CN112876538B - 靶向新生血管标记物cd105的多肽及其应用 - Google Patents
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Abstract
本发明提供一种靶向新生血管标记物CD105的多肽及其应用。本发明提供的多肽CD1051、CD1052可特异性结合CD105蛋白,该多肽可替代CD105抗体应用于CD105阳性的血管内皮细胞等,还可用于制备在体内靶向肿瘤成像或可视化的制剂。将该多肽与抗肿瘤药物或其他试剂形成缀合物,可靶向CD105蛋白用于抗血管生成的肿瘤治疗。此外,还可以反映心血管系统疾病中新生血管的变化情况,在动脉粥样硬化的诊断和预后判断中发挥重要作用。本发明多肽特异性、敏感性好,制备方法简单,成本低,实用性强。本发明为实时监控患者体内CD105表达和肿瘤分级、分期、转移和预后的相关性提供了简便快捷、经济、准确的检测手段。
Description
技术领域
本发明涉及生物医学检测领域,具体地说,涉及一种靶向新生血管标记物CD105的多肽及其应用。
背景技术
恶性肿瘤是严重危及人类生命的疾病之一,近几十年来,世界范围内肿瘤的发病率和病死率越来越高。现在对于多数恶性肿瘤可以采用手术、化疗与放射、靶向和免疫治疗等方法杀灭大部分肿瘤细胞,但绝大多数肿瘤患者仍无法获得根治,容易复发和转移,恶性肿瘤患者的生存时间和生活质量始终较低。
CD105又名Endoglin,是一个特异表达于新生血管内皮的Ⅰ型跨膜糖蛋白,其胞外区部分有4个活性位点,在有活性血管生成区域及肿瘤组织中特异性高表达,而在正常组织血管内皮细胞不表达或低表达。CD105编码基因位于人染色体9q34,编码的蛋白是一种同型二聚体的跨膜糖蛋白,含633个氨基酸,胞外区含有561个氨基酸,分子量为180KD,存在于细胞膜表面。CD105是转化生长因子β(transforming growth factorβ,TGF-β)受体复合物成分,参与心血管发育、机体稳态等生理过程,并与肿瘤的发展、浸润及转移有关。Endoglin在心血管系统发育过程中的表达表明其与心血管发育有关,主要作用是促进血管的生成、发展及平衡。近年研究发现,CD105是多种肿瘤新生血管内皮的标记物,其在肿瘤新生血管内皮细胞的表达强度显著强于CD34、FⅧR,而且与肿瘤分级、分期、转移和预后相关,可成为抗血管生成治疗肿瘤的新靶标。在实性肿瘤体中,CD105高表达于肿瘤实质、肿瘤附近的血管内皮细胞和肿瘤的间质成分中,参与血管生成,尤其是在胶质瘤、乳腺癌、肝癌、结直肠癌、前列腺癌、胃癌和肺癌中。可作为肿瘤诊断、转移、复发和判断预后的早期指标,是治疗人体恶性肿瘤的理想分子靶点。
肿瘤的血管生长是实体肿瘤代谢的关键途径,是肿瘤生长和转移的重要基础,肿瘤实体内微血管的数量与肿瘤的生长转移密切相关,因此一种可靠的肿瘤微血管标志物将为临床的诊断治疗及预后的判断提供重要的信息。然而,不同于CD31和CD34等泛血管内皮标志物,CD105高表达于重塑或新生的血管中,更能准确反映内皮细胞的增殖状态,可作为衡量内皮细胞增殖状态的指标之一。CD105作为内皮细胞增殖的标志,与肿瘤细胞的增殖活性有关,其所标记的新生血管在恶性肿瘤的发生、发展及预后中具有重要意义。
近年来,人们一直对以血管为靶点的肿瘤治疗寄予极大地关注。目前,抑制血管生成的靶向治疗已成为研究热点,CD105是一种在增殖细胞上特异性表达的跨膜糖蛋白,在正常静止的内皮细胞或正常器官中不易检测到,在分子成像和癌症治疗中是肿瘤血管生成(即新血管形成)的理想靶点。CD105参与血管生成过程,其抗体能够特异地与处于增殖状态的肿瘤组织的血管内皮细胞结合,因此CD105成为理想的抑制肿瘤血管生成的靶点。