CN116410264A - 一种特异性靶向白喉毒素受体的dtx多肽及其应用 - Google Patents
一种特异性靶向白喉毒素受体的dtx多肽及其应用 Download PDFInfo
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- CN116410264A CN116410264A CN202310189961.0A CN202310189961A CN116410264A CN 116410264 A CN116410264 A CN 116410264A CN 202310189961 A CN202310189961 A CN 202310189961A CN 116410264 A CN116410264 A CN 116410264A
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Abstract
本发明涉及药学领域,具体是一种特异性靶向白喉毒素受体,跨生物膜屏障,特别是血脑屏障的DTX多肽,其修饰的复合物、递药系统,以及在脑部肿瘤等脑部疾病的诊断与治疗中的用途。本发明采用计算机辅助设计手段制备了与白喉毒素受体具有高结合活性、跨生物膜屏障能力的多功能靶向多肽分子DTX,并涉及DTX修饰的荧光素和药物、高分子载体材料的制备及其在脑部影像和靶向治疗递药系统构建中的应用。DTX多肽的最大优势在于具有血脑屏障和脑肿瘤的双级靶向能力。本发明结果显示,DTX可介导药物或纳米递药系统跨生物膜屏障、主动寻靶、在脑内肿瘤等脑内疾病的诊断和靶向治疗中具备良好的应用前景。
Description
技术领域
本发明涉及药学领域,具体地说,是一种特异性靶向白喉毒素受体的及其应用。
背景技术
由于血脑屏障(blood-brain barrier,BBB)的存在,传统药物很难进入脑内,脑内疾病的治疗仍是临床上的一大难题。主动靶向递药系统在脑内疾病的治疗中至关重要。构成血脑屏障的脑毛细血管内皮细胞上存在多种特异性的受体。包括低密度脂蛋白受体、转铁蛋白受体和白喉毒素受体(diphtheria toxin receptor,DTR)等。其中DTR也称为肝素结合表皮生长因子的膜结合前体(pro heparinbinding-epidermal growth factor,proHB-EGF),是一种跨膜糖蛋白,在平滑肌细胞、血管内皮细胞、血脑屏障、神经元上均有表达。此外,在脑胶质瘤等病变条件下,DTR在BBB中的表达大幅度上调。而且脑胶质瘤细胞表面也高表达DTR,与病理程度成正相关性。
利用多肽、蛋白或抗体与其相应的配体或抗原相互作用,介导药物入脑是目前主动靶向递药的主要策略。其中多肽类具有合成方便、结构简单、高亲和性和低免疫原性等特征,目前应用最广。
发明内容
本发明的目的在于提供一种可以靶向DTR,具有跨生物膜屏障能力,特别是血脑屏障的靶向多肽分子DTX,及其修饰的复合物、递药系统在脑部肿瘤等脑部疾病的诊断与治疗中的应用,达到良好的肿瘤靶向效果。
本发明利用计算机辅助设计方法将可以靶向DTR的DTX多肽,以DTX及其衍生物构建载药复合物、纳米递药系统,可通过受体介导的胞吞转运作用跨越BBB,增加药物入脑效率,实现在脑部肿瘤等脑内疾病的诊断与治疗中的用途,也为后续靶向多肽的设计提供参照。涉及DTX设计与制备,并以DTX修饰药物分子或高分子载体材料,构建DTX复合物及DTX修饰的递药系统,以提高药物对肿瘤的靶向诊疗效果。
本发明的第一方面,提供一种特异性靶向白喉毒素受体的DTX多肽,所述的DTX多肽具有血脑屏障和脑肿瘤的双级靶向能力。
进一步的,所述的DTX多肽是由天冬氨酸、组氨酸、苏氨酸、赖氨酸、缬氨酸、天冬酰胺、丝氨酸、赖氨酸、亮氨酸、丝氨酸、亮氨酸、苯丙氨酸和苯丙氨酸13个连续氨基酸构成的多肽,所述的多肽为L构型多肽(即LDLHLTLKLVLNLSLKLLLSLLLFLF,SEQ ID NO.1)。其中,氨基酸构型可以改变,或者为L构型多肽的逆序D构型多肽(即DFDFDLDSDLDKDSDNDVDKDTDHDD,SEQ IDNO.2)。
本发明的第二方面,提供一种如上所述的DTX多肽以共价键与影像物质X连接得到的DTX-X复合物,用于高表达白喉毒素受体的血脑屏障、外周实体瘤和脑肿瘤的影像诊断和示踪。
