CN111592663B - 一种冠醚修饰的嵌段共聚物胶束的制备方法及其应用 - Google Patents
一种冠醚修饰的嵌段共聚物胶束的制备方法及其应用 Download PDFInfo
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Abstract
本发明制作了一种未见报道过的胶束。该种胶束是由冠醚修饰的PEG5000‑PCL嵌段共聚物胶束。采用开环聚合合成COOH‑PEG5000‑PCL后,然后通过酯化反应将冠醚键合到COOH‑PEG5000‑PCL上,分子量大约为10000。通过溶剂挥发法值得所需胶束,最后采用MTT比色法测定该胶束对于HeLa细胞的细胞毒性。该种胶束的创新在于利用冠醚对于金属离子的络合性能,通过络合细胞内金属离子,破坏肿瘤细胞微环境,达到抑制癌细胞活性的作用。
Description
技术领域
本发明涉及到一种聚合物胶束的制备领域,具体涉及一种冠醚修饰的嵌段共聚物胶束的制备方法。
背景技术
长期以来,癌症一直威胁着人类的生命,化疗等传统治疗可能会产生严重的副作用。此外,传统化疗药物的疗效受到非特异性吸收和过早解释的限制。近年来,纳米材料的出现带来了好消息,传统治疗方法的缺点,和一些纳米材料已被批准用于临床使用和聚合物越来越用作人们来封装或结合抗肿瘤药物,包括蛋白质药物小分子化疗药物、核酸药物和小干扰RNA(siRNA)。改善药物的药代动力学和肿瘤部位的积累,从而减少药物的副作用,提高药物的疗效。如嵌段共聚物胶束、脂质体、固体脂质纳米颗粒、纳米球等。两亲性嵌段共聚物可形成多种纳米粒子,具有巨大的医疗潜力,成为抗癌药物的重要纳米载体之一,通过EPR效应传递药物。在相同的聚合物链中,具有疏水性和亲水性嵌段的两亲性共聚物可以自组装并在水介质中形成不同类型的纳米颗粒结构。据报道,两亲性嵌段共聚物形成的纳米颗粒结构可能是胶束。胶束具有核-壳结构,具有疏水核和亲水壳,可以封装各种疏水药物而不改变化学药物的结构。常见的两亲性嵌段共聚物胶束为PCEC。它们包括FDA批准的疏水性PCL和亲水聚乙二醇。PCEC胶束可在水中自组装形成,是本研究的重要辅料。
此外,离子稳态在细胞增殖和凋亡中也起着重要作用。离子载体作为离子平衡干扰剂已被广泛应用。研究表明,离子稳态的干扰可诱导细胞凋亡,用于肿瘤治疗。早在上个世纪,Pedersen就合成并发现了一类叫做冠醚的新化合物,并发现了这种化合物和阳离子的络合性质。这些杂环化合物因强非共价阳离子结合性质而受到广泛的关注。典型的冠醚是一种环状聚醚,含有3-20个氧原子,每个氧原子由两个或两个以上的碳原子隔开,对碱金属和碱土阳离子的亲和力最大。冠醚在选择性结合一系列金属离子和各种中性和离子有机物方面有着广泛的应用。而且已经有报道MarkoMarjanovic等人用研究了14种冠醚及其类似物抗肿瘤细胞增殖能力与结构活性的关系。Iva等进行了冠醚化合物潜在的抑制P-gp外排工作。在本研究中,我们试图利用胶束的特点以及冠醚对于金属离子的结合作用,二者结合来抑制癌细胞的活性。我们分别使用18-冠-6醚、15-冠-5醚和12-冠-4醚,将冠醚与PEG-PCL结合自组装形成未见报道的胶束。
发明内容
因此,本发明要解决的技术问题在于克服现有技术中冠醚作为药物潜力的缺陷,从而提供一种冠醚修饰的嵌段共聚物胶束的方法,所述的方法采用开环聚合合成COOH-PEG5000-PCL后,然后通过酯化反应将冠醚键合到COOH-PEG5000-PCL上,分子量9000-11000。通过溶剂挥发法值得所需胶束,最后采用MTT比色法测定该胶束对于HeLa细胞的细胞毒性。
为解决上述技术问题,本发明采用下述技术方案:
一种冠醚修饰的嵌段共聚物胶束的方法,包括下述步骤:
S1、将羧基-聚乙二醇5000-羟基COOH-PEG5000-OH溶解于甲苯中得到浓度为9-11mg/ml的第一反应液;
S2、将第一反应液升温到95-100℃,先后加入第一催化剂后和己内酯缓慢搅拌条件下反应20-26h,离心沉降洗涤得到羧基-聚乙二醇5000-聚己内酯COOH-PEG5000-PCL;
S3、COOH-PEG5000-PCL进行冠醚改性获得冠醚-PEG5000-PCL
将COOH-PEG5000-PCL溶解于二氯甲烷中,然后加入第二催化剂搅拌均匀,再加入羟甲基冠醚,所述羟甲基冠醚和COOH-PEG5000-PCL的摩尔比为1.9:1-2.1:1,搅拌条件下室温反应20h-28h,然后经过透析后冷冻干燥,得到0.18克的冠醚-PEG5000-PCL;
S4、将冠醚-PEG5000-PCL充分溶解再四氢呋喃中后,在搅拌条件下缓慢滴入超纯水中,最后置于通风橱中将四氢呋喃挥发干净即可得到所需胶束。
所述步骤S2中的第一催化剂为异辛酸亚锡,所述异辛酸亚锡为COOH-PEG5000-OH的10wt%-15wt%,所述的己内酯为COOH-PEG5000-OH的110wt%-120wt%。
所述步骤S3中的所述第二催化剂为二环己基碳二亚胺(DCC)和4-二甲氨基吡啶(DMAP),DCC:DMAP的摩尔比为2.8:1-3.2:1。
所述的羟甲基冠醚为2-羟甲基-12-冠-4-醚、2-羟甲基-15-冠-5-醚、2-羟甲基-18-冠-6-醚中的一种。
步骤S2中的离心沉降洗涤为:将反应产物倒入冰乙醚中,离心沉降后用乙醚反复洗涤三次,然后冷冻干燥后得到羧基-聚乙二醇5000-聚己内酯(COOH-PEG5000-PCL)。
一种所述的方法制备得到的冠醚修饰的胶束,所述胶束粒径水合粒径为110-130nm,透射电子显微镜TEM下观察平均粒径为50nm。
一种所述的冠醚修饰的胶束在制备抑制HeLa细胞活性药物方面的应用,具有抑制作用的浓度在500ug/ml。
所述冠醚修饰的胶束的临界胶束浓度(CMC)为40ug/ml。
与现有技术相比,本发明具有如下有益效果:
本发明提供了一种冠醚修饰的嵌段共聚物胶束的方法,该种胶束是由冠醚修饰的PEG5000-PCL嵌段共聚物胶束。首先在异辛酸亚锡的催化下通过开环聚合,将COOH-PEG5000-OH和己内酯,合成为分子量9000-11000的COOH-PEG5000-PCL后,然后通过酯化反应将冠醚键合到COOH-PEG5000-PCL上,分子量为9000-11000。通过溶剂挥发法制得所需胶束,最后采用MTT比色法测定该胶束对于HeLa细胞的细胞毒性。该种胶束的创新在于利用冠醚对于金属离子的络合性能,通过络合细胞内金属离子,破坏肿瘤细胞微环境,达到抑制癌细胞活性的作用。
本发明所用的COOH-PEG5000-PCL的分子量在9000-11000,亲水段(PEG)和疏水段(PCL)的比例接近1:1,可以使得材料能够较好的通过自组装行为形成胶束。经测量,所述胶束粒径水合粒径为110-130nm,透射电子显微镜TEM下观察平均粒径为50nm;临界胶束浓度(CMC)为40ug/ml左右,在较低的CMC值下,意味着可在较低浓度下胶束保持稳定而不解体,在500ug/ml左右时,该种类型的胶束对于癌细胞活性有抑制作用。
本发明所用的胶束纳米颗粒粒径在110-130nm,较小的粒径有利于EPR效应和细胞内吞,胶束和细胞内金属离子络合,有利于进入细胞实现诱导细胞凋亡的目的。
由于冠醚分子具有毒性,本发明通过将冠醚分子其制备成胶束后,可以通过EPR效应累积在肿瘤部位,并且降低生物毒性。冠醚对于金属离子具有络合作用,当胶束进入细胞后可以络合胞内金属离子,破坏细胞微环境,引起细胞凋亡。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1是实施例1-4的核磁表征图;