利用CD105与肿瘤血管的特异性亲和力,将放射性物质与抗CD105单抗结合,可以进行肿瘤的造影,Bredow等将111In标记CD105抗鼠单克隆抗体注射到荷瘤的C57BL/6小鼠静脉内,显示放射物质在肿瘤组织处蓄积。这些研究均表明,CD105单克隆抗体具有很好的靶向作用,利用其靶向作用可以用来筛选高CD105的患者作抗血管生成的靶向治疗,监测抗血管治疗后的效果,判断肿瘤是否复发及手术或放疗后的效果。基于Endoglin的靶向显像可以了解恶性肿瘤的血管生成情况,且可以作为抗肿瘤治疗观察的可靠指标,并为新生血管靶向治疗提供依据。CD105独特的分布提示它可作为治疗恶性肿瘤理想的靶点。应用CD105的单克隆抗体联合化疗药物、生物毒素、放射性核素进行抗肿瘤的靶向治疗将具有广阔的应用前景。
近年来许多研究表明Endoglin与心血管疾病存在密切关系,根据目前的研究分析其可能成为诊断心血管疾病的生物标志物,以及可能成为预防和治疗心
血管疾病的潜在靶点。研究证明血清CD105水平及CD105-TGF-β复合物水平与动脉粥样硬化性心脏病密切相关,研究发现CD105在动脉粥样硬化斑块中有高水平的表达。由于CD105表达的特点,它可作为动脉粥样硬化的一个检测标志物,探讨它在动脉粥样硬化发病过程中的表达与信号转导的关系,可为研究动脉粥样硬化的发病机理及防治提供新的途径,在动脉粥样硬化的诊断和预后判断中发挥重要作用。
分子影像在免疫治疗和个性化医学中发挥越来越重要的作用。其中分子探针的制备是分子影像的关键,由于CD105与肿瘤血管生成有关,将CD105应用于无创分子成像技术,可以观察到全身肿瘤CD105表达水平,成为当前分子成像领域的研究热点。在乳腺癌小鼠中应用89Zr和IRDye 800CW标记的CD105抗体,同时将近红外荧光技术和正电子发射型计算机断层显像(PET)结合,可使癌细胞迅速、持久地显影。越来越多的研究表明CD105在MRI、PET等显像技术中具有潜在的应用前景,这将为恶性肿瘤的早期诊断、治疗和疗效评估等方面提供重要的参考依据。
但由于抗体药物存在着制备繁琐、体外稳定性较差、分子较大、标记困难、穿透力弱,翻译后修饰且费用昂贵等原因使其进一步应用受到限制。因此,为了提高癌症诊断和治疗的特异性和准确性,弥补抗体的缺陷,迫切需要寻求针对新的肿瘤标志物设计小分子探针,以作为检测和治疗癌症的有效方法。多肽类靶向小分子药物及诊断探针以成本低、分子量小、生物相容性好、穿透性强、无免疫原性、并有较快的血液清除速率、且制备简单等特点,在肿瘤靶向给药、癌症诊断等方面彰显出很强的优越性。因此,亟需开发一种针对CD105的分子探针,这对于肿瘤的诊断具有重要意义。
发明内容
本发明的目的是提供一种靶向新生血管标记物CD105的多肽及其应用。
为了实现本发明目的,第一方面,本发明提供一种靶向新生血管标记物CD105的多肽,其为多肽CD1051或CD1052。
多肽CD1051包含如下的氨基酸序列或由其组成:
i)如SEQ ID NO:1所示的氨基酸序列;或
ii)在i)的N端和/或C端连接标签得到的氨基酸序列;或
iii)i)或ii)的氨基酸序列经取代、缺失和/或增加一个或多个氨基酸得到的具有相同功能的多肽。
多肽CD1052包含如下的氨基酸序列或由其组成:
iv)如SEQ ID NO:2所示的氨基酸序列;或
v)在iv)的N端和/或C端连接标签得到的氨基酸序列;或
vi)iv)或v)的氨基酸序列经取代、缺失和/或增加一个或多个氨基酸得到的具有相同功能的多肽。
第二方面,本发明提供编码所述多肽的核酸分子。
第三方面,本发明提供含有所述核酸分子的生物材料,所述生物材料包括但不限于重组DNA、表达盒、转座子、质粒载体、病毒载体、工程菌或转基因细胞系。
第四方面,本发明提供所述多肽的以下任一应用:
1)用于制备治疗或预防CD105表达异常相关疾病的药物;
2)用于制备CD105表达异常相关疾病的检测试剂;
3)用于制备CD105表达异常相关疾病的分子显像剂。
