| FK506 is an immunosuppressive agent which is widely used for prevention and treatment of allograft rejection.At present,heart transplant recipients are given drugs by long-term oral administration or intravenous injection,that drugs are distributed throughout the whole body.However,drugs usually cannot focus on target organs effectively,on the other hand,the toxic side effects of whole body are generated.After heart transplantation,the way to release immunosuppressive agent to target organ becomes a hotspot and a difficult topic in recent years.Ultrasound-targeted microbubble destruction(UTMD)technology can trigger microbubbles rupture locally in targeted organs,allowing drugs to concentrate in local tissues.The aim of this research is to prepare a FK506-loaded microbubbles(FK506-MBs),which could achieve targeted drug delivery and release by UTMD,and improve local drug concentration of transplanted heart and enhance therapeutic effect.It is expected to decrease side effects of immunosuppressive agents.This study consists of the following three parts:Part 1 Preparation and characterization of FK506-MBsObjective Preparation of Blank MBs(Blank microbubbles,Blank MBs)and FK506-MBs,and evaluation of their characterization.Methods(1)FK506-MBs and Blank MBs were prepared by thin-film hydration method.The drug encapsulation efficiency and drug loading efficiency of FK506-MBs were evaluated by high performance liquid chromatography(HPLC).(2)The particle sizes and concentration of FK506-MBs and Blank MBs were evaluated by a zeta potential analyzer and a hemocytometer,respectively.FK506-MBs and Blank MBs were visualized with an inverted optical microscope.(3)The drug stability of FK506-MBs was evaluated by measuring the FK506 leakage percentage after FK506-MBs was placed at different time points at 4 ° C and 37 ° C.(4)Ultrasonic transfection apparatus(NEPA GENE,Japan)was used to trigger the FK506 release from FK506-MBs,and the drug releasing efficiency under different ultrasound irradiation time was detected.Results(1)During the preparation of FK506-MBs,when the initial dose of FK506 was 2 mg,the drug encapsulation efficiency and drug loading efficiency of FK506-MBs was 75.8 ± 4.09% and 33.48 ± 1.21%,respectively.(2)FK506-MBs and Blank MBs were mixture of white milky suspension.The microscopic images revealed that all FK506-MBs and Blank MBs were spherical and most of them separated from each other without aggregation.The particle size and the concentration of FK506-MBs and Blank MBs were 1.65 ± 0.32 ?m,(4.35 ± 0.18)× 109 /ml and 1.20 ± 0.20 ?m,(5.71 ± 0.15)× 109 /ml,respectively.(3)When FK506-MBs were stored at 4 °C and 37 °C,the leakage of the drug was increased with time.After being stored 24 h at 4 °C,the FK506 leakage percentage was 6.0 ± 0.57%.While after being stored 24 h at 37 °C,the FK506 leakage percentage reached 15.67 ± 1.17%.(4)UTMD can trigger the release of FK506 from FK506-MBs in vitro.When the ultrasound irradiation time was 2 min,the drug releasing efficiency was 72 ± 1.15%.Conclusion In this study,FK506-MBs with high drug encapsulation efficiency and drug loading efficiency could be successfully prepared by thin-film hydration method.They could be kept relatively stable after storage at 4 ° C for 24 hours,and UTMD was able to trigger drug release.Part 2 Optimization of Ultrasound-targeted microbubble destruction(UTMD)parameters and evaluation the safety of MBs and UTMDObjective To prove whether UTMD could be able to increase the permeability of vessel wall for targeted drug delivery,we also optimized the ultrasound irradiation time and proved the safety of MBs and UTMD.Methods(1)Heterotopic heart transplantation models in rats were prepared.After the injection of Blank MBs through the tail vein,the Sonitron 2000 V ultrasonic transfection system was used with a probe frequency of 1 MHz for UTMD.The parameters of the ultrasonic irradiation were as follows: the ultrasonic intensity was 2 w/cm2,the duty cycle was 50%,and the ultrasonic irradiation time was set to 1 min,2 min,3 min,respectively.The irradiation area was the intraperitoneal transplanted heart.The control group(0 min)was injected with the same microbubbles,but the ultrasonic irradiation time was 0 min.Evans blue(EB)staining was performed immediately after UTMD to observe the blue staining of the transplanted heart under different ultrasound irradiation time.(2)By comparing the heart rate changes of transplanted hearts before and after UTMD,the damage of UTMD to transplanted hearts under different ultrasound irradiation time was evaluated.The model rats were euthanasia after UTMD,and the transplanted heart specimens were obtained and stained with HE(haematoxylin and eosin,HE)to observe the heart tissue damage.