Plusieurs agents anticancéreux très puissants sont caractérisés par une solubilité aqueuse limitée et une toxicité systémique importante. Cette dernière serait liée d’une part à la solubilisation des agents anticancéreux à l’aide de surfactifs de bas poids moléculaire, connus pour leur toxicité intrinsèque, et d’autre part, par le manque de spécificité tissulaire des anticancéreux. Les vecteurs colloïdaux à base de polymères permettraient de résoudre certains défis liés à la formulation d’agents anticancéreux hydrophobes. D’abord, les polymères peuvent être sélectionnés afin de répondre à des critères précis de compatibilité, de dégradation et d’affinité pour le médicament à formuler. Ensuite, le fait d’encapsuler l’agent anticancéreux dans un vecteur peut améliorer son efficacité thérapeutique en favorisant son accumulation au niveau du tissu cible, i.e. la tumeur, et ainsi limiter sa distribution au niveau des tissus sains. Des travaux antérieurs menés au sein de notre laboratoire ont mené à la mise au point de micelles à base de poly(N-vinyl-pyrrolidone)-bloc-poly(D,L-lactide) (PVP-b-PDLLA) capables de solubiliser des agents anticancéreux faiblement hydrosolubles dont le PTX. Ce dernier est commercialisé sous le nom de Taxol® et formulé à l’aide du Crémophor EL (CrEL), un surfactif de bas poids moléculaire pouvant provoquer, entre autres, des réactions d’hypersensibilité sévères. Bien que les micelles de PVP-b-PDLLA chargées de PTX aient démontré une meilleure tolérance comparée au Taxol®, leur potentiel de ciblage tumoral et leur efficacité thérapeutique étaient similaires à la forme commerciale à doses égales. Ceci était possiblement dû au fait que les micelles étaient rapidement déstabilisées et ne pouvaient retenir leur cargo suite à leur administration intraveineuse. Nous avons donc décidé de poursuivre les travaux avec un autre type de vecteur, soit des nanoparticules, qui possèdent une stabilité intrinsèque supérieure aux micelles. L’objectif principal de cette thèse de doctorat était donc de mettre au point des nanoparticules polymères pour l’administration parentérale d’agents anticancéreux faiblement solubles dans l’eau. Les nanoparticules devaient permettre d’encapsuler des agents anticancéreux hydrophobes et de les libérer de manière contrôlée sur plusieurs jours. De plus, elles devaient démontrer un temps de circulation plasmatique prolongée afin de favoriser l’accumulation passive du médicament encapsulé au niveau de la tumeur. La première partie du travail visait à employer pour la première fois le copolymère amphiphile PVP-b-PDLLA comme émulsifiant dans la préparation de nanoparticules polymères. Ainsi, une méthode de fabrication des nanoparticules par émulsion huile-dans-eau a été appliquée afin de produire des nanoparticules à base de PDLLA de taille inférieure à 250 nm. Grâce aux propriétés lyoprotectrices de la couronne de PVP présente à la surface des nanoparticules, celles-ci pouvaient retrouver leur distribution de taille initiale après lyophilisation et redispersion en milieu aqueux. Deux anticancéreux hydrophobes, soit le PTX et l’étoposide (ETO), ont été encapsulés dans les nanoparticules et libérés de ces dernières de façon contrôlée sur plusieurs jours in vitro. Une procédure de « salting-out » a été appliquée afin d’améliorer le taux d’incorporation de l’ETO initialement faible étant donnée sa solubilité aqueuse légèrement supérieure à celle du PTX. Le second volet des travaux visait à comparer le PVP comme polymère de surface des nanoparticules au PEG, le polymère le plus fréquemment employé à cette fin en vectorisation. Par le biais d’études d’adsorption de protéines, de capture par les macrophages et de biodistribution chez le rat, nous avons établi une corrélation in vitro/in vivo démontrant que le PVP n’était pas un agent de surface aussi efficace que le PEG. Ainsi, malgré la présence du PVP à la surface des nanoparticules de PDLLA, ces dernières étaient rapidement éliminées de la circulation sanguine suite à leur capture par le système des phagocytes mononucléés. Par conséquent, dans le troisième volet de cette thèse, le PEG a été retenu comme agent de surface, tandis que différents polymères biodégradables de la famille des polyesters, certains synthétiques (PDLLA et copolymères d’acide lactique/acide glycolique), d’autres de source naturelle (poly(hydroxyalkanoates)(PHAs)), ont été investiguées comme matériaux formant le cœur des nanoparticules. Il en est ressorti que les propriétés physicochimiques des polyesters avaient un impact majeur sur l’efficacité d’encapsulation du PTX et son profil de libération des nanoparticules in vitro. Contrairement aux PHAs, les polymères synthétiques ont démontré des taux d’incorporation élevés ainsi qu’une libération contrôlée de leur cargo. Des études de pharmacocinétique et de biodistribution ont démontré que les nanoparticules de PDLLA dotées d’une couronne de PEG conféraient un temps de circulation