CN113262308A - 叶酸受体介导紫杉醇和羟基喜树碱超顺磁氧化铁纳米粒及其制备方法及用途 - Google Patents
叶酸受体介导紫杉醇和羟基喜树碱超顺磁氧化铁纳米粒及其制备方法及用途 Download PDFInfo
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Abstract
本发明公开一种叶酸受体介导紫杉醇和羟基喜树碱超顺磁氧化铁纳米粒及其制备方法。所述的纳米粒为:p‑PS/ FA@ PEG/PEI‑SPIONs @PTX +HCPT。本发明的载药纳米粒系统,具有协同增效作用,能增强抗肿瘤作用,并且毒副作用小,是极具开发价值的治疗肿瘤的纳米粒制剂。
Description
技术领域
本发明属于医药技术领域,具体为一种叶酸受体介导紫杉醇和羟基喜树碱超顺磁氧化铁纳米粒及其制备方法。
背景技术
威胁人类生命健康的最大杀手之一是肿瘤,化学药物是治疗鼻咽癌不可缺少的手段,但化学药物对癌变细胞和人体正常细胞的非特异性,使其在治疗过程中不仅杀伤癌变细胞,同时也杀伤正常细胞,造成了全身严重的毒副作用。近几年以来,寻找安全有效的靶向药物是治疗肿瘤研究的一个热点。
纳米给药系统可实现在分子水平控制对药物递送和释放,通过载体修饰可实现药物在肿瘤部位的被动或主动靶向,从而大大增强治疗效果,并显著改善毒副作用。因此,构建一种抗肿瘤药物(PTX)肿瘤靶向给药系统,将PTX递送于肿瘤细胞内,对于提高治疗肿瘤的疗效、减少对其它组织的毒副作用具有重要的意义。
超顺磁氧化铁纳米粒子(SPIONs)可以作为磁性药物靶向载体,在有外加磁场存在时,表现出较强的磁性。SPIONs在体内既安全又易于控制,作为抗肿瘤药物载体应用前景广阔。经过水溶性聚合物修饰的氧化铁纳米粒水分散性好,可将药物和铁磁性物质包裹起来,增加药物在水中的溶解度,通过物理作用发挥靶向性,利用体外磁场效应引导药物在体内定向移动和定位集中。
现有技术中超顺磁氧化铁纳米粒子(SPIONs)用于难溶抗肿瘤药物已有应用,如发明人在之前的专利申请(一种叶酸介导抗肿瘤药物超顺磁肿瘤靶向纳米粒及其制备方法2017106087746)中记载的技术那样。然而,目前的纳米粒只能和单一的药物进行接枝,尚未出现同时将两种抗肿瘤药物共同接枝的报道。
发明内容
本发明针对上述问题提供一种叶酸受体介导紫杉醇和羟基喜树碱超顺磁氧化铁纳米粒及其制备方法。
为达到上述目的,本发明的技术方案如下:
叶酸受体介导紫杉醇和羟基喜树碱超顺磁氧化铁纳米粒为:p-PS/FA@PEG/PEI-SPIONs @PTX+HCPT。
所述的叶酸受体介导紫杉醇和羟基喜树碱超顺磁氧化铁纳米粒,其制备方法包括以下步骤:
A、将FA接枝在PEG/PEI-SPIONs纳米粒的表面上,获得FA@PEG/PEI-SPIONs;
B、将p-PS接枝到FA@PEG/PEI-SPIONs上,获得p-PS/FA@PEG/PEI-SPIONs;
C、将PTX和HCPT接枝到p-PS/FA@PEG/PEI-SPIONs上,获得p-PS/FA@ PEG/PEI-SPIONs@PTX+HCPT。
所述的步骤A中,PEG/PEI-SPIONs纳米粒的制备方法包括以下步骤:
将PEG-1000和PEI-1800混合均匀,在8-12min内升温至78-82℃,得混合液;将Fe(acac)3添加到混合液中,在78-82℃下继续搅拌8-12min后,加热至250-270℃,保温0.5-1.5h后停止加热,冷却至50-70℃,得到含有PEG/PEI-SPIONs的混合液,分离得到PEG/PEI-SPIONs 纳米粒;所述的PEG-1000、PEI-1800、Fe(acac)3的质量比为12-17:0.1-0.5:0.5-1;
上述整个过程在磁力搅拌下进行,并通入氩气除氧。
所述的分离PEG/PEI-SPIONs纳米粒的方法包括以下步骤:
向冷却后的含有PEG/PEI-SPIONs纳米粒的混合液中加入甲苯进行超声分散,用磁铁在烧杯底部进行吸附,当黑色磁性物质基本被吸附至烧杯底部后,弃去上清夜,重复该清洗过程1-3次后,黑色沉淀物再用丙酮清洗1-3次,即得。
所述的步骤A中将FA接枝在PEG/PEI-SPIONs纳米粒的表面的方法包括以下步骤:
将PEG/PEI-SPIONs分散于去离子水中,获得PEG/PEI-SPIONs分散液,然后在快速搅拌的条件下,将PEG/PEI-SPIONs分散液缓慢滴加到FA活性酯中,在磁力搅拌下反应8-36h后,然后用LS磁选柱对样品进行分离,最后用透析袋MWCO 8000-14000Da透析,去除游离叶酸,直到透析液没有紫外吸收,得到FA@PEG/PEI-SPIONs分散液。
所述的FA活性酯的制备方法包括以下步骤:将FA溶解在DMSO中活化,然后加入EDC和sulfo-NHS,搅拌2-5h进行叶酸活化,得到FA活性酯;
所述的FA、DMSO、EDC、sulfo-NHS的摩尔比为:0.1-0.3:300-400:0.3-0.5:0.3-0.5。
所述的步骤B中,将p-PS接枝到FA@PEG/PEI-SPIONs上的方法包括以下步骤:
取p-PS溶解于DMF中,在搅拌状态下逐滴滴加FA@PEG/PEI-SPIONs,在800-1500rpm转速下磁力搅拌12-36h,然后离心取沉淀;其中,p-PS、DMF、FA@PEG/PEI-SPIONs的重量比为:5-15:3000-7000:1-5;然后用CHCl3洗涤多次除去游离聚合物p-PS,得到p-PS/FA@ PEG/PEI-SPIONs。
