Fabrication Of Polymer-Protein Conjugates With Core-shell Structures And Their Applications In Biological&Medical Science

Nanosystems using proteins as the primary component commonly possess satisfied biocompatibility and degradability,low off-target effect,and highly refined functionalities,therefore they have broad applications in many biomedical research fields such as drug/gene vectors,protein therapeutics,biosensors and nanoreactors.At present,many kinds of protein-based nanomaterials with diverse structures have been developed to meet different demands in various biomedical fields.Synthetic polymers are considered as ideal materials for fabricating protein-based nanosystems due to their high controllability,and can be used for improving proteins’ functionality and stability.Thus,conjugates formed from the combination of polymers and proteins via some methods such as covalent connection have been studied considerably in recent years.In this dissertation,we employed proteins and polymerizable monomers as template and surface components respectively to fabricate a series of core-shell polymer-protein conjugates by means of polymer growth in situ and polymer grafting.The obtained conjugates were endowed with different core-shell relationships by monitoring the functionalities of protein core and polymer shell.Moreover,their applications in some biomedical fields like anti-cancer drug nanocarriers and biosignal sensors were also explored.There are three major sections in this dissertation,and the main research contents and conclusions are shown as follows:1.Fabrication of inert polymer-protein conjugates for long-circulating drug carriers in vivoExogenous proteins usually suffer from some limitations including rapid degradation and clearance in vivo.To address this problem,researchers commonly utilize hydrophilic polymers for surface modification of the proteins to improve their stabilities.In this work,by using gelatin nanoparticles(GNPs)and monomer N-vinyl-2-pyrrolidinone(NVP)as protein core template and outer polymeric component respectively,we encapsulated GNPs with protective poly(N-vinyl-2pyrrolidinone)(PVP)shell formed by in situ radical polymerization process,to obtain core-shell polymer-protein conjugates.Since both core and shell were composed of inert substances and simply combined with each other,their interactions were considered as being negligible.Here the anti-fouling PVP shell protected inner core from rapid clearance in vivo,thus enhanced its stability.On the other hand,internalization of GNPs by non-immune cells was difficult because GNPs’ essence was collagen partially hydrolysate.Moreover,the PVP shell around GNPs core as physical barrier further hindered its internalization.Compared with native GNPs,the conjugates possessed lowered non-specific protein adsorption ratio and prolonged blood retention time.However,being limited to its low cellular internalized efficiency,conjugates loaded with anti-cancer drug fluorouracil(5-FU)exhibited relatively poor cytotoxicity to caner cells.2.Fabrication of polymer-protein conjugates with functionalized shell for cancer therapyTo endow GNPs long-acting properties in vivo while improve its internalized efficiency by cancer cells,here we changed the polymeric shell of conjugates by functionalized modifications.Cationic gelatin nanoparticles(+)GNPs were firstly prepared by in situ radical polymerization process and used as core templates.Then it was decorated with anti-cancer drug doxorubicin(DOX)terminated poly(2-methylacryloyl oxyethyl phosphorylcholine)(DOX-pMPC)to make surface positive charge and obtain core-shell polymer-protein conjugates.The hydrophilic polymeric shell could inhibit non-specific protein adsorption effectively,thus prolong GNPs’ blood retention time.When the conjugates were enriched in tumor sites via passive targeting effect,the acyl hydrazone linkage between DOX and pMPC chain was inclined to be broken by hydrolysis,inducing the removal of polymeric shell and re-exposure of core’s positive charge.Cationic core loading with drug could be uptaken by cancer cells efficiently,thereby generating its therapeutic potency.By comparison to GNPs conjugated with drug directly,the polymer-protein conjugates exhibited significantly enhanced anti-cancer efficacy.3.Fabrication of dual-functionalized polymer-protein conjugates for enzyme biosignal sensingEnzyme catalytic metabolism is an important way for organism maintaining their normal functions.However,it is still lack of real-time testing method for the process of enzyme catalysis.In this work,we synthesized a cross-linkable molecule DAA-Flavin,which had similar structure with the cofactor of glucose oxidase(GOx).DAA-Flavin was then copolymerized with main monomer acrylamide(AAm)around GOx surface to fabricate core-shell GOx nanocapsules.The enzyme serving as core was encapsulated in the functional polymer shell,which protected the enzyme from denaturation and improved its environmental stability.Moreover,cofactor-like fluorescent molecules DAA-Flavin distributed in the polymer shell could act as mediators to accept electron given by the enzymatic active center,thus endowing the nanocapsules redox fluorescent property.In the presence of substrate,fluorescence intensity of the nanocapsules was attenuated gradually due to the continuous reduction of DAA-Flavin,and there was a highly linear correlation between the fluorescence decay rate and enzyme activity(R2～0.990).Furthermore,attenuated fluorescence intensity at reducing state could restore under oxidation condition,making the redox fluorescence reversible.The fabrication of this novel kind of core-shell enzyme nanocapsules enabled the conversion from biochemical signals to photoelectric signals,and it was expected to have prominent prosepect in the field of biosensors and biofuel cells.