进一步的,所述的复合物中的影像物质X选自荧光物质Fluorescein,近红外染料Cy5、IR820、DiR等,磁共振影像剂Gd-DTPA等,放射影像剂99mTc-DTPA等,可用作脑、脑部肿瘤或外周肿瘤的影像诊断和示踪。
本发明通过计算机辅助设计方法设计了DTX多肽,通过Boc固相多肽合成法制备了DTX多肽,并通过引入巯基后,可与马来酰亚胺功能化影像物质(如荧光物质Fluorescein、近红外染料Cy5、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。
本发明的第三方面,提供一种如上所述的DTX多肽以共价键与抗肿瘤药物Y连接得到的DTX-Y复合物,用于高表达白喉毒素受体的外周肿瘤和脑肿瘤的靶向治疗。
进一步的,所述的复合物中的抗肿瘤药物Y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、蛋白降解靶向嵌合体、p53激活肽等多肽类药物,可用作脑部肿瘤或外周肿瘤的靶向治疗。
本发明所设计的DTX多肽修饰药物,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、卡巴他赛、喜树碱、羟基喜树碱、9-硝基喜树碱、伊立替康、长春新碱和长春瑞滨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激活肽等多肽药物)、或通过连接键合成蛋白降解靶向嵌合体等DTX多肽-药物复合物。
本发明的第四方面,提供一种如上所述的DTX多肽以共价键与聚乙二醇-Z复合物连接得到DTX-聚乙二醇-Z复合物,用于纳米递药系统的制备。
进一步的,所述的复合物中Z是磷脂、聚乳酸(PLA)、乳酸羟基乙酸共聚物(PLGA)、聚己内酯(PCL)。
更进一步的,DTX-聚乙二醇-磷脂复合物,可用于脂质体递药系统、聚合物胶束递药系统、聚合物圆盘递药系统的制备;DTX-聚乙二醇-聚乳酸复合物、DTX-聚乙二醇-乳酸羟基乙酸共聚物复合物、DTX-聚乙二醇-聚己内酯复合物,可用于聚合物胶束递药系统、纳米粒递药系统的制备。
进一步的,所述的脂质体递药系统、聚合物胶束递药系统、脂质圆盘递药系统和纳米粒递药系统,可用于包载诊断药物或肿瘤治疗药物。
更进一步的,所述的诊断药物,选自荧光物质如香豆素6、罗丹明B、Cy7、IR820、DiR、DiD,磁共振影像剂Gd-DTPA,可用于脑、脑部肿瘤或外周肿瘤的影像诊断和示踪。
更进一步的,所述的肿瘤治疗药物,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、蛋白降解靶向嵌合体、p53激活肽和蜂毒肽、蝎毒肽和抗菌肽等多肽类药物,可用于脑部肿瘤或外周肿瘤的靶向治疗。
本发明所设计的DTX多肽引入巯基后,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DTX多肽修饰的脂质体、脂质圆盘、聚合物胶束、纳米粒等纳米递药系统的构建。
本发明所设计的DTX多肽修饰的纳米递药系统可包载阿霉素、表阿霉素、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、硼替佐米、卡非佐米、环磷酰胺、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、顺铂、奥沙利铂、p53激活肽、蜂毒肽、蝎毒肽、贝伐单抗、曲妥单抗等;也可包载荧光物质和磁共振影像剂,如FAM、近红外染料Cy5.5、IR820、DiR、DiD、Gd-DTPA等。
本发明提供了DTX多肽制备和性质考察以及上述所修饰的药物复合物和纳米递药系统用于肿瘤诊疗的物质基础。本发明的试验结果表明:DTX多肽具有跨BBB,靶向肿瘤细胞的多功能靶向作用,在模型动物体内表现出更优肿瘤靶向能力;DTX修饰的纳米递药系统显示出了良好的肿瘤靶向性能和更强的抗肿瘤效果。
为了实现上述目的,本发明采用以下技术方案:
1.DTX多肽的设计、合成与表征
通过计算机辅助设计方法,模拟白喉毒素与白喉毒素受体的结合,截取亲和力较强的白喉毒素的多肽片段。采用Boc保护固相合成法制备引入半胱氨酸的DTX-Cys。
