Philosophiae Doctor - PhD / The prevalence of obesity amongst South Africans is alarming, with more than 29% of men and 56% of women considered to be obese. Angiogenesis, a process for development of new blood vessels play a major role in growth and survival of the adipose tissues. Pharmacological inhibitors of angiogenesis are therefore a sensible strategy to reduce excess body weight. Current anti-obesity drugs have limitations because of their lack of selectivity and specificity, which lead to undesirable side effects and reduced drug efficacy. Future anti-obesity therapeutic strategies should be target-specific, with minimal toxicity towards healthy tissues will be more appropriate for obesity treatment. Targeted nano-therapeutic agents are currently being developed to overcome the drawbacks associated with conventional drug therapies. The nano-based delivery vehicles that specifically target diseased cells are appealing as they could reduce drug toxicity towards healthy tissues and be more effective at lower dosages. The main aim of this study was to develop a receptor-mediated nanotherapy that specifically targets the white adipose tissue vasculature and trigger the death of these cells through apoptosis. The 14 nm gold nanoparticles (AuNPs) were synthesized using theTurkevich method following reduction of gold aurate by sodium citrate salt. Different chemistries were used to functionalise the AuNPs for biological application by conjugating with either vascular targeting peptide or pro-apoptotic peptide on their surface or both. The nanomaterials were characterised by UV-Vis, Zeta potential and transmission electron microscopy (TEM). The sensitivity and specificity of various AuNP conjugates were tested in vitro on colon and breast cancer cell lines. A human (Caco-2) cell line that expresses the receptor for the adipose homing peptide was chosen as an in vitro model system. Cellular toxicity and uptake of the nanoparticles was evaluated using the WST-1 assay, Inductively Coupled Plasma-Optical Emission Spectra (ICP-OES) and TEM. The induction of apoptosis following exposure to the nanoparticles was examined by Western blot and flow cytometric analysis. The anti-proliferative activity of the targeted therapeutic nanoparticles on the cells was more pronounced on the cells expressing the receptor for the adipose homing peptide. The uptake of unfunctionalised AuNPs was higher compared to functionalised nanoparticles, but this did not impair cell viability. The activity of the therapeutic peptide was retained and enhanced following conjugation to AuNPs as shown by Western blot and flow cytometric analysis. The nanotherapy under study demonstrated receptor mediated targeting, and enhanced activity on the cells expressing the receptor. However, the therapeutic and efficacy of the targeted nanotherapy still need to be tested in animal models of obesity to confirm the treatment specificity.

<p>Antivascular targeting is a promising strategy for tumor therapy. This strategy overcomes many of the transport barriers and has shown efficacy in many preclinical models, but targeting epitopes on tumor vasculature can also promote accumulation in healthy tissues. We used Elastin-like Polypeptide (ELP) to form block copolymers (BCs) consisting of two separate ELP blocks seamlessly fused at the genetic level. ELPBCs self-assemble into spherical micelles at a critical micelle temperature (CMT), allowing external control over monovalent unimer and multivalent micelle forms. We hypothesized that thermal self-assembly could trigger specific binding of ligand-ELPBC to target receptors via the multivalency effect as a method to spatially restrict high-avidity interactions. We termed this approach Dynamic Affinity Modulation (DAM). The objectives of this study were to design, identify, and evaluate protein-based drug carriers that specifically bind to target receptors through static or dynamic multivalent ligand presentation.</p> <p>ELPBCs were modified to include a low-affinity GRGDS or GNGRG ligand and a unique conjugation site for hydrophobic compounds. This addition did not disrupt micelle self-assembly and facilitated thermally-controlled multivalency. The ability of ligand-ELPBC to specifically interact with isolated AvB3 or CD13 was tested using an in vitro binding assay incorporating an engineered cell line. RGD-ELPBC promoted specific receptor binding in response to multivalent presentation but NGR-ELPBC did not. Enhanced binding with multivalent presentation was also observed only with constructs exhibiting CMT < body temperature. This study establishes proof-of-principle of DAM, but ELPBC requires thermal optimization for use with applied hyperthermia. Static affinity targeting of fluorescent ligand-ELPBC was then analyzed in vivo using intravital microscopy (IM), immunohistochemistry (IHC), and custom image processing algorithms. IM showed increased accumulation of NGR-ELPBC in tumor tissue relative to normal tissue while RGD-ELPBC and non-ligand ELPBC did not, and IHC verified these observations. This study shows (1) multivalent NGR presentation is suitable for static multivalent targeting of tumors and tumor vasculature, (2) multivalent RGD presentation may be suitable for DAM with thermal optimization, and (3) ELPBC micelles may selectively target proteins at the tumor margin.</p> / Dissertation

