Experimental Animal Study On New Strategy And Mechanism Of TLR4 Agonist Sustained-release Nanoparticlesin Sepsis Protection

Sepsis is one of the most common causes of death in hospitalized patients.According to the investigation,more than 19 million people suffer fromsepsis-related diseasesin the world each year.And a mortality rate induced by sepsis is more than 40%.Furthermore,approximately 3 million of the living patients suffered from sepsishave organ dysfunction or cognitive impairment such as infective cardiomyopathy.Sepsis is now defined as a life-threatening disease of organ dysfunction caused by a dysregulated host immune response to infection.The host response in sepsis patients is usually uncontrolled,overwhelming and finally leads to multi-organ failure.The pathogenesis of sepsis is a very complex pathological process involving hyperactivation and suppression of the immune response.And the later stage of immunosuppression is detrimental to the host and makessepsis patients susceptible to secondary infections.It is found in the studies that the death in sepsis patients,63%of sepsis patients dying after 5 days in initial infection,is closely associated with in vivoimmunosuppression.Furthermore,it is found that immunomodulators can regulate the release of cytokines in vivo by activating multiple signaling pathways,improve the immune disorder in sepsis patients,maintain normal immune function,reduce organ damage,and ultimately reducesepsis mortality.Toll-like receptors(TLR)area kind of important intrinsic immune pattern receptor in vivo,which can be activated to initiate anti-infective defense after pathogens invasion.TLR family plays an important role in recognition of microbial pathogens.TLR4 signaling pathway has an important role in the prevention and treatment of sepsis,especially in regulating in vivoimmune system to achieve sepsis protection.The activation of TLR4 signaling pathwaycould reduce the use of antibiotics and other drugs and further decreaseside effect induced by treatment.Therefore,the therapeutic method based on TLR4 signaling pathwaymay provide a new strategy for the treatment of sepsis.In recent years,the development of nanomedicine has provided new therapies and strategies for the prevention and treatment of sepsis.Therefore,we use a kind of TLR4 agonist sustained-release nanoparticleto investigate the sepsis protection and the in vivomechanism in this study for providing new therapeutic strategyin sepsis treatment.Part Ⅰ:Synthesis and characterization of MPLA@PLGA nanoparticlesPurpose:Activation of TLR4 signaling pathway is important for sepsis treatment.Due to the poor water solubility,monophosphate lipid A(MPLA),a TLR4 agonist,cannot be used directly.Therefore,MPLA was loaded into poly(lactic-co-glycolic acid)(PLGA)nanoparticles to form the nanoparticles(MPLA@PLGA)with TLR4 activity.Furthermore,MPLA@PLGAnanoparticles were characterized for their physicochemical properties.Methods:By oil-in-water(O/W)emulsion method,we prepared a kind of nanoparticles,namely MPLA@PLGA nanoparticles,using PLGA nanoparticles loaded with hydrophobic TLR4 agonist MPLA,and characterized the prepared nanoparticles by the transmission electron microscopy to observe the shape,the particle size and zeta analyzer to detect the hydrated size of nanoparticles,the endotoxin assay to measure the loading rate and release of MPLA,the particle size and zeta analyzer to measure the dispersion and stability of MPLA@PLGA nanoparticles in different solutions,and the fluorescence imagingof small animalto analysizethein vivo retention of MPLA@PLGA nanoparticles.Results:The results showed that the nanoparticles had a uniform spherical structure under transmission electron microscopy.It was showed that the average particle size of MPLA@PLGA nanoparticles was about 110 nm.The loading rate of MPLA in PLGA nanoparticles was 49.43%measured by endotoxin detection kit.Furthermore,MPLA loaded intoPLGA nanoparticles achieved sustained release.In addition,MPLA@PLGA nanoparticles were stable in deionized water,phosphate buffer saline(PBS)solution and Dulbecco’s modified eagle medium(DMEM)in different time periods indicates that the nanoparticles have good stability in various solutions.PLGA nanoparticles loaded with fluorescent dye DID(DID@PLGA)were used to simulate the retention behavior of MPLA@PLGA by in vivo fluorescence imaging of small animals,and the results showed that the PLGA nanoparticles loaded with MPLA had a long retention timein vivo.Conclusion:MPLA@PLGA nanoparticles were successfully prepared,and the prepared MPLA@PLGA nanoparticles have good biocompatibility,stability and sustained release and can be used for subsequent research analysis.Part Ⅱ:Cytokine changes associated with nanoparticles(MPLA@PLGA)in septic mice and the mechanism of the enhanced natural immune systemPurpose:To investigate the changes of cytokines and natural immune cellsinduced by the nanoparticles(MPLA@PLGA)intwoinfection models,including escherichia coli(E.coli)infectionmodel andmultibacterial infection model,for analyzing the mechanism of theenhanced natural immune system in order to provide new ideas for immune regulation of sepsis.Methods:Firstly,the optimal action of MPLA@PLGAnanoparticles was studied.The mice were randomly divided into PBS-treated group,MPLA@PLGA 6h-treated group,MPLA@PLGA 24h-treated group,and MPLA@PLGA 48h-treated