Matthias Hartlieb

Covalently cross-linked poly(2-oxazoline) materials for biomedical applications - from hydrogels to self-assembled and templated structures

Covalently cross-linked polymeric materials play an important role in life science. Hydrogels produced from multifunctional polymers can be utilized in numerous (bio)applications, such as drug delivery, tissue engineering and (bio)sensing. Also nano-/micro-scale assemblies benefit from a covalent linkage for instance to prevent premature disassembly or to generate a passive tissue specificity when used as a drug delivery agent. In both cases there is a need for biocompatible polymers with manifold (orthogonal) functionalization possibilities. By using the cationic ring-opening polymerization of 2-oxazolines it is possible to accomplish both tasks. In this review we summarize covalently cross-linked structures consisting of poly(2-oxazoline)s including three dimensional scaffolds, micellar systems as well as multilayer capsules. We focus on the cross-linking chemistry and the impact of the addressed systems regarding biological application.

Amine end-functionalized poly(2-ethyl-2-oxazoline) as promising coating material for antifouling applications

The antifouling behavior of different poly(2-ethyl-2-oxazoline) (PEtOx) coatings was investigated under real live conditions. Amine end-functionalized PEtOx of different molar masses have been prepared using a new and straightforward, two step synthesis method. Subsequently, the PEtOx were attached to glass surfaces via a tetraether lipid and a common silane, respectively. The polymers and coatings were characterized using techniques such as 1H NMR spectroscopy and MALDI-TOF-MS as well as XPS and contact angle measurements. In a next step, the coatings were exposed to the simultaneous attack of five different bacteria in synthetic river water. A clear reduction of the biofilm formation was observed. In addition, the stability of the coatings against thermal, mechanical, and chemical stress was studied.

Cationic poly(2-oxazoline) hydrogels for reversible DNA binding

A new 2-oxazoline monomer with a Boc-protected amino group in the side chain (BocOx) was synthesized. Homopolymerization as well as copolymerization with 2-ethyl-2-oxazoline (EtOx) revealed a pseudo first order kinetic. A series of homopolymers was synthesized, deprotected and characterized regarding their structure and thermal properties. The copolymerization with EtOx yielded a series of water soluble polymers with varying amino contents. After deprotection it was shown by the ethidium bromide assay that these polymers were able to form complexes with DNA. Treatment with epichlorohydrin leads to the formation of hydrogels. The swelling properties of the gels were investigated and it could be demonstrated that also the polymeric scaffolds were able to immobilize DNA from aqueous solution. Furthermore, the release of the DNA was accomplished using heparin.

Linear Poly(ethylene imine)-Based Hydrogels for Effective Binding and Release of DNA

A series of copolymers containing both amine groups of linear poly(ethylene imine) (LPEI) and double bonds of poly(2-(3-butenyl)-2-oxazoline) (PButEnOx) was prepared. To this end, a poly(2-ethyl-2-oxazoline) (PEtOx) precursor was hydrolyzed to the respective LPEI and functionalized in an amidation reaction with butenyl groups resulting in the double bond containing poly(2-(3-butenyl-2-oxazoline)-co-ethylene imine) (P(ButEnOx-co-EI)). Hydrogels were obtained by cross-linking with dithiols under UV-irradiation resulting in networks with different properties in dependence of the content of double bonds. The developed method allows the exact control of the amount of ethylene imine units within the copolymer and, thus, within the resulting hydrogels. The gel structures were characterized by solid state NMR and infrared spectroscopy. In addition the water uptake behavior from the liquid and the gas phase was investigated. It was shown by an ethidium bromide assay (EBA) that the copolymers and the respective hydrogels were able to bind and release DNA. Furthermore, the influence of the ethylene imine content on this interaction was investigated.

Matrix Supported Poly(2-oxazoline)-Based Hydrogels for DNA Catch and Release

We describe the synthesis of matrix supported hydrogel structures based on amine containing poly(2-oxazoline)s and their use to bind and release genetic material for potential applications in diagnostics or pathogen detection. Amine containing poly(2-oxazoline)s were synthesized by copolymerization of 2-ethyl-2-oxazoline with a monomer bearing a tert-butyl oxycarbonyl (Boc) protected amine group in the 2-position and subsequent deprotection. The statistical copolymers were used to generate hydrogels and matrix supported hydrogels by cross-linking of a certain fraction of the amine groups with epichlorhydrin. Supported structures were prepared by soaking porous polyethylene (PE) or polypropylene (PP) filter materials in a copolymer/epichlorhydrin solution, which was cross-linked upon heating. Scanning electron microscopy (SEM) of the composites revealed a bead like structure of the gel phase, which could be attributed to a lower critical solution temperature (LCST) behavior of the initial polymer prior to gelation. The dependency of the LCST behavior on the content of amine groups was investigated. Swelling values and the ratio of hydrogel per composite was determined using water sorption analysis. Subsequently, the ability of the systems to absorb and release labeled DNA was tested. Uptake and stimulated release, triggered by changes in pH, temperature, and heparin concentration, were investigated using fluorescence microscopy. Polymerase chain reaction (PCR) proved the successful recovery of the DNA, demonstrating the potential of the presented system for a broad range of molecular biological applications.

