CN110114367B - Vap多肽及其在制备靶向诊疗肿瘤药物中的应用 - Google Patents
Vap多肽及其在制备靶向诊疗肿瘤药物中的应用 Download PDFInfo
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- CN110114367B CN110114367B CN201780069749.3A CN201780069749A CN110114367B CN 110114367 B CN110114367 B CN 110114367B CN 201780069749 A CN201780069749 A CN 201780069749A CN 110114367 B CN110114367 B CN 110114367B
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Abstract
本发明提供了与GRP78蛋白具有高结合活性的高稳定性D构型多肽DVAP和SVAP,还提供了L构型多肽LVAP以及DVAP、SVAP修饰的模型药物和高分子载体材料,以及其在肿瘤影像和靶向治疗用递药系统构建中的应用。
Description
技术领域
本发明属药学领域,涉及VAP多肽及其在制药中的应用,具体涉及高度稳定且可靶向葡萄糖调节蛋白GRP78的多功能D构型多肽,及L构型多肽和稳定性D构型多肽的药物复合物和修饰的纳米递药系统,尤其涉及D构型多肽DVAP(D构型氨基酸序列DPDADVDRDTDNDS)和SVAP(D构型氨基酸序列DSDNDTDRDVDADP),及L构型多肽LVAP(氨基酸序列SNTRVAP)和稳定性D构型多肽的诊断和治疗药物复合物、修饰的高分子载体材料及其所构建的脂质体、聚合物胶束、聚合物圆盘、纳米粒等纳米递药系统,以及在制备肿瘤诊断和靶向治疗药物中的应用。
背景技术
文献报道了肿瘤是严重威胁人类生命和健康的疾病,死亡率高居所有疾病死亡率首位。传统的化疗作为肿瘤药物治疗的主要手段,存在对肿瘤组织选择性差、毒性大、治疗窗窄、易产生多药耐药等缺陷。因此,为克服传统治疗手段的局限性,近年来,主动靶向成为提高肿瘤组织靶向效率的重要策略。资料公开了主动靶向策略主要针对肿瘤组织中高表达的受体或转运体,利用与特异性受体或转运体具有识别、结合能力的对应配体,将药物或纳米递药系统递送至肿瘤组织或细胞中,常用的对应配体包括单克隆抗体、多肽、核酸适体、小分子化合物等;配体修饰后的药物或纳米递药系统可通过细胞表面受体或转运体与配体的特异性识别、结合、内化,将药物递送至肿瘤组织和细胞内,从而实现药物对肿瘤的主动靶向目标。
葡萄糖调节蛋白GRP78又名免疫球蛋白重链结合蛋白(Bip),是内质网主要的分子伴侣之一,在蛋白质的折叠以及内质网应激反应中发挥着重要的作用。研究显示,若干肿瘤细胞株,实体瘤以及人癌组织活检样本中GRP78的表达明显升高,研究表明,GRP78蛋白在乳腺癌、肝癌、结肠癌、胃癌等多种肿瘤中均高表达,并且与这些肿瘤的发生、进展、预后和耐药密切相关。最近有研究表明,GRP78在肿瘤细胞中可以转移到细胞膜表面,而在正常细胞的膜表面却没有观察到这种转移;研究还表明GRP78在肿瘤干细胞的调控中发挥关键作用,通过Cripto/GRP78信号通路调节成体干细胞以及肿瘤干细胞的功能,维持干细胞干性,因此,通过GRP78受体介导途径将药物或纳米递药系统特异性递送到肿瘤组织,对提高药物的肿瘤诊断与治疗效果将具有重要价值。
LVAP(L构型氨基酸序列SNTRVAP)是通过噬菌体展示技术筛选出的一条七肽,研究证明其对GRP78具有高亲和活性的,但迄今尚未见有在肿瘤靶向诊疗方面的研究报道。
基于现有技术的现状,本申请的发明人拟提供VAP多肽在肿瘤靶向诊治中的应用并进一步优化已有多肽的稳定性,达到更好的体内肿瘤靶向效果。
发明内容
本发明的目的是针对现有技术的缺陷,提供VAP多肽及其肿瘤靶向诊治中的应用,并进一步优化已有多肽的稳定性,达到更好的体内肿瘤靶向效果。
本发明的第一方面提供了一种D构型多肽,所述D构型多肽为DVAP和/或SVAP,且所述DVAP的氨基酸序列为DPDADVDRDTDNDS,所述SVAP的氨基酸序列DSDNDTDRDVDADP。
本发明的第二方面提供了一种DVAP和/或SVAP多肽复合物,所述DVAP和/或SVAP多肽复合物为权利要求1所述的DVAP和/或SVAP多肽修饰含有马来酰亚胺基团的影像物质,其中,所述DVAP和/或SVAP多肽复合物的结构为DVAP-X和/或SVAP-X,X为所述影像物质;优选地,所述X选自荧光物质、近红外染料、磁共振影像剂和放射影像剂中的一种或多种;
更优选地,所述荧光物质为Fluorescein,近红外染料为Cy7、IR820、DiR,所述磁共振影像剂为Gd-DTPA,所述放射影像剂99mTc-DTPA。
本发明的第三方面提供了一种L构型的LVAP多肽复合物,所述LVAP多肽复合物为LVAP多肽修饰含有马来酰亚胺基团的影像物质,其中,所述LVAP多肽的氨基酸序列为SNTRVAP,且所述LVAP多肽复合物的结构为LVAP-X,X为所述影像物质;
优选地,所述X选自荧光素、近红外染料、磁共振影像剂和放射影像剂中的一种或多种;
更优选地,所述荧光素为Fluorescein,近红外染料为Cy7、IR820、DiR,所述磁共振影像剂为Gd-DTPA,所述放射影像剂99mTc-DTPA。
本发明的第四方面提供了一种DVAP和/或SVAP多肽复合物,所述DVAP和/或SVAP多肽复合物为权利要求1所述的DVAP和/或SVAP多肽修饰抗肿瘤药物,其中,所述DVAP和/或SVAP多肽复合物的结构为DVAP-Y和/或SVAP-Y,Y为所述抗肿瘤药物;
优选地,所述抗肿瘤药物选自阿霉素和表阿霉素等蒽环类药物、紫杉醇和多烯紫杉醇和卡巴他赛等紫杉烷类药物、喜树碱和羟基喜树碱和伊立替康等喜树碱类药物、长春新碱和长春瑞滨等长春花碱类药物、硼替佐米和卡非佐米等蛋白酶体抑制剂、小白菊内酯等内酯类药物、p53激活肽和蜂毒肽、蝎毒肽和抗菌肽等多肽类药物中的一种或多种;
更优选地:所述抗肿瘤药物选自含酮或醛基的阿霉素或表阿霉素,含羟基或氨基的紫杉醇、多烯紫杉醇、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱、依托泊苷、吉西他滨、阿糖胞苷、5-氟尿嘧啶、替尼泊苷、莫立替尼、埃博霉素、长春瑞滨、放线菌素D、米托蒽醌、丝裂霉素、博来霉素、伊立替康,含硼酸基团的硼替佐米或卡非佐米,和/或多肽药物p53激活肽、蜂毒肽和蝎毒肽中的一种或多种。
本发明的第五方面提供了一种L构型的LVAP多肽复合物,所述LVAP多肽复合物为LVAP多肽修饰抗肿瘤药物,其中,所述LVAP多肽的氨基酸序列为SNTRVAP,且所述LVAP多肽复合物的结构为LVAP-Y,Y为所述抗肿瘤药物;
