CN117298288A - 多糖抗肿瘤小分子药物共聚物及其转铁蛋白修饰自组装纳米粒和制法与应用 - Google Patents
多糖抗肿瘤小分子药物共聚物及其转铁蛋白修饰自组装纳米粒和制法与应用 Download PDFInfo
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Abstract
一种多糖抗肿瘤小分子药物共聚物及其转铁蛋白修饰自组装纳米粒和制法与应用,属于药物领域。将多糖通过氨基化修饰后通过二硫键连接子与抗肿瘤小分子药物连接,得到氧化还原敏感的多糖抗肿瘤小分子药物共聚物,利用纳米沉淀法制备成前药纳米粒后与转铁蛋白共孵育获得靶向功能化修饰氧化还原敏感性的转铁蛋白修饰自组装纳米粒。其制备方法和成分简单,反应条件温和,适合抗肿瘤小分子药物种类多,粒径可控,载药量高,可广泛用于肺癌,肝癌等多种实体瘤治疗。转铁蛋白修饰自组装纳米粒在系统给药中利用靶向功能主动识别到肿瘤组织,并在肿瘤细胞中以氧化还原触发的形式快速释放化疗药物,降低了化疗药物的全身毒性并提高了化疗药物的治疗效果。
Description
技术领域
本发明涉及药物技术领域,涉及一种多糖抗肿瘤小分子药物共聚物及其转铁蛋白修饰自组装纳米粒和制法与应用,更具体的涉及普鲁兰紫杉醇共聚物,还有多糖抗肿瘤小分子药物共聚物自组装形成前药纳米粒,再通过转铁蛋白修饰得到转铁蛋白修饰自组装纳米粒,及其制备方法和在用于制备抗肺部实体瘤药剂中的用途。该普鲁兰紫杉醇共聚物作为一种前药,具有抗肿瘤作用,具有氧化还原敏感性,其形成的转铁蛋白修饰自组装纳米粒能够主动靶向到肿瘤组织,显著抑制肿瘤的生长。
背景技术
癌症是一种严重危害人体健康的常见多发病,而肿瘤化疗一直是国内外研究的热点问题。化疗是一种系统性治疗肿瘤的方法,抗肿瘤小分子药物大多为细胞毒性药物,游离药物无选择性全身分布且存在溶解性差、治疗窗窄等问题,其中,作为抗肿瘤小分子药物的代表之一-紫杉醇(PTX)是肺癌治疗的基石化疗药物,常与其他抗癌药物联合使用。PTX通过阻止细胞分裂过程中的解聚来稳定微管。然而,PTX的固有溶解度较差,口服生物利用度有限。是一种商业静脉注射PTX制剂,由脱水乙醇和(聚氧乙烯蓖麻油EL)CremophorEL的混合物构成,通常用于治疗肺癌。遗憾的是,由于它们对癌细胞的靶向效率相当低,它表现出致命的剂量依赖性副作用,并且紫杉醇极低的水溶性往往要求其与表面活性剂(如聚氧乙烯蓖麻油)、乙醇等混合使用,极大的增强了其对人体的毒副作用。因此,迫切需要开发新的药物递送系统来规避这些阻碍PTX递送的问题。
多糖聚合物是天然高分子材料,具有生物相容性好、生物可降解性能,且具有无免疫原性、易改性的特点,有些材料甚至具有靶向某些组织的能力,现已成为药物递送系统研究的热点材料。普鲁兰(PULL)是一种包含麦芽三糖残基的胞外多糖,由于其出色的生物相容性和可修饰性而被广泛用于制造药物。现对多糖聚合物进行了疏水性改性,制成两亲性多糖聚合物进而自组装形成纳米粒已经成为多糖聚合物药物递送系统的研究焦点。但是这类纳米载体通常是将药物装载于纳米载体的疏水内核中,无法将药物直接搭载在多糖聚合物分子上。将药物装载于纳米载体的疏水内核中存在药物装载过程复杂、周期长、释放速度难以控制或释放行为无环境响应性等问题。因此,需要寻求一种新的装载工艺。
纳米药物靶向递送系统可有效提高药物在肿瘤处积累效果和降低系统毒性的副作用,在肿瘤治疗中受到越来越多的关注。纳米药物靶向递送体系可充分利用肿瘤组织的结构特性,依靠实体瘤组织对纳米粒特有的高通透性和EPR滞留效应产生被动靶向,依靠特定的肿瘤细胞靶向配体实现肿瘤组织的主动靶向能力,将纳米药物富集在肿瘤组织中,发挥抗肿瘤作用。因此,纳米药物靶向递送体系也是研究的热点问题。转铁蛋白(TF)是一种内源性血糖蛋白,长期以来一直被是细胞上转铁蛋白受体(TFR),且部分癌细胞表面此类受体显示高水平表达。因此,通过战略性地开发TF修饰的PULL/PTX前药纳米颗粒可以达到靶向递送抗癌药物的作用。
发明内容
本发明的目的是提供一种多糖抗肿瘤小分子药物共聚物及其转铁蛋白修饰自组装纳米粒和制法与应用,该多糖抗肿瘤小分子药物共聚物作为一种前药,其能够抗肿瘤细胞生长,且具有氧化还原敏感性和低毒性,其通过纳米沉淀法自组装得到的前药纳米粒,再经过转铁蛋白(TF)修饰,得到转铁蛋白修饰自组装纳米粒。在一些肿瘤细胞高水平表达的转铁蛋白受体(TFR)基础上,转铁蛋白(TF)修饰自组装纳米粒,是以天然PULL通过二硫键搭载紫杉醇并通过TF修饰的纳米粒,其是一种靶向性氧化还原敏感性前药纳米粒。在研究中,建立小鼠肺部转移瘤,通过静脉给药,显示出明显的肿瘤内积聚,显著抑制了肺部肿瘤的生长。本发明克服了现有紫杉醇类抗肿瘤药物装载周期长、缺乏刺激响应性、具有剂量依赖的系统毒性等问题。在体外,通过TF与TFR的相互作用,显示出更多的细胞摄取能力。在体内,转铁蛋白修饰自组装纳米粒显示出明显的肿瘤组织积聚,在肿瘤细胞高水平的谷胱甘肽(GSH)及活性氧(ROS)下,释放出游离的抗肿瘤小分子药物,显示出显著抑制肿瘤生长的能力,同时与阳性对照市售紫杉醇制剂相比,具有更高的安全性。本发明证实了氧化还原敏感性多糖抗肿瘤小分子药物共聚物及其转铁蛋白修饰自组装纳米粒抑制转移性肺癌的可信度。
具体地,本发明的第一个目的是提供了一种多糖抗肿瘤小分子药物共聚物,其能够克服传统药物载药量低,毒副作用大,肿瘤组织递送效率低的缺点,并且具有氧化还原敏感性,并且通过多糖抗肿瘤小分子药物共聚物形成的转铁蛋白修饰自组装纳米粒具有肿瘤组织靶向性。
本发明的第二个目的,提供一种多糖抗肿瘤小分子药物共聚物,及其形成的转铁蛋白修饰自组装纳米粒的制备方法。
