CN105664174A - Lf-HA-DOX大分子前药复合物及其构建方法和在治疗神经胶质瘤中的应用 - Google Patents
Lf-HA-DOX大分子前药复合物及其构建方法和在治疗神经胶质瘤中的应用 Download PDFInfo
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Abstract
本发明公开了Lf-HA-DOX大分子前药复合物及其构建方法和在治疗神经胶质瘤中的应用。本发明所述Lf-HA-DOX大分子前药复合物是以乳铁蛋白Lf作为靶向修饰的配体,以阿霉素DOX作为治疗药物,以透明质酸HA作为载体骨架,同时利用透明质酸的肿瘤靶向性,得到双重靶向的大分子前药复合物。本发明所述Lf-HA-DOX大分子前药复合物既可以透过血脑屏障,还具有很好的神经胶质瘤靶向性,同时可以实现缓慢释药的效果,是神经胶质瘤的治疗中具有很好的应用前景。
Description
技术领域
本发明涉及药物载体领域,具体涉及一种既可以通过血脑屏障,又能双重靶向作用于神经胶质瘤的载体Lf-HA-DOX大分子前药复合物及其构建方法和应用。
背景技术
胶质瘤是最常见和恶性程度最强的原发性颅内肿瘤。但是由于其弥漫浸润性生长以及对放化疗的抵抗,胶质瘤很难被传统的治疗方式完全治愈。因此,神经胶质瘤的临床治疗仍是一个需要研究和探索的难题。
传统的化疗失败的一个重要原因是因为血脑屏障的存在导致抗肿瘤药物很难达到脑实质。而传统的载体,包括纳米载体,不具有透过血脑屏障的能力,这严重限制了其在胶质瘤中的应用。另外,胶质瘤有大多数肿瘤共有的特性,如低pH,低pO2等。这也导致传统载体在肿瘤部位释放药物受限。因此,研究者们一直在寻找可以通过静脉给药途径进入脑并且对pH敏感的药物载体。
透明质酸(hyaluronicacid,HA)是CD44受体的一个重要的配体,而CD44在包括胶质瘤在内的多种肿瘤中均是高表达,由此以HA为骨架的运输系统可以用于抗肿瘤治疗。受体介导的转胞吞作用(Receptor-mediatedtranscytosis,RMT)是药物载体通过血脑屏障的一种转运途径。内皮细胞上有很多种这样的转运受体,如:转铁蛋白受体,胰岛素受体,脂蛋白受体。乳铁蛋白(lactoferrin,Lf)是转铁蛋白中一员,其受体(Lfreceptor,LfR)属于低密度脂蛋白受体相关蛋白(low-densitylipoproteinreceptor-relatedprotein,LRP)。之前已有多项研究显示Lf可以通过LRP介导的转胞吞作用运输到大脑,另外有些研究显示LfR在血脑屏障上高表达。而且有研究显示Lf对于血脑屏障的通透性明显优于转铁蛋白。而且,LfR不仅在血脑屏障中高表达而且在胶质瘤细胞表面高表达。因此,Lf可以成为一种有效的血脑屏障和胶质瘤双靶向配体。
阿霉素(Doxorubicin,DOX)是一种非常好的广谱的抗癌药物,已经广泛用于抵抗多种实体肿瘤。但是由于其差的靶向性以及严重的副作用,尤其是心肌毒性,所以其在颅内肿瘤中的应用是有限的。
发明内容
本发明的目的在于根据目前用于治疗神经胶质瘤的药物难以通过血脑屏障、脑内局部注射法在临床应用中存在困难等诸多缺陷,提供一种可以通过静脉给药的、既可以透过血脑屏障,又对神经胶质瘤具有靶向性作用,且对pH有响应性的Lf-HA-DOX大分子前药复合物,该复合物可以安全有效的治疗神经胶质瘤。
本发明另一目的在于提供上述Lf-HA-DOX大分子前药复合物的构建方法。
本发明还有一个目的在于提供上述Lf-HA-DOX大分子前药复合物在制备治疗神经胶质瘤药物中的应用。
本发明上述目的通过以下技术方案予以实现:
Lf-HA-DOX大分子前药复合物,是以乳铁蛋白Lf作为靶向修饰的配体,以阿霉素DOX作为神经胶质瘤的治疗药物,以透明质酸HA作为载体骨架得到的大分子前药。本发明所述Lf-HA-DOX大分子前药复合物具有双重靶向性,具体是指乳铁蛋白和透明质酸两种分子通过受体介导转运将药物递送至脑内。所述透明质酸骨架本身也具备肿瘤靶向性,能与肿瘤细胞表面高表达的CD44受体特异性结合,通过受体介导转运的胞吞作用,增加药物病灶区的富集浓度,达到肿瘤靶向的目的。该复合物的构建方法是本发明的关键。
