxal-bis(4-methyl-4-phenyl-3-thiosemicarbazone) are found to be most cytotoxic (GI50 <0.1 µM to 2.1 µM) in five cancer cell lines tested. Moreover, the cytotoxicity is similar to that of adriamycin, a known anticancer drug, in all cell lines. However, it is less potent than a copper bis(thiosemicarbazone) analog, copper biacetyl-bis(4-methyl-3-thiosemicarbazone) Cu(ATSM), a well studied anticancer agent in many cell lines. Cellular studies were carried out for the selected complexes Cu(GTSC) and Cu(GTSCHCl) along with Cu(ATSM) on HCT116 colon cancer cells. The order of lipophilicity and cellular uptake as studied by ICP-OES are correlated with their cytotoxicity. Based on the interaction of these complexes with DNA using the ethidium bromide displacement assay, DNA -melting, -viscosity and -cleavage studies, it is suggested that these complexes intercalate partially with DNA. DNA cleavage studies using pBR322 DNA revealed that only Cu(GTSCHCl) complex cleaves DNA. Mechanistic discrimination studies suggest that the complex cleaves DNA through the hydrolytic pathway. Since the topoisomerase IIα (Topo IIα), a nuclear enzyme resolving topological problems of DNA, is considered as one of the possible molecular targets for a number of anticancer drugs including some of the copper thiosemicarbazone complexes, Topo IIαinhibition studies were carried out in human Topo IIα. Interestingly, many copper bis(thiosemicarbazone) complexes are able to inhibit Topo IIα activity by acting as Topo IIα poison. Cu(GTSCHCl) complex was found to be the most active in this series of complexes (90 % inhibition at 100 µM) and it inhibits the enzyme in a dose dependant manner. Based on the results, it was concluded that the cell death may be mediated, at least in part, through DNA cleavage and Topo IIαinhibition. Severe side effects, poor distribution profiles and or organ specific toxicity make the conventional chemotherapy of limited value with metal based drugs. Therefore, developing cancer-specific drug delivery systems is an urgent need in cancer therapy. Among the many strategies available to target cancer, targeting the receptors that are overexpressed in the cancer cell membrane is a novel strategy being used in recent studies. Therefore the last part my work, “Copper bis(thiosemicarbazone) complexes linked to poly(ethylene glycol) and multiwalled carbon nanotubes for targeted delivery to cancer cells ” was designed to target cancer cells. Here, copper complexes (therapeutic molecule) were attached with PEG and MWCNT (carrier) along with folic acid or biotin (targeting molecule). First, CuATSM–A was functionalized with a disulfide linker and connected with folic acid via a poly(ethylene glycol) (PEG600) linker. This was synthesized to target KB (human nasopharyngeal carcinoma) cells, a cell line that overexpresses the folate receptor on the cell surface. In order to investigate the targeting efficacy, the corresponding fluorescent labeled analogs and non-targeted PEG conjugates were synthesized. Flow cytometry studies with fluorescent marker (fluorescein isothiocyanate) labeled PEG analogs showed the targeting efficacy on KB cells. The copper complex, CuATSM–A, attached with biotin–PEG2000 also was synthesized to target high-biotin-using HeLa (human cervical carcinoma) cells. Multiwalled carbon nanotubes (MWCNT) were also used as nanocarriers. Here, the MWCNT was decorated with PEG600 diamine and then functionalized with the copper complex (therapeutic molecule), folic acid (targeting molecule), and FITC (fluorescent molecule). The conjugation of all the molecules with MWCNT is characterized by various spectroscopic techniques.