本发明的第三个目的,提供一种多糖抗肿瘤小分子药物共聚物,及其形成的转铁蛋白修饰自组装纳米粒用于制备抗肿瘤药剂的应用。
为实现上述目的,本发明采取的技术方案是:
本发明的一种多糖抗肿瘤小分子药物共聚物,是多糖通过连接子共轭连接抗肿瘤小分子药物形成的多糖抗肿瘤小分子药物共聚物;
其中,连接子为具有二硫键的连接子,其原料优选为2,2'-二硫代二乙酸、3,3'-二硫代二丙酸或4,4'-二硫代二丁酸中的一种,更优选为3,3'-二硫代二丙酸酐(DTPA-A)。
所述的多糖优选为能够进行氨基化修饰的天然多糖,更优选为普鲁兰(PULL),PULL的平均分子量范围为5000~200000Da,更优选为200000Da。
所述的抗肿瘤小分子药物为具有活性羟基的抗肿瘤化疗药物或具有活性氨基的抗肿瘤化疗药物,更优选为紫杉烷类、蒽环类化合物、喜树碱类化合物中的一种;其中,紫杉烷类选自多西他赛、卡巴他赛、紫杉醇(PTX);蒽环类化合物选自阿霉素,更优选为紫杉醇(PTX)。
本发明的一种转铁蛋白修饰自组装纳米粒,是转铁蛋白(TF)通过酰胺键连接多糖抗肿瘤小分子药物共聚物形成。
进一步的,每10mg转铁蛋白修饰自组装纳米粒上有3.63~3.87mg抗肿瘤小分子药物,0.027~0.033mg转铁蛋白,余量为多糖。
进一步的,转铁蛋白修饰自组装纳米粒的粒径为120~160nm,载药量高达36%~40%。
作为优选,转铁蛋白优选为铁离子饱和的牛转铁蛋白。
本发明的一种多糖抗肿瘤小分子药物共聚物的制备方法,是将多糖和抗肿瘤小分子药物通过二硫键连接合成,更优选为将PULL和PTX通过二硫键连接合成多糖抗肿瘤小分子药物共聚物(PULL/PTX共聚物)。
本发明的一种转铁蛋白修饰自组装纳米粒的制备方法,是采用上述多糖抗肿瘤小分子药物共聚物,经纳米沉淀法自组装形成前药纳米粒,加入转铁蛋白与前药纳米粒共孵育,最终TF通过酰胺反应连接在前药纳米粒外部,制备完成得到转铁蛋白修饰自组装纳米粒。
更优选为,多糖抗肿瘤小分子药物共聚物为PULL-SS-PTX共聚物,转铁蛋白修饰自组装纳米粒为TF-PULL-SS-PTX NPs。
所述的多糖抗肿瘤小分子药物共聚物的制备方法,包括以下步骤:
S1:乙酸化多糖聚合物(PULL-CM)
将活化的多糖、氢氧化钠溶液和氯乙酸溶液混合,搅拌均匀,在60~80℃反应5~8h,固液分离,洗涤,干燥,得到乙酸化多糖聚合物(PULL-CM);按摩尔比,活化的多糖中单糖单元:氢氧化钠:氯乙酸=(1~2):(1~3):(2~6);
S2:氨基修饰
以水作为溶剂,将乙酸化多糖聚合物(PULL-CM)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC)、N-羟基琥珀酰亚胺(NHS)、三乙胺混合搅拌,活化10~12h,再加入乙二胺,反应24h以上,加入甲醇作为沉淀析出剂,沉淀析出后,固液分离,洗涤固体,干燥,得到化合物PULL-ED;
按摩尔比,乙酸化多糖聚合物中单糖单元:EDC:NHS:三乙胺:乙二胺=(4~5):(2~4):(1.5~3.5):(2~3):(15~25)。
S3:连接二硫键
将抗肿瘤小分子药物和具有二硫键的连接子的原料混合,以EDC为催化剂,以二氯甲烷(DCM)为溶剂,室温活化1~3h,加入4-二甲氨基吡啶(DMAP)后,反应40~50h,得到反应物;其中,按摩尔比,抗肿瘤小分子药物:具有二硫键的连接子的原料:EDC:4-二甲氨基吡啶(DMAP)=(1~1.5):(1~2.5):(4~6):(1~1.5);
将反应物去除溶剂,再以稀盐酸作为沉淀析出剂,沉淀产物,固液分离,水洗至中性,干燥,得到化合物PTX-DTPA;
S4:共聚
将化合物PTX-DTPA、NHS、EDC、三乙胺混合,室温活化后,得到PTX-DTPA溶液;
向PTX-DTPA溶液中滴加化合物PULL-ED溶液,再加入缓冲液调整pH值为7.8~8.0,然后室温反应20~25h,得到反应液;
将反应液进行透析,得到白色絮状沉淀物,干燥,得到PULL-SS-PTX共聚物。
所述的S1中,活化的多糖为将多糖置于异丙醇中,浸泡10~24h。
所述的S1中,混合的温度为25~45℃,搅拌的速度为800~1000rpm/min,其中,作为优选,氢氧化钠溶液和氯乙酸溶液,滴加入活化的多糖中,滴加的速率为1~1.5mL/min。
所述的S1中,氢氧化钠溶液的质量浓度为10~15%;氯乙酸溶液的质量浓度为50~60%。
所述的S1中,洗涤采用甲醇和水的混合液,按体积比,甲醇:水=(8~8.5):(2~1.5)。
所述的S1中,干燥优选为冷冻干燥。
所述的S2中,活化过程中,伴随搅拌,搅拌速率优选为800rpm~1000rpm/min。
所述的S2中,洗涤采用甲醇和水的混合液,按体积比,甲醇:水=(8~8.5):(2~1.5)。
所述的S3中,具有二硫键的连接子的原料优选为2,2'-二硫代二乙酸、3,3'-二硫代二丙酸或4,4'-二硫代二丁酸,更优选为3,3'-二硫代二丙酸酐(DTPA-A)。
所述的S3中,所述的3,3'-二硫代二丙酸酐(DTPA-A),采用以下制备方法制得:
将3,3'-二硫代二丙酸(DTPA)和乙酰氯混合,在60~70℃下冷凝回流3.5~5h,减压去除乙酰氯,用冷乙醚作为沉淀析出剂,得到化合物3,3'-二硫代二丙酸酐(DTPA-A);
按固液比,3,3'-二硫代二丙酸在乙酰氯中浓度为=(0.04~0.24)g/mL。
所述的S3中,稀盐酸优选为0.1~0.2mol/L的盐酸水溶液。
所述的S4中,室温活化的时间为20~30h。