图2是实施例1-4中步骤S3产物的羧基和电位关系图;
图3是实施例1-4中步骤S3产物的临界胶束浓度图;
图4是实施例1-4中步骤S4产物的粒径表征图;
图5是实施例1-4中步骤S4产物的胶束稳定性图;
图6是实施例1-4中步骤S4图产物的胶束透射电镜图;
图7是实施例1-4HeLa细胞毒性测试图
图8是实施例1-4HeLa细胞内吞激光共聚焦图。
具体实施方式
下面将结合附图对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明实施例中透射电镜照片由JEOL型扫描电镜测得。
粒径信息由马尔文激光粒度仪测试得到;
激光共聚焦图片由FLS 800型激光共聚焦显微镜拍摄;
细胞毒性试验由MTT法得到,并用酶标仪测试吸光度计算细胞存活率;
小动物成像图片由小动物活体成像仪器拍摄得到。
为解决上述技术问题,本发明采用下述技术方案:
一种冠醚修饰的嵌段共聚物胶束的方法,包括下述步骤:
S1、将羧基-聚乙二醇5000-羟基COOH-PEG5000-OH溶解于甲苯中得到浓度为9-11mg/ml的第一反应液;
S2、将第一反应液升温到95-100℃,加入第一催化剂后,加入己内酯缓慢搅拌条件下反应20-26小时,离心沉降洗涤得到羧基-聚乙二醇5000-聚己内酯(COOH-PEG5000-PCL);
S3、COOH-PEG5000-PCL进行冠醚改性获得冠醚-PEG5000-PCL
将COOH-PEG5000-PCL溶解于二氯甲烷中,然后加入第二催化剂搅拌均匀,再加入羟甲基冠醚,所述羟甲基冠醚和COOH-PEG5000-PCL的摩尔比为1.9:1-2.1:1,搅拌条件下室温反应20h-28h,然后经过透析后冷冻干燥,得到0.18克的冠醚-PEG5000-PCL;
S4、将冠醚-PEG5000-PCL充分溶解再四氢呋喃中后,在搅拌条件下缓慢滴入超纯水中,最后置于通风橱中将四氢呋喃挥发干净即可得到所需胶束。
所述步骤S2中的第一催化剂为异辛酸亚锡,所述异辛酸亚锡为COOH-PEG5000-OH的10wt%-15wt%,所述的己内酯为COOH-PEG5000-OH的110wt%-120wt%。
所述步骤S3中的所述第二催化剂为二环己基碳二亚胺(DCC)和4-二甲氨基吡啶(DMAP),DCC:DMAP的摩尔比为2.8:1-3.2:1。
所述的羟甲基冠醚为2-羟甲基-12-冠-4-醚、2-羟甲基-15-冠-5-醚、2-羟甲基-18-冠-6-醚中的一种。
步骤S2中的离心沉降洗涤为:将反应产物倒入冰乙醚中,离心沉降后用乙醚反复洗涤三次,然后冷冻干燥后得到羧基-聚乙二醇5000-聚己内酯(COOH-PEG5000-PCL)。
一种所述的方法制备得到的冠醚修饰的胶束,所述胶束粒径水合粒径为110-130nm,透射电子显微镜TEM下观察平均粒径为50nm。
一种所述的冠醚修饰的胶束在制备抑制HeLa细胞活性药物方面的应用,产生抑制作用的浓度为500ug/ml。
所述冠醚修饰的胶束的临界胶束浓度(CMC)为40ug/ml。为详细说明上述方案,本发明提供如下实施例:
实施例1
本实施例的冠醚修饰的胶束的制备方法,包括下述步骤:
S1、将1.0克羧基-聚乙二醇5000-羟基(COOH-PEG5000-OH)溶解于10ml甲苯中得到浓度为10mg/ml的第一反应液;
S2、将第一反应液升温到100℃,以此加入80微升(约0.1g)的异辛酸亚锡,然后加入1.2克的己内酯,缓慢搅拌条件下反应24小时,将反应产物倒入冰乙醚中,离心沉降后用乙醚反复洗涤三次,然后冷冻干燥后得到大约1.9克COOH-PEG5000-PCL(羧基-聚乙二醇5000-聚己内酯),产率为86.36%;本实施例制备的羧基-聚乙二醇5000-聚己内酯核磁表征图见图1。
