Functionalized polymer nanocontainers for targeted drug delivery

Abstract

Self-assembling amphiphilic ABA triblock-copolymers, forming vesicular structures in aqueous solutions were the core material used in this thesis. The interest in amphiphilic blockcopolymers resides basically in their improved characteristics in comparison to low molecular weight amphiphiles, such as higher mechanical stability. Moreover, the polymer brush coating of liposome-polymer hybrids (Stealth liposomes) is known to be effective on reducing uptake by reticuloendothelial system (RES). This led to the idea of avoiding the use of lipids at all in these structures and work with self-assembling polymers. This has been done in our group through the use of an ABA amphiphilic triblock-copolymer, consisting of poly(2-methyl-2-oxazoline)-bpoly( dimethylsiloxane)-b-poly(2-methyl-2-oxazoline) (PMOXA-PDMS-PMOXA), which surface contains a hydrophilic polymer with similar characteristics to poly(ethylene glycol). These two advantages of amphiphilic polymer vesicles, i.e. higher stability and the ability to avoid reticuloendothelial uptake led to two main research interests which were addressed in this work: the use of nanocarriers based on synthetic self-assembling polymers as active targeting drug delivery systems and the immobilization of vesicles on surfaces. Both research topics shared the need to attach the polymer vesicles via docking sites. To address this a series of ABA triblock-copolymers were synthesized and functionalized to promote their interaction either with other molecules or with surfaces. The synthesis of the polymers was carried out via cationic ring opening polymerization of 2-methyl-2-oxazoline departing from a telechelic poly(dimethylsiloxane). Langmuir films proved the surface activity of the amphiphilic polymers obtained. From the functionalization point of view, biotinylated blockcopolymers proved to be the most versatile modification, through the use of the wide spread biotin-streptavidin specific interaction. Vesicles were prepared by already stablished methods (solvent injection extrusion method) and new developed ones (direct dissolution), and were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), and fluorescence correlation spectroscopy (FCS). Moreover, the density of the vesicles was determined by ultracentrifugation techniques. To obtain active targeting drug-delivery systems, ligands were introduced onto the nanocontainer surface via coupling with streptavidin to the biotin moieties present on the nanocontainers surface. In vitro studies were performed to investigate the interactions of these ligand-functionalized nanocontainers with cells, in which uptake was followed via fluorescence microscopy. The selectivity of the interaction was also investigated with mixed cell cultures. Only ligand-functionalized nanocontainers were able of specific and selective targeting of receptor expressing cells. Absence of ligand resulted in no uptake by cells expressing targeting receptors, supporting thus the stealth characteristics of the polymer brushc constituting the nanocontainer wall. Therefore, specific type of receptor targeting can be concluded for ligandfunctionalized nanocontainers. Moreover, no cytotoxicity was observed for these artificial vesicles in preliminary studies. In order to follow the pathway of the nanocontainers into the cells, gold nanoparticle encapsulation was tested. Neither pre-formed nor in situ formation of gold nanoparticles rendered gold encapsulation in polymer vesicles. Another approach was the use of a fluorescently labeled block-copolymer, for which the hydroxyl end groups of the triblock-copolymer were coupled covalently with a fluorescent molecule. However, for detection purposes encapsulation of fluorescent dyes was sufficient and proved more flexible in terms of concentration range and choice of dye. To render surface-immobilized polymer vesicles, biotinylated polymers were used. Their adsorption onto a streptavidin decorated surface was studied with a quartz crystal microbalance with dissipation (QCM-D). The results were not conclusive since an unexpected adsorption was observed for vesicles without biotin moieties. This adsorption could be ascribed to unspecific polymer-protein interactions. Other hydrophobic blocks with low Tg, such as poly(propylene oxide) and poly(tetrahydrofuran) were investigated as replacements for poly(dimethylsiloxane) in poly(2- methyl-2-oxazoline)-b-poly(dimethylsiloxane)-b-poly(2-methyl-2-oxazoline) block-copolymers. Their surface activity was studied with Langmuir films, which rendered typical isotherms. Their aggregation was assessed by transmission electron microscopy (TEM) and dynamic light scattering (DLS), mainly finding spherical aggregates that can be ascribed as vesicular structures. Finally, these nanocontainers were studied as vehicles to entrap small solutes. By insertion of membrane proteins, the permeability characteristics of the nanocontainers can be improved. Langmuir film techniques showed protein-polymer interaction, which can be interpreted as insertion in the polymer membrane. Insertion was further confirmed by entrapment of small solutes in the cavity of the nanocontainers by forming a complex with an already encapsulated counterion. This post-encapsulation approach results interesting as a recovery system, in which a substance could be easily removed from a complex matrix system, allowing the concentration of the species of interest.