The Application Of FK506-loaded PLGA Nanoparticles In The Treatment Of Cardiac Acute Rejection

Part 1: Preparation and characterization of FK506-loaded PLGA nanoparticlesObjective: Fabrication and evaluation of FK506-loaded PLGA nanoparticles(PLGA-FK506-NPs)in vitro and in vivo.Methods:(1)PLGA-FK506-NPs were prepared by using ultrasonic emulsification solvent evaporation method.(2)The morphology of nanoparticles was observed by scanning electron microscopy(SEM)and transmission electron microscopy(TEM).(3)The mean size and stability of nanoparticles was determined by dynamic light scattering technique.(4)High-performance liquid chromatography(HPLC)was used to measure the drug entrapment and loading efficiency as well as the release rate in vitro.(5)The whole blood,serum and tissues were taken from rats for blood analysis,blood biochemical analysis and hematoxylin-eosin(HE)staining to evaluate the safety of PLGA-FK506-NPs.Results:(1)The PLGA-FK506-NPs were monodispersed and spherical under SEM and TEM.(2)The mean size and zeta potential of PLGA-FK506-NPs were 110 ± 1.3 nm and-20.56 ± 3.65 m V,respectively.(3)The PLGA-FK506-NPs showed excellent stability within 48 h in both PBS and water.(4)The FK506 entrapment and loading efficiency were94.46 ± 1.88% and 5.38 ± 0.24%,respectively.(5)Approximately 50.99 ± 1.87% of FK506 was released from PLGA-FK506-NPs within 7 days,while free FK506 solution exhibited a rapid release behavior and 50.37 ± 1.05% of FK506 was released within 4 h.(6)The toxicity evaluation showed that the counts of red blood cells,white blood cells,platelets, lymphocytes,neutrophils and monocytes were normal in PLGA-FK506-NPs group,and there was no significant statistical difference between PBS group and FK506 group.Moreover,HE staining results showed that there was no bleeding and inflammatory cell exudation in major organs(heart,liver,spleen,lung,kidney)in all groups.Conclusion: In this study,PLGA-FK506-NPs were successfully prepared by ultrasonic emulsification solvent evaporation method,with uniform particle size,high encapsulation rate,sustained release effect,excellent stability and safety.Part 2: Establishment and monitoring of rat heterotopic heart transplantation modelObjective: The study was aimed to prepare rat heterotopic heart transplantation model and to evaluate the perfusion changes of acute rejection using contrast-enhanced ultrasonic myocardial perfusion imaging(CEUS-MPI);The survival time of allografts were monitored in allograft and isograft groups.Methods:(1)Rat heterotopic heart transplantation model was performed using a microsurgical technique.(2)In allograft,Brown Norway(BN)rats were donors and Lewis rats were recipients,while in isograft,Lewis rats were both donors and recipients.(3)CEUS-MPI was performed on 3,5 and 7 days after the operation and the mean peak signal intensity was analyzed.Then the grafts were taken for HE staining,CD3,CD68 and CD31 immunohistochemical staining.(4)Meanwhile,6 allografts and isografts were established,respectively.The survival time was evaluated by visual examination,palpation and echocardiography.Results:(1)The rat heterotopic transplantation model was successfully established with a success rate of 91.3%.(2)In the allograft group,the grade of allograft rejection was aggravated,the infiltration of T cells and macrophages was increased,and the microvascular density was gradually reduced with a decreased mean peak signal intensity.(3)However,the grade of allograft rejection in the isograft group has no obvious changes over time as well as T cells infiltration,macrophages infiltration,microvascular density and the mean peak intensity.(4)During observation and a follow-up time of 28 days,the mean survival time was only 7.4 ± 1.0 days in the allograft group,while the mean survival time was more than 28 days in the isograft group.Conclusion: The rat heterotopic transplantation model has been successfully established and the acute rejection was progressively aggravated after operation.CEUS-MPI can be used to evaluate the grade of acute rejection.Part 3: The application of PLGA-FK506-NPs in treatment of cardiac acute rejectionObjective: To compare the pharmacokinetic characteristics between free FK506 and PLGA-FK506-NPs and evaluate the immunosuppressive efficacy of free FK506 and PLGA-FK506-NPs in treatment of cardiac acute rejection.Methods:(1)Di R dye was used to simulate FK506,the biodistribution of Di R dye and Di R-labeled PLGA-NPs(PLGA-Di R-NPs)were performed by a small animal imaging system.(2)the pharmacokinetics of PLGA-FK506-NPs and free FK506 were further evaluated by measuring the concentration of FK506 in whole blood,spleen,mesenteric lymph nodes(MLN),inguinal lymph nodes(ILN)and axillary lymph nodes(ALN)by using the HPLC/MS system at desired time points.(3)PBS,free FK506,and PLGA-FK506-NPs were injected intravenously(1 mg/kg)to evaluate the treatments of the established rat heterotopic heart transplantation model,HE staining of the grafts were taken for the AR grading.CD3 immunohistochemical staining was used to evaluate the infiltration of T lymphocytes.Immunofluorescence staining was used to evaluate the secretion of IFN-? and IL-2.(4)The survival time was evaluated by visual examination,palpation and echocardiography.Results:(1)The fluorescence intensity of PLGA-Di R-NPs in the spleen and lymph nodes was increased compared with the pure Di R dye.(2)Pharmacokinetics study revealed that the PLGA-FK506-NPs behaved significantly different from free FK506 and exhibited a higher area under curve(1.69-fold).(3)Notably,the FK506 concentrations of in the spleen and MLN of the PLGA-FK506-NPs group were 3.1-fold and 2.9-fold higher than those of free FK506 group.(4)The mean survival time of the grafts was 7.6 ± 1.1 days in the PBS group and 17.1 ± 2.0 days in the PLGA-FK506-NPs group,which was significantly different from that in the FK506 group(13.3 ± 1.7 days)(P < 0.01).(5)The grade of acute rejection in the PLGA-FK506-NPs group was 1R(40%),2R(40%)and 3R(20%),while the rejection in the FK506 group was 1R(20%),2R(60%),3R(20%)and 3R(100%)in the PBS group.(6)T cell infiltration was significantly reduced in the FK506 treatment group and the PLGA-FK506-NPs group compared with PBS group.(7)The secretion of IL-2 and IFN-? was decreased in the FK506 treatment group and the PLGA-FK506-NPs group,while the secretion of IL-2 was the least in the PLGA-FK506-NPs treatment group as well as IFN-?.Conclusion: Compared with free FK506,PLGA-FK506-NPs increased the concentration of FK506 in spleen and MLN,and it showed better pharmacokinetic parameters.Moreover,it prolonged the graft survival time with the decreased grade of acute rejection and the reduced secretion of inflammatory cytokines.