优选地,所述抗肿瘤药物选自阿霉素和表阿霉素等蒽环类药物、紫杉醇和多烯紫杉醇和卡巴他赛等紫杉烷类药物、喜树碱和羟基喜树碱和伊立替康等喜树碱类药物、长春新碱和长春瑞滨等长春花碱类药物、硼替佐米和卡非佐米等蛋白酶体抑制剂、小白菊内酯等内酯类药物、p53激活肽和蜂毒肽、蝎毒肽和抗菌肽等多肽类药物中的一种或多种;
更优选地:所述抗肿瘤药物选自含酮或醛基的阿霉素或表阿霉素,含羟基或氨基的紫杉醇、多烯紫杉醇、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱、依托泊苷、吉西他滨、阿糖胞苷、5-氟尿嘧啶、替尼泊苷、莫立替尼、埃博霉素、长春瑞滨、放线菌素D、米托蒽醌、丝裂霉素、博来霉素、伊立替康,含硼酸基团的硼替佐米或卡非佐米,和/或多肽药物p53激活肽、蜂毒肽和蝎毒肽中的一种或多种。
本发明的第六方面提供了一种DVAP和/或SVAP多肽复合物,所述DVAP和/或SVAP多肽复合物为权利要求1所述的DVAP和/或SVAP多肽修饰高分子载体材料,其中,所述DVAP和/或SVAP多肽复合物的结构为DVAP-聚乙二醇-Z和/或SVAP-聚乙二醇-Z,Z为所述高分子载体材料;
优选地,所述高分子载体材料选自磷脂、聚乳酸、乳酸羟基乙酸共聚物和聚己内酯中的一种或多种。
本发明的第七方面提供了一种L构型的LVAP多肽复合物,所述LVAP多肽复合物为LVAP多肽修饰高分子载体材料,其中,所述,所述LVAP多肽的氨基酸序列为SNTRVAP,且所述LVAP多肽复合物的结构为LVAP-聚乙二醇-Z,Z为所述高分子载体材料;
优选地,所述高分子载体材料选自磷脂、聚乳酸、乳酸羟基乙酸共聚物和聚己内酯中的一种或多种。
本发明的第八方面提供了一种递药系统,所述递药系统包括第六方面或第七方面所述的复合物;优选地,所述递药系统为脂质体递药系统、聚合物胶束递药系统、聚合物圆盘递药系统或纳米粒递药系统。
根据本发明第八方面所述的递药系统,所述递药系统还包括所述DVAP、SVAP和/或LVAP多肽复合物以外的(1)诊断药物和/或(2)抗肿瘤药物;优选地:
所述(1)诊断药物选自荧光物质、近红外染料和磁共振影像剂中的一种或多种,更优选地,所述荧光物质为Fluorescein,所述近红外染料选自Cy7、IR820、DiR,和/或所述磁共振影像剂为Gd-DTPA,和/或
所述(2)抗肿瘤药物选自:阿霉素和表阿霉素等蒽环类药物、紫杉醇和多烯紫杉醇和卡巴他赛等紫杉烷类药物、喜树碱和羟基喜树碱和伊立替康等喜树碱类药物、长春新碱和长春瑞滨等长春花碱类药物、硼替佐米和卡非佐米等蛋白酶体抑制剂、小白菊内酯等内酯类药物、p53激活肽和蜂毒肽、蝎毒肽和抗菌肽等多肽类药物中的一种或多种。
本发明第九方面提供了第一方面所述的DVAP和/或SVAP多肽、第二方面至第七方面所述的DVAP、SVAP和/或LVAP多肽复合物、第八方面所述的递药系统在制备用于诊断、示踪和/或治疗肿瘤的药品或医疗产品中的应用,优选地:
所述肿瘤为高表达葡萄糖调节蛋白GRP78肿瘤。
本发明的第十方面提供了一种用于肿瘤诊断和/或靶向治疗的方法,向有需要的受试者给予:
根据第一方面所述的D构型多肽;
根据第二方面至第七方面任一项所述的DVAP、SVAP和/或LVAP多肽复合物;和/或
第八方面所述的递药系统。
本发明的稳定化D构型VAP多肽,所述D构型VAP多肽为DVAP和/或SVAP,且所述DVAP的氨基酸序列为DPDADVDRDTDNDS,所述SVAP的氨基酸序列DSDNDTDRDVDADP。
具体的,本发明设计并制备了D构型多肽DVAP和/或SVAP,两条多肽均对血清具有高稳定性、与GRP78具有高亲和力。
本发明的D构型多肽或文献报道的L构型多肽(LVAP,氨基酸序列为SNTRVAP)复合物,所述的DVAP、SVAP、LVAP多肽复合物为DVAP、SVAP、LVAP修饰影像物质,其中,所述DVAP、SVAP、LVAP多肽复合物的结构为DVAP-X、SVAP-X和/或LVAP-X,X为所述影像物质。
优选地,所述X选自荧光素、近红外染料、磁共振影像剂和放射影像剂中的一种或多种,更优选地,所述荧光素为Fluorescein,所述近红外染料选自cy7、IR820和DiR中的一种或多种,所述磁共振影像剂为Gd-DTPA,所述放射影像剂99mTc-DTPA。
具体地,本发明的DVAP、SVAP或文献报道的LVAP巯基化后,可利用其分子中巯基与马来酰亚胺功能化荧光物质(如Fluorescein、近红外染料Cy7、IR820、DiR等)、磁共振影像剂Gd-DTPA和放射影像剂99mTc-DTPA,反应而形成复合物。
本发明的DVAP、SVAP或文献报道的LVAP复合物,所述DVAP、SVAP、LVAP多肽复合物为DVAP、SVAP、LVAP修饰抗肿瘤药物,其中,所述DVAP、SVAP、LVAP多肽复合物的结构为DVAP-Y、SVAP-y和/或LVAP-Y,Y为所述抗肿瘤药物。
优选地,所述抗肿瘤药物选自阿霉素和表阿霉素等蒽环类药物、紫杉醇和多烯紫杉醇和卡巴他赛等紫杉烷类药物、喜树碱和羟基喜树碱和伊立替康等喜树碱类药物、长春新碱和长春瑞滨等长春花碱类药物、硼替佐米和卡非佐米等蛋白酶体抑制剂、小白菊内酯等内酯类药物、p53激活肽和蜂毒肽、蝎毒肽和抗菌肽等多肽类药物中的一种或多种。
更优选地:所述抗肿瘤药物选自含酮或醛基的阿霉素或表阿霉素,含羟基或氨基的紫杉醇、多烯紫杉醇、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱、依托泊苷、吉西他滨、阿糖胞苷、5-氟尿嘧啶、替尼泊苷、莫立替尼、埃博霉素、长春瑞滨、放线菌素D、米托蒽醌、丝裂霉素、博来霉素、伊立替康,含硼酸基团的硼替佐米或卡非佐米,和/或多肽药物p53激活肽、蜂毒肽和蝎毒肽中的一种或多种。
具体地,本发明的DVAP、SVAP或文献报道的LVAP修饰药物,包括通过马来酰亚胺己肼衍生物反应形成pH敏感腙键(涉及阿霉素、表阿霉素等含酮或醛基的药物)、或通过3-(2-吡啶二巯基)丙酸衍生物反应形成二硫键(涉及紫杉醇、多烯紫杉醇、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱、依托泊苷、吉西他滨、阿糖胞苷、5-氟尿嘧啶、替尼泊苷、莫立替尼、埃博霉素、长春瑞滨、放线菌素D、米托蒽醌、丝裂霉素、博来霉素、伊立替康等含羟基或氨基的药物)、或通过多巴胺与药物中硼酸基团反应形成pH敏感硼酸脂(涉及硼替佐米等含硼酸基团的药物)、或通过固相合成直接形成酰胺键(涉及p53激活肽、抗菌肽、多肽毒素等多肽药物)的多肽-药物复合物。
本发明的DVAP、SVAP或文献报道的LVAP复合物,所述DVAP、SVAP、LVAP多肽复合物为DVAP、SVAP、LVAP修饰高分子载体材料,其中,所述DVAP、SVAP、LVAP多肽复合物的结构为DVAP-聚乙二醇-Z、SVAP-聚乙二醇-Z和/或LVAP-聚乙二醇-Z,Z为所述高分子载体材料。
优选地,所述高分子载体材料选自磷脂、聚乳酸、乳酸羟基乙酸共聚物和聚己内酯中的一种或多种。