2.DTX多肽体内外靶向能力评价
考察荧光标记DTX多肽对脑毛细血管内皮细胞(大鼠原代毛细血管内皮细胞BCEC和bEND.3细胞)、模型肿瘤细胞(脑胶质瘤细胞U87)以及U87肿瘤球模型的体外靶向性;通过正常小鼠、荷U87皮下瘤模型裸鼠和荷U87原位瘤模型裸鼠尾静脉注射荧光标记DTX多肽考察其在体内脑组织和肿瘤部位的靶向性。
3.DTX多肽-DTPA-Gd与DTX多肽-DTPA-99mTc的合成
通过马来酰亚胺基团与巯基的Michael加成反应合成了DTX多肽-DTPA,螯合Gd或99mTc得DTX多肽-DTPA-Gd或DTX多肽-DTPA-99mTc。
4.DTX多肽-药物的制备
引入半胱氨酸后的DTX多肽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。
引入半胱氨酸后的DTX多肽与药物的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、卡巴他赛、喜树碱、羟基喜树碱、9-硝基喜树碱、伊立替康、长春新碱、长春瑞滨等含羟基或氨基的药物。
DTX多肽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。
DTX多肽通过固相合成直接与多肽药物缩合,其中所涉及药物包括p53激活肽等多肽药物。
5.DTX多肽修饰的纳米递药系统的构建与表征
首先合成DTX多肽修饰的高分子材料DTX多肽-PEG-DSPE、DTX多肽-PEG-PLA、DTX多肽-PEG-PLGA、DTX多肽-PEG-PCL等。通过引入半胱氨酸的DTX多肽上游离巯基与Mal-PEG-DSPE、Mal-PEG-PLA、Mal-PEG-PLGA、Mal-PEG-PCL等所含马来酰亚胺的反应实现材料的合成,即:将Mal-PEG-DSPE、Mal-PEG-PLA、Mal-PEG-PLGA、Mal-PEG-PCL等分别溶解在乙腈中,旋转蒸发,成膜,加入含巯基AG的PBS(pH 8.0)反应制备得到DTX多肽修饰的高分子材料,1H-NMR表征。
然后制备DTX多肽修饰的纳米递药系统。一定量的DTX多肽-PEG-DSPE和mPEG-DSPE和磷脂和胆固醇、或DTX多肽-PEG-DSPE和mPEG-DSPE、或DTX多肽-PEG-PLA和mPEG-PLA、或DTX多肽-PEG-PLGA和mPEG-PLGA、或DTX多肽-PEG-PCL和mPEG-PCL,以及一定量药物,采用成膜水化等方法分别制备相应的DTX多肽修饰脂质体、聚合物胶束、聚合物圆盘、聚合物纳米粒等纳米递药系统;激光散射粒度仪表征纳米递药系统粒径和粒径分布。
6.DTX多肽修饰纳米递药系统的体内靶向性评价
考察bEND.3细胞、U87细胞、U87肿瘤球以及体外BBB/U87肿瘤球模型对DTX多肽修饰纳米递药系统的摄取情况。
通过荷U87皮下移植瘤模型裸鼠尾静脉注射载DiR的DTX多肽修饰纳米递药系统,考察其对肿瘤的靶向能力。
通过荷U87原位移植瘤模型鼠尾静脉注射载DiR的DTX多肽修饰纳米递药系统,考察不同时间点其在脑肿瘤部位的分布。
7.DTX多肽修饰药物的体外抗肿瘤效果评价
以MTT法考察DTX多肽修饰药物对U87细胞的体外生长抑制;通过荷U87原位移植瘤模型裸鼠尾静脉注射DTX多肽修饰药物,以裸鼠中位生存期、肿瘤组织细胞凋亡等为指标评价体内抗肿瘤效果。
8.DTX多肽修饰纳米递药系统的体外抗肿瘤效果评价
以MTT法考察包载肿瘤治疗药物的DTX多肽修饰纳米递药系统对U87细胞的体外生长抑制;通过荷U87原位移植瘤模型裸鼠尾静脉注射包载肿瘤治疗药物的DTX多肽修饰纳米递药系统,以裸鼠中位生存期、肿瘤组织细胞凋亡、新生血管和拟态血管数量为指标评价体内抗肿瘤效果。
本发明优点在于:
1、本发明采用计算机辅助设计手段制备了与白喉毒素受体具有高结合活性、跨生物膜屏障能力的多功能靶向多肽分子DTX,并涉及DTX修饰的荧光素和药物、高分子载体材料的制备及其在脑部影像和靶向治疗递药系统构建中的应用。结果显示:本发明DTX及其修饰的模型药物可被表达白喉毒素受体的阳性细胞(如人脑毛细血管内皮细胞,胶质瘤细胞)特异性结合或摄取,具有跨越生物膜屏障组成细胞(如血脑屏障内皮细胞)的能力以及靶向肿瘤细胞的能力。DTX修饰的高分子载体材料所构建的纳米递药系统(如脂质体、聚合物胶束、聚合物圆盘、纳米粒等)可更有效地将所包载模型药物递送至脑组织及表达白喉毒素受体的细胞内,显著提高脑内疾病的治疗效果。本发明的DTX多肽的最大优势在于具有血脑屏障和脑肿瘤的双级靶向能力。
