CN104774247A - 与整合素受体ανβ3相关的5肽 - Google Patents
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Abstract
本发明属于生物工程制药技术领域或蛋白质多肽类药物及生物医学工程领域，具体涉及一种对整合素受体具有高亲和力的多肽及其应用，例如用于癌症诊断和治疗中。本发明所述的多肽由5个氨基酸组成其序列为精氨酸-色氨酸-精氨酸-天冬酰胺-蛋氨酸(Arg-Trp-Arg-Asn-Met)。体外流式细胞术测定亲和力实验表明本发明的多肽对整合素受体高表达的细胞有很强的靶向性；体外激光共聚焦细胞摄取实验表明本发明的多肽能够靶向到整合素受体αvβ3高表达的肿瘤细胞上，而对整合素受体αvβ3低表达的肿瘤细胞和正常细胞基本无靶向作用。体外细胞毒性结果显示本发明的5肽对细胞基本无毒性，在癌症的诊断和治疗中，有着很好的应用前景。
Description
技术领域
本发明属于生物工程制药技术领域和蛋白质多肽类药物及生物医学工程领域，具体涉及针对整合素受体αvβ3具有高亲和力多肽及其应用，例如用于癌症诊断和治疗中。
背景技术
目前，癌症已经成为引起人类死亡的主要原因之一，而化疗仍然是治疗癌症的主要方法，但是，由于传统的化药缺少针对肿瘤细胞的特异性，导致了对病人的一些器官毒性。
整合素在体内细胞中都有广泛的表达，大多数细胞表面都可表达一种以上的整合素，这些整合素不仅参与着一些细胞的生长、分化及凋亡等生理功能，而H在多种病理过程中也发挥着极为重要的作用。整合素家族包括18种α亚型和8种β亚型，而αβ亚型的配对决定了配体的特异性。整合素超级家族中，整合素受体αvβ3在成像与靶向治疗中起着主要的作用。由于整合素受体αvβ3在多种肿瘤如胶质瘤，乳腺癌，黑色素瘤，前列腺癌及卵巢癌中都是高表达的，因此整合素受体αvβ3成为比较重要的药物靶点。目前3肽(序列为精氨酸-色氨酸-精氨酸)是证明对整合素αvβ3具有高亲和力的多肽(详见专利：与整合素αvβ3受体具有高亲和力的多肽，申请号201310019870.9)。
发明内容
本发明的目的在于提供能够与整合素受体αvβ3相关的靶向多肽，使其能够对整合素受体αvβ3高表达的肿瘤进行诊断。
本发明所述的5肽是在3肽基础上由5个氨基酸组成，其具有精氨酸-色氨酸-精氨酸-天冬酰胺-蛋氨酸的氨基酸序列。
为了检测本发明多肽的药理活性，体外实验通过荧光染料罗丹明B对多肽进行标记，以检测其对肿瘤的靶向性。在体外细胞亲和力实验和摄取实验中，本发明的5肽展现出了其良好的对肿瘤细胞整合素受体αvβ3的靶向能力，且能力强于3肽。在体外细胞毒性实验中，本发明的5肽为表现出对细胞的毒性。
下面是本发明部分药效学实验：
一、标记荧光染料
本发明应用于体外细胞实验的荧光染料为罗丹明B(Rhodamine B)，简称RhB，分别与上述3肽，及本发明5肽通过酰胺键连接，简称分别为RWr-RhB与RWrNM-RhB，连接方法具体为取1mg罗丹明B(详见参考文献：Cao，J.，et al.，Fast clearing RGD-based near-infrared fluorescent probes for in vivo tumor diagnosis.Contrast Media Mol Imaging，2012.7(4)：p. 390-402)溶于1ml PBS(pH为7.4)中，加入1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐，N-羟基琥珀酰亚胺(EDC/NHS)(摩尔比Rhodamine B∶EDC∶NHS＝1∶1.5∶1.5)，避光反应4h，活化。分别称取1mmol 3肽及5肽溶入含有荧光染料罗丹明B的1ml PBS缓冲液(pH7.4)中，室温避光搅拌过夜。反应结束后，反应液浓缩后通过葡聚糖凝胶G-25柱，PBS缓冲液(pH7.4)作洗脱液，得到纯化的RWr-RhB及RWrNM-RhB，-20℃储存备用。环状RGD作为对照也使用相同的方法标记简称为c(RGDyK)-RhB。
二、体外细胞亲和力实验
将培养好的人脑胶质瘤U87MG细胞从12孔板上洗脱后重悬于PBS溶液，分别与罗丹明B标记的3肽，5肽(500umol/L)共孵育2小时，并通过流式细胞术检测其平均荧光强度，荧光强度越强证明对细胞的亲和力越强。当探针与细胞上的受体亲和力强时，流式细胞仪检测出的细胞平均荧光强度值高。体外亲和力实验结果显示浓度相同的标记了RhB的3肽，5肽的探针分别与整合素受体高表达的U87MG细胞孵育后，在U87MG细胞中3肽探针平均荧光强度为98，5肽探针平均荧光强度为123，空白染料平均荧光强度为25，结果表明这些探针在与U87MG细胞亲和力强弱对比中，本发明5肽的强度最大。