plasmatique prolongé au PTX et favorisaient son accumulation tumorale. Les nanoparticules polymères représentent donc une alternative intéressante au Taxol®. / Many highly potent anticancer drugs are characterized by poor aqueous solubility and can impart significant systemic toxicity. This toxicity can be attributed in part to the solubilisation of these anticancer agents with low molecular weight surfactants that are known to cause serious biological side effects on their own. Moreover, following their intravenous (IV) injection, the anticancer agents distribute throughout the body, causing deleterious effects in healthy organs and tissues. Colloidal polymeric drug carriers have been investigated as a means to circumvent these drawbacks. First, polymeric materials can be tailored to meet specific requirements in terms of biocompatibility, biodegradability and affinity for the cargo molecule. Second, associating a drug to a carrier system can drastically alter its distribution throughout the body, enhancing its deposition at the target site, e.g. the tumour, while sparing healthy tissues, thus minimizing systemic toxicity. Previous work in our group has led to the design of block copolymer micelles based on poly(N-vinyl-pyrrolidone)-block-poly(D,L-lactide) (PVP-b-PDLLA) that were shown to solubilise hydrophobic anticancer agents such as paclitaxel (PTX). PTX is commercially available as Taxol®, a Cremophor EL (CrEL)-based formulation. CrEL is a low molecular weight surfactant that has been linked to severe side effects including life-threatening hypersensitivity reactions. Although PTX-loaded PVP-b-PDLLA micelles have demonstrated much improved tolerability compared to Taxol®, they did not increase PTX tumoral concentrations and exhibited anticancer efficacy similar to Taxol® at equivalent dosage. This was attributed to rapid destabilisation of the micelles and release of their cargo following IV administration. We chose to pursue our work with a colloidal drug carrier that exhibits greater stability compared to block copolymer micelles, i.e. polymeric nanoparticles. The main objective of this project was to develop polymeric nanoparticles for the parenteral delivery of hydrophobic anticancer drugs. The nanoparticles had to meet certain requirements such as be able to encapsulate hydrophobic anticancer drugs and release them in a controlled fashion over several days. Furthermore, the nanoparticles should confer prolonged plasma residence times to the encapsulated drug and favour its passive accumulation at its intended site of action, i.e. the tumour. The first part of this work focussed on applying PVP-b-PDLLA for the first time as polymeric emulsifier for the preparation of PDLLA nanoparticles with appropriate mean diameters (250 nm) using an oil-in-water emulsion method. Two hydrophobic anticancer drugs, PTX and etoposide (ETO), were successfully incorporated into the nanoparticles. A salting-out method was applied to enhance the loading efficiency of ETO, which was initially low given its slightly higher aqueous solubility compared to PTX. Both drugs were released in a controlled fashion from the PDLLA nanoparticles in vitro. Because of the lyoprotective effect of PVP, the polymer corona allowed for the particles to be easily redispersed in aqueous media following lyophilisation. The second part of the thesis aimed at evaluating whether the PVP coating could confer “stealth” properties to the PDLLA nanoparticles. Our study provided direct comparison between PVP and PEG, the most widely employed surface agent in drug delivery. In vitro protein adsorption and phagocytosis studies corroborated the in vivo findings, which showed that PVP-coated nanoparticles were rapidly cleared from circulation following their uptake by the mononuclear phagocyte system. Hence, our results indicated that PVP as coating materiel is not as efficient as PEG in conferring “stealth” properties to polymeric nanoparticles. Consequently, in the last section of this thesis, PEG was selected as coating agent while various biodegradable polymers were investigated as core-forming materials. Both synthetic (PDLLA and lactide/glycolide copolymers) and natural (polyhydroxyalkanoates (PHAs)) polyesters were tested. Our results demonstrated that the physicochemical properties of the polyesters significantly influenced the loading efficiency and release kinetics of PTX. While nanoparticles based on synthetic polyesters exhibited high encapsulation levels and controlled PTX release in vitro, PHA-based nanoparticles exhibited immediate unloading of their cargo. Pharmacokinetic and biodistribution studies in rodents revealed that encapsulating PTX in PEG-coated PDLLA-based nanoparticles led to enhanced plasma residence time and tumour deposition of the drug compared to Taxol®. Polymeric nanoparticles thus represent an appealing alternative to Taxol®.

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)