所述的步骤C中将PTX和HCPT接枝到p-PS/FA@PEG/PEI-SPIONs上的方法包括以下步骤:
a、将制得的p-PS/FA@PEG/PEI-SPIONs溶解在CHCl3中,然后向该溶液中加入PTX和HCPT,获得混合液a;
b、将混合液a加入相当于DMF重量一半的浓度为2-8mg/ml的SDS溶液中,在120-200w 功率下超声下乳化1-10分钟时间,获得乳化液;
c、将乳化液在室温下缓慢搅拌直至将CHCl3至完全挥干,在1500-3000rpm/min转速下离心3-10min,收集沉淀和,用CHCl3和蒸馏水交替清洗2-5次,最终获得p-PS/FA@ PEG/PEI-SPIONs@PTX+HCPT纳米粒。
本发明中各英文缩写代表的化合物为:
p-PS:磷酸根聚苯乙烯;
FA:叶酸;
PTX:紫杉醇;
DMF:二甲基甲酰胺;
HCPT:羟基喜树碱;
SPIONs:超顺磁性氧化铁纳米粒;
DMAP:二甲氨基吡啶;
PTX:紫杉醇;
HCPT:羟基喜树碱;
PEG:聚乙二醇;
PEI:聚乙烯亚胺;
FA:叶酸;
sulfo-NHS:N-羟基硫代琥珀酰亚胺;
EDC:1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐;
DMSO:二甲基亚砜。
本发明的有益效果为:
本发明实现了将抗癌药物PTX和HCPT通过p-PS作用,共同接枝到 FA@PEG/PEI-SPIONs,从而制备出一种紫杉醇和羟基喜树碱超顺磁氧化铁纳米自组装体纳米粒,该纳米粒可在肿瘤酸性条件下同时缓慢释放这两种药物,实现药物的靶向性。并且,这种同时接枝的PTX和HCPT与单一使用PTX或HCPT相比,PTX+HCPT联合用药导致细胞毒性增强,说明PTX和HCPT联合具有协同作用。
本发明的p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT纳米粒在磁场作用下,具有双重靶向作用,经本发明实验证明,能够发挥更强的抑制鼻咽癌细胞增殖的能力。
附图说明
图1为p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT纳米粒的水和动力学粒径及Zeat电位图;
图2为PEG/PEI-SPIONs、p-PS-SPIONs和HCPT/PTX@p-PS-SPIONs纳米粒透射电镜图;
图3为p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT纳米粒紫外吸收及PTX和HCPT标准曲线;
图4为p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT红外吸收图谱;
图5为PTX(A)和HCPT(B)的标准曲线及在37℃,pH分别为5.0和7.3条件下 HCPT/PTX@FA@p-PS-SPIONs药物的释放曲线(C)
图6为空白纳米乳FA@p-PS-SPIONs对CNE-1、CNE-2、HNE-1和C666-1作用24h(A) 和48h(B)的细胞毒性,均值±标准差,n=6,与正常组比*P<0.05;
图7为TX、HCPT、PTX+HCPT混合物(摩尔比=1:1)、p-PS/FA@PEG/PEI-SPIONs@PTX +HCPT和p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT+MF作用48h后对不同鼻咽癌细胞的抑制率(*p<0.01,n=3);
图8为鼻咽癌细胞系(CNE-1(A)、CNE-2(B)、HNE-1(C)和C666-1(D))经过不同的方式处理后的荧光显微镜图像(200×);
图9为HCPT/PTX@FA@p-PS-SPIONs纳米粒被鼻咽癌细胞CNE-1摄取后释放药物的荧光图 (200×);
图9-1为HCPT/PTX@FA@p-PS-SPIONs纳米粒被鼻咽癌细胞CNE-2摄取后释放药物的荧光图(200×);
图9-2为HCPT/PTX@FA@p-PS-SPIONs纳米粒被鼻咽癌细胞HNE-1摄取后释放药物的荧光图(200×);
图9-3为HCPT/PTX@FA@p-PS-SPIONs纳米粒被鼻咽癌细胞C666-1摄取后释放药物的荧光图(200×)。
具体实施方式
下面通过具体实施例对本发明进行详细说明。
实施例1
本发明的叶酸受体介导紫杉醇和羟基喜树碱超顺磁氧化铁纳米粒,包括以下步骤:
A、将FA接枝在PEG/PEI-SPIONs纳米粒的表面上,获得FA@PEG/PEI-SPIONs:
将PEG-1000和PEI-1800混合均匀,在8min内升温至78℃,得混合液;将Fe(acac)3添加到混合液中,在78℃下继续搅拌8min后,加热至250℃,保温0.5h后停止加热,冷却至50℃,得到含有PEG/PEI-SPIONs的混合液;向含有PEG/PEI-SPIONs纳米粒的混合液中加入甲苯进行超声分散,用磁铁在烧杯底部进行吸附,当黑色磁性物质基本被吸附至烧杯底部后,弃去上清夜,重复该清洗过程1次后,黑色沉淀物再用丙酮清洗1次,得到PEG/PEI-SPIONs纳米粒;所述的PEG-1000、PEI-1800、Fe(acac)3的质量比为12:0.1:0.5;上述整个过程在磁力搅拌下进行,并通入氩气除氧;
