CN110064061A - 一种靶向标记mmp-2的磁共振分子探针的构建方法 - Google Patents

一种靶向标记mmp-2的磁共振分子探针的构建方法 Download PDF

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CN110064061A
CN110064061A CN201910344346.6A CN201910344346A CN110064061A CN 110064061 A CN110064061 A CN 110064061A CN 201910344346 A CN201910344346 A CN 201910344346A CN 110064061 A CN110064061 A CN 110064061A
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钟士江
单煜恒
张哲�
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    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
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    • A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
    • A61K49/1875—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle coated or functionalised with an antibody

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Abstract

本发明公开了一种靶向标记MMP‑2的磁共振分子探针的构建方法，所述MRI诊断神经胶质瘤的新型分子探针由超顺磁氧化铁纳米颗粒(superparamagneticironoxidenanoparticles，USPIO)和基质金属蛋白酶‑2抗体(MMP2Ab)通碳二亚胺法合成。本发明与现有技术相比的优点在于：USPIO‑PEG‑MMP2Ab不仅具有更高的磁饱和强度及弛豫率，且具有良好的生物相容性，经静脉给药后能主动靶向胶质瘤细胞，在磁共振T2WI上产生明显的“阴性对比”效果，并通过磁共振分子成像完成对脑胶质瘤的定性与分级诊断。

Description

一种靶向标记MMP-2的磁共振分子探针的构建方法
技术领域
涉及一种分子探针及其制作方法，特别是一种利用核磁共振技术(MRI)诊断神经胶质瘤的分子探针及其制作方法，属于医疗卫生领域。
背景技术
胶质瘤是原发性人脑肿瘤中的一类恶性疾病，罹患者大多不到5年的生存率，恶性程度高。胶质瘤的治疗是世界医学难题，尽管近年来胶质瘤在手术、化疗与放疗方面取得很大进步，但其治疗效果并没有得到显著提高，很大一部分原因是由于其发病部位位于中枢神经系统，且其相当一部分会浸润迁移至外周组织，所以了解胶质瘤侵袭的分子机制对其早期诊断、明确分级、确定治疗方案有着关键性的作用。目前，脑胶质瘤的定性诊断主要依靠病理切片检查，根据病理结果评估患者预后并选择治疗方法。然而，这种侵入性检查手段不仅给病人带来痛苦同时还会引起出血、脑损伤等并发症。传统的影像学检查方法如CT、MRI所提供的诊断信息有限，不仅不能做到准确分级，甚至难以将脑胶质瘤同脱髓鞘性疾病、炎性假瘤、感染等中枢神经系统病变相鉴别。
分子影像学是近年来兴起的，利用现有影像学技术对人体内部生理或病理过程在分子水平上进行的非侵入性的、实时监测的成像技术。寻找不同疾病中异常变化的标志性生物分子，开发针对疾病靶标的特异性探针是实现分子水平诊断的基础和关键。基质金属蛋白酶(MMPs)家族是一类结构中含Zn2+和Ca2+的蛋白水解酶类，主要分解细胞外基质蛋白酶类成分，包括发育、组织塑形、炎症、退行性疾病、肿瘤生长、侵袭与转移、血管生成等生理病理过程，其中当过表达MMP2时能促进胶质瘤的转移，因此利用分子影像学技术可以实现对胶质瘤的特异性靶向。
发明内容
本发明要解决的技术问题是，为解决以上现有难题，实现对胶质瘤的主动靶向，发明了一种新型的MRI分子探针USPIO-PEG-MMP2Ab。
为解决上述技术问题，本发明提供的技术方案为：一种靶向标记MMP-2的磁共振分子探针的构建方法，所述MRI诊断神经胶质瘤的新型分子探针由超顺磁氧化铁纳米颗粒(superparamagneticironoxidenanoparticles，USPIO)和基质金属蛋白酶-2抗体(MMP2Ab)通碳二亚胺法合成。
本发明与现有技术相比的优点在于：USPIO-PEG-MMP2Ab不仅具有更高的磁饱和强度及弛豫率，且具有良好的生物相容性，经静脉给药后能主动靶向胶质瘤细胞，在磁共振T2WI上产生明显的“阴性对比”效果，并通过磁共振分子成像完成对脑胶质瘤的定性与分级诊断。
作为改进，高温分解法合成USPIO-PEG超顺磁氧化铁纳米颗粒：
1.称取15gPEG加入50ml三口圆底烧瓶中；
2.200ml/min的氩气环境下，磁力搅拌10min匀速升温至80℃；
3.加入0.7g乙酰丙酮铁后保温10分钟，然后以7.2℃/min，25min内升温至260℃，保温1h，停止加热，自然冷却至60℃左右；
4.所得产物用60ml甲苯超声清洗，并置于磁铁上吸附，使用甲苯、丙酮依次超声清洗取下层黑色沉淀即为USPIO-PEG。
作为改进，合成USPIO-PEG-MMP2Ab探针
1.取USPIO-PEG(1mgFe，3.5mg/ml)加入2mLEP管中；
2.称取0.5mgEDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)及1.4mgN-羟基硫代琥珀酰亚胺(sulfo-NHS)，加入100μl去离子水溶解后，迅速转移至USPIO溶液中，室温下反应15min；
3.采用PD-10交换柱对活化后的USPIO进行缓冲液交换，洗脱液采用NaHCO3(0.1M)，然后加入0.5mgMMP2Ab在室温下进行偶联反应，4h后，采用PBS缓冲液进行透析过夜；
4.偶联完毕后，将偶联物置于4℃冰箱储存备用，使用前用无菌PBS洗2次，1次5min。
具体实施方式
本发明在具体实施时，一种靶向标记MMP-2的磁共振分子探针的构建方法，所述MRI诊断神经胶质瘤的新型分子探针由超顺磁氧化铁纳米颗粒(superparamagneticironoxidenanoparticles，USPIO)和基质金属蛋白酶-2抗体(MMP2Ab)通碳二亚胺法合成。
高温分解法合成USPIO-PEG超顺磁氧化铁纳米颗粒：
1.称取15gPEG加入50ml三口圆底烧瓶中；
2.200ml/min的氩气环境下，磁力搅拌10min匀速升温至80℃；
3.加入0.7g乙酰丙酮铁后保温10分钟，然后以7.2℃/min，25min内升温至260℃，保温1h，停止加热，自然冷却至60℃左右；
4.所得产物用60ml甲苯超声清洗，并置于磁铁上吸附，使用甲苯、丙酮依次超声清洗取下层黑色沉淀即为USPIO-PEG。
合成USPIO-PEG-MMP2Ab探针
1.取USPIO-PEG(1mgFe，3.5mg/ml)加入2mLEP管中；
2.称取0.5mgEDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)及1.4mgN-羟基硫代琥珀酰亚胺(sulfo-NHS)，加入100μl去离子水溶解后，迅速转移至USPIO溶液中，室温下反应15min；
3.采用PD-10交换柱对活化后的USPIO进行缓冲液交换，洗脱液采用NaHCO3(0.1M)，然后加入0.5mgMMP2Ab在室温下进行偶联反应，4h后，采用PBS缓冲液进行透析过夜；
4.偶联完毕后，将偶联物置于4℃冰箱储存备用，使用前用无菌PBS洗2次，1次5min。
本发明的工作原理：本发明所制分子探针是一种通过缩短病变组织质子弛豫时间而增强磁共振成像对比度的新型磁共振对比剂，与传统的钆对比剂相比，USPIO-PEG-MMP2Ab不仅具有更高的磁饱和强度及弛豫率，且具有良好的生物相容性。经静脉给药后能主动靶向胶质瘤细胞，在磁共振T2WI上产生明显的“阴性对比”效果，并通过磁共振分子成像完成对脑胶质瘤的定性与分级诊断。
以上对本发明及其实施方式进行了描述，这种描述没有限制性，以上所示的也只是本发明的实施方式之一，实际的结构并不局限于此。总而言之如果本领域的普通技术人员受其启示，在不脱离本发明创造宗旨的情况下，不经创造性的设计出与该技术方案相似的结构方式及实施例，均应属于本发明的保护范围。