第五方面,本发明提供一种靶向新生血管标记物CD105的分子探针,由所述多肽连接信号单元而成。
所述信号单元选自放射性同位素、荧光染料、量子点、磁性纳米粒子、超顺磁性材料、超声微泡等中的至少一种。
可选地,多肽与信号单元之间通过连接分子进行连接;所述连接分子为6-叔丁氧羰肼基烟酸(HYNIC)、1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺(EDC)或N-羟基琥珀酰亚胺(NHS)等。6-叔丁氧羰肼基烟酸与多肽C端的Cys上的巯基反应;1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺或N-羟基琥珀酰亚胺与多肽C端的羧基反应。
可选地,多肽与信号单元通过螯合剂进行连接;所述螯合剂为DTPA、DOTA、NOTA或TETA等。通过羧基与多肽N端的氨基反应进行偶联。
进一步地,多肽与6-叔丁氧羰肼基烟酸偶联物可以螯合99mTc进行SPECT成像;多肽与螯合剂DTPA和DOTA的偶联物可以螯合顺磁性Gd进行MRI成像;多肽与螯合剂DTPA、DOTA、NOTA或TETA的偶联物可以螯合68Ga或64Cu进行PET成像。
多肽可以与多聚体如聚乙二醇(PEG)、聚乙烯醇(PVA)、环糊精、聚酰胺-胺型树枝状高分子(PAMAM)、聚乳酸(PLA)、聚乳酸-乙醇胺(PLGA)中的任意一种或至少两种进行组合通过亲疏水非共价连接形成组装体(如二价体或多价体)。
第六方面,本发明提供所述分子探针在制备CD105表达异常相关疾病的分子显像剂中的应用,所述分子显像剂可用于荧光成像、正电子发射断层成像、单光子发射断层成像、磁共振成像、光声成像、超声成像或其他活体影像学融合成像技术等,优选荧光成像、PET/CT或PET/MRI。
第七方面,本发明提供一种组合物,包括所述多肽或多肽组装体以及用于杀伤癌细胞的制剂,任选包含药物载体。
所述用于杀伤癌细胞的制剂为能杀伤癌细胞的化学药物、生物药物、纳米药物、放射性药物、光热治疗或光动力治疗药物等;优选地,所述用于杀伤癌细胞的制剂为烷化剂、抗代谢药物、抗肿瘤天然药物、抗肿瘤抗生素、抗肿瘤新生血管药、金属络合物或肿瘤放射靶向标记物等。
所述药物载体为纳米材料、脂质体或胶束等。
其中,多肽组装体是指多肽与多聚体混合通过亲疏水非共价连接形成的组装体(如二价体或多价体);所述多聚体选自聚乙二醇(PEG)、聚乙烯醇(PVA)、聚乳酸-乙醇胺(PLGA)和脂质体或胶束等中的至少一种,显像制剂通过组装体中多肽N端修饰的螯合剂螯合上去进行体内显像。
第八方面,本发明提供所述组合物在制备用于治疗或预防CD105表达异常相关疾病的药物中的应用。
本发明中,所述CD105表达异常相关疾病为肿瘤或心血管系统疾病。
优选地,所述肿瘤选自神经胶质瘤、乳腺癌、非小细胞肺癌、结直肠癌、肝癌、骨肉瘤、血管肉瘤、白血病、胃癌或前列腺癌等。
优选地,所述心血管系统疾病选自冠状动脉粥样硬化症、心肌纤维化、心房颤动、高血压病或心力衰竭等。
借由上述技术方案,本发明至少具有下列优点及有益效果:
本发明提供的多肽CD1051、CD1052具有靶向CD105阳性肿瘤细胞的特性,在实际应用中,可以将本发明多肽作为分子探针用于非侵入性的影像学技术检测肿瘤进展预测评估接受治疗的病人耐药反应和疗效评估。本发明提供的多肽探针选择性强,纯度高,分子量小,特异性强,无免疫原性,安全可靠,可以采用化学合成的方法制备,简单易行,适于作为化疗的预测和伴随诊断试剂。
本发明提供的多肽CD1051、CD1052可替代CD105抗体应用于CD105阳性的血管内皮细胞等,还可以用于制备在体内靶向肿瘤成像或可视化的制剂。将该多肽与抗肿瘤药物或其他试剂形成缀合物,可靶向CD105蛋白用于抗血管生成的肿瘤治疗。此外,还可以反映心血管系统疾病(尤其是动脉粥样硬化斑块)中新生血管的变化情况,在动脉粥样硬化的诊断和预后判断中发挥重要作用。本发明多肽特异性、敏感性好,制备方法简单,成本低,实用性强。本发明为实时监控患者体内CD105表达和肿瘤分级、分期、转移和预后的相关性提供了简便快捷、经济、准确的检测手段。