Use of metal based anticancer medication began with the clinical approval of cisplatin in 1978. Research led to the development of six platinum based drug candidates which are in use around the world. However there is a great need to develop better treatment strategies. The present work entitled “Cytotoxicity of Metal Based Anticancer Active Complexes and Their Targeted Delivery Using Nanoparticles” is an effort to prepare cytotoxic metal complexes based on platinum(IV) and copper(II) and deliver them selectively to cancer cells using a targeting ligand, biotin, with two different delivery vehicles, viz. PEGylated polyamidoamine dendrimer (PAMAM) and gold nanoparticles (AuNPs). Chapter 1 provides a brief introduction to cancer and its characteristic features, followed by a short description about different treatment modalities in clinical practice. An account of the development of anticancer drugs starting from purely organic drugs to the field of metal based anticancer drugs is discussed. An overview of the available targeting strategies are discussed with specific examples. The section ends with the scope of the present work. Platinum based anticancer drugs currently in use contain platinum in the +2 oxidation state. These drugs showed side effects and are often ineffective against resistant cells, especially in the latter stages of treatment. A recent focus of metal based anticancer drug research is the development of platinum(IV) systems which shows promise to have greater activity in cancer cells in a reducing environment. Reported platinum(IV) dual drugs contain the components of “cisplatin” or an analogue along with an active organic drug. But there are no known dual drugs based on platinum(IV) that would generate a cytotoxic metal complex along with cisplatin. In Chapter 2, a bimetallic dual drug (M4) (Figure 1), the first of its kind, with components of cisplatin and copper bis(thiosemicarbazone) has been prepared (Figure 1). The components and the bimetallic complex were characterized using several spectroscopic techniques. The dual drug M4 was found to be highly cytotoxic (IC50 1.3 M) against HeLa cells and was better than cisplatin (IC50 6.8 M). The bimetallic complex turned out to be better than the mixture (IC50 7.2 M) of individual drugs which indicated possible synergism of the released cisplatin and the copper bis(thiosemicarbazone) from the dual drug. Figure 1: Structure of the platinum(IV) and copper bis(thiosemicarbazone) complexes. A novel approach towards conjugation of platinum(IV) drugs to a carrier has been developed using a malonate moiety (Figure 2). The bis(butyric acid) complex, Pt(NH3)2(OCOC3H7)2Cl2 (M1), was taken as model complex to demonstrate the conjugation strategy. The complex M4 was also conjugated to the partially PEGylated 5th generation PAMAM dendrimers. Figure 2: Schematic representation of the platinum(IV) drug conjugated PAMAM dendrimer. The cytotoxicity of M4 was reduced to a small extent on conjugation to the dendrimer. In the presence of 5 mM sodium ascorbate as a reducing agent, sustained release (40 %) of the drug was shown to occur over a period of 48 h by the drug release study. The reduction in cytotoxicity of the dendrimer conjugates could be due to incomplete release of the active drug. Unfortunately, no enhanced activity was observed with the additional targeting ligand, biotin. The drug uptake study revealed that the dendrimer conjugates were successful in entering cancer cells. There was no preferential uptake with biotin conjugated dendrimers which explained the similar cytotoxicity of dendrimer conjugates with and without biotin. Different delivery vehicles showed varied efficiency in delivering the pay load (drugs) to the cancer site. In this connection, PEGylated gold nanoparticles have shown good promise as a drug delivery vehicle. In Chapter 3, M1 and M4 are both conjugated to malonate functionalized PEGylated gold nanoparticles (30 nm). Biotin was also attached to the AuNPs for targeting HeLa cells. Figure 3: Schematic representation of the platinum(IV) drug and biotin conjugated AuNPs. The AuNPs were highly stable in water without agglomeration. There was no shift in the Surface Plasmon Resonance (SPR) band after conjugation of the drug molecules and targeting ligands. TEM images and DLS measurements showed there was no change in particle size. Drug conjugated AuNPs