Iron platinum (FePt) has attracted growing interest because of its high Curie temperature, magneto-crystalline anisotropy and chemical stability. Nanoparticles (NPs) made of this alloy are promising candidates for a wide range of biomedical applications including magnetic separation, magnetic targeted drug delivery, hyperthermia for cancer therapy and also as magnetic resonance imaging (MRI) contrast agents. This thesis presents the synthesis, functionalization and characterization of FePt NPs along with a toxicity study and an investigation into their application as MRI contrast agents. Regarding their synthesis, different approaches have been explored including the co-reduction of Fe and Pt precursors in an aqueous media, the thermal decomposition in a conventional high-boiling solvent such as benzyl ether, and in low-melting organic salts (ionic liquids). The data revealed an inhomogeneous composition distribution of Fe and Pt between particles obtained in aqueous media, due to the iron salts hydrolysis, and a mismatch in the co-reduction kinetic of the two metal precursors. While the iron content in the NPs could be increased by using more hydrolytically stable iron precursors or stronger reducing agents, there are remaining limiting parameters which prevent further Fe content increase in NPs. In contrast, by excluding the water from the reaction system and using a Fe²⁻ iron precursor, homogenous 1:1 Fe to Pt ratio NPs can be obtained through a modified thermal decomposition pathway in benzyl ether. Based on the study of synthesis in this conventional chemical, the potential of ionic liquids (ILs) to be used as novel solvents for FePt NPs synthesis was further explored. It was then demonstrated that ionic liquids (ILs) can not only be used as a solvent for synthesis of FePt NPs, but also can provide an exciting alternative pathway to direct synthesis fct-FePt NPs. In the context of the bioapplication of FePt NPs, a family of FePt NPs was specifically designed to enhance their MRI contrast agents properties. In contrast with previous reports, this thesis demonstrates that FePt NPs can be made non-toxic and provides the first data on their cellular uptake mechanisms. A six times increase in the FePt based T₂ contrast properties compared to clinical iron oxide NPs is reported. The relationship between the MRI contrast properties and the NPs architecture is explored and rationalised as the basis for the design of NPs as enhanced MRI contrast agents. Finally, the first observations of cellular and in vivo MR imaging with FePt NPs is also reported. This study opens the way for several applications of FePt NPs such as regenerative medicine and stem cell therapy, thus providing a bio-platform to develop novel diagnostic and therapeutic agents.

Les copolymères à blocs amphiphiles s’auto-assemblent en solution aqueuse grâce à l’association de leurs segments hydrophobes. Les nanoparticules formées à partir de copolymères biocompatibles et biodégradables tels que les poly(hydroxyalkanoates) (PHAs) sont particulièrement attractives pour la conception de systèmes à libération prolongée de principes actifs. La relation entre la composition/structure chimique du copolymère, ses propriétés d’auto-assemblage et ses effets sur les cellules in-vitro doit être étudiée. Des copolymères à blocs poly(acide malique)-b-poly(3-hydroxybutyrate) (PMLA-b-PHB), PMLA-b-PHB-b-PMLA et poly(triméthylène carbonate)-b-poly(acide-malique) (PTMC-b-PMLA) ont été synthétisés par polymérisation par ouverture cycle (ROP) des monomères correspondants, suivie d’une hydrogénolyse. Une gamme de copolymères bien définis, caractérisés par spectroscopie RMN 1H, 13C{1H}, HSQC, HMBC, et DOSY, par analyses SEC, DSC, TGA, et mesure des angles de contact, présentant des balances hydrophile/hydrophobe modulables, a été obtenue grâce au control précis de la fraction hydrophile f (11-82%). Des auto-assemblages modulables ont été formés par nanoprécipitation des copolymères en l’absence d’agent tensio-actif. De larges agrégats ainsi que des micelles cœur-couronne (Rh = 16-335 nm) ont été obtenus en fonction du copolymère utilisé (dibloc vs. tribloc). Des micelles stables pendant 10 jours à 37 °C en solution aqueuse ont été obtenues pour les copolymères avec f allant jusqu’à 50%. Les copolymères PMLA-b-PHB et PTMC-b-PMLA n’ont pas révélé de toxicité aigüe in-vitro. De plus, l’utilisation du PHB a avantageusement permis de diminuer la captation des nano-objets par les macrophages et d’augmenter la captation par les cellules hépatiques. / Amphiphilic block copolymers are able to form self-assembled systems in aqueous solution by association of their hydrophobic segments. Nanoparticles formed from biodegradable and biocompatible polymers such as poly(hydroxyalkanoate) copolymers are particularly attractive for drug delivery applications. The relationship between the chemical structure/composition of the macromolecule, its self-assembly properties and its effect on cells in-vitro has to be studied.The synthesis of poly(-malic acid)-b-poly(3-hydroxybutyrate) (PMLA-b-PHB), PMLA-b-PHB-b-PMLA, and poly(trimethylene carbonate)-b-poly(-malic acid) (PTMC-b-PMLA) was established through the ring-opening polymerization (ROP) of the corresponding monomers followed by hydrogenolysis. A range of well-defined copolymers characterized by 1H, 13C{1H}, HSQC, HMBC, DOSY NMR spectroscopy, SEC, DSC, TGA, contact angle analyses, with tunable hydrophilic/hydrophobic balance were thus obtained through the precise control of the hydrophilic weight fraction f (11-82%). Tunable self-assembled systems were obtained by nanoprecipitation of the amphiphilic PHA-based copolymers without the use of a surfactant. Large aggregates and core-shell micelles (Rh = 16-335nm) were obtained depending on the polymer topology. PHB-based copolymers with f up to 50% formed highly stable micelles at 37 °C over a period of 10 days in aqueous solution. PMLA-b-PHB as well as PTMC-b-PMLA copolymers revealed no acute in-vitro cytotoxicity. The use of PHB as hydrophobic segment enabled to minimize the non-specific scavenging by macrophages cells while the cellular uptake by hepatocytes was favored.

