Construction Of NIR-responsive Multifunctional Nano-Delivery Systems And Their Applications In Rheumatoid Arthritis Therapy

Rheumatoid arthritis(RA)is a systemic autoimmune disease characterized by inflammation and hyperplasia of the joint synovial membranes,which seriously affects the life quality of patients.During the occurrence and progression of RA,highly activated macrophages in the synovial membrane accelerate the process of inflammation by secreting large amounts of pro-inflammatory cytokines,making them ideal target cells for the treatment of RA.RA is currently treated clinically mainly with chemical drugs,but most of these traditional drugs have poor selectivity,and their long-term administration may induce toxic side effects on normal tissues and organs,which cause serious systemic complications,including infection,viral hepatitis,or bone marrow dysfunction.Moreover,the pathogenesis of RA is complex,and a single drug for a certain target is not enough to completely inhibit the process of RA,which is the main reason why most RA patients are difficult to be completely cured clinically.To reduce the administration of chemical drugs and compensate for the insufficiency of monotherapy,the combination of multiple targeted treatment regimens with different mechanisms is widely recognized.Small interfering RNA(si RNA),which acts on myeloid cell leukemia-1(Mcl-1),specifically inhibits the expression of the anti-apoptotic protein Mcl-1 and induces apoptosis of activated macrophages in RA synovial membranes.Near infrared(NIR)laser-induced phototherapy,including photothermal therapy(PTT)and photodynamic therapy(PDT),employ phototherapeutic agents that produce local hyperthermia and cytotoxic reactive oxygen species(ROS)under NIR to precisely kill activated macrophages.However,insufficient oxygen supply of RA inflammatory site limits the in vivo anti-inflammatory effect of PDT,but the hypoxic environment caused by its consumption of oxygen can activate the cytotoxicity of the hypoxia-activated prodrug Tirapazamine(TPZ).Given that chemical drugs,nucleic acid drugs and phototherapeutic agents all need to be effectively delivered to the cells to be effective,in this study,we used folate receptor-?(FR-?)overexpressed on the surface of activated macrophages as targets and constructed nano-carriers to co-deliver TPZ,Mcl-1 si RNA,and semiconducting polymer PCPDTBT quantum dots(SPs)into activated macrophages.The combination of chemotherapy,nucleic acid drugs and phototherapeutics for the treatment of RA provides a safe and efficient anti-RA effect.The details are as follows.1.Construction and evaluation of NIR-responsive targeted hybrid nanocarrier for co-delivery of SPs and TPZFirstly,we prepared PCPDTBT with dual functions of PTT and PDT into CTAB-stabilized SPs(CTAB-SPs)to serve as phototherapeutic agents,and then used CTAB as a template to prepare SPs/mesoporous silica hybrid nanoparticles(SMs).Next,PEG-FA was modified on the surface of the nanoparticles to prepare PEG-FA functionalized SMs(SMPFs),which achieved the targeting ability to activated macrophages as well as enhanced in vitro stability and hemocompatibility of the nanoparticles.To address the limitation of the overall efficacy caused by insufficient molecular O₂supply,we loaded the hypoxia-activated prodrug TPZ in the mesopores of SMs(TPZ loaded SMPFs,SMPFs/T),thus taking advantage of the hypoxic microenvironment caused by PDT to activate the hypoxia activated chemotherapy(Ha CT)cytotoxicity of TPZ.By evaluating the physicochemical properties,in vitro targeting ability and anti-inflammatory effect of the constructed nanoparticles,we demonstrated that SMPFs/T can effectively kill activated macrophages,showing the ability to combined treatment of RA with PTT,PDT and Ha CT under NIR laser at808 nm.2.Construction and evaluation of NIR-responsive targeted nanocarrier for co-delivery of SPs,TPZ and Mcl-1 si RNAThrough the analysis of the in vitro release behavior of SMPFs/T,we found that the open mesoporous structure of nanoparticles exposed the loaded drug to the surrounding environment,resulting in the leakage of the loaded drug.To solve this problem and improve the delivery efficiency of nanoparticles,we used FA modified branched polyethyleneimine(PEI-FA)to block the mesoporous of nanoparticles to obtain PEI-FA-coated drug-loaded SPs/mesoporous silica hybrid nanoparticles(PFHNs/T),simultaneously achieve the targeted ability to activated macrophages and reduce the release of loaded drugs during in vivo circulation.Mcl-1 si RNA can interfere with the expression of anti-apoptotic protein Mcl-1 in activated macrophages,thereby inducing apoptosis of the cells.PEI in nanocarriers is the gold standard polymer for nucleic acid drug transfection.Using PEI-si RNA complexes,we realized the combined delivery of nucleic acid drugs(PFHNs/TM).It was found that the targeted combined killing of activated macrophages by PTT,PDT and Ha CT by PFHNs/TM and the RNA interfering effect of Mcl-1 si RNA caused down-regulation of pro-inflammatory cytokines(TNF-?,IL-1?and IL-6)and up-regulation of anti-inflammatory cytokines(IL-10)in AIA rats serum levels,and the symptoms of RA were significantly alleviated.In summary,the multifunctional nano-delivery systems were constructed in this project,which combined with PTT,PDT,Ha CT,and RNAi for targeted killing activated macrophages,has shown superior therapeutic effects for RA in vitro and in vivo compared to monotherapy.We also demonstrate the potential of the proposed multifunctional nano-delivery system for the co-delivery of phototherapeutics,chemotherapeutics and nucleic acid drugs,which provides an experimental basis for the treatment of other inflammatory or tumour conditions.