Preparation Of Polyglutamic Acid Peptide Dendrimers And Their Application In Dissolving Thrombus

Thrombosis is one of the major causes of cardiovascular diseases,that results in millions of death every year worldwide.Nattokinase?NK?is a member of the subtilisin family of serine proteases produced by Bacillus subtilis natto,the enzyme directly cleaves cross-linked fibrins,catalyzes the conversion of plasminogen to plasmin or inactivates the fibrinolysis inhibitor.Most importantly,NK has high safety,low cost,simple production process and low side effects.However,NK is sensitive to variations in temperature and pH so that it is easy to lose the enzyme activity,it also has not the ability to target thrombus.Therefore,the design and preparation of NK drug carrier can not only protect the enzyme activities of NK,but also give it the properties of targeted thrombus.Polyglutamic acid peptide dendrimer possesses functional groups on its surface,adjustable sizes,biodegradability,biocompatibility,and low toxicity,whichcould be used as ideal carrier for drug protection and delivery.Based on the above considerations,in this study,we prepared a series of polyglutamic acid peptide dendrimers,and studied their chemical and physical properties as a drug carrier-loaded NK.The main research contents and results as follows:1.Second generations,third generations and fourth generations ofpolyglutamic acid peptide dendrimers?G_n,n=2,3,4?were prepared,and then PEGylated polyglutamic acid peptide dendrimers?G_n-PEG-G_n,n=2,3,4?,which loaded NK to form nanocomposites were prepared.The enzyme activity of nanocomposites showed that the optimal dendrimer product is G₃-PEG-G₃ and the best composite is G₃-PEG-G₃/NK with a ratio of 6/1.Zeta potential,TEM and DLS revealed that G₃-PEG-G₃ load NK via the electrostatic interactions between the negative charges of G₃-PEG-G₃ and positive charges of NK.MTT experiments demonstrated that G₃-PEG-G₃ have good cell viability and biocompatibility.In vitro thrombolysis experiments showed G₃-PEG-G₃/NK?6/1?nanoparticle has the excellent property in dissolving thrombus.2.The azide terminated multiarm-polyethylene glycol?x-PEG?N₃?_x,x=2,4,6,8?were prepared,and then multiarm-polyethylene glycol-polyglutamic acid peptide dendrimer?x-PEG?G₃?_x,x=2,4,6,8?,which loaded NK to form nanocomposites were prepared.The enzyme activity of nanocomposites showed that the optimal dendrimer product is 4-PEG?G₃?₄ and the best composite is 4-PEG?G₃?₄/NK with a ratio of 1/1.Zeta potential,TEM and DLS revealed that4-PEG?G₃?₄ load NK via the electrostatic interactions between the negative charges of 4-PEG?G₃?₄and positive charges of NK.MTT experiments demonstrated that 4-PEG?G₃?₄ have good cell viability and biocompatibility.In vitro and in vivo thrombolysis experiments showed4-PEG?G₃?₄/NK?1/1?nanoparticle has the excellent property in dissolving thrombus.3.The azide terminated multiarm-polylactic acid?x-PLA?N₃?_x,x=3,4,6?were prepared,and then multiarm-polylactic acid-polyglutamic acid peptide dendrimer?x-PLA?G₃?_x,x=3,4,6?,which loaded NK to form nanocomposites were prepared.The enzyme activity of nanocomposites showed that the optimal dendrimer product is 4-PLA?G₃?₄ and the best composite is4-PLA?G₃?₄/NK with a ratio of 1/1.We prepared Fe₃O₄-4-PLA?G₃?₄-RGD nanoparticles,TEM and LCSM demonstrated that Fe₃O₄-4-PLA?G₃?₄-RGD was prepared successfully,VSM showed that Fe₃O₄-4-PLA?G₃?₄-RGD has good magnetic property.MTT experiments demonstrated that Fe₃O₄-4-PLA?G₃?₄-RGD have good cell viability and biocompatibility.In vitro and in vivo thrombolysis experiments showed Fe₃O₄-4-PLA?G₃?₄-RGD/NK?1/1?nanoparticle has the excellent property in dual targeted thrombolysis.4.Silicon dioxide-polyglutamic acid peptide dendrimer?M-MSNs-G₃-RGD?was perpared,and then FTIR,TGA,TEM and LSCM proved that M-MSNs-G₃-RGD was prepared successfully,VSM showed M-MSNs-G₃-RGD has good magnetic property.M-MSNs-G₃-RGD loaded NK to form nanocomposite,the enzyme activity of nanocomposite showed that the best composite is M-MSNs-G₃-RGD/NKwitharatioof4/1.MTTexperimentsdemonstratedthat M-MSNs-G₃-RGD have good cell viability and biocompatibility.In vitro and in vivo thrombolysis experiments showed M-MSNs-G₃-RGD/NK?4/1?nanoparticle has the excellent property in dual targeted thrombolysis.