iRGD靶向声学脂质体在类风湿关节炎诊疗中的作用

Efficacy of internalized RGD-modified echogenic liposomes in diagnosis and treatment in a mouse model of rheumatoid arthritis

目的制备集治疗与成像为一体的载甲氨喋呤(MTX)和吲哚菁绿(ICG)iRGD靶向载药声学脂质体(iRGD-MTX-ICGELIP),观察其靶向性及联合低频超声体外抑制滑膜细胞(MH7A)增殖的效果。方法采用薄膜水化法和冷冻冻干法制备iRGD-MTX-ICG-ELIP,检测其一般特性及声学响应性,通过细胞摄取实验验证iRGD-MTX-ICG-ELIP的体外靶向结合性能,构建类风湿关节炎小鼠模型,通过靶组织的药物荧光强度验证iRGD-MTX-ICG-ELIP的体内靶向性;CCK8法检测iRGD-MTXICG-ELIP联合超声体外抑制MH7A增殖的效果。结果制备的iRGD-MTX-ICG-ELIP粒径为134.4±17.6 nm,电位为-10.07±4.28 mv,iRGD-MTX-ICG-ELIP中MTX和ICG的包封率分别为(62.56±0.77)%和(95.13±0.82)%;其联合低频超声控释药物发现,随超声作用强度增加和作用时间的延长,MTX与ICG释放均增多。细胞摄取实验表明血管内皮细胞HUVECs对iRGDMTX-ICG-ELIP的摄取效率比对MTX-ICG-ELIP摄取效率高1.89倍,差异有统计学意义(P<0.05),活体成像实验显示iRGDMTX-ICG-ELIP在RA发病关节的荧光强度明显强于非靶向组;CCK8检测结果显示,iRGD-MTX-ICG-ELIP联合超声组的MH7A存活率为(32.49±3.04)%,与未联合超声组比较差异有统计学意义(P<0.05)。结论本研究制备的iRGD-MTX-ICGELIP粒径小、均一性好,对HUVECs和RA病变关节组织具有较好的靶向性。较佳的药物包封率和声学响应性增强了iRGDMTX-ICG-ELIP联合超声抑制MH7A的增殖作用,为后续更精准有效的治疗类风湿关节炎提供前期基础。

Objective To prepare internalized RGD（iRGD）-modified echogenic liposomes containing methotrexate（MTX） and indocyanine green（ICG）（iRGD-MTX-ICG-ELIP） and evaluate its targeting efficiency and inhibitory effect combined with ultrasound on synovial cells.Methods iRGD-MTX-ICG-ELIP was prepared by the thin-film rehydration and freezelyophilization method and its general characteristics and acoustic responsiveness were assessed.The targeting effect of the prepared liposome was observed by assessing its cell uptake in vitro.In a mouse model of rheumatiod arthritis,the targeting effect of the prepared liposome was determined by detecting the fluorescence intensity of the drug in arthrosis.The inhibitory effect of iRGD-MTX-ICG-ELIP combined with ultrasound on synovial MH7 A cells in vitro were investigated using CCK8 test.Results The average diameter and zeta potential of iRGD-MTX-ICG-ELIP was 134.4±17.61 nm and-10.07±4.28 mV,and the entrapment efficiency of MTX and ICG was（62.56±0.77）% and（95.13±0.82）%,respectively.With ultrasound exposure,the release of MTX and ICG from iRGD-MTX-ICG-ELIP increased with the ultrasound intensity and with the exposure time.In HUVECs,the uptake efficiency of iRGD-MTX-ICG-ELIP was 1.89 times higher than that of non-targeted MTX-ICG-ELIP（P〈0.05）.In vivo imaging of mouse joint with rheumatiod arthritis showed that the fluorescence intensity of iRGD-MTX-ICG-ELIP was significantly stronger than that of the non-targeted liposome.CCK8 assay showed that iRGD-MTX-ICG-ELIP combined with ultrasound resulted in a survival rate of MH7 A cells of（32.49±3.04）%,significantly lower than the rate of cells treated with iRGD-MTX-ICG-ELIP but without ultrasound（P〈0.05）.Conclusions iRGD-MTX-ICG-ELIP has a suitable particle size and can effectively target HUVECs and the joints with rheumatiod arthritis.With a good drug entrapment efficiency and acoustic responsiveness,the drug-loaded liposome shows enhanced inhibitory effect on MH7 A cells combined with ultrasound in vitro,suggesting its potential in the treatment of rheumatoid arthritis.