载125I 标记三螺旋形成寡核苷酸纳米粒的制备及初步应用

摘要

目的：构建载125 I标记反雄激素受体（AR）基因三螺旋形成寡核苷酸（125 I-TFO）纳米粒，并初步探讨其在体外对前列腺癌细胞的抑制作用。方法应用 Bolton-Hunter 法对三螺旋形成寡核苷酸进行125 I 标记，通过复乳化溶剂挥发法制备包载125 I-TFO 的纳米粒（125 I-TFO-NP），测定其粒径、形态、包封率、载药量及体外释放特点。用γ计数器测定前列腺癌细胞 LNCaP 对125 I-TFO-NP 的摄取情况，CCK-8法检测纳米粒对前列腺癌细胞增殖的抑制作用、荧光定量 PCR 和蛋白免疫印迹法检测纳米粒中 AR mRNA 表达水平。结果所获125 I-TFO 的标记率和放射化学纯度分别为（86．87±0．45）％、（96．21％±0．97）％。125 I-TFO-NP 的平均粒径为（298．2±0．5）nm，包封率与载药量分别为（70．6±6．6）％和（1．89±0．13）μg／mg，缓释作用明显。前列腺癌细胞对125 I-TFO-NP 的摄取率高于未包裹125 I-TFO （P ＜0．01）。与空白对照组及 TFO-NP 组比较，125 I-TFO-NP 组中前列腺癌细胞的增殖抑制率升高（P ＜0．01），AR mRNA 及蛋白表达水平均下降（P ＜0．05）。结论制备的载125 I-TFO-NP性状良好，可通过下调 AR 的表达水平，抑制前列腺癌细胞的增殖。纳米粒可为反基因放射治疗提供理想的载体。%Objective Preparing the 1 25 I labeled anti-androgen receptor (AR)gene triplex-forming oligonucleotide loaded nanoparticles,To investigate the inhibitory effects on prostate cancer cells in vitro. Methods Synthesizing the 1 25 I labeled triplex-forming oligonucleotide by Bolton-Hunter Method,then prepar-ing the 1 25 I labeled triplex-forming oligonucleotide loaded nanoparticles (1 25 I-TFO-NP)by double emulsion sol-vent evaporation method.Its size,shape,embedding ratio and drug loading and release characteristics were i-dentified.The uptake of 1 25 I-TFO-NP by prostate cancer cells was measured by γradiation counter.The inhibi-tory effect on cells was test by CCK-8.AR gene expression was detected by Quantitative Real-time PCR and Western Blotting.Results The labeling efficiency and radiochemical purity were (86.87 ±0.45 )% and (96.21 ±0.97)% respectively.The size of 1 25 I-TFO-NP was(298.2 ±0.5)nm in average.The embedding ra-tio and drug loading were(70.6 ±6.6)% and (1 .89 ±0.1 3)μg/mg.The release test in vitro show signifi-cant controlled-release.The uptake of 1 25 I-TFO-NP by prostate cancer cells was significantly higher than that of non-embed 1 25 I-TFO (P <0.01 ).The cellular inhibitory rates of 1 25 I-TFO-NP were significant higher than that of blank control group (P <0.01 ).The AR mRNA expression level of 1 25 I-TFO-NP group were decreased sig-nificantly (P <0.05).Comparing with blank control group and TPO-NP group,the relative expression of AR 1 25 I-TFO-NP group were decreased significantly (P <0.01 ).Conclusions The character of 1 25 I-TFO labeled nanoparticles was fine.The nanoparticles are able to inhibit the proliferation the prostate cancer cells though in-hibiting androgen receptor relative gene and provide ideal carrier for genetic radiotherapy.