Preparation Of Uricase-containing Coacervate Microdroplet Protocells And Their Application To Removal Of Uric Acid In Serum

Biological enzymes have many advantages such as low toxicity,specific recognition ability and high catalytic efficiency.They are also ideal medical antidotes.The loading of bio enzymes is especially expected to improve their catalytic efficiency and extend the shelf life of the enzymes.The key of the technology for the biological enzymes' immobilization lies in the research and development of new functional carrier materials.The coacervate microdroplets have good fluidity,excellent biocompatibility,high permeability and substrate enrichment capacity,and are easy to synthesize and modify.Based on the unique liquid-liquid phase separation synthesis method of primitive non-membrane cells in organisms,the agglomerated phase carrier can be used as a model of artificial membrane-free cells.In our work,coacervate is used to facilitate the synthesis and loading of the UOx,and the function of for the resultant UOx-coacervate hybrid particles for the biological detoxification is achieved through the degradation of uric acid in actual serum samples.The details are summarized as follows:(1)At present,various biological carrier materials have emerged endlessly,but most of which show certain biological toxicity.In Chapter 2,this paper first proposed the use of PDDA and CM-Dextran to construct coacervate with adjust size of condensed phase particles,and successfully realized the loading of urate oxidase(UOx).Anionic polymer(PDDA)and cationic polymer(CM-Dextran)were adopted as precursors to build coacervate microdroplet via electrostatic interactions.The coacervate microdroplet was prepared as a carrier and a membrane-free primitive artificial cell model.The zeta surface charge of the aggregate particles is affected by the proportion of the polymer precursor.When the weight ratio of PDDA and CM-Dextran was 0.27,the zeta potential of the coacervate carrier approached the isoelectric point.The coacervate microdroplet also displayed good adsorption performance,and showed a high internalization capacity for dye molecules such as Hochest.The coacervate kept stable at pH ranging from 7.4 to 10.3,or in sodium chloride solution at level up to 20 mg/mL.In the biotoxicity experiment,it was found that coacervate did not have obvious hemolysis at low concentration(6 mg/mL),and showed good biocompatibility.Finally,based on the simultaneous assembly and embedding method,a coacervate carrier loaded with UOx,namely theUOx-coacervate was successfully constructed,and the effective coating of UOx was achieved,thereby successfully realizing the biological fixation of urate oxidase.(2)Through the successful loading of UOx by the coacervate microdroplet in Chapter 2,the biological stability of UOx is significantly improved.The activity of UOx loaded in the agglomerate coacervate was slightly lower than that of pure UOx.However,it was advantageous in that in the presence of organic reagents(DMSO)or environment change(5 repeated freeze-thaw cycles),the activity of the loaded enzymes maintained.In addition,because of the adsorption of the uric acid on the coacervate microdroplet(K=108),the process of UOx catalyzed oxidation of uric acid mainly occurred inside the coacervates,which indicated that the aggregate particles could be used as a microreactor.The particles loaded with UOx were stable after being diluted 10 times with serum or left in the serum for 24 hours.This indicated that using the coacervate microdroplets to load the UOx for human metabolism could keep ideal function.Besides,when the loading amount was 50.0 ?g/mL,the uric acid concentration in serum decreased from 100% to 3% after 7.5 h of reaction,and the uric acid was eliminated after 24 h.The ideal effect of removing uric acid in serum was successfully achieved.