将PEG/PEI-SPIONs分散于去离子水中,获得PEG/PEI-SPIONs分散液,然后在快速搅拌的条件下,将PEG/PEI-SPIONs分散液缓慢滴加到FA活性酯中,在磁力搅拌下反应8h后,然后用LS磁选柱对样品进行分离,最后用透析袋MWCO 8000-14000Da透析,去除游离叶酸,直到透析液没有紫外吸收,得到FA@PEG/PEI-SPIONs分散液;
所述的FA活性酯的制备方法包括以下步骤:将FA溶解在DMSO中活化,然后加入EDC和sulfo-NHS,搅拌2h进行叶酸活化,得到FA活性酯;所述的FA、DMSO、EDC、sulfo-NHS 的摩尔比为:0.1:300:0.3:0.3。
B、将p-PS接枝到FA@PEG/PEI-SPIONs上,获得p-PS/FA@PEG/PEI-SPIONs:
取p-PS溶解于DMF中,在搅拌状态下逐滴滴加FA@PEG/PEI-SPIONs,在800-1500rpm转速下磁力搅拌12h,然后离心取沉淀;其中,p-PS、DMF、FA@PEG/PEI-SPIONs的重量比为:5:3000:1;然后用CHCl3洗涤多次除去游离聚合物p-PS,得到p-PS/FA@PEG/PEI-SPIONs。
C、将PTX和HCPT接枝到p-PS/FA@PEG/PEI-SPIONs上,获得p-PS/FA@ PEG/PEI-SPIONs@PTX+HCPT:
a、将制得的p-PS/FA@PEG/PEI-SPIONs溶解在CHCl3中,然后向该溶液中加入PTX和HCPT,获得混合液a;
b、将混合液a加入相当于DMF重量一半的浓度为2mg/ml的SDS溶液中,在120w功率下超声下乳化1分钟时间,获得乳化液;
c、将乳化液在室温下缓慢搅拌直至将CHCl3至完全挥干,在1500rpm/min转速下离心 3min,收集沉淀和,用CHCl3和蒸馏水交替清洗2次,最终获得p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT纳米粒。
实施例2
本发明的叶酸受体介导紫杉醇和羟基喜树碱超顺磁氧化铁纳米粒,包括以下步骤:
A、将FA接枝在PEG/PEI-SPIONs纳米粒的表面上,获得FA@PEG/PEI-SPIONs:
将PEG-1000和PEI-1800混合均匀,在12min内升温至82℃,得混合液;将Fe(acac)3添加到混合液中,在82℃下继续搅拌12min后,加热至270℃,保温1.5h后停止加热,冷却至70℃,得到含有PEG/PEI-SPIONs的混合液;向含有PEG/PEI-SPIONs纳米粒的混合液中加入甲苯进行超声分散,用磁铁在烧杯底部进行吸附,当黑色磁性物质基本被吸附至烧杯底部后,弃去上清夜,重复该清洗过程3次后,黑色沉淀物再用丙酮清洗3次,得到 PEG/PEI-SPIONs纳米粒;所述的PEG-1000、PEI-1800、Fe(acac)3的质量比为17:0.5:1;上述整个过程在磁力搅拌下进行,并通入氩气除氧;
将PEG/PEI-SPIONs分散于去离子水中,获得PEG/PEI-SPIONs分散液,然后在快速搅拌的条件下,将PEG/PEI-SPIONs分散液缓慢滴加到FA活性酯中,在磁力搅拌下反应36h后,然后用LS磁选柱对样品进行分离,最后用透析袋MWCO 8000-14000Da透析,去除游离叶酸,直到透析液没有紫外吸收,得到FA@PEG/PEI-SPIONs分散液;
所述的FA活性酯的制备方法包括以下步骤:将FA溶解在DMSO中活化,然后加入EDC和sulfo-NHS,搅拌5h进行叶酸活化,得到FA活性酯;所述的FA、DMSO、EDC、sulfo-NHS 的摩尔比为:0.3:400:0.5:0.5。
B、将p-PS接枝到FA@PEG/PEI-SPIONs上,获得p-PS/FA@PEG/PEI-SPIONs:
取p-PS溶解于DMF中,在搅拌状态下逐滴滴加FA@PEG/PEI-SPIONs,在800-1500rpm转速下磁力搅拌36h,然后离心取沉淀;其中,p-PS、DMF、FA@PEG/PEI-SPIONs的重量比为:15:7000:5;然后用CHCl3洗涤多次除去游离聚合物p-PS,得到p-PS/FA@ PEG/PEI-SPIONs。
C、将PTX和HCPT接枝到p-PS/FA@PEG/PEI-SPIONs上,获得p-PS/FA@ PEG/PEI-SPIONs@PTX+HCPT:
a、将制得的p-PS/FA@PEG/PEI-SPIONs溶解在CHCl3中,然后向该溶液中加入PTX和HCPT,获得混合液a;
b、将混合液a加入相当于DMF重量一半的浓度为8mg/ml的SDS溶液中,在200w功率下超声下乳化10分钟时间,获得乳化液;
c、将乳化液在室温下缓慢搅拌直至将CHCl3至完全挥干,在3000rpm/min转速下离心 10min,收集沉淀和,用CHCl3和蒸馏水交替清洗5次,最终获得p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT纳米粒。
实施例3
本发明的叶酸受体介导紫杉醇和羟基喜树碱超顺磁氧化铁纳米粒,包括以下步骤:
A、将FA接枝在PEG/PEI-SPIONs纳米粒的表面上,获得FA@PEG/PEI-SPIONs:
将PEG-1000和PEI-1800混合均匀,在10min内升温至80℃,得混合液;将Fe(acac)3添加到混合液中,在80℃下继续搅拌10min后,加热至260℃,保温1h后停止加热,冷却至 60℃,得到含有PEG/PEI-SPIONs的混合液;向含有PEG/PEI-SPIONs纳米粒的混合液中加入甲苯进行超声分散,用磁铁在烧杯底部进行吸附,当黑色磁性物质基本被吸附至烧杯底部后,弃去上清夜,重复该清洗过程2次后,黑色沉淀物再用丙酮清洗2次,得到PEG/PEI-SPIONs纳米粒;所述的PEG-1000、PEI-1800、Fe(acac)3的质量比为15:0.3:0.8;上述整个过程在磁力搅拌下进行,并通入氩气除氧;
将PEG/PEI-SPIONs分散于去离子水中,获得PEG/PEI-SPIONs分散液,然后在快速搅拌的条件下,将PEG/PEI-SPIONs分散液缓慢滴加到FA活性酯中,在磁力搅拌下反应24h后,然后用LS磁选柱对样品进行分离,最后用透析袋MWCO 8000-14000Da透析,去除游离叶酸,直到透析液没有紫外吸收,得到FA@PEG/PEI-SPIONs分散液;
所述的FA活性酯的制备方法包括以下步骤:将FA溶解在DMSO中活化,然后加入EDC和sulfo-NHS,搅拌3h进行叶酸活化,得到FA活性酯;所述的FA、DMSO、EDC、sulfo-NHS 的摩尔比为:0.2:360:0.4:0.4。
B、将p-PS接枝到FA@PEG/PEI-SPIONs上,获得p-PS/FA@PEG/PEI-SPIONs:
取p-PS溶解于DMF中,在搅拌状态下逐滴滴加FA@PEG/PEI-SPIONs,在1200rpm转速下磁力搅拌18h,然后离心取沉淀;其中,p-PS、DMF、FA@PEG/PEI-SPIONs的重量比为: 10:5000:3;然后用CHCl3洗涤多次除去游离聚合物p-PS,得到p-PS/FA@PEG/PEI-SPIONs。
C、将PTX和HCPT接枝到p-PS/FA@PEG/PEI-SPIONs上,获得p-PS/FA@ PEG/PEI-SPIONs@PTX+HCPT:
a、将制得的p-PS/FA@PEG/PEI-SPIONs溶解在CHCl3中,然后向该溶液中加入PTX和HCPT,获得混合液a;
b、将混合液a加入相当于DMF重量一半的浓度为5mg/ml的SDS溶液中,在160w功率下超声下乳化6分钟时间,获得乳化液;
c、将乳化液在室温下缓慢搅拌直至将CHCl3至完全挥干,在2000rpm/min转速下离心6 min,收集沉淀和,用CHCl3和蒸馏水交替清洗3次,最终获得p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT纳米粒。
实施例4
本发明的叶酸受体介导紫杉醇和羟基喜树碱超顺磁氧化铁纳米粒,包括以下步骤:
A、将FA接枝在PEG/PEI-SPIONs纳米粒的表面上,获得FA@PEG/PEI-SPIONs:
将PEG-1000和PEI-1800混合均匀,在11min内升温至79℃,得混合液;将Fe(acac)3添加到混合液中,在79℃下继续搅拌8-12min后,加热至255℃,保温0.8h后停止加热,冷却至65℃,得到含有PEG/PEI-SPIONs的混合液;向含有PEG/PEI-SPIONs纳米粒的混合液中加入甲苯进行超声分散,用磁铁在烧杯底部进行吸附,当黑色磁性物质基本被吸附至烧杯底部后,弃去上清夜,重复该清洗过程3次后,黑色沉淀物再用丙酮清洗1次,得到 PEG/PEI-SPIONs纳米粒;所述的PEG-1000、PEI-1800、Fe(acac)3的质量比为13:0.2:0.9;上述整个过程在磁力搅拌下进行,并通入氩气除氧;
将PEG/PEI-SPIONs分散于去离子水中,获得PEG/PEI-SPIONs分散液,然后在快速搅拌的条件下,将PEG/PEI-SPIONs分散液缓慢滴加到FA活性酯中,在磁力搅拌下反应8-36h后,然后用LS磁选柱对样品进行分离,最后用透析袋MWCO 8000-14000Da透析,去除游离叶酸,直到透析液没有紫外吸收,得到FA@PEG/PEI-SPIONs分散液;
所述的FA活性酯的制备方法包括以下步骤:将FA溶解在DMSO中活化,然后加入EDC和sulfo-NHS,搅拌2-5h进行叶酸活化,得到FA活性酯;所述的FA、DMSO、EDC、sulfo-NHS 的摩尔比为:0.2:350:0.4:0.3。
B、将p-PS接枝到FA@PEG/PEI-SPIONs上,获得p-PS/FA@PEG/PEI-SPIONs:
取p-PS溶解于DMF中,在搅拌状态下逐滴滴加FA@PEG/PEI-SPIONs,在1200rpm转速下磁力搅拌30h,然后离心取沉淀;其中,p-PS、DMF、FA@PEG/PEI-SPIONs的重量比为: 11:4000:4;然后用CHCl3洗涤多次除去游离聚合物p-PS,得到p-PS/FA@PEG/PEI-SPIONs。
C、将PTX和HCPT接枝到p-PS/FA@PEG/PEI-SPIONs上,获得p-PS/FA@ PEG/PEI-SPIONs@PTX+HCPT:
a、将制得的p-PS/FA@PEG/PEI-SPIONs溶解在CHCl3中,然后向该溶液中加入PTX和HCPT,获得混合液a;
b、将混合液a加入相当于DMF重量一半的浓度为4mg/ml的SDS溶液中,在120-200w功率下超声下乳化3分钟时间,获得乳化液;