具体地,本发明的DVAP、SVAP或文献报道的LVAP巯基化后,可修饰在含马来酰亚胺功能基的聚乙二醇-二硬脂酰基磷脂酰乙醇胺(PEG-DSPE)、聚乙二醇-聚乳酸(PEG-PLA)、聚乙二醇-乳酸羟基乙酸共聚物(PEG-PLGA)、聚乙二醇-聚己内酯(PEG-PCL)等高分子载体材料上,可用于DVAP、SVAP、LVAP修饰的脂质体、聚合物胶束、聚合物圆盘、纳米粒等纳米递药系统的构建。
本发明的递药系统,所述递药系统包括前述的DVAP、SVAP、LVAP多肽复合物。优选地,所述递药系统为脂质体递药系统、聚合物胶束递药系统、聚合物圆盘递药系统或纳米粒递药系统。
作为优选实施方式,本发明还提供了包括所述DVAP、SVAP、LVAP多肽复合物以外的(1)诊断药物和/或(2)抗肿瘤药物的前述递药系统。
优选地,所述(1)诊断药物选自荧光物质、近红外染料和磁共振影像剂中的一种或多种。更优选地,所述荧光物质为Fluorescein,所述近红外染料选自Cy7、IR820、DiR中的一种或多种,和/或磁共振影像剂为Gd-DTPA。和/或所述(2)抗肿瘤药物选自阿霉素和表阿霉素等蒽环类药物、紫杉醇和多烯紫杉醇和卡巴他赛等紫杉烷类药物、喜树碱和羟基喜树碱和伊立替康等喜树碱类药物、长春新碱和长春瑞滨等长春花碱类药物、硼替佐米和卡非佐米等蛋白酶体抑制剂、小白菊内酯等内酯类药物、p53激活肽和蜂毒肽、蝎毒肽和抗菌肽等多肽类药物中的一种或多种。
具体地,本发明所设计的DVAP、SVAP、LVAP修饰的纳米递药系统可包载阿霉素和表阿霉素等蒽环类药物、紫杉醇和多烯紫杉醇和卡巴他赛等紫杉烷类药物、喜树碱和羟基喜树碱和伊立替康等喜树碱类药物、长春新碱和长春瑞滨等长春花碱类药物、硼替佐米和卡非佐米等蛋白酶体抑制剂、小白菊内酯等内酯类药物、p53激活肽和蜂毒肽、蝎毒肽和抗菌肽等多肽类药物等;也可包载荧光物质、近红外染料和磁共振影像剂,如Fluorescein、Cy7、IR820、DiR、Gd-DTPA等。
本发明还提供了前述的DVAP、SVAP多肽、前述的DVAP、SVAP、LVAP多肽复合物、前述的递药系统在制备用于诊断、示踪和/或治疗肿瘤的药品或医疗产品中的应用。
优选地:
所述肿瘤为高表达GRP78肿瘤。
具体地,本发明第一方面的DVAP、SVAP或文献报道的LVAP可介导药物或纳米递药系统靶向GRP78高表达的细胞及其组织,用于肿瘤的靶向诊断和治疗。
本发明还提供了一种用于诊断、示踪和/或治疗肿瘤的组合产品,所述组合产品包括选自以下的一种或多种成分:前述的DVAP、SVAP、LVAP多肽复合物和前述的递药系统。
优选地,所述组合产品为试剂盒,和/或
所述肿瘤为高表达GRP78肿瘤。
本发明的一种诊断、示踪和/或治疗肿瘤的方法,包括对患有所述肿瘤或疑患有所述肿瘤的患者通过口服或非口服途径给予有效剂量的选自以下的一种或多种物质:前述的DVAP、SVAP、LVAP多肽复合物、前述的递药系统和前述的组合产品。
优选地,所述肿瘤为高表达GRP78肿瘤;和/或
优选地,所述口服或非口服途径可以为通过口服、注射、贴片、喷雾和其他已知的一种或多种递送给所述患者。所述有效量可以包括对治疗、降低、缓和、减轻、消除或状况的一种或多种症状有效的量,所述状况寻求被治疗,或可选地,所述状况寻求被避免,或另外在所述状况或其效果中产生临床上可确认的有利变化。
本发明提供了前述的DVAP、SVAP、LVAP多肽在制备肿瘤靶向产品中的应用。优选地:所述肿瘤靶向产品用于靶向GRP78高表达的肿瘤;和/或所述肿瘤靶向产品为用于诊断、示踪和/或治疗肿瘤的药品、实验试剂和/或医疗产品。本发明提供了LVAP多肽(SNTRVAP)修饰的药物复合物和修饰的纳米递药系统;同时针对L构型多肽的体内稳定性差,在血液中易降解,可能导致肿瘤靶向能力降低的问题,提供了高度稳定且与GRP78有高度结合活性的D构型多肽靶向分子DVAP(D构型氨基酸序列DPDADVDRDTDNDS)与SVAP(D构型氨基酸序列DSDNDTDRDVDADP),并构建其药物复合物和修饰的纳米递药系统,能实现肿瘤的靶向诊断和治疗,获得更好的体内肿瘤靶向效果。
具体的,本发明制备了具有高稳定性的D构型多肽DVAP(D构型氨基酸序列DPDADVDRDTDNDS)和SVAP(D构型氨基酸序列DSDNDTDRDVDADP),并以LVAP(L构型氨基酸序列SNTRVAP)、DVAP和SVAP修饰药物分子和高分子载体材料,构建VAP药物复合物、VAP修饰的纳米递药系统。
本发明中,利用固相多肽合成技术,设计并制备了D构型多肽靶向分子DVAP(D构型氨基酸序列DPDADVDRDTDNDS)与SVAP(D构型氨基酸序列DSDNDTDRDVDADP),两条多肽均对血清具有高稳定性、与GRP78具有高亲和力。
本发明中,LVAP和所设计的DVAP、SVAP连接半胱氨酸后,可利用其分子中巯基与马来酰亚胺功能化影像物质(荧光物质Fluorescein、近红外染料Cy7、IR820、DiR、磁共振影像剂Gd-DTPA、放射影像剂99mTc-DTPA等)反应而形成复合物。
本发明中,LVAP和所设计的DVAP、SVAP修饰药物,包括通过马来酰亚胺己肼衍生物反应形成pH敏感腙键(涉及阿霉素、表阿霉素等含酮或醛基的药物)、或通过3-(2-吡啶二巯基)丙酸衍生物反应形成二硫键(涉及紫杉醇、多烯紫杉醇、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱等含羟基或氨基的药物)、或通过多巴胺与药物中硼酸基团反应形成pH敏感硼酸脂(涉及硼替佐米等含硼酸基团的药物)、或通过固相合成直接形成酰胺键(涉及p53激活肽、抗菌肽、多肽毒素等多肽药物)的多肽-药物复合物。
本发明中,LVAP和所设计的DVAP以及SVAP连接半胱氨酸后,可修饰在含马来酰亚胺功能基的聚乙二醇-二硬脂酰基磷脂酰乙醇胺(PEG-DSPE)、聚乙二醇-聚乳酸(PEG-PLA)、聚乙二醇-乳酸羟基乙酸共聚物(PEG-PLGA)、聚乙二醇-聚己内酯(PEG-PCL)等高分子载体材料上,用于LVAP、DVAP和SVAP修饰的脂质体、聚合物胶束、聚合物圆盘、纳米粒等纳米递药系统的构建。
本发明中,所设计的LVAP、DVAP和SVAP修饰的纳米递药系统包载阿霉素和表阿霉素等蒽环类、紫杉醇和多烯紫杉醇和卡巴他赛等紫杉烷类、喜树碱和羟基喜树碱和伊立替康等喜树碱类、长春新碱和长春瑞滨等长春花碱类、硼替佐米和卡非佐米等蛋白酶体抑制剂类、小白菊内酯等内酯类、p53激活肽和蜂毒肽、蝎毒肽和抗菌肽等多肽类等抗肿瘤药物;或包载影像物质,如Fluorescein,近红外染料Cy7、IR820、DiR、磁共振影像剂Gd-DTPA等。
本发明所述的DVAP、SVAP和LVAP可介导药物或纳米递药系统靶向葡萄糖调节蛋白GRP78高表达的细胞及其组织,用于肿瘤的靶向诊断和治疗。
本发明提供了DVAP、SVAP制备和性质考察以及上述LVAP、DVAP和SVAP所修饰的药物复合物和纳米递药系统用于制备肿瘤诊疗药物的物质基础;以及进行了LVAP、DVAP和SVAP介导的体内主动靶向的试验,其中包括,
