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In this work, we report the synthesis of [Ru(phen)3 2+]-based complexes and their use as photosensitizers for photodynamic therapy (PDT), a treatment of pathological conditions based on the photoactivation of bioactive compounds, which are not harmful in the absence of light irradiation. Of these complexes, Ru-PhenISA and Ru-PhenAN are polymer conjugates containing less than 5%, (on a molar basis), photoactive units. Their performance is compared with that of a small [Ru(phen)3 2+] compound, [Ru(phen)2BAP](OTf)2 (BAP = 4-(4′-aminobutyl)-1,10-phenanthroline, OTf = triflate anion), used as a model of the photoactive units. The polymer ligands, PhenISA and PhenAN, are polyamidoamines with different acid-base properties. At physiological pH, the former is zwitterionic, the latter moderately cationic, and both intrinsically cytocompatible. The photophysical characterizations show that the complexation to macromolecules does not hamper the Ru(phen)3 2+ ability to generate toxic singlet oxygen upon irradiation, and phosphorescence lifetimes and quantum yields are similar in all cases. All three compounds are internalized by HeLa cells and can induce cell death upon visible light irradiation. However, their relative PDT efficiency is different: the zwitterionic PhenISA endowed with the Ru-complex lowers the PDT efficiency of the free complex, while conversely, the cationic PhenAN boosts it. Flow cytometry demonstrates that the uptake efficiency of the three agents reflects the observed differences in PDT efficacy. Additionally, intracellular localization studies show that while [Ru(phen)2BAP](OTf)2 remains confined in vesicular structures, Ru-PhenISA localization is hard to determine due to the very low uptake efficiency. Very interestingly, instead, the cationic Ru-PhenAN accumulates inside the nucleus in all treated cells. Overall, the results indicate that the complexation of [Ru(phen)2BAP](OTf)2 with a cationic polyamidoamine to give the Ru-PhenAN complex is an excellent strategy to increase the Ru-complex cell uptake and, additionally, to achieve accumulation at the nuclear level. These unique features together make this compound an excellent photosensitizer with very high PDT efficiency.

A synthetic strategy was established for decorating and stabilizing superparamagnetic iron oxide nanoparticles (SPIONs) with a zwitterionic linear polyamidoamine (PAA). The strategy was successfully tested with a PAA coded ISA23 previously found endowed with interesting biological properties, such as biocompatibility, degradability in aqueous media and stealth-like properties when injected in test animals. A post-synthetic functionalization with catechol-bearing moieties of a preformed PAA was successfully carried out. ISA23 was obtained by polyaddition reactions of methyl-piperazine and 2,2-bis(acrylamidoacetic) acid. It was functionalized using nitrodopamine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as coupling agent, to randomly form amide bonds with 17% of ISA23 carboxylic groups (ISA23-ND). SPIONs were prepared by a thermal decomposition synthesis in 1-octadecene with oleic acid, and then transferred in water by two distinct ligand exchange procedures: i) the direct displacement of oleate molecules from SPION surface by ISA23 in a biphasic (n-hexane/water) environment; ii) the two-step method involving an intermediate small molecule, tetramethylammonium hydroxide, used as a transient transfer agent, which was in turn exchanged with ISA23-ND in a second exchange step occurring in water. The two-step procedure provided a SPION@PAA nanocomposite more stable than that obtained by the one-step procedure in the presence of an applied external magnetic field. ATR-FTIR spectroscopy, ζ-potential and thermogravimetric analysis (TGA) showed the presence of the ISA23 on the SPION surface. In particular, TGA showed that the ISA23-ND amount on the NPs accounted for 26% of the overall nanocomposite mass. The nanocomposite size was determined by both TEM (21.1±2.9 nm) and DLS measurements (hydrodynamic size 100±28 nm). SPION@ISA23-ND were re-suspended after lyophilization reverting to their pristine dimensions. The SPION@ISA23-ND adsorption of BSA in water, considered as the first stage of phagocytosis, was very low, suggesting that ISA23 could impart stealthiness to SPION@ISA23-ND. 1H-NMR relaxivity measurements showed an r2 value of 158 s-1 mmol-1 L (vs 100 s-1 mmol-1L for Endorem®) at relevant clinical fields for magnetic resonance imaging (from 0.2 to 1.5 T). SPION@ISA23-ND was tested on HeLa cells and their internalization was visualized by reflectance microscopy. Finally, with the aim of prepare a new dual magneto-optical system, a synthetic procedure to decorate SPION@ISA23-ND with a fluorescent dye was devised, even though the emission intensity of the resultant conjugate was lower than expected, possibly due to luminescence quenching caused by the closeness of emitting moieties to the SPION surface.