2、本发明结果显示,DTX可介导药物或纳米递药系统跨生物膜屏障、主动寻靶、在脑内肿瘤等脑内疾病的诊断和靶向治疗中具备良好的应用前景。
附图说明
图1.白喉毒素与白喉毒素受体结合复合物模拟图。利用计算机辅助技术,模拟白喉毒素中与白喉毒素受体(PDB:1XDT)的结合,截取结合的多肽片段,命名为DTX,氨基酸序列为DHTKVNSKLSLFF。
图2.DTX-Cys多肽的HPLC及ESI-MS图谱。A图为HPLC谱图。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:28%B~53%B 20min;流速:1mL/min;柱温:40℃;检测:UV 214nm。其纯度为98.12%,符合要求。B图为ESI-MS谱图。DTX-Cys分子量为1638.79道尔顿,与理论分子量相符合。
图3.DTX-Cy5的ESI-MS图谱。DTX-Cy5实测分子量为2243.8道尔顿,与理论分子量2244.59相符合。
图4.DTX-DOX的ESI-MS图谱。DTX-DOX分子量为2173.4道尔顿,与理论分子量相符合。
图5.DTX-PEG-DSPE的1H-NMR图谱。Mal-PEG-DSPE中马来酰亚胺(Mal)基团的特征吸收峰消失,提示成功合成DTX-PEG-DSPE。
图6.脂质体的粒径分布图。A图为负载阿霉素的空白脂质体(sLip/DOX)的水合粒径图。B图为负载阿霉素的DTX修饰的脂质体粒径分布图(DTX-sLip/DOX)。结果可知,sLip/DOX水合粒径为92.56nm,分散系数为0.136。DTX-sLip/DOX水合粒径为97.76nm,分散系数为0.186,提示脂质体粒径合适、粒径分布均一。
图7.脑毛细血管内皮细胞bEND.3对荧光标记DTX多肽的摄取。A图为bEND.3细胞与DTX-FAM摄取的代表性激光共聚焦显微照片。蓝色为DAPI标记的细胞核,绿色为荧光素FAM的荧光,比例尺为20μm。B图和C图分别为bEND.3细胞与DTX-FAM摄取的流式结果及其荧光定量统计分析。ns:无统计学差异;***p<0.001。结果提示bEND.3细胞可有效摄取DTX多肽。Mean±SD,n=3。
图8.Cy5标记的DTX多肽对正常小鼠的脑靶向性。A图为正常ICR小鼠尾静脉注射游离Cy5以及Cy5标记DTX多肽后1h的成像照片。B图为对对应荧光半定量统计分析结果。结果可见DTX-Cy5在小鼠脑内具有很强的荧光,与游离Cy5具有显著性差异,说明的DTX多肽具有良好的脑靶向能力。Mean±SD,n=3
图9.脑毛细血管内皮细胞bEND.3摄取荧光标记DTX多肽修饰脂质体(DTX-sLip)的代表性激光共聚焦显微照片。蓝色为DAPI标记的细胞核,绿色为荧光素FITC的荧光,比例尺为10μm。
图10.脑胶质瘤细胞U87对荧光标记DTX多肽的摄取。A图为U87细胞与DTX-FAM摄取的代表性激光共聚焦显微照片。蓝色为DAPI标记的细胞核,绿色为荧光素FAM的荧光,比例尺为20μm。B图和C图分别为U87细胞与DTX-FAM摄取的流式结果及其荧光定量统计分析。ns:无统计学差异;***p<0.001。结果提示U87细胞可有效摄取DTX多肽。Mean±SD,n=3。
图11.U87肿瘤球与荧光标记DTX多肽共孵育4h后的激光共聚焦断层扫描照片。结果提示,DTX多肽可有效被U87肿瘤摄取,且可深入渗透图肿瘤球内部。比例尺=100μm。
图12.Cy5标记的DTX多肽对U87原位荷瘤小鼠的脑靶向性。图为U87脑原位荷瘤裸鼠经尾静脉注射游离Cy5以及Cy5标记DTX多肽后1h在个脏器的成像照片。结果提示DTX多肽可有效在原位肿瘤处蓄积,具有较好的体内脑肿瘤靶向能力。
图13.脑肿瘤细胞U87摄取荧光标记DTX多肽修饰脂质体(DTX-sLip)的代表性激光共聚焦显微照片。蓝色为DAPI标记的细胞核,绿色为荧光素FITC的荧光,比例尺为10μm。
