CN114099713A - 活体内检测cdk4/6激酶活性的分子探针及其应用 - Google Patents
活体内检测cdk4/6激酶活性的分子探针及其应用 Download PDFInfo
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Abstract
活体内检测CDK4/6激酶活性的分子探针及其应用,涉及生物医药技术领域。该分子探针包括传感肽CDKACT4和脂质体CPP30‑Lipo,分子式为CPP30‑Lipo/CDKACT4;以该分子探针的荧光变化来反应CDK4/6激酶活性变化及评估激酶活性抑制剂抗肿瘤药物疗效,在活体小动物水平CPP30‑Lipo/CDKACT4分子探针能在palbociclib治疗后肿瘤大小尚未发生变化时就检测到palbociclib对CDK4/6活性的抑制作用,并且实现在活体水平直接监测CDK4/6激酶活性。为临床早期、准确评价激酶活性抑制剂抗肿瘤药物疗效提供一种潜在的方法。
Description
技术领域
本发明涉及生物医药技术领域,尤其是涉及基于荧光成像监测的活体内检测CDK4/6激酶活性的分子探针及其应用。
背景技术
肿瘤的发生发展与细胞周期有关,细胞周期的异常激活导致癌细胞的失控性增殖。细胞周期蛋白依赖性激酶(Cyclin-dependent kinases,CDKs)是细胞周期调控机制的核心部分,一直被作为抗肿瘤药物治疗的重要靶点,CDK抑制剂也是近十年药物研究的热门。CDK4/6抑制剂的临床应用首先在乳腺癌的治疗方面获得突破,palbociclib和ribociclib因为其显著的抗乳腺癌疗效,分别于2015年和2017年获得美国食品和药物管理局(FDA)快速批准上市,用于激素受体阳性Her2阴性复发转移性乳腺癌的一线治疗。我国国家食品药品监督管理局也于2018年7月31日正式批准palbociclib在中国上市。除乳腺癌以外,CDK4/6选择性抑制剂治疗非小细胞肺癌、肝癌、黑色素瘤、卵巢癌、胶质母细胞瘤、套细胞淋巴瘤等的临床试验正在进行中。,CDK4/6抑制剂是极具临床应用前景的新型靶向药物,因此如何早期、精准判定CDK4/6的活性及抗肿瘤药物疗效是摆在临床实践中的重要问题。
目前,临床上的常规的做法是通过测量肿瘤病灶的大小来评价抗肿瘤药物的疗效,如使用游标卡尺的直接测量和应用电子计算机断层扫描、磁共振成像、超声、X光等影像学技术。但以上的评价方法需等到病灶大小变化明显时才可以判定,具有一定的滞后性,容易导致几个疗程结束后才发现治疗无效,不仅延误治疗,还可能使患者不得不承受药物的毒副反应。研究发现,肿瘤大小发生变化之前,大多会出现分子、基因或蛋白水平的变化,会导致微环境、代谢及生理功能的改变。分子影像技术可以在体检测以上的变化,为尽早、准确地判定抗肿瘤药物的疗效提供可能。近二十年来,科学家一直在探索可以直接靶向药物关键靶点的分子影像学技术,以达到快速、精准检测抗肿瘤药物疗效的目的。
监测激酶活性是评价激酶靶向药物疗效最直接的方法。荧光是一种荧光基团通过吸收光子被激发到更高能的状态,并在一定时间后通过释放光子从激发态松弛到基态的现象,已被广泛用于激酶活性传感器的设计。法国Morris研究团队使用具有溶剂化显色效应的荧光基团5-TAMRA标记Cyclin D/CDK4特异性底物肽构建CDKACT4荧光生物传感器,用于监测CDK4激酶活性。但是,该传感器因为多肽的不稳定性及不具备穿膜能力,只能在细胞裂解液水平监测CDK4激酶的活性,未能在活细胞或小动物进一步得到证实。
因此,本发明基于上述CDKACT4生物传感器,运用可进行稳定活细胞递送及溶酶体逃逸的靶向脂质体作为该传感器的载体,构建能特异性靶向乳腺肿瘤细胞并能反应CDK4/6激酶活性变化的分子探针CPP30-Lipo/CDKACT4,实现在活细胞及活体水平精准监测CDK4/6激酶活性及CDK4/6抑制剂Palbociclib的抗肿瘤疗效。