1.VAP、VAP-Cys及其荧光标记物(VAP-Fluorescein、VAP-Cy7)的合成
采用固相合成方法制备LVAP、LVAP-Cys、DVAP、DVAP-Cys、SVAP、SVAP-Cys。通过马来酰亚胺基团与巯基的Michael加成反应合成了LVAP-Fluorescein、DVAP-Fluorescein、SVAP-Fluorescein、LVAP-Cy7、DVAP-Cy7、SVAP-Cy7。HPLC、MS表征结构。
2.VAP的稳定性和受体亲和性评价
从血清稳定性、与葡萄糖调节蛋白GRP78结合能力和与高表达这种蛋白的细胞摄取能力三方面进行DVAP、SVAP性质的考察。将DVAP、SVAP和LVAP分别与小鼠血清在37℃进行孵育,在不同时间点检测多肽的浓度进行稳定性的比较。采用表面等离子共振法评价DVAP、SVAP和LVAP与GRP78的结合能力,比较DVAP-Fluorescein、SVAP-Fluorescein、LVAP-Fluorescein对GRP78高表达的细胞(如:脐静脉内皮细胞HUVEC)和模型肿瘤细胞(如:脑胶质瘤细胞U87)的体外靶向性,比较体外3D肿瘤球模型对DVAP-Fluorescein、SVAP-Fluorescein、LVAP-Fluorescein的摄取能力。
3.VAP药物复合物的制备
连接半胱氨酸后的LVAP、DVAP和SVAP与药物的马来酰亚胺己肼衍生物反应,形成含pH敏感腙键的多肽-药物复合物,其中所涉及药物包括阿霉素、表阿霉素等含酮或醛基的药物;
连接半胱氨酸后的LVAP、DVAP和SVAP与药物的3-(2-吡啶二巯基)丙酸衍生物反应,形成含二硫键的多肽-药物复合物,其中所涉及药物包括紫杉醇、多烯紫杉醇、喜树碱、羟基喜树碱、9-硝基喜树碱、长春新碱等含羟基或氨基的药物;
LVAP、DVAP和SVAP通过修饰上多巴胺进而与药物的硼酸基团反应,形成含pH敏感硼酸脂的多肽-药物复合物,其中所涉及药物包括硼替佐米等含硼酸基团的药物;
LVAP、DVAP和SVAP通过固相合成直接与多肽药物缩合制成融合多肽,其中所涉及药物包括p53激活肽、抗菌肽、多肽毒素等多肽药物。
4、VAP-阿霉素复合物的体内药效与药动学
连接半胱氨酸后的DVAP与阿霉素马来酰亚胺己肼衍生物(MAL-DOX)缩合得的DVAP-阿霉素复合物(DVAP-DOX),通过荷U87皮下移植瘤模型裸鼠尾静脉给药,以瘤体积、瘤重和抑瘤率为指标评价其体内抗肿瘤效果;通过荷U87原位瘤模型裸鼠尾静脉给药,以中位生存时间为指标评价其体内抗肿瘤效果;通过小鼠尾静脉注射给药,荧光法定量绘制药物动力学曲线与体内分布。
5.VAP-PEG-PLA胶束递药系统的构建与表征
首先合成LVAP、DVAP和SVAP修饰的高分子材料LVAP-PEG-PLA、DVAP-PEG-PLA和SVAP-PEG-PLA。通过连接半胱氨酸的多肽上的游离巯基与Mal-PEG-PLA所含马来酰亚胺的反应实现材料的合成。将Mal-PEG-PLA溶解在乙腈中,旋转蒸发,成膜,加入含多肽的PBS(pH 8.0)反应制备得到LVAP-PEG-PLA、DVAP-PEG-PLA和SVAP-PEG-PLA;
然后分别制备LVAP、DVAP、SVAP修饰的胶束(LVAP-Micelle、DVAP-Micelle、SVAP-Micelle)。一定量的VAP-PEG-PLA、mPEG-PLA和药物(香豆素C6、DiR或者紫杉醇)采用成膜法制备胶束,激光散射粒度仪表征胶束粒径和粒径分布。
6.VAP-Micelle的体内外肿瘤靶向性评价
考察U87细胞、HUVEC细胞和U87肿瘤球体外模型对LVAP-Micelle/C6、DVAP-Micelle/C6、SVAP-Micelle/C6和mPEG-Micelle/C6的摄取情况;
通过荷U87皮下移植瘤模型裸鼠尾静脉分别注射LVAP-Micelle/DiR、DVAP-Micelle/DiR、SVAP-Micelle/DiR和mPEG-Micelle/DiR,比较不同组在各时间点的肿瘤内分布。
7.VAP-Micelle/PTX的体内抗肿瘤效果评价
通过荷U87皮下移植瘤模型裸鼠尾静脉分别注射LVAP-Micelle/PTX、SVAP-Micelle/PTX、DVAP-Micelle/PTX和mPEG-Micelle/PTX、临床用制剂泰素、生理盐水,以瘤体积、瘤重和肿瘤组织细胞凋亡、新生血管和拟态血管数量为指标评价不同载紫杉醇递药系统的体内抗肿瘤效果。
本发明提供了DVAP、SVAP制备和性质考察以及上述LVAP、DVAP和SVAP所修饰的药物复合物和纳米递药系统用于制备肿瘤诊疗药物的物质基础;本发明的试验结果表明:LVAP、DVAP和SVAP均可介导的体内主动靶向;与LVAP相比,DVAP和SVAP血清中稳定性更好,因而其介导的体内主动靶向效果更优。
附图的简要说明
图1示出了DVAP的HPLC和ESI-MS图谱:
色谱方法:色谱柱(YMC,C18):150×4.6mm;流动相A:水(含0.1%三氟乙酸),流动相B:乙腈(含0.1%三氟乙酸);洗脱程序:0-30min 5%B-65%B;流速:0.7mL/min;柱温:40℃;检测:UV 214nm,保留时间:11.1min。ESI-MS:743.5,与理论分子量相符合。
图2示出了DVAP-Cys的HPLC和ESI-MS图谱:
色谱方法同上,保留时间:12.0min。ESI-MS:846.5,与理论分子量相符合。
图3示出了SVAP的HPLC和ESI-MS图谱:
色谱方法同上,保留时间:10.8min。ESI-MS:743.5,与理论分子量相符合。
图4示出了SVAP-Cys的HPLC和ESI-MS图谱:
色谱方法同上,保留时间:11.5min。ESI-MS:846.5,与理论分子量相符合。
图5示出了LVAP的HPLC和ESI-MS图谱:
色谱方法同上,保留时间:9.5min。ESI-MS:743.5,与理论分子量相符合。
图6示出了LVAP-Cys的HPLC和ESI-MS图谱,
色谱方法同上,保留时间:9.6min。ESI-MS:846.5,与理论分子量相符合。
图7示出了DVAP-Fluorescein的HPLC和ESI-MS图谱:
色谱方法同上,保留时间:15.3min。ESI-MS:1274.4,与理论分子量相符合。
图8示出了SVAP-Fluorescein的HPLC和ESI-MS图谱:
色谱方法同上,保留时间:16.9min。ESI-MS:1274.4,与理论分子量相符合。
图9示出了LVAP-Fluorescein的HPLC和ESI-MS图谱:
色谱方法同上,保留时间:16.5min。ESI-MS:1274.4,与理论分子量相符合。
图10示出了DVAP-Cy7的HPLC和ESI-MS图谱:
色谱方法同上,保留时间:26.5min。ESI-MS:1535.8,与理论分子量相符合.