本发明所述Lf-HA-DOX大分子前药复合物的具体构建方法如下:
(1)含肼基透明质酸(HA-ADH)的制备:称取HA(分子量6500)溶于去离子水中,加入过量的己二酸二酰肼ADH,搅拌使其溶解,用HCl调节反应液pH至6.8。称取等摩尔量的1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐EDC·HCl及1-羟基苯并三唑(HOBt),溶于DMSO与水的混合溶液中(1:1,v/v),并将其滴入上述反应液中,室温下反应12~24h。反应结束后,调节溶液pH至7.4,在去离子水中透析3-5d(截留分子量为6000),冻干得HA-ADH;
(2)称取HA-ADH于三口烧瓶中,加入去离子水在氮气氛围下搅拌使之溶解,加入冰乙酸调节pH至5,称取盐酸阿霉素加入其中,待其溶解后,继续通氮气0.5~4h,密封,避光继续在室温反应12~24h。反应结束后,将反应液滴入大量的无水乙醇中沉淀,抽滤,固体用无水乙醇洗涤后干燥24~48h得产物透明质酸-阿霉素大分子前药(HA-DOX);
(3)透明质酸-阿霉素大分子前药(HA-DOX)在0.1M的MES缓冲液(pH=5)中溶解,称取等摩尔量的EDC和NHS加入,待其溶解后,再继续反应0.5~2h,用0.1MNaOH调节pH至7,加入乳铁蛋白,室温反应12~24h。反应结束后,将反应液转移至截留分子量为10000的透析袋中透析3~5d。将透析后的溶液冻干得到乳铁蛋白修饰的透明质酸-阿霉素大分子前药复合物(Lf-HA-DOX)。
(4)采用凝胶电泳法,确证Lf与透明质酸(HA)的偶联反应是否成功。
采用免疫荧光法观察对U87细胞(人恶性胶质母细胞瘤细胞)和C6细胞(大鼠神经胶质瘤细胞)表面CD44受体表达进行了情况验证,CD44受体是一种跨膜糖蛋白,由细胞外、跨膜及膜内三部分组成,透明质酸(Hyaluronicacid,HA)通过与CD44受体的特异性结合来调节细胞的移动、黏附、分化和聚集等生理功能,在肿瘤部位高表达。证实了透明质酸在胶质瘤上也有一定的靶向功能,它主要是通过受体介导作用,增加病灶区的富集浓度,达到肿瘤靶向的目的。
通过细胞实验,考察了所得多糖基大分子前药在U87细胞(人恶性胶质母细胞瘤细胞)和C6细胞(大鼠神经胶质瘤细胞)中的摄入情况;与阿霉素原药对比,在相同药物浓度下测定U87和C6细胞中的荧光强度。
基于裸鼠原位脑胶质瘤模型,使用cy5.5分子标记大分子前药并通过尾静脉注射,借助小动物活体荧光成像仪观察大分子前药的体内代谢分布,发现本发明大分子前药(Lf-HA-DOX)具有良好的穿透BBB到达肿瘤的靶向能力及在肿瘤处的聚积能力。
与现有技术相比,本发明具有如下有益效果:
(1)本发明通过化学作用偶联了乳铁蛋白,作为一种低密度脂蛋白的配体,使得抗肿瘤药物能够跨跃血脑屏障,从而将药物递送至脑内。透明质酸本身具有肿瘤靶向性,透明质酸通过与CD44受体的特异性结合来调节细胞的移动、黏附、分化和聚集等生理功能,对肿瘤生长起到重要作用,以透明质酸作为大分子前药的主体骨架,可以增加病灶区药物的富集浓度,达到肿瘤靶向的目的。实现了对胶质瘤的双重靶向;
(2)肿瘤部分微环境偏酸性,采用酸敏感的酰腙键键合抗肿瘤阿霉素,可以赋予其肿瘤响应性,使得抗肿瘤药物在未到达病兆部位时不释放,降低其毒副作用;
(3)本发明复合物与现有的神经胶质瘤靶向研究相比,双靶向递送为其突出特征,既可通过主动靶向作用跨过血脑屏障,又可靶向作用于脑胶质瘤,减少了目的基因在其他细胞中非特异性表达,提高了药物利用率;
(4)本发明采用静脉注射给药途径符合纳米载体的发展方向,避免了脑内局部注射的创伤,方便临床推广应用;