were also very stable in high salt concentrations as well as over a large range of pH. AuNPs with M1 were found to be less cytotoxic than the parent drug. Biotinylated AuNPs with M1 were more potent than non-biotinylated nanoparticles and increased cytotoxicity (35 %) was observed with biotin conjugation. Surprisingly, the enhanced activity of biotinylated AuNPs could not be correlated to the drug uptake study. The cytotoxicity of the bimetallic dual drug containing AuNPs were about 10-fold less and no increased activity was observed with the biotinylated conjugates. The reduced activity of AuNPs with the bimetallic drug was due to incomplete release from the AuNPs (20 % release after 48 h). But the release kinetics was very slow and sustained which might increase in vivo activity. The unexpected lower activity of biotinylated conjugates with copper bis(thiosemicarbazone) was suggestive of interference between bis(thiosemicarbazone) complex and the biotin receptor resulting in reduced drug uptake. Copper bis(thiosemicarbazone) complexes hold very good promise as a class of non-platinum anticancer drug candidates. However, they lack selectivity towards malignant cells. Recently, CuATSM has shown hypoxia selectivity and very good cytotoxicity resulting in 64CuATSM being used in advanced stages of clinical trials for imaging hypoxic cells. In Chapter 4, a copper bis(thiosemicarbazone) complex analogous to Cu(ATSM) with a redox active cleavable disulfide linker and a terminal carboxylic acid group (CuATSM-SS-COOH) was synthesised and characterised spectroscopically. The complex was highly cytotoxic and has an IC50 value (6.9 M) similar to that of cisplatin against HeLa cells. The complex was conjugated to PEGylated gold nanoparticles by amide coupling between the acid group from the drug molecule and the amine on the AuNPs (20 nm) for smart drug delivery. The gold nanoparticles were decorated with biotin for targeted delivery to the HeLa cells. Figure 4: Schematic representation of the CuATSM-SS-COOH and biotin decorated AuNPs. The CuATSM-SS-COOH was insoluble in water but conjugation to PEGylated gold nanoparticles made it water soluble. The drug molecules and biotin conjugated AuNPs were highly stable which was confirmed by TEM and DLS measurements. Similar to the study described in the previous chapter, these AuNPs were also stable in a wide range of pH and salt concentrations. In vitro glutathione (GSH) triggered release study demonstrated substantial release of the cytotoxic agent from the AuNPs (60 %) over a period of 48 h. In vitro cell viability study with HeLa cells showed reduced cytotoxicity (IC50 15 M) of AuNPs with and without biotin containing drug conjugates relative to the parent copper complex (IC50 6.9 M). The reduction of the cytotoxicity correlated well with the released amount of the active drug from the nanoconjugates over the same time period. In vivo studies demonstrated the effectiveness of these nanoparticle carriers as suitable vehicles as they exhibited nearly four-fold reduction of tumor volume without significant loss in body weight. Moreover, the biotin targeted nanoparticle showed significant (p < 0.5) reduction in tumor volume compared to the non-targeted gold nanoparticles. Thus, this smart linking strategy Can be extended to other cytotoxic complexes that suffer from non-specificity, low aqueous solubility and toxicity. Multinuclear anticancer active complexes do not act in the same way as that of their corresponding mononuclear analogues. In the case of multinuclear platinum complexes, the activity not only depends on the active moiety but also on the spacer length between the moieties. In Chapter 5, a series of multinuclear copper bis(thiosemicarbazone) complexes were prepared and characterised using different techniques. Figure 5: General structures of binuclear copper bis(thiosemicarbazone) complexes. All the complexes showed redox activity and have a very high negative reduction potential, i.e. these compounds would not be easily reduced in the biological medium and would remain as copper(II) species. As the concentration of the reducing agents are more within cancer cells, once these complexes are inside cells they would be reduced to Cu(I). These compounds were shown to be highly lipophilic from the large log P values. Unfortunately, these binuclear complexes were less active than similar