c、将乳化液在室温下缓慢搅拌直至将CHCl3至完全挥干,在2200rpm/min转速下离心 6min,收集沉淀和,用CHCl3和蒸馏水交替清洗4次,最终获得p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT纳米粒。
实施例5
本发明的叶酸受体介导紫杉醇和羟基喜树碱超顺磁氧化铁纳米粒,包括以下步骤:
A、将FA接枝在PEG/PEI-SPIONs纳米粒的表面上,获得FA@PEG/PEI-SPIONs:
将PEG-1000和PEI-1800混合均匀,在11min内升温至81℃,得混合液;将Fe(acac)3添加到混合液中,在81℃下继续搅拌11min后,加热至252℃,保温0.6h后停止加热,冷却至66℃,得到含有PEG/PEI-SPIONs的混合液;向含有PEG/PEI-SPIONs纳米粒的混合液中加入甲苯进行超声分散,用磁铁在烧杯底部进行吸附,当黑色磁性物质基本被吸附至烧杯底部后,弃去上清夜,重复该清洗过程2次后,黑色沉淀物再用丙酮清洗3次,得到 PEG/PEI-SPIONs纳米粒;所述的PEG-1000、PEI-1800、Fe(acac)3的质量比为16:0.4:0.9;上述整个过程在磁力搅拌下进行,并通入氩气除氧;
将PEG/PEI-SPIONs分散于去离子水中,获得PEG/PEI-SPIONs分散液,然后在快速搅拌的条件下,将PEG/PEI-SPIONs分散液缓慢滴加到FA活性酯中,在磁力搅拌下反应30h后,然后用LS磁选柱对样品进行分离,最后用透析袋MWCO 8000-14000Da透析,去除游离叶酸,直到透析液没有紫外吸收,得到FA@PEG/PEI-SPIONs分散液;
所述的FA活性酯的制备方法包括以下步骤:将FA溶解在DMSO中活化,然后加入EDC和sulfo-NHS,搅拌4h进行叶酸活化,得到FA活性酯;所述的FA、DMSO、EDC、sulfo-NHS 的摩尔比为:0.2:380:0.5:0.3。
B、将p-PS接枝到FA@PEG/PEI-SPIONs上,获得p-PS/FA@PEG/PEI-SPIONs:
取p-PS溶解于DMF中,在搅拌状态下逐滴滴加FA@PEG/PEI-SPIONs,在800-1500rpm转速下磁力搅拌16h,然后离心取沉淀;其中,p-PS、DMF、FA@PEG/PEI-SPIONs的重量比为:11:6000:2;然后用CHCl3洗涤多次除去游离聚合物p-PS,得到p-PS/FA@ PEG/PEI-SPIONs。
C、将PTX和HCPT接枝到p-PS/FA@PEG/PEI-SPIONs上,获得p-PS/FA@ PEG/PEI-SPIONs@PTX+HCPT:
a、将制得的p-PS/FA@PEG/PEI-SPIONs溶解在CHCl3中,然后向该溶液中加入PTX和HCPT,获得混合液a;
b、将混合液a加入相当于DMF重量一半的浓度为7mg/ml的SDS溶液中,在120-200w功率下超声下乳化8分钟时间,获得乳化液;
c、将乳化液在室温下缓慢搅拌直至将CHCl3至完全挥干,在1800rpm/min转速下离心4 min,收集沉淀和,用CHCl3和蒸馏水交替清洗2次,最终获得p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT纳米粒。
实施例6
本发明的叶酸受体介导紫杉醇和羟基喜树碱超顺磁氧化铁纳米粒,包括以下步骤:
A、将FA接枝在PEG/PEI-SPIONs纳米粒的表面上,获得FA@PEG/PEI-SPIONs:
将PEG-1000和PEI-1800混合均匀,在9min内升温至79℃,得混合液;将Fe(acac)3添加到混合液中,在79℃下继续搅拌11min后,加热至250-270℃,保温1.3h后停止加热,冷却至58℃,得到含有PEG/PEI-SPIONs的混合液;向含有PEG/PEI-SPIONs纳米粒的混合液中加入甲苯进行超声分散,用磁铁在烧杯底部进行吸附,当黑色磁性物质基本被吸附至烧杯底部后,弃去上清夜,重复该清洗过程3次后,黑色沉淀物再用丙酮清洗2次,得到 PEG/PEI-SPIONs纳米粒;所述的PEG-1000、PEI-1800、Fe(acac)3的质量比为13:0.2:0.8;上述整个过程在磁力搅拌下进行,并通入氩气除氧;
将PEG/PEI-SPIONs分散于去离子水中,获得PEG/PEI-SPIONs分散液,然后在快速搅拌的条件下,将PEG/PEI-SPIONs分散液缓慢滴加到FA活性酯中,在磁力搅拌下反应12h后,然后用LS磁选柱对样品进行分离,最后用透析袋MWCO 8000-14000Da透析,去除游离叶酸,直到透析液没有紫外吸收,得到FA@PEG/PEI-SPIONs分散液;
所述的FA活性酯的制备方法包括以下步骤:将FA溶解在DMSO中活化,然后加入EDC和sulfo-NHS,搅拌4h进行叶酸活化,得到FA活性酯;所述的FA、DMSO、EDC、sulfo-NHS 的摩尔比为:0.15:360:0.4:0.5。
B、将p-PS接枝到FA@PEG/PEI-SPIONs上,获得p-PS/FA@PEG/PEI-SPIONs:
取p-PS溶解于DMF中,在搅拌状态下逐滴滴加FA@PEG/PEI-SPIONs,在1300rpm转速下磁力搅拌26h,然后离心取沉淀;其中,p-PS、DMF、FA@PEG/PEI-SPIONs的重量比为: 12:4500:2;然后用CHCl3洗涤多次除去游离聚合物p-PS,得到p-PS/FA@PEG/PEI-SPIONs。
