Design and Synthesis of Responsive Nanocarriers Using Reversible Deactivation Radical Polymerization for Biomedical Applications

Tese de doutoramento em Engenharia Química, apresentada ao Departamento de Engenharia Química da Faculdade de Ciências e Tecnologia da Universidade de Coimbra The aim of this work was the development of well-defined co(polymers) based on stimuli-responsive segments to further use as building blocks in the development of nanocarriers for biomedical applications. The synthesis strategy involved the use of reversible deactivation radical polymerization (RDRP) methods, as well as “click” coupling reactions that led to the preparation of well-defined polymers, with controlled molecular weight, narrow molecular weight distributions, and well-defined chain-end functionalities. The homopolymerization studies of the 2-(diisopropylamino)ethyl methacrylate (DPA) by atom transfer radical polymerization (ATRP) involving an eco-friendly catalytic system that uses sodium dithionite (Na2S2O4) as a reducing agent and supplemental activator originated well-defined pH-responsive polymers with controlled properties. The detailed mechanism of such reaction system was investigated and the structure of the initiator, solvent, concentration of the catalyst, and the ratios of Na2S2O4 were adjusted to optimize the polymerization and afford polymers with narrow molecular weight distribution (Đ < 1.15) even at high monomer conversion (~ 90%). The slow and continuous feed of Na2S2O4 solution to the reaction mixture allowed the polymerization to be carried out in the presence of only 100 ppm of CuBr2 when the ligand tris(2-pyridylmethyl)-amine (TPMA) was used. This system was successfully extended to the polymerization of oligo(ethylene oxide) methyl ether methacrylate (OEOMA). The residual metal catalyst concentrations used and non-toxic nature of the Na2S2O4 make this SARA ATRP method very attractive for the well-controlled synthesis of water soluble polymers for biomedical applications. The high conversion and preservation of the chain-end functionality allowed the direct synthesis of POEOMA-b-PDPA block copolymers through one-pot polymerization approach. The pH-dependent self-assembly behavior of these brush-like copolymers in aqueous solutions was studied and the preliminary results suggested that the preparation method plays an important role on the final morphology of the nanoaggregates. Due to the pH critical value of the DPA block, these block copolymers form stable nanostructures at physiological pH, but disassemble at pH < 6.2. Copolymers composed by longer PDPA segments were found to originate larger self-assembled particles with critical micelle concentration (CMC) in the range of 1.0 x 10-3 mg.mL-1. Despite the high versatility of the ATRP method, it is not very efficient in the polymerization of the so called non-activated monomers, namely vinyl acetate (VAc) and N-vinyl caprolactam (NVCL). The reversible addition fragmentation chain transfer (RAFT) was proposed as an alternative RDRP method for the polymerization of such monomers and two new xanthates with alkyne functionalities were designed and synthesized. The kinetic studies revealed that the protected alkyne-terminated RAFT agent (PAT-X1) was able to conduct the RAFT polymerization of both VAc and NVCL in 1,4-dioxane at 60 °C, with a good control over the molecular weight and relatively narrow MW distributions (Đ < 1.4) up to high monomer conversions. The linear evolution of Mn,GPC with conversion as well as the close agreement between Mn,th and Mn,GPC values confirmed the controlled feature of the RAFT system. The poly(N-vinyl caprolactam) (PNVCL) is a temperature-responsive polymer and its solution behaviour was fully investigated under different conditions. The stringent control over the polymer molecular weight allows the development of PNVCL with tunable phase transition temperatures around 37 °C. The deprotection of the alkyne functionality of the polymers synthesized by RAFT, allowed a further copper catalyzed azide–alkyne [3+2] dipolar cycloaddition (CuAAC) to obtain new linear block copolymers. This “click” coupling reaction allowed the conjugation of the alkyne-terminated PNVCL synthesized by RAFT with and azide-terminated POEOMA synthesized by ATRP, originating POEOMA-b-PNVCL copolymers. Such block copolymers are hydrophilic but, due to the temperature responsive nature of the PNVCL segment, they become amphiphilic at temperatures above its low critical solution temperature (LCST) and self-assemble into spherical vesicular aggregates with narrow size distributions. A small drop in the solution temperature caused the disruption of the nanostructures and induced the fast release of nile red (NR), an hydrophobic small molecule used as a model drug. Moreover, the sharp and reversible solution properties of the PNVCL block turn those copolymers interesting candidates for the development of temperature-triggered drug delivery systems (DDS). The CuAAC coupling reaction was extended for the development of responsive polymers with linear-dendritic architectures. The synthesized linear pH-responsive and temperature-responsive polymers were conjugated to polyester dendritic structures based on the monomer 2,2-bis(hydroxymethyl) propionic acid (bis-MPA), functionalized with poly(ethylene glycol) (PEG) segments, to obtain linear dendritic block copolymers (LDBC). This thesis contributed to the development of methods that allow the synthesis of new block copolymers having stimuli-responsive segments, and intended to extend the application of these structures to the development of tailor made nanocarriers to be used as DDS for cancer therapy. FCT - SFRH/BD69635/2010