(5)本发明的工艺简单,操作方便,所需设备及原材料廉价。
附图说明
图1为Lf-HA-DOX前药的分子结构;
图2为用PBS,DOX,HA-DOX和Lf-HA-DOX处理后的C6胶质瘤裸鼠的K-M生存曲线;
图3为C6胶质瘤裸鼠脑组织(1)和心脏组织(2)HE染色。其中,A(A1,A2):PBS对照组;B(B1,B2):DOX组;C(C1,C2):HA-DOX组;D(D1,D2):Lf-HA-DOX组。
具体实施方式
以下结合具体实施例来进一步解释本发明,但实施例并不对本发明做任何形式的限定。
以下实施例中所提到的药物、设备或器材,如无特别说明,均为本领域技术人员常用的药物、设备或器材。
实验例中所用到的实验动物为Balb/c裸鼠,体重18~22g,购自中山大学实验动物中心。动物在SPF(specificpathogenfree)级、每12小时明暗交替的环境饲养,室温25℃,水和饲料均充足。
实施例Lf-HA-DOX大分子前药复合物的构建
(1)透明质酸-阿霉素大分子前药(HA-DOX)的制备方法申请人已经在中国专利(CN104689338A)中公布,在此简单描述如下:称取0.5gHA溶于50mL去离子水中,加入过量的己二酸二酰肼ADH6.5325g,搅拌使其溶解,调节溶溶液pH至6.8。称取EDC及HOBt,溶于DMSO与水的混合溶液中(1:1,v/v,10mL),滴入上述反应液中,室温下反应24h。透析冻干得肼化透明质酸(HA-ADH)。称取0.50gHA-ADH加入10mL去离子水,在氮气氛围下搅拌使之溶解,加入冰乙酸调节pH至5,称取一定量的盐酸阿霉素加入其中,待其溶解后,继续通氮气30min,密封,室温反应24h。反应结束后,将反应液滴入100mL无水乙醇中沉淀,用布氏漏斗抽滤,固体用20mL无水乙醇洗涤后于真空烘箱中40℃下干燥48h得产物。
(2)称取0.32gHA-DOX在10mL0.1M的MES缓冲液(pH=5)中溶解,称取0.216gEDC和0.298gNHS,待其溶解后,再继续反应90min,用0.1MNaOH调节pH至7左右,称取20mg乳铁蛋白Lf加入上述反应液,室温反应24h。反应结束后,将反应液转移至截留分子量为10000的透析袋中透析3d,冻干后得到乳铁蛋白修饰的透明质酸阿霉素大分子前药(Lf-HA-DOX)。
(3)将所制备的大分子前药溶于PBS中,分别选取pH5.0醋酸-醋酸钠缓冲液(0.02M);pH6.0及pH7.4磷酸二氢钠-磷酸氢二钠缓冲液(0.02M);作为释放介质,取制备好的前药注射液(5mg/mL)装入到截留分子量为3000的透析袋中,然后置于装有20mL释放溶液的离心管内,于37℃恒温振荡器避光震荡,在设定的时间间隔内,从透析袋中取出3mL溶液并补加3mL新鲜释放液,取出的溶液用紫外分光光度计检测480nm处的吸收值,根据之前测定的DOX在不同pH缓冲液中的标准曲线计算在此段时间内的药物释放量。
实验结果:Lf-HA-DOX的分子结构如图1所示。拟合药物释放曲线中可以看出前药具有明显的酸响应特性,阿霉素在三种介质中的释放速率明显不同,随缓冲介质pH升高,阿霉素的释放速率明显减慢,这与酰腙键的特点相符。在24h时,在pH5.0时累计释放量已达到45%,而在pH7.4时释放量还不足15%。
实验例Lf-HA-DOX对原位胶质瘤裸鼠的治疗效果评价
原位脑胶质瘤模型建立:用4%水合氯醛(10ml/kg)腹腔注射麻醉Balb/c裸鼠后,将其头部固定在脑立体定位仪上。根据小鼠头部立体定向解剖图谱确定右尾状核为靶点,于前囟前1mm,中线右旁开3mm,用牙科钻先钻一小孔,然后再立体定位仪引导下,25μl微量注射器抽取C6细胞悬液5μl(5*105个细胞),沿骨孔缓慢垂直进针至硬脑膜下4mm,再退回1mm。缓慢注射5分钟,注射完毕后留针3min,缓慢拔针,之后用骨蜡封闭骨孔,4号缝线缝合切口。