mononuclear complexes. One possible reason for the reduced cytotoxicity of these complexes could be adherence of the complexes to the cell membrane due to the high lipophilicity of these complexes. Out of five different methylene spacers between two bis(thiosemicrarbazone) moieties, the complex with a three carbon spacer was shown to be the most active against HeLa cells. The complexes with five and six methylene spacers turn out to be noncytotoxic. Further experiments are necessary to reveal the mechanism of action in these complexes. In summary, bimetallic complexes can be very active and may be a way of overcoming drug resistance in platinum based therapy. A dual drug can be delivered using a malonate moiety and a disulfide linker. Gold nanoparticles are good delivery vehicles for these dual drugs and show great potential for improvement and translation to the next stage. (For figures pl refer the abstract pdf file)

Submitted in complete fulfillment for the Degree of Doctorate of Philosophy in Biotechnology, Durban University of Technology, Durban, South Africa, 2016. / Plants have provided a source of medicine from the beginning of human history and are the core of modern medicine. Moreover, plant based drug discovery has led to the development of various anticancer drugs (such as vincristine, vinblastine, etoposide, paclitaxel, camptothecin, topotecan and irinotecan). The use of botanical, phytochemical, biological and molecular techniques have facilitated the discovery of anthraquinones from Ceratotheca triloba that can inhibit the human topoisomerase II enzyme (target for anticancer drugs) and kill cancer cells. However, the C. triloba plant has not been extensively studied for its anticancer activity. Therefore, the aim of this study was to further investigate the anticancer activity of C. triloba and determine the classes of compounds that contributed towards its activity. In this study the leaf and root extracts were prepared by using hexane, DCM, hexane: DCM (1:1), methanol and/or water. These extracts were examined for their growth inhibitory potential on three cancer cell lines (A375 [melanoma], MDA-MB-231[breast] and WHCO1 [esophageal]) by using the MTT assay. Then, different mobile phases were prepared for optimizing the separation of the compounds of the active extract by TLC. Column chromatography was performed with the active extract by using five mobile phases (hexane : DCM [60 : 40, 40 : 60], DCM, DCM : ethyl acetate [90 : 10, 70 : 30, 60 : 40, 50 : 50, 50 : 60, 30: 60, 20 : 80], ethyl acetate and ethyl acetate: methanol [80 : 20, 70 : 30, 50 : 50]). The fractions collected from the column were examined for their growth inhibitory potential on two melanoma cell lines (A375 and UACC-62). The IC50 and TGI (total growth inhibition) values of the active fractions were determined. Also, the apoptosis inducing effects of the active fractions and standards (camptothecin and doxorubicin) were determined by using flow cytometer based assays (FITC annexin assay, PE active caspase 3 assay and BD MitoScreen assay). Subsequently, the chemical structures of the compounds that contributed towards the activity of these fractions were obtained by EI-LC-MS analysis. The results demonstrated that the hexane root extract exhibited the best percentage of growth inhibition (%GI) on all three cancer cell lines. The separation of the compounds of the hexane root extract was optimized on TLC plates by using different ratios of hexane and DCM. Column chromatography allowed for fractionation of this extract. Purified compounds were not obtained due to co-elution. Further research would have to be conducted to obtain purified compounds. This may involve the use of mini-column chromatography and PTLC. Overall a total of ten combined fractions were collected from the column. Four of these fractions (F2, F4, F5 and F8) displayed a high %GI on the A375 and UACC-62 cell lines. Moreover, fraction F4 was the most active fraction as it had the lowest IC50 (0.70 µg.ml-1 [A375] and 0.39 µg.ml-1 [UACC-62]) and TGI (12.50 µg.ml-1[A375] and 25 µg.ml-1 [UACC-62]) values in comparison to the other fractions. All four fractions induced depolarization of the mitochondria membrane potential (ΔΨ), caspase 3 activation, early apoptosis (phospholipid phosphatidylserine exposure) and/or late apoptosis in the melanoma cells. The results also revealed that fraction