Core cross-linked nanogels based on the self-assembly of double hydrophilic poly(2-oxazoline) block copolymers

The synthesis of poly(2-oxazoline)-based block copolymers consisting of a cationic and a hydrophilic segment is described. The self-assembly of these macromolecules in organic solvents results in the formation of micelles and vesicles, respectively, depending on the solvent used. To transfer the systems into water, cross-linking using glutaraldehyde was applied, followed by the consumption of excessive aldehyde functions by either diethylamine or 6-aminofluorescein (6AF). The cross-linked assemblies were analyzed regarding their size and shape by electron microscopy and light scattering methods, as well as for their chemical composition by solid state NMR spectroscopy. 6AF associated samples were examined with respect to their absorption and fluorescence behavior in aqueous environment, revealing an alkaline microenvironment within the presented nanostructures. The toxicity of the systems against mouse fibroblast cell line L929 was examined by the XTT assay and was found to be insignificant for concentrations of up to 2.5 mg mL-1. Flow cytometry and fluorescence microscopy analysis revealed an efficient concentration and time dependent cellular uptake of the nanogels.

3rd generation poly(ethylene imine)s for gene delivery

Cationic polymers play a crucial role within the field of gene delivery offering the possibility to circumvent (biological) barriers in an elegant way. However, polymers are accompanied either by a high cytotoxicity or low efficiency. In this study, a series of high molar mass poly(2-oxazoline)-based copolymers was synthesized introducing 2-ethyl-2-oxazoline, ethylene imine, and primary amine bearing monomer units representing a new generation of poly(ethylene imine) (PEI). The potential of these modified PEIs as non-viral gene delivery agents was assessed and compared to linear PEI by studying the cytotoxicity, the polyplex characteristics, the transfection efficiency, and the cellular uptake using plasmid DNA (pDNA) as well as small interfering RNA (siRNA). High transfection efficiencies, even in serum containing media, were achieved using pDNA without revealing any cytotoxic effects on the cell viability at concentrations up to 1 mg mL-1. The delivery potential for siRNA was further investigated showing the importance of polymer composition for different genetic materials. To elucidate the origins for this superior performance, super-resolution and electron microscopy of transfected cells were used, identifying the endosomal release of the polymers as well as a reduced protein interaction as the main difference to PEI-based transfection processes. In this respect, the investigated copolymers represent remarkable alternatives as non-viral gene delivery agents.

Toward Anisotropic Hybrid Materials: Directional Crystallization of Amphiphilic Polyoxazoline-Based Triblock Terpolymers

We present the design and synthesis of a linear ABC triblock terpolymer for the bottom-up synthesis of anisotropic organic/inorganic hybrid materials: polyethylene-block-poly(2-(4-(tert-butoxycarbonyl)amino)butyl-2-oxazoline)-block-poly(2-iso-propyl-2-oxazoline) (PE-b-PBocAmOx-b-PiPrOx). The synthesis was realized via the covalent linkage of azide-functionalized polyethylene and alkyne functionalized poly(2-alkyl-2-oxazoline) (POx)-based diblock copolymers exploiting copper-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry. After purification of the resulting triblock terpolymer, the middle block was deprotected, resulting in a primary amine in the side chain. In the next step, solution self-assembly into core-shell-corona micelles in aqueous solution was investigated by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Subsequent directional crystallization of the corona-forming block, poly(2-iso-propyl-2-oxazoline), led to the formation of anisotropic superstructures as demonstrated by electron microscopy (SEM and TEM). We present hypotheses concerning the aggregation mechanism as well as first promising results regarding the selective loading of individual domains within such anisotropic nanostructures with metal nanoparticles (Au, Fe3O4).

Well-defined hyperstar copolymers based on a thiol–yne hyperbranched core and a poly(2-oxazoline) shell for biomedical applications

Well defined ‘hyperstar’ copolymers were synthesized by combining hyperbranched polymers produced by thiol–yne chemistry with poly(oxazoline)s. The hyperbranched core was prepared using an AB2 monomer and a trifunctional alkene, applying a monomer feeding approach. The degree of branching was high (0.9) while maintaining low dispersities (1.3). Poly(2-ethyl-2-oxazoline) (PEtOx) functionalized with a thiol end group was coupled to the surface of the hyperbranched structure accessing terminal alkyne units. PEtOx-SH was produced by the termination of the living polymerization with ethyl xanthate and subsequent conversion to thiol under alkaline conditions. The degree of polymerization was varied producing PEtOx with 23 or 42 repeating units, respectively with a dispersity of around 1.1. After conjugation of the polymer arms, hyperstar copolymers were characterized by SEC, NMR spectroscopy, light scattering, and AFM. The polymers were able to encapsulate the hydrophobic dye Nile red within the core of the structure with loading efficiencies between 0.3 and 0.9 wt%. Cytotoxicity of the hyperstars was assessed using A2780 human ovarian carcinoma cells resulting in IC50 values of around 0.7 mg ml−1. Successful internalization and colocalization with lysosomal compartments was observed by confocal microscopy studies.

Looped flow RAFT polymerization for multiblock copolymer synthesis

A looped flow process was designed for the synthesis of well-defined multiblock copolymers using reversible addition–fragmentation chain transfer (RAFT) polymerization. The reaction conditions were optimized to reach high conversions whilst maintaining a high end-group fidelity. The loop set-up proved to be a flexible, robust and time-efficient process for scaling-up multiblock copolymers.