图14.DTX-DOX对U87细胞的体外抑瘤药效。图为U87细胞与不同浓度药物作用72h后的生长曲线。其中,DTX修饰的阿霉素(DTX-DOX)的IC50为0.0134μM,远低于游离DOX的IC50(1.3μM)。提示DTX修饰可显著提升药物DOX抑制U87细胞的活性。Mean±SD,n=3。
图15.负载阿霉素的DTX修饰脂质体对U87细胞的体外抑瘤药效。图为U87细胞与不同浓度药物作用72h后的生长曲线。其中,负载阿霉素的空白脂质体(sLip/DOX)以及负载阿霉素的DTX修饰脂质体(DTX-sLip/DOX)的IC50值分别为119和93.32nM。提示DTX修饰可提升阿霉素脂质体抑制U87细胞的活性。Mean±SD,n=3。
具体实施方式
下面结合实施例对本发明提供的具体实施方式作详细说明。
实施例1:DTX、DTX-显像剂、DTX-抗肿瘤药物、DTX-高分子复合物及其修饰的递送系统的制备
1.DTX多肽的设计与合成
通过计算机辅助设计方法,模拟白喉毒素与白喉毒素受体(PDB:1XDT)的结合(图1)。截取亲和力较强的白喉毒素的多肽片段,并命名为DTX,氨基酸序列为DHTKVNSKLSLFF(SEQ ID NO.1)。
采用Boc保护固相合成法制备引入半胱氨酸的DTX-Cys。经制备液相纯化、冻干后得到多肽纯品。HPLC和ESI-MS表征多肽纯度和分子量,结果见图2。其纯度为98.12%,符合要求。分子量为1638.79道尔顿,与理论分子量相符合。
2.DTX-FAM与DTX-Cy5的制备
通过DTX-Cys的巯基与荧光素的马来酰亚胺的加成反应,合成DTX-FAM或DTX-Cy5。将多肽DTX与1.5倍过量的马来酰亚胺荧光素(MAL-FAM)或马来酰亚胺Cy5(MAL-Cy5),溶解于少量DMF中,加1%体积DIEA,反应2h,高效液相监测反应,待多肽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FAM标记或Cy5标记的DTX(DTX-FAM、DTX-Cy5)。ESI-MS质谱表征(图3)。
3.DTX-抗肿瘤药物的制备
以DTX-阿霉素复合物制备作为DTX连接含酮或醛基药物的实施例。取一定量的DTX-Cys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。加入4倍摩尔量的阿霉素6-马来酰亚胺己肼衍生物,于室温避光反应1h。反应液用C18制备柱进行分离(色谱柱:waters X bridge 19×300mm;流动相:A纯水,B乙腈;洗脱方法:100%A~100%B线性梯度),收集相应组分,冷冻干燥得DTX-阿霉素复合物。ESI-MS质谱表征(图4)。
以DTX-紫杉醇复合物作为DTX以二硫键连接药物的实施例。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5mLDMF中,1.5倍摩尔量的DTX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,经制备液相制备冻干得多肽-紫杉醇复合物。
以DTX-硼替佐咪复合物作为DTX氮端修饰药物的实施例。依照DTX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DTX-p53激活肽PMI复合物作DTX融合多肽药物的实施例。直接通过固相多肽合成法制得,具体方法为:确定DTX-PMI多肽序列后,按与制备DTX相同的方法依次接入氨基酸,经氢氟酸切割并纯化后得DTX-PMI融合多肽。
4.DTX-DTPA-Gd和DTX-DTPA-99mTc的制备
马来酰亚胺-DTPA溶于DMF,同上与DTX-Cys多肽的PBS溶液混合搅拌反应,制备液相纯化,冷冻干燥得DTX-DTPA纯品,螯合Gd或99mTc即得DTX-DTPA-Gd或DTX-DTPA-99mTc。
5.DTX-高分子复合物的制备