三、体外细胞摄取实验
将培养好的人脑胶质瘤细胞U87MG，人乳腺癌细胞MDA-MB-231，人乳腺癌细胞MCF-7和正常肝细胞L02转移到共聚焦皿中，过夜孵育后分别加入相同浓度的罗丹明B标记的5肽(500umol/L)，加入罗丹明B标记的环状RGD作为阳性对照，37℃孵育2小时后，加入染核试剂12μg/mL Hochest33342染色，最后用激光共聚焦显微镜观察细胞内探针的摄取情况。在整合素受体αvβ3高表达的U87MG和MDA-MB-231细胞中(图1所示)，环状RGD和5肽探针显示出了很强的荧光强度，而在整合素受体αvβ3低表达的MCF-7和L02细胞(图2所示)，这两种探针的荧光强度不强，这一结果说明5肽探针对整合素受体αvβ3高表达的细胞有靶向性而对整合素受体αvβ3低表达的细胞靶向性不强。
四、体外细胞毒性实验
U87MG细胞、MCF-7细胞与正常肝L02细胞，待长至90％以上时，用0.25％胰蛋白酶液消化后，制备成单细胞悬液(完全生长培养基)，以3000个细胞/孔铺96孔板，于37℃，含5％CO2的培养箱中培养，在培养24h后换为1％FBS的低血清培养液，然后加入不同浓度梯度的5肽，使他们的终浓度为2.5，5，10，20，40，80，100和200μM。结果显示(图3所示)5肽对这几种细胞的存活率均在80％以上，说明5肽对这些细胞基本无毒。
综上所述，本发明的5肽能够靶向到整合素受体αvβ3高表达的细胞上，而对整合素受体 αvβ3低表达的细胞基本无靶向性，并且5肽对细胞基本无毒性。由此可见5肽能够成为对整合素受体αvβ3高表达肿瘤进行体外诊断。
本发明的5肽可以优选用实施例1固相合成的方法制备。
附图说明
图1激光共聚焦细胞摄取实验观察罗丹明B标记的探针对不同肿瘤细胞的摄取(A为U87MG细胞，B为MDA-MB-231细胞)
图2激光共聚焦细胞摄取实验观察罗丹明B标记的探针对不同肿瘤细胞的摄取(A为MCF-7细胞，B为L02细胞)
图3体外细胞毒性实验考察5肽对不同细胞的毒性
具体实施方式
实施例1
5肽的合成
1：偶联
先将Fmoc-Met-Rink amide MBHA resin(芴甲氧羰酰基-苯丙氨酸-rink氨基树脂)0.33/2mmol6.06g溶胀于二甲基甲酰胺中，10ml/g树脂，接着用20％六氢吡啶/二甲基甲酰胺溶液脱去Fmoc保护基(以下称之为脱帽)，用二甲基甲酰胺洗涤脱帽子后的树脂，加入原料Fmoc-Asn(tBU)-OH 2.3g，缩合剂用HBTU 1.44g，反应时间30分钟，kaiser法检测，如检测结果为阴性，说明偶联反应完成，则接下去进行脱帽，时间30分钟，如检测结果仍为阳性，则说明偶联反应未完成或反应不完全，需要延长偶联时间或重新投料偶联，直至偶联反应完成。重复上述操作，依次偶联；Fmoc-D-Arg(Pbf)-OH；Fmoc-Trp-OH；Fmoc-Arg(Pbf)-OH，完成所有直链肽合成后，用甲醇洗涤所得到的peptidyl resin，真空干燥箱里充分干燥。
2：树脂与肽的分离裂解
将120ml裂解液(10ml/g peptidyl resin)加入树脂中，25℃条件下，磁力搅拌2.5h后用砂芯漏斗将裂解液与树脂分离，弃去树脂，保留滤液。将滤液缓慢滴加到10eq体积的冰无水乙醚中，滴加完毕后，自然沉降30min。然后用高速离心机离心10min(4000rpm)，弃去上清液，保留沉淀，将所得沉淀在干燥箱中干燥8-10h，得到干粉粗品。
3：纯化
取上述干粉粗品1g，用0.1％三氟乙酸/水溶解。经过滤后，上样到C18制备柱，用高效液相进行梯度洗脱，收集目标肽的洗脱液体，检测液体纯度。把合格的样品混合后旋蒸，最后用冻干机冻干，得到Arg-Trp-Arg-Asn-Met，纯度大于98％。
序列为：Arg-Trp-Arg-Asn-Met
质谱图中可以看出Arg-Trp-Arg-Asn-Met分子量是381.7*2-2＝761.4
紫外光谱图中273nm处有肽的吸收峰。
Claims (3)
1.一种与整合素αvβ3具有高亲和力的多肽，其序列为：Arg-Trp-Arg-Asn-Met。
2.权利要求1的多肽用于诊断肿瘤疾病的用途。
3.权利要求1的多肽用于治疗肿瘤疾病的用途。
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| CN111233975A (zh) * | 2018-11-28 | 2020-06-05 | 复旦大学 | 可靶向整合素的多肽mn及其在制备肿瘤靶向药物中的应用 |
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