CN113082221A - Tyr/Ang双重修饰的青蒿琥酯纳米靶向制剂的制备方法和在颅脑肿瘤治疗中的应用 - Google Patents
Tyr/Ang双重修饰的青蒿琥酯纳米靶向制剂的制备方法和在颅脑肿瘤治疗中的应用 Download PDFInfo
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Abstract
本发明涉及一种Tyr/Ang双重修饰的青蒿琥酯纳米靶向制剂的制备方法和在颅脑肿瘤治疗中的应用,属于医药技术领域。本发明所制备的纳米靶向制剂是以青蒿琥酯作为原料,以聚乙二醇‑聚己内酯为载体材料,以Tyr/Ang为双靶头修饰载体,采用溶剂挥发法制备。本发明的Tyr/Ang双重修饰的青蒿琥酯纳米制剂可靶向富集于血脑屏障,穿过血脑屏障进入脑实质后又可靶向定位于脑胶质瘤细胞从而对脑胶质瘤起到靶向杀伤作用,具有良好的靶向性,及较好的肿瘤生长抑制作用,毒副作用低,使用较安全。将抗疟药青蒿琥酯制备成为双靶头修饰的靶向纳米制剂为新型脑胶质瘤等颅脑肿瘤的治疗提供了新思路,具有重要的临床意义。
Description
技术领域
本发明属于医药技术领域,涉及一种Tyr/Ang双重修饰的青蒿琥酯纳米靶向制剂的制备方法和在颅脑肿瘤治疗中的应用。
背景技术
脑胶质瘤是最常见的原发性颅脑肿瘤,约占颅内肿瘤的46%,死亡率高。手术联合放化疗的综合治疗是脑胶质瘤常用的标准治疗方案。由于恶性脑胶质瘤的侵袭性生长特性及解剖位置的特殊性,手术难以彻底清除。放疗虽有一定疗效,但容易造成放射性坏死,且缓解期通常不超过8个月。一般的抗肿瘤药物穿透血脑屏障(BBB)能力不足而肿瘤局部药物浓度较低,且容易发生耐药性,导致脑胶质瘤化疗失败而复发。因此,在保证对正常组织无伤害的前提下,开发可迅速穿透BBB,靶向入脑并定位于脑胶质瘤后快速释放药物,对脑胶质瘤多重耐药具有良好抑制作用的精准靶向药物,对耐药脑胶质瘤的治疗具有重要意义。
PEG-PCL存在疏水和亲水两部分材料,可以自组装形成纳米粒。青蒿琥酯是抗疟药青蒿素的衍生物,是一种具有过氧桥结构的倍半萜内酯类化合物。除抗疟作用外,其抗肿瘤作用近年来逐渐被人们认识和引起广泛关注。其抗肿瘤作用机制可能与抑制肿瘤细胞增殖、诱导细胞分化和凋亡、抑制肿瘤血管新生、抑制肿瘤侵袭转移、增敏抗肿瘤化疗药物及逆转耐药等多种机制有关。但有关将青蒿琥酯制成双靶头修饰的脑靶向纳米制剂并用于脑胶质瘤等颅脑肿瘤的治疗尚未见报道。
发明内容
有鉴于此,本发明的目的之一在于提供一种Tyr/Ang双重修饰的青蒿琥酯纳米靶向制剂的制备方法,本发明的目的之二在于提供一种Tyr/Ang双重修饰的青蒿琥酯纳米靶向制剂,本发明的目的之三在于提供Tyr/Ang双重修饰的青蒿琥酯纳米靶向制剂在治疗多重耐药的脑胶质瘤中的应用,本发明的目的之四在于提供一种药物制剂,本发明的目的之五在于提供一种药物制剂在治疗多重耐药的脑胶质瘤中的应用。
为达到上述目的,本发明提供如下技术方案:
1、一种Tyr/Ang双重修饰的青蒿琥酯纳米靶向制剂的制备方法,所述方法为,将聚乙二醇-聚己内酯、酪氨酸修饰的聚乙二醇-聚己内酯和Angiopep2修饰的聚乙二醇-聚己内酯溶解在四氢呋喃中,加入青蒿琥酯,200~600rpm的速度下搅拌,再逐滴加入水中,继续搅拌4~8h,即得Tyr/Ang双重修饰的青蒿琥酯纳米靶向制剂,得到Tyr/Ang双重修饰的青蒿琥酯纳米靶向制剂。