所述异辛酸亚锡为COOH-PEG5000-OH的10wt%,所述的己内酯为COOH-PEG5000的120wt%;
S3、COOH-PEG5000-PCL进行冠醚改性获得冠醚-PEG5000-PCL
将0.2克COOH-PEG5000-PCL溶解于10ml的二氯甲烷中,然后加入催化剂入6.18mg二环己基碳二亚胺(DCC)和1.2mg 4-二甲氨基吡啶(DMAP),DCC:DMAP的摩尔比为3:1,然后加入2-羟甲基-12-冠-4-醚,所述2-羟甲基-12-冠-4-醚和COOH-PEG5000-PCL的摩尔比为2:1,搅拌条件下室温反应24h,然后经过透析后冷冻干燥,得到0.1876克的冠醚-PEG5000-PCL。
S4、将30毫克冠醚-PEG5000-PCL充分溶解在6毫升四氢呋喃中后,充分溶解后,在搅拌条件下缓慢滴入30ml的超纯水中,然后置于通风橱中,待四氢呋喃完全挥发干净之后即得所需胶束。
本实施例所述胶束粒径水合粒径为110-130nm,透射电子显微镜TEM下观察平均粒径为50nm,所述冠醚修饰的胶束的临界胶束浓度(CMC)为42.35ug/ml。
实施例2(对比例)
本实施例的冠醚修饰的胶束的制备方法,其中步骤S1和S2同实施例1,步骤S3和S4为:
S3、COOH-PEG5000-PCL进行改性获得醇醚-PEG5000-PCL
将0.2克COOH-PEG5000-PCL溶解于10ml的二氯甲烷中,然后加入催化剂入6.18mg二环己基碳二亚胺(DCC)和1.2mg 4-二甲氨基吡啶(DMAP),DCC:DMAP的摩尔比为3:1,然后加入四乙二醇单甲醚,使其和COOH-PEG5000-PCL的摩尔比为2:1,搅拌条件下室温反应24h,然后经过透析后冷冻干燥,得到0.1763克的醇醚-PEG5000-PCL,作为另外的对比例,将四乙二醇单甲醚替换为五乙二醇单甲醚或六乙二醇单甲醚,其他步骤及试剂均相同
S4、将30mg醇醚-PEG5000-PCL充分溶解在6ml四氢呋喃中后,充分溶解后,在搅拌条件下缓慢滴入30ml的超纯水中,然后置于通风橱中,待四氢呋喃完全挥发干净之后即得所需胶束。
图2是实施例1和实施例2中步骤S3制备得到胶束中羧基和电位关系图,通过图2可以发现随着羧基含量的提高,电位也随之下降。表1是实施例1-4的电位图,结合图2,可以得出冠醚以及醇醚和PEG-PCL的反应率为80%以上。
表1
聚合物电位表
图3是实施例1-4中步骤S3制备得到的临界胶束浓度图,通过图3发现,我们所制得的嵌段共聚物胶束的临界胶束的临界胶束浓度在40ug/ml,这样一个较低的临界胶束浓度能够保证胶束在体内不会解开。
图4是实施例1-4中步骤S4制备得到水合粒径表征图,通过粒径表征,我们可以看到所制备胶束的粒径在120nm,这保证了其能够受益于EPR效应。
图5是实施例1-4中步骤S4制备得到胶束稳定性图,通过连续七天的粒径测试,我们发现粒径没有明显的改变,这说明所制备的胶束稳定性良好。
图6是实施例1-4中步骤S4制备得到胶束透射电镜图(Scale bar:200nm);通过投射电镜我们发现胶束粒径为50nm,进一步说明所制备胶束粒径足够小,可以保证受益于EPR效应。
实施例3
本实施例的冠醚修饰的胶束的制备方法,包括下述步骤:
S1、将0.9g COOH-PEG5000-OH(羧基-聚乙二醇5000-羟基)溶解于10ml甲苯中得到浓度为9mg/ml的第一反应液;
