Construction And Application Of Nano-scavenging Systems For The Treatment Of Osteoporosis And Autoimmune Diseases

External stimulation(e.g.,pathogens,melittin)and internal stimulation(e.g.,proinflammatory cytokines)are important pathogenic factors and pathological molecules in a series of diseases like inflammation,autoimmune diseases(AIDs),and metabolismrelated diseases,which can trigger systemic immune disorder or local immune microenvironment disorder and then cause the pathogenesis and progression of diseases.Current drugs clinically used to treat diseases associated with immunity and metabolism mainly include glucocorticoids,immunosuppressive agents,antibodies,and so on.Despite their therapeutic effects to some extent in a variety of diseases,they will inevitably affect the body’s immune network and interfere the normal communication between cells,thus bringing uncontrollable side effects.For example,glucocorticoids such as dexamethasone have promising therapeutic effects for several inflammations and AIDs,but their broad-spectrum inhibitory effects on immune cells can impair the recognition and response to pathogen-associated molecular patterns,and the long-term use may induce concurrent infection of multiple bacteria,fungi,and viruses,seriously threatening organism safety.Immunosuppressive agents such as methotrexate can target specific immune cells to exert suppressive efects,but their nonspecific side effects are significant and long-term administration is toxic,and can easily cause damage to bone marrow,liver,kidney,and gastrointestinal.Antibodies can bind and scavenge pathogenic antigens with high efficiency and specificity,thereby exerting therapeutic efficacy.However,many problems remain for antibodies,such as immunogenicity,suboptimal biodistribution,complicated preparation process,high cost,and susceptibility to antibody resistance after repeated administration,resulting in limited clinical efficiency.At the same time,individual antibody can only be directed against a single target,resulting in limited therapeutic efficacy in diseases with complex pathological backgrounds.Therefore,there is a great need for the development of effective and safe immunomodulatory drugs to meet the urgent clinical needs.Nano-scavenging system based on nanomaterial is an emerging therapeutic technique,which is characterized by utilizing the physicochemical characteristics of nanomaterial itself to neutralize or scavenge harmful substances in the body through interactions(like receptor recognition,electrostatic adsorption,and coordination binding)or chemical degradation,respectively.Compared with conventional treatments,nanoscavenging systems can avoid uncontrollable toxic side effects to the normal immune network.In particular,for diseases such as osteoporosis and AIDs that require long-term administration,the effects of traditional medicines on the immune system accumulate with prolonged treatment and eventually destroy the body’s immune system.In contrast,nano-scavenging systems can maximally avoid immune disorders while exerting therapeutic efficacy,thus allowing long-term applications.In this thesis,aiming at important pathogenic factors and pathological molecules involved in diseases such as osteoporosis and AIDs,a series of efficient and safe nanoscavenging systems were designed through polymer structure modulation,screening,and biomimetic modification,for effective scavenging of cell-free DNA(cfDNA),proinflammatory cytokines,and reactive oxygen species(ROS).The nano-scavenging systems achieved excellent efficacy in animal models of osteoporosis and multiple AIDs.The research content of this thesis is listed as follows:Chapter 1 illustrated the concept of nano-scavenging systems,introduced the structures and functions of multiple nano-scavenging systems,detailed the challenges,advantages,and pitfalls of nano-scavenging systems in the treatment of diseases such as inflammation,AID,tumor,and infection by comparison with traditional therapeutic means,and finally introduced the advances of nano-scavenging systems.In Chapter 2,pre-osteoclast(RAW 264.7)membrane-coated poly(lactic-coglycolic acid)nanodecoys(denoted as RAW-PLGA nanodecoys)were designed and synthesized for RANKL and TNF-α scavenging to treat osteoporosis.Abundant RANK and TNF-α receptor on cell membrane could effectively neutralize RANKL and TNF-α,respectively,thus preventing RANKL-induced osteoclastogenesis and TNF-α-induced osteoblast apoptosis,which in turn promoted osteoblastogenesis and mineralization,and finally restored the balance between osteoclasts and osteoblasts.In pre-osteoclasts,RAWPLGA nanodecoys could effectively inhibit RANKL-triggered NF-κB and MAPK pathway activation,thereby preventing osteoclastogenesis.In addition,in pre-osteoblasts,RAW-PLGA nanodecoys inhibited TNF-α-induced early and late apoptosis by 93 and 91%,respectively,thus promoting osteoblastogenesis and mineralization.After systemic administration,RAW-PLGA nanodecoys were able to effectively escape the capture of reticuloendothelial