group.After E.coli challenge,the survival rate and weight change of mice were recorded.The protective effect of MPLA@PLGA nanoparticles in the internal organs of septic mice was subsequently studied.The main organs of the mice were collected and stained with Hematoxylin-eosin(HE)at 16h after E.coliinfection.Furthermore,the mechanism of the immune system enhanced by MPLA@PLGA was investigated.The mice in the PBS and MPLA@PLGA groups were analyzed at 24 hours(h)after E.coliinfection.The various cytokines and immune cells,including F4/80+CD206+ cells,CD4+ T cells,CD8+ T cells and NK cells,were studied.After that,we studiedthe protective effect of MPLA@PLGAnanoparticles in the cecum ligation and puncture(CLP)model.The mice were randomly divided into sham group,PBS group and MPLA@PLGA group.After CLP challenge,the survival rate and weight changes of mice in the three groups were recorded.Finally,we studied the effect of MPLA@PLGA nanoparticles on macrophages in vitro.Raw 264.7 cellswere firstly treated with PBS or MPLA@PLGA.The treated cells were mixed with E.colior fluorescent dye Cy5.5 labeled E.coli toanylysize the polarizationand phagocytosis of macrophages.Results:The results showed that MPLA@PLGA 24h-treatedgroup had the best protective effect.The results of the HE staining and LB plates of the major organs showed that the inflammationand bacterial colony number in MPLA@PLGA-treated mice were lower than those of PBS-treated mice.At24 h after intradermal injection of MPLA@PLGA nanoparticles and PBS,the percentages of NK cells,monocytes and F4/80+CD80+cells were significantly higher in the MPLA@PLGA-treated mice,while the percentages of CD8+T cells,B cells,CD4+T cells,F4/80+CD206+ cells and neutrophils did not change significantly.The various cytokines did not change significantly.After E.coliinfection,the percentages of NK cells,neutrophils and F4/80+CD80+cells were significantly increasedin the MPLA@PLGA-treated mice.And the secretion of various cytokines,including TNF-α,IFN-γ,IL-6,IL-17A and MCP-1,was significantly increased in the MPLA@PLGA-treated mice,while the level of IL-10 was decreased in the MPLA@PLGA-treated mice.The survival rate of mice treated with MPLA@PLGA nanoparticles in the polymicrobial model was higher than that of mice in the PBS-treated group.In vitro experiments,the results confirmed that our prepared MPLA@PLGA nanoparticles enhanced the bactericidal ability of Raw 264.7 cells by promoting their polarization toward F4/80+CD80+and enhancing the uptake of E.coli.Conclusion:The prepared MPLA@PLGA nanoparticles can safely activate TLR4 signaling pathway and pre-activate the natural immune system without "cytokine storm",rapidly up-regulate the levels of various cytokines and the percentages of natural immune cell after bacterial infection.It could effectively enhance the in vivo immune response in the septic mice,protect the major organs in the septic mice,and increase the survival rate of septic mice.It also improved the bacterial phagocytosis and bactericidal activityof macrophages in order toachievesepsis protection in E.coliinfection and CLP models.Part Ⅲ:Long-term protection and specific protection of MPLA@PLGA nanoparticles against E.coliinfectionPurpose:To study the long-lasting protection and specific protection of MPLA@PLGAnanoparticles against E.coli infection,and to provide new ideas for clinical sepsis protection.Methods:The levels of natural immune cells and cytokines in surviving mice treated with MPLA@PLGA at 30 days were analyzed to studythe immune system in the immunosuppressed phase of sepsis.After that,high-dose E.coliinfection was administered to survived mice and healthy mice.The survival rate and body weight changes in the two groups were observed.The bacterial colony in LB platesfrom the main organs of mice were evaluated at 6 h afterE.coliinfection.The specificprotection against E.coli infection in the survived mice treated with MPLA@PLGA was assessed by lethal Staphylococcus aureus(S.a.u.)infection.RESULTS:It was found that compared with healthy mice,the percentages of CD4+T cells,NK cells,monocytes,B cells and CD4+ memory T cells weresignificantly decreased in survivedmice treated with MPLA@PLGA.The percentages of neutrophils,CD8+T cells and CD8+ memory T cells weresignificantly increased.Thefunctional cytokines,especially IL-1β,IL 27,IL-17A,INF-β and IL-10,had more significant increases.All the MPLA@PLGA-treated mice that survived from the first E.coli infection were livedafter the second high-dose E.coli infection.Furthermore,bacterial counts in the major organs of survived mice were significantly decreased.The body weight of survived mice could return to pre-infection levels about 5 days after the second E.coli-induced sepsis.No difference was observed in the survival rate of MPLA@PLGA-treated mice from the first infection andhealthy mice after lethal infection induced by S.a.u.The survival rate in the two groupswas 20%.Conclusion:Immunoparalysis occurs inthe immunosuppressive phase of sepsis.the prepared MPLA@PLGA nanoparticles altered the immune homeostasis of sepsis mice during the immunosuppressive phase of sepsis.It could provide long-lasting protective effect,increase the number of memory immune cells,promote immune memory,and finally enhancerepeated protection against bacterial infection.Furthermore,the protection against bacterial infection had specificity.