Claims (3)

1.一种靶向标记MMP-2的磁共振分子探针的构建方法，其特征在于：所述MRI诊断神经胶质瘤的新型分子探针由超顺磁氧化铁纳米颗粒(superparamagneticironoxidenanoparticles，USPIO)和基质金属蛋白酶-2抗体(MMP2Ab)通碳二亚胺法合成。
2.根据权利要求1所述的一种靶向标记MMP-2的磁共振分子探针的构建方法，其特征在于：高温分解法合成USPIO-PEG超顺磁氧化铁纳米颗粒：
1.称取15gPEG加入50ml三口圆底烧瓶中；
2.200ml/min的氩气环境下，磁力搅拌10min匀速升温至80℃；
3.加入0.7g乙酰丙酮铁后保温10分钟，然后以7.2℃/min，25min内升温至260℃，保温1h，停止加热，自然冷却至60℃左右；
4.所得产物用60ml甲苯超声清洗，并置于磁铁上吸附，使用甲苯、丙酮依次超声清洗取下层黑色沉淀即为USPIO-PEG。
3.根据权利要求1所述的一种靶向标记MMP-2的磁共振分子探针的构建方法，其特征在于：合成USPIO-PEG-MMP2Ab探针
1.取USPIO-PEG(1mgFe，3.5mg/ml)加入2mLEP管中；
2.称取0.5mgEDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)及1.4mgN-羟基硫代琥珀酰亚胺(sulfo-NHS)，加入100μl去离子水溶解后，迅速转移至USPIO溶液中，室温下反应15min；
3.采用PD-10交换柱对活化后的USPIO进行缓冲液交换，洗脱液采用NaHCO3(0.1M)，然后加入0.5mgMMP2Ab在室温下进行偶联反应，4h后，采用PBS缓冲液进行透析过夜；
4.偶联完毕后，将偶联物置于4℃冰箱储存备用，使用前用无菌PBS洗2次，1次5min。
CN201910344346.6A 2019-04-26 2019-04-26 一种靶向标记mmp-2的磁共振分子探针的构建方法 Pending CN110064061A (zh)

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