S2、将第一反应液升温到105℃,以此加入催化剂0.117g异辛酸亚锡和0.99g己内酯,缓慢搅拌条件下反应20小时,将反应产物加入50ml乙醚,离心沉降后继续用乙醚反复洗涤三次,然后冷冻干燥后得到羧基-聚乙二醇5000-聚己内酯(COOH-PEG5000-PCL);所述异辛酸亚锡为COOH-PEG5000-OH的13wt%,所用的己内酯为COOH-PEG5000-OH的110wt%;
S3、COOH-PEG5000-PCL进行冠醚改性获得冠醚-PEG5000-PCL
将0.2g COOH-PEG5000-PCL溶解于二氯甲烷中,然后加入催化剂,5.76mg二环己基碳二亚胺(DCC)和1.2mg 4-二甲氨基吡啶(DMAP),DCC:DMAP的摩尔比为2.8:1,然后加入羟甲基冠醚,所述羟甲基冠醚和COOH-PEG5000-PCL的摩尔比为1.9:1,搅拌条件下室温反应28h,然后经过透析后冷冻干燥,得到0.1895g的冠醚-PEG5000-PCL;所述羟甲基冠醚为2-羟甲基-18-冠-6-醚;
S4、将0.05g冠醚-PEG5000-PCL充分溶解再四氢呋喃中后,在搅拌条件下缓慢滴入50ml超纯水中,最后置于通风橱中将四氢呋喃挥发干净即可得到所需胶束。
本实施例所述胶束粒径水合粒径为110-130nm,透射电子显微镜TEM下观察平均粒径为50nm,所述冠醚修饰的胶束的临界胶束浓度(CMC)为39.71ug/ml。
实施例4
本实施例的冠醚修饰的胶束的制备方法,包括下述步骤:
S1、将1.1g COOH-PEG5000-OH(羧基-聚乙二醇5000-羟基)溶解于10ml甲苯中得到浓度为11mg/ml的第一反应液;
S2、将第一反应液升温到95℃,以此加入催化剂0.165g异辛酸亚锡和1.265g己内酯,缓慢搅拌条件下反应26小时,将反应产物中倒入50ml乙醚,离心沉降后继续用乙醚反复洗涤三次,然后冷冻干燥后得到COOH-PEG5000-PCL(羧基-聚乙二醇5000-聚己内酯);所述异辛酸亚锡为COOH-PEG5000-OH的15wt%,所述的己内酯为COOH-PEG5000-OH的115wt%;
S3、COOH-PEG5000-PCL进行冠醚改性获得冠醚-PEG5000-PCL
将0.2COOH-PEG5000-PCL溶解于二氯甲烷中,然后加入催化剂6.59mg二环己基碳二亚胺(DCC)和1.2mg 4-二甲氨基吡啶(DMAP),DCC:DMAP的摩尔比为3.2:1,然后加入羟甲基冠醚,所述羟甲基冠醚和COOH-PEG5000-PCL的摩尔比为2.1:1,搅拌条件下室温反应20h,然后经过透析后冷冻干燥,得到0.1798g的冠醚-PEG5000-PCL;所述羟甲基冠醚为2-羟甲基-15-冠-5-醚;
S4、将0.05g冠醚-PEG5000-PCL充分溶解再四氢呋喃中后,在搅拌条件下缓慢滴入50ml超纯水中,最后置于通风橱中将四氢呋喃挥发干净即可得到所需胶束。
本实施例所述胶束粒径水合粒径为110-130nm,透射电子显微镜TEM下观察平均粒径为50nm,所述冠醚修饰的胶束的临界胶束浓度(CMC)为41.58ug/ml。
所述冠醚修饰的胶束的临界胶束浓度的测定方法:
S5、称取一定量的尼罗红染料,溶解于20ml的二氯甲烷中,使得最终浓度为1umol/ml。然后依次取2ml溶液置于10个样品瓶中,通风橱中避光条件下使二氯甲烷完全挥发。
S6、取所得胶束(1mg/ml)5ml,梯度稀释,使得浓度依次为1000ug/ml、500ug/ml、250ug/ml、125ug/ml、62.5ug/ml、31.25ug/ml、15.63ug/ml、7.81ug/ml、3.91ug/ml、1.95ug/ml,分别加入到S1中10个样品瓶中,通风橱中避光、搅拌过夜即可(8-12h)。
S7、用S6所得溶液进行应该测试,所用激发波长为543nm,发射波长为620nm,记录所得荧光强度。
