CN107773757A - 一种多靶点逆转多药耐药的肿瘤双重靶向磁性纳米颗粒、制备方法及应用 - Google Patents
一种多靶点逆转多药耐药的肿瘤双重靶向磁性纳米颗粒、制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种多靶点逆转多药耐药的肿瘤双重靶向磁性纳米颗粒、制备方法及应用,包括Fe3O4的磁性内核,包裹在内核上的带有正电荷的聚合物,以及位于聚合物外的透明质酸构成的修饰层。本发明设计了一种可多靶点逆转肿瘤多药耐药的双重靶向磁性抗肿瘤给药系统,制备简单,构思巧妙,解决了单一靶点逆转多药耐药疗效差的问题,并且具有双重靶向作用,获得了较好的治疗效果。
Description
技术领域
本发明涉及一种磁性纳米材料,尤其涉及的是一种多靶点逆转多药耐药的肿瘤双重靶向磁性纳米颗粒、制备方法及应用。
背景技术
癌症由复杂的多种基因突变所致,其发病过程涉及基因的动态变化、复杂的信号网络以及避免细胞编程性死亡等多重机制,因此肿瘤的治疗极具挑战性。近年来,联合治疗的策略在各类肿瘤治疗领域中取得了令人瞩目的成就,无论是化学药物之间的联合还是化疗和其他治疗方式比如放疗的联合,其疗效提高的原因都在于同时影响肿瘤的多重发病机制。
小分子化疗药物作用于肿瘤细胞生长繁殖的不同环节,抑制或杀死肿瘤细胞,是目前治疗肿瘤的主要手段之一。然而,许多肿瘤常规化疗效果差,预后不良是困扰临床的重要难题。对于90%的原发性和转移性肿瘤的治疗,细胞多药耐药性是一个非常重要且持续的障碍。经治疗后,残存的肿瘤干细胞耐药性形成,常导致对多种药物敏感性降低,并引起肿瘤复发甚至转移,因此肿瘤多药耐药性(MDR)则是肿瘤化疗失败的关键因素,已成为当今医学界研究的热点。根据肿瘤细胞多药耐药机制,人们先后应用钙离子通道阻滞剂、免疫抑制剂、蛋白激酶抑制剂以及中药等作为逆转剂与小分子化疗药物联合应用,均取得了不同程度的作用效果,但是这些药物的副作用较大,因此临床应用受到限制。还有研究者制备了各类纳米载体,载体被细胞通过内吞的形式摄入,虽然药物可以顺利进入细胞,但是细胞的多药耐药蛋白仍很活跃,药物还是能够被大量外排出来,使疗效受到一定影响。
发明内容
本发明的目的在于克服现有技术的不足,提供了一种多靶点逆转多药耐药的肿瘤双重靶向磁性纳米颗粒、制备方法及应用,能够提高抗肿瘤效果。
本发明是通过以下技术方案实现的,本发明的一种多靶点逆转多药耐药的肿瘤双重靶向磁性纳米颗粒,包括Fe3O4的磁性内核,包裹在内核上的带有正电荷的聚合物,以及位于聚合物外的透明质酸构成的修饰层。
作为本发明的优选方式之一,所述透明质酸的分子量为5000~105Da。
作为本发明的优选方式之一,所述透明质酸上装载有siRNA。
作为本发明的优选方式之一,所述siRNA选自Pgp-siRNA、VEGF-siRNA、MRP-siRNA、BCRP-siRNA至少一种。MRP-siRNA选自MRP1-siRNA、MRP2-siRNA、MRP3-siRNA、MRP4-siRNA、MRP5-siRNA、MRP6-siRNA、MRP7-siRNA、MRP8-siRNA。
作为本发明的优选方式之一,所述siRNA与磁性纳米颗粒的重量比为1:4~1:10。
作为本发明的优选方式之一,所述带有正电荷的聚合物为聚乙烯亚胺。
作为本发明的优选方式之一,所述聚乙烯亚胺分子量范围为1800~150000Da。
作为本发明的优选方式之一,聚乙烯亚胺为分枝状或者线状。
一种多靶点逆转多药耐药的肿瘤双重靶向磁性纳米颗粒的制备方法,包括以下步骤:
(1)将四水合氯化亚铁与六水合氯化铁溶于去离子水,剧烈搅拌,将氨水快速加入,调整反应体系的pH值至9~10,分别在60℃温度下持续搅拌2h;
(2)反应结束后,磁铁分离,用蒸馏水反复洗涤直至PH=7,除去上层清液,真空干燥,研磨即得纳米磁性Fe3O4粒子,密闭保存;
(3)取适量产物分散在双蒸水中,将带正电的聚合物分散在蒸馏水中,然后缓慢滴入上述溶液,继续搅拌2h,产物磁性分离,蒸馏水和乙醇洗涤至中性,真空干燥;
(4)取上一步产物超声分散在双蒸水中;
(5)将透明质酸溶解在双蒸水中,缓慢滴加入上述溶液,搅拌,反应结束后,磁铁分离,用蒸馏水反复洗涤直至PH=7,除去上层清液,真空干燥,研磨即可。
一种所述的磁性纳米颗粒在制备抗肿瘤药物中的应用。
一种所述的磁性纳米颗粒组合小分子化疗药物在制备抗肿瘤药物中的应用。
作为本发明的优选方式之一,所述小分子化疗药物为阿霉素。
本发明先制备Fe3O4纳米核心,然后包覆带正电荷的聚合物,之后再采用透明质酸修饰纳米粒,用于装载小干扰RNA。
本发明采用磁性纳米粒的被动靶向性和透明质酸与肿瘤上高度表达的CD44受体特异性结合介导的主动靶向性相结合的双重靶向机理,使载体获得高靶向性,减少对正常细胞的伤害。
本发明采用带正电荷的聚合物对磁性纳米粒进行修饰,利用正电荷包载核苷类物质形成复合物,可保护核苷类物质不被破坏,且该类物质具有质子海绵的效应,有利于核苷酸从细胞内涵体中逃离,以获得高的转染效率和治疗效果。
本发明采用小干扰RNA降低逆转多药耐药基因的表达,也可起通过透明质酸起到逆转多药耐药的作用,因此具有多靶点逆转多药耐药的作用。
本发明相比现有技术具有以下优点:本发明设计了一种可多靶点逆转肿瘤多药耐药的双重靶向磁性抗肿瘤给药系统,制备简单,构思巧妙,解决了单一靶点逆转多药耐药疗效差的问题,并且具有双重靶向作用,获得了较好的治疗效果。
附图说明
图1是未包裹前的Fe3O4纳米颗粒透射电镜图;
图2是PEI-Fe3O4纳米颗粒透射电镜图;
图3是HA-PEI-Fe3O4纳米颗粒透射电镜图;
图4是PEI-Fe3O4纳米粒子红外光谱图;
图5是Fe3O4纳米颗粒X射线衍射图;
图6是PEI-Fe3O4热重分析图;
图7是转染效率考察图,a为空白细胞组;b为Fe3O4组;c为PEI-Fe3O4组;d为HA-PEI-Fe3O4组;
图8是胞内阿霉素浓度考察图,a为BEL-7402组,b为BEL/5-FU组,c为BEL/5-FU+PEI-Fe3O4/siRNA组,BEL/5-FU+HA-PEI-Fe3O4/siRNA组;
图9是流式细胞仪检测细胞内ADR表达率分析图。
具体实施方式
下面对本发明的实施例作详细说明,本实施例在以本发明技术方案为前提下进行实施,给出了详细的实施方式和具体的操作过程,但本发明的保护范围不限于下述的实施例。
实施例1
本实施例制备一种多靶点逆转多药耐药的肿瘤双重靶向磁性纳米颗粒的过程如下:
将0.8900g四水合氯化亚铁与2.1000g六水合氯化铁溶于30ml去离子水,超声分散6min,0.22μm微孔滤膜过滤后加入到三口烧瓶中,在氮气保护下冰水冷却到2-4℃,剧烈搅拌下,将氨水快速加入到三口烧瓶中,加入4.0ml、氨水,调整反应体系的pH值至9-10,分别在60℃温度下持续搅拌2h。反应结束后,磁铁分离,用蒸馏水反复洗涤直至PH=7,除去上层清液,真空干燥1h,研磨即得纳米磁性Fe3O4粒子,密闭保存。取100mg产物分散在20ml双蒸水中,50mg PEI分散在10ml蒸馏水中,然后缓慢滴入上述溶液,继续搅拌2h,产物磁铁分离,顺序采用蒸馏水和乙醇洗涤至中性,真空干燥。取100mg的PEI-Fe3O4纳米颗粒,分散在50ml双蒸水中,超声分散;分别将HA(MW 10KDa)、HA(MW 50KDa)100mg溶解在50ml双蒸水中,缓慢滴加入上述PEI-Fe3O4溶液,搅拌8h,反应结束后,磁铁分离,用蒸馏水反复洗涤直至PH=7,除去上层清液,真空干燥1h,研磨即得HA-PEI-Fe3O4粒子,密闭保存。
