Aptamer And Urokinase Based Nanobubbles’ Ultrasound Imaging And Therapeutic Study On Acute DVT

Objective:Porous PLGA nanobubbles（PLGA NBs）were prepared by modified double emulsion solvent volatilization method.The best preparation scheme of porous PLGA NBs was verified by characterization and ultrasonic imaging.The aptamer H（Apt H）and urokinase（UK）were piggybacked on porous PLGA NBs to prepare targeted drug-loaded nanobubbles（Apt H@PLGA@UK NBs）,used for the treatment of acute deep venous thrombosis（DVT）.To explore a safe,effective and low toxic and side effects treatment modality,and lay a foundation for the clinical diagnosis and treatment of acute DVT.Methods:1.The groups were divided into Control group,single-porogenic agent group and dual-porogenic agent group according to different porogenic agent schemes;2%NH₄HCO₃group,4%NH₄HCO₃ group,6%NH₄HCO₃ group,8%NH₄HCO₃ group and 10%NH₄HCO₃ group according to different porogenic agent concentrations.PLGA NBs of each group were prepared and examined from optical microscopy,electron microscopy,Zeta potential and particle size and stability,respectively.2.Ultrasound imaging of each group of PLGA NBs was performed using a homemade in vitro ultrasound imaging equipment.Compare the Peak Intensity（PI）of ultrasonography for each group of PLGA NBs to determine the optimal porogenic agent scheme and porogenic agent concentration,and to determine the optimal developing concentration of PLGA NBs.3.Prepare drug-loaded microbubbles（PLGA@UK NBs）and targeted drug-loaded microbubbles（Apt H@PLGA@UK NBs）,respectively.The encapsulation rate and drug loading of UK,the binding rate of Apt H were detected.The biosafety of Apt H@PLGA@UK NBs was verified using human umbilical vein endothelial cell toxicity assay and in vivo toxicity assay in experimental rabbits.4.40 acute DVT animal models were prepared by the stenosis method and divided equally into 5 groups according to different treatment modalities.Control group:no any treatment;US group:low power focused ultrasound irradiation alone;UP group:low power focused ultrasound irradiation+PLGA NBs;UPU group:low power focused ultrasound irradiation+PLGA@UK NBs;UAPU group:low power focused ultrasound irradiation+Apt H@PLGA@UK NBs.5.Two-dimensional ultrasound（2DUS）,color doppler flow imaging（CDFI）,and contrast enhanced ultrasound（CEUS）were used to assess acute DVT after each treatment,and the maximum length and maximum thickness of the thrombus were recorded as indicators for quantitative evaluation of the effect of thrombolysis.6.The four coagulation tests:thrombin time（TT）,prothrombin time（PT）,activated partial thromboplastin time（APTT）,fibrinogen（FIB）content,and D-dimer（D-dimer）content were measured in the peripheral blood of rabbits before modeling,before treatment,and after the last treatment.Finally,the inferior vena cava thrombus tissue was taken for pathological HE staining.Results:1.The yield of PLGA NBs lyophilized powder produced by different porogenic agent schemes and different porogenic agent concentrations reached 80%.Optical microscopy showed that the PLGA NBs were uniformly sized and well dispersed round microbubbles,and the surface of the microbubbles in the group with high-porogenic agent concentration was rougher.The electron microscope showed that PLGA NBs in each group were regular spherical.The surface of the PLGA NBs sphere in the dual-porogenic agent group has a pore-like structure,and the interior has a cavity structure.The surface of the PLGA NBs sphere in the high-porogenic agent group has more pore structures,larger pore channels,and larger interior cavity structure.The particle size of PLGA NBs in 10%NH₄HCO₃ group was smaller than that in other groups,and the Zeta potential was larger（all P<0.05）.There was no statistical difference between the particle size distribution and Zeta potential of PLGA NBs in other groups（all P>0.05）.2.The ultrasonographic imaging of porous PLGA NBs was good.The ultrasonographic PI of porous PLGA NBs gradually increased with the addition of porogenic agent and the increase of porogenic agent concentration（all P<0.05）.The ultrasonographic PI of PLGA NBs in the 10%NH₄HCO₃ and 8%NH₄HCO₃ groups were not significantly different（P>0.05）.Ultrasonographic PI increased with increasing concentration of PLGA NBs,and the highest PI was observed when the concentration reached 15 mg/m L.3.The particle size of PLGA@UK NBs,Apt H@PLGA@UK NBs were higher than that of PLGA NBs（all P<0.05）,and the Zeta potential of microbubbles in each group was not significantly different（all P>0.05）.The encapsulation rate of UK in PLGA@UK NBs was 78.66%,and the drug loading was 30.25%.The binding rate of Apt H in Apt H@PLGA@UK NBs was 66%.The survival rate of human umbilical vein endothelial cells incubated with media containing different concentrations of Apt H@PLGA@UK NBs was more than 90%.The biochemical indexes of the experimental rabbits injected with the same concentration of Apt H@PLGA@UK NBs were all within the normal range,and all the important organ sections were not significantly different from the normal control group,with normal cell structure and no damage.4.The maximum length of thrombus in the Control group and US group gradually increased with time（all P<0.05）;the thrombus in the UP group grew at Day 3 compared with Day 1（P<0.05）,and there was no significant change in the length of thrombus at Day 6（P>0.05）;the thrombus in the UPU group grew at Day 3（P<0.05）and shortened to the same length as Day 1 at Day 6 length（P>0.05）;the maximum length of thrombus in the UAPU group gradually shortened with time（all P<0.05）.The maximum thickness of the thrombus in the Control group did not change significantly with time extension（all P>0.05）;the maximum thickness of the thrombus in the US and UP groups did not change at Day 3（all P>0.05）and shrank at Day 6（all P<0.05）;the maximum thickness of the thrombus in the UPU and UAPU groups gradually shrank with time（all P<0.05）.5.There was no statistically significant difference of the four items of coagulation and D-dimer content of rabbits in each group before modeling and treatment（all P>0.05）;after treatment,PT,APTT,and TT were prolonged in the UPU and UAPU groups compared with the other three groups（all P<0.05）,TT and PT were prolonged in the UAPU group compared with the UPU group;FIB content and D-dimer content were reduced in the UPU and UAPU groups compared with the other three groups（all P<0.05）,and FIB content and D-dimer content were reduced in the UAPU group compared with the UPU group（all P<0.05）.PT,APTT and TT in Control group,US group and UP group had no significant changes before and after treatment（all P>0.05）,while FIB content and D-dimer content were higher before and after treatment than before modeling（all P<0.05）;the PT,APTT and TT of UPU group and UAPU group after treatment were longer than those before treatment and modeling（all P<0.05）,and the content of FIB and D-dimer before treatment were higher than those before modeling and after treatment（all P<0.05）.Conclusions:1.The modified double emulsification solvent volatilization method can prepare porous nanobubble contrast agents with uniform particle size and good dispersion,good ultrasound imaging effect and high yield.2.PLGA NBs with an internal aqueous phase porogenic agent concentration of8%and a contrast concentration of 15 mg/m L had the highest peak ultrasound contrast intensity and the best visualization effect.3.Apt H@PLGA@UK NBs conform to the nano-microbubble standard with high encapsulation rate and high drug loading capacity of UK and high binding rate of Apt H,as well as high cell safety and good biocompatibility.4.Apt H@PLGA@UK NBs can be used for the diagnosis and therapeutic evaluation of acute DVT.It is effective in the treatment of acute DVT and meet the standard of clinical thrombolytic therapy,while improving the abnormalities of coagulation indexes due to thrombosis.