Preparation And Properties Of Molecularly Imprinted Temperature-responsive Enzyme Inhibitors

Enzymes are involved in almost all biochemical reactions related to life activities and are an indispensable class of protein molecules for biochemical processes such as metabolism,signalling and cell regulation.In the body,endogenous enzyme inhibitors（EIs）self-regulate enzyme activity by specifically binding to and blocking the active site of the enzyme,not only to correct metabolic imbalances caused by enzyme overactivity or overexpression,but also to protect against pathogenic invasion.Inspired by the mechanism and function of natural EIs,the development of artificial enzyme inhibitors has received widespread attention due to their multiple advantages in the biomedical field.Molecularly imprinted polymers（MIPs）are one of the most promising alternatives to artificial enzyme inhibitors,which have high affinity and selective with low cost,easy preparation and storage.Although MIPs have made great progress as molecular recognition materials in the fields of separation and adsorption,the development of MIPs that mimic natural enzyme inhibition mechanisms for biomedical applications is still in its infancy.Currently,molecularly imprinted polymer nanoparticles（MIP-NPs）with a particle size of less than 200 nm and a homogeneous and controlled morphology have become an important research direction for the preparation and application of molecularly imprinted enzyme inhibitors.MIP-NPs have the advantages of good dispersion,high specific surface and easy elution of template molecules.However,nearly all the MIP-NPs prepared by conventional methods have rigid structures that can only recognize target molecules simply and mechanically and cannot actively respond to external stimuli.For this reason,stimulus-responsive MIP-NPs have been developed,which can be used to mimic the functions of biological molecules in vivo.In this thesis,the preparation methods and performance of different enzymes imprinted polymer nanoparticles and their inhibition mechanisms on the corresponding enzymes were investigated in depth from the perspective of molecularly imprinted enzyme inhibitors.This work will provide a basis for the further development and application of MIEIs in the field of trypsin-assisted cancer therapy and will be of great importance in expanding the way molecularly imprinted polymers are used for the slow/controlled release of drugs.Details of the research are as follows:（1）The preparation optimization of thermo-responsive lysozyme-imprinted nanoparticles and the study of their performanceIn this chapter,two kinds of thermo-responsive lysozyme-imprinted nanoparticles were prepared by precipitation polymerization and solid-phase imprinting as molecular imprinted enzyme inhibitors.The morphology,particle size and temperature-responsive binding capacity of the Ly-MIP-NPs prepared by the two methods were compared and investigated;the influence of different monomer ratios on the performance of the Ly-MIP-NPs prepared by solid-phase imprinting was discussed in detail to obtain the optimal ratios for the preparation of molecularly imprinted enzyme inhibitors.This paper provides theoretical support for the design and preparation of molecularly imprinted enzyme inhibitors.（2）Preparation of thermo-responsive lipase-imprinted inhibitors by solid-phase imprinting and their controlled drug release propertiesIn this chapter,Li-MIP-NPs were prepared by solid-phase imprinting using lipase as a template molecule,and their inhibitory effect on the activity of lipase was also investigated.Lipase can break ester bonds and release ibuprofen molecules grafted onto the surface of Si O₂nanoparticles via ester bonds.Li-MIP-NPs were added at different temperatures（37℃,25℃）to study the inhibition effect of Lipase activity and drug controlled release.In this study,we demonstrated the various forms of MIPs involved in controlled release of drugs,indicating that molecular imprinted enzyme inhibitors have great development potential as biomedical therapeutic agents.（3）Preparation of thermo-responsive trypsin-imprinted inhibitors by solid-phase imprinting and their manipulation of cell behaviorsThis chapter used solid-phase imprinting to prepare trypsin-imprinted nanoparticles and we also investigated their thermo-responsive binding ability and inhibitory effect on trypsin.The manipulation experiment of Tr-MIP-NPs on cell behavior was further designed.By controlling the binding temperature（37℃,25℃）,the inhibition of molecular imprinting enzyme inhibitors on the digestion performance of Trypsin and the regulation of the adhesion behavior of L929 cells were studied.This study shows very important utility in the treatment of cell damage depending on trypsin activity,and holds promise for the application of Tr-MIP-NPs to the regulation of pathological cells.