(3)Twenty-seven normal SD rats were divided into three groups: PBS group(9),MBs group(9)and UTMD group(9).The MBs group only received intravenous Blank MBs.Three rats in each group were randomly euthanasia at 30 min,1 day,and 7 days after intervention.To evaluate the security of MBs and UTMD,the whole blood and heart,liver,spleen,lung,and kidney of the rat were obtained for blood cell analysis,serum biochemical analysis,and organ HE staining.Results(1)After UTMD,the myocardial tissues were stained with EB.The blue stained area of the heart was increased and the degree of blue staining was deepened as ultrasonic irradiation time was prolonged.(2)There was no significant change in heart rate before and after UTMD in 1 min group and 2 min group.When the ultrasound irradiation time was 3 min,the heart rate of transplanted hearts decreased significantly after UTMD.(3)There were no obvious myocardial cell necrosis and abnormal myocardial fiber changes in 1 min group and 2 min group.However,HE staining showed slight vacuolization of the heart fiber in 3 min group.(4)Compared with the PBS group,rats in the MBs group and UTMD groups had no abnormal changes in blood cell count,liver and kidney function,and myocardial enzymes.HE staining showed that there was no obvious tissue damage of each organ.Conclusion This study demonstrated that UTMD ultrasound frequency of 1 MHz,ultrasonic intensity of 2 w/cm~2,duty cycle of 50%,irradiation time of 2 min can significantly enhance the vascular permeability of the transplanted heart,and no damage to the transplanted hearts.Part 3 Efficacy evaluation of FK506-MBs ultrasound targeted release inhibiting acute heart transplantation rejection in ratsObjective To verify the effectiveness of FK506-MBs combined with UTMD targeted drug delivery in the treatment of acute cardiac allograft rejection.Methods(1)Rats after heart transplantation were given by intravenous injection of Di R(1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyaine iodide,Di R)or by Di R-loaded microbubbles(Di R-MBs)+ UTMD.Then,the fluorescence intensity of transplanted hearts in Di R group and Di R-MBs + UTMD group were performed by a small animal imaging system.(2)Rats after heart transplantation were given by intravenous injection of Di I(1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyaine perchlorate,Di I)or by Di I-loaded microbubbles(Di I-MBs)+ UTMD.Then,the transplanted hearts were obtained for tissue section,and the intensity of Di I in the transplanted heart was observed by a confocal laser scanning microscopy.(3)Rats after heart transplantation were given by intravenous injection of FK506 or by FK506-MBs + UTMD.After 30 min,the model rats were euthanasia and the transplanted heart was obtained.The concentration of the drug in the transplanted heart tissue was measured by ultra-high performance liquid chromatography tandem mass spectrometry(UPLC-MS)in FK506 group and FK506-MBs + UTMD group.(4)The survival time of the transplanted heart was evaluated by heartbeat of the transplanted heart.AR degree was evaluated by HE staining,and the infiltration of T lymphocytes and the secretion of IFN-? and IL-2 were evaluated by immunofluorescence staining and western blot.Results(1)After treatment,Di R was distributed throughout the whole body,mainly in the liver and spleen.The fluorescence intensity of the cardiac graft was the highest at 30 min.Compared with the Di R group,the fluorescence intensity of the transplanted hearts in the Di R-MBs + UTMD group was higher.(2)The red fluorescence was detected in the grafts section in Di I-MBs + UTMD group.But,the red fluorescence was not discovered in the myocardial interstitium in Di I group.(3)The concentration of FK506 in FK506-MB + UTMD group was 3.17 ± 0.38 ?g/g,while in FK506 group was 1.93 ± 0.29 ?g/g.(4)HE staining showed that the acute rejection of the FK506-MBs + UTMD group was significantly weaker than the other groups.(5)Immunofluorescence staining and western blot results showed that the infiltration of CD3 positive cells in the myocardial tissues of transplanted heart of the FK506-MBs + UTMD group was significantly decreased compared with the other groups,and the secretion of IFN-? and IL-2 was markedly decreased.(6)Survival evaluation showed that the survival time of the transplanted heart in the PBS group was 6.66 ± 1.36 days,the survival time of the transplanted heart in the FK506 group was 12.83 ± 1.17 days,and the survival time of the transplanted heart in the FK506-MBs + UTMD group was 16.00 ± 0.89 days(p<0.05).Conclusion This study showed that FK506-MBs combined with UTMD targeted drug delivery significantly increased drug concentration in the transplanted heart,enhanced therapeutic effect,and provided a new sight for the treatment of organ transplantation rejection. |
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