system and prolong blood circulation.In ovariectomy-induced osteoporosis mouse model,RAW-PLGA nanodecoys effectively inhibited serum levels of RANKL and TNF-α by 57 and 69%,respectively,returned them to near normal levels.In addition,RAW-PLGA nanodecoys significantly improved the levels of multiple osteoporosis-related physiological indexes.The bone morphology and indexes of mice treated with RAW-PLGA nanodecoys were basically restored to normal levels,as shown by micro-CT.In Chapter 3,a series of spherical,α-helical,cationic polypeptides with different degrees of polymerization and guanidine-rich side chains were synthesized using different generations of dendrimers as initiators for effective cfDNA scavenging to treat a variety of AIDs including rheumatoid arthritis,systemic sclerosis,and systemic lupus erythematosus.The cationic polypeptide G3-8 with highest cfDNA-scavenging efficiency and lowest cytotoxicity and immunostimulatory was selected through comparison of their properties including cfDNA-binding ability,cytotoxicity,and immunostimulatory properties.Compared with linear polypeptides,spherical G3-8 bound more tightly to cfDNA in serum.Compared with amino-modified polypeptides,guanidine-modified G38 could form salt bridges with the phosphate backbone of cfDNA,resulting in improved cfDNA-binding efficiency.Compared with random-coil polypeptides,α-helical G3-8 possessed higher bonding constant and binding sites,enabling tighter binding to cfDNA.In collagen-induced arthritis mouse model,intra-articularly injected G3-8 significantly alleviated collagen deposition-induced joint swelling while effectively inhibiting the expression of multiple pro-inflammatory cytokines.Thereby,G3-8 inhibited cartilage erosion and joint tissue fibrosis of mice,as well as improving exercise capacity.In systemic sclerosis mouse model,subcutaneously injected G3-8 obviously downregulated the levels of multiple pro-inflammatory cytokines and immunoglobulins,exhibiting excellent anti-inflammatory capacity.After G3-8 treatment,systemic sclerosis mice showed rapid skin tissue recovery,significant fibrosis reduction,and collagen deposition decrease in skin tissues.Finally,in systemic lupus erythematosus mouse model,intravenously injected G3-8 downregulated the levels of multiple autoantibodies,significantly improved renal function,and successfully suppressed the inflammatory cytokine storm triggered by tape stripping injury.Based on the macrophage membrane-coated biomimetic nanodecoys in Chapter 2 and cfDNA nano-scavengers in Chapter 3,Chapter 4 developed a multi-target nanoscavenging system targeting pro-inflammatory cytokines,cfDNA,and ROS,which achieved multi-dimensional regulation of the AID immune microenvironment and inflammatory response.Based on G3-8 preferred by Chapter 3,a composite nanoscavenging system was constructed by adsorbing catalase(CAT)via electrostatic interaction and further encapsulating macrophage(RAW 264.7)membrane on the surface.Macrophage membrane obviously improved blood circulation and increased inflammation-targeting efficiency of G3-8 cationic carrier,and the blood circulation halflife was prolonged from 1.89 h to 13.03 h.In addition,macrophage membrane could efficiently scavenge a variety of pro-inflammatory cytokines(such as TNF-α,IL-6,and IL-1β).At the inflammatory sites,CAT could decompose H2O2 to relieve oxidative stress.In addition,the oxygen bubbles generated by H2O2 decomposition could promotemacrophage membrane shedding and G3-8 exposure,thus efficiently scavenging cfDNA.In collagen-induced arthritis and inflammatory bowel disease mouse models,composite nano-scavenging system almost restored the serum levels of cfDNA to normal level,significantly reduced the concentrations of multiple pro-inflammatory cytokines,as well as reducing the concentrations of ROS and oxidative stress-related molecules such as myeloperoxidase and malondialdehyde.In addition,the composite nano-scavenging system effectively inhibited inflammatory cell infiltration in synovium and colon,reduced clinical scores,and promoted organ and tissue repair.In Chapter 5,we summarized the doctoral thesis and provided perspectives for future work.In this doctoral thesis,a series of nano-scavenging systems based on in vivo harmful substance-scavenging strategies have been exploratively designed for the treatment of osteoporosis and AIDs.This thesis expands the application of nano-scavenging systems for disease treatment and provides a new strategy for the scavenging of multiple harmful substances in immune microenvironment.In addition,synergistic therapeutic strategies for scavenging against multiple targets can help to deeply understand the complex regulatory mechanisms of the immune microenvironment and provide instructive strategies for immunotherapy based on microenvironment regulation.Meanwhile,the systematic study and regularity findings in this study on cell membranes and polypeptides will also lay the theoretical and experimental foundation for developing novel nanoscavenging systems.