作为优选的技术方案之一,所述聚乙二醇-聚己内酯、酪氨酸修饰的聚乙二醇-聚己内酯、Angiopep2修饰的聚乙二醇-聚己内酯、青蒿琥酯、四氢呋喃和水的质量体积比为13~17:1~3:1~3:0.5~2:0.5~2:3~7,mg:mg:mg:mg:mL:mL。
作为优选的技术方案之一,所述酪氨酸修饰的聚乙二醇-聚己内酯的制备方法为:
1)将Boc-PEG2000-OH、己内酯和辛酸亚锡催化剂按照2~8:16~22:1~5的比例,110~150℃下聚合18~30h,二氯甲烷溶解,乙醚沉淀,得到反应物a;
2)将反应物a溶于二氯甲烷,加入三氟乙酸,室温下搅拌1h,洗涤、干燥、乙醚沉淀,得到反应物b,所述反应物a与三氟乙酸的质量体积比为0.5~2.5:3~7,g:mL;
3)取反应物b溶于氯仿,加入Boc-L-Tyr、EDC和DMAP,室温下反应9~12h,洗涤、干燥,乙醚沉淀,再加入三氟乙酸,室温下搅拌0.2~1.8h,洗涤、干燥,乙醚再沉淀,得到酪氨酸修饰的聚乙二醇-聚己内酯,所述反应物b、Boc-L-Tyr、EDC和DMAP的摩尔比为:0.5~2:1~3:1~3:12~18。
作为优选的技术方案之一,所述Angiopep2修饰的聚乙二醇-聚己内酯的制备方法为:
1)将Mal-PEG2000-OH、己内酯和辛酸亚锡催化剂按照2~8:16~22:1~5的比例,110~150℃下聚合18~30h,二氯甲烷溶解,乙醚沉淀,得到反应物1;
2)将反应物1溶于DMSO,加入Angiopep2多肽搅拌反应24h,透析,得到Angiopep2修饰的聚乙二醇-聚己内酯,所述反应物1和Angiopep2多肽的摩尔比为:0.5~2:0.5~2。
2、利用所述制备方法所制备的Tyr/Ang双重修饰的青蒿琥酯纳米靶向制剂。
3、所述Tyr/Ang双重修饰的青蒿琥酯纳米靶向制剂在治疗多重耐药的脑胶质瘤中的应用。
4、一种药物制剂,所述药物制剂包括所述Tyr/Ang双重修饰的青蒿琥酯纳米靶向制剂和一种或多种抗颅脑肿瘤药物。
作为优选的技术方案之一,所述颅脑肿瘤为脑胶质瘤、脑膜瘤、颅咽管瘤、神经鞘瘤、神经节胶质细胞瘤、脑下垂体肿瘤或脉络丛肿瘤。
5、所述的药物制剂在治疗多重耐药的脑胶质瘤中的应用。
本发明的有益效果在于:
本发明所制备的Tyr/Ang双重修饰的青蒿琥酯纳米制剂可靶向富集于BBB,穿过BBB进入脑实质后又可靶向定位于脑胶质瘤细胞从而对脑胶质瘤等颅脑肿瘤起到高效和靶向的杀伤作用。体内外研究显示,该双靶头靶向纳米制剂相比单纯的青蒿琥酯溶液和单靶头修饰的纳米制剂对胶质瘤U251细胞有良好的靶向性,且对其增殖具有更强的抑制作用。本发明的靶向纳米制剂针对性强,对BBB穿透性和脑胶质瘤靶向杀伤作用强,毒副作用低,使用较安全,将抗疟药青蒿琥酯制备成为双靶头修饰的靶向纳米制剂为新型脑胶质瘤等颅脑肿瘤的治疗提供了新思路,并拓宽了青蒿琥酯的应用范围,提高了其市场价值。
附图说明
为了使本发明的目的、技术方案和优点更加清楚,下面将结合附图对本发明作优选的详细描述,其中:
图1为PCL-PEG系列纳米粒的核磁氢谱,a为PCL-PEG-Tyr,b为PCL-PEG-Mal,c、d为Ang和PCL-PEG-Ang。
图2为PCL-PEG系列纳米粒的粒径分布,a为PCL-PEG,b为PCL-PEG-Tyr,c为PCL-PEG-Ang,d为PCL-PEG-Tyr/Ang。