实施例2
1、透射电镜表征
将实施例1制备得到的Fe3O4、PEI-Fe3O4、HA-PEI-Fe3O4按照0.1mg/ml的浓度超声分散在水中并滴在覆盖碳膜的铜网上晾干。如图1~3所示,用透射电镜(TEM,JEOL-2010)观察其分散形貌、粒径大小及包覆情况。可见Fe3O4颗粒粒径为10nm左右,呈现一定聚集度;PEI-Fe3O4颗粒聚集度减小,外包覆一层半透明的薄膜;HA-PEI-Fe3O4颗粒在HA的交联下颗粒粒径增大,外包覆的薄膜增厚,说明HA形成了一定的交联作用,颗粒有所增大。
2、X射线衍射
采用X射线衍射分析仪(XRD)对PEI-Fe3O4、HA-PEI-Fe3O4磁性纳米粒进行物相分析。如图5所示,2θ=30.26°,35.61°,43.64°,53.91°,57.33°,62.84°处的六个明显衍射峰分别对应于(220),(311),(400),(422),(511),和(440)的六个晶面,这表明通过改进化学共沉淀法制备所得的产物为Fe3O4,并具有立方晶系结构,表面修饰PEI并没有改变Fe3O4的晶体结构。采用谢乐公式计算β=Kλ/D cosθ,其中β为半峰宽,K为峰形因子(一般取0.89),λ为X射线波长,D为晶体的半径,计算的粒子的结晶大小约为12nm。从衍射谱图看,峰的位置、强度结果与粉末衍射PDF卡上的纯的Fe3O4标准数据基本吻合。
3、红外光谱分析(FT-IR)
采用红外光谱仪FT-IR检测PEI-Fe3O4纳米粒子的结构,其红外光谱如图4所示。600cm-1出现的是Fe3O4的特征吸收峰;1388-1637cm-1出现的数个吸收峰是聚乙烯亚胺的-NH2的剪切振动峰(1560cm-1左右)造成的;3453cm-1对应于PEI-Fe3O4磁性纳米粒中PEI亚甲基(CH2)不对称伸缩振动峰νas,由此可见PEI已经成功包覆于Fe3O4磁性纳米粒表面。
4、热重分析
为了了解PEI-Fe3O4、HA-PEI-Fe3O4磁性纳米粒子表面PEI的包覆情况,采用TGA分析仪对PEI-Fe3O4在氮气氛围内进行热重分析,如图6所示,10℃/min的速度升温到600℃,失重分为两个阶段,第一阶段温度在173.39℃以下失重2.44%,显示为PEI-Fe3O4磁性纳米粒子表层中水的蒸发;第二阶段是到458.08℃的失重占5.946%,这是由于PEI分解造成的失重。从失重曲线上计算看出PEI在PEI-Fe3O4磁性纳米粒子颗粒上的包覆率为5.946%。
5、粒径分析
采用马尔文ZS90激光纳米粒度仪对Fe3O4、PEI-Fe3O4、HA-PEI-Fe3O4溶液进行粒度检测,结果显示见下表1。结果显示Fe3O4粒子稍有团聚,粒径为159nm左右,表面电荷呈现弱的正电荷;包覆上PEI后电荷变为+35mv,粒子间斥力增加,体系趋向稳定;包载上HA后电荷转变为-56mv左右,表面电荷发生转变,说明HA较好地包覆住PEI-Fe3O4粒子表面,颗粒显电负性,且粒子由于HA的架桥作用呈现颗粒增大的趋势。
表1粒度检测结果
实施例3
