PhD Thesis
Polyelectrolyte Complexes and their Therapeutic Potential
Christopher V. Synatschke (01/2013-06/2013)
Support: Axel H. E. Müller
Summary
This thesis describes the preparation of polyelectrolyte nanostructures, the characterization of interpolyelectrolyte complexes (IPECs) made from these structures and their use in a therapeutic context. The therapeutic use of such IPECs connects the two major topics of this work: First, the delivery of genes into eukaryotic cells in vitro by means of new star-shaped polycations was explored. Second, the structure of ionic multicompartment micelles (MCMs) when complexed with polyions was studied and the performance of these nanostructures as delivery agents of anti-cancer drugs both in vitro and in vivo was tested. To better understand structure-property relationships of polycations relevant for gene delivery, a library of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) homopolymers was synthesized. Star-shaped polymers with a different number of arms and molecular weights were created by using sugar-based or inorganic nanoparticles as core molecules with varying number of initiation sites. The cytotoxicity as well as transfection performance of polyplexes from these polymers and plasmid DNA was determined for different PDMAEMA-nitrogen/DNA phosphate ratios in CHO-K1 cells. A decrease of the cytotoxicity of polymers with a given molecular weight was observed with increasing degree of branching, i.e., with increased arm-number. Star-shaped PDMAEMA with roughly 20 arms (Si-PDMAEMA) showed exceptionally high transfection efficiency. The superior transfection behavior of this specific polymer was demonstrated in non-dividing or differentiated (C2C12 and human T lymphocytes) cell lines. Additionally, polymeric micelles were produced from a polybutadiene-block-PDMAEMA diblock copolymer in aqueous solution and subsequently used for gene transfection. Their transfection efficiency was in the same range as that of Si-PDMAEMA, hinting towards a general design principle for highly effective gene vectors. This consists of a star-shaped architecture of PDMAEMA chains emanating from a common center. The second major part of this thesis deals with the structure of ionic MCMs complexed with diverse polycations as well as the drug delivery capabilities of some of these complex micellar structures. MCMs from polybutadiene-block-poly(1-methyl-2-vinyl pyridinium)-block-poly(methacrylic acid) (BVqMAA) triblock terpolymers were used as the basis for further structural modifications. These micelles exhibit a core-shell-corona morphology, where PB forms the core of the micelles, a discontinuous (patchy) shell consisting of an IPEC between P2VPq and PMAA is present and finally a corona of excess PMAA stabilizes the micelles in aqueous solution. At sufficiently high pH a portion of the corona carries negative charges, which were then used to form further IPECs with either cationic homopolymers or double-hydrophilic block copolymers featuring one positively charged block. If polycations other than poly(2-vinyl pyridine) such as quaternized PDMAEMA were used for the complexation, a new and distinguishable IPEC compartment was formed on top of the already existing P2VPq/PMAA IPEC. In the case of MCMs with a short to moderate block length of the corona, i.e., the degree of polymerization (DP) of PMAA was between 345 to 550 units, a layered arrangement of the newly formed IPEC compartment was found. For BVqMAA micelles with a long PMAA corona (DP of PMAA = 1350) complexed with different quaternized homopolymers, a patchy arrangement of the newly formed IPEC compartment instead of a layered one was found. The formation of this new structure is due to an interfacial energy minimization between the new IPEC and the compartmentalized core of the micelles that became possible due to the exceptionally long PMAA corona of the precursor micelles. Finally, MCMs from BVqMAA were tested for their capacity to deliver a hydrophobic anti-cancer drug for photodynamic therapy to lung cancer cells in vitro and in vivo. The influence of the corona composition was studied by forming complexes with a double-hydrophilic diblock copolymer, poly(L-lysine)-block-poly(ethylene glycol) (PLL-b-PEG). A new cylinder-on-sphere morphology was observed in electron microscopy for BVqMAA/PLL-b-PEG complex micelles at high complexation ratios between lysine and MAA units. Highest cytotoxicity and uptake were found for pure BVqMAA micelles, both decreasing with increasing amount of PLL-b-PEG attached to the micelles. In mice, a prolonged blood circulation time in the range of several hours was exclusively observed when fully PEGylated micelles were injected. Those micelles were the only ones showing an enhanced accumulation in a subcutaneous tumor model 24 h after intravenous injection. The amount of drug delivered to the tumor tissue by the micelles suppressed tumor growth for up to 21 days after a single dose injection and photoirradiation step. The potential of complex micelles on the basis of BVqMAA MCMs could thus be proven.