图3为纳米粒的透射电镜图,a为PCL-PEG,b为PCL-PEG-Tyr,c为PCL-PEG-Ang,d为PCL-PEG-Tyr/Ang。
图4为负载青蒿琥酯的PCL-PEG系列纳米粒的体外释放行为结果。
图5为负载青蒿琥酯的PCL-PEG系列纳米粒的体外U251细胞毒性结果。
图6为U251细胞体外摄取负载香豆素-6的PCL-PEG系列纳米粒的效率结果。
图7为Tyr/Ang双重修饰的青蒿琥酯纳米粒对裸鼠原位移植瘤模型肿瘤生长的影响结果。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式,本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用,本说明书中的各项细节也可以基于不同观点与应用,在没有背离本发明的精神下进行各种修饰或改变。需要说明的是,以下实施例中所提供的图示仅以示意方式说明本发明的基本构想,在不冲突的情况下,以下实施例及实施例中的特征可以相互组合。
实施例1
聚乙二醇-聚己内酯(PEG2000-PCL7500,简称为PEG-PCL)为载体材料,制备PCL-PEG-Tyr和PCL-PEG-Ang。
(1)PCL-PEG-Tyr合成与表征
取Boc-PEG2000-OH 0.5g,加入1.9g己内酯及0.3g辛酸亚锡催化剂,130℃下聚合反应24h,加入10mL二氯甲烷溶解完全,再倒入大量乙醚中沉淀得到Boc-PEG2000-PCL7500产物。取1.5g Boc-PEG2000-PCL7500溶于6mL二氯甲烷中,加入5mL三氟乙酸,室温下搅拌1h,纯水洗涤3次,无水硫酸钠干燥,然后倒入大量乙醚中沉淀得到NH2-PEG2000-PCL7500产物。取1g NH2-PEG2000-PCL7500溶于5mL氯仿,加入Boc-L-Tyr(2eq.)、EDC(2eq.)和0.2g DMAP,室温下反应10h,纯水洗涤3次,无水硫酸钠干燥。然后加入5mL三氟乙酸,室温下搅拌1h,纯水洗涤3次,无水硫酸钠干燥,然后倒入大量乙醚中沉淀,得到Tyr-PEG2000-PCL7500产物(PCL-PEG-Tyr)。将产物溶解在DMSO-d6中并采用核磁氢谱分析。
(2)Ang-PEG2000-PCL7500合成与表征
取Mal-PEG2000-OH 0.5g,加入1.9g己内酯及0.3g辛酸亚锡催化剂,于130℃下聚合反应24h,加入10mL二氯甲烷溶解完全,倒入大量乙醚中沉淀得到Mal-PEG2000-PCL7500产物。取0.5g Mal-PEG2000-PCL7500溶于4mL DMSO中,加入Angiopep2多肽(1.1eq.),氨基酸序列如SEQ ID NO:1所示,室温下搅拌反应24h,然后转移至透析袋中(截留分子量8000-14000),纯水透析纯化除去未反应的多肽和有机溶剂,收集溶液冷冻干燥即得Ang-PEG2000-PCL7500产物(PCL-PEG-Ang)。将产物溶解在DMSO-d6中采用核磁氢谱分析。
结果如图1、2所示,PCL-PEG-Tyr和PCL-PEG-Ang载体材料的合成采用1H-NMR验证分析,PCL-PEG-Tyr在6.65和7.0ppm处有酪氨酸(Tyr)的特征质子信号(图1中a),表明Tyr连接成功。对于PCL-PEG-Ang,在6.7ppm处能够观察到明显的Mal基团特征峰(图1中b),但是在Ang连接后,该特征峰消失,提示多肽Ang连接成功(图1中b和图1中d)。另外,PCL-PEG-Ang在6.65,7.05 and 7.20ppm处有多肽Ang的质子信号,同样提示连接成功(图1中c和图1中d)。
