Study On Preparation Of Enzyme-responsive Tacrolimus Nanoparticle And Their Prevention Of Acute Rejection Of Liver Transplantation

Background:Liver transplantation（LT）is the only effective treatment for end-stage liver disease and the best choice for acute liver failure,immunological and metabolic liver diseases.Mature surgical transplantation techniques,standardized perioperative management,and reasonable immunosuppressive regimens have significantly prolonged the postoperative survival of recipients in recent years.However,T cell-mediated acute rejection remains an important risk factor that affecting recipient’s quality of life.Therefore,high doses of immunosuppressants are often used to prevent acute rejection and protect graft function,such as calcineurin inhibitors（cyclosporine and tacrolimus（TAC or FK506））,anti-proliferative drugs（mycophenolate mofetil）,mammalian target protein inhibitor of rapamycin（sirolimus）,and glucocorticoids.Among them,TAC binds to FK506-binding protein in T cells to form a complex and inhibit the activity of calcineurin,thereby affecting the expression of cytokines such as IL-2,IFN-γ,TNF-α,and inhibiting the generation of cytotoxic T cells.TAC has a powerful ability to suppress immune function compared to other immunosuppressants and has become a first-line treatment for organ transplantation.However,TAC has poor water solubility（4～12μg/ml）,low bioavailability（～19%）,and narrow therapeutic window（5～15 ng/ml）.Long-term high-dose use of TAC can cause serious side effects due to the distribution of non-target organs,such as neurotoxicity,hypertension,hyperglycemia,chronic renal inefficiency,opportunistic infections,tumor recurrence,etc.Therefore,it is urgent to develop an innovational TAC delivery system to increase the accumulation of immunosuppressants in the allograft and reduce systemic dosage.Nanoparticle-based drug delivery systems can improve the therapeutic efficacy of drugs and reduce the side effects caused by off-target effects.Importantly,nanoparticles are designed to selectively accumulate in tissues such as tumors or inflammation in a variety of ways,such as particle size is controlled to pass through the inter-endothelial gaps of target organs,targeting ligands are modified to increase cellular uptake,and microenvironment-responsive sensitive fragments are linked to achieve environmentally responsive release or aggregation.Among the above targeting strategies,the method that highly expressed enzymes in disease microenvironment cause morphological changes of nanoparticles to increase the concentration of drugs in target tissues is a promising therapeutic approach.Nanoparticles undergo nano-to-micron scale morphological changes under the cleavage of related enzymes in the microenvironment of target organs,thereby increasing the accumulation of nanoparticles in diseased tissues and slowly releasing drugs to enhance therapeutic efficacy.Enzyme-responsive nanocarriers have achieved great success in the field of cancer therapy in recent years.However,the strategies that enhance the therapeutic efficacy of acute rejection reaction by increasing the accumulation of nanomaterials in allograft are rarely investigated.Therefore,we designed an enzyme-responsive tacrolimus nanoparticle to increase aggregation in the allograft and enhance the treatment of rejection.This nanoparticle has a core-shell structure,with a hydrophobic core delivering TAC and a hydrophilic shell containing a substrate peptide fragment of matrix metalloproteinase 9（MMP9）.Upon exposure to overexpressed MMP9enzyme in the liver allograft,the target on the surface of the tacrolimus nanoparticle is cleaved,resulting in the structural change from nanomicelles to micrometer aggregates,which accumulated in the allograft for sustained release of TAC to enhance the therapeutic efficacy of rejection.Purpose:We aim to develop a novel nanomedicine for the treatment of acute rejection after LT,with the goal of improving the tissue distribution of immunosuppressants,increasing drug concentration at inflammatory sites,reducing off-target side effects,enhancing therapeutic efficacy for acute rejection,and improving patient compliance.Our ultimate objective is to provide a new therapeutic option for the prevention of rejection after organ transplantation.Method:1.TAC and MMP9 enzyme-responsive peptides（L-amino acids）-m PEG are linked to the carboxyl groups of poly（L-glutamic acid）（PLG）through esterification and amidation respectively.The successful synthesis of the products at each stage is confirmed by ¹H NMR and gel permeation chromatography（GPC）.In addition,MMP9 enzyme non-responsive peptides（D-amino acids）are also synthesized as a control for the study.The