C、将PTX和HCPT接枝到p-PS/FA@PEG/PEI-SPIONs上,获得p-PS/FA@ PEG/PEI-SPIONs@PTX+HCPT:
a、将制得的p-PS/FA@PEG/PEI-SPIONs溶解在CHCl3中,然后向该溶液中加入PTX和HCPT,获得混合液a;
b、将混合液a加入相当于DMF重量一半的浓度为7mg/ml的SDS溶液中,在130w功率下超声下乳化3分钟时间,获得乳化液;
c、将乳化液在室温下缓慢搅拌直至将CHCl3至完全挥干,在2700rpm/min转速下离心 7min,收集沉淀和,用CHCl3和蒸馏水交替清洗4次,最终获得p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT纳米粒。
实施例7
用高效液相色谱法(HPLC)测定实施例3未载入的PTX含量
PTX-紫杉醇;FA-叶酸;PEI-聚乙烯亚胺;PEG-聚乙二醇;SPIONs-氧化铁纳米粒子。
取不同浓度的PTX溶液,测定不同浓度的PTX在228nm处的峰面积,然后以浓度为横坐标,对应的峰面积为纵坐标作图,得到标准曲线,将测定的未载入的PTX的峰面积带入标准曲线中,计算出未载入纳米粒子的PTX的量。药物的载药量和包封率公式计算如下:
载药量=载入纳米粒子的PTX的量/(PTX@FA/PEI/PEG-SPIONs重量);
包封率=载入纳米粒子的PTX的量/加入的PTX的总量。
实验例1
p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT纳米粒的表征
a.p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT粒径&Zeta电位及形貌
如图1和下表1所示,p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT水分散性良好,具有较高的稳定性,水和动力学粒径为186.7 9.9nm(PDI=0.29 0.01),Zeta电位为-10.35 2.39,在透射电子显微镜(TEM)中观察到的粒径为100.2±3.5nm。如图2所示,透射电镜下,p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT纳米粒呈球状纳米结构,分散性良好,表明组装效果良好。
表1不同时间p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT纳米组装体粒径变化
b、p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT的紫外光谱、标准曲线及红外光谱
紫外光谱和红外光谱图如图3-4所示,结合前期的数据分析,PTX和HCPT成功共载于 p-PS/FA@PEG/PEI-SPIONs纳米粒上。
实验例2
p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT的载药能力、释放能力及稳定性研究
通过药物累积释放能力的测定结果如图5所示,当p-PS/FA@PEG/PEI -SPIONs@PTX+HCPT纳米粒在pH=7.4体外环境下,释放时间达到20h时,仅39.5%的PTX 和41.3%的HCPT释放;在pH=5.0体外环境下,PTX和HCPT的累积释放量分别达到了78.3%和79.8%,结果表明该递药纳米粒可在肿瘤酸性条件下大量释放药物,实现药物的靶向性;在4℃放置2个月无明显的沉淀,粒径无明显变化,均在190nm左右,稳定性较好;p-PS/FA@ PEG/PEI-SPIONs@PTX+HCPT包封率达80%,载药量为15%左右。
实施例3
4)p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT纳米粒的体外靶向性研究
a.材料安全性评价
如图6所示,空白纳米粒p-PS/FA@PEG/PEI-SPIONs分别与鼻咽癌细胞CNE-1、CNE-2、 HNE-1和C666-1孵育48h后,细胞生存率均在80%以上,随着纳米粒p-PS/FA@ PEG/PEI-SPIONs浓度增加,细胞存活率略有降低但无显著性差异;体外安全性实验结果表明, p-PS/FA@PEG/PEI-SPIONs对鼻咽癌细胞无毒性。
b.p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT纳米粒对鼻咽癌细胞生长抑制率的研究