发明目的
本发明的目的在于针对现有技术存在的上述缺点与不足,提供活体内检测CDK4/6激酶活性的分子探针及其制备方法。
本发明的另一目的在于提供所述分子探针在活细胞及活体早期、精准监测CDK4/6抑制剂抗肿瘤药物疗效的应用。
所述活体内检测CDK4/6激酶活性的分子探针,包括传感肽CDKACT4和脂质体CPP30-Lipo;该分子探针的通式为CPP30-Lipo/CDKACT4,所述传感肽CDKACT4包括靶向CDK4/6的多肽序列与荧光标记物5-TAMRA连接而成的功能域、肽脯氨酰基顺反异构酶Pin1的色氨酸-色氨酸中心区(WW)衍生而来的磷酸氨基酸识别域,功能域与磷酸氨基酸识别域通过linker连接,所述传感肽CDKACT4用于靶向CDK4/6激酶活性;所述脂质体CPP30-Lipo作为载体,用于活细胞递送和溶酶体逃逸。
所述活体内检测CDK4/6激酶活性的分子探针的制备方法,包括以下步骤:
1)传感肽CDKACT4的合成;
取衍自抑癌基因Rb的CDK4/CyclinD特异性磷酸化位点S795附近序列作为靶向CDK4/6的多肽序列,并在该多肽序列S795磷酸化位点-2位赖氨酸位点用连接臂使该多肽序列与具有溶剂化显色效应的荧光标记物5-TAMRA相连,得到传感肽的功能域;肽脯氨酰基顺反异构酶Pin1的色氨酸-色氨酸中心区(WW)是一个特异性的磷酸化丝氨酸/苏氨酸模体基序的连接区,Pin1通过该区域与底物中磷酸化蛋白结合,短WW肽是从WW晶体结构连接到磷酸肽界面的氨基酸残基衍生而来的一段多肽序列,命名为磷酸氨基酸识别域;将功能域与磷酸氨基酸识别域通过linker连接,得到CDK4/6传感肽CDKACT4。
2)DOPE-PEG2000-Mal-CPP30的合成与纯化;
(1)称取10mg CPP30-cys多肽溶解于0.3mL DMF和1~1.2mL PBS(pH 7.4)混合溶液中,加入12~15mg DOPE-PEG2000-Maleimide,室温持续搅拌反应10~15h;
(2)将终产物转移入透析袋中(截留分子量3500),用ddH2O作为透析液,4℃透析过夜,除去未反应的DOPE-PEG2000-Maleimide和CPP30,得到DOPE-PEG2000-Mal-CPP30溶液。
3)CPP30修饰的DOPE长循环阳离子脂质体包载CDKACT4传感肽的制备与纯化;
(1)取2mg CDKACT4传感肽溶解在2mL ddH2O中;
(2)取DOPE/胆固醇/DOTAP按照质量比10mg/1mg/1mg比例溶解于3mL氯仿中,充分混匀后转移至茄形烧瓶中,37℃缓慢真空旋转蒸发,使氯仿挥发完全,在瓶底形成脂质薄膜;
(3)茄形瓶中加入0.2mL上述合成的DOPE-PEG2000-CPP30溶液和1.2~1.5mLCDKACT4多肽水溶液,并补充ddH2O 1mL,而后加入转子震荡,使脂质膜充分水化;
(4)将上述混合溶液装于试管中,利用超声清洗机以100W功率冰浴超声15~25min,直到看见脂质体溶液澄清状;
(5)脂质体挤出器中放置好聚碳酸酯膜,将上述脂质体样品先通过200nm的聚碳酸酯膜至少3次,后通过100nm的聚碳酸酯膜至少3次,使脂质体粒径均匀且大小合适;
(6)将步骤(5)中产物转移入孔径50nm的透析装置中,用ddH2O作为透析液,4℃透析过夜,除去未负载的DOPE-PEG2000-CPP30和CDKACT4,最后补充去离子水定容至4mL,得到CPP30-Lipo/CDKACT4水溶液。
所述分子探针可在活细胞及活体早期、监测CDK4/6激酶抑制剂抗肿瘤药物疗效的应用。
所述应用的具体步骤可为:
1)药物治疗前对小鼠肿瘤进行一次核磁共振成像及荧光活体成像;荧光成像前于小鼠尾静脉按体重注射探针,于注射后不同时间点将小鼠麻醉后,置于IVIS Lumina II光学成像系统中成像;而后随机分为Palbociclib(150mg/kg/d)、超纯水两组,口服管饲给药,每天测量肿瘤大小;药物治疗后的观察点再进行一次核磁共振成像和尾静脉注射探针进行荧光活体成像。
2)组织标本的体外分析:将治疗前穿刺的组织及治疗后取出的肿瘤组织用4%多聚甲醛于4℃固定24h后,进行常规脱水、包埋、切片,厚度为4μm。再进行pRb及Ki67免疫组化染色,对比荧光成像仪所检测肿瘤部位荧光强度的变化与病理水平分子变化的相关性,验证该探针监测CDK4/6抑制剂疗效的精确性与灵敏性。
与现有技术相比,本发明具有以下突出的优点和有益效果:
本发明突破传统的以测量肿瘤直径大小来评估疗效的局限性,以CPP30-Lipo/CDKACT4分子探针的荧光变化来反应CDK4/6激酶活性变化及评估激酶活性抑制剂抗肿瘤药物疗效,与传统成像方法相比,在活体小动物水平CPP30-Lipo/CDKACT4分子探针能在palbociclib治疗后肿瘤大小尚未发生变化时就检测到palbociclib对CDK4/6活性的抑制作用,并且是首次实现在活体水平直接监测CDK4/6激酶活性。该发明为在临床早期、准确评价激酶活性抑制剂抗肿瘤药物疗效提供一种潜在的方法,对改善肿瘤患者的药物治疗效果和预后、提高生活质量具有重要意义。
附图说明
图1为CPP30-Lipo/CDKACT4分子探针的结构组成示意图。
图2为CPP30-Lipo/CDKACT4分子探针的电镜图。
图3为CPP30-Lipo/CDKACT4分子探针的粒径分布。
图4为CPP30-Lipo/CDKACT4分子探针的吸收和荧光发射光谱。
图5为MCF-7细胞对CPP30-Lipo/CDKACT4分子探针的时间依赖性摄取。
图6为CPP30-Lipo/CDKACT4分子探针的细胞靶向性。
图7为CPP30-Lipo/CDKACT4分子探针的细胞毒性。
图8为CPP30-Lipo/CDKACT4分子探针在活细胞水平监测CDK4/6激酶活性(左:经不同浓度palbociclib处理后的MCF-7细胞与探针共孵育后的荧光成像图;右:左图的荧光强度定量分析图)。
图9为CPP30-Lipo/CDKACT4分子探针在活体小动物水平监测CDK4/6抑制剂Palbociclib的疗效(左:小鼠摄取探针后肿瘤部位的荧光成像图;右:同一小鼠的核磁成像图)。
图10为CPP30-Lipo/CDKACT4分子探针对健康小鼠体重的影响。
图11为CPP30-Lipo/CDKACT4分子探针对健康小鼠血液系统的影响。
图12为CPP30-Lipo/CDKACT4分子探针对健康小鼠肝肾功能的影响。
图13为CPP30-Lipo/CDKACT4分子探针对健康小鼠重要脏器的影响。
具体实施方式
为使本发明的目的及优点更加清楚明白,以下结合附图及实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
实施例1CPP30-Lipo/CDKACT4探针的结构
所述活体内检测CDK4/6激酶活性的分子探针,包括传感肽CDKACT4和脂质体CPP30-Lipo;该分子探针的通式为CPP30-Lipo/CDKACT4,所述传感肽CDKACT4包括靶向CDK4/6的多肽序列与荧光标记物5-TAMRA连接而成的功能域、肽脯氨酰基顺反异构酶Pin1的色氨酸-色氨酸中心区(WW)衍生而来的磷酸氨基酸识别域,功能域与磷酸氨基酸识别域通过linker连接,所述传感肽CDKACT4用于靶向CDK4/6激酶活性;所述脂质体CPP30-Lipo作为载体,用于活细胞递送和溶酶体逃逸。图1为CPP30-Lipo/CDKACT4分子探针的结构组成示意图。
