CN1317037C - 一种磁共振肿瘤靶向对比剂及其制备方法 - Google Patents
一种磁共振肿瘤靶向对比剂及其制备方法 Download PDFInfo
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Abstract
本发明涉及医疗检测试剂技术领域，尤其涉及一种在磁共振成像中能特异性使肿瘤显像的对比剂及其制备方法。本发明的目的在于利用叶酸和叶酸受体高亲和力的特性来实现靶向显影，提供一种特异性、选择性、增加效果好、毒副作用低的磁共振肿瘤靶向对比剂及其制备方法。本发明提供一种磁共振肿瘤靶向对比剂，它是以DTPA-Poly-L-Lysine为磁共振顺磁性钆对比剂的载体，与叶酸偶联成大分子多聚体，分子量为30,000～70,000，该大分子多聚体具有载带Gd3+浓度高、化学性质稳定、纯度高、与叶酸受体高亲和力结合等特性。本发明还提供上述磁共振肿瘤靶向对比剂的制备方法。
Description
技术领域
本发明涉及医疗检测试剂技术领域，尤其涉及一种在磁共振成像中能特异性使肿瘤显像的对比剂及其制备方法。
背景技术
临床磁共振成像(Magnetic Resonance Imag-ing，MRI)中应用普通对比剂进行动态增强能够提高肺癌诊断的准确性，但它是以结节或肿块内血管密度为基础的，反映的是病变的血供特点，不是特征性的；单抗型靶向对比剂应用于人体会产生人抗鼠抗体反应，加上肿瘤间质内压力升高，偶联的大分子通透性差的原因使得单抗型对比剂的靶向效果并不如人们所期望。Unger等利用抗CEA抗体与二乙二胺五醋酸钆(Gd-DTPA)偶联成抗CEA抗体-Gd-DTPA共扼化合物用于结肠腺癌的MR成像研究，结果发现存在着抗体用量大、增强效果差等缺点，且在人体中发生免疫反应，产生抗-抗体限制了重复使用(参考文献：E.C.Unger，W.G.Totty，D.M.Neufeld et al.，Magneticresonance imaging using gadolinium labeled monoclonal antibody.Invest.Radiol.20(1985)，693-700)。
发明内容
本发明的目的在于利用叶酸和叶酸受体高亲和力的特性来实现靶向显影，提供一种特异性、选择性、增强效果好、毒副作用低的磁共振肿瘤靶向对比剂及其制备方法。
本发明提供一种磁共振肿瘤靶向对比剂，它是以二乙三胺五乙酸聚赖氨酸DTPA-Poly-L-Lysine为磁共振顺磁性钆对比剂的载体，与叶酸偶联成大分子多聚体，分子量为30,000～70,000道尔顿。
一种磁共振肿瘤靶向对比剂，它是叶酸与聚赖氨酸、钆喷酸葡胺(Gadolinium diethylenetriamine pentacetic acid，GD-DTPA)的联结体，即叶酸-PL-Gd-DTPA。
本发明还提供上述磁共振肿瘤靶向对比剂的制备方法，包括以下步骤：
①DTPA双酸酐(CaDTPA)的合成
10g二乙三胺五乙酸(DTPA)悬浮于30ml乙酸酐和15ml吡啶的混合液中，于60℃搅拌反应6h，冷却后加适量乙醇过滤，乙醚洗涤，干燥，得白色固体DTPA双酸酐(CaDTPA)粗品。
②DTPA-Poly-L-Lysine制备
取聚赖氨酸(Poly-L-Lysine)10mg，溶解于3ml 0.2mol/LNaH2CO3/Na2HCO3缓冲液(pH＝9.6)中，在冰浴搅拌状态下加入DTPA双酸酐(CaDTPA)溶液，室温下再搅拌反应16小时后，取出反应混合液，用平衡透析法除去游离的CaDTPA，并转换到0.2M NaAc/HAc(醋酸钠/醋酸)缓冲液(pH＝5)。取出透析袋中溶液，减压浓缩后放4℃保存，用于下步反应。
