Enzyme responsive polymers preparation

While the previous section dealt with the structural elements that define enzyme-responsive polymers, here we will introduce different methods that can be used to integrate an enzyme-responsive functionality with a polymeric material. These methods are placed into three groups. The first method deals with the preparation of enzymatically degradable polymers, the second introduces strategies to incorporate enzyme-responsive linkers into the polymer and the third explores ways to prepare enzyme-responsive polymers enzymatically. 

The introduction of enzyme-sensitive cross-links in an otherwise nonenzyme-responsive polymer such as PEG is frequently used to prepare enzyme-responsive polymer hydrogels and polymer particles. Because of their versatility and natural predisposition as enzyme substrates, short peptide sequences are almost exclusively used as cross-linkers, although dex-tran has also been used (Klinger et al., 2012). They can be readily changed to respond to a variety of proteases such as matrix metalloproteinases,plasmin or trypsin (Lutolf et al., 2003a Yang et al, 2010). In most cases, the peptides have to be modified at the termini to introduce reactive groups that are able to react with the polymer. 

Enzyme-responsive polymer conjugates are typically block polymers of an artihdal polymer and a polypeptide. Three strategies have been explored to prepare polymer-polypeptide conjugates. Firstly, the artificial polymer (e.g., PEG/PEO) can be immobilised on a solid support and terminated with an amine group (Fig. 6.11). This makes direct SPPS of the polypeptide... 

Characterisation of the enzyme-responsive material prepared through the methods outlined above is essential not only to test the enzyme responsiveness of the polymer but also to characterise the material s overall performance under the conditions in which it will be used in its ultimate application. This section will provide a brief overview of standard and specialised techniques that have been employed for this purpose and covers mechanical, chemical, physical and biological properties as well as enzyme responsiveness. While we will discuss the reason for choosing particular techniques and explain their advantages and Umitations in the context of the analysis of enzyme-responsive materials, explanations of the working principles of the techniques will not be provided and the reader is referred to other specialised textbooks on this topic instead. 

By definition, ERMs are very well suited to bridge the gap between manmade materials and biological processes (see Table 6.1) (Ulijn, 2006 Zelzer et al., 2013). Responsiveness to enzymes provides not only a means of communication between the material and its biological environment, it also presents the possibility of designing materials that display reversible and dynamic responses to a stimulus. While reversibility is not an uncommon feature for smart polymers, a dynamic interaction wherein response of a material only persists in the presence of the stimulus or a cofactor is rare. Several reversible enzyme-responsive polymer systems have been prepared so far (Ku et al, 2011), but truly dynamic or fuelled polymer-based systems have yet to be developed. 

Xi Zhang and co-workers employed chitosan and adenosines -triphosphate (ATP) as building blocks to fabricate polymeric supra-amphiphiles based on electrostatic interactions, which can self-assemble to form spherical aggregates [73]. The spherical a egates inherit the phosphatase responsiveness of ATP. This enzyme-responsive system can be more biocompatible and block polymers are not needed in preparation, which makes it possible to fabricate the chitosan-based enz nne-responsive assemblies in a large-scale, cheap way. 

Directions for preparing a potentiometric biosensor for penicillin are provided in this experiment. The enzyme penicillinase is immobilized in a polyacrylamide polymer formed on the surface of a glass pH electrode. The electrode shows a linear response to penicillin G over a concentration range of 10 M to 10 M. 

Tor [7] developed a new method for the preparation of thin, uniform, self-mounted enzyme membrane, directly coating the surface of glass pH electrodes. The enzyme was dissolved in a solution containing synthetic prepolymers. The electrode was dipped in the solution, dried, and drained carefully. The backbone polymer was then cross-linked under controlled conditions to generate a thin enzyme membrane. The method was demonstrated and characterized by the determination of acetylcholine by an acetylcholine esterase electrode, urea by a urease electrode, and penicillin G by a penicillinase electrode. Linear response in a wide range of substrate concentrations and high storage and operational stability were recorded for all the enzymes tested. 

Polymers derived from natural sources such as proteins, DNA, and polyhy-droxyalkanoates are optically pure, making the biocatalysts responsible for their synthesis highly appealing for the preparation of chiral synthetic polymers. In recent years, enzymes have been explored successfully as catalysts for the preparation of polymers from natural or synthetic monomers. Moreover, the extraordinary enantioselectivity of lipases is exploited on an industrial scale for kinetic resolutions of secondary alcohols and amines, affording chiral intermediates for the pharmaceutical and agrochemical industry. It is therefore not surprising that more recent research has focused on the use of lipases for synthesis of chiral polymers from racemic monomers. 

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