Preparation Of Nucleoside-based Polymeric Nanoparticles And Evaluation Of Their Affinity To Endotoxins

Endotoxin is an exogenous pyrogen.Endotoxin entering the body can easily lead to symptoms such as inflammation and fever,which can endanger life in severe cases.Therefore,it is necessary to detect and remove endotoxin in environment,food,medicine and other fields.A good affinity reagent is essential for the detection and removal of endotoxin.As an affinity reagent with high specific surface area and high adsorption capacity,polymer nanoparticles are widely used in the detection and removal of biomolecules.Their superior performance depends on the functional monomers in the side chain structure.Nucleic acid aptamer has been used in the detection of endotoxin as an affinity probe with high adsorption capacity and high specificity.Therefore,this project aims to simulate the affinity between aptamer and endotoxin to prepare a polymer nanoparticle with nucleoside as the functional monomer as the affinity reagent for detecting and removing endotoxin.The main research results are as follows:1.Five 5'-acrylacyl-nucleoside monomers were successfully synthesized by acylation of five natural nucleosides using lipase as catalyst.Respectively,5'-propylene acylthymidine nucleoside(5'-AT),5'-propylene acyl-nucleoside uracil(5'-AU),5'-propylene acyl cytosine nucleoside(5'-AC),5'-propylene acyl-adenine nucleoside(5'-AA)and 5'-propylene acyl-guanine nucleoside(5'-AG).The key to successful synthesis is to control the anhydrous and anaerobic reaction conditions strictly,and the highest yield was obtained by using anhydrous THF or 1,4-dioxane as the reaction solvent.Controlling the droplet acceleration,temperature(60-65°C)and reaction time(24-48 h)can maintain the enzyme activity to the maximum extent and reduce the generation of by-products.2.Using 5'-acrylacylnucleoside,isopropylacrylamide(NIPAm)and tertbutylacrylamide(TBAm)as functional monomers,a library containing more than 100 nucleoside NPs was synthesized by free radical polymerization by adjusting the types and ratio of various functional monomers,including 5'-AT-NPs,5'-AA-NPs,5'-AC-NPs,5'-AG-NPs and 5'-AU-NPs five series.According to the characteristics of nucleoside monomers,the functional monomers,initiators and emulsifiers in the reaction system were optimized.The results showed that the 5'-acrylylnucleoside monomer was hydrophilic,and when it was combined with the hydrophobic monomer TBAm,the nucleoside NPs had good properties.These five nucleosides monomers are all negatively charged,so it is suitable to choose anionic surfactant SDS as dispersant,otherwise some nucleoside NPs will agglomerate.The yield of NPs is related to the type and concentration of initiator,reaction temperature and reaction time.Excessive initiation efficiency may lead to excessive polymerization of NPs and agglomeration,while the concentration of nanoparticles in solution is very low.For example,this problem existed in the synthesis of5'-AA-NPs using APS as initiator.The optimal synthesis conditions are as follows: total concentration of monomer 16.25 mmol,initiator AIBN 1.2 mg/m L,dispersant SDS 0.4mg/m L,temperature 65°C,anaerobic reaction for 3 h.The physical and chemical properties of the synthesized nucleoside NPs were characterized.The results showed that the particle size,monodispersion and Zeta potential were not affected by the buffer p H.Among them,the yield of 5'-AT-NPs,5'-AA-NPs and 5'-AU-NPs were high,the particle size was uniform,and the Zeta potential was stable below-20 m V.3.A high throughput screening method for bionic affinity ligands of endotoxin based on fluorescence quantitative detection was established,and the affinity screening of the synthesized NPs library was performed.The results showed that the adsorption of endotoxin on the NPs synthesized with TBAm and nucleoside as functional monomers was positively proportional to the ratio of nucleoside monomers,indicating that the hydrogen bonding provided by nucleoside monomers played a key role in the binding of endotoxin to polymer nanoparticles.The optimized NPs were 5'-AT-NP 21(40% 5'-AT,58% TABm,2% BIS),which not only had a high affinity for endotoxin,but also had a uniform particle size and high yield.The results showed that the p H value of buffer solution and PBS concentration had no significant effect on the affinity effect of 5'-AT-NP 21,indicating that nucleoside NPs were not sensitive to the external environment,which was completely different from the affinity ligand with positive charge.Therefore,it can be used for the removal of endotoxin in different water and more complex environments such as biological agents.The results of adsorption thermodynamics and kinetics experiment showed that 5'-AT-NP 21 had a high affinity for endotoxin,the maximum theoretical adsorption capacity was 0.51 mg/g,and it was fast to achieve the adsorption equilibrium.