Charged hydrogels for post-loading, release, and molecular imprinting of proteins

Abstract

Molecular imprinting is a technique to create template-shaped cavities in polymer matrices with memory of the template molecules, to be used in molecular recognition. Molecular imprinting of low molecular weight compounds is a well established technique used to create high affinity materials. On the other hand, molecular imprinting of high molecular weight compounds such as proteins is not nearly as successful for various reasons. Nevertheless, the use of hydrogels has led to some encouraging protein imprinting results. A hydrogel consists of a three dimensional polymer network that is able to retain large amounts of water with conservation of the network structure. As a result of the high water content, hydrogels are generally considered as biocompatible materials, which makes them particularly interesting for biomedical and pharmaceutical applications. In this thesis, an innovative method to prepare hydrogel nanoparticles with protein imprints on their surface is proposed. Polyacrylamide nanoparticles were prepared inside the internal aqueous compartment of liposomes, prepared by detergent removal. Macroscopic hydrogel formation due to polymerization of the monomers outside the liposomes was prevented by inhibition of free-radical polymerization of non-encapsulated monomers by addition of ascorbic acid. This method offers straightforward control over the shape and size of the particles. Moreover, the liposomal bilayer offers the possibility to incorporate membrane proteins, which can then be used as templates for surface imprinting. It is shown that SN/GpA is successfully incorporated in the liposomal bilayer. Importantly, the hydrophilic domain of SN/GpA was oriented towards the internal compartment of the liposome, and therefore would be available for the formation of imprints. However, despite many attempts towards the synthesis of PINAPLES, a proof of concept was not obtained. This is partially due to the current state of the art of protein imprinting. Protein imprinting is still a relatively new scientific field. Although the number of research papers that describe protein imprinting is increasing, the actual scientific proof of the feasibility of selective recognition of proteins by molecular imprinted polymers in aqueous environment is not convincing. Electrostatic interaction between protein and hydrogel network can cause high aspecific binding, and may therefore not be suited for molecular imprinting. However, this strong interaction may be useful as a tool for controlled release of therapeutic proteins from pharmaceutical formulations. Charged hydrogels were obtained by copolymerization of methacrylated dextran (Dex-MA) with either methacrylic acid (MA) or 2-N,N-dimethylaminoethyl methacrylate (DMAEMA). The effect of incorporation of the charged monomers on hydrogel charge, equilibrium swelling and release of model proteins was assessed both at low (10 mM HEPES) and physiological ionic strength (HEPES buffered saline, HBS). It was concluded that incorporation of charge in a hydrogel network can be used for the immobilization of proteins and triggered release by increasing ionic strength. Next, it was shown that these reversible charge interactions can be exploited for efficient post-loading and release of proteins that are negative, positive or neutral at physiological pH. Importantly, as opposed to conventional loading during preparation of the hydrogel, no chemical modification to the protein occurred using this loading method.