Biomimetic Yeast Capsules System For Diagnosis And Treatment Of Transplant Rejection

Transplantation is an effective treatment for patients with end-stage organ diseases.Rejection is the major complication and the main cause of mortality after transplantation.Non-invasive monitoring and effective treatment of transplantation rejection facilitate the survival of grafts,but challenges remain due to inferiority in specificity and sensitivity.Therefore,it is urgent to develop novel diagnostic methods and treatment strategies to improve the survival time and living quality of postoperative patients.It has been reported that macrophages infiltrate into allograft during early rejection,and the infiltration of macrophages in the graft are closely related to the pathological degree of rejection,suggesting that macrophages is an ideal candidate for diagnosis and treatment of transplant rejection.Additionaly,yeast capsules(YC)extracted from yeast can target macrophages by simulating the path of microbial gastrointestinal infection after oral administration,which can be used as biomimetic carrier for tracking macrophages.Fluorescence imaging is a powerful tool for non-invasive imaging of macrophages due to its exceptional sensitivity and high temporal resolution.However,conventional organic fluorophores suffer from poor photostability,small Stokes shift,and aggregation-caused quenching(ACQ),which restricts its application for long-term tracking of living cells.Compared with conventional fluorophores,aggregation-induced emission luminogens(AIEgen)have several extraordinary properties,such as high brightness,excellent photostability,and high-contrast imaging,indicating that AIEgen are ideal for long term,real-time tracking of macrophages.Herein,we hypothesized that the biomimetic properties of YC can be used to track macrophages in vivo for non-invasive and real-time monitoring of transplant rejection.Tacrolimus(FK506),as a first-line immunosuppressant can be used to prevent and treat acute rejection after transplantation.However,the long-term systemic administration of FK506 would induce complications such as opportunistic infections and malignancies,making it challenging to balance its efficacy and toxicity in the clinic.FK506 can inhibit T lymphocyte proliferation and activation efficiently.And it is well known that the proliferation and activation of T lymphocytes occurs primarily in the lymph nodes.Thus,we speculate that delivering FK506 to lymph nodes would improve the therapeutic index of FK506.Our previous study found that macrophages carried YC can migrate to lymph nodes and lesion site.Herein,we hypothesized that YC can deliver FK506 to lymph nodes for managing rejection.Thus,the aim of this study was to fabricate a biomimetic YC system for diagnosis and treatment of transplant rejection.Part 1 Preparation and characterization of yeast capsulesObjective: Preparation and characterization of yeast capsules.Methods:(1)YC were extracted from yeast using hot acid base organic solvent menthod.(2)The main components,morphologies and size of YC were observed via fourier transform infrared spectroscopy(FTIR),thermogravimetric analysis(TGA),transmission electron microscopy(TEM),scanning electron microscopy(SEM),and confocal laser-scanning microscopy(CLSM).(3)The safety of YC was evaluated by blood biochemical analysis and hematoxylin-eosin(HE)staining.Results:(1)YC were successfully extracted with diameter of 3.74 ?m.The TEM images showed that several cytoplasmic components were lost in YC compared to intact yeast cells.And the wrinkled surfaces and pore structure were observed on the surface of YC from the SEM.(2)YC were mainly comprised of ?-(1,3)-D-glucan,which could be recognized by the dectin-1 receptor.(3)The function biomarkers of liver and kidneys,including alanine transaminase(ALT),aspartate aminotransferase(AST),blood urea nitrogen(BUN),and creatinine(CR),remained unchanged after oral administration of YC.(4)After oral administration of YC,no significant changes were observed in the content of white blood cells,lymphocytes,monocytes,neutrophils,red blood cells and hemoglobin compared with the control group.(5)The results of HE staining showed that the main organs including the heart,lungs,liver,spleen,and kidneys had no obvious tissue damage after oral administration of YC.Conclusion: The YC were successfully extracted with an excellent safety profile,providing an ideal and cost-effective candidate for tracking macrophage in vivo.Part 2 Yeast capsules with aggregation induced emission characteristics for non-invasive monitoring of transplant rejectionObjective: Fabricating AIEgen/YC to monitor the progress of rejection and evaluate the therapeutic effect of immunosuppressive therapy in vivo.Methods:(1)Fabrication of AIEgen/YC by encapsulating AIEgen(HBTTPIP,HBTTPAP,HBTTPEP)into the hollow structure of YC.Then AIEgen/YC were characterized by TEM,SEM and confocal laser scanning microscopy(CLSM).In addition,molecular dynamics simulation was performed to elucidate the interaction between HBTTPEP and ?-glucan.(2)The intracellular uptake of AIEgen/ YC were evaluated by TEM,CLSM and flow cytometry(FCM).