The Experimental Study Of Preparation Of Nanoscale AMH-targeted Ultrasound Contrast Agents And Rat Transplanted Ovary Targeted Imaging

Objective:The targeted imaging of ovarian transplantation in rats was achieved by preparing AMH targeted contrast agents,and the expression level of AMH molecules in ovarian tissue was further evaluated with the NB_AMHultrasound molecular imaging to achieve early detection and quantitative evaluation of the survival status of transplanted ovaries.Methods:1)Part I:the lipid materials were mixed in a certain proportion,and the pure nanometer particle size contrast agent（NBs）was prepared by"thin film hydration method"and"mechanical shock method".The"avidin-biotin"bridging method was used to conjugate the anti-AMH antibody to the surface of the nanobubble in varying proportions to produce NB_AMHor NB_{Ig G}.The morphology of nanobubble was observed by inverted microscope and scanning electron microscope.The Zetasizer system was used to measure the particle size,dispersion and stability of nanobubbles.The conjugation efficiency of NBs with AMH antibody was measured by fluorescence microscopy and flow cytometry;2)Part II:Before the experiment of ovarian granulosa cells binding assay in vitro,we first tested the cells viability with 1×10⁴,10⁵,10⁶,10⁷,or 10⁸NBs/m L.Cell viability was measured using a Cell Counting Kit-8（CCK-8）.We established a hypoxia/reoxygenation（H/R）model in vitro.After the intervention was completed,the supernatant was collected and subjected to AMH analysis by ELISA.The H/R cells were incubated with NB_AMH,and the adhesion of NB_AMHto ovarian granulosa cells was observed under microscope and the adhesion efficiency was detected by flow cytometry;3)Part III:Rat ovarian transplantation models were established.Twenty rats（10 normal rats and 10 ovarian transplantation model rats）were randomly injected with contrast agents NB_AMHand NBs through the tail vein.Ultrasound diagnostic instrument was used to observe the effect of contrast enhanced imaging and quantitative analysis of contrast intensity.Fluorescence imaging system was used to observe the distribution of NB_AMHin the important organs in vivo.The targeted distribution of NB_AMHin ovaries were observed by laser confocal microscopy.After the in vivo targeting experiment,a rat ovarian transplantation model was established again.Sixty hours after the transplantation,40 rats were randomly divided into 4 groups(3,5,7 and 10 day group.NB_AMHultrasound molecular imaging was performed and AMH molecular immunofluorescence and Western Blot were used to further detect AMH molecular expression levels at different time points after ovarian transplantation.Results:1)Part I:the particle size of the NBs was 478.24±28.02nm and NB_AMHwas549.33±28.53nm,the nanometer hollow particles were uniform in size under the microscope and scanning electron microscope.The particle size and concentration remained stable for at least 60min at room temperature.Flow cytometer showed that the optimal binding rate of AMH antibody to NBs was 65.3±2.7%,and the optimal ratio was1:5;2)Part ii:No obvious cytotoxicity was observed in NBs.Through the H/R experiment,ELISA results showed that AMH expressed in both ovarian granulosa cells under normal and hypoxic conditions.Targeting experiments in vitro showed that the binding rate of NB_AMHto cells was 23.97±0.15%,which was significantly higher than that of NBs group and NB_{Ig G}group（P<0.001）;3)Part iii:In vivo ultrasound molecular imaging showed that NB_AMHsignificantly enhanced contrast imaging of transplanted ovaries compared with NBs.Fluorescence imaging system further confirmed the specific distribution of NB_AMHin ovarian tissue.The AMH immunofluorescence showed that NB_AMHwas able to enter the interspace of transplanted ovarian tissue and co-locate with AMH molecules.In addition,through molecular molecular imaging monitoring of AMH,the intensity of angiographic signals at 3,5,7,and 10 days after ovarian transplantation increased with the increase of transplantation time;AMH molecular immunofluorescence and Western Blot results showed the same trend,similar to the results of ultrasound molecular imaging.Conclusion:1.In this study,AMH-targeted nanobubbles were successfully prepared with optimal ratio.2.NB_AMHhas obvious targeted adhesion to ovarian granulosa cells in vitro.3.NB_AMHcan significantly enhance the contrast imaging of ovarian transplantation in vivo.4.NB_AMHcan be used for in vivo imaging and quantitative evaluation of AMH molecules to reflect the survival status of transplanted ovaries.Ultrasound molecular imaging provides a new method for in vivo,noninvasive and dynamic monitoring of the survival status of transplanted ovaries.