F4 (25 µg.ml-1) induced depolarization of the ΔΨ in a higher percentage of A375 (78.11%) and UACC-62 (87.4%) cells than the other fractions and standards. This fraction also induced caspase 3 activation in a high percentage of A375 (90.56%) and UACC-62 (96.78%) cells. Therefore fraction F4 was also the most active fraction in terms of apoptosis activity. Based on our results and literature findings we can deduce that the active fractions induced the intrinsic or extrinsic (type II) apoptosis pathway in the melanoma cells. Six classes of compounds were identified from the four active fractions. These were: benzothiophenones, benzopyranones, naphthoquinones, anthraquinones, androstanes and quinazolines. In conclusion, this is the first study that evaluated the growth inhibition potential of the leaf and root extracts of C. triloba on a panel of cancer cells. This research indicated that the hexane root extract displayed the best levels of cell growth inhibition. The active constituents of this extract were isolated into four fractions which elicited apoptosis inducing effects that promoted the extrinsic (type II) or intrinsic apoptosis pathway in the melanoma cells. Furthermore, fraction F4 contained the most active compounds from C. triloba as it had the lowest IC50 and TGI values (in comparison to the other fractions) and induced depolarization of the ΔΨ in the highest percentage of melanoma cells. It was confirmed that six classes of compounds were accountable for the anticancer activity of these fractions. Thus, the C. triloba plant is a rich source of anticancer compounds. / D

Development of Non-Traditional Platinum Anticancer Agents: trans-Platinum Planar Amine Compounds and Polynuclear Platinum Compounds By Daniel E. Lee, Ph.D. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. Virginia Commonwealth University, 2015 Major Director: Nicholas P Farrell, Ph.D., Professor, Department of Chemistry Platinum anticancer compounds with cis geometry, similar to cisplatin, have been explored to circumvent the cisplatin resistance; however, they were not considered broadly active in cisplatin cells due to exhibiting similar or same cell death mechanism as cisplatin. Platinum compounds with trans geometry were less studied due to transplatin being clinically inactive; but with few structural modifications, they resulted in unaffected cytotoxic activities in cisplatin resistant cells with structural modification by exhibiting different modes of DNA binding. This research focused on further exploring and establishing structure-activity relationship of two promising non-classical series of platinum compounds with trans-geometry: trans-platinum planar amine (TPA) compounds and noncovalently binding polynuclear platinum compounds (PPC-NC). During this research, further optimizations of the reactivity of TPA compounds were accomplished by modifying the leaving carboxylate groups. The effects of modified reactivity were probed by a systematic combination of chemical and biophysical assays, followed by evaluating their biological effects in cells. To establish the structural-activity relationship of PPC-NCs, Mono-, Di-, Tri-, and Tetraplatin NC with charge of 4+, 6+, 8+, and 10+ were synthesized and evaluated by utilizing biophysical and biological assays. Lastly, a new class of polynuclear platinum compounds, Hybrid-PPCs, were synthesized and evaluated to overcome the pharmacokinetic problems of BBR3464, phase II clinical trial anticancer drug developed previously in our laboratory.

Les bienfaits des acides gras oméga-3, essentiellement l’EPA (C20:5n-3) et le DHA (C22:6n-3) sont bien élucidés dans la littérature. Ils jouent en effet, un rôle essentiel dans la prévention de nombreuses maladies neurodégénératives et cardiovasculaires. Ces acides gras polyinsaturés à longue chaîne sont majoritairement retrouvés dans des sources d’origines marines. Dans ce contexte, nous nous sommes intéressés d’une part à l’effet du complexe phospholipopeptidique provenant de l’hydrolyse enzymatique des têtes de saumon, sur l’anxiété et le stress oxydant dans le cadre d’une étude comportementale effectuée sur un modèle murin. D’autre part, nous avons mis en place une approche de double vectorisation, sous formes de nanoémulsions, visant à augmenter la biodisponibilité de deux molécules hydrophobes et bioactives (coenzyme Q10 et curcumine) en utilisant les