The nano/micro particles have been widely used as a carrier of therapeutic and contrast imaging agents. The nano/micro particles have many advantages, such as, specificity, controlled release, multifunctionality and engineerability. By tuning the chemical, physical and geometrical properties, the efficacy of delivery of nano/micro particle can be improved. In this study, by analyzing the effect of physical and geometrical properties of particle, such as, size, shape, material property and flow condition, the optimal condition for particle delivery will be explored. The objectives of this study are (1) to develop predictive mathematical models and (2) experimental models for particle margination and adhesion, and (3) to find optimal particle geometry in terms of size and shape to enhance the efficiency of its delivery. The effect of particle size expressed in terms of Stokes number and shape, namely, spherical, ellipsoidal, hemispherical, discoidal and cylindrical particle on the particle trajectory is investigated. For discoidal and cylindrical particles, the effect of aspect ratio is also considered. To calculate particle trajectory in the linear shear flow near the substrate, Newton's law of motion is decomposed into hydrodynamic drag and resistance induced by particle motion. The drag and resistance is estimated through finite volume formulation using Fluent v6.3. Particle behavior in the linear shear flow does strongly depend on Stokes number. Spherical particle is transported following the streamline in the absence of external body force. However, non-spherical particles could across the streamline and marginate to the substrate. For non-spherical particles, the optimal [Stokes number] in terms of particle margination is observed; [Stokes number almost equal to] 20 for ellipsoidal, hemispherical and discoidal particle; [Stokes number almost equal to] 10 for cylindrical particle. For discoidal particle with [gamma subscript d]=0.2 shows fastest margination to the substrate. The effect of gravitational force is also considered with respect to the fluid direction. When the gravitational force is applied, mostly, gravitational force plays a dominant role for particle margination. However, using small particle aspect ratio ([gamma subscript d]=0.2 and 0.33), spontaneous drift induced by particle-fluid-substrate interaction could overcome gravitational effect in some cases ([Stokes number]=10, G=0.1). In addition the adhesion characteristic of spherical particle has been studied using in vitro micro fluidic chamber system with different particle size and flow condition. The experimental results are compared to the mathematical model developed by Decuzzi and Ferrari (Decuzzi and Ferrari, 2006) and in vivo test (Decuzzi et al., 2010). The optimal particle size for S=75 and 90 is found to be 4-5 [micrometer] through the in vitro non-specific interaction of spherical particle on the biological substrate. The suggested mathematical model has proven to be valid for current experimental condition. At the end, the mathematical model, in vitro flow chamber results and in vivo test have been compared and the scaling law for particle adhesion on the vessel wall has been confirmed. / text

Pour améliorer la sélectivité cellulaire tumorale et/ou l'efficacité anti-tumorale de la thérapie photodynamique (PDT), des molécules actives (peptides, pseudopeptides, agent alkylant) ont été conjuguées à la molécule photo-activable. Dans une 1ère partie, l'effet vasculaire de la PDT a été favorisé par le ciblage de la vascularisation tumorale de phénotype angiogénique. Un photosensibilisateur de type chlorine a été couplé à un peptide ligand de neuropiline-1 (NRP-1) et a montré son efficacité anti-tumorale in vitro et in vivo en PDT. Cependant, la partie peptidique s'est avérée instable in vivo. Des pseudopeptides modifiés au niveau du site de clivage du peptide initial ont été synthétisés et testés in vitro, conjugués ou non à la chlorine. L'incorporation cellulaire de la chlorine a été améliorée après son couplage aux pseudopeptides. Une technique d'ARN interférence, visant à éteindre l'expression de NRP-1 dans les cellules MDA-MB-231, surexprimant NRP-1, a mis en évidence l'incorporation cellulaire récepteur-dépendante des conjugués et leur sélectivité cellulaire. Les pseudopeptides et conjugués se sont révélés affins pour NRP-1 et plus stables in vivo. Le ruthénium (Ru) s'affiche comme une molécule d'intérêt thérapeutique en oncologie. Dans une 2ème partie, nous avons appréhendé l'intérêt in vivo de coupler cet agent alkylant à des photosensibilisateurs de type porphyrine et chlorine. Les études d'efficacité in vivo sur un modèle de souris nude xénogreffées en ectopique avec des cellules de carcinome oral humain KB, ont été menées par une méthodologie de plans d'expérience. Deux conjugués [Ru(_6-p-PriC6H4Me)(5-(3-pyridyl)-10,15,20-triphenylporphyrin)Cl2, ou Rut1, et Ru4(_6-p-PriC6H4Me)4(5,10,15,20-tetra(3-pyridyl)porphyrin)Cl8, ou Rut4] efficaces in vitro ont été comparés in vivo. Le composé Rut4 est apparu comme le plus efficace. Des études de biodistribution par spectrofluorimétrie fibrée et de pharmacocinétique classique ont montré un intervalle drogue-lumière optimal de 24h. Néanmoins, malgré l'optimisation des modalités thérapeutiques, le couplage du Ru avec une molécule photo-activable (porphyrine ou chlorine) n'a pas conduit à une efficacité anti-tumorale significative in vivo / To improve tumor cells selectivity and/or photodynamic therapy (PDT) anti-tumor efficiency, active molecules (peptides, pseudopeptides, alkylating agents) have been conjugated to the photo-activable molecule. In a first part, vascular effect of PDT has been promoted by targeting the tumor vasculature with angiogenic phenotype. A chlorine-type photosensitizer have been coupled to a neuropilin-1 (NRP-1)-targeting peptide and has shown its in vitro and in vivo anti-tumor efficiency for PDT. However, the peptidic sequence has been degraded in vivo. Clivage site-modified pseudopeptides have been synthesized and tested in vitro, conjugated to TPC or not. Chlorine cellular uptake has been improved after coupling with pseudopeptides. A technique of RNA interference-mediated silencing of NRP-1 in MDA-MB-231, over-expressing NRP-1, has highlighted the receptor-dependent uptake of the conjugates and their cellular selectivity. Pseudopeptides and conjugates have shown their NRP-1 affinity and their in vivo stability. Ruthenium (Ru) is considered as an interest therapeutic molecule in oncology. In a second part, we have studied the in vivo interest of coupling an alkylating agent with porphyrine and chlorine-type photosensitizers. The in vivo efficiency studies on nude mice xenografted ectopically with KB cells have been carried using experimental design approach. Two in vitro efficient conjugates [Ru(_6-p-PriC6H4Me)(5-(3-pyridyl)-10,15,20-triphenylporphyrin)Cl2, or Rut1, and Ru4(_6-p-PriC6H4Me)4(5,10,15,20-tetra(3-pyridyl)porphyrin)Cl8, or Rut4] have been compared in vivo. Rut4 conjugate has appeared to be more efficient than Rut1. Biodistribution studies using fiber spectrofluorimeter and classic pharmacokinetic studies have shown an optimal drug-light interval of 24h. However, after PDT parameters optimization, the Ru coupling with a photo-activable molecule (porphyrin or chlorine) hasn?t shown any significant in vivo anti-tumor efficiency

