CN112791194A - 一种peg/pei修饰的磁性纳米颗粒的制备方法 - Google Patents

一种peg/pei修饰的磁性纳米颗粒的制备方法 Download PDF

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CN112791194A
CN112791194A CN202110024603.5A CN202110024603A CN112791194A CN 112791194 A CN112791194 A CN 112791194A CN 202110024603 A CN202110024603 A CN 202110024603A CN 112791194 A CN112791194 A CN 112791194A
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peg
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magnetic nanoparticle
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刘艳
李翔
陆云姝
顾芬芬
李若男
达先鸿
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XinHua Hospital Affiliated To Shanghai JiaoTong University School of Medicine
University of Shanghai for Science and Technology
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Abstract

本发明提供了一种磁性纳米颗粒的制备方法，其特征在于，由如下步骤制备而成:S1.向涡旋铁颗粒中加入醇后，超声使其分散；S2.经磁吸附分离，去除醇；S3.添加PEG和/或PEI、水后，超声乳化；S4.弃上清液后，用水和醇分别洗涤，收集沉淀干燥后即得到PEG和/或PEI修饰的磁性纳米颗粒。该纳米载体毒性低，效果好。

Description

一种PEG/PEI修饰的磁性纳米颗粒的制备方法
技术领域
本发明涉及生物医药领域，具体地，