lipides totaux (phospholipides et triacylglycérols) du complexe phospholipopeptidique riche en EPA et en DHA. Les résultats de ce travail ont montré que le CPLP, sa fraction lipidique et peptidique ont un effet anxiolytique à une dose de 600 mg de CPLP/jour pendant 14 jours de traitement. Il a également été démontré dans cette étude que l’hydrolysat peptidique du CPLP diminue significativement, à double dose, le stress oxydant en baissant le niveau endogène des espèces réactives de l’oxygène (ROS) dans les neurones. D’autre part, pour une utilisation thérapeutique, la biodisponibilité du CoQ10 vectorisé à forte dose est améliorée jusqu’à 38 fois par la formulation huileuse composée de lipides polaires du CPLP. Concernant la supplémentation classique en CoQ10 en tant que complément alimentaire, la formulation émulsionnée présente une meilleure disponibilité à dose aigüe, avec une concentration plasmatique deux fois plus élevée que la formulation de référence. Malgré une activité anticancéreuse reconnue pour la curcumine, sa faible solubilité diminue sa biodisponibilité et limite de ce fait son utilisation. La formulation nanoémulsionnée de curcumine contribue à inhiber la prolifération de cellules cancéreuses (MCF7) / The benefits of omega 3 fatty acids, mainly EPA (C20:5n-3) and DHA (C22:6n-3) are well understood in the literature. They indeed play an essential role in the prevention of many neurodegenerative and cardiovascular diseases. These polyunsaturated fatty acids are mostly found in marine sources. In this context, we were interested on the effects of phospholipopeptidic complex from the enzymatic hydrolysis of salmon heads on anxiety and oxidative stress using a behavioural study (mouse model). On the other hand, we have developed a double vectorization operating nanoemulsions, to increase the bioavailability of two hydrophobic and bioactive molecules (conenzyme Q10 and curcumine) by total lipids (phospholipids and triacylglycerols) from the phospholipopeptidic complex rich in EPA and DHA. The results of this study showed that the CPLP, its lipid and peptide fractions have an anxiolytic effect at a dose of 600 mg of CPLP / day for 14 days of treatment. It was also demonstrated that the peptide’s hydrolyzate ingested at double dose decreases significantly the oxidative stress by lowering the endogenous level of reactive oxygen species (ROS) in neurons. For therapeutic uses, the bioavailability of CoQ10 increased up to 38 times compared to referential formulation when verctorized at high dose in the oily formulation composed of CPLP’s total lipids. Regarding conventional CoQ10 supplementation as a dietary supplement, the emulsified formulation has a better availability at single dose, with plasma concentrations two times higher than the reference formulation. Although the anti-cancer activity of curcumine is highlighted, its low solubility and hence its low bioavailability, are factors limiting its use. The formulation of nanoemulsified curcumine allows a significant reduction in the proliferation of cancer cells (MCF7)

5 Abstract The formerly reported hydroxyamide Ph2PfcC(O)NHCH2CH2OH 1 and its respective novel congeneric analogues Ph2PfcC(O)NHCH3−n(CH2OH)n (3: n = 2; 4: n = 3) were used to prepare a series of arene-ruthenium(II) complexes [(η6 -arene)RuCl2(L-κP)] 6-8 (arene = C6H6, p-cymene, C6Me6; L = 1, 3 or 4). These complexes were studied as pre-catalysts in redox isomerization of allylic alcohols to carbonyl compounds. Among the compounds prepared, complex 6b [(η6 -p-cymene)RuCl2(1-κP)] showed best results. The solid state structure of the product of photolytic decomposition of complex [(η6 - C6Me6)RuCl2(2-κP)] (2 = Ph2PfcC(O)N(CH2CH2OH)2), viz, [(µ-Cl)3{Ru(η6 -C6Me6)}2] [FeCl4] 9, was determined by single-crystal X-ray diffraction analysis. The bis-phosphane complexes [MII Cl2(1-κP)2] (M = trans-Pd (10), cis-Pt (11) and trans- Pt (12)) together with chalcogenide derivatives Ph2P(O)fcC(O)NH(CH2)2OH (13) and Ph2P(S)fcC(O)NH(CH2)2OH (14) derived from hydroxyamide 1 were tested in vitro for their cytotoxicity against human ovarian A2780 cancer cell line. Complexes tested showed moderate cytotoxicity. Triol-amide FcC(O)NHC(CH2OH)3 15 (Fc = ferrocenyl) reacted with decavanadate (Bu4N)2[H3V10O28] at elevated temperature and prolonged reaction time yielding the hybrid hexavanadate (Bu4N)2[{FcC(O)NHC(CH2O)3}2V6O13] 16...

