Functionalized polymer nanocontainers for targeted drug delivery

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.