金属有机框架负载小干扰RNA克服乳腺癌的基质屏障  

Study of metal organic framework with siRNA for overcoming matrix barrierin breast cancer

作　　者：曾铖 张剑 Zeng Cheng;Zhang Jian(Department of Medical Oncology,Fudan University Shanghai Cancer Center Department of Oncology,Shanghai Medical College,Fudan University,Shanghai 200032,China)

机构地区：[1]复旦大学附属肿瘤医院肿瘤内科,复旦大学上海医学院肿瘤学系,上海200032

出　　处：《中华肿瘤杂志》2024年第5期409-418,共10页Chinese Journal of Oncology

摘　　要：目的开发一种新型递送策略,利用金属有机框架(MOF)负载针对整合素αv(ITGAV)的小干扰RNA siITGAV,以克服基质屏障,进而增强乳腺癌内药物递送及免疫可及性。方法构建MOF@siITGAV颗粒,对其进行材料表征。通过细胞摄取实验观察乳腺癌4T1细胞对MOF@siITGAV的摄取情况,细胞计数试剂盒8法检测MOF@siITGAV的细胞毒性。设空白对照组、游离siITGAV组和MOF@siITGAV组,采用实时荧光定量聚合酶链反应检测各组4T1细胞中ITGAV mRNA的表达,采用Western blot检测ITGAV蛋白的表达,采用酶联免疫吸附试验检测TGF-β1的含量。采用3D球体穿透实验观察MOF@siITGAV穿透4T1细胞球的能力。建立三阴性乳腺癌小鼠模型,观察MOF@siITGAV对移植瘤生长及小鼠心、肝、脾、肺、肾等主要脏器的影响,采用免疫组化染色检测肿瘤组织中Ⅰ型胶原蛋白和CD8的表达。结果成功构建MOF@siITGAV颗粒,粒径为(198.0±3.3)nm,电位为-(20.2±0.4)mV。4T1细胞能有效摄取MOF@siITGAV,并触发siRNA的有效释放,MOF@siITGAV组细胞中ITGAV mRNA和蛋白表达水平、TGF-β1的表达水平均显著下调[相对于空白对照组,游离siITGAV组和MOF@siITGAV组4T1细胞中ITGAV mRNA相对表达水平分别为(109.9±19.0)%和(46.5±11.3)%;空白对照组、MOF@siNC组和MOF@siITGAV组TGF-β1浓度分别为(474.5±34.4)pg/ml、(437.2±16.5)pg/ml和(388.4±14.4)pg/ml]。MOF@siITGAV在4T1细胞球中的穿透能力更强,对4T1细胞无明显杀伤效应,0、10、20、40、80和160μg/ml MOF@siITGAV分别处理4T1细胞24 h,细胞活性分别为(99.7±3.5)%、(98.2±5.2)%、(97.3±6.6)%、(92.1±8.1)%和(92.4±4.1)%。MOF@siITGAV能有效抑制小鼠乳腺癌移植瘤的生长,给药15 d后,空白对照组、MOF@siNC组和MOF@siITGAV组小鼠的肿瘤体积分别为(691.1±193.0)mm^(3)、(652.7±306.5)mm^(3)和(135.3±41.9)mm^(3),MOF@siITGAV组小鼠的肿瘤体积小于空白对照组(P=0.025)。与空白对照组和MOF@siNC组相比,MOF@siITGAV组小鼠肿瘤组织�Objective This study aimed to develop a new delivery strategy that utilized metal organic framework(MOF)loaded with small-interfering RNA(siRNA)targeting ITGAV to overcome tumor matrix barrier,and thus enhance drug penetration and immune accessibility in breast cancer.Methods MOF@siITGAV particles were constructed and characterized.The uptake of MOF@siITGAV in breast cancer cell line 4T1 was observed by the cellular uptake assay.The toxicity of MOF@siITGAV was detected by cell counting kit 8(CCK-8).The blank control group,naked siITGAV group and MOF@siITGAV group were set.Real-time fluorescent quantitative polymerase chain reaction(RT-qPCR)and Western blot were used to detect the expressions of ITGAV.The level of transforming growth factorβ1(TGF-β1)in the cell culture medium was detected by enzyme-linked immunosorbent assay(ELISA).The penetration of MOF@siITGAV in 4T1 cells was tested by constructing 3D spheroids.Mouse models of triple negative breast cancer were established.The effect of MOF@siITGAV on the growth of transplanted tumors and main organs was verified.Imminohistochemical(IHC)was used to test the expression of collagen and CD8.Results MOF@siITGAV particles were constructed with sizes of(198.0±3.3)nm and zeta potential of-(20.2±0.4)mV.MOF@siITGAV could be engulfed by 4T1 cells and triggered to release siRNA.Compared to the blank control group,the expression of ITGAV in the MOF@siITGAV group[(46.5±11.3)%]and the naked siITGAV group[(109.9±19.0)%]was lower.TGF-β1 in the cell culture medium of the blank control group,naked siITGAV group,and MOF@siITGAV group was(474.5±34.4)pg/ml,(437.2±16.5)pg/ml,and(388.4±14.4)pg/ml,respectively.MOF@siITGAV could better penetrate into 4T1 spheroids and exhibit no obvious toxicity.The cell viability was(99.7±3.5)%,(98.2±5.2)%,(97.3±6.6)%,(92.1±8.1)%,and(92.4±4.1)%,respectively,after MOF@siITGAV treatment with the concentration of 0,10,20,40,80,and 160μg/ml,respectively,for 24 h.The tumor growth in the MOF@siITGAV group was suppressed significantly.After 1

关 键 词：乳腺肿瘤 金属有机框架 整合素ΑV 基因转染 小干扰RNA 细胞外基质 

分 类 号：R737.9[医药卫生—肿瘤]