Tumour-associated vasculature provides an accessible target for systemic gene therapy using targeted adenoviruses. The aim of this thesis is to develop strategies for targeting adenovirus infection to tumour-associated endothelium. Adenovirus expressing luciferase (Adluc) was coated with an amino-reactive polymer based on poly [N-(2-hydroxypropyl) methacrylamide] [pHPMA] to ablate normal infection pathways¬. This was a pre-requisite to redirecting virus tropism to infect endothelial cells via specific receptors. Direct attachment to the pHPMA-adenovirus (pcAdluc) of ligands including vascular endothelial growth factor (VEGF165) and a monoclonal antibody (RAFL) recognising VEGF receptor 2 (VEGFR-2) retargeted infectivity to VEGFR-2-positive endothelial cells and not to receptor-negative cells. Specificity of transduction in vitro was shown by competition with excess antibody. In vivo however, the VEGF165-retargeted virus failed to transduce tumour-associated endothelia following systemic administration. Similarly, direct linkage of a monoclonal antibody against E-Selectin (MHES) demonstrated E-Selectin-specific transduction of tumour necrosis factor-α (TNF-α)-activated endothelial cells, although overall levels of infection were not increased compared to unmodified Adluc. A two-component targeting system using protein A or protein G as ‘bridging’ agents was developed to ensure the required orientation of targeting antibodies. Using this system MHES mediated greater transduction of TNF-α-activated endothelial cells than Adluc. Conjugation using protein A also gave non-specific effects which were not seen with protein G. Whereas the unmodified Adluc virus failed to transduce TNF-α-activated endothelium in an umbilical vein model ex vivo, the MHES-protein G-pHPMA-adenovirus (MHES-StrepGpcAdluc) mediated good transduction. Similarly, StrepGpcAdluc retargeted with a chimeric P-Selectin Glycoprotein Ligand-1 (PSGL-1)-Fc fusion protein, showed good circulation kinetics and significant uptake into HepG2 xenografts following intravenous administration. Histological studies suggested selective targeting to tumour-associated endothelial cells. Overall these findings support the assertion that tumour-associated vasculature is an accessible target for systemic gene delivery, and the use of protein G as bridging agent facilitates rapid screening of Fc-bearing ligands for retargeting pcAd infection to tumour-associated endothelium.

Ce travail rapporte la synthèse et l'évaluation biologique des molécules hybrides de type isocombrétastatine A-4/belinostat. L'évaluation biologique de cette nouvelle série nous a permis d'identifier deux molécules inhibitrices de la polymérisation de la tubuline ainsi que de la HDAC8 possédant une puissante activité anti-proliférative dans la gamme du nanomolaire. De plus, nous démontrons que le système de catalyseur PtO2/PTSA-MeOH/H2O est très efficace pour convertir les alcynes internes et terminaux en cétones et qu’il est compatible avec une grande variété de groupes fonctionnels. / In this work, we report the synthesis and biological evaluation of isocombretastatin A-4/belinostat hybrid molecules. The biological evaluation of these new series has identified two molecules with potent anti-proliferative activity in the nanomolar range, which exhibit inhibitory activity on tubulin assembly and HDAC8. Second, we demonstrate that the PtO2/PTSA-MeOH/H2O catalyst system is very efficient in converting internal and terminal alkynes to ketones and that it is compatible with a wide variety of functional groups.