图2为CPP30-Lipo/CDKACT4分子探针的电镜图,图3为CPP30-Lipo/CDKACT4分子探针的粒径分布,图4为CPP30-Lipo/CDKACT4分子探针的吸收和荧光发射光谱。本发明实施例构建的CPP30-Lipo/CDKACT4纳米探针,在电镜下呈圆球形,颗粒均一分散,粒径较为均一,纳米粒度仪检测其平均粒径为161nm,酶标仪检测吸收光谱与荧光发射光谱与荧光标记物5-TAMRA基本一致。
实施例2CPP30-Lipo/CDKACT4探针的制备
一、所述靶向CDK4/6激酶活性的传感肽CDKACT4的设计合成方法如下:
取衍自抑癌基因Rb的CDK4/CyclinD特异性磷酸化位点S795附近序列(GGYKFKSSPLRIPG)作为靶向CDK4/6的多肽序列,并在其S795磷酸化位点-2位赖氨酸位点用连接臂使其与具有溶剂化显色效应的荧光标记物5-TAMRA相连,得到传感肽的功能域。肽脯氨酰基顺反异构酶Pin1的色氨酸-色氨酸中心区(WW)是一个特异性的磷酸化丝氨酸/苏氨酸模体基序的连接区,Pin1通过该区域与底物中磷酸化蛋白结合,短WW肽(GFARVYMSRSSGWERPS GG)是从WW晶体结构连接到磷酸肽界面的氨基酸残基衍生而来的一段多肽序列,命名为磷酸氨基酸识别域。将上述功能域与磷酸氨基酸识别域通过linker连接,则得到CDK4/6传感肽CDKACT4。该多肽的氨基酸序列如SEQIDNo.1。
二、DOPE-PEG2000-Mal-CPP30的合成与纯化
1、称取10mg CPP30-cys(GYRRTTPSYYRMYLRC)多肽溶解于0.3mL DMF和1.2mL PBS(pH 7.4)混合溶液中,加入15mg DOPE-PEG2000-Maleimide,室温持续搅拌反应12h;
2、将终产物转移入透析袋中(截留分子量3500),用ddH2O作为透析液,4℃透析过夜,除去未反应的DOPE-PEG2000-Maleimide和CPP30,得到DOPE-PEG2000-Mal-CPP30溶液。
三、CPP30修饰的DOPE长循环阳离子脂质体包载CDKACT4传感肽的制备与纯化
1、取2mg CDKACT4传感肽溶解在2mL ddH2O中;
2、取DOPE/胆固醇/DOTAP按照质量比10mg/1mg/1mg比例溶解于3mL氯仿中,充分混匀后转移至茄形烧瓶中,37℃缓慢真空旋转蒸发,使氯仿挥发完全,在瓶底形成脂质薄膜;
3、茄形瓶中加入0.2mL上述合成的DOPE-PEG2000-CPP30溶液和1.5mL CDKACT4多肽水溶液,并补充ddH2O 1mL,而后加入转子震荡,使脂质膜充分水化;
4、将上述混合溶液装于试管中,利用超声清洗机以100W功率冰浴超声20min,直到看见脂质体溶液澄清状;
5、脂质体挤出器中放置好聚碳酸酯膜,将上述脂质体样品先通过200nm的聚碳酸酯膜5次,后通过100nm的聚碳酸酯膜5次,使脂质体粒径均匀且大小合适;
6、将步骤5中产物转移入孔径50nm的透析装置(自制)中,用ddH2O作为透析液,4℃透析过夜,除去未负载的DOPE-PEG2000-CPP30和CDKACT4,最后补充去离子水定容至4mL,得到CPP30-Lipo/CDKACT4水溶液。
实施例3CPP30-Lipo/CDKACT4分子探针的细胞时间依赖性摄取、细胞靶向性和细胞毒性验证
将本发明所合成的探针用于孵育乳腺癌MCF-7细胞,收集细胞后使用流式细胞仪进行荧光检测,从图5、6可以看出,本发明探针对MCF-7细胞具有良好的靶向性,且呈时间依赖性摄取。图7可见,不同浓度的探针孵育MCF-7细胞72h后,CCK8实验分析细胞活性,证明探针无细胞毒性。
实施例4CPP30-Lipo/CDKACT4探针在活细胞水平监测CDK4/6激酶活性