通过MTT实验进行了PTX、HCPT、PTX+HCPT混合物(摩尔比=1:1)、p-PS/FA@ PEG/PEI-SPIONs@PTX+HCPT和p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT+MF(0.3T)对鼻咽癌细胞系生长抑制率的研究。结果如图7所示。由于不同鼻咽癌细胞系的异质性以及对不同药物的敏感性差异,导致不同药物对不同鼻咽癌细胞的生长抑制程度不同。PTX和HCPT 通过自由扩散进入细胞后可直接作用于肿瘤细胞,而p-PS/FA@ PEG/PEI-SPIONs@PTX+HCPT经细胞摄取进入肿瘤细胞后,释放PTX和HCPT才能抑制肿瘤细胞的增殖,这是一个时间依赖和细胞内环境依赖的缓慢过程。与单一PTX或HCPT相比, PTX联合HCPT对CNE-1、CNE-2、HNE-1和C666-1细胞增殖的抑制作用明显优于单一药物,同时p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT纳米粒对细胞表现出比PTX+HCPT更强的抑制作用。通过与叶酸受体阴性细胞和阳性细胞对比发现,p-PS/FA@ PEG/PEI-SPIONs@PTX+HCPT纳米粒可通过受体-配体介导的途径显著靶向叶酸受体阳性癌细胞HNE-1,从而产生有效的细胞毒活性;在外加磁场(MF)的作用下p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT对四种鼻咽癌细胞生长均有更强的抑制作用。因此,p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT纳米粒可实现对鼻咽癌细胞的双重靶向作用,增强其抑制鼻咽癌细胞生长的作用。
c.Calcein/PI双染色评估PTX和HCPT的协同作用
Calcein/PI双染色实验中,空白纳米粒p-PS/FA@PEG/PEI-SPIONs处理的鼻咽癌细胞 CNE-1、CNE-2、HNE-1和C666-1均显示出鲜艳的绿色,见图8,这表明空白纳米粒对鼻咽癌细胞没有明显的细胞毒性。当鼻咽癌细胞被药物干预后,明显抑制鼻咽癌细胞的生长。同时,药物对不同鼻咽癌细胞呈现不同的作用。与单一使用PTX或HCPT相比,PTX+HCPT 联合用药导致细胞毒性增强,说明PTX和HCPT联合具有协同作用。而p-PS/FA@ PEG/PEI-SPIONs@PTX+HCPT纳米粒在磁场作用下,具有双重靶向作用,发挥更强的抑制鼻咽癌细胞增殖的能力,结果如图9所示。
d.p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT细胞摄取研究
为了确定p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT纳米粒被细胞摄取情况,p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT与鼻咽癌细胞细胞CNE-1、CNE-2、HNE-1和C666-1孵育不同时间(1h、2h和6h)后,通过DAPI染色液进行细胞核染色定位,如图9、图9-1、图9-2 和图9-3所示,可以观察到p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT纳米粒在与鼻咽癌细胞共同孵育1h后被细胞摄取的药物较少,其荧光强度较弱,零星分布于细胞中;共孵育2h后,药物PTX和HCPT集中分布于细胞核中,并且2h荧光强度高于孵育1h;在孵育6h后,鼻咽癌细胞细胞核处的药物累积量更多,其荧光强度高于1h和2h的荧光强度。同时,在外加磁场的作用下,细胞能摄取更多的药物,促进药物富集于鼻咽癌细胞。
e.p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT药代动力学研究
将20只雄性SD大鼠,根据体重随机分为4组,每组5只。分别为PTX组,HCPT组, PTX+HCPT组,p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT组,实验前禁食12h,自由饮水。分别尾静脉注射PTX(5mg/kg)、HCPT(2mg/kg)、PTX+HCPT(PTX:5mg/kg,HCPT:2mg/kg) 和p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT(PTX:5mg/kg,HCPT:2mg/kg)给药,分别于 0.038、0.083、0.25h、0.5h、1h、2h、4h、6h、8h、12h、24h、36h眼眶静脉取血0.3ml并置于预先肝素钠化的离心管中,在5000rpm转速下离心10min,分离血浆并保存于-20℃冰箱备用。血浆样品采用甲醇沉淀法处理,建立HPLC法测定血浆中PTX和HCPT的浓度并进行方法学验证实验,数据处理采用DAS2.0药代动力学软件,进行房室模型的拟合与药代动力学参数的计算,结果如下:
p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT中药物PTX和HCPT浓度-时间曲线下面积(ACU)为40.167 2.647μg·h/ml和9.267 3.14μg·h/ml,是PTX+HCPT混合物(PTX: 14.3671.291,HCPT:1.783 0.204)中药物的2.8倍和5.2倍。p-PS/FA@PEG/PEI- SPIONs@PTX+HCPT中PTX和HCPT的半衰期t1/2分别为6.279 0.627h和8.773 3.248h,而PTX+HCPT混合物中PTX和HCPT的半衰期t1/2分别为0.867 0.667h和0.457 0.0461,明显小于p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT半衰期。p-PS/FA@ PEG/PEI-SPIONs@PTX+HCPT中PTX和HCPT的平均停留时间分别为4.667 2.583h和 2.368 0.167,而PTX+HCPT混合物中PTX和HCPT平均停留时间分别为1.064 0.035h和 0.336 0.029h。因此,p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT纳米粒不仅能延长大鼠体内药物PTX和HCPT的有效浓度及作用时间,而且能有效降低体内清除率。本研究中药代动力学信息有助于p-PS/FA@PEG/PEI-SPIONs@PTX+HCPT在大鼠体内的评价,为该递药系统的开发应用提供科学依据。
