ABSTRACT
The demand to develop convergent technology platforms, such as biofunctional medical devices, is rapidly increasing. However, immobilization on medical device scaffolds, drying, sterilization, and rehydration of biomolecules may alter the three-dimensional folding of the effector molecule and may lead to the loss of function. In this report, we describe a method that allows the monitoring of the threedimensional structure of immobilized anti-Fas IgM on polyurethane, which may be used for the extracorporeal blood treatment to limit systemic inflammation in severely ill patients. We further report on
the stabilizing features of a nano-coating that efficiently protects immobilized proteins from stress-mediated damage. 7.1 IntroductionThe implantation of metals or polymers into living tissues requires biocompatibility and effective functionality. Today, implantable materials combined with biological effector molecules are developed to improve the biocompatibility of the product, achieve a prolonged lifetime of the product, and promote specific physiological reactions within the host. The efforts to combine biological materials with implant materials for use in patients have been hampered because biologicals, such as immobilized effector proteins, are denatured after sterilization, thus requiring expensive and time-consuming aseptic production steps. We, therefore, used a nano-coating technology for the three-dimensional stabilization of immobilized proteins that preserves the function of biologic-device combination products during and after standard terminal sterilization (Tscheliessnig et al., 2012). The most common sterilization procedures for the production of medical devices are β-or γ-irradiation and ethylene oxide (EtO) gas sterilization. Each of these procedures occurs in conjunction with high energy transfer and thus leads to modified protein folding attributable to loading shifts, redistribution of charges within the protein, breakdown of hydrogen bonds, and eventually disruption of covalent bonds within the protein (Drake et al., 1957; Gianfreda and Scarfi, 1991; Kapoor and Priyadarsini, 2001). Especially with β-and γ-irradiation, the presence of oxygen and water facilitates the formation of destructive oxygen radicals (Garrison et al., 1962), which may lead to undesirable chain reactions within the protein for prolonged periods of time (e.g., during the storage of terminally sterilized material). In the past, many efforts were made to identify ideal protein protectors. For example, the protective effects of sugars (e.g., trehalose and saccharose), sugar alcohols (e.g., mannitol), and proteins (e.g., albumin) are well known (Arakawa et al, 2007; Jain and Roy, 2009; Jorgensen et al., 2009). However, during freezing or lyophilization and reconstitution, sugars may result in unappreciated damage of the biomolecule (Han et al., 2007). Therefore, a sugar-free formulation, comprising small molecules such as amino acids in
combination with glycyrrhizic acid, was used to stabilize and protect immobilized biomolecules. This method presupposes that at least three prerequisites must be met to allow the irradiation of immobilized proteins without significant loss of function: (i) removal of water molecules, (ii) establishment of hydrogen bonds between co-solvent and protein, and (iii) avoidance of crystallization-mediated protein damage during dehydration/hydration. According to the concept of preferential exclusion (Timasheff et al., 1993; Arakawa et al., 2001), co-solvents that substitute for the water molecules, which normally constitute the hydrogen bonds in the hydrated form, were used in combination with glycosidic saponins (Fuchs et al., 2009). Glycyrrhizic acid, a member of glycosidic saponins, was selected because of its unique feature to form amorphous gels while chemically interacting with stabilizing amino acids. The amorphous nature of the dried films of the amino acid/glycyrrhizic acid solution on a suitable carrier material was shown by wide-angle X-ray diffraction (XRD). Thermal analysis of the frozen amino acid/glycyrrhizic acid solution by differential scanning calorimetry revealed two thermal transitions of the freeze-concentrated non-ice phase (Tg¢ is glass transitions of maximally freeze-concentrated phases): Tg1¢ at −60°C and Tg2¢ at −49°C, suggesting two different amorphous glass states in the frozen solution. The physical properties of the nano-coating enable the coating of any surface to reach homogenous thin films with variable thickness in the nanometer range (Tscheliessnig et al., 2012). This saponin-mediated glassy state in conjunction with selected amino acids proved to be highly protective for the immobilized proteins, possibly because of the high numbers of hydroxyl groups present in the glycoside part of the saponin. 7.2 Nano-CoatingFigure 7.1 schematically illustrates the mode of function of the nano-coating that fulfils the three aforementioned prerequisites. The stabilizing solution comprises a specifically selected mixture of small molecules, such as amino acids, combined with saponin-type glycosides. The solution covers the medical device surface material, including the immobilized proteins. During careful drying and removal of water molecules, the small-molecule co-solutes
subsequently interact with the protein, thereby maintaining its native conformation. At the end of the drying process, the glassy stabilizing and protective layer consists of a molecular film in the nanometer range that protects the functionality of the surface during sterilization by β-or γ-irradiation and EtO gas sterilization. After sterilization, the functionalized material can be applied to its intended use. Alternatively, because of the prolonged shelf life achieved by nano-coating, the materials can be used for long-term storage.