③Gd-DTPA-Poly-L-Lysine制备
Gd2O3加入到0.1mol/L HCl中，加热至60℃，持续反应10min，完全溶解后，蒸发多余的HCl即得到GdCl3的溶液。将制备的GdCl3溶液加入到经过减压浓缩的DTPA-Poly-L-Lysine溶液中，然后在室温下再搅拌反应24～30小时后，用平衡透析法除去游离的Gd3+。制成Gd-DTPA-Poly-L-Lysine。采用高频电感藕合等离子体发射光谱法(ICP-AES)测量样品Gd3+的含量。
④叶酸与Gd-DTPA-Poly-L-Lysine的偶联
把叶酸溶解在0.1mol/L NaH2CO3/Na2HCO3缓冲液(pH＝9.6)，然后加入ECD(双半胱乙酯)和Gd-DTPA-Poly-L-Lysine，在室温下再搅拌反应24～30小时后，用Sephadex CL-4B层析柱除去没联结上的叶酸，收集叶酸与Gd-DTPA-Poly-L-Lysine的联结体，冷冻浓缩即得叶酸-PL-Gd-DTPA。
叶酸-PL-Gd-DTPA体外表征：
1)Gd3+含量测定：采用高频电感藕合等离子体发射光谱法(ICP-AES)测量。用于动物试验的每分子叶酸与Gd-DTPA-Poly-L-Lysine联结体的Gd3+含量是56个。
2)大分子的纯度测定：使用TSK3000(Bio-Rad)分子筛凝胶分析柱，用0.01mol/l，pH＝7.4的PB液洗脱的高压液相色谱(HPLC)分析，出现一个大峰(＞95％)，较小分子量峰＜2％。醋酸纤维薄膜电泳和分子筛柱层析Sepharose CL-4B柱层析分析也没发现大量的较小分子量物质，见图1。
3)活性测定：使用细胞叶酸受体竞争结合法证明叶酸(folate)-PL-Gd-DTPA活性，结果得到与叶酸类似的竞争曲线，说明靶向对比剂folate-PL-Gd-DTPA保持了叶酸与叶酸受体高亲和力结合的特性(KD～10-10M)，见图2。
叶酸-PL-Gd-DTPA荷瘤动物实验：
1)肿瘤动物模型的制备
在无菌条件下，将传代于裸鼠皮下的人肺腺癌瘤体(H460人肺癌细胞株)切成约2mm大小的瘤块，移植于裸鼠(BALB/C小鼠，6-7周龄)右侧偏背侧肩胛下区皮下，约30天后瘤径达到1.0-1.5cm用于物成像实验；
2)实验动物MR成像
将荷人肺癌裸鼠模型(n＝3)用于MRI实验。实验前一天，停止啮齿类动物正常饮食，给予无叶酸饮食。实验时，先用1.5T超导高场MR扫描仪，行平扫。在完成平扫后，将裸鼠固定于注射装置中，用1.0ml注射器经鼠尾静脉注入制备的靶向对比剂叶酸-PL-Gd-DTPA(0.2mmol/kg体重)；注射成功后，再次将裸鼠固定于固定板上，行各时间点扫描。注射试剂后扫描时间点选择：30’、3h、6h、14h、24h、48h、72h。
3)MR信号值测量方法
尽量取相同层面瘤体最大径上的5个圆形兴趣区，面积为2mm，测其ROI值，然后计算其平均值。同时测量肝脏、腿部肌肉ROI值，方法同上。
4)数据处理
采用SAS 6.12版本统计软件，用随机区组设计资料的方差分析方法对数据进行处理，之后用SNK-q检验进行两两间比较。
结果发现，瘤体信号值在注射叶酸-PL-Gd-DTPA后，呈明显强化效果，并随着观察时间的延长，试剂叶酸-PL-Gd-DTPA有向瘤体局部聚集的趋势，峰值出现在约增强后48小时，最大强化率126.0％；而在肝脏中，亦成明显强化效果，峰值出现在约增强后6小时，之后即成下降趋势，见图3至图6、表1。
表1 裸鼠各组织峰值强化率
| ROI值 | 峰值强化率 | ||
| 注射前 | 注射后(峰值) | ||
| 瘤体 | 192.5±15.2 | 521.4±61.9 | 170.8％ |