synthesized TAC prodrug polymers are self-assembled into nanoparticles,LNPs and DNPs,by a nano-precipitation method.The drug loading of TAC in LNPs and DNPs is detected by high-performance liquid chromatography（HPLC）.The particle size,zeta potential,and morphology of LNPs and DNPs is determined by dynamic light scattering（DLS）,and transmission electron microscopy（TEM）.The changes in particle size and morphology of LNPs and DNPs under the action of MMP9 enzyme are detected by DLS and TEM.2.T-cell activation–induced marker assays:Evaluating the inhibitory effect of LNPs on T cell activation in vitro through flow cytometry.3.The acute rejection model of rat liver transplantation is established and the recipient’s liver tissues are obtained at different time periods.The degree of rejection is evaluated by hematoxylin-eosin staining（H&E）and the expression level and m RNA transcription of MMP9 enzyme in liver tissues are determined through Western blot and PCR.The accumulation of Cy7-labeled nanoparticles in various organs is observed by small animal imaging system.4.The therapeutic effects of LNPs on acute rejection after liver transplantation are evaluated:the degree of acute rejection is assessed by H&E staining of liver tissue;the inflammatory cell infiltration and proliferation in liver tissue are investigated through CD3,Foxp3 immunofluorescence,and Ki-67 immunohistochemistry;the systemic immune status of rats is evaluated by analyzing the number and ratio of CD4⁺and CD8⁺T cells in peripheral blood using flow cytometry;the protective ability of LNPs on liver cells is investigated through TUNEL staining,reactive oxygen species（ROS）staining,and liver function tests;the safety in vivo and comprehensive therapeutic effects of LNPs are evaluated through changes in body weight,survival time,H&E staining of major organ on postoperative day（POD）60 d,and Sirius Red staining of liver tissue.Result:1.We have successfully synthesized LNPs and DNPs with particle sizes of 84.8±10 nm and 104±1 nm,respectively,as measured by DLS.The NPs can maintain stable particle size for 48 hours in PBS solution.The Zeta potentials are-14.5±4.0m V and-13.4±3.1 m V,respectively.TEM images show that the NPs have regular spherical structures with sizes ranging from 40 to 80 nm.The drug loading of TAC is measured by HPLC is 18.5%and 20%for LNPs and DNPs,respectively.2.The DLS and TEM analyses confirmed that LNPs undergo micrometer-scale deformations under MMP9 enzyme,while DNPs only undergo slight changes.Western Blot and PCR analysis confirmed that the content of MMP9 enzyme in the allograft with acute rejection was significantly higher than that in the normal liver.In vitro imaging analysis revealed that LNPs accumulate more in allograft than DNPs,and increased over time.3.The expression of activation marker CD69 on T cell surface was analyzed by flow cytometry,which confirmed that both LNPs and DNPs can inhibit the activation of T cells.4.An acute rejection model of LT was established with DA rats as donor and Lewis rats as recipient;Histological analysis of liver tissue stained with H&E confirmed that LNPs can alleviate inflammation in the portal area,as well as damage to bile ducts and veins;Immunofluorescence and immunohistochemistry of liver tissue also showed that LNPs can reduce infiltration of T lymphocytes and increase the proportion of regulatory T cells(T_regs),while decreasing the proliferation of inflammatory cells;Flow cytometry analysis of lymphocyte subsets in the blood on POD 10 confirmed that LNPs can reduce the number of CD8⁺cytotoxic T cells,maintain the CD4⁺/CD8⁺ratio,and thus stabilize the systemic immune microenvironment;LNPs can reduce the generation of ROS in liver tissue and avoid hepatocyte apoptosis,thereby improving liver function;Furthermore,LNPs can promote rapid weight recovery in recipients and prolong their survival time;The pathological analysis of major organs on POD 60 showed that LNPs had good biosafety and can retard liver fibrosis.Conclusion:A novel MMP9 enzyme-responsive TAC nanoparticle has been successfully synthesized in this study,which undergoes a nano-to-micro scale structural change upon MMP9 enzyme activation,thereby increase the aggregation in allograft.In a rat model of acute rejection of LT,the LNPs significantly suppressed the infiltration and proliferation of inflammatory cells in allograft,alleviated graft injury,improved liver function,stabilized the systemic immune microenvironment,promoted body weight recovery,and prolonged recipient survival.This experiment demonstrates that enzyme-responsive nanomedicine with morphological transformation ability is a promising therapeutic strategy for the prevention of acute rejection after LT.