合成DTX多肽修饰的高分子材料DTX多肽-PEG-DSPE、DTX多肽-PEG-PLA、DTX多肽-PEG-PLGA、DTX多肽-PEG-PCL等。通过引入半胱氨酸的DTX多肽上游离巯基与Mal-PEG-DSPE、Mal-PEG-PLA、Mal-PEG-PLGA、Mal-PEG-PCL等所含马来酰亚胺的反应实现材料的合成,1H-NMR表征。以DTX-PEG-DSPE的制备为例,将DTX-Cys溶于0.1M的PBS溶液中(pH7.2),Mal-PEG-DSPE溶于DMF,两者混合后磁力搅拌反应,HPLC监测,待Mal-PEG-DSPE反应完全后停止反应,过量的DTX-Cys和DMF透析(截留分子量3.5kDa)除去,冷冻干燥得DTX-PEG-DSPE,1H-NMR表征其结构,MAL-PEG-DSPE中MAL的特征吸收峰消失,提示成功合成DTX-PEG-DSPE(见图5)。
6.DTX多肽修饰的纳米递药系统的构建与表征
制备DTX多肽修饰的纳米递药系统。一定量的DTX多肽-PEG-DSPE和mPEG-DSPE和磷脂和胆固醇、或DTX多肽-PEG-DSPE和mPEG-DSPE、或DTX多肽-PEG-PLA和mPEG-PLA、或DTX多肽-PEG-PLGA和mPEG-PLGA、或DTX多肽-PEG-PCL和mPEG-PCL,以及一定量药物,采用成膜水化等方法分别制备相应的DTX多肽修饰脂质体、聚合物胶束、聚合物圆盘、聚合物纳米粒等纳米递药系统;激光散射粒度仪表征纳米递药系统粒径和粒径分布。
以DTX修饰的负载阿霉素的脂质体(DTX-sLip/DOX)为例,PEG-脂质体膜材料处方组成为HSPC/Chol/mPEG2000-DSPE(52:43:5,mol/mol),DTX修饰的PEG脂质体膜材料处方为HSPC/Chol/mPEG2000-DSPE/DTX-PEG-DSPE(52:43:3:2,mol/mol)。分别称取上述膜材料溶于氯仿,减压旋转蒸发除去氯仿,得均匀脂质膜,真空干燥24h。加入一定体积0.32M硫酸铵溶液,60℃水浴震荡2h,得脂质体混悬液。在60℃水浴中,使用高压均质机(若脂质体体积少于10mL则改用微型挤出器)依次将脂质体挤压过400、200、100核孔膜,得空白脂质体。将上述空白脂质体用生理盐水洗脱通过Sephadex G-50凝胶柱更换外水相。按照药脂比1:10(w/w)加入阿霉素生理盐水溶液,60℃水浴20min。以生理盐水洗脱通过Sephadex G-50凝胶柱,除去游离药物,得阿霉素脂质体。图6展示了脂质体的粒径分布图。结果可知,DTX-sLip/DOX水合粒径为97.76nm,分散系数为0.186,提示脂质体粒径合适、分散均一。
实施例2:DTX及其修饰的纳米载体的脑靶向能力评价
1.DTX多肽体外脑靶向能力评价
将白喉毒素受体阳性表达的脑毛细血管内皮细胞(bEND.3细胞)均匀铺在共聚焦培养皿上,孵育24h后显微镜观察细胞融合度和形态。用含10%FBS的DMEM配置浓度为5μM的荧光标记DTX多肽(DTX-FAM)溶液。吸弃培养基,向每孔细胞中加入上述多肽分子溶液,37℃孵育4h,PBS(pH=7.4)洗涤3次,4%多聚甲醛固定10min后,DAPI(4',6-diamidino-2-phenylindole)染色,用共聚焦显微镜观察。为了定量分析,细胞经相同条件处理后,用胰酶消化收集细胞,用流式细胞仪检测细胞摄取量。bEND.3细胞对DTX多肽的摄取结果如图7所示,结果显示bEND.3细胞可有效摄取DTX多肽。
2.DTX多肽体内脑靶向能力考察
将Cy5标记的DTX多肽分子(DTX-Cy5)配置成100μM的溶液,ICR小鼠尾静脉注射100μL,1h后麻醉小鼠,分别用生理盐水和4%多聚甲醛溶液灌流,解剖小鼠,取脑组织,在小动物活体荧光成像系统下观察多肽分子的脑内分布情况。如图8所示,DTX-Cy5在小鼠脑内具有很强的荧光,与游离Cy5具有显著性差异,说明的DTX多肽具有良好的体内脑靶向能力。
3.DTX多肽修饰纳米递药系统的脑靶向性评价
利用氨基与异硫氰基的特异反应合成功能性材料FITC-PEG3400-DSPE。合成步骤大致如下:称取适量NH2-PEG3400-DSPE与1.2倍当量的异硫氰酸荧光素(FITC),混匀后溶于DMF,加入三乙胺,于室温下避光搅拌反应,采用茚三酮检测反应,待溶液不再变蓝,提示NH2-PEG3400-DSPE反应完全。过量的FITC和DMF通过透析(截留分子量3500)去除,经冷冻干燥后得到NH2-PEG3400-DSPE。进一步利用薄膜水化法制备荧光标记的DTX多肽修饰脂质体。