图11示出了SVAP-Cy7的HPLC和ESI-MS图谱:
色谱方法同上,保留时间:26.5min。ESI-MS:1535.8,与理论分子量相符合。
图12示出了LVAP-Cy7的HPLC和ESI-MS图谱,
色谱方法同上,保留时间:26.5min。ESI-MS:1535.8,与理论分子量相符合。
图13示出了DVAP-DOX的HPLC和ESI-MS图谱:
色谱方法除流动相中0.1%三氟乙酸替换成0.1%甲酸外,其他方法同上,保留时间:15.5min。ESI-MS:1599.6,与理论分子量相符合。
图14示出了DVAP-PEG3000-PLA2000、SVAP-PEG3000-PLA2000和LVAP-PEG3000-PLA2000的1H-NMR图谱:
Mal-PEG-PLA的核磁图谱于6.7ppm显示出马来酰亚胺峰,而VAP-PEG-PLA的核磁图谱中该峰消失,显示Mal-PEG-PLA中的马来酰亚胺基团已反应完全。
图15示出了DVAP、SVAP和LVAP的血清稳定性:
图纵坐标为完整多肽的残留百分比,可见DVAP和SVAP在50%小鼠血清中的稳定性显著高于LVAP,孵育2h,LVAP完全降解、DVAP和SVAP几乎不降解。
图16示出了DVAP、SVAP和LVAP与GRP78结合活性:
DVAP、SVAP和LVAP与GRP78的结合活性相似,稍弱于LVAP,KD值分别为4.010μM和5.223μM、2.696μM。三条多肽的解离模式相似,Kd值分别为0.02091 1/s、0.02826 1/s、0.01898 1/s。
图17示出了GRP78的细胞内分布:
在肿瘤细胞U87细胞上,及脐静脉细胞HUVEC上,GRP78广泛分布于细胞膜上及细胞内。而在正常细胞HEK293细胞膜上未见分布,膜内有少量GRP78分布。
图18示出了脑胶质瘤细胞U87对Fluorescein标记多肽的摄取:
图A和图B分别为Fluorescein标记的DVAP、SVAP和LVAP与U87细胞作用4h后的激光共聚焦照片和流式细胞荧光检测结果。可见U87细胞对DVAP、SVAP和LVAP的摄取明显高于游离荧光素,对DVAP、SVAP的摄取没有明显差异,稍弱于LVAP。
图19示出了脐静脉内皮细胞HUVEC对Fluorescein标记多肽的摄取:
图A和图B分别为Fluorescein标记的DVAP、SVAP和LVAP与HUVEC细胞作用4h后的激光共聚焦照片和流式细胞荧光检测结果。可见HUVEC细胞对DVAP、SVAP和LVAP的摄取明显高于游离荧光素,对DVAP、SVAP的摄取没有明显差异,稍弱于LVAP。
图20示出了Fluorescein标记多肽及VAP-Micelle/C6的U87肿瘤球摄取:
图为各Fluorescein标记多肽及VAP胶束被U87肿瘤球摄取情况,由图可知,各Fluorescein标记多肽及VAP胶束均能很好的被U87肿瘤球摄取,与无靶胶束和FAM有明显差异。
图21示出了DVAP、SVAP和LVAP竞争抑制实验:
可见DVAP、SVAP和LVAP三条多肽能够相互完全抑制,证明三条多肽均结合在GRP78蛋白的同一位点。
图22示出了Cy7标记多肽的皮下移植瘤内分布:
图A为荷U87皮下移植瘤裸鼠尾静脉注射Cy7标记多肽1h后的离体肿瘤影像分布结果;图B为给药后各个时间点荧光半定量结果;图C为离体肿瘤和脏器的荧光分布图像;图D为离体肿瘤荧光半定量结果,Cy7标记的DVAP、SVAP和LVAP在肿瘤内的蓄积均显著高于游离Cy7(***p<0.001),且肿瘤靶向效果依次为:DVAP≈SVAP>LVAP。
图23示出了DVAP-DOX皮下瘤抑制实验:
图A为各组裸鼠肿瘤体积随时间变化的曲线,与生理盐水组相比,各给药组对肿瘤生长均有抑制作用。同剂量DVAP-DOX的药效显著优于DOX和MAL-DOX,明显优于RGD-DOX。图B为将裸鼠处死取出肿瘤组织后称重并进行统计分析结果,图C为离体肿瘤组织照片,可见同剂量DVAP-DOX组肿瘤大小与瘤重显著低于其它各组。
图24示出了DVAP-DOX抗原位脑胶质瘤模型裸鼠的生存曲线:
PBS组、DOX组、MAL-DOX组、DVAP-DOX组(高、中、低)和DVAP组的平均生存时间分别为36、36、48、58、52、48.5和45.5天。结果表明,与其余各组相比,DVAP-DOX延长脑胶质瘤模型裸鼠的生存时间效果最显著,其最低剂量即可达到4倍剂量的MAL-DOX抗脑胶质瘤效果。
图25示出了DVAP-DOX的小鼠体内药物动力学曲线与分布:
图A为药动学曲线,图B为体内主要脏器分布。结果表明,DVAP-DOX的AUC虽然仍显著低于MAL-DOX,但比游离DOX有明显增加。而且,从体内主要脏器的分布结果可见,DVAP-DOX可显著降低药物在心脏中的分布,这提示DVAP-DOX可能可以降低阿霉素用药后对心脏的毒副作用。
图26示出了载DiR的VAP胶束的皮下瘤内分布:
图A为尾静脉注射24h后的在体荧光分布图像,从左到右依次为PBS组,mPEG-Micelle/DiR、LVAP-Micelle/DiR、DVAP-Micelle/DiR和SVAP-Micelle/DiR。图B为脏器的荧光分布图像。结果表明DVAP或SVAP修饰的胶束能更好地靶向至肿瘤部位。
图27示出了载紫杉醇胶束的粒径:
图为各紫杉醇胶束的粒径图片。由图可知,各处方胶束大小均无显著差异。
图28示出了载紫杉醇胶束体外抗U87细胞和HUVEC细胞活性曲线:
图A和图B分别为mPEG-Micelle/PTX、DVAP-Micelle/PTX、SVAP-Micelle/PTX、LVAP-Micelle/PTX和泰素抗U87细胞和HUVEC细胞的活性曲线,图A表明U87细胞给药4h培养72h后,其IC50分别为0.73、0.09、0.12、0.59和0.75μM,四种胶束均能抑制U87细胞的体外生长,其中DVAP-Micelle/PTX、SVAP-Micelle/PTX的体外活性分别为LVAP-Micelle/PTX的6.56倍和4.92倍,图B表明HUVEC细胞给药4h培养72h后,其IC50分别为0.52、0.04、0.04、0.23和0.74μM,四种胶束均能抑制HUVEC细胞的体外生长,其中DVAP-Micelle/PTX、SVAP-Micelle/PTX的体外活性均为LVAP-Micelle/PTX的5.75。
图29示出了载紫杉醇胶束体外对新生血管形成的抑制:
图为mPEG-Micelle/PTX、DVAP-Micelle/PTX、SVAP-Micelle/PTX、LVAP-Micelle/PTX和泰素对新生血管体外模型的抑制照片,相比于LVAP-Micelle/PTX,DVAP-Micelle/PTX和SVAP-Micelle/PTX抑制新生血管的形成更显著。
图30示出了载紫杉醇胶束体外对拟态血管形成的抑制:
图为mPEG-Micelle/PTX、DVAP-Micelle/PTX、SVAP-Micelle/PTX、LVAP-Micelle/PTX和泰素对拟态血管体外模型的抑制照片,相比于LVAP-Micelle/PTX,DVAP-Micelle/PTX和SVAP-Micelle/PTX抑制拟态血管的形成更显著。
图31示出了载紫杉醇胶束皮下瘤抑制实验:
图A为各组裸鼠肿瘤体积随时间变化的曲线,与生理盐水组相比,各给药组对肿瘤生长均有抑制作用。DVAP-Micelle/PTX、SVAP-Micelle/PTX与LVAP-Micelle/PTX相比具有极显著性差异(n=8,***p<0.001)。将裸鼠处死取出肿瘤组织后称重并进行统计分析(图B),发现DVAP-Micelle/PTX和SVAP-Micelle/PTX组瘤重显著低于LVAP-Micelle/PTX(n=8,***p<0.001)。
图32示出了TUNEL染色结果:
图为mPEG-Micelle/PTX、DVAP-Micelle/PTX、SVAP-Micelle/PTX、LVAP-Micelle/PTX促进皮下瘤凋亡的TUNEL染色照片(bar=50μm),其中凋亡的阳性细胞核呈棕黄色或棕褐色。
图33示出了CD31/PAS双染色结果:
图为mPEG-Micelle/PTX、DVAP-Micelle/PTX、SVAP-Micelle/PTX、LVAP-Micelle/PTX抑制新生血管形成的CD31/PAS双染色照片(bar=100μm),其中新生血管细胞核呈棕黄色或棕褐色。
实施发明的最佳方式
通过下述实施例将有助于进一步理解本发明,但本发明不局限于如下描述范围。
实施例1:VAP、VAP-Fluorescein、VAP-Cy7、VAP-药物、VAP-PEG-PLA的合成与表征
1.DVAP、SVAP和LVAP,DVAP-Cys、SVAP-Cys和LVAP-Cys的合成与表征
采用固相多肽合成法,设计并合成由D构型氨基酸所构成的DVAP(序列为DPDADVDRDTDNDS)、DVAP-Cys(序列为DCDPDADVDRDTDNDS)和SVAP(序列为DSDNDTDRDVDADP)、SVAP-Cys(序列为DSDNDTDRDVDADPDC)以及L构型氨基酸所构成的LVAP(序列为SNTRVAP)和LVAP-Cys(序列为SNTRVAPC)。
具体方法:以Boc固相多肽合成法,在PAM树脂上按序列依次接入氨基酸,以HBTU/DIEA为缩合剂、TFA为脱保护剂进行反应。反应完成后,将树脂用含P-cresol的氟化氢进行切割,冰浴搅拌反应1h。反应结束后减压抽去管中氟化氢,冰乙醚沉淀并洗涤沉淀3次,沉淀以20%乙腈重新溶解,收集滤液后旋蒸,得到多肽粗品溶液。多肽粗品用乙腈/水(含0.1%TFA)体系分离纯化。HPLC和ESI-MS表征DVAP、DVAP-Cys、SVAP、SVAP-Cys、LVAP及LVAP-Cys的纯度和分子量(Mw)。HPLC图谱、质谱图见附图1、图2、图3、图4、图5和图6。
2.VAP-Fluorescein与VAP-Cy7的合成与表征
将上述步骤得到的DVAP-Cys、SVAP-Cys或LVAP-Cys溶于0.1M的PBS溶液中(pH7.2),取Fluorescein-5-maleimide溶于DMF,两者混合后磁力搅拌反应,HPLC监测,待DVAP-Cys、SVAP-Cys或LVAP-Cys反应完全后停止反应,制备液相纯化,用乙腈/水(含0.1%TFA)体系分离纯化。冷冻干燥得DVAP-Fluorescein、SVAP-Fluorescein或LVAP-Fluorescein纯品。HPLC图谱、质谱图见附图7、8、9。
VAP-Cy7的制备方法同上。HPLC图谱、质谱图见附图10、11、12。
3.VAP-DTPA-Gd与VAP-DTPA-99mTc的制备
maleimide-DTPA溶于DMF,同上与DVAP-Cys、SVAP-Cys或LVAP-Cys溶于的PBS溶液混合搅拌反应,制备液相纯化,冷冻干燥得DVAP-DTPA、SVAP-DTPA或LVAP-DTPA纯品,螯合Gd或99mTc即得VAP-DTPA-Gd或VAP-DTPA-99mTc。
4.VAP-药物复合物的制备
以VAP-阿霉素复合物制备作为VAP连接含酮或醛基药物的实施例。9.4mg巯基化VAP(DVAP或SVAP或LVAP)多肽溶于磷酸盐3mL缓冲液(0.1mM,pH 7.0),加入10倍摩尔量的三(2-羧乙基)膦(TCEP),于4℃搅拌20min。然后加入4倍摩尔量的阿霉素6-马来酰亚胺己肼衍生物(MAL-DOX),于室温避光反应1h。反应液用制备液相纯化,冷冻干燥得LVAP或DVAP或SVAP-阿霉素复合物,HPLC、MS表征结构,结果见附图13。
以VAP-紫杉醇复合物作为VAP以二硫键连接含羟基或氨基药物的实施例:200mg紫杉醇溶于10mL氯仿中,冷却至0-5℃,先后加入39.99mg DCC及60.4mg 3-(2-吡啶二巯基)丙酸,加料完毕后,升至室温反应过夜。反应液过滤,经柱层析纯化(CHCl3/MeOH=50∶1-15∶1,V/V洗脱)得紫杉醇3-(2-吡啶二巯基)丙酸衍生物。紫杉醇3-(2-吡啶二巯基)丙酸衍生物溶解在5mL DMF中,1.5倍摩尔量的VAP-Cys溶解在PBS/DMF中,溶液pH值保持4~5将紫杉醇3-(2-吡啶二巯基)丙酸衍生物滴加至巯基多肽溶液中,于室温反应6h,经制备液相纯化冻干得多肽-紫杉醇复合物。
以VAP-硼替佐咪复合物作为VAP连接含硼酸基团药物的实施例:依照VAP的合成在树脂上依次接入氨基酸,待多肽的所有氨基酸残基接入完毕,三氟乙酸脱去氮端的Boc保护。加入含3倍摩尔量的丁二酸酐与DIEA的DMF溶液,于室温反应30min。洗涤树脂后,加入5倍摩尔量的三甲基氯硅烷保护多巴胺,并以HBTU/DIEA为缩合剂,于室温反应1h。树脂用HF切割,并经制备型HPLC纯化得多肽-多巴胺衍生物。在pH7.4的缓冲液中,多肽-多巴胺衍生物与硼替佐咪以摩尔比1∶1混合即得多肽-硼替佐咪复合物。
以VAP-PMI融合多肽作为VAP连接多肽药物的实施例:直接通过固相多肽合成法制得,具体方法为:确定VAP-PMI多肽序列后,按与制备VAP相同的方法依次接入氨基酸,经HF切割并纯化后得VAP-PMI融合多肽。
5.VAP-PEG-PLA的合成与表征
通过多肽的游离巯基与Mal-PEG-PLA所含马来酰亚胺的反应实现膜材料的合成。将40mg Mal-PEG-PLA溶解在5mL乙腈中,旋转蒸发,成膜,加入3mL PBS(pH8.0,0.2M)在37℃水化形成胶束,8h内加入9.6mg VAP-Cys并反应过夜,HPLC检测反应。过量的VAP-Cys通过透析除去,冻干,1H-NMR表征(图14)。
实施例2:VAP的血清稳定性考察
将DVAP、SVAP及LVAP配成1mg/mL水溶液,取0.1mL加入0.9mL的25%小鼠血清中,37℃孵育,分别于0和15min,0.5、1、2和4h取出100μL反应液,加入20μL三氯乙酸(TCA)沉淀血清中蛋白,4℃静置20min,12000转/分钟离心10min,取上清液20μL进行HPLC分析。血清稳定性结果(图15)表明,DVAP和SVAP具有比LVAP更好的血清稳定性。
实施例3:VAP与葡萄糖调节蛋白GRP78的结合活性实验
通过biacore系统进行预结合分析,选取pH5.0为最佳GRP78与CM5芯片结合pH。将重组人GRP78偶联至CM5芯片上,RU值达到目标值。将DVAP、SVAP及LVAP分别配置成浓度为0.3125、0.625、1.25、2.5、5、10和20μM的样品溶液。从低到高依次进样,用BiacoreT200Evaluation software软件分析DVAP、SVAP及LVAP与蛋白的结合活性,并分别计算其KD值及Kd值(图16)。