附图说明
图1为本发明较佳实施例中CD105阳性多肽与蛋白磁珠的结合。
图2为本发明较佳实施例中表面等离子共振(SPRi)检测CD1051和CD1052多肽与人CD105蛋白的亲合力。其中,人CD105蛋白在GenBank上的登录号为AAH14271.1。
图3为本发明较佳实施例中CD1051多肽标记FITC的液相色谱和质谱检测图。
图4为本发明较佳实施例中CD1052多肽标记FITC的液相色谱和质谱检测图。
图5为本发明较佳实施例中FITC标记多肽CD1051和CD1052多肽探针对CD105阳性的HUVEC细胞的成像图。
图6为本发明较佳实施例中CD1051-ICG近红外探针的质谱检测图。
图7为本发明较佳实施例中CD1051-ICG近红外探针对体内微小肿瘤的高灵敏活体成像图。
具体实施方式
本发明旨在提供一种对CD105具有特异性结合的多肽及其应用,所提供的多肽为肿瘤复发、转移和预后评估提供了简便快捷、经济、准确的检测手段。所述多肽不仅特异性、敏感性好,并且制备方法简单,成本低,具有很强的实用性。
本发明采用以下技术方案:
第一方面,本发明采用氨基修饰的TentaGel树脂作为固相载体,利用Fmoc合成策略进行混合均分合成库容量为106的一珠一物肽库。利用微流控芯片的方法进行高通量一珠一物多肽库筛选,阳性肽珠经MALDI-TOF-MS鉴定,获得了一系列能特异性结合CD105的活性多肽。
作为优选技术方案,本发明所述多肽为表1中CD1051、CD1052之一所示:
表1
CD1051 | HYKYWDEDYEC | SEQ ID NO:1 |
CD1052 | HHEYWTERHRC | SEQ ID NO:2 |
本发明中,所述CD1051和CD1052所示的氨基酸序列对CD105高特异性亲和。
第二方面,本发明提供一种DNA片段,其包含编码上述本发明第一方面所述多肽的氨基酸序列。
作为优选技术方案,所述DNA片段包含编码本发明上述CD1051和CD1052之一的氨基酸序列。
第三方面,本发明提供一种活体分子成像的方法,所述方法包括:提供CD105靶向性分子探针,所述CD105靶向性分子探针由信号组分、CD105靶向亲和组分以及连接体三部分组成,所述信号组分为可供影像学设备检测的部分,所述靶向亲和组分为与CD105特异性结合的多肽部分,所述连接体是将信号分子和靶向组分连接起来;用所述的CD105靶向性分子探针对所述患者进行荧学成像、正电子发射断层成像、单光子发射断层成像、磁共振成像、光声成像、超声成像或其他活体影像学融合成像技术优选荧光成像,PET/CT或PET/MRI。
第四方面,本发明还提供一种分子成像探针的组合物,所述连接体是通过与多聚体混合、非共价连接形成的;或利用其他直接的化学反应将信号组件和CD105多肽直接连接。所述CD105靶向性探针的信号组分选自放射性同位素、荧光染料、量子点、磁性纳米粒子、超顺磁性材料、超声微泡的一种或几种材料组合。
第五方面,本发明所述的连接分子为6-叔丁氧羰肼基烟酸(HYNIC)、1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺(EDC)、或N-羟基琥珀酰亚胺(NHS);或DTPA、DOTA、NOTA、TETA等螯合剂;和/或,所述多聚体为聚乙二醇(PEG)、聚乙烯醇(PVA)、环糊精、聚酰胺-胺型树枝状高分子(PAMAM)、聚乳酸(PLA)、聚乳酸-乙醇胺(PLGA)中的任意一种或至少两种的组合。
第六方面,本发明还提供一种药物组合物,包括本发明第一方面所述的多肽(CD1051和CD1052)。
