| Rheumatoid arthritis（RA）is a kind of autoimmune disease（AD）characterized by the systemic abnormal inflammatory immune response that affects the joints,connective tissues,muscle,tendons,and fibrous tissue.The main symptoms include erythema,swelling,pain,stiffness of systemic multiple joints.In serious cases,it will lead to irreversible organ damage and disability eventually.The crucial triggers for the onset of articular disease are unknown but they probably associate with environmental factors（smoking,microorganisms,and stress）and genetic factors（HLA-DR4 alleles,etc）.The body’s immune system may produce abnormal immune response to autoantigens and tissues under the combined factors mentioned above,which further leads to inflammation and tissue damage.Most of the existing clinical treatment drugs are symptomatic treatment,requiring long-term medication,and can only partially eliminate inflammation,rather than relieve the autoimmune attack of RA from the root cause.Immune tolerance means that the body’s immune system can accurately recognize but do not attack its own antigen,which is an important mechanism to maintain immune homeostasis.However,this mechanism is destroyed in RA,and the immune balance is broken.Antigen specific immune tolerance induced by autoantigen or autoantigen combined with small molecule immunomodulators is a promising treatment strategy.The peripheral immune organs（second lymphoid organs）such as lymph node and spleen are the natural residences for dendritic cells,macrophages,T cells and B cells,and are also important sites for immune response.In immunotherapy,the effective enrichment of therapeutic drugs in peripheral immune organs is conducive to their full interaction with immune cells and can achieve a multiplier therapeutic effect.Therefore,targeted delivery of therapeutic molecules to peripheral immune organs via appropriate pharmacological means is very important to induce strong immune tolerance and inhibit the inflammation of RA.In addition to the secondary lymphoid organs,the peripheral immune system of patients with chronic rheumatic autoimmune diseases usually forms a temporary third lymphoid structures,also known as ectopic lymphoid structures（ELSs）.ELSs refer to the mononuclear lymphocyte clusters formed in peripheral non lymphoid organs or inflammatory sites.ELSs in inflamed joints have the same immune cell composition as lymph nodes,and partly bear the immune response in the lesion.Theoretically,ELSs in rheumatic autoimmune diseases reproduce the structural composition and immune response of secondary lymphoid organs in chronic inflammation sites,and can even express the characteristics of functional germinal centers.For example,the ectopic germinal centers of ELSs maintain and promote autoimmune inflammation by producing disease-specific autoantibodies.According to this feature,the delivery of therapeutic molecules such as autoantigens to the ELSs in the joint synovium may help to induce robust immune tolerance and rebuild immune homeostasis.It has been recently reported that nano-carriers,after intravenous administration,preferably accumulated in inflamed joints owing to the passive targeting caused by extravasation through leaky vasculature and subsequent inflammatory cell-mediated sequestration（ELVIS）.Then,the nanomedicine residing in the inflamed joints will be captured by the antigen-presenting cells in ELSs,and the ELSs targeting will be achieved.Additionally,nanotechnology can also reduce allergic reactions caused by peptide or protein drugs,reduce the toxicity of chemotherapeutic drugs,improve the pharmacokinetic characteristics of drugs,and increase bioavailability and efficacy.Therefore,this study intends to construct a novel nanocarriers with ELSs targeting ability to make therapeutic molecules fully interact with lymphocyte clusters in ELSs,thereby inducing immune tolerance to resist the development of inflammation.In this study,considering the clinical transformation potential of the designed nanocarriers,we designed the novel nano-emulsions（NEs）by using commercialized pharmaceutical excipients（Pharmaceutical excipients in Chinese pharmacopoeia or DMF registration database of FDA）.We envisioned that such a nanocarrier co-loaded with a citrullinated multiepitope self-peptide（CitP）and rapamycin（a small molecule immunomodulator）could co-deliver both cargos to ELSs after intravenous administration,and APCs in ELSs would preferentially capture those nanocarriers for superior tolerance induction.Therefore,the injectable lipid emulsions（ILEs）,widely used in clinic for parenteral nutrition,is used to encapsulate hydrophobic rapamycin in the oil phase.Then,polyethylene glycol（PEG）is modified on the surface of ILEs to increase the stability and long circulation capability of the formulation.The surface of the PEGylation ILEs is decorated with a small amount of iron hydroxide to improve the encapsulation rate of CitP.After intravenous injection,this NEs delivery system can co-deliver citrullinated autoantigen peptides and low-dose rapamycin to the ELSs in inflamed joints.At the stage of preparing the nano-carriers,we chose the same formulation composition as the commercially available ILEs（the same type of excipients but different quality,Lipovenoes MCT）to prepare the oil-in-water emulsion core.Firstly,ILEs,erving as the core of the nano-emulsions（NEs）,were prepared by a high-pressure homogenizer using soybean oil（SO）,medium chain triglyceride oil（MCT）,and egg yolk lecithin.Then a PEGylated lipid DSPE-m PEG2000（N-（carbonyl-methoxypolyethyleneglycol2000）-1,2-distearoyl-sn-glycero-3-phosphoethanolamine-sodium salt）and ferric hydroxide were mixed with the obtained ILEs for surface coating via the