为验证本发明所合成的分子探针是否可以在活细胞水平监测CDK4/6激酶活性,先用0,10,100,1000nm四个不同浓度梯度的Palbociclib预处理MCF-7细胞24小时,而后用CPP30-Lipo/CDKACT4与已预处理细胞共孵育12小时,活细胞微孔板荧光成像(图8)观察到,药物浓度越高,细胞的荧光强度越低。即CPP30-Lipo/CDKACT4分子探针可监测不同浓度CDK4/6激酶抑制剂Palbociclib用药后乳腺癌MCF-7中CDK4/6激酶活性的差异。
实施例5CPP30-Lipo/CDKACT4分子探针用于活体小动物水平CDK4/6激酶抑制剂疗效的监测
药物治疗前对小鼠肿瘤进行一次核磁共振成像及荧光活体成像。荧光成像前于小鼠尾静脉按体重注射探针,于注射后不同时间点将小鼠麻醉后,置于IVIS Lumina II光学成像系统中成像。而后随机分为Palbociclib(150mg/kg/d)、超纯水两组,口服管饲给药,每天测量肿瘤大小。药物治疗7天后的观察点再进行一次核磁共振成像和尾静脉注射探针进行荧光活体成像。结果显示(图9),用传统影像学技术MRI监测疗效时,治疗前后肿瘤最长径并没有发生明显变,用CPP30-Lipo/CDKACT4分子探针监测疗效时显示,未经治疗组,肿瘤部位荧光强度相较于7天前显著增强,而经过7天Palbociclib治疗后的小鼠,治疗后肿瘤部位荧光信号强度相较于治疗前降低。以上结果说明CPP30-Lipo/CDKACT4分子探针能在活体小动物水平早期、实时、精准监测Palbociclib抗肿瘤疗效。
实施例6CPP30-Lipo/CDKACT4分子探针的动物安全性验证
将两组正常健康小鼠分别尾静脉注射CPP30-Lipo/CDKACT4探针和PBS,每天称量小鼠体重直至28天,两组小鼠体重无明显差异(图10);分别在1天、3天、7天和28天眼眶取血收集小鼠外周血全血及血清,检测全血中WBC、Lymph#、RBC、PLT各指标,及血清中ALT、AST、ALP、BUN、CR各指标,两组间各项指标无明显差异(图11、12);取血后的小鼠颈椎脱臼处死,取主要脏器做H&E染色,图13显示对两组小鼠心、肝、脾、肺、肾HE染色观察均为正常,不存在组织病理学上认为的异常、退化或损伤。因此说明,本发明制备的CPP30-Lipo/CDKACT4探针具有良好的活体生物安全性。
上述实施例仅为本发明的较佳实施例,不能被认为用于限定本发明的实施范围。凡依本发明申请范围所作的均等变化与改进等,均应仍归属于本发明的专利涵盖范围之内。
序列表
<110> 厦门大学附属翔安医院
<120> 活体内检测CDK4/6激酶活性的分子探针及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 31
<212> PRT
<213> 人工序列(Artificial Sequence)
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Gly Phe Ala Arg Val Tyr Met Ser Arg Ser Ser Gly Trp Glu Arg Pro
1 5 10 15
Ser Gly Gly Tyr Lys Phe Lys Ser Ser Pro Leu Arg Ile Pro Gly
20 25 30
Claims (7)
1.活体内检测CDK4/6激酶活性的分子探针,其特征在于包括传感肽CDKACT4和脂质体CPP30-Lipo;该分子探针的通式为CPP30-Lipo/CDKACT4。