所述的S4中,滴加化合物PULL-ED滴加速度为1~2mL/min。
所述的S4中,缓冲液优选为碳酸盐溶液,更优选为碳酸钠溶液或碳酸氢钠溶液。
所述的S4中,按摩尔比,PTX-DTPA:NHS:EDC:三乙胺:PULL-ED的单糖单元=(1~1.5):(1~3):(2~4):(1~3):(4~8)。
所述的S4中,透析采用的MW=8000~14000的透析袋,以水为介质,每10~12h换液,总透析时间为48~72h。
本发明的多糖抗肿瘤小分子药物共聚物的合成路线如下:
本发明的转铁蛋白修饰自组装纳米粒的制备方法,为纳米沉淀法,具体包括以下步骤:
步骤(1):将PULL-SS-PTX共聚物溶于DMSO中,得到共聚物溶液;
在搅拌速度为400~1000rpm/min的持续搅拌下,将共聚物溶液以1~1.5mL/min的速度加入水中,滴加完成后,施加间断超声,反应,得到粒径均匀的前药纳米粒(PULL-SS-PTX NPs);
其中,按固液比,PULL-SS-PTX共聚物:DMSO:水=(9.5~10.5)mg:(0.2~0.6)mL:(1.8~2.2)mL;
步骤(2):将前药纳米粒、EDC溶液、NHS溶液和三乙胺混合,室温活化后,再加入转铁蛋白溶液,室温孵育12~24h,然后透析,除去游离转铁蛋白,得到转铁蛋白修饰自组装纳米粒(TF-PULL-SS-PTX NPs);
其中,按质量比,前药纳米粒:EDC:NHS:三乙胺:转铁蛋白=(5~10):(0.8~1.2):(0.5~0.7):(0.4~0.6):(0.5~1)。
所述的步骤(1)中,所述的超声,其功率为50~200W,超声持续2~3s,间断时间1~2s;反应时间为2~3min。
所述的步骤(1)中,前药纳米粒的粒径为100~160nm,粒径分布为0.100~0.300。
所述的步骤(2)中,EDC溶液的质量体积浓度为10~16mg/mL,NHS溶液的质量体积浓度为5.5-9.2mg/mL,转铁蛋白溶液的质量体积浓度为2~4mg/mL。
所述的步骤(2)中,室温活化时间优选为20~40min。
所述的步骤(2)中,透析采用的MW=100000Da的透析袋,以水为介质,每10~12h换液,总透析时间为48~72h。
本发明的第三个目的是:一种多糖抗肿瘤小分子药物共聚物,及其形成的转铁蛋白修饰自组装纳米粒用于制备抗肿瘤药剂的应用,更优选为用于制备靶向抗肿瘤药物中的应用。
所述的转铁蛋白修饰自组装纳米粒具有肿瘤组织靶向性,氧化还原敏感性性和抑制肿瘤生长的应用。
所述的肿瘤尤其指肺部肿瘤或肝部肿瘤。
所述靶点为转铁蛋白受体。
所述的氧化还原敏感键为二硫键。
本发明的多糖抗肿瘤小分子药物共聚物及其转铁蛋白修饰自组装纳米粒和制法与应用,其有益效果在于:
本发明将抗肿瘤小分子药物直接通过肿瘤细胞敏感性化学键共轭于多糖聚合物分子上,将改善药物的装载工艺,改善药物的稳定性并赋予药物刺激响应性释放的特性。
本发明制备的PULL-SS-PTX共聚物,可以自组装形成均匀的纳米体系形成自组装的前药纳米粒,该纳米体系经实验证明具有优势在于:(1)粒径小且均一(100~160nm);(2)采用纳米沉淀法,制备工艺简单,易于产业化;(3)高载药量(36%~40%),利于药物的装载,降低毒副作用;(4)易于表面修饰,经转铁蛋白修饰后具有肿瘤靶向性,提高肿瘤部位积聚能力,提高药效;(5)通过氧化还原敏感连接子实现肿瘤部位细胞内环境的药物释放,提高药效的同时降低了毒副作用。
通过研究结果表明,本发明所制备的PULL-SS-PTX纳米粒在抑制肺部肿瘤生长上具有明显的效果,经转铁蛋白修饰后的TF-PULL-SS-PTX纳米粒在具有转铁蛋白受体高水平表达的肿瘤组织上具有明显提高的聚积能力,进一步提高了肿瘤的治疗。可以用于制备抗肿瘤药物,尤其是用于制备靶向于转铁蛋白受体的抗肿瘤药物。
附图说明
图1为本发明实施例制备的多糖抗肿瘤小分子药物共聚物的核磁共振氢谱和红外表征。
A为PULL的核磁共振氢谱图,溶剂为氧化氘;
B为PULL-CM的核磁共振氢谱图,溶剂为氧化氘;
C为PULL-ED的核磁共振氢谱图,溶剂为氧化氘;
D为DTPA的核磁共振氢谱图,溶剂为氘代DMSO;
E为DTDP-A的核磁共振氢谱图,溶剂为氘代DMSO;
F为PTX的核磁共振氢谱图,溶剂为氘代DMSO;
G为PTX-SA的核磁共振氢谱图,溶剂为氘代DMSO;
H为PTX-DTPA-A的核磁共振氢谱图,溶剂为氘代DMSO;
I为PULL-CC-PTX的核磁共振氢谱图,溶剂为氘代DMSO;
J为PULL-SS-PTX的核磁共振氢谱图,溶剂为氘代DMSO;
K为TF-PULL-SS-PTX NPs的核磁共振氢谱图,溶剂为氘代DMSO;
图2为共聚物的傅立叶变换红外光谱(FT-IR)的图。
图3为PTX自组装前药纳米制剂的照片。
图4为PTX自组装前药纳米制剂的稳定性。
A为4℃下储存一周纳米制剂粒径大小变化情况;
B为4℃下储存一周纳米制剂粒径PDI变化情况;
C为10%血清存在下37℃震荡孵育72小时内纳米制剂粒径大小变化情况;
D为10%血清存在下37℃震荡孵育72小时内纳米制剂粒径PDI变化情况。
图5为PTX自组装前药纳米制剂的热力学性质。
A为纳米制剂TGA结果;
B为纳米制剂DSC结果;
C为纳米制剂P-XRD结果。
图6为PTX自组装前药纳米制剂的氧化还原敏感性及其释药行为考察。
A为DTT介导的药物释放示意图;
B为10mM DTT存在下的三种PTX自组装前药纳米粒的UV-Vis光谱图;
C为10mM DTT存在下的三种PTX自组装前药纳米粒的透射电镜图;
D为10mM DTT存在下的三种PTX自组装前药纳米粒的药物释放速率图。