实施例2
负载青蒿琥酯的PCL-PEG系列纳米粒的制备
制备负载青蒿琥酯的未修饰PCL-PEG纳米粒:20.0mg PCL-PEG和1.0mg的青蒿琥酯溶解在1.0mL的四氢呋喃中,然后在400rpm速度下搅拌,再逐滴加入5mL的纯化水中,继续搅拌6小时挥发掉四氢呋喃,即得载药PCL-PEG纳米粒。
负载青蒿琥酯的单靶头修饰纳米粒:17.97mg未修饰的PCL-PEG纳米粒,2.03mgTyr修饰的载体材料PCL-PEG-Tyr(或17.51mg未修饰的PCL-PEG纳米粒,2.49mg Ang修饰的载体材料PCL-PEG-Ang)溶解在1.0mL的四氢呋喃中,加入1.0mg的青蒿琥酯药物,然后在400rpm速度下搅拌,再逐滴加入5.0mL的纯化水中,继续搅拌6小时挥发掉四氢呋喃后,即得载药PCL-PEG-Tyr或PCL-PEG-Ang纳米粒。
制备负载青蒿琥酯的Tyr/Ang双修饰的纳米粒即双靶头修饰纳米粒(PCL-PEG-Tyr/Ang):15.53mg无修饰的载体材料PCL-PEG,1.98mg Tyr修饰的载体材料PCL-PEG-Tyr以及2.49mg Ang修饰的载体材料PCL-PEG-Ang溶解在1.0mL的四氢呋喃中,加入1.0mg的青蒿琥酯药物,400rpm的速度下搅拌,再逐滴加入5.0mL的纯化水中,继续搅拌6小时挥发掉四氢呋喃后,即得载药PCL-PEG-Tyr/Ang。
负载青蒿琥酯的PCL-PEG系列纳米粒的表征:
1.粒径分析、Zeta电位测定和透射电镜观察
纳米粒的粒径和Zeta电位采用Malvern Zetasizer Nano(Malvern,UK)分析测定。Z-average值用来评价纳米粒的粒径,多分散指数(PDI)用来衡量粒径的分布。纳米粒的形貌采用透射电镜来观察分析。
2.青蒿琥酯的包封率测定
青蒿琥酯的包封率(EE%)采用如下方法测定:5.0mL的载药纳米粒在13,000rpm离心10min,上清液溶解在50.0mL(±0.05mL)二甲基亚砜中。采用反相C18高效液相色谱柱(200mm×4.6mm,particle size:5μm)进行分析测定。进样量10μL,柱温30℃,流动相是乙腈和pH3.0的磷酸盐缓冲液(60:40,v/v),检测波长210nm,流速1.0mL/min。药物的保留时间在10.05–10.20min之间。EE%定义公式为:
本发明使用溶剂挥发法制备载药的PCL-PEG纳米粒。结果如表1、图2和图3所示,所有处方均自组装形成了粒径在60nm左右的纳米粒,PCL-PEG-Ang和PCL-PEG-Tyr/Ang纳米粒的粒径稍大一些(65nm),可能是由于连接了大分子Ang的原因。对于<200nm的纳米粒来说,能够通过EPR效应蓄积于肿瘤部位并且表现出网状内皮系统摄取减少的现象。PCL-PEG和PCL-PEG-Tyr纳米粒的多分散指数(PDI)分别为0.129±0.009和0.133±0.008,提示该纳米粒具有较窄的粒径分布,粒径大小更均一。而PCL-PEG-Ang和PCL-PEG-Tyr/Ang纳米粒的PDI增加了一点(0.204±0.014和0.207±0.013),可能是由于连接了大分子Ang的原因。另外,PCL-PEG-Ang和PCL-PEG-Tyr/Ang纳米粒的Zeta电位相比于PCL-PEG和PCL-PEG-Tyr纳米粒的Zeta电位要稍高一些,这也可能是由于连接了大分子Ang的原因,所有PCL-PEG纳米粒的包封率都较高,大于75%(表1)。