The Gram-negative bacterium Pseudomonas aeruginosa is a life-threatening nosocomial pathogen due to its generally low susceptibility toward antibiotics. Furthermore, many strains have acquired resistance mechanisms requiring new antimicrobials with novel mechanisms to enhance treatment options. Proline-rich antimicrobial peptides, such as the apidaecin analog Api137, are highly efficient against various Enterobacteriaceae infections in mice, but less active against P. aeruginosa in vitro. Here, we extended our recent work by optimizing lead peptides Api755 (gu-OIORPVYOPRPRPPHPRL-OH; gu = N,N,N′,N′-tetramethylguanidino, O = L-ornithine) and Api760 (gu-OWORPVYOPRPRPPHPRL-OH) by incorporation of Ile-Orn- and Trp-Orn-motifs, respectively. Api795 (gu-O(IO)2RPVYOPRPRPPHPRL-OH) and Api794 (gu-O(WO)3RPVYOPRPRPPHPRL-OH) were highly active against P. aeruginosa with minimal inhibitory concentrations of 8–16 and 8–32 μg/mL against Escherichia coli and Klebsiella pneumoniae. Assessed using a quartz crystal microbalance, these peptides inserted into a membrane layer and the surface activity increased gradually from Api137, over Api795, to Api794. This mode of action was confirmed by transmission electron microscopy indicating some membrane damage only at the high peptide concentrations. Api794 and Api795 were highly stable against serum proteases (half-life times >5 h) and non-hemolytic to human erythrocytes at peptide concentrations of 0.6 g/L. At this concentration, Api795 reduced the cell viability of HeLa cells only slightly, whereas the IC50 of Api794 was 0.23 ± 0.09 g/L. Confocal fluorescence microscopy revealed no colocalization of 5(6)-carboxyfluorescein-labeled Api794 or Api795 with the mitochondria, excluding interactions with the mitochondrial membrane. Interestingly, Api795 was localized in endosomes, whereas Api794 was present in endosomes and the cytosol. This was verified using flow cytometry showing a 50% higher uptake of Api794 in HeLa cells compared with Api795. The uptake was reduced for both peptides by 50 and 80%, respectively, after inhibiting endocytotic uptake with dynasore. In summary, Api794 and Api795 were highly active against P. aeruginosa in vitro. Both peptides passed across the bacterial membrane efficiently, most likely then disturbing the ribosome assembly, and resulting in further intracellular damage. Api795 with its IOIO-motif, which was particularly active and only slightly toxic in vitro, appears to represent a promising third generation lead compound for the development of novel antibiotics against P. aeruginosa.