| 肝脏 | 253.0±23.8 | 476.2±44.2 | 88.2％ |
| 肌肉 | 185.3±19.0 | 262.8±38.8 | 41.8％ |
本发明利用叶酸和叶酸受体高亲和力的特性来实现靶向显影，是配体—受体导向系统，其结合具有特异性、选择性、饱和性、亲合力强和生物效应明显等特点。利用配体为Gd-DTPA的载体，通过受体介导作用，增加药物在病灶局部的浓度、提高疗效，降低毒副作用，达到靶向肿瘤显影目的。
附图说明
图1是试剂纯化后高压液相色谱图：出现一个大峰(＞95％)，较小分子量峰＜2％
图2是使用细胞叶酸受体竞争结合法证明folate-PL-Gd-DTPA活性，结果得到与叶酸类似的竞争曲线
图3是平扫时间点裸鼠横断位图像
图4是30’时间点裸鼠横断位图像
图5是48h时间点裸鼠横断位图像
图6是72h时间点裸鼠横断位图像
具体实施方式
实施例1：一种磁共振肿瘤靶向对比剂，它是叶酸与聚赖氨酸、钆喷酸葡胺GD-DTPA的联结体，即叶酸-PL-Gd-DTPA，分子量为30,000～70,000道尔顿。
实施例2：一种制备实施例1所述的磁共振肿瘤靶向对比剂的方法，它包括以下步骤：
①DTPA双酸酐(CaDTPA)的合成
10g二乙三胺五乙酸(DTPA)悬浮于30ml乙酸酐和15ml吡啶的混合液中，于60℃搅拌反应6h，冷却后加适量乙醇过滤，乙醚洗涤，干燥，得白色固体DTPA双酸酐(CaDTPA)粗品；
②DTPA-Poly-L-Lysine制备
取聚赖氨酸(Poly-L-Lysine)10mg，溶解于3ml 0.2mol/LNaH2CO3/Na2HCO3缓冲液(pH＝9.6)中，在冰浴搅拌状态下加入DTPA双酸酐(CaDTPA)溶液，室温下再搅拌反应16小时后，取出反应混合液，用平衡透析法除去游离的CaDTPA，并转换到0.2M NaAc/HAc(醋酸钠/醋酸)缓冲液(pH＝5)。取出透析袋中溶液，减压浓缩后放4℃保存，用于下步反应；
③Gd-DTPA-Poly-L-Lysine制备
Gd2O3加入到0.1mol/L HCl中，加热至60℃，持续反应10min，完全溶解后，蒸发多余的HCl即得到GdCl3的溶液。将制备的GdCl3溶液加入到经过减压浓缩的DTPA-Poly-L-Lysine溶液中，然后在室温下再搅拌反应24～30小时后，用平衡透析法除去游离的Gd3+。制成Gd-DTPA-Poly-L-Lysine。采用高频电感藕合等离子体发射光谱法(ICP-AES)测量样品Gd3+的含量；
④叶酸与Gd-DTPA-Poly-L-Lysine的偶联
把叶酸溶解在0.1mol/L NaH2CO3/Na2HCO3缓冲液(pH＝9.6)，然后加入ECD(双半胱乙酯)和Gd-DTPA-Poly-L-Lysine，在室温下再搅拌反应24～30小时后，用Sephadex CL-4B层析柱除去没联结上的叶酸，收集叶酸与Gd-DTPA-Poly-L-Lysine的联结体，冷冻浓缩即得叶酸-PL-Gd-DTPA。
Claims (2)
1、一种磁共振肿瘤靶向对比剂，其特征在于它是以二乙三胺五乙酸聚赖氨酸DTPA-Poly-L-Lysine为磁共振顺磁性钆对比剂的载体，与叶酸偶联成大分子多聚体，分子量为30,000～70,000道尔顿。
2、一种制备权利要求1所述的磁共振肿瘤靶向对比剂的方法，其特征在于它包括以下步骤：
①DTPA双酸酐CaDTPA的合成
10g二乙三胺五乙酸DTPA悬浮于30ml乙酸酐和15ml吡啶的混合液中，于60℃搅拌反应6小时，冷却后加适量乙醇过滤，乙醚洗涤，干燥，得白色固体DTPA双酸酐CaDTPA粗品；
②DTPA-Poly-L-Lysine制备