图7为PTX自组装前药纳米制剂的体外B16-F10细胞随时间摄取能力共聚焦成像考察。
图8为PTX自组装前药纳米制剂的体外B16-F10细胞随时间摄取能力流式细胞术考察。
图9为PTX自组装前药纳米制剂的细胞毒性。
图10为细胞转铁蛋白受体蛋白表达情况考察。
A为细胞中转铁蛋白受体蛋白Western Blot考察结果;
B为A图结果量化结果;
C为细胞膜表面转铁蛋白受体蛋白流式细胞术考察结果量化图。
图11为LLC细胞靶向能力考察。
A为PULL-SS-PTX NPs,TF-PULL-SS-PTX NPs及外加TF的TF-PULL-SS-PTX NPs在LLC细胞的细胞摄取共聚焦成像图;
B为PULL-SS-PTX NPs,TF-PULL-SS-PTX NPs及外加TF的TF-PULL-SS-PTX NPs在LLC细胞上细胞摄取的流式细胞术结果图及量化结果图;;
图12为PULL-SS-PTX NPs、TF-PULL-SS-PTX NPs在LLC细胞小鼠肺部转移瘤模型上的药物分布。
A为药物在小鼠心脏、肝脏、脾脏、肾脏、肺部肿瘤上的荧光成像图;
B为A图中荧光值量化图。
图13为PULL/PTX自组装前药纳米制剂对B16-F10细胞小鼠肺部转移肿瘤模型的抑制能力考察。
A为动物实验流程图;
B为药效图;
C为肺部重量图;
D为肺部组织H&E染色结果图。
图14为PULL-SS-PTX NPs、TF-PULL-SS-PTX NPs在LLC细胞小鼠肺部转移瘤模型上的肿瘤抑制能力考察。
A为动物实验流程设计图;
B为小鼠活体成像药效结果图;
C为小鼠离体肺组织成像图及量化结果图;
D为肺部解剖照片,肺组织H&E染色及肺组织ki-67染色结果图。
图15为PULL/PTX自组装前药纳米制剂的系统安全性考察。
A为LLC-luc肺癌模型小鼠给药期间体重变强情况;
B为三种PTX自组装前药纳米粒溶血性;
C为LLC肺癌模型小鼠血液UREA检测结果;
D为LLC肺癌模型小鼠血液CREA检测结果;
E为LLC肺癌模型小鼠血液ALT检测结果;
F为LLC肺癌模型小鼠血液AST检测结果;
G为LLC肺癌模型小鼠心、肝脏、脾脏、肾脏组织H&E染色结果。
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将发明限制在所述的实施例范围之内。
以下实施例中,具有二硫键的连接子的原料采用3,3'-二硫代二丙酸酐(DTPA-A),其制备方法为:称取1.0g 3,3'-二硫代二丙酸(DTPA),向圆底烧瓶中加入5mL乙酰氯并溶解,65℃下冷凝回流4h。减压旋蒸除去有机溶剂,50mL冷乙醚沉淀产物,过滤,真空干燥后得到白色固体粉末,即为产物3,3'-二硫代二丙酸酐(DTPA-A)。
实施1:
一种多糖抗肿瘤小分子药物共聚物的制备方法,包括如下步骤:
S1:称取2.0g PULL于干燥的圆底烧瓶中,加入12mL的异丙醇,放置12小时,得到活化的PULL。在40℃下,以1.5mL/min速率滴加NaOH溶液(6mL,10%w/v(g/mL)),氯乙酸溶液(6mL,55%w/v(g/mL)),升温至70℃,800~1000rpm/min搅拌下继续反应6h。反应结束后,过滤后,以10倍反应液体积甲醇/水(甲醇占比80%,v/v)洗涤固体,纯化3次。冷冻干燥,得到产物PULL-CM。所述普鲁兰多糖分子量为200000Da。
S2:称取700mg PULL-CM溶解于30mL H2O中,按下列顺序加入460mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC),280mg N-羟基琥珀酰亚胺(NHS),222mg三乙胺,800rpm~1000rpm/min搅拌下活化10~12小时。然后加入1.08g乙二胺,25℃下反应24h。以过10倍反应液体积的甲醇沉淀产物,过滤,用甲醇/水(甲醇体积占比80%,v/v)洗涤2到3次,冷冻干燥后即得产物PULL-ED。
S3:称取0.311g PTX,0.115g 3,3'二硫代二丙酸酐(DTPA-A)或溶解于10mL二氯甲烷(DCM)中,然后加入0.211g 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC),于800~1000rpm/min搅拌下25℃活化2h后,向反应液中加入45.0mg的4-二甲氨基吡啶(DMAP),室温下反应48h。反应结束后,减压旋蒸除去DCM,以0.1mol/L盐酸沉淀得到产物,过滤,以水洗至中性,置于真空干燥箱干燥,得到白色粉末产物紫杉醇-3,3'-二硫代二丙酸连接体(PTX-DTPA)。
S4:精密称取150mg PTX-DTPA,33.8mg NHS和56.6mg EDC溶于3mL DMSO中,随后滴加32.5μL三乙胺,25℃下活化24h后,得到PTX-DTPA溶液。将137mg PULL-ED溶于2mL DMSO中,1mL/min滴加到上述的PTX-DTPA溶液中,并以5mmol/L碳酸钠/碳酸氢钠缓冲液调节反应液pH至8.0,室温下反应24h后,将反应液转移至透析袋(MW=8000~14000),以H2O为透析介质透析72h,每12h换液一次,冷冻干燥后得到PULL-SS-PTX共聚物。
对比例
一种的PULL-CC-PTX共聚物制备方法,同实例1的步骤S1-S2,不同之处在于:
S3:称取0.311g PTX,0.06g丁二酸酐(SA)溶解于10mL二氯甲烷中,然后加入0.211g1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC),于800~1000rpm/min搅拌下25℃活化2h后,向反应液中加入45.0mg 4-二甲氨基吡啶(DMAP),室温下反应48h。反应结束后,减压旋蒸除去DCM,以0.1mol/L盐酸沉淀得到产物,过滤,以水洗至中性,置于真空干燥箱干燥,得到白色粉末产物紫杉醇-丁二酸(PTX-SA)。
S4:精密称取136.78mg PTX-SA,33.8mg NHS和56.6mg EDC溶于3mL DMSO中,随后滴加32.5μL三乙胺,25℃下活化24h,得到PTX-SA溶液。将137mg PULL-ED溶于2mL DMSO中,1mL/min滴加到上述的PTX-SA溶液中,并以5mmol/L碳酸钠/碳酸氢钠缓冲液调节反应液pH至8.0,室温下反应24h后,将反应液转移至透析袋(MW=8000~14000),以H2O为透析介质透析72h,每12h换液一次,冷冻干燥后得到PULL-CC-PTX共聚物。
对上述实例1制备的PULL-SS-PTX共聚物和对比例1制备的PULL-CC-PTX共聚物进行表征:
(1)对原始PULL、PULL-CM、PULL-ED、DTPA、DTPA–A、PTX、PTX-SA、PTX-DTPA-A及其PULL-SS-PTX共聚物和PULL-CC-PTX共聚物的1H NMR(600MHz)光谱进行了比较,如图1A所示,天然PULL显示异头质子和羟基质子在δ5.5-4.4ppm和糖环质子在δ3.8-3.0ppm之间共振,在图1B的PULL-CM光谱中,亚甲基质子(-CH2-)的信号出现在δ4.2ppm,说明PULL-CM的成功合成;图1C的PULL-ED光谱中,亚甲基质子(-CH2-)的信号出现在δ3.2ppm,说明PULL-ED的成功合成。
DTPA在图1D中δ12.4ppm处的羧酸(-COOH)信号在图1E的DTPA-A的1H NMR光谱中消失了,表明DTPA-A合成成功。
在图1G的PTX-SA和图1H的PTX-DTPA的1H NMR光谱中,纯PTX的2'-CH的特征信号从δ4.6ppm偏移到δ5.6ppm。此外,亚甲基(-CH2-)质子峰(δ3.0-2.6ppm)羧基质子峰(δ12.4ppm)的出现验证了PTX-SA和PTX-DTPA的结构。
图1F中PTX的芳香质子(δ8.0–7.0ppm)和PULL的糖环质子(δ5.0–3.5ppm)的1H NMR信号在1I和1J中的存在支持了PULL-CC-PTX和PULL-SS-PTX的成功合成。
图2所示,为天然PULL及其结合物的FT-IR光谱。在未修饰的PULL的IR光谱中,在3420cm-1、2923cm-1处可以看到各种不同的吸收信号,例如OH拉伸、CH2拉伸和糖苷(α-(1,4)和α-(1,6))拉伸和1104cm-1。PULL-CM在1640cm-1处有一个新峰,这归因于羧酸根(COO-)基团的对称伸缩振动。在PULL-ED的FT-IR光谱中,-C=O的特征峰蓝移到1631cm-1,C-N伸缩信号出现在1385cm-1。原始PTX的FT-IR光谱呈现出其独特的OH、CH、C=O酯、C=O酰胺、C-O-C吸收带,在3440cm-1、2943cm-1、1732cm-1、分别为1646cm-1和1244cm-1。大多数这些PTX峰在PTX-SA和PTX-DTPA的FT-IR光谱中保持不变。二酸接头与PTX分子的共轭增强了C-H伸缩信号,并将O-H伸缩带移至较低频率。PULL-CC-PTX和PULL-SS-PTX的FT-IR光谱显示出来自PTX(~1655cm-1)和PULL(~3420cm-1)的典型吸收峰,表明PTX成功地与PULL骨架结合。
实施例2:
前药纳米粒的制备方法及优化:
制备方法:称取10mg PULL-CC-PTX或PULL-SS-PTX溶于0.5mL二甲基亚砜中,800rpm/min搅拌速度下,使用100uL量程移液器在搅拌下逐滴滴加入到去离子水中;滴加完毕后,使用探头超声在195W功率下超声3s停2s的工作模式下超声3分钟使粒径均匀。最后用透析袋(MW8000~14000Da)透析48小时,每12小时换一次水以除去二甲基亚砜溶剂,得到如图3所示紫杉醇前药纳米粒溶液(1mg/mL),分别得到PULL-CC-PTX NPs和PULL-SS-PTX NPs。
本实施例中采用纳米沉淀法制备前药纳米粒,利用单因素分析法对制备的过程参数:DMSO与水体积比,搅拌速度,超声功率进行考察,通过对前药纳米粒粒径和PDI的影响分析,得出优化的制备方法(见表1)。
表1前药纳米粒制备的工艺优化参数
由表1可知,DMSO与水的比例对胶束的粒径影响较大,三种前药胶束的粒径随着DMSO和H2O比例的降低而增大。随着搅拌速度的增加,三种前药胶束的粒径逐渐减小。随着超声功率的增大,三种胶束的粒径均有所减小。最终得到的最优制备参数为DMSO与H2O的体积比为0.5:2,搅拌速度为800rpm/min,超声功率为195W,时间3min,脉冲工作3s,中断2s。此条件下,PULL-CC-PTX(对比例1),PULL-SS-PTX(实施例1)平均粒径分别为117.7±3.2nm,133.5±2.0nm,PDI分别为0.183,0.149。
实施例3:
转铁蛋白自组装纳米粒的制备及其表征:
将实施例2中方法制备的PULL-SS-PTX NPs溶液中加入68μL 1-(3-二甲氨基丙基)-3-乙基碳二亚胺(14mg/mL),68μL N-羟基琥珀酰亚胺活化羧基(8mg/mL),1.06μL三乙胺室温活化半小时后,加入250μL(4mg/mL)转铁蛋白水溶液,室温孵育12h,最后用透析袋(MW=100000Da)透析48小时,以水为介质,每12小时换一次水除去二甲基亚砜溶剂及游离的转铁蛋白,得到粒径为160纳米,转铁蛋白修饰自组装纳米粒溶液(1mg/mL)。