Le laboratoire a récemment identifié la protéine Damaged-DNA-Binding 2 (DDB2), connue à l’origine pour son rôle dans la réparation de l’ADN comme une protéine impliquée dans la tumorigenèse mammaire. En effet, nous avons montré son rôle dans la croissance et la progression des tumeurs mammaires via la régulation transcriptionnelle de gènes cibles. Dans ce travail, nous avons montré que la surexpression de DDB2 augmente la sensibilité des cellules tumorales MDA-MB231 et SKBr3 traitées à la doxorubicine et au 5-fluorouracile (5-FU). Inversement, l’inhibition de l’expression de DDB2 dans les cellules T47D qui l’expriment naturellement s’accompagne d’une baisse de la sensibilité à ces agents anticancéreux. Nos résultats montrent que la sensibilité des cellules au 5-FU mais pas à la doxorubicine, lorsque DDB2 est surexprimée, dépend en partie du contrôle négatif qu’exerce cette dernière sur l’activité de NF-κB, en régulant positivement l’expression d’IκBα. Enfin, la recherche d’autres gènes cibles de DDB2, impliqués dans l’apoptose, nous a conduits à celui codant le facteur anti-apoptotique Bcl-2. DDB2 agit négativement sur l’expression de Bcl-2 en interagissant avec une région de l’ADN localisée dans le promoteur P2 du gène correspondant. De part, son rôle anti-apoptotique, la régulation de son expression pourrait bien être à l’origine de la sensibilité aux agents anticancéreux induite par DDB2. L’ensemble de ces résultats met donc en évidence l’intérêt clinique de DDB2 comme marqueur prédictif de la réponse aux agents anticancéreux, et devrait contribuer à une meilleure compréhension des mécanismes impliqués dans l’échappement des cellules tumorales aux thérapies / The laboratory has recently identified the Damaged-DNA Binding 2 protein (DDB2), a protein involved in DNA repair, as an important actor in breast tumorigenesis. Our laboratory has shown that DDB2 is involved in breast tumor growth and progression through the transcriptional regulation of target genes. Thus, the first aim of this work was to study the role of DDB2 and its target genes in the response of breast cancer cells to anticancer drugs. We showed that DDB2 overexpressed in breast cancer cell lines, such as MDA-MB231 and SKBr3, increased the cells sensitivity to apoptosis induced by doxorubicin and 5-Fluorouracil (5-FU). Conversely, the inhibition of DDB2 expression in T47D cells, which express endogenously this protein, decreased cell sensitivity to anticancer agents. Our results showed that cell sensitivity induced by DDB2 expression to 5-FU but not doxorubicin depended on its ability to repress NF-κB activity via the regulation of IκBα expression. At last, the search of potential DDB2 target genes implicated in apoptosis has led us to identify the anti-apoptotic factor Bcl-2. We showed the ability of DDB2 to downregulate Bcl-2 expression via its interaction with DNA region located in P2 promoter of the corresponding gene. Results suggest that Bcl-2 dowregulation by DDB2 could be a major event that explains the enhanced sensitivity of cancer cells to therapeutic agents. Altogether, these data highlight the clinical interest of DDB2, as a predictive marker of the response to anticancer agents. A better understanding of its mode of action will contribute to improve therapeutic treatments and avoid their failure in resistant patients

Ce doctorat portait sur le développement de nanoparticules (NPs) photosensibles constituées d’un cœur photolysablepoly(acrylate d'o-nitrobenzyle) (polymère hydrophobe biocompatible - PANB) et d’une couronne basée sur un dérivé du dextrane (polysaccharide bactérien, hydrophile et biodégradable). Dans un premier temps, le PANB-N3 a été synthétisé par i) polymérisation radicalaire contrôlée (SET-LRP) de l’acrylate d’o-nitrobenzyle puis ii) modification chimique de l’extrémité de chaîne par une fonction azoture. En parallèle, le dextrane a été hydrophobisé par quelques chaînes grasses dotées d’un groupe alcyne (obtention du DexAlcyne-15). Ces polymères précurseurs peuvent alors réagir par chimie click CuAAC (Cycloaddition azide-alcyne catalysée par Cu(I)) pour engendrer divers glycopolymères