表1 PCL-PEG系列纳米粒的表征结果
实施例3
负载青蒿琥酯的PCL-PEG系列纳米粒药物性能研究
(1)体外释放考察
20.0mg冻干负载青蒿琥酯的纳米粒重分散在5.0mL磷酸盐缓冲液(PBS,0.1M,pH7.4)中,然后装入透析袋中(截留分子量3.5–5kDa)。将透析袋浸润在15.0mL的释放介质(含有0.5%w/v SDS的PBS)中,在80rpm振荡空气浴(37.0±0.5℃)中孵化96小时。在指定的时间点(2,4,6,8,10,24,48,72,96h),1.0mL的释放介质被取出用于HPLC分析药物含量,同时加入等体积的新鲜释放介质以满足漏槽条件。
青蒿琥酯的体外释放行为结果如图4所示,青蒿琥酯包载于PCL-PEG纳米粒中,在释放过程中满足漏槽条件,体外释放测定了96小时,整体上PCL-PEG系列纳米粒的释放行为相似,不受载体材料上配体(Tyr和Ang)的影响。经过起初24小时释放后,青蒿琥酯的释放速率随着释放时间略有下降,整体呈缓释状态。前24小时的释放了45%左右的药量,在到达96小时,释放量在65%左右。基于PCL-PEG共聚物的药物释放在其他文献中也观察到了类似的释放行为。缓慢释放行为能够减少药物的体内清除,从而维持足够的药物浓度以抑制肿瘤生长。
(2)体外细胞毒性
消化收集生长状态正佳的U251细胞,按5×103cells/mL的密度接种于96孔板中,贴壁培养48h备用。制备载有青蒿琥酯的PCL-PEG,PCL-PEG-Tyr,PCL-PEG-Ang,PCL-PEG-Tyr/Ang纳米粒;用青蒿琥酯的乙醇溶液作为药物的溶液剂。各纳米粒及溶液剂稀释成浓度为5、10、20、40、80μg/mL的药物浓度。接种好细胞的96孔板中弃掉培养基,加入上述浓度的制剂,每个浓度5孔,放入培养箱培育24h。取出加药的96孔板,吸去药液,每孔加入100uL培养液和10uL CCK-8,放入培养箱继续孵育3h。取出孵育好的96孔板,采用酶标仪在450nm波长下测定吸光度值。
负载青蒿琥酯的PCL-PEG纳米粒对U251细胞的体外细胞毒性结果如图5所示,游离青蒿琥酯溶液可对U251细胞产生明显的抗增殖作用,且呈浓度依赖性,从而证明其对此类肿瘤具有抗癌作用。另外,游离青蒿琥酯溶液组对U251细胞的细胞毒性最大。结果表明,在体外条件下,自由药物可以通过高浓度梯度的被动扩散快速进入细胞内。相反,载药PCL-PEG纳米粒进入细胞内后经历了药物缓释过程。在体外释放研究中(图4),仅约45%的游离青蒿琥酯在24小时内从PCL-PEG纳米粒中释放。因此,与各纳米制剂相比,青蒿琥酯溶液对单层U251细胞的增殖具有更强的抑制作用。在这些纳米粒中,细胞摄取的改善导致预期增强的抗增殖效果。结果表明,与无靶头的PCL-PEG纳米粒相比,靶头修饰的纳米粒改善了细胞摄取,导致预期增强的抗增殖效果。但单靶头修饰纳米粒与双靶头修饰纳米粒的细胞毒性无明显差异,可能是由于Ang的修饰更多的是靶向血脑屏障的原因。为了更好的显示双靶头修饰的意义,最好是建立体外血脑屏障细胞模型用于细胞毒性的考察。