为了验证转铁蛋白修饰自组装纳米粒(TF-PULL-SS-PTX NPs)的表面功能化,采用1HNMR和FT-IR进行分析验证,如图1的(K)所示,在TF-PULL-SS-PTX NPs的核磁共振氢谱中,在δ2.1ppm和δ0.8ppm处出现的新峰分别证实了TF酰胺(-CONH)和甲基(-CH3)基团的存在,赋予了TF在PULL-SS-PTX NPs的表面上。在图2中TF-PULL-SS-PTX NPs的FT-IR光谱中,与C=O伸缩振动(1650cm-1)和N-H弯曲振动(1549cm-1)匹配的谱带代表TF蛋白的肽键。相对于非功能化的NP,这些峰更加密集和明显,为PULL/PTX前药纳米粒的功能化提供了强有力的证据。
实施例4:
三种紫杉醇前药纳米粒的表征:
如图3所示为PULL-CC-PTX;PULL-SS-PTX;TF-PULL-SS-PTX前药纳米制剂的照片,为评估上述三种前药纳米粒的稳定性,本实施例分别在4℃孵育7天检测纳米粒的粒径变化以获得纳米粒储存稳定性,在含有10%FBS的PBS中孵育72小时以获得纳米粒的胶体稳定性。如图4所示,前药纳米粒在测试期间没有明显的粒径变化,具有较好的储存稳定性及胶体稳定性。
本实施例对PULL,PTX,以及前药纳米粒的热力学行为和XRD进行了表征,图5A所示的TGA结果显示,同PULL-SS-PTX纳米粒相比,TF-PULL-SS-PTX NPs显示出更高的热稳定性和更高的炭残基百分比。表面附着TF的键增加了纳米粒的热稳定性。如图5B所示DSC结果显示在PULL-CC-PTX;PULL-SS-PTX;TF-PULL-SS-PTX纳米粒中PTX的热学特征几乎消失,TF-PULL-SS-PTX NPs的水解吸收信号变得更加明显。同样提示TF的功能化增加了纳米粒的热稳定性。XRD结果如图5C所示,原始PULL未描绘出尖锐的XRD峰,表明其无定形性质。然而,它在2θ值为20°时显示出相对强烈和广泛的信号,这归因于多糖链的氢键辅助结构重排,PTX在5.8°、9.0°、11.3°和12.6°的2θ处显示出几个分辨良好且清晰的衍射信号,这证实了其结晶特性,在PULL-CC-PTX NPs;PULL-SS-PTX NPs;TF-PULL-SS-PTX NPs状态下,天然PULL的特定XRD峰变得相对更宽,这意味着PULL链的分子内氢键网络在PTX残基缀合后被破坏,PTX独特的结晶信号在其嫁接到PULL主链后也消失了。这可归因于NPs内PTX分子的结晶到无定形相变。该结果与DSC结果显示出良好的一致性。将结晶药物转化为无定形状态可能会提高其溶解度和治疗效果。
实施例5:
PULL/PTX前药纳米粒的氧化还原敏感性及药物释放行为:
为了考察前药纳米粒的还原触发释药行为,本实施例使用10mmol/L二硫苏糖醇(DTT)模拟细胞质和细胞核中的GSH还原环境考察纳米粒的还原触发释药行为。图6A为DTT触发的药物释放行为机制。本实施例对其敏感性通过UV-Vis光谱、DLS和TEM研究进行考查,如图6B所示,PULL-CC-PTX NPs的UV-Vis吸收光谱在DTT处理后保持不变,表明它们的氧化还原不敏感特性。相反,PULL-SS-PTX NPs和TF-PULL-SS-PTX NPs在模拟的非还原状态下表现出约200nm的特征吸收带,对应于PTX残基。暴露于DTT后,它们的吸收峰的吸收最大值急剧下降。这归因于在DTT的还原性巯基存在下二硫键断裂,导致PTX分子从这些支架上部分脱离。如图6C所示的TEM图像可知,与DTT孵育4小时后,PULL-SS-PTX NPs和TF-PULL-SS-PTXNPs的流体动力学直径显着增加,而对照支架的粒径没有明显变化(即PULL-CC-PTX NPs)。
本实施例对前药纳米粒在10mmol/L的DTT中的释放行为及与不敏感支架的纳米粒进行比较,结果如图6D所示,在没有DTT的情况下,敏感支架和不敏感支架的纳米粒均表现出显着降低的PTX释放率(约14-26%的药物在24小时内释放)。与PULL-CC-PTX NPs相比,敏感支架纳米粒在10mM DTT存在下表现出极大的药物洗脱率,十小时时药物释放已超过50%,由此可见敏感支架均表现出突出的还原敏感性。
实验结果表明,PULL-SS-PTX NPs和TF-PULL-SS-PTX NPs的二硫键在还原条件下可被还原成巯基而发生断裂,具有良好的还原响应能力因此相同时间下还原环境中释放的紫杉醇较多。二硫键的这种特性可降低紫杉醇药物在血液循环系统中的泄露,提高其在肿瘤部位的响应能力,改善递送效率,实现紫杉醇药物在肿瘤细胞内的还原响应型释放。
实施例6:
具有抗肿瘤作用的PULL/PTX前药纳米粒的体外细胞毒性及细胞摄取能力及靶向性考察:
为了评估PULL/PTX自组装前药纳米粒的细胞毒性,本实施例采用MTT法考察了LLC细胞,B16-F10细胞,BEAS-2B细胞的细胞毒性。利用流式细胞术和共聚焦成像技术考察了PULL-CC-PTX NPs;PULL-SS-PTX NPs;TF-PULL-SS-PTX NPs在B16-F10细胞上的细胞摄取能力。为探究不同细胞的受体表达情况,通过Western Blot及流式细胞术分别探究了BEAS-2B,B16-F10,LLC细胞的整体及细胞膜表面的转铁蛋白受体表达情况。利用流式细胞术和共聚焦成像技术考察了TF功能化紫杉醇药物前药纳米粒在LLC细胞上的靶向性摄取能力。
