| Objective:To explore the feasibility of direct preparation of targeted microbubbles containing Arg-Gly-Asp-Ser(RGDS)peptide fragments and carrying urokinase(UK);to detect the general physicochemical properties of targeted contrast agents;and to study the possible factors influencing the carrying rate of targeted contrast agents.Methods:(1)UK was labeled with fluorescent dye FITC to make FITC-UK,and RGDS was labeled with fluorescent dye 5-TAMRA to make 5-TAMRA-RGDS.(2)The general physicochemical properties of targeted microbubbles were determined by direct configuration of targeted contrast agents,and by observation of the appearance of targeted microbubbles,determination of PH,optical microscopy and particle size cytometry.(3)Flow cytometry was used to detect the carrying rate of fluorescent labeled targeted contrast agents and find out the possible factors.? Effects of composition ratio and incubation time:Group by component ratio,SonoVue:urokinase:RGDS were divided into group A(1:1:1),group B(1:2:1),group C(1:1:2),group D(2:1:1),group E(2:2:1),group F(2:1:2)and group G(1:2:2).Solutions of different groups were added into 5 mltest tubes respectively,and then incubated at room temperature in the absence of light after the solution was fully mixed.Each component is divided into three parts.They were incubated for 0 hours,1 hour and 2 hours respectively.Flow cytometry was used to detect the UK,RGDS and UK,RGDS double-carrying rates at 0h,1h and 2h of incubation.?The influence of adding order:The experiment was divided into group A(Add the UK first group),group B(Add RGDS first group)and control group(Add both UK and RGDS groups)according to different adding order.Flow cytometry was used to detect the carrying rate of UK,the carrying rate of RGDS,double carry rates for UK and RGDS in three groups of solutions with different dosing sequence.?The effect of washing:After washing seven groups of target contrast agent solutions with different proportions after incubation by suspension washing method,the UK carrying rate,RGDS carrying rate,UK and RGDS double carrying rate of seven groups with different proportions of target contrast agent after washing were detected by flow cytometry,and compared with those before washing.Results:(1)The FITC-UK solution was bright golden yellow,and the 5-TAMRA-RGDS solution was bright dark rosy red.(2)General physicochemical properties of RGDS-urokinase targeted contrast agent:?Targeted contrast agent prepared by naked eye observation is a white emulsion suspension,which can be divided into two layers at rest.The upper layer is white,delicate and uniform microbubbles,which mostly gather around the tube wall.The lower layer is a transparent slightly white solution.With gentle vibration,the solution immediately turns into a white emulsion suspension.Seven groups of targeted microbubbles contrast agents prepared according to different proportions of SonoVue,urokinase and RGDS all have pink curd-like appearance,opaque,easily stratified after stationary.The upper layer is white,delicate and uniform curd-like microbubbles,and the lower layer is slightly turbid,translucent and light pink liquid with different brightness.?The pH value of SonoVue contrast agent was 6.98.Targeted contrast agent pH was 7.3,neutral solution.?Under the optical microscope,the target contrast agent microbubbles are circular structure with central light transmission and dark film-like shell wrapped around them.The distribution of the microbubbles is concentrated and the size is uniform.?The average particle size of targeted contrast agent is 1415 nm,which is larger than that of SonoVue contrast agent,but still at nanometer level.The Zata potential was-5.86 mv,and the targeted contrast agent solution was negatively charged.?SonoVue is configured in different proportion with FITC-UK and 5-TAMRA-RGDS.The target contrast agent labeled with double fluorescence can be stimulated green fluorescence and red fluorescence under fluorescence microscope.(3)Influencing factors of carrying rate of urokinase targeted contrast agent with RGDS:? There was no significant difference in UK carrying rate measured by seven groups of targeted contrast agents with different proportions at different incubation time(0 h,1 h,2 h)(Ftime?2.362,P=0.210),and RGDS carrying rate(Ftime?0.530,P=0.640),UK and RGDS double carrying rate had no significant difference meaning(Ftime=0.521,P=0.629).There were significant differences in UK carrying rates of target contrast agents prepared by different proportions of SonoVue,UK and RGDS(Fgrouping=11.533,P=0.000),RGDS carrying rates(Fgrouping=18.835,P=0.000),UK and RGDS double carrying rates(Fgrouping=8.272,P=0.001).When the mixing ratio of SonoVue:UK:RGDS is 1:2:1,UK has the highest carrying rate.When the mixing ratio of SonoVue:UK:RGDS is 1:2:2,the carrying rate of RGDS is the highest.When the mixing ratio of SonoVue:UK:RGDS is 1:2:1,the double carrying rate of UK and RGDS is the highest.(2)The influence of dosing sequence:There were significant differences in UK carrying rate(F=26.861,P=0.00),RGDS carrying rate(F=17.392,P=0.00),UK and RGDS double carrying rate(F=31.240,P=0.00).The carrying rate of UK in control group was higher than that in group A and B,and that in group B was higher than that in group A.The difference was statistically significant(P<0.05).The carrying rate of RGDS in group B was higher than that in group A and control group(P<0.05).The double carryings of UK and RGDS in control group were higher than those in group A and B,and the double carryings of UK and RGDS in group B were higher than those in group A.The difference was statistically significant(P<0.05).At the same time,the UK carrying rate and the dual carrying rate of UK and RGDS were higher than those of the other two drugs.The RGDS carrying rate of the targeted contrast agent prepared by adding RGDS in the first order is higher than that of the other two kinds of targeted contrast agents prepared by adding RGDS in the second order.(3)The effect of washing on the carrying rate:7 groups of targeted contrast agents with different proportions of SonoVue,UK and RGDS were washed by suspension washing after incubation for 2 hours.The changes of UK carrying rate,UK and RGDS double carrying rate before and after washing in each group were statistically significant(P<0.05).The UK carrying rate,UK and RGDS double carrying rate after washing in each group were lower than before washing.There was no significant difference in RGDS carrying rate between group A,group C,group D,group E,group F and group G before and after washing(P>0.05).Conclusion:(1)Direct preparation of urokinase-targeted contrast agent with RGDS is feasible.(2)The ratio of SonoVue,UK and RGDS has influence on UK carrying rate,RGDS carrying rate,UK and RGDS double carrying rate.The final result of this experiment is that the carrying rate of UK,UK and RGDS is the highest when the ratio of UK:RGDE is 1:2:1.When the mixing ratio of SonoVue:UK:RGDS is 1:2:2,the carrying rate of RGDS is the highest.(3)Incubation time had no effect on UK carrying rate,RGDS carrying rate,UK and RGDS dual carrying rate.(4)The sequence of SonoVue,UK and RGDS addition has influence on UK carrying rate,RGDS carrying rate,UK and RGDS dual carrying rate of targeted contrast agent.The results of this experiment show that the UK carrying rate,UK and RGDS double carrying rate of targeted contrast agent prepared by adding UK and RGDS in the order of adding drugs simultaneously are higher than the other two order of adding drugs.The RGDS carrying rate of target contrast agents prepared by adding RGDS in the first order was higher than that of the other two orders.(5)The influence of washing on UK carrying rate,UK and RGDS double carrying rate.After washing,the UK carrying rate,UK and RGDS double carrying rate were all reduced,which indirectly indicated that the stability of the targeted contrast agent prepared by direct method was poor,and the drug loaded on the contrast agent was easy to fall off when confronted with water flow and other shocks. |
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