hydrophobic interaction between DSPE and egg yolk lecithin as well as the electrostatic interaction between PEGylated ILEs（with zeta-potential around-35 m V）and Fe（OH）3,respectively.To prepare NEs@CitP/Rapa,rapamycin was incorporated into oil phase by the“like-dissolves-like”principle,while the CitP peptide was absorbed on the surface via coordinate-covalent bonds and electrostatic interactions.The size and zeta-potential of NEs@CitP/Rapa measured by dynamic light scattering（DLS）were 129.2±4.62 nm and-33.7±2.1 m V,respectively.Transmission electron microscopy（TEM）images showed that the NEs were uniformly spherical.Then,the ability of NEs to encapsulate hydrophobic rapamycin and hydrophilic CitP was studied.The encapsulation efficiency（EE）of CitP and rapamycin measured by high performance liquid chromatography（HPLC）was 84.5±5.2%and 88.3±5.8%,respectively.In vitro cell experiment was used to investigate the uptake efficiency and toxicity of different components in our preparation.Compared with free drug,the NEs could significantly improve the uptake of drug on DC2.4（a dendritic cell line）and bone marrow-derived dendritic cell（BMDC）.The uptake efficiency reached 88.6%and 50%respectively,while free drug group was only 8.9%and 9.9%respectively.Moreover,both free form and NEs were rarely internalized by 3T3 cell.Furthermore,the results of in vitro cytotoxicity experiments showed that all components in NEs had no obvious cytotoxic effect,and NEs encapsulating could reduce the toxicity of rapamycin.At the same time,in vitro and in vivo experiments were conducted to investigate the potential of the formulation in inducing tolerogenic APCs.The results on BMDCs showed that both NEs@CitP/Rapa and NEs@Rapa effectively reversed the maturation of BMDCs caused by LPS in vitro,and the preparations significantly down-regulated the expression of costimulatory molecules CD40 and CD86.The in vivo results on AIA also showed that NEs@CitP/Rapa treatment significantly inhibited the expression of costimulatory molecules on APCs in draining lymph nodes.These results suggested that NEs@CitP/Rapa could effectively inhibit the costimulatory molecules on APCs both in vivo and in vitro.Then,we investigated the inflamed joints and ELSs targeting capability of NEs on the AIA mouse model.After intravenous administration,NEs entrapment significantly improved the drug concentration in the inflamed joints,compared with free form.At the same time,the inflammatory paws were removed at 36 h after the administration and prepared into single-cell suspension,and the ELSs targeting capability was quantitatively assessed by using flow cytometry.The results showed that the uptake efficiency of NEs by follicular dendritic cells（FDCs）in ELSs on AIA mice was significantly higher than that of free group on AIA mice and NEs group on healthy mice,with positive rate was 24.0%,9.3%,7.7%respectively.The uptake efficiency of NEs on other APCs such as DCs and macrophages was also significantly higher than that of free form drugs.In vivo biodistribution results showed that our well-designed NEs could passively accumulate in inflamed joints and then preferably be captured by APCs in ELSs.Finally,we investigate the therapeutic efficacy of our formulation in both acute and chronic RA animal models,and the mechanism of immune tolerance was also studied.The therapeutic efficacy results showed that NEs@CitP/Rapa effectively inhibited the development of inflammation in both AIA and CIA mice.As for therapeutic mechanism,NEs@CitP/Rapa treatment was associated with reducing the production of pro-inflammatory cytokines（i.e.,TNF-α,IL-1β,IFN-γ,IL-6 and IL-17A）,increasing the level of anti-inflammatory cytokines（i.e.,TGF-βand IL-10）,promoting polarization of T and B cells toward regulatory phenotypes（Tregs and Bregs）,decreasing the level of type II collagen specific antibodies in CIA models.Those functions eventually reshaped homeostasis of both cellular and humoral immune responses and produced subsequent excellent therapeutic effects for RA,and thus effectively inhibited the development of inflammation.In conclusion,we have constructed a novel NEs by the commercialized pharmaceutical excipients,and the NEs could simultaneously load hydrophobic rapamycin and water-soluble RA self-antigen peptide.After intravenous injection,this delivery system can co-deliver self-antigens and rapamycin to ELSs in inflamed joints of mouse RA models.Outcomes of therapeutic efficacy demonstrated that NEs@CitP/Rapa successfully induced immune tolerance in the presence of a low-dose rapamycin and thus effectively inhibited the progression of both acute and chronic arthritis.This ectopic lymphoid targeted NEs serves as a promising therapy for RA treatment.Therefore,we summarized three innovations of this research topic:Firstly,this study proposed to target ELSs in inflamed joints to treat inflammation for the first time.We have used nanotechnology to successfully deliver therapeutic molecules into such a structure for inducing immune tolerance,and then effectively suppressed the development of inflammation in RA models.Secondly,this novel NEs is made of commercialized pharmaceutical excipients,and it has the ability to simultaneously loading hydrophobic and hydrophilic drugs.Therefore,this nanocarrier has great potential to be harnessed as delivery vehicles in clinical application owing to the non-toxicity and simple manufacture merits.Finally,we have explored the possibility of a combination therapy of RA autoantigen and rapamycin to treat RA for the first time,and the results showed that the combination therapy effectively inhibited the progression of inflammation in RA mice. |
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