如图7所示,前药纳米粒显示出随时间依赖的摄取能力。其中,PULL-CC-PTX NPs显示出更高的荧光信号,可能由于暴露于细胞内升高水平的GSH后,不同的氧化还原响应NPs可以快速分解,洗脱的香豆素6在癌细胞的酸性微环境下发生质子化,显着降低其荧光强度。相反,不敏感的纳米粒在细胞环境中表现出更高的稳定性,香豆素6仍被加载到这些纳米囊泡中,导致荧光信号显着增加。B16-F10细胞上氧化还原敏感和不敏感的NPs的细胞摄取效率也通过流式细胞仪分析进行了量化,结果如图8所示,与CLSM的结果非常吻合。
细胞毒性结果如图9所示。与相比,PULL-CC-PTX NPs;PULL-SS-PTX NPs;TF-PULL-SS-PTX NPs的细胞毒性明显降低。这是由于普鲁兰多糖为一种由短梗霉发酵产生的天然存在的多糖,是具有安全性的药物辅料,且较癌细胞相比,人正常肺上皮细胞中具有氧化还原敏感的紫杉醇前药纳米粒的细胞毒性显著低于非氧化还原敏感的紫杉醇纳米粒,这是因为癌细胞中具有高水平的GSH以及ROS,使得前药胶束中紫杉醇更快被释放到细胞内,从而达到更强的细胞杀伤性能。
如图10A、B所示,Western Blot结果显示,癌细胞上转铁蛋白受体表达整体高于人正常肺上皮细胞(BEAS-2B)。与图10C流式细胞术显示的细胞膜蛋白表达含量结果相一致。
为了评估紫杉醇前药纳米制剂的靶向性细胞摄取能力,本实施例在小鼠路易斯肺癌细胞(LLC-luc)上进行了细胞靶向摄取能力的考察。将LLC细胞分别与PULL-SS-PTX NPs及TF-PULL-PTX NPs溶液共孵育2小时,采用流式细胞术和共聚焦荧光显微镜成像技术进行定量和成像分析,并加入TF饱和的TF-PULL-PTX NPs制剂以探究转铁蛋白功能化前药纳米粒经受体介导的靶向性摄取能力。如图11B所示,流式细胞术结果显示TF-PULL-SS-PTX NPs的摄取能力高于PULL-SS-PTX NPs,且外加TF饱和受体后,TF-PULL-SS-PTX NPs的细胞摄取能力降低。此结果与图11A所示共聚焦图像的摄取情况相一致,提示我们TF功能化紫杉醇前药纳米粒能够提高制剂在癌细胞上的摄取能力,其高摄取能力由转铁蛋白受体介导。
实施例7:
PULL/PTX前药纳米粒对肺部转移瘤的抑制情况及肿瘤组织靶向性研究:
为了评价TF功能化紫杉醇前药纳米粒的体内肿瘤组织靶向能力,本实施例利用尾静脉注射建立了小鼠LLC-luc细胞肺部转移瘤模型,利用DiR标记的纳米粒尾静脉给药,于给药24小时后通过小动物活体成像荧光拍照系统进行了体内分布研究。如图12A、B所示,PULL-SS-PTX NPs主要分布在肝脏和脾脏,没有显示在肺部积聚。肝脏和脾脏的摄取可归因于单核吞噬细胞系统介导的全身递送外来粒的清除,且纳米粒的高度肝脏组织聚积性也可能由于PULL具有天然的肝脏组织靶向性。在用TF-PULL-SS-PTX NPs处理的小鼠肺中检测到了强烈的荧光信号。相对于PULL-SS-PTX NPs,TF-PULL-SS-PTX NPs在原位建立的肿瘤组织上有明显的积聚荧光信号,这可归因于它们靶向的能力。
为考察抗肿瘤能力,本实施例通过B16-F10细胞及LLC-luc细胞的肺部转移模型对PULL-CC-PTX NPs;PULL-SS-PTX NPs;TF-PULL-SS-PTX NPs进行了抗肿瘤能力考察,图13A为B16-F10细胞肺部转移模型抗肿瘤能力考察流程图,如图13B、C、D所示,不敏感的支架(PULL-CC-PTX NPs)在B16-F10转移性肺癌小鼠模型上显示出极其有限的化学治疗效果,此外与PULL-SS-PTX NPs和相比,TF-PULL-SS-PTX NPs的TFR靶向性在改善体内抗癌潜力方面的优点并未在B16-F10肺癌模型上建立,这可能归结于在B16-F10细胞上没有表现出高出正常细胞很多的受体表达水平。
在B16-F10细胞小鼠肺癌模型的抗肿瘤结果及LLC细胞小鼠肺癌模型的肿瘤靶向能力考察的基础上,排除了不敏感支架(PULL-CC-PTX NPs),将PULL-SS-PTX NPs及TF-PULL-SS-PTX NPs用于LLC细胞小鼠肺癌模型上进行抗肿瘤作用的研究。图14A为LLC-luc细胞肺部转移模型抗肿瘤能力考察流程图,如图14B、C所示,与对照组相比,和PULL-SS-PTX NPs处理组的细胞增殖缓慢。TF-PULL-SS-PTX NPs可以更有效地抑制肿瘤生长。与用生理盐水、/>和PULL-SS-PTX NPs处理的小鼠相比,信号强度降低了20倍、10倍和5倍。TF-PULL-SS-PTX NPs显示出优越的肿瘤抑制能力。此外通过肺组织切片H&E染色及Ki-67染色分析进一步评估,如图14D所示,H&E染色分析显示对照组癌细胞肺叶上广泛转移,TF-PULL-SS-PTX NPs显示出减少的转移性癌细胞及炎性细胞。与其他组相比,细胞增殖标志物Ki-67免疫荧光分析显示出TF-PULL-PTX NPs组明显低的阳性率。同样表明靶向制剂组在抑制肿瘤细胞增殖方面的明显优势。
实施例7:
前药纳米粒的系统安全性:
为评估PULL-CC-PTX NPs;PULL-SS-PTX NPs;TF-PULL-SS-PTX NPs的系统安全性,本实施例对研究期间动物体重的进行性变化进行记录,如图15A所示,生理盐水治疗组的严重肿瘤发展和紫杉醇的高赋形剂相关毒性导致动物体重减轻。相比之下,前药纳米粒治疗的小鼠体重在研究期间较为稳定。此外,如图15B所示,纳米制剂的溶血性结果显示,在考察浓度范围内(4-80μg/mL),PULL-SS-PTX NPs和TF-PULL-SS-PTX NPs表现出最小的溶血,而在4到80μg/mL的等效PTX浓度范围内严重破坏红细胞膜。