greffés Dex-g-PANB. Dans un deuxième temps, les NPs ont été formulées par deux procédés puis caractérisées en termes de taille, recouvrement en dextrane (quantité par gramme de PANB, épaisseur de la couche surfacique) et stabilité colloïdale en milieu salin, en présence de tensioactif compétitif ou dans un milieu de culture (DMEM). Le procédé de nanoprécipitation a été appliqué aux Dex-g-PANB présentant de fortes fractions massiques en PANB (>40%) alors que le procédé d’émulsion-évaporation de solvant organique a été mis en œuvre en utilisant le DexAlcyne-15 comme tensioactif hydrosoluble et le PANB-N3 comme matériau hydrophobe. Grâce à leurs fonctionnalités complémentaires, une réaction CuAAC peut (ou non) avoir lieu à l’interface liquide/liquide pendant l’élaboration des NPs et conduire à l’obtention de NPs « non clickées » ou « clickées ». Enfin, le caractère photosensible des NPs a été validé par irradiation UV en observant une disparition progressive des NPs résultant de la photolyse des PANB. Afin d’utiliser ces NPs comme systèmes stimulables de délivrance de médicaments, un anticancéreux (Doxorubicine - Dox) a été encapsulé au sein des NPs, pendant leur élaboration. Cette encapsulation a été optimisée et les NPs chargées de DOX ont été caractérisées en termes de taille et d’efficacité d’encapsulation. La libération de la DOX hors des NPs a ensuite été suivie par simple diffusion, ou provoquéepar irradiation UV. Enfin, le potentiel biologique de ces NPs a été évalué vis-à-vis d’une lignée cellulaire tumorale humaine d'origine intestinale isolée d'un adénocarcinome colique (Caco-2). Après vérification de leur biocompatibilité et de la résistance des Caco-2 aux irradiations UV, nous avons pu montrer que les NPs chargées pouvaient libérer suffisamment de DOX en seulement 30 secondes d’irradiation (puissane: 54mW/cm2) pour éradiquer plus de 50% de ces cellules cancéreuses. / This work was focused on the development of light-sensitive nanoparticles (NPs) based on a photodegradable poly(o-nitrobenzyl acrylate) core(PNBA, hydrophobic and biocompatible polymer) and a dextran derivative shell (dextran is a biodegradable and hydrophilic bacterial polysaccharide). Firstly, PNBA-N3 was synthesized by i) Single-Electron Transfer Living Radical Polymerization (SET-LRP) of o-nitrobenzyl acrylate then ii) introduction of one azide end-function. In the same time, DexAlkyne-15 carrying several alkyne groups was produced by hydrophobization of dextran. Such DexAlkyne-15 and PNBA-N3 can react by CuAAC (Cu(I)-catalyzed azide-alkyne cycloaddition) click chemistry leading to Dex-g-PNBA glycopolymers with various macromolecular parameters. Secondly, NPs were produced by comparing two processes then characterized in terms of size, dextran amount, shell thickness and colloidal stability in NaCl or cell culture media, or in presence of one strong surfactant. On one hand, NPs were made by nanoprecipitation of Dex-g-PNBA exhibiting high PNBA weight fractions (>40 %). On the other hand, NPs were produced by emulsion-evaporation of the organic solvent using DexAlkyne-15 as water-soluble surfactant and PNBA-N3 as hydrophobic materials. In this case, in situ CuAAC occurred (or not) at the liquid/liquid interface during the NPs formulation, leading to “clicked” and “not-clicked” NPs. Finally, NPs disruption was studied by UV irradiation according the PNBA chains photolysis. To use such NPs as smart drug delivery systems, Doxorubicin (DOX - an anticancer agent), was loaded inside the NPs during their elaboration. The experimental conditions were optimized to enhance the DOX encapsulation. The kinetics release of encapsulated DOX were studied by diffusion or under UV irradiation. Finally, the biological potential of these NPs was estimated towards Caco-2 (continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells). After checking the NPs biocompatibility and theCaco-2 strength under UV irradiation, we proved that such loaded NPs can release enough DOX under 30 second irradiation (power: 54mW/cm2) to decrease the Caco-2 viability about more than 50%.

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.