Ultrasound Molecular Imaging Of Acute Cardiac Transplantation Rejection Using Nanobubbles Targeted To T Lymphocytes

Clinical surveillance of acute heart transplantation rejection requires repeated endomyocardial biopsies and noninvasive evaluation of AR remains challenging.The aim of the present study was to prepare for a T lymphocyte-targeted nanobubbles(NBs),and to clarify the value of T lymphocyte-targeted ultrasound molecular imaging in the diagnosis of AR.The present study includes three parts:Part 1 Preparation and characterization of targeted nanobubbles conjugated with anti-CD3 antibodyObjective Prepare for nanobubbles conjugated with anti-CD3 antibody or isotype antibody and evaluate the characterization of the NBs.Methods The NBs were prepared by thin-film hydration method and separated by appropriate centrifugation speed and centrifugation time.Biotin-avidin-biotin linking was used in the preparation of the anti-CD3 antibody(NBCD3)or isotype antibody(NBcon)conjugated NBs.The morphology,distribution,particle size,zeta potential,concentration,stability and contrast ability of the two kinds of NBs were evaluated in vitro.Fluorescence microscopy and flow cytometry analysis were used to evaluate the antibody conjugation efficiency of the NBs.Results(1)The prepared ultrasound contrast agents with thin-film hydration and mechanical oscillation method were a mixture of MBs and NBs.In the separation study,higher centrifugation speed will lead to a decrease in concentration and longer centrifugation time will not diminish the mean diameter of the NBs.Therefore,centrifugation at 300 rpm,3 min can ensure that the particle size of NBs is relatively small,and the concentration of NBs can also be guaranteed.(2)The NBs conjugated with biotinylated antibodies appeared as a milky white suspension and exhibited no obvious delamination after static at room temperature.They were uniformed in size and well distributed.The mean diameter and concentration of the NBCD3 and NBcon were 457.06 ± 52.68 nm,(9.12 ± 0.46)× 109/ml and 460.64 ± 45.51 nm,(9.22 ± 0.63)× 109/ml respectively.There was no significant difference of the mean diameter,concentration,zeta potential and PDI between the NBCD3 and NBcon.(3)The NBs showed excellent stability in diameter and concentration,and could achieve good ultrasonic contrast effect in vitro.(4)The flow cytometry results indicated that more than 90% of the NBCD3(91.5 ± 3.0%)or NBcon(90.5 ± 3.5%)was successfully conjugated with either anti-CD3 antibody or control Ig G.Conclusion In this study,we prepared anti-CD3 antibody conjugated NBs successfully.They were uniform in size and showed excellent stability and contrast effects.Part 2 Attachment studies of targeted NBs conjugated with anti-CD3 antibody to T lymphocytesObjective To verify the binding ability of NBCD3 to T lymphocytes and evaluate its toxicity to T lymphocytes.Methods T lymphocytes were selected by immunomagnetic isolation kit(negative selection),and the purity of the isolated T lymphocytes was verified by flow cytometry analysis.The attachment ability of the NBCD3 and NBcon to T lymphocytes were observed by optical and confocal laser scanning microscope respectively,and the attachment efficiency of the NBs were verified by flow cytometry.The cytotoxicity of the NBs to T lymphocytes were determined by MTT assay.Results(1)Flow cytometry results showed that the purity of the T lymphocytes was more than 95%.(2)There were a number of NBCD3 attached to the T lymphocytes after incubation with NBCD3,whereas,there were only a few NBcon bound to the surface of the T lymphocytes.The flow cytometry analysis demonstrated the binding rate of the NBCD3 to the T lymphocytes(35.4 ± 9.7%)was higher than the NBcon group(0.33 ± 0.2%)(P < 0.05).(3)The viabilities of the T lymphocytes were not affected after incubation with different amount of NBCD3 and NBcon.Conclusion The NBCD3 prepared in this study can adhere to T lymphocytes specifically and exhibited no obvious toxicity to T lymphocytes.The successful preparation of NBCD3 makes it possible to achieve T lymphocyte-targeted molecular imaging in subsequent animal experiments.Part 3 Ultrasound molecular imaging of acute cardiac transplantation rejection in rats with targeted NBs conjugated with anti-CD3 antibodyObjective To clarify the value of T lymphocyte-targeted ultrasound molecular imaging in the diagnosis of acute rejection in heart transplantation.Methods Heterotopic heart transplantation models in rats were prepared.Ultrasound molecular imaging with NBCD3 and NBcon were performed respectively and the destruction-replenishment method was used to quantify the signal of attached NBs.The biodistribution of the NBs in vivo was evaluated by a small animal imaging system.The penetrability of NBs was confirmed by fluorescence imaging of frozen sections of myocardium after injection of Di I-labeled NBs under CLSM.HE staining,immunohistochemical staining and Western Blot were used to detect acute rejection and assess the infiltration of T lymphocytes.Furthermore,the degree of T lymphocyte infiltration and ultrasound molecular imaging results were correlated and analyzed.Results(1)Ultrasound molecular imaging was performed in 33 rats(16 allografts and 17 isografts)completely.(2)The NBs distributed to the whole body after intravenous injection via the tail vein and mainly accumulated in liver and spleen.(3)After injection,the Di I-labeled NBCD3 were present in the myocardial interstitium in the allografts.In contrast,the red fluorescence of the NBCD3 was hardly detected in the isografts.(4)The signal intensity(SI)of NBCD3 was significantly higher compared with NBcon in allograft rats(2.86 ± 2.05 d B versus 0.85 ± 0.47 d B,P < 0.05),as well as higher than that of the NBCD3 in isograft rats(2.86 ± 2.05 d B versus 0.71 ± 0.29 d B,P < 0.05).However,there was no significant difference for NBCD3 and NBcon in isograft rats(0.71 ± 0.29 d B versus 0.69 ± 0.29 d B,P > 0.05).(5)Moreover,the SI of NBCD3 was positively correlated with the number of infiltrated T lymphocytes(R2 = 0.67,P < 0.01),but the correlation was not observed for NBcon in the same rats(R2 = 0.03,P > 0.05).Conclusion T lymphocyte-targeted ultrasound molecular imaging can detect acute rejection in heart transplantation and it could be a noninvasive approach in the evaluation of AR.