将白喉毒素受体阳性表达的脑毛细血管内皮细胞(bEND.3细胞)均匀铺在共聚焦培养皿上,孵育24h后显微镜观察细胞融合度和形态。吸弃培养基,向每孔细胞中加入荧光标记的DTX多肽修饰脂质体,37℃孵育4h,PBS(pH=7.4)洗涤3次,4%多聚甲醛固定10min后,DAPI染色,用共聚焦显微镜观察。bEND.3细胞的摄取结果如图9所示,提示DTX修饰可显著提高脂质体在bEND.3细胞的摄取。
实施例3:DTX及其修饰的纳米载体对脑肿瘤体内外靶向能力评价
1.DTX多肽体外靶向能力评价
将白喉毒素受体阳性表达的模型肿瘤细胞(脑胶质瘤细胞U87)均匀铺在共聚焦培养皿上,孵育24h后显微镜观察细胞融合度和形态。用含10%FBS的DMEM配置浓度为5μM的荧光标记DTX多肽(FAM-DTX)溶液。吸弃培养基,向每孔细胞中加入上述多肽分子溶液,37℃孵育4h,PBS(pH=7.4)洗涤3次,4%多聚甲醛固定10min后,DAPI染色,用共聚焦显微镜观察。为了定量分析,细胞经相同条件处理后,用胰酶消化收集细胞,用流式细胞仪检测细胞摄取量。U87细胞对DTX多肽的摄取结果如图10所示,结果显示U87细胞可有效摄取DTX多肽。
2.DTX多肽肿瘤球渗透能力评价
用无血清配液配置2%的低分子琼脂糖溶液,经高压灭菌30min后,趁热铺入48孔板中,每孔150μL,置于紫外下继续照射30min并使之降温凝固。消化U87细胞,每孔接种4000个细胞,每孔400μL。沿同一方向振摇孔板使细胞成团。将48孔板置于培养箱中继续培养7天,即可形成U87肿瘤球。吸弃肿瘤球的培养液,加入相同荧光强度的DTX-FAM和游离FAM溶液。于37℃孵育4h后取出肿瘤球,PBS洗涤3次后,用4%多聚甲醛固定30min,将肿瘤球置于共聚焦皿中,采用激光共聚焦显微镜断层扫面拍照。如图11所示,DTX多肽可有效被U87肿瘤摄取,且可渗透进肿瘤球深部。
3.DTX多肽体内脑肿瘤靶向能力考察
将20g左右的裸鼠麻醉后,固定于脑立体定位仪上,微量注射器吸取5μL的U87细胞悬液(6×105个细胞),接种于小鼠纹状体部位(前囟向前0.6mm,向右1.8mm,纵深3mm),构建胶质瘤原位小鼠模型。种瘤第7天后开始实验,分别尾静脉给予小鼠相同荧光强度的DTX-Cy5和游离Cy5溶液。1h后,麻醉小鼠,用小动物活体成像仪观察脑内的荧光分布。结果如图12所示,DTX多肽可有效在原位肿瘤处蓄积,具有较好的体内脑肿瘤靶向能力。
4.DTX多肽修饰纳米递药系统的体内靶向性评价
将U87细胞均匀铺在共聚焦培养皿上,孵育24h后显微镜观察细胞融合度和形态。吸弃培养基,向每孔细胞中加入荧光标记的DTX多肽修饰脂质体(如实施例2.3的方法制备),37℃孵育4h,PBS(pH=7.4)洗涤3次,4%多聚甲醛固定10min后,DAPI染色,用共聚焦显微镜观察。U87细胞的摄取结果如图13所示,提示DTX修饰可显著提高脂质体在bEND.3和U87细胞的摄取。
实施例4:DTX-抗肿瘤药物及DTX修饰的纳米递送系统的抗肿瘤药效评价
1.DTX多肽修饰药物的体外抗肿瘤效果评价
采用MTT法评价DTX多肽修饰药物对U87细胞的抑制作用。取对数期生长的U87细胞,消化并计数,以每孔5000个细胞铺于96孔板中,于培养箱中孵育24h。加入不同浓度的游离阿霉素(DOX)、药物复合物(DTX-DOX)、物理混合物(DTX+DOX)以及游离的多肽(DTX)。每个浓度设立三个复孔,同时预留不加任何样品的细胞培养液作为阴性对照。将96孔板置于培养箱中继续孵育72h,每孔加入20μL 5mg/mL MTT溶液,继续孵育4h,吸弃孔内培养液并在每孔中加入150μL DMSO,置于空气摇床中低速振荡15min使孔底部蓝紫色结晶充分溶解,酶标仪测定各孔在490nm波长下的吸光度,计算各孔细胞存活率。用GraphPad Prism软件将存活率对药物浓度对数值做图,计算半数抑制浓度(IC50)。结果如图14所示,DTX修饰的阿霉素(DTX-DOX)的IC50为0.0134μM,远低于游离DOX的IC50(1.3μm)。提示DTX修饰可显著提升药物DOX抑制U87细胞的活性。
2.DTX多肽修饰纳米递药系统的体外抗肿瘤效果评价