实施例4:GRP78的细胞内分布考察
选用三种细胞U87,HUVEC,HEK293做GRP78膜定位实验,选用破膜不破膜两种方式考察GRP78在细胞中的分布。将三种细胞分别接种到共聚焦小皿上,用PBS洗三次后用10μM的DIO多聚甲醛溶液固定8min,用1%BSA于37℃封闭30min后用PBS洗三次,加入1%BSA稀释的Anti-GRP78抗体,37℃孵育4h,PBS洗两次,加入1%BSA稀释的二抗,37℃孵育2h,PBS洗两次,每次5min。DAPI染色10min,PBS洗三次,甘油封片,激光共聚焦观察,结果如图17所示。
实施例5:VAP的体外细胞靶向性验证
1.VAP对脑胶质瘤细胞U87的体外靶向性
取对数生长期的单层培养的脑胶质瘤细胞(U87细胞),用0.25%胰蛋白酶消化单层培养细胞,用含10%胎牛血清的DMEM培养液配成单细胞悬液,以每孔1×105个细胞接种于12孔培养板中,每孔体积1mL,将培养板移入二氧化碳培养箱中,37℃、5%CO2及饱和湿度条件下培养24h后,用含10%胎牛血清的DMEM培养液配制浓度为5μM的FAM、DVAP-Fluorescein、SVAP-Fluorescein及LVAP-Fluorescein溶液,将培养板中的培养液吸出,分别加入上述溶液,37℃孵育4h,吸弃上清液。用PBS溶液洗三次,甲醛固定液固定细胞,DAPI进行细胞核染色后,激光共聚焦观察,细胞内化照片如图18A所示,另用PBS洗三次后,进行流式细胞仪分析,结果如图18B所示。
2.VAP对人脐静脉内皮细胞HUVEC的体外靶向性
取对数生长期的单层培养的人脐静脉内皮细胞(HUVEC细胞),同上试验,细胞内化照片如图19A所示,流式细胞仪分析结果如图19B所示。
3.VAP对体外U87肿瘤球的靶向性
将2%的低分子琼脂糖溶液趁热加入48孔板中,每孔150μL,室温放置冷却凝固后,每孔接种400μL U87细胞悬液,细胞密度为2×103个/孔。置于二氧化碳培养箱中,37℃、5%CO2及饱和湿度条件下培养7天即形成肿瘤球。用含10%胎牛血清的DMEM培养液配制浓度为5μM的FITC、DVAP-Fluorescein、SVAP-Fluorescein及LVAP-Fluorescein溶液。将培养板中的培养液吸出,分别加入上述溶液,37℃孵育4h,吸弃上清液,PBS洗三次,多聚甲醛固定15min后,置于共聚焦显微镜下观察,照片如图20A所示。
4.VAP相互竞争抑制试验
设9组,每组3复管进行VAP间相互竞争抑制的考察,分组分别为空白对照组、LVAP-Fluorescein、DVAP-Fluorescein、SVAP-Fluorescein、LVAP-Fluorescein(+LVAP)、LVAP-Fluorescein(+DVAP)、LVAP-Fluorescein(+SVAP)、DVAP-Fluorescein(+LVAP)、DVAP-Fluorescein(+DVAP)、DVAP-Fluorescein(+SVAP)、SVAP-Fluorescein(+LVAP)、SVAP-Fluorescein(+DVAP)、SVAP-Fluorescein(+SVAP),将U87细胞用胰酶消化后转移到EP管内,PBS洗三次去除胰酶,低温4℃预处理20min,配好的多肽溶液(非荧光标记)也放置在4℃低温预处理,随后将多肽溶液与细胞悬液混合震荡处理2h,使细胞表面的受体蛋白饱和,然后加入荧光素标记的多肽溶液,4℃放置过夜后用PBS洗涤3次,流式测细胞摄取量(如图21所示)。
实施例6:VAP体内肿瘤靶向性验证
首先构建皮下瘤动物模型,将处于对数生长期的U87细胞胰酶消化,调整细胞浓度为3×107个/mL,接种100μL至裸小鼠右背侧近腋部皮下,接种后饲养于SPF级,定期观察肿瘤大小,待肿瘤大小为200mm3时,筛选出无坏死、肿瘤形状规则的荷瘤裸鼠,分组进行试验,以0.15μmoL/只的剂量将Cy7、DVAP-Cy7、SVAP-Cy7及LVAP-Cy7溶液通过尾静脉注入荷瘤裸鼠动物模型体内,2h后处死裸鼠,取出肿瘤,用活体成像仪检测肿瘤的荧光分布(如图22A所示)并进行荧光半定量计算(如图22B所示)。
实施例7: DVAP-DOX体内药效学和药动学试验
1.U87皮下瘤药效试验
构建的U87皮下瘤动物模型,待肿瘤大小为100mm3时,分组进行试验。皮下瘤模型鼠尾静脉分别注射生理盐水、DOX、MAL-DOX(含高、低剂量)和DVAP-DOX(含高、低剂量)以及RGD-DOX(整合素受体配体c(RGDyK)与MAL-DOX按照VAP-DOX相同方法制备的多肽药物复合物)各100μl。给药组除低剂量含DOX总给药剂量为1.25mg/kg,其它均为2.5mg/kg,分为五次给药,每次给药间隔为两天。隔天以游标卡尺测量肿瘤的长径(a)及短径(b)。根据公式计算各组裸鼠肿瘤体积,绘制肿瘤体积随时间的变化曲线,计算各组统计学差异。按照以下公式计算肿瘤体积:
V瘤体积=0.5(a×b2)
给药21天后,断颈处死所有裸鼠,取出皮下肿瘤称重,并计算各组统计学差异与抑瘤率(附图23)。
2.U87原位瘤药效试验
构建U87原位脑胶质瘤模型裸鼠:取对数生长期的U87细胞,每只裸小鼠接种5×105个细胞(分散于5μL PBS缓冲液中)。裸小鼠麻醉后,用脑立体定位仪固定,细胞接种于纹状体右部(前囟前0.6mm,侧1.8mm,深3mm)。定期观察裸小鼠状态。尾静脉分别注射PBS、DOX、MAL-DOX、DVAP、DVAP-DOX(含高、中、低剂量),给药组除DVAP-DOX低剂量含DOX总给药剂量为2.5mg/kg,中剂量为5mg/kg,其它均为10mg/kg,DVAP剂量为DVAP-DOX高剂量中所含的DVAP量,分别在肿瘤种植后第10、13、16、19和22天给药,记录裸鼠的生存时间(附图24)。
3.DVAP-DOX体内药动学试验
ICR小鼠分别尾静脉注射200μL DOX、DVAP-DOX和MAL-DOX(其中含DOX为10mg/kg),于1、5、15、30和45min,1、2、4和6h分别取全血50μL,以PBS稀释4倍,在荧光Ex 485/Em 590测定,绘制药物浓度-时间曲线与药物的体内分布(附图25)。
实施例8:VAP胶束对体外U87肿瘤球的靶向性
1.载香豆素6胶束的制备
称取lmg VAP-PEG-PLA,9mg mPEG-PLA和5ug香豆素6(C6),溶解在2mL乙腈中,37℃水浴,减压(~0.085MPa)蒸干,成膜,室温真空干燥过夜,加入2mL生理盐水水化,CL-4B柱层析除去游离香豆素,制得包载香豆素6的胶束(VAP-Micelle/C6)。
2.VAP-Micelle/C6胶束体外U87肿瘤球靶向性验证
将2%的低分子琼脂糖溶液趁热加入48孔板中,每孔150μL,室温放置冷却凝固后,每孔接种400μL U87细胞悬液,细胞密度为2×103个/孔。置于二氧化碳培养箱中,37℃、5%CO2及饱和湿度条件下培养7天即形成肿瘤球,用含10%胎牛血清的DMEM培养液配制浓度为5ng/mL的Micelle/C6、DVAP Micelle/C6、SVAP Micelle/C6及LVAP Micelle/C6溶液,将培养板中的培养液吸出,分别加入上述溶液,37℃孵育4h,吸弃上清液,PBS洗三次,多聚甲醛固定15min后,DAPI染色,置于共聚焦显微镜下观察,照片如图20B所示。
