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甲状腺未分化癌(anaplastic thyroid cancer,ATC)是目前人类致死性最高的实体恶性肿瘤之一,虽然它的发病率低,但恶性度高,且目前尚无有效治疗手段[1]。131I核素治疗是经典的治疗分化型甲状腺癌的有效方法之一,但在非摄碘性肿瘤中的应用明显受限[2]。既往研究结果证实,热休克蛋白90抑制剂17-丙烯胺基-17-去甲氧基格尔德霉素(17-allylamino-17-demethoxygeldanamycin,17-AAG)和哺乳动物雷帕霉素靶蛋白(mammalian rapamycin target protein,mTOR)抑制剂Torin2均可明显抑制ATC增殖并诱导其凋亡[3-4]。但将多种水溶性差的靶向药物溶解并最大限度地降低正常组织毒性仍为亟待解决的问题。通过纳米载体将多种靶向治疗药物及核素共载,可有望解决上述难题并实现同时作用于多分子靶点的目的,增强其抗肿瘤活性。本研究拟构建一种新型的表面修饰胎牛血清白蛋白(bovine serum albumin,BSA)、标记131I、共载17-AAG与Torin2的多功能介孔二氧化硅(mesoporous silica,mSiO2)纳米载体,并对其表征、释药动力学及胞内核素摄取定量等方面进行相关研究。
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RPMI 1640培养基购自美国Corning公司,胎牛血清、Torin2购自美国Sigma公司,17-AAG购自美国Selleck公司。131I由北京原子高科股份有限公司提供。ATC细胞系FRO为日本长崎大学山下俊一教授惠赠。JEOL-100CXⅡ型透射电子显微镜产自日本电子光学公司,已经过计量质控的1261型γ计数仪产自澳大利亚LKB公司,1260 Infinity型高效液相色谱仪产自美国安捷伦公司。
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采用传统的模板法制作mSiO2纳米载体[5]。按照浓度比为1:1对17-AAG与Torin2两种药物进行装载,将硅球进行氨基化修饰后连接BSA。制备好的纳米载体置于-20℃保存备用。
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通过透射电镜和动态光散射对纳米载体的形貌、粒径和粒径分布进行表征分析。
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将浓度为0.03125、0.06250、0.12500、0.25000、0.50000 mg/mL的17-AAG溶液及浓度为0.03125、0.06250、0.25000、0.50000、1.00000 mg/mL的Torin2溶液分别在335 nm及317 nm处测定特征紫外吸光度值,建立两种药物的标准曲线并通过拟合方程计算得出纳米载体的载药量及包封率,计算方法如下。
$ 载药量(\% ) = (投药量-上清液药量 )/纳米载体的质量\times 100\% $
$ 包封率 (\% ) = ( 投药量-上清液药量 )/ 投药量 \times 100\% $
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将包载17-AAG和Torin2的纳米载体置于释放介质(pH7.4的PBS缓冲液)中,间隔特定时间后每次取样1 mL,用高效液相色谱法分析并计算17-AAG和Torin2的浓度,以纯药的释放度作对照。每组平行3个样本,取平均值。
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采用氯胺T法进行131I标记[6]。取适量10 mg/mL载药纳米载体加入50 μL(5 mg/mL)的氯胺T及37 MBq Na131I溶液,反应60 s,加入50 μL(5 mg/mL)偏重亚硫酸钠终止反应。测定混合物的放射性活度后,以5000 r/min离心8 min,离心半径5 cm,重复3次以提纯标记物。测定产物的标记率及放化纯度。
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将FRO细胞以2.5×105个/孔接种于24孔板,每孔分别加入1.11 MBq的131I-NaI、(17-AAG+Torin2)@mSiO2-BSA-131I,分别培养0.5、1、3、5、7、9、11 h后冲洗并裂解细胞,用γ计数仪测量放射性计数。
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采用SPSS19.0统计学软件进行分析,对符合正态性分布的实验数据采用x±s表示,核素胞内摄取定量实验所得数据经检测为方差不齐,组间均数比较采用独立样本t′检验。P<0.05为差异有统计学意义。
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本研究成功构建了多功能纳米载体(17-AAG+Torin2)@mSiO2-BSA,并经动态光散射分析显示,mSiO2和(17-AAG+Torin2)@mSiO2-BSA的有效直径约为170~250 nm和200~300 nm;透射电镜结果显示纳米载体的分散性好,具有理想的球形形态(图 1)。