采用MTT法评价DTX多肽修饰纳米递药系统对U87细胞的抑制作用。取对数期生长的U87细胞,消化并计数,以每孔5000个细胞铺于96孔板中,于培养箱中孵育24h。加入不同浓度的DTX修饰的载阿霉素脂质体(DTX-sLip/DOX)和负载阿霉素的空白脂质体(sLip/DOX)。每个浓度设立三个复孔,同时预留不加任何样品的细胞培养液作为阴性对照。将96孔板置于培养箱中继续孵育72h,每孔加入20μL 5mg/mL MTT溶液,继续孵育4h,吸弃孔内培养液并在每孔中加入150μL DMSO,置于空气摇床中低速振荡15min使孔底部蓝紫色结晶充分溶解,酶标仪测定各孔在490nm波长下的吸光度,计算各孔细胞存活率。用GraphPad Prism软件将存活率对药物浓度对数值做图,计算半数抑制浓度(IC50)。结果如图15所示,sLip/DOX)以及DTX-sLip/DOX的IC50值分别为119和93.32nM。提示DTX修饰可提升阿霉素脂质体抑制U87细胞的活性。
以上已对本发明创造的较佳实施例进行了具体说明,但本发明创造并不限于所述实施例,熟悉本领域的技术人员在不违背本发明创造精神的前提下还可做出种种的等同的变型或替换,这些等同的变型或替换均包含在本申请权利要求所限定的范围内。
Claims (12)
1.一种特异性靶向白喉毒素受体的DTX多肽,其特征在于,所述的DTX多肽是由天冬氨酸、组氨酸、苏氨酸、赖氨酸、缬氨酸、天冬酰胺、丝氨酸、赖氨酸、亮氨酸、丝氨酸、亮氨酸、苯丙氨酸和苯丙氨酸13个连续氨基酸构成的多肽,所述的多肽为L构型多肽LDLHLTLKLVLNLSLKLLLSLLLFLF,氨基酸序列如SEQ ID NO.1所示;或者为L构型多肽的逆序D构型多肽DFDFDLDSDLDKDSDNDVDKDTDHDD,氨基酸序列如SEQ ID NO.2所示。
2.一种如权利要求1所述的DTX多肽以共价键与影像物质X连接得到的DTX-X复合物。
3.根据权利要求2所述的DTX-X复合物,其特征在于,所述的影像物质X选自荧光物质Fluorescein,近红外染料Cy5、IR820或DiR,磁共振影像剂Gd-DTPA,放射影像剂99mTc-DTPA。
4.一种如权利要求1所述的DTX多肽、或一种如权利要求2或3所述的DTX-X复合物在制备脑、脑部肿瘤或外周肿瘤的影像诊断和示踪试剂中的应用。
5.一种如权利要求1所述的DTX多肽以共价键与抗肿瘤药物Y连接得到的DTX-Y复合物。
6.根据权利要求5所述的DTX-Y复合物,其特征在于,所述的抗肿瘤药物Y选自蒽环类药物阿霉素或表阿霉素,紫杉烷类药物紫杉醇、多烯紫杉醇或卡巴他赛,喜树碱类药物喜树碱、羟基喜树碱或伊立替康,长春花碱类药物长春新碱或长春瑞滨,蛋白酶体抑制剂硼替佐米或卡非佐米,内酯类药物小白菊内酯,蛋白降解靶向嵌合体,多肽类药物p53激活肽、蜂毒肽、蝎毒肽或抗菌肽。
7.一种如权利要求1所述的DTX多肽、或一种如权利要求5或6所述的DTX-Y复合物在制备脑部肿瘤或外周肿瘤的靶向治疗药物中的应用。
8.一种如权利要求1所述的DTX多肽以共价键与聚乙二醇-Z复合物连接得到DTX-聚乙二醇-Z复合物;所述的复合物中的Z选自磷脂、聚乳酸、乳酸羟基乙酸共聚物、聚己内酯。
9.一种如权利要求1所述的DTX多肽、或一种如权利要求8所述的DTX-聚乙二醇-Z复合物在制备脂质体递药系统、聚合物胶束递药系统、聚合物圆盘递药系统、纳米粒递药系统中的应用。
10.根据权利要求9所述的应用,其特征在于,所述的递药系统包载诊断药物或肿瘤治疗药物。
11.根据权利要求10所述的应用,其特征在于,所述的诊断药物选自荧光物质香豆素6、罗丹明B、Cy7、IR820、DiR、DiD,或磁共振影像剂Gd-DTPA。
12.根据权利要求10所述的应用,其特征在于,所述的肿瘤治疗药物选自蒽环类药物阿霉素或表阿霉素,紫杉烷类药物紫杉醇、多烯紫杉醇或卡巴他赛,喜树碱类药物喜树碱、羟基喜树碱或伊立替康,长春花碱类药物长春新碱或长春瑞滨,蛋白酶体抑制剂硼替佐米或卡非佐米,内酯类药物小白菊内酯,蛋白降解靶向嵌合体,多肽类药物p53激活肽、蜂毒肽、蝎毒肽或抗菌肽。
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