[Formulae and special characters can only be approximated here. Please see the pdf version of the Abstract for an accurate reproduction.] Several inert platinum complexes were synthesised: [(en)Pt([special character]-dpzm)2Pt(en)]4+, [{Pt(dien)}2[special character]-dpzm]4+, [{Pt(dien)}2[special character]-H2N-(CH2)6-NH2]4+, cis-[(NH3)2Pt([special character]--dpzm)2Pt(NH3)2]4+, trans-[Pt(NH3)2([special character]-dpzm)2]2+. Three active complexes, all with chloro ligands, were also synthesised: trans-[{Pt(NH3)Cl2}2[special character]-dpzm)], trans-[{Pt(NH3)2Cl}2[special character]-dpzm]2+ (di-Pt) and trans-[trans-{Pt(NH3)2Cl}2{trans-[Pt(NH3)2([special character]-dpzm)2]}]4+ (tri-Pt). 1H NMR established that multi-nuclear platinum complexes will preferentially associate in the DNA minor groove with a preference for A/T sequences, and with a binding constant [special character]-105 M-1, regardless of the charge, linking ligand, length or shape. Using [(en)Pt([special character]-dpzm)2Pt(en)]4+ and the oligonucleotide d(GC)5 it was determined that the metal complex binds G/C rich sequences also in the minor groove, but with a much reduced binding constant, 103 M-1. CD studies showed [(en)Pt([special character]-dpzm)2Pt(en)]4+ was able to induce a DNA conformation change from B-type to what appeared to be a partial Z-type. Transcription assays showed that even though the metal complex does not bind DNA covalently, it is still able to inhibit DNA transcription at particular sites. The complexes di-Pt, tri-Pt, [{Pt(dien)}2[special character]-dpzm]4+ and trans-[Pt(NH3)2([special character]-dpzm)2]2+ were tested for anti-cancer activity in the L1210 murine leukaemia cell line, and gave values of 3.8, 2.5, [special character]200 and 64 [special character]M respectively. In the cisplatin resistant line (L1210/DDP), trans-[Pt(NH3)2([special character]-dpzm)2]2+ showed an increase in activity with a drop to 32 [special character]M, while both di-Pt and tri-Pt showed decreases in activity to values of 8.8 and 3.6 [special character]M. In the human ovarian carcinoma 2008 cell line and its cisplatin resistant derivative C13[special character]5, both complexes showed good activity with values of 2.5 and 20.9 [special character]M respectively, but again both showed decreases in activity in the resistant line with values of 17.8 and 37.7 [special character]M respectively. To help explain the difference between activity of these complexes and the complexes BBR3464 and BBR3005, cell uptake and DNA interstrand cross-linking experiments were performed. The cell uptake studies showed that both di-Pt and tri-Pt are taken up by cells at very high levels, when administered at 100 [special character]M, thus indicating that the difference is unlikely to be due to large differences in cell uptake. The DNA interstrand cross-linking studies showed both complexes readily form interstrand adducts (50% interstrand cross-linking at 12 nM and 22 nM respectively, c.f cisplatin 3 [special character]M). These results suggest that the rigid nature of the dpzm linker may be affecting the DNA adducts formed, with more interstrand links being formed than BBR3464. Possibly, it is this that causes the large differences in cytotoxicity. The DNA binding of di-Pt and tri-Pt was examined with the nucleosides adenosine and guanosine and the dinucleotide d(GpG). Both complexes bound at the N7 of guanosine, but 2-fold slower than cisplatin. In addition, di-Pt bound at the N7 and either the N1 or N3 of adenosine, 7-fold slower than guanosine. Di-Pt forms a large variety of cross-links between two d(GpG) molecules, however it could not be established whether the 1,2-intrastrand adduct could be formed. Di-Pt, however, forms a 1,2-GG interstrand adduct with the oligonucleotide d(ATGCAT)2 resulting in a conformation change away from B-type DNA. The sugar pucker of the G3 nucleoside changes from 2[special character]-endo towards 3[special character]-endo, and the position of the nucleotide relative to the sugar changes from anti to syn. The ability of multi-nuclear platinum complexes to form covalent adducts in the DNA minor groove remains unclear. It appears that di-Pt can form up to 33% minor groove adducts with the oligonucleotide d(AT)5, but when added to the oligonucleotide d(GCCAAATTTCCG)2 no definite minor groove adducts are seen and the major adduct appears to be a 1,2-interstrand cross-link between the two A6's or between the G1 and G11. Finally, a study of the encapsulation of platinum complexes within cucurbit[7]uril (Q7) as a means of reducing drug toxicity was made. For complex A and di-Pt, encapsulation of the linker ligand occurred. The effect of Q7 on the rate of hydrolysis of di-Pt was at least a 3-fold reduction as compared to free di-Pt with guanosine. Studies with [{Pt(dien)}2[special character]-dpzm]4+/Q7 and the oligonucleotide d(CGCGAATTCGCG)2 showed that the metal complex could dissociate from the Q7 and associate with the oligonucleotide, where an equilibrium is achieved with 15 % of the metal complex bound to the oligonucleotide and 75 % encapsulated in Q7. Tests in the L1210 and L1210/DDP cancer cell lines showed that di-Pt/Q7 has almost the same activity compared to free di-Pt.