将所测不同浓度17-AAG及Torin2的紫外吸光度值(A)与浓度(C)建立药物的标准曲线,通过线性拟合所得17-AAG及Torin2的标准曲线拟合方程分别为C=-0.00416+0.32348A、R2=0.99958及C=-0.05936+0.88709A、R2=0.99038,证明两种药物的标准曲线有较高的线性相关程度(图 2),代入公式(1)、(2)得到mSiO2仅装载单药时分别对17-AAG及Torin2的载药量及包封率,并通过计算间接得到(17-AAG+Torin2)@mSiO2-BSA对17-AAG及Torin2的载药量分别为(7.31±0.22)%和(6.04±0.79)%,包封率分别为(86.21±1.32)%和(85.17±2.05)%。
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体外药物释放曲线(图 3)显示,17-AAG和Torin2、纳米载体包裹的17-AAG和Torin2在480 min时的累积释放量分别为(38.69±0.15)%、(20.20±0.13)%、(15.26±0.28)%和(51.20±0.09)%,且基本均在180 min后保持缓慢平稳释放。
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经薄层层析法测定证实131I已成功标记到(17-AAG+Torin2)@mSiO2-BSA上,且所得产物的标记率为66.31%~78.25%,放化纯度高达98.80%~99.42%。
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(17-AAG+Torin2)@mSiO2-BSA-131I可被ATC细胞快速摄取,且于3 h时达摄取高峰,5 h后基本保持动态平衡,FRO细胞对纳米载体携带的131I摄取量明显高于Na131I溶液(t=32.63~109.31,均P < 0.01)(表 1)。
组别 0.5 h 1h 3 h 5h 7h 9h 11 h Na131I 4.40±0.98 4.42±0.77 4.45±0.51 4.50±0.73 4.39±0.55 4.32±0.41 4.21±0.58 (17-AAG+Torin2)@mSio2-BSA-131I 481.64±9.76 516.21±10.81 532.41±8.35 383.32±13.17 307.18±8.95 254.72±11.21 243.56±12.69 t值 84.27 81.80 109.31 49.74 58.49 38.66 32.63 P值 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 注:表中,ATC:甲状腺未分化癌;17-AAG:17-丙烯胺基-17-去甲氧基格尔德霉素;mSiO2:介孔二氧化硅;BSA:胎牛血清白蛋白。 表 1 ATC细胞系FRO在不同时间点的胞内放射性计数(x±s)(×102 CPM)
Table 1. Counts per minute of 131I in the FRO cells at different times(x±s)(×102 CPM)
131I标记共载两种靶向药物的多功能纳米载体的构建
Construction of 131I-labeled and dual-drug loaded multifunctional nanoparticles
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摘要:
目的构建131I标记的、共载17-丙烯胺基-17-去甲氧基格尔德霉素(17-AAG)与Torin2两种分子靶向性药物的新型多功能介孔二氧化硅(mSiO2)纳米载体,测定其表征,了解其释药动力学,以及甲状腺未分化癌(ATC)细胞对其摄取的情况。 方法采用传统的模板法制作mSiO2,按照浓度比为1:1的比例装载两种靶向药物17-AAG与Torin2,并在其表面进行氨基化修饰后连接胎牛血清白蛋白(BSA),测定其基本表征、药物的载药量及包封率。采用高效液相色谱法分析纳米载体所载药物的体外释放情况。采用氯胺T法对纳米载体进行131I标记,测定其标记率及放化纯度。通过核素胞内摄取定量实验了解该纳米载体被ATC细胞的摄取及滞留情况。采用SPSS19.0统计学软件,对数据进行独立样本t'检验。 结果成功制备mSiO2并完成装载(17-AAG+Torin2)@mSiO2-BSA-131I纳米载体,经测定得到两者的有效直径分别约为170~250 nm和200~300 nm,并证实所得纳米载体的分散性好且具有理想的球形形态。131I标记率为66.31%~78.25%,放化纯度为98.80%~99.42%,mSiO2对17-AAG及Torin2的载药量分别为(7.31±0.22)%和(6.04±0.79)%,包封率分别为(86.21±1.32)%和(85.17±2.05)%。证实所得纳米载体对17-AAG与Torin2均具有一定的缓释效果。ATC细胞内核素摄取定量实验结果显示(17-AAG+Torin2)@mSiO2-BSA-131I可被细胞快速摄取且于3 h时达摄取高峰,其细胞摄碘量明显高于Na131I溶液,差异有统计学意义(t=32.63~109.31,均P < 0.01)。 结论mSiO2纳米载体可以实现两种靶向药物的共载及131I的标记,且具有一定的药物缓释作用,ATC细胞能够明显、快速地摄取(17-AAG+Torin2)@mSiO2-BSA-131I。 