(3)体外细胞摄取效率
消化收集生长状态正佳的U251细胞,按3×105cells/mL的密度接种于6孔板中,贴壁培养48h备用。制备载有香豆素-6的PCL-PEG,PCL-PEG-Tyr,PCL-PEG-Ang,PCL-PEG-Tyr/Ang纳米粒备用。上述各纳米粒按照香豆素-6 5μg/mL用DMEM培养液稀释后,加入贴壁后的细胞中(吸去贴壁细胞原有的培养液),孵化2h。等待孵化相应时间后,弃去制剂,PBS清洗3遍,每孔加入0.5mL胰酶消化,加入2.5mL培养液,离心收集细胞。收集的细胞用300uL含有2%(v/v)血清的PBS重悬,采用NovoCyte流式细胞仪测定荧光强度。
U251细胞的体外摄取效率结果如图6所示,荧光强度被认为是与细胞中香豆素-6的量成正比;然而,对照组几乎未检测到荧光。结果表明,与香豆素-6溶液相比,PCL-PEG纳米粒中被摄取的香豆素-6的含量明显增加。这个结果可以用纳米粒子的内吞作用来解释。在基于PCL-EPG的纳米粒中,与未经修饰的PCL-PEG纳米粒相比,经Tyr或Ang修饰的PCL-PEG纳米粒(PCL-PEG-Tyr、PCL-PEG-Ang和PCL-PEG-Tyr/Ang)显示U251细胞摄取的香豆素-6的量显著增加。双修饰的PCL-PEG纳米粒(PCL-PEG-Tyr/Ang)的香豆素-6摄取量最高。PCL-PEG-Tyr/Ang纳米粒可以通过增加药物的吸收和保留时间来诱导协同效应,这在体内抗肿瘤疗效研究中可以显示出来。
(4)体内抑瘤活性
取对数生长期的的U251细胞,调节细胞浓度为2×106个细胞/mL。裸鼠麻醉后头部固定于立体定向仪上,剪开头皮并打孔,于颅骨中线右侧2mm、冠状缝前0.5mm交点处,微量注射器缓慢注射细胞悬液5μL/20min制备脑胶质瘤裸鼠原位移植瘤模型。将小鼠随机分为4组:对照组(磷酸盐缓冲液)、青蒿琥酯溶液组(ATN)、负载青蒿琥酯的PCL-PEG纳米粒组(PCL-PEG)和负载青蒿琥酯的PCL-PEG-Tyr/Ang纳米粒组(PCL-PEG-Tyr/Ang)。每3天尾静脉注射青蒿琥酯溶液或青蒿琥酯纳米粒,剂量为30mg/kg。分别于15天和30天后处死裸鼠,分别测量移植瘤的长径(a)及与之垂直的短径(b),计算出肿瘤体积(V=a×b2/2)。
采用裸鼠原位移植瘤模型模型评价Tyr/Ang双重修饰的青蒿琥酯纳米靶向制剂对脑胶质瘤的体内抑制效果。结果如图7所示,15天和30天时青蒿琥酯单独注射组(ATN)和单独的PCL-PEG纳米粒组(PCL-PEG)原位移植瘤体积较对照组同时间点比较均有一定程度的降低,而负载青蒿琥酯的PCL-PEG-Tyr/Ang纳米粒组(PCL-PEG-Tyr/Ang)的肿瘤体积分别较上述两组又显著缩小。结果显示,Tyr/Ang双重修饰的青蒿琥酯纳米粒可显著提高其胶质瘤靶向作用,并对原位脑胶质瘤产生更加强大的抑制作用。
本发明的Tyr/Ang双重修饰的青蒿琥酯纳米靶向制剂除了单独用于颅脑肿瘤的治疗,还可与其他抗颅脑肿瘤药物联合应用,用于克服或逆转颅脑肿瘤耐药等情况。
最后说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本技术方案的宗旨和范围,其均应涵盖在本发明的权利要求范围当中。
序列表
<110> 中国人民解放军陆军特色医学中心