Claims (9)
1.一种叶酸受体介导紫杉醇和羟基喜树碱超顺磁氧化铁纳米粒,其特征在于,所述的纳米粒为:p-PS/ FA@ PEG/PEI-SPIONs @PTX +HCPT。
2.如权利要求1所述的叶酸受体介导紫杉醇和羟基喜树碱超顺磁氧化铁纳米粒,其特征在于,其制备方法包括以下步骤:
A、将FA接枝在PEG/PEI-SPIONs纳米粒的表面上,获得FA@PEG/PEI-SPIONs;
B、将p-PS接枝到FA@PEG/PEI-SPIONs上,获得p-PS/ FA@ PEG/PEI-SPIONs;
C、将PTX和HCPT接枝到p-PS/ FA@ PEG/PEI-SPIONs上,获得p-PS/ FA@ PEG/PEI-SPIONs @PTX +HCPT。
3.如权利要求2所述的叶酸受体介导紫杉醇和羟基喜树碱超顺磁氧化铁纳米粒,其特征在于,所述的步骤A中,PEG/PEI-SPIONs纳米粒的制备方法包括以下步骤:
将PEG-1000和PEI-1800混合均匀,在8-12min内升温至78-82℃,得混合液;将Fe(acac)3添加到混合液中,在78-82℃下继续搅拌8-12min后,加热至250-270℃,保温0.5-1.5h后停止加热,冷却至50-70℃,得到含有PEG/PEI-SPIONs的混合液,分离得到PEG/PEI-SPIONs纳米粒;所述的PEG-1000、PEI-1800、Fe(acac)3的质量比为12-17:0.1-0.5:0.5-1;
上述整个过程在磁力搅拌下进行,并通入氩气除氧。
4.如权利要求3所述的叶酸受体介导紫杉醇和羟基喜树碱超顺磁氧化铁纳米粒,其特征在于,所述的分离PEG/PEI-SPIONs纳米粒的方法包括以下步骤:
向冷却后的含有PEG/PEI-SPIONs纳米粒的混合液中加入甲苯进行超声分散,用磁铁在烧杯底部进行吸附,当黑色磁性物质基本被吸附至烧杯底部后,弃去上清夜,重复该清洗过程1-3次后,黑色沉淀物再用丙酮清洗1-3次,即得。
5.如权利要求2所述的叶酸受体介导紫杉醇和羟基喜树碱超顺磁氧化铁纳米粒,其特征在于,所述的步骤A中将FA接枝在PEG/PEI-SPIONs纳米粒的表面的方法包括以下步骤:
将PEG/PEI-SPIONs分散于去离子水中,获得PEG/PEI-SPIONs分散液,然后在快速搅拌的条件下,将PEG/PEI-SPIONs分散液缓慢滴加到FA活性酯中,在磁力搅拌下反应8-36 h后,然后用LS磁选柱对样品进行分离,最后用透析袋MWCO 8000-14000 Da透析,去除游离叶酸,直到透析液没有紫外吸收,得到FA@PEG/PEI-SPIONs分散液。
6.如权利要求5所述的叶酸受体介导紫杉醇和羟基喜树碱超顺磁氧化铁纳米粒,其特征在于:
所述的FA活性酯的制备方法包括以下步骤:将FA溶解在DMSO中活化,然后加入EDC和sulfo-NHS,搅拌2-5h进行叶酸活化,得到FA活性酯;
所述的FA、DMSO、EDC、sulfo-NHS的摩尔比为:0.1-0.3:300-400:0.3-0.5: 0.3-0.5。
7.如权利要求2所述的叶酸受体介导紫杉醇和羟基喜树碱超顺磁氧化铁纳米粒,其特征在于:
所述的步骤B中,将p-PS接枝到FA@PEG/PEI-SPIONs上的方法包括以下步骤:
取p-PS溶解于DMF中,在搅拌状态下逐滴滴加FA@PEG/PEI-SPIONs,在800-1500 rpm转速下磁力搅拌12-36h,然后离心取沉淀;其中,p-PS、DMF、FA@PEG/PEI-SPIONs的重量比为:5-15:3000-7000:1-5;然后用CHCl3洗涤多次除去游离聚合物p-PS,得到p-PS/ FA@ PEG/PEI-SPIONs。
8.如权利要求7所述的叶酸受体介导紫杉醇和羟基喜树碱超顺磁氧化铁纳米粒,其特征在于:
所述的步骤C中将PTX和HCPT接枝到p-PS/ FA@ PEG/PEI-SPIONs上的方法包括以下步骤:
a、将制得的p-PS/ FA@ PEG/PEI-SPIONs溶解在CHCl3中,然后向该溶液中加入PTX和HCPT,获得混合液a;
b、将混合液a加入相当于DMF重量一半的浓度为2-8mg/ml的SDS溶液中,在120-200w功率下超声下乳化1-10分钟时间,获得乳化液;
c、将乳化液在室温下缓慢搅拌直至将CHCl3至完全挥干,在1500-3000 rpm/min转速下离心3-10 min,收集沉淀和,用CHCl3和蒸馏水交替清洗2-5次,最终获得p-PS/ FA@ PEG/PEI-SPIONs @PTX +HCPT纳米粒。
9.如权利要求1-8任何一项所述的叶酸受体介导紫杉醇和羟基喜树碱超顺磁氧化铁纳米粒在制备治疗鼻咽癌药物中的用途。
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