2.如权利要求1所述活体内检测CDK4/6激酶活性的分子探针,其特征在于所述传感肽CDKACT4包括靶向CDK4/6的多肽序列与荧光标记物5-TAMRA连接而成的功能域、肽脯氨酰基顺反异构酶Pin1的色氨酸-色氨酸中心区WW衍生而来的磷酸氨基酸识别域,功能域与磷酸氨基酸识别域通过linker连接,所述传感肽CDKACT4用于靶向CDK4/6激酶活性;所述脂质体CPP30-Lipo作为载体,用于活细胞递送和溶酶体逃逸。
3.活体内检测CDK4/6激酶活性的分子探针的制备方法,其特征在于包括以下步骤:
1)传感肽CDKACT4的合成;
2)DOPE-PEG2000-Mal-CPP30的合成与纯化;
3)CPP30修饰的DOPE长循环阳离子脂质体包载CDKACT4传感肽的制备与纯化。
4.如权利要求3所述活体内检测CDK4/6激酶活性的分子探针的制备方法,其特征在于在步骤1)中,所述传感肽CDKACT4的合成,具体步骤为:取衍自抑癌基因Rb的CDK4/CyclinD特异性磷酸化位点S795附近序列作为靶向CDK4/6的多肽序列,并在该多肽序列S795磷酸化位点-2位赖氨酸位点用连接臂使该多肽序列与具有溶剂化显色效应的荧光标记物5-TAMRA相连,得到传感肽的功能域;肽脯氨酰基顺反异构酶Pin1的色氨酸-色氨酸中心区WW是一个特异性的磷酸化丝氨酸/苏氨酸模体基序的连接区,Pin1通过该区域与底物中磷酸化蛋白结合,短WW肽是从WW晶体结构连接到磷酸肽界面的氨基酸残基衍生而来的一段多肽序列,命名为磷酸氨基酸识别域;将功能域与磷酸氨基酸识别域通过linker连接,得到CDK4/6传感肽CDKACT4。
5.如权利要求3所述活体内检测CDK4/6激酶活性的分子探针的制备方法,其特征在于在步骤2)中,所述DOPE-PEG2000-Mal-CPP30的合成与纯化,具体步骤为:
(1)称取10mg CPP30-cys多肽溶解于0.3mL DMF和1~1.2mL pH 7.4的PBS混合溶液中,加入12~15mg DOPE-PEG2000-Maleimide,室温持续搅拌反应10~15h;
(2)将终产物转移入透析袋中,截留分子量3500,用ddH2O作为透析液,4℃透析过夜,除去未反应的DOPE-PEG2000-Maleimide和CPP30,得到DOPE-PEG2000-Mal-CPP30溶液。
6.如权利要求3所述活体内检测CDK4/6激酶活性的分子探针的制备方法,其特征在于在步骤3)中,所述CPP30修饰的DOPE长循环阳离子脂质体包载CDKACT4传感肽的制备与纯化,具体步骤为:
(1)取2mg CDKACT4传感肽溶解在2mL ddH2O中;
(2)取DOPE/胆固醇/DOTAP按照质量比10mg/1mg/1mg比例溶解于3ml氯仿中,充分混匀后转移至茄形烧瓶中,37℃缓慢真空旋转蒸发,使氯仿挥发完全,在瓶底形成脂质薄膜;
(3)茄形瓶中加入0.2mL上述合成的DOPE-PEG2000-CPP30溶液和1.2~1.5mL CDKACT4多肽水溶液,并补充ddH2O 1mL,而后加入转子震荡,使脂质膜充分水化;
(4)将上述混合溶液装于试管中,利用超声清洗机以100W功率冰浴超声15~25min,直到看见脂质体溶液澄清状;
(5)脂质体挤出器中放置好聚碳酸酯膜,将上述脂质体样品先通过200nm的聚碳酸酯膜至少3次,后通过100nm的聚碳酸酯膜至少3次,使脂质体粒径均匀且大小合适;
(6)将步骤(5)中产物转移入透析装置中,用ddH2O作为透析液,4℃透析过夜,除去未负载的DOPE-PEG2000-CPP30和CDKACT4,最后补充去离子水定容至4mL,得到CPP30-Lipo/CDKACT4水溶液。
7.如权利要求1所述分子探针在活细胞及活体早期、监测CDK4/6激酶抑制剂抗肿瘤药物疗效中的应用。
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