<120> Tyr/Ang双重修饰的青蒿琥酯纳米靶向制剂的制备方法和在颅脑肿瘤治疗中的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Thr Phe Phe Tyr Gly Gly Ser Arg Gly Lys Arg Asn Asn Phe Lys Thr
1 5 10 15
Glu Glu Tyr Cys
20
Claims (9)
1.一种Tyr/Ang双重修饰的青蒿琥酯纳米靶向制剂的制备方法,其特征在于,所述方法为,将聚乙二醇-聚己内酯、酪氨酸修饰的聚乙二醇-聚己内酯和Angiopep2修饰的聚乙二醇-聚己内酯溶解在四氢呋喃中,加入青蒿琥酯,200~600rpm的速度下搅拌,再逐滴加入水中,继续搅拌4~8h,即得Tyr/Ang双重修饰的青蒿琥酯纳米靶向制剂,得到Tyr/Ang双重修饰的青蒿琥酯纳米靶向制剂。
2.根据权利要求1所述的制备方法,其特征在于,所述聚乙二醇-聚己内酯、酪氨酸修饰的聚乙二醇-聚己内酯、Angiopep2修饰的聚乙二醇-聚己内酯、青蒿琥酯、四氢呋喃和水的质量体积比为13~17:1~3:1~3:0.5~2:0.5~2:3~7,mg:mg:mg:mg:mL:mL。
3.根据权利要求1或2所述的制备方法,其特征在于,所述酪氨酸修饰的聚乙二醇-聚己内酯的制备方法为:
1)将Boc-PEG2000-OH、己内酯和辛酸亚锡催化剂按照2~8:16~22:1~5的比例,110~150℃下聚合18~30h,二氯甲烷溶解,乙醚沉淀,得到反应物a;
2)将反应物a溶于二氯甲烷,加入三氟乙酸,室温下搅拌1h,洗涤、干燥、乙醚沉淀,得到反应物b,所述反应物a与三氟乙酸的质量体积比为0.5~2.5:3~7,g:mL;
3)取反应物b溶于氯仿,加入Boc-L-Tyr、EDC和DMAP,室温下反应9~12h,洗涤、干燥,乙醚沉淀,再加入三氟乙酸,室温下搅拌0.2~1.8h,洗涤、干燥,乙醚再沉淀,得到酪氨酸修饰的聚乙二醇-聚己内酯,所述反应物b、Boc-L-Tyr、EDC和DMAP的摩尔比为:0.5~2:1~3:1~3:12~18。
4.根据权利要求1或2所述的制备方法,其特征在于,所述Angiopep2修饰的聚乙二醇-聚己内酯的制备方法为:
1)将Mal-PEG2000-OH、己内酯和辛酸亚锡催化剂按照2~8:16~22:1~5的比例,110~150℃下聚合18~30h,二氯甲烷溶解,乙醚沉淀,得到反应物1;
2)将反应物1溶于DMSO,加入Angiopep2多肽搅拌反应24h,透析,得到Angiopep2修饰的聚乙二醇-聚己内酯,所述反应物1和Angiopep2多肽的摩尔比为:0.5~2:0.5~2。
5.利用权利要求1~5任一项所述的制备方法所制备的Tyr/Ang双重修饰的青蒿琥酯纳米靶向制剂。
6.权利要求5所述的Tyr/Ang双重修饰的青蒿琥酯纳米靶向制剂在治疗多重耐药的脑胶质瘤中的应用。
7.一种药物制剂,其特征在于,所述药物制剂包括权利要求5所述的Tyr/Ang双重修饰的青蒿琥酯纳米靶向制剂和一种或多种抗颅脑肿瘤药物。
8.根据权利要求7所述的药物制剂,其特征在于,所述颅脑肿瘤为脑胶质瘤、脑膜瘤、颅咽管瘤、神经鞘瘤、神经节胶质细胞瘤、脑下垂体肿瘤或脉络丛肿瘤。
9.权利要求7或8所述的药物制剂在治疗多重耐药的脑胶质瘤中的应用。
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