| Rheumatoid arthritis(RA)is a systemic autoimmune disease that leads to synovial inflammation and bone destruction.Although significant progress has been made in the clinical treatment of RA,long-term use of anti-rheumatic drugs still has many drawbacks,including gastrointestinal bleeding,liver and kidney toxicity.In addition,surgical treatment has disadvantages such as easy adhesion after major trauma.Therefore,the exploration of RA treatment has been a research hotspot and difficulty in this field.RA inflammatory process occurs mainly in synovial tissue,and macrophages play a crucial role in the pathophysiological responses.Macrophages exist in two different phenotypes:a pro-inflammatory classical(M1)phenotype and an anti-inflammatory(M2)phenotype.In RA,macrophages are primarily of M1 phenotype,causing inflammatory response and cause damage of joint bone and cartilage by producing a series of pro-inflammatory cytokines.Although the pathogenesis of RA has not been clearly elucidated,the mechanism involves unwanted immune responses that attack the joints.Therefore,the M1 macrophages have been considered as a vital therapeutic target to relive symptoms of RA.Nowadays,phototherapy including photodynamic therapy(PDT)and photothermal therapy(PTT)is paid ever-growing attention in cancer treatment.RA synovial tissues have rapid malignant proliferation,strong metabolism and aggressiveness,which are similar to the pathological environment of tumor cell growth.Phototherapy can induce the necrosis and apoptosis of cells by generating cytotoxic reactive oxygen species(ROS)and heat.However,hypoxia microenvironment of RA joints and PDT-mediated oxygen consumption,limiting the application of phototherapy in RA treatment.Therefore,combining phototherapy with oxygen carriers could be a promising strategy for RA treatment.In this study,we prepared the M1 macrophages-targeted liposomes(Cy I&Hb/FA-Lipo)to simultaneously delivery phototherapeutic agent Cy I and oxygen carrier Hb for RA treatment.Cy I,an iodinated cyanine dye,which obtained excellent photodynamic and photothermal effects,as well as fluorescence imaging function.Hemoglobin(Hb),an efficient and safe oxygen carrier,with inherent reversible oxygen-binding capability and can transport oxygen molecules to tissues.After folic acid(FA)-mediated internalization,Cy I and Hb were simultaneously released upon near-infrared light(NIR)irradiation,and synergistic PDT and PTT mediated by Cy I could induce M1 macrophages apoptosis at the lesion area.Meanwhile,the co-delivered Hb supplied oxygen to facilitate ROS generation and subsequently enhance PDT efficiency.Moreover,the relieved hypoxia environment promoted the transformation of M1 macrophages into anti-inflammatory M2 macrophages,achieving a synergistic anti-rheumatoid arthritis effect.In this study,the encapsulation and fluorescence properties of Cy I&Hb/FA-Lipo were identified by absorption and fluorescence spectra.The oxygen-carrying capacity of Cy I&Hb/FA-Lipo was investigated by dissolved oxygen meter.The singlet oxygen sensor green(SOSG)probe and the expression of HIF-1αwere used to investigate the~1O2 production and the hypoxia levels in the joint tissue.The macrophage polarization was investigated by immunofluorescence staining.Thermocouple thermometer was used to evaluate the PTT effect;MTT experiments and flow cytometry were used to investigate the inhibition ability and the percentage of apoptosis and necrosis of M1 macrophages.Confocal laser scanning microscope(CLSM)was used to observe the targeting of Cy I&Hb/FA-Lipo in vivo.Collagen-induced arthritis(CIA)joints was established and used to evaluate the therapeutic effect of Cy I&Hb/FA-Lipo.The experimental results showed that the Cy I&Hb/FA-Lipo prepared by the thin film hydration method had good serum stability and storage stability.The results of UV absorption spectra and fluorescence spectra showed that Cy I and Hb were successfully encapsulated in liposomes and retained the fluorescent properties of Cy I,which could be excited by NIR for real-time monitoring and imaging in vivo.The results of in vitro photodynamic and oxygen-carrying capacity experiments showed that Hb liposomes could continuously transport oxygen into hypoxic environment,thus acting as an oxygen-carrying liposome for enhancing photodynamic effects and increasing anti-inflammatory M2 macrophages.The results of in vitro cell studies showed that Cy I&Hb/FA-Lipo could be efficiently taken up by M1 macrophages through the uptake pathway mediated by FA and FRβ.Meanwhile,the oxygen carrier Hb could provide oxygen and improve the yield of ~1O2,exerting the combining anti-inflammatory effect of PDT/PTT/oxygen.The results of immunofluorescence experiments showed that Cy I&Hb/FA-Lipo could effectively induce the apoptosis of pro-inflammatory M1 macrophages and the repolarization of M1macrophages to anti-inflammatory M2 macrophages.The in vivo treatment and anti-inflammatory results in mice showed that the Cy I&Hb/FA-Lipo group had a better therapeutic effect on RA under NIR irradiation,and could exert an anti-inflammatory effect by increasing the M2 macrophages in the inflammatory tissue of RA.The prepared liposomes(Cy I&Hb/FA-Lipo)could effectively induce pro-inflammatory M1 macrophages apoptosis and increase anti-inflammatory M2macrophages via PDT/PTT/oxygen combining strategy.This iodinated cyanine dye-based oxygen-carrying phototherapy liposome is expected to provide new ideas and strategies for RA-targeted phototherapy and macrophage repolarization. |
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