Abstract:ObjectiveTo construct 131I-labeled and dual-drug-loaded multifunctional mesoporous silica(mSiO2) nanoparticles, and to investigate its characteristics, drug release kinetics, and uptake by anaplastic thyroid cancer(ATC) cells. MethodsThe mSiO2 nanoparticles were constructed through the conventional template method. 17-allylamino-17-demethoxygeldanamycin(17-AAG) and Torin2 were loaded at equal concentrations, and bovine serum albumin was connected to the amino-modified nanoparticles. The characteristics, encapsulation efficiency, and drug loading rates of the nanoparticles were then investigated. Drug release was analyzed by high-performance liquid chromatography. The nanoparticles were labeled with 131I using the chloramine-T method. The time-dependent cellular uptake of 131I-labeled nanoparticles was also analyzed to evaluate the uptake and retention of the nanoparticles in ATC cells. T'-test was used for data analysis using SPSS19.0 software. ResultsThe mSiO2 nanoparticles and (17-AAG+Torin2)@mSiO2-BSA-131I samples were successfully constructed. The average diameters were 170 nm to 250 nm and 200 nm to 300 nm, respectively, with good dispersibility and spherical shape. The labeling rate and radiochemical purity of the 131I-nanoparticles were 66.31% to 78.25% and 98.80% to 99.42%, respectively. The drug loading rates of 17-AAG and Torin2 were 7.31%±0.22% and 6.04%±0.79%, and their encapsulation efficiencies were 86.21%±1.32% and 85.17%±2.05%, respectively. The nanoparticles loaded with 17-AAG and Torin2 exhibited slow drug release behavior. The uptake of(17-AAG+Torin2)@mSiO2-BSA-131I was higher than that of Na131I(t=32.63-109.31, all P < 0.01) and could reach its maximum level at 3 h after incubation. ConclusionsThe mSiO2 nanoparticles loaded with 17-AAG and Torin2 and labeled with 131I exhibited slow drug release behavior. ATC cells could uptake(17-AAG+Torin2)@mSiO2-BSA-131I rapidly. -
表 1 ATC细胞系FRO在不同时间点的胞内放射性计数(x±s)(×102 CPM)
Table 1. Counts per minute of 131I in the FRO cells at different times(x±s)(×102 CPM)
组别 0.5 h 1h 3 h 5h 7h 9h 11 h Na131I 4.40±0.98 4.42±0.77 4.45±0.51 4.50±0.73 4.39±0.55 4.32±0.41 4.21±0.58 (17-AAG+Torin2)@mSio2-BSA-131I 481.64±9.76 516.21±10.81 532.41±8.35 383.32±13.17 307.18±8.95 254.72±11.21 243.56±12.69 t值 84.27 81.80 109.31 49.74 58.49 38.66 32.63 P值 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 注:表中,ATC:甲状腺未分化癌;17-AAG:17-丙烯胺基-17-去甲氧基格尔德霉素;mSiO2:介孔二氧化硅;BSA:胎牛血清白蛋白。 -
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