移植后第10天,随机选取6只裸鼠分为2组:分别给与近红外染料Cy5.5标记的HA-DOX和Lf-HA-DOX(n=3),然后用活体成像系统记录不同时间荧光的分布情况。另外的裸鼠随机分为4组,每组10只:1)PBS对照组,2)DOX处理组,3)HA-DOX处理组,4)Lf-HA-DOX处理组。分别用PBS,DOX,HA-DOX,Lf-HA-DOX复合物溶液处理各组裸鼠。采用尾静脉注射的给药方式,每3天给药一次,每次剂量为含5mgDOX/kg的复合物,共给予4次。最后一次给药后1天,从每组中取3只裸鼠,灌注固定,取心脑组织进行HE染色。剩余动物用来观察其生存期并做K-M生存曲线分析。
实验结果:
1.颅内药物的分布
通过尾静脉注射带有近红外荧光染料标记的复合物后,与HA-DOX组相比,Lf-HA-DOX组颅内荧光信号从2h到24h是逐渐增强,这暗示由Lf介导的转运确实是可以通过血脑屏障。并且24h后取材发现,Lf-HA-DOX组脑部荧光信号明显增强,这主要是由于Lf和血脑屏障上表达的LfR特异性的结合,从而介导了血脑屏障的转胞吞作用。而HA-DOX组荧光主要聚集在肺脏和肾脏。这可能主要是由于CD44在肺脏和肾脏处高表达。
2.各处理组生存曲线变化
对裸鼠的疗效观察也能从其K-M生存曲线中得以反应。Lf-HA-DOX处理组40天的中位生存期较PBS组(20天,p=0.005),DOX处理组(25天,p=0.007),HA-DOX处理组(28天,p=0.027)明显延长(图2)。
3.HE染色分析
瘤细胞胞浆丰富,核大,形状不规则,核深染,部分可见大核仁。在Lf-HA-DOX处理组,胶质瘤细胞密度降低,有时可见小的散在坏死灶;另外,DOX处理组可以看到明显的心肌毒性,而连了HA或者Lf-HA靶向的复合物则没有观察到心肌毒性(图3)。
结论:Lf-HA-DOX对裸鼠疗效实验证实了其体内的双靶向效应。结果显示,Lf-HA-DOX能够改善对裸鼠的治疗效果,而具有双靶向作用的Lf-HA-DOX能产生最显著的肿瘤抑制和最明显的中位生存期延长。组织HE染色分析进一步显示Lf-HA-DOX可以明显的减少DOX的心肌毒性作用。
Claims (3)
1.Lf-HA-DOX大分子前药复合物,其特征在于是以乳铁蛋白作为靶向修饰的配体,以阿霉素作为治疗药物,以透明质酸作为载体骨架,得到的双重靶向的大分子前药复合物。
2.权利要求1所述Lf-HA-DOX大分子前药复合物的构建方法,其特征在于包括如下步骤:
(1)含肼基透明质酸的制备:将透明质酸溶于去离子水中,加入过量的己二酸二酰肼,搅拌使其溶解,用HCl调节反应液pH至6.8;称取等摩尔量的1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐及1-羟基苯并三唑,溶于DMSO与水的混合溶液中(1:1,v/v),并将其滴入上述反应液中,室温下反应12~24h;反应结束后,调节溶液pH至7.4,在去离子水中透析3~5d(截留分子量为6000),冻干得含肼基透明质酸;
(2)将含肼基透明质酸置于三口烧瓶中,加入去离子水在氮气氛围下搅拌使之溶解,加入冰乙酸调节pH至5,加入盐酸阿霉素,待其溶解后,继续通氮气0.5~4h,密封,避光继续在室温反应12~24h;反应结束后,将反应液滴入大量的无水乙醇中沉淀,抽滤,固体用无水乙醇洗涤后干燥24~48h得到的产物为透明质酸-阿霉素大分子前药;
(3)透明质酸-阿霉素大分子前药在0.1M、pH=5的MES缓冲液中溶解,称取等摩尔量的EDC和NHS加入,待其溶解后,再继续反应0.5~2h,用0.1MNaOH调节pH至7,加入乳铁蛋白,室温反应12~24h;反应结束后,将反应液转移至截留分子量为10000的透析袋中透析3~5d;将透析后的溶液冻干得到Lf-HA-DOX大分子前药复合物。
3.权利要求1所述Lf-HA-DOX大分子前药复合物在制备治疗神经胶质瘤的药物中的应用。
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