优选地,所述的制剂为能杀伤癌细胞的化学药物、生物药物、纳米药物、放射性药物、光热治疗或光动力治疗药物或包裹这些药物的载体中的任意一种;更优选地,所述的制剂为烷化剂、抗代谢药物、抗肿瘤天然药物、抗肿瘤抗生素、抗肿瘤新生血管药、金属络合物或肿瘤放射靶向标记物中的任意一种。
第七方面,本发明还提供了另外一种药物组合物,所述药物组合物包括本发明第一方面所述的多肽(CD1051和CD1052)及显像制剂。
优选地,所述的显像制剂为放射性核素、放射性核素标记物或分子影像制剂中的任意一种。
第八方面,本发明还提供如本发明第一方面所述的多肽(CD1051和CD1052)或本发明第四方面所述的组合体在制备诊断CD105表达异常相关疾病的试剂用于免疫治疗、预防或诊断癌症的药物或显像制剂中的用途。优选所述CD105表达异常相关疾病为肿瘤和心血管系统疾病。
优选地,所述癌症为神经胶质瘤、乳腺癌、非小细胞肺癌、结直肠癌、肝癌、骨肉瘤、血管肉瘤、白血病、胃癌和前列腺癌中的任意一种。心血管系统疾病优选为冠状动脉粥样硬化症、心肌纤维化、心房颤动、高血压病、心力衰竭。
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段,所用原料均为市售商品。
实施例1 CD105靶向多肽筛选系统的构建和筛选
1、实验仪器与材料
N-甲基吗啉(NMM),哌啶,三氟乙酸(TFA),二氯甲烷(DCM),茚三酮,维生素C,苯酚,四甲基脲六氟磷酸盐(HBTU),六氢吡啶,三异丙基硅烷(TIS),乙二硫醇(EDT),N,N二甲基甲酰胺(DMF),无水乙醚,树脂,甲醇,各种Fmoc保护氨基酸,MB-Streptavidin(链霉亲和素磁珠),多肽合成管,摇床,真空水泵,旋转蒸发仪,激光共聚焦显微镜(ZEISS LSM 710),上述试剂和材料均可通过商业途径获得。
2、CD105“一珠一物”多肽文库的合成
采用Fmoc固相肽合成方法合成多肽文库,具体方法如下:称取200mg的Tentagel-NH2树脂,按照上述固相多肽合成程序循环,依次加入Met、Gly、Cys依次进行反应,脱保护后进行混合均分合成;待反应完成后,把树脂均分2份,向每管分别加入Tyr、His与等量的HBTU进行偶联,待偶联完毕后,把3管树脂脱保护后混合,再把树脂相应均分为若干份,依次循环合成;
①分别加入Asp、Asn、Arg偶联方法同上;
②分别加入Glu、Tyr偶联方法同上;
③分别加入Thr、His、Asp偶联方法同上;
④分别加入Tyr、Phe、Trp偶联方法同上;
⑤分别加入Gln、Tyr、Arg偶联方法同上;
⑥分别加入Glu、Lys、Val偶联方法同上;
⑦分别加入Asn、Tyr、His偶联方法同上;
⑧分别加入Tyr、His偶联方法同上;
待偶联完毕后,树脂TFA脱保护后混合。经过甲醇置换和收缩步骤,真空抽干,得到加载有肽库的干燥树脂备用。
3、CD105阳性多肽的筛选
(1)取干燥肽库用1×PBS洗3次,加入5%的脱脂牛奶在混旋仪上37℃对肽珠表面封闭2h,再用1×PBS洗3次;
(2)取生物素标记的CD105蛋白与多肽库混合,37℃孵育2h后用1×PBS洗3次;
(3)然后分别取100μL MB-Streptavidin加入肽库在混旋仪上37℃避光混合孵育2h。把孵育后含多肽库EP管置于磁力架上。阳性多肽受磁力影响吸附于EP管侧壁,而阴性多肽由于重力沉降在EP管底。
由图1可以看出阳性肽珠与CD105受体蛋白孵育后,阳性肽珠特异性识别蛋白,阳性肽珠表面将包覆一层磁珠具有磁性从而被磁场捕获。将阳性肽珠转移到微芯片阵列中,滴加溴化氢原位裂解,用MALDI-TOF-MS鉴定通过Mascot数据库解出相应序列信息,得到两条多肽分别为:CD1051和CD1052(SEQ ID NO:1和2)。