(3)Experiments of sequential cellular endocytosis were performed to assess the cellular endocytosis function of macrophages,and assay of Transwell was used to evaluate the migration function of macrophages.(4)The cytotoxicity of AIEgen/YC was determined by CCK-8 kit.(5)The skin graft model was established between BALB/c and C57BL/6 mice.(6)Allografts were collected at 1,3,5,and 7 d after transplantation for HE staining and immunohistochemical staining(IHC).(7)The diagnosis of rejection was obtained using an in vivo imaging system after administration of PBS,AIEgen and AIEgen/YC,then the major organs and lympho nodes were harvested for ex vivo imaging.(8)The therapeutic effect of rejection after FK506 treatment was assessed by oral AIEgen/YC.Results:(1)AIEgen/YC were successfully prepared with good stability.Further,the encapsulation efficiency of HBTTPIP,HBTTPAP and HBTTPEP was determined to be 79.0%,86.9% and 91.6%,respectively.(2)The confocal fluorescent imaging,morphology analysis,and molecular dynamics simulations indicated that AIEgen was trapped in the cavities of YC and bonded with the triple helical structure of ?-glucan,restricting the intramolecular rotation and activation of NIR emission.(3)AIEgen/YC can be endocytosed by macrophages through the dectin-1 receptor,without affecting the macrophagic phagocytosis and chemotaxis.(4)AIEgen/YC can track macrophages for up to 7 days.(5)The skin transplantation models were established with success rate more than 95%.(6)The signals of AIEgen/YC were validated to correlate well with the degree of rejection.(7)A significantly stronger fluorescence signal was detected in the allograft skin than that of isograft skin after oral administration of HBTTPEP/YC.(8)The fluorescent intensities of allograft skins were increased from POD 1 to POD 7 after single oral HBTTPEP/YC,indicating that HBTTPEP/YC can be leveraged to monitor the progress of rejection.(9)After treatment with FK506,the fluorescent signals of HBTTPEP/YC were significantly decreased in the graft compared to the control,suggesting that administration of HBTTPEP/YC can assess the efficacy of immunosuppression therapeutics.Conclusion: We have successfully engineered AIEgen/YC by encapsulating AIEgen into the hollow structure of YC.Among them,HBTTPEP/YC can be endocytosed by macrophages with the highest efficiency,and realize long-term tracking of macrophages without hampering the macrophagic function.Furthemore,oral HBTTPEP/YC can be leveraged to monitor the progress of rejection and evaluate the prognosis of immunosuppressive therapy.This work offers a novel and universal strategy for tracking macrophages in vivo,which can dynamically monitor the recipient's immune response to the graft.Part 3 Yeast capsules deliver FK506 to lymph nodes for treatment of rejectionObjective: Preparing biomimetic FK506/YC for rejection therapy after heart transplantation.Methods:(1)FK506/YC were prepared by self-deposition,then FK506/YC were characterized by TEM,SEM and CLSM.(2)The in vitro release tests were performed using centrifuge method.(3)Heterotopic heart transplantation(HT)was accomplished by using a microsurgical technique.(4)The survival of the grafts was monitored by daily palpation,and the endpoint was defined as complete cessation of palpable cardiac graft beats.(5)The HT rats received different treatments on postoperative day(POD)1 to 5 by gavage.Allografts and isografts were harvested on POD 7 to assess the pathology and T lymphocytes infiltration.The secretion of IL-2 and IFN-? were assessed by ELISA analysis and real-time PCR.(6)Blood urea nitrogen(BUN)and serum creatinine(CR)were examined by an automatic biochemical analyser to monitor the nephrotoxicity of the experimental groups.(7)After treatment with fluorescent probe labeled YC,Peyer's patches,MLNs and allograft were collected to obtain the oral route of YC by CLSM and in vivo imaging system.Results:(1)FK506 was successfully loaded into the YC with diameter of 3.56 ?m.The encapsulation efficiency of FK506/YC was about 80%,and the release profiles of FK506 showed that about 60% of the encapsulated FK506 was released within 24 h from the YC.(2)The FK506 in MLNs were much higher for FK506/YC compared with that of FK506 after a 24 h treatment.(3)The HT models were established with success rate more than 90%.(4)The FK506/YC significantly prolonged allograft survival compared with the PBS group(mean survival time,17.8 ± 1.9 versus 7.3 ± 1.0 days;P < 0.01).(5)The representative HE stained allograft sections at POD7 showed that myocardial tissues of FK506/YC group resembled those of isografts,which represented little lymphocyte infiltration and myocyte necrosis.Furthermore,T cell infiltration,and secretion of IL-2 and IFN-? were dramatically reduced in the FK506/YC group.(6)No nephrotoxicity was observed after five consecutive administrations of FK506/YC.(7)YC can be disseminated to the MLNs from the Peyer's patches,and they can accumulate in MLNs.Conclusion: FK506/YC were successfully developed to achieve lymph nodes specific targeting delivery.Treatment of rejection with FK506/YC significantly prolonged the survival of cardiac allografts,indicating that FK506/YC is a promising strategy for the treatment of cardiac allograft rejection.