实施例9:VAP胶束体内靶向性验证
1.载DiR胶束的制备
1mg VAP-PEG-PLA,9mg mPEG-PLA和1mg DiR以制备载香豆素6胶束的方法制得载DiR胶束(VAP-Micelle/DiR)。
2.VAP-Micelle/DiR体内靶向性验证
U87皮下瘤模型裸鼠分别尾静脉注射100μL的PBS,mPEG-Micelle/DiR、DVAP-Micelle/DiR、SVAP-Micelle/DiR及LVAP-Micelle/DiR,分别在注射后2、4、8、12及24h时麻醉裸鼠,用活体成像仪记录DiR荧光在裸鼠体内的分布情况并进行荧光半定量计算(如图26所示)。
实施例10:载紫杉醇VAP胶束的体外药效学试验
1.载紫杉醇胶束的制备及表征
称取1mg VAP-PEG-PLA,9mg mPEG-PLA和2mg紫杉醇以制备载香豆素6胶束的方法制得载紫杉醇的VAP胶束(VAP-Micelle/PTX),粒径与分布如图27所示。
2.VAP-Micelle/PTX体外药效试验
以4.0×103个/孔将U87细胞接种于96孔板,24h后,将培养液吸出,加200μL一系列浓度的DVAP-Micelle/PTX、SVAP-Micelle/PTX、LVAP-Micelle/PTX和mPEG-Micelle/PTX和泰素,共培养72h,后加入MTT溶液继续培养4h,弃去培养液,加入150μL DMSO,振荡至紫色颗粒溶解,用酶标仪在590nm处测定吸光度值,采用MTT法测定细胞存活率,计算细胞存活率和半数致死剂量(如图28所示)。
3.VAP-Micelle/PTX对新生血管形成的抑制试验
取24孔培养板每孔加入50μL基质胶,平铺于24孔板内,37℃培养箱内孵育30min待其凝固,0.25%胰酶消化HUVEC细胞,用含1μM紫杉醇VAP胶束或游离紫杉醇药液的DMEM培养液配成单细胞悬液,以每孔1×105个细胞接种于24孔培养板中,37℃、5%CO2及饱和湿度条件下培养12h后观察血管样结构形成(如图29所示)。
4.VAP-Micelle/PTX对拟态血管形成的抑制试验
取24孔培养板每孔加入50μL基质胶,平铺于24孔板内,37℃培养箱内孵育30min待其凝固,0.25%胰酶消化U87细胞,用含1μM紫杉醇VAP胶束或游离紫杉醇药液的DMEM培养液配成单细胞悬液,以每孔1×105个细胞接种于24孔培养板中,37℃、5%CO2及饱和湿度条件下培养12h后观察血管样结构形成(如图30所示)。
实施例11:VAP-Micelle/PTX体内药效学试验
1.VAP-Micelle/PTX体内药效学试验
构建的U87皮下瘤动物模型,待肿瘤大小为100mm3时,分组进行试验,皮下瘤模型鼠尾静脉分别注射生理盐水、泰素、LVAP-Micelle/PTX、DVAP-Micelle/PTX、SVAP-Micelle/PTX和mPEG-Micelle/PTX各100μl,给药组紫杉醇总给药剂量为25mg/kg,分为五次,每次给药间隔为两天,隔天以游标卡尺测量肿瘤的长径(a)及短径(b),根据公式计算各组裸鼠肿瘤体积,绘制肿瘤体积随时间的变化曲线,计算各组统计学差异。按照以下公式计算肿瘤体积:
V瘤体积=0.5(a×b2)
给药18天后(接种后24天),常规处理裸鼠,取皮下肿瘤称重,并计算各组统计学差异(如图31所示)。
2.VAP-Micelle/PTX促凋亡试验
荷瘤裸鼠在给药完成后的第14天,处死取出瘤组织进行固定,作冰冻切片,通过TUNEL法检测肿瘤凋亡情况,采用末端脱氧核苷酸转移酶(TDT)介导的dUTP缺口末端标记法(Terminal deoxynucleotidyl Transferase-mediated dUTP nick end labeling,TUNEL)检测肿瘤细胞的凋亡程度,按步骤:石蜡切片常规脱蜡至水;PBS漂洗3次,每次3min;0.3%H2O2溶液室温处理20min;20μg/mL蛋白酶K 37℃消化20min;PBS漂洗3次,每次3min;每张切片滴加TUNEL混合液(TDT和biotin-dNTP)30μL置于湿盒中37℃孵育60min;阳性结果为细胞核呈棕黄色或棕褐色,细胞核内棕色颗粒阳性即判定为凋亡细胞。在普通光学显微镜下连续观察5个高倍视野计数阳性细胞数,视野内细胞中阳性细胞数所占的百分比为凋亡指数,结果如图32所示。
3.VAP-Micelle/PTX对肿瘤血管抑制试验
荷瘤裸鼠在给药完成后的第14天,处死取出瘤组织固定,石蜡包埋切片,进行CD31免疫组化染色与PAS双染。在普通光学显微镜下连续观察3个高倍视野计数CD31阳性血管数,结果如图33所示。
Claims (7)
1.一种DVAP和/或SVAP多肽复合物,其特征在于,所述DVAP和/或SVAP多肽复合物为DVAP和/或SVAP多肽修饰抗肿瘤药物,其中,所述DVAP的氨基酸序列为DPDADVDRDTDNDS,所述SVAP的氨基酸序列为DSDNDTDRDVDADP,所述DVAP和/或SVAP多肽复合物的结构为DVAP-Y和/或SVAP-Y,Y为所述抗肿瘤药物;并且,所述抗肿瘤药物选自喜树碱类药物、长春花碱类药物、内酯类药物、多肽类药物中的一种或多种。
2.根据权利要求1所述的DVAP和/或SVAP多肽复合物,其特征在于,所述抗肿瘤药物选自喜树碱、羟基喜树碱、伊立替康、长春新碱、长春瑞滨、小白菊内酯、p53激活肽、蜂毒肽、蝎毒肽、抗菌肽中的一种或多种。
3.一种递药系统,其特征在于,所述递药系统包括DVAP和/或SVAP多肽复合物、以及所述DVAP和/或SVAP多肽复合物以外的(1)诊断药物和/或(2)抗肿瘤药物;其中:
所述DVAP的氨基酸序列为DPDADVDRDTDNDS,所述SVAP的氨基酸序列为DSDNDTDRDVDADP;
所述DVAP和/或SVAP多肽复合物为DVAP和/或SVAP多肽修饰高分子载体材料;所述DVAP和/或SVAP多肽复合物的结构为DVAP-聚乙二醇-Z和/或SVAP-聚乙二醇-Z,Z为所述高分子载体材料;
所述(1)诊断药物选自荧光物质、近红外染料和磁共振影像剂中的一种或多种;和/或
所述(2)抗肿瘤药物选自:喜树碱类药物、长春花碱类药物、内酯类药物、多肽类药物中的一种或多种。
4.根据权利要求3所述的递药系统,其特征在于,
所述荧光物质为Fluorescein,所述近红外染料选自Cy7、IR820、DiR,和/或所述磁共振影像剂为Gd-DTPA。
5.根据权利要求3所述的递药系统,其特征在于,
所述抗肿瘤药物选自喜树碱、羟基喜树碱、伊立替康、长春新碱、长春瑞滨、小白菊内酯、p53激活肽、蜂毒肽、蝎毒肽、抗菌肽中的一种或多种。
6.权利要求1或2所述的DVAP和/或SVAP多肽复合物、权利要求3-5中任一项所述的递药系统在制备用于诊断、示踪和/或治疗肿瘤的药品或医疗产品中的应用。
7.根据权利要求6所述的应用,其特征在于,所述肿瘤为高表达葡萄糖调节蛋白GRP78肿瘤。
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