实施例2通过表面等离子共振(SPRi)方法检测CD1051和CD1052多肽与CD105蛋白的亲和作用
将1mg/mL的CD1051和CD1052多肽及1×PBS点到芯片上,在4℃湿润条件下孵育过夜,然后用10×PBS清洗10min,再用1×PBS清洗10min,最后用去离子水清洗2次,每次10min,浸入含5%牛奶的1×PBS中,4℃条件下孵育过夜,然后用10×PBS清洗10min,1×PBS清洗10min,最后用去离子水清洗2次,每次10min,用氮气吹干,装芯片上机(PlexeraHT表面等离子共振成像系统)。
流动相依次通过1×PBS、2×PBS、0.625μg/mL、1.25μg/mL、2.5μg/mL、5μg/mL和10μg/mL的人CD105纯化蛋白,记录分析SPRi信号。
由图2可以看出CD1051和CD1052的SPRi信号随着蛋白浓度的增加逐渐增强,说明本发明的CD1051和CD1052多肽对CD105都有强结合的,而且亲和解离常数分别达到1.35×10-8M和1.09×10-7M接近抗体的亲和力。可以作为探针靶向CD105阳性血管内皮细胞,用于相关的检测研究和靶向治疗应用。
实施例3 CD1051和CD1052多肽偶联物的制备和荧光检测
本实施例以多肽荧光偶联物制备为例,示例性地展示多肽CD1051和CD1052的偶联物的制备过程,具体如下:
称取100mg的wang-Cys树脂,按照固相多肽合成策略根据CD1051和CD1052的序列依次循环。偶联反应结束后,脱保护,清洗。加入ε-氨基己酸进行室温反应2h,脱保护后,在吡啶、N,N二甲基甲酰胺和二氯甲烷体积比为1∶2∶4的溶液中,将异硫氰酸荧光素(FITC)与50mg树脂混合避光室温反应过夜,FITC与树脂的摩尔比为1∶1。反应结束后,异丙醇清洗甲醇脱水。最后向上述树脂中裂解液,脱出侧链保护基团,真空抽干,得到粗品多肽FITC荧光偶联物。采用MALDI-TOF鉴定和HPLC纯化用于后续实验。结果如图3和图4所示得到纯度95%以上的FITC-CD1051和FITC-CD1052多肽。
实施例4 CD1051和CD1052多肽与CD105高表达细胞HUVEC和低表达细胞293T的亲和分析
HUVEC细胞和293T细胞用含10%胎牛血清的DMEM培养基培养,以1×103/mL的细胞浓度接入圆形玻底培养皿(35mm),37℃,5%CO2细胞培养箱中培养24h后,弃去培养液,两种细胞中分别加入含1μmol/L Hoechst 33342,4℃避光孵育15min后,用预冷1×PBS洗涤2次,分别加入50μM的FITC标记多肽,4℃避光孵育20min后,用预冷1×PBS洗涤3次。用激光扫描共聚焦显微镜(ZEISS LSM 710)检测细胞中的荧光分布。结果如图5所示,加入CD1051和CD1052多肽在HUVEC细胞观测到有很强绿色荧光,而且特异性与靶标蛋白的表达量呈正相关,但阴性细胞293T基本没有绿色荧光,表明靶向探针具有很好的特异性和亲和力,可以作为靶向分子替代抗体用于CD105阳性相关细胞的诊断和检测。
实施例5 CD105多肽显像制剂的制备及其功能验证
本实施例制备CD1051多肽近红外体内成像显像制剂并验证了其功能,具体方法如下:称取1mg的前面已合成的CD1051多肽溶于1×PBS中,称取0.5mg吲哚菁绿-马来酰亚胺(ICG-MAL)溶于500μL去离子水中,将二者混合调pH至7.4左右,室温振荡反应24h,冷冻干燥后进行质谱检测和HPLC纯化。得到的产物经过透析之后,冻干,得到ICG-多肽显像制剂,用于小鼠荧光成像。非小细胞肺癌细胞系A549用含10%胎牛血清的DMEM培养基培养,按1×106通过皮下注射入Balb/c裸鼠右后肢,待肿瘤长至50mm3。称取1mg ICG-CD1051多肽溶于1mL 1×PBS中,尾静脉注射150μL ICG-多肽半小时后,使用IVIS Spectrum小动物活体光学三维成像系统进行信号采集。24h解剖后取主要器官进行体外荧光分布成像。