取聚赖氨酸Poly-L-Lysine10mg，溶解于pH＝9.6的3ml 0.2mol/LNaH2CO3/Na2HCO3缓冲液中，在冰浴搅拌状态下加入CaDTPA溶液，室温下再搅拌反应16小时后，取出反应混合液，用平衡透析法除去游离的CaDTPA，并转换到0.2M NaAc/HAc缓冲液，pH＝5，取出透析袋中溶液，减压浓缩后放4℃保存；
③Gd-DTPA-Poly-L-Lysine制备
Gd2O3加入到0.1mol/L HCl中，加热至60℃，持续反应10min，完全溶解后，蒸发多余的HCl即得到GdCl3的溶液，将制备的GdCl3溶液加入到经过减压浓缩的DTPA-Poly-L-Lysine溶液中，然后在室温下再搅拌反应30小时后，用平衡透析法除去游离的GD3+，制成Gd-DTPA-Poly-L-Lysine，采用高频电感藕合等离子体发射光谱法测量样品Gd3+的含量；
④叶酸与Gd-DTPA-Poly-L-Lysine的偶联
把叶酸溶解在pH＝9.6的0.1mol/L NaH2CO3/Na2HCO3缓冲液，然后加入双半胱乙酯ECD和Gd-DTPA-Poly-L-Lysine，在室温下再搅拌反应30小时后，用Sephadex CL-4B层析柱除去没联结上的叶酸，收集叶酸与Gd-DTPA-Poly-L-Lysine的联结体，冷冻浓缩即得叶酸-PL-Gd-DTPA。
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| KR101059285B1 (ko) * | 2009-05-07 | 2011-08-24 | 가톨릭대학교 산학협력단 | 가돌리늄 착물, 그 제조방법, 및 그것을 포함하는 ｍｒｉ 조영제 |
| CN101757642A (zh) * | 2010-03-03 | 2010-06-30 | 天津科技大学 | 一种含钆纳米粒子的制备方法 |
| CN102989015B (zh) * | 2012-12-10 | 2014-12-10 | 西安交通大学 | 一种叶酸受体靶向肿瘤的ct成像分子探针的制备方法 |
| US10265398B2 (en) * | 2015-11-06 | 2019-04-23 | Wisconsin Alumni Research Foundation | Long-lived gadolinium based tumor targeted imaging and therapy agents |
| CN107349434A (zh) * | 2016-05-09 | 2017-11-17 | 中国科学院苏州纳米技术与纳米仿生研究所 | 一种超支化聚合物及其制备方法和应用 |
| CN113514588B (zh) * | 2021-06-02 | 2023-02-10 | 原子高科股份有限公司 | 注射用双半胱乙酯有关物质的高效液相色谱分析方法 |
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| ～(99)Tc～m-DTPA-Folate的制备及其荷瘤裸鼠体内分布 万丹晶等，核技术，第4卷 2004 * |
| ～(99)Tc～m-DTPA-Folate的制备及其荷瘤裸鼠体内分布 万丹晶等，核技术，第4卷 2004;氨基苯甲酸修饰的DTPA螯合物及其弛豫性能 魏俊发等，高等学校化学学报，第19卷第10期 1998 * |
| 氨基苯甲酸修饰的DTPA螯合物及其弛豫性能 魏俊发等，高等学校化学学报，第19卷第10期 1998 * |
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