This thesis deals with the design of injectable hydrogels that can be used in minimally invasive surgery, such as endoscopic submucosal dissection (ESD), percutaneous hernia repair or fistulas closure.Polyamidoamines (PAAm) constitute a class of hydrogel of special interest for these purposes. After studying the different factors that affect their properties, we show that it is also possible to obtain PAAM-based microgels for applications in drug delivery or cell encapsulation.It is possible to synthesize redox-responsive nanocomposite degradable PAAm that can be injected into the submucosa of the stomach to facilitate the ESD.We show that hybrid alginate/PAAm hydrogels can be used for the percutaneous treatment of direct inguinal hernia and hydrogel-based creams have been prepared for use in fistulas closure. The last chapter of the thesis is devoted to the development of a new crosslinking strategy for hyaluronic acid in cosmetic surgery., Cette thèse porte sur la conception d'hydrogels injectables pouvant être utilisés en chirurgie mini-invasive, par exemple en dissection endoscopique sous-muqueuse (ESD) ou en réparation de hernie.Les polyamidoamines (PAAm) constituent une classe d'hydrogel intéressante à ces fins. Après avoir étudié les différents facteurs qui affectent leurs propriétés, nous montrons qu'il est également possible d'obtenir des microgels à base de PAAm pour la délivrance de médicaments ou l'encapsulation de cellules. Il est possible de synthétiser des PAAm dégradables qui peuvent être injectés dans la sous-muqueuse de l'estomac pour la ESD.Nous avons montré que les hydrogels hybrides alginate / PAAm peuvent être utilisés pour le traitement percutané de la hernie inguinale directe et des crèmes à base d'hydrogel ont été préparées pour être utilisées pour le colmatage des fistules. Le dernier chapitre de la thèse est consacré à la réticulation de l'acide hyaluronique pour la chirurgie esthétique.

The pH-responsive poly(amidoamine)s (PAAs) have been previously described. Whereas ISA23 enhances transfection in vitro and ISA1 promotes the cytosolic delivery of the non-permeant toxins this process shows poor efficiency. The aim of this study was to prepare and evaluate PAA conjugates containing the membrane disrupting peptide melittin (MLT). It was hypothesised that PAA conjugation would reduce the haemolytic activity of MLT at pH 7.4, however, upon delivery to tumours by the EPR effect, the polymer would uncoil in an acidic environment exposing MLT and allowing it to interact with membranes. PAA-MLT conjugates were prepared using MLT as a comonomer together with bis-acryloylpiperazine, 2-methylpiperazine and bis-hydroxyethylethylenediamine (ISA1-like), or bis-acrylamidoacetic acid and 2-methylpiperazine (ISA23-like). The melittin content of the conjugates was 6-19% (w/w). Although ISA1-MLT improved gelonin delivery compared to the parent polymer ISA1 (alpha 13-fold increase) and showed pH-dependent haemolytic activity at a polymer concentration of 0.05 mg/ml, this conjugate also displayed high haemolytic activity at pH 7.4. In contrast, ISA23-MLT like the parent compound ISA23 did not deliver gelonin. However, this conjugate could have potential as a novel polymeric anticancer conjugate due to its lack of haemolytic activity at pH 7.4 and retention of cytotoxicity. (c) 2005 Elsevier B.V. All rights reserved.