结果如图6所示,经MALDI-TOF-MS鉴定ICG成功标记在CD1051多肽上。将上述制备得到的多肽显像制剂经尾静脉注射入CD105高表达的荷瘤小鼠体内,如图7所示,在1-24h之内,肿瘤部位荧光逐渐增强,表明该多肽探针具有很好的靶向性,可靶向CD105蛋白用于抗血管生成的肿瘤治疗。
综上,本发明提供的多肽CD1051、CD1052具有靶向表达CD105阳性肿瘤血管组织的特性,在临床上为相关肿瘤分级、分期、转移和预后的相关性提供了简便快捷、经济、准确的检测手段。此外还可以反映心血管系统疾病(尤其是动脉粥样硬化斑块)中新生血管的变化情况,在动脉粥样硬化的诊断和预后判断中发挥重要作用。本发明的多肽特异性、敏感性好,制备方法简单,成本低,实用性强。因而在实际应用中,可以将本发明的多肽作为靶向多肽,与能杀伤癌细胞的制剂相缀合或混合,用于肿瘤的靶向治疗和成像。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之做一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
<110> 福建医科大学
<120> 靶向新生血管标记物CD105的多肽及其应用
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Claims (10)
1.靶向新生血管标记物CD105的多肽,其特征在于,其为多肽CD1051或CD1052,氨基酸序列分别如SEQ ID NO:1、2所示。
2.编码权利要求1所述多肽的核酸分子。
3.含有权利要求2所述核酸分子的生物材料,所述生物材料为重组DNA、表达盒、转座子、质粒载体、病毒载体、工程菌或转基因细胞系。
4.权利要求1所述多肽的以下任一应用:
1)用于制备CD105表达异常相关疾病的检测试剂;
2)用于制备CD105表达异常相关疾病的分子显像剂。
5.靶向新生血管标记物CD105的分子探针,其特征在于,由权利要求1所述多肽连接信号单元而成;
所述信号单元选自放射性同位素、荧光染料、量子点、磁性纳米粒子、超顺磁性材料、超声微泡中的至少一种。
6.根据权利要求5所述的分子探针,其特征在于,多肽与信号单元之间通过连接分子进行连接;
所述连接分子为6-叔丁氧羰肼基烟酸、1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺或N-羟基琥珀酰亚胺;或者,
多肽与信号单元通过螯合剂进行连接;所述螯合剂为DTPA、DOTA、NOTA或TETA。
7.根据权利要求5或6所述分子探针在制备CD105表达异常相关疾病的分子显像剂中的应用,所述分子显像剂用于荧光成像、正电子发射断层成像、单光子发射断层成像、磁共振成像、光声成像或超声成像。
8.根据权利要求7所述的应用,其特征在于,所述分子显像剂用于荧光成像、PET/CT或PET/MRI。
9.一种组合物,其特征在于,包括权利要求1所述多肽或多肽组装体以及用于杀伤癌细胞的制剂,任选包含药物载体;
所述用于杀伤癌细胞的制剂为能杀伤癌细胞的化学药物、生物药物、纳米药物、放射性药物、光热治疗或光动力治疗药物;
所述药物载体为纳米材料、脂质体或胶束;
其中,多肽组装体是指多肽与多聚体混合通过亲疏水非共价连接形成的组装体;所述多聚体选自聚乙二醇、聚乙烯醇、环糊精、聚酰胺-胺型树枝状高分子、聚乳酸、聚乳酸-乙醇胺中的至少一种。
10.根据权利要求9所述的组合物,其特征在于,所述用于杀伤癌细胞的制剂为烷化剂、抗代谢药物、抗肿瘤天然药物、抗肿瘤抗生素、抗肿瘤新生血管药、金属络合物或肿瘤放射靶向标记物。
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