Synthesis Of Polylysine Dendrimers And Its Applications As Nattokinase Carriers In Targeting Thrombolysis

Nattokinase（NK）,as a novel type of thrombolytic agent,has aroused researchers’ attention,which is attributed to its attractive properties,such as direct dissolving thrombosis,activating the human body to produce urokinase and endogenous tissue plasminogen activator.However,NK as a protease drug,there are still some shortcomings,such as vulnerable to the impact of the external environment,sensitive to changes in temperature and p H.Compared with the traditional thrombolytic therapy,the use of drug carrier embedded thrombolytic drugs has attracted the attention of researchers.After encapsulation of NK by drug carrier,it is possible to better protect NK from inactivation and improve its resistance to temperature and pH stability.Polylysine dendrimers are considered to be an ideal drug carrier because of their controlled size,functional surface functional,good water solubility,biodegradability and biocompatibility.Therefore,a series of polylysine dendrimers with different generations were prepared,and their properties as a drug carrier-loaded NK in thrombolysis were studied.In order to achieve target thrombolysis ability,we introduced magnetic targeting and arginine-glycine-aspartate thrombosis site targeting sequence（RGD）.The former is the use of external magnetic field to achieve the purpose of targeting thrombosis,the latter is because the RGD tripeptide is capable of specifically binding to the platelet GP IIb/IIIa receptor and thus has thrombus targeting.The thesis mainly includes the following two aspects:1.Second generations,third generations and fo urth generations of polylysine dendrimers（PLLD Gn）were prepared by divergence-convergence method,and then to form nanocomposites with NK.When NK and PLLD G4 molar ratio was 1:30,the formation of NK/PLLD nanocomposites had the highest nattokinase activity（up to 117%）,and NK/PLLD nanocomposites had good temperature and p H stability.The results of fluorescence spectroscopy showed that the interaction between NK and PLLD was hydrogen bonding and van der waals force.In in vitro thrombolysis results showed that the thrombolysis rate of NK/PLLD nanocomposites reached 50% within 12 h.NK/PLLD nanocomposites can play a role in the slow release of NK compared to free NK.Hemolysis and MTT experiments showed that PLLD had lower cytotoxicity and good biocompatibility.2.The Fe₃O₄ magnetic nanoparticles were prepared by coprecipitation method.The periphery were modified with 3-aminopropyltriethoxysilane（APTES）,and then modified with third generations of polylysine dendrimers（PLLD）.Finally,Fe₃O₄-PLLD-RGD were prepared by the modification of arginine-glycine-aspartic acid（RGD）tripeptide.Then Fe₃O₄-PLLD-RGD/NK were prepared via ion adsorption fixing NK to the surface of Fe₃O₄-PLLD-RGD.The results of in vitro targeting showed that RGD-modified magnetic nanoparticles had good thrombus targeting ability and magnetic targeting capability.In in vitro thrombolysis experiments showed that the thrombolysis rate of Fe₃O₄-PLLD-RGD/NK magnetic nanoparticles reached 50% within 2.5 h,the thrombolysis rate of free NK and Fe₃O₄-PLLD/NK reached 50% within 5.5 h.Fe₃O₄-PLLD-RGD/NK magnetic nanoparticles had good thrombotic targeting ability and rapid thrombolytic capability.Hemolysis and MTT experiments showed that Fe₃O₄-PLLD-RGD had lower cytotoxicity and good biocompatibility.