Design of polymer /ca Co3 hybrid particles via the supercritical cacoon dioxyde technology for use in regenerative

par Zeynep Yilmaz (Ergül)

Thèse de doctorat en Biomolécules, pharmacologie, thérapeuthique

Sous la direction de Christine Jérôme et de Frank Boury.

Thèses en préparation à Angers en cotutelle avec l'Université de Liège (BELGIQUE) , dans le cadre de École doctorale 502 Biologie-Santé (Nantes-Angers) , en partenariat avec Micro et nanomédecines biomimétiques (UMR_S 1066) (laboratoire) depuis le 10-01-2013 .


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  • Résumé

    In the frame of this work, we investigated the synthesis of polyphosphoester-based copolymers and explored their potential for the development of both organic and inorganic drug delivery systems. A great advantage of polyphosphoesters is the possible functionalization of the side-chains due to the pentavalency of the phosphorus atom, allowing to introduce some pendant groups for modifying their physical and chemical properties. In this perspective, we introduced different types of unsaturations like butenyl, butynyl, and allyl groups on the cyclic phospholane monomers and polymerized them in order to allow further derivatizations and introduce functional moieties along the PPE backbone. In order to avoid the contamination of final products by metallic residues, we favored the use of organocatalysts for the synthesis of the defined PPEs (co)polymers by ring opening polymerization. After optimization of the homopolymerization and of the statistical copolymer of various cyclic phospholane monomers, we synthesized a series of PPE block copolymers with either alkyl alkene or alkyne moieties by ring opening polymerization of functional cyclic phospholane monomers initiated from PEO-OH monomethyl ether. Post-polymerization modifications of the resulting PEO-b-PPE with pendant alkene and alkyne groups produced two types of well-defined double hydrophilic block copolymers. First, mercaptopropionic acid was added by a photochemical thiol–yne click reaction onto the pendant alkyne groups of the PEO-b-polybutynyl phosphate copolymer (PEO-b-PBYP). The accordingly prepared PPE-based copolymers (PEO-b-PBYPCOOH) exhibit carboxylic groups as side-chains which are known for their high capacity to complex calcium ions. On the other hand, deprotection of the pendant allyl groups of the PPE-b-poly(allyl phospholane) copolymer (PEO-b-PAllP) by nucleophilic substitution gave access to the negatively charged polyphosphodiesters (PEO-b-PPDO-). While the newly formed copolymers have been used for a specific purpose in the field of drug delivery mentioned below, the latter represent a very versatile platform for many other applications. Indeed, many other type of functions could be added to the PPE backbone via their pendant unsaturation. Moreover, only one type of “click” reaction was demonstrated in this work but other grafting reactions could be envisioned such as thiol-ene radical addition or azide-alkyne Huisgen cycloaddition. In other words, the scope of this copolymer could be improved in the future and several type of reactions could be applied to this PPE segment in order to bring the expected functionalities. Next, PEO-b-PPE copolymers with various densities of pendant butenyl groups grafted on to PPE backbone were used for developing organic drug delivery systems. The density of pendant unsaturated functions was adjusted by copolymerizing the alkene bearing cyclic phospholane with the isobutylphosphoester (iBP). Then, pendant double bonds of the amphiphilic copolymers were cross-linked by UV irradiation and doxorubicin was loaded in the core cross-linked micelles by impregnation. For the sake of the comparison, doxorubicin was encapsulated by nanoprecipitation in the corresponding non-cross-linked micelles. As a rule the loading capacities of the core cross-linked micelles are superior to the ones of the non-cross-linked micelles, except when the polyphosphoester block is exclusively constituted of polyBP. In the last part of the manuscript, novel double hydrophilic block copolymers, i.e. PEO-b-PBYPCOOH and PEO-b-PPDO-, were used as templating agents of CaCO3 particles, a common type of inorganic DDS. Both pendant carboxylic groups and negatively charged oxygen pendant on the backbone have been used to interact with calcium for directing the CaCO3 nucleation process and control the morphology of the particles. A classical chemical pathway and a process based on the supercritical carbon dioxide (scCO2) technology were successfully applied to formulate CaCO3 particles in the presence of copolymers used as templating agents. As a reference, we also formulated particles with hyaluronic acid (HA). Whatever the process used led to the spherical CaCO3 particles in the vaterite form in the presence of the charged PPE-based copolymers. However, scCO2 formulation process provided less aggregated particles. CaCO3 particles exhibited low size dispersity and smaller size when PEO-b-PBYPCOOH copolymer was used as a templating agent. It is worth noting that it is the smallest vaterite particles ever formulated in scCO2. The internal structure of the particles was also proved porous with an internal cavity in their center, which is of particular interest for encapsulation of biomolecules and delivery applications. This was a strong incentive for us to investigate the protein loading of the accordingly prepared particles. Lysozyme was used as a model protein. The size of the lysozyme loaded particles formulated with PEO-b-PBYPCOOH was almost unchanged compared to their corresponding counterparts, remained spherical and obtained in the vaterite polymorph. However, a significant decrease in size was observed upon loading the HA-templated particles probably due to the competition of the lysozyme with the HA at the surface of the CaCO3 particles. The active protein loading of CaCO3 particles templated with the PEO-b-PBYPCOOH was almost found twice higher than with the HA. This observation supports the hypothesis that our PPE-based templating agents induce a better resistance of the protein towards denaturation as compared to the traditionally used HA. Confocal microscopy images evidenced the encapsulation and core–shell distribution of lysozyme into CaCO3. The released profile reached a steady state at 59 % of release after 90 mins.