本实施例检测双重靶向肿瘤的磁性纳米颗粒多靶点逆转多药耐药的作用,选择BEL7402、BEL7402/5-FU耐药细胞为实验对象。荧光标记针对多药耐药蛋白的Pgp-FAMsiRNA和Pgp-siRNA,以及其他siRNA均购买于上海吉玛生物有限公司,按照siRNA与载体重量比1:8的比例制备得到磁性纳米载体负载siRNA的PEI-Fe3O4/Fam-siRNA、HA-PEI-Fe3O4/Fam-siRNA、PEI-Fe3O4/siRNA、HA-PEI-Fe3O4/siRNA复合体系;其中复合材料含量和siRNA的负载量可通过调节原溶液的浓度调整。
所述Pgp-siRNA的序列分别为:
Pgp-siRNA:Sense strand:5’-AAGAAGGAAAAGAAACCAACUdTdT-3’;
Antisense strand:5’-dTdTUUCUUCCUUUUCUUUG GUUGA-3’。
1、转染效率的考察
取细胞浓度为1×104/mL的BEL7402、BEL7402/5-FU细胞接种在培养皿中,置于37℃,5%CO2培养箱中培养24h。待贴壁后,每个孔中分别加入上述PEI-Fe3O4/Fam-siRNA、HA-PEI-Fe3O4/Fam-siRNA,使体系中含20μM纳米载体和200nM的FAM-siRNA,分别在37℃下孵育细胞不同时间间隔。弃去培养基,细胞用PBS冲洗3次,并用Lyso Tracker溶酶体荧光探针染色1h后,再次用PBS洗涤3次。共聚焦显微镜免疫荧光法观测对照组、Naked Fam-siRNA、PEI-Fe3O4/Fam-siRNA组、HA-PEI-Fe3O4/Fam-siRNA组在BEL7402肝癌细胞中荧光蛋白表达,考察载体对体外转染效率的影响。如图7所示,结果显示,PEI-Fe3O4及HA-PEI-Fe3O4均可提高Fam-siRNA的荧光表达率。
2、逆转多药耐药作用考察
将对数生长期的BEL7402、BEL7402/5-FU细胞,消化,单细胞重悬与RPM1-1604培养基中,以5×105/mL接种到培养瓶中。分别将BEL7402、BEL7402/5-FU细胞分别采用10μM的阿霉素孵育2h,其中BEL7402/5-FU耐药肝癌细胞分为空白BEL7402/5-FU细胞组、BEL7402/5-FU+PEI-Fe3O4/siRNA组、BEL7402/5-FU+HA-PEI-Fe3O4/siRNA组,体系中含20μM纳米载体和200nM的FAM-siRNA。流式细胞仪检测各组细胞中荧光表达,考察载体对胞内阿霉素浓度的影响,ADR激发波长480nm,发射波长575nm。如图8和图9所示,结果显示,BEL7402/5-FU细胞中荧光很弱;敏感细胞中荧光较强。BEL7402/5-FU细胞内ADR的荧光强弱顺序依次为:BEL7402组>HA-PEI-Fe3O4组>PEI-Fe3O4组>BEL7402/5-FU组。可见PEI-Fe3O4可显著提高胞内阿霉素浓度,且HA的加入进一步增加胞内阿霉素浓度,说明该载体体系可多靶点逆转多药耐药,提高胞内阿霉素的浓度,增加治疗效果。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (12)
1.一种多靶点逆转多药耐药的肿瘤双重靶向磁性纳米颗粒,其特征在于,包括Fe3O4的磁性内核,包裹在内核上的带有正电荷的聚合物,以及位于聚合物外的透明质酸构成的修饰层。
2.根据权利要求1所述的一种多靶点逆转多药耐药的肿瘤双重靶向磁性纳米颗粒,其特征在于,所述透明质酸的分子量为5000~105Da。
3.根据权利要求1所述的一种多靶点逆转多药耐药的肿瘤双重靶向磁性纳米颗粒,其特征在于,所述透明质酸上装载有siRNA。