S8、以荧光强度为纵坐标,相应的胶束浓度为横坐标绘制曲线,两条曲线的切线的交叉点即为临界胶束浓度。
实施例5
采用3-(4,5-二甲基噻唑-2-基)-5-(3-羧基甲氧基苯基)-2-(4-磺苯基)-2H-四唑仑(MTT)法测定了实施1-4制备得到的冠醚-PEG5000-PCL聚合物胶束对HeLa的细胞毒性。HeLa细胞接种到96孔板上,密度为每孔1-2万个细胞,培养24小时。分别加入不同浓度(31.25、62.5、125、250、500ug/mL)的聚合物胶束溶液。然后在37℃的培养箱中培养24h(5%的CO2大气)。MTT孵育4h,DMSO溶解,酶标测定吸光度,计算细胞存活率。
图7是实施例1-4HeLa细胞毒性测试图通过细胞毒性测试我们发现,所制备胶束从31.25ug/ml提高到500ug/ml时,对于HeLa细胞活性的抑制明显增强。
图8是实施例1-4HeLa细胞内吞激光共聚焦图通过激光共聚焦我们发现,细胞在2h内即可进入细胞,这说明了胶束的进胞速率较为可观。
显然,上述实施例仅仅是为清楚地说明所作的举例,而并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (6)
1.一种冠醚修饰的嵌段共聚物胶束的方法,其特征在于,包括下述步骤:
S1、将羧基-聚乙二醇5000-羟基COOH-PEG5000-OH溶解于甲苯中得到浓度为9-11mg/ml的第一反应液;
S2、将第一反应液升温到95-105℃,先后第一催化剂和己内酯缓慢搅拌条件下反应20-26小时,离心沉降洗涤得到羧基-聚乙二醇5000-聚己内酯COOH-PEG5000-PCL,所述COOH-PEG5000-PCL的分子量为9000-11000;
S3、COOH-PEG5000-PCL进行冠醚改性获得冠醚-PEG5000-PCL:
将COOH-PEG5000-PCL溶解于二氯甲烷中,然后加入第二催化剂搅拌均匀,再加入羟甲基冠醚,所述羟甲基冠醚和COOH-PEG5000-PCL的摩尔比为1.9:1-2.1:1,搅拌条件下室温反应20h-28h,然后经过透析后冷冻干燥,得到0.18克的冠醚-PEG5000-PCL,所述的羟甲基冠醚为2-羟甲基-12-冠-4-醚、2-羟甲基-15-冠-5-醚、2-羟甲基-18-冠-6-醚中的一种;
S4、将冠醚-PEG5000-PCL充分溶解在四氢呋喃中后,在搅拌条件下缓慢滴入超纯水中,最后置于通风橱中将四氢呋喃挥发干净即可得到所需胶束。
2.根据权利要求1所述的冠醚修饰的嵌段共聚物胶束的方法,其特征在于,
所述步骤S2中的第一催化剂为异辛酸亚锡,所述异辛酸亚锡为COOH-PEG5000-OH的10wt%-15wt%,所述己内酯为COOH-PEG5000-OH的110wt%-120wt%。
3.根据权利要求2所述的冠醚修饰的嵌段共聚物胶束的方法,其特征在于,
所述步骤S3中的所述第二催化剂为二环己基碳二亚胺DCC和4-二甲氨基吡啶DMAP,DCC:DMAP的摩尔比为2.8:1-3.2:1。
4.根据权利要求1所述的冠醚修饰的嵌段共聚物胶束的方法,其特征在于,
步骤S2中的离心沉降洗涤为:将反应产物倒入冰乙醚中,离心沉降后用乙醚反复洗涤三次,然后冷冻干燥后得到羧基-聚乙二醇5000-聚己内酯COOH-PEG5000-PCL。
5.一种权利要求1-4任一项所述的方法制备得到的冠醚修饰的胶束,所述胶束粒径水合粒径为110-130nm,透射电子显微镜TEM下观察平均粒径为50nm。
6.一种权利要求5所述的冠醚修饰的胶束在制备抑制HeLa细胞活性药物方面的应用,其特征在于,所述冠醚修饰的胶束的产生抑制活性的浓度在500ug/ml。
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