为评估制剂的体内安全性,在实验最后一天对小鼠实施安乐死后收集小鼠的心脏、肝脏、脾脏、肾脏以及血液,将组织切片进行H&E染色,血液进行肝肾功能检测。如图15C-F所示,与相比,纳米粒的血清检测未显示出明显的肝肾毒性。图15G所示,前药纳米粒治疗组的各种重要器官组织的组织学检查表明没有细胞坏死、变性、炎症反应和其他病理变化。相反,/>静脉注射治疗组在心脏和脾脏组织中表现出明显的坏死和炎症。
研究表明氧化还原敏感的反应性前药纳米制剂的安全性得到了改善。
Claims (10)
1.一种多糖抗肿瘤小分子药物共聚物,其特征在于,该多糖抗肿瘤小分子药物共聚物是多糖通过连接子共轭连接抗肿瘤小分子药物形成的多糖抗肿瘤小分子药物共聚物。
2.根据权利要求1所述的多糖抗肿瘤小分子药物共聚物,其特征在于,连接子为具有二硫键的连接子,其原料为2,2'-二硫代二乙酸、3,3'-二硫代二丙酸或4,4'-二硫代二丁酸中的一种;
和/或,所述的多糖为能够进行氨基化修饰的天然多糖;
和/或,所述的抗肿瘤小分子药物为具有活性羟基的抗肿瘤化疗药物或具有活性氨基的抗肿瘤化疗药物。
3.一种转铁蛋白修饰自组装纳米粒,其特征在于,是转铁蛋白通过酰胺键连接权利要求1所述的多糖抗肿瘤小分子药物共聚物形成。
4.根据权利要求3所述的转铁蛋白修饰自组装纳米粒,其特征在于,每10mg转铁蛋白修饰自组装纳米粒上有3.63~3.87mg抗肿瘤小分子药物,0.027~0.033mg转铁蛋白,余量为多糖;
转铁蛋白修饰自组装纳米粒的粒径为120~160nm,载药量高达36%~40%。
5.权利要求1或2所述的多糖抗肿瘤小分子药物共聚物的制备方法,其特征在于,将多糖和抗肿瘤小分子药物通过二硫键连接合成。
6.权利要求或4所述的转铁蛋白修饰自组装纳米粒的制备方法,其特征在于,采用权利要求1或2所述的多糖抗肿瘤小分子药物共聚物,经纳米沉淀法自组装形成前药纳米粒,加入转铁蛋白与前药纳米粒共孵育,最终转铁蛋白通过酰胺反应连接在前药纳米粒外部,制备完成得到转铁蛋白修饰自组装纳米粒。
7.权利要求5所述的多糖抗肿瘤小分子药物共聚物的制备方法,其特征在于,包括以下步骤:
S1:乙酸化多糖聚合物
将活化的多糖、氢氧化钠溶液和氯乙酸溶液混合,搅拌均匀,在60~80℃反应5~8h,固液分离,洗涤,干燥,得到乙酸化多糖聚合物;按摩尔比,活化的多糖中单糖单元:氢氧化钠:氯乙酸=(1~2):(1~3):(2~6);
S2:氨基修饰
以水作为溶剂,将乙酸化多糖聚合物、1-(3-二甲氨基丙基)-3-乙基碳二亚胺、N-羟基琥珀酰亚胺、三乙胺混合搅拌,活化10~12h,再加入乙二胺,反应24h以上,加入甲醇作为沉淀析出剂,沉淀析出后,固液分离,洗涤固体,干燥,得到化合物PULL-ED;
按摩尔比,乙酸化多糖聚合物中单糖单元:EDC:NHS:三乙胺:乙二胺=(4~5):(2~4):(1.5~3.5):(2~3):(15~25);
S3:连接二硫键
将抗肿瘤小分子药物和具有二硫键的连接子的原料混合,以EDC为催化剂,以二氯甲烷为溶剂,室温活化1~3h,加入4-二甲氨基吡啶后,反应40~50h,得到反应物;其中,按摩尔比,抗肿瘤小分子药物:具有二硫键的连接子的原料:EDC:4-二甲氨基吡啶=(1~1.5):(1~2.5):(4~6):(1~1.5);
将反应物去除溶剂,再以稀盐酸作为沉淀析出剂,沉淀产物,固液分离,水洗至中性,干燥,得到化合物PTX-DTPA;
S4:共聚
将化合物PTX-DTPA、NHS、EDC、三乙胺混合,室温活化后,得到PTX-DTPA溶液;
向PTX-DTPA溶液中滴加化合物PULL-ED溶液,再加入缓冲液调整pH值为7.8~8.0,然后室温反应20~25h,得到反应液;
其中,按摩尔比,PTX-DTPA:NHS:EDC:三乙胺:PULL-ED的单糖单元=(1~1.5):(1~3):(2~4):(1~3):(4~8);
将反应液进行透析,得到白色絮状沉淀物,干燥,得到PULL-SS-PTX共聚物。
8.权利要求7所述的多糖抗肿瘤小分子药物共聚物的制备方法,其特征在于,所述的S1中,混合的温度为25~45℃,搅拌的速度为800~1000rpm/min,其中,氢氧化钠溶液和氯乙酸溶液,滴加入活化的多糖中,滴加的速率为1~1.5mL/min。
9.权利要求3或4转铁蛋白修饰自组装纳米粒的制备方法,其特征在于,包括以下步骤:
步骤(1):将PULL-SS-PTX共聚物溶于DMSO中,得到共聚物溶液;
在搅拌速度为400~1000rpm/min的持续搅拌下,将共聚物溶液以1~1.5mL/min的速度加入水中,滴加完成后,施加间断超声,反应,得到粒径均匀的前药纳米粒(PULL-SS-PTXNPs);
其中,按固液比,PULL-SS-PTX共聚物:DMSO:水=(9.5~10.5)mg:(0.2~0.6)mL:(1.8~2.2)mL;超声的功率为50~200W,超声持续2~3s,间断时间1~2s;反应时间为2~3min;
步骤(2):将前药纳米粒、EDC溶液、NHS溶液和三乙胺混合,室温活化后,再加入转铁蛋白溶液,室温孵育12~24h,然后透析,除去游离转铁蛋白,得到转铁蛋白修饰自组装纳米粒(TF-PULL-SS-PTX NPs);
其中,按质量比,前药纳米粒:EDC:NHS:三乙胺:转铁蛋白=(5~10):(0.8~1.2):(0.5~0.7):(0.4~0.6):(0.5~1)。
10.权利要求1或2所述的多糖抗肿瘤小分子药物共聚物和权利要求3或4所述的转铁蛋白修饰自组装纳米粒用于制备抗肿瘤药剂的应用。
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