4.根据权利要求3所述的一种多靶点逆转多药耐药的肿瘤双重靶向磁性纳米颗粒,其特征在于,所述siRNA选自Pgp-siRNA、VEGF-siRNA、MRP-siRNA、BCRP-siRNA至少一种。
5.根据权利要求3所述的一种多靶点逆转多药耐药的肿瘤双重靶向磁性纳米颗粒,其特征在于,所述siRNA与磁性纳米颗粒的重量比为1:4~1:10。
6.根据权利要求3所述的一种多靶点逆转多药耐药的肿瘤双重靶向磁性纳米颗粒,其特征在于,所述带有正电荷的聚合物为聚乙烯亚胺。
7.根据权利要求6所述的一种多靶点逆转多药耐药的肿瘤双重靶向磁性纳米颗粒,其特征在于,所述聚乙烯亚胺分子量范围为1800~150000Da。
8.根据权利要求6所述的一种多靶点逆转多药耐药的肿瘤双重靶向磁性纳米颗粒,其特征在于,聚乙烯亚胺为分枝状或者线状。
9.如权利要求1~8任一项所述的一种多靶点逆转多药耐药的肿瘤双重靶向磁性纳米颗粒的制备方法,其特征在于,包括以下步骤:
(1)将四水合氯化亚铁与六水合氯化铁溶于去离子水,剧烈搅拌,将氨水快速加入,调整反应体系的pH值至9~10,分别在60℃温度下持续搅拌2h;
(2)反应结束后,磁铁分离,用蒸馏水反复洗涤直至PH=7,除去上层清液,真空干燥,研磨即得纳米磁性Fe3O4粒子,密闭保存;
(3)取适量产物分散在双蒸水中,将带正电的聚合物分散在蒸馏水中,然后缓慢滴入上述溶液,继续搅拌2h,产物磁性分离,蒸馏水和乙醇洗涤至中性,真空干燥;
(4)取上一步产物超声分散在双蒸水中;
(5)将透明质酸溶解在双蒸水中,缓慢滴加入上述溶液,搅拌,反应结束后,磁铁分离,用蒸馏水反复洗涤直至PH=7,除去上层清液,真空干燥,研磨即可。
10.一种如权利要求1所述的磁性纳米颗粒在制备抗肿瘤药物中的应用。
11.一种如权利要求1所述的磁性纳米颗粒组合小分子化疗药物在制备抗肿瘤药物中的应用。
12.根据权利要求11所述的一种应用,其特征在于,所述小分子化疗药物为阿霉素。
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CN108543071A (zh) * | 2018-04-23 | 2018-09-18 | 闫金银 | 用于乳腺癌联合精准治疗的递药系统及其应用 |
CN109620969A (zh) * | 2019-01-30 | 2019-04-16 | 广东药科大学 | 一种针对耐药肿瘤细胞的靶向抗肿瘤药物体系及其构建方法 |
CN110246641A (zh) * | 2019-05-05 | 2019-09-17 | 桂林理工大学 | 一种制备良好分散性的降解透明质酸修饰的超顺磁性氧化铁纳米粒子的方法 |
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CN108543071A (zh) * | 2018-04-23 | 2018-09-18 | 闫金银 | 用于乳腺癌联合精准治疗的递药系统及其应用 |
CN109620969A (zh) * | 2019-01-30 | 2019-04-16 | 广东药科大学 | 一种针对耐药肿瘤细胞的靶向抗肿瘤药物体系及其构建方法 |
CN110246641A (zh) * | 2019-05-05 | 2019-09-17 | 桂林理工大学 | 一种制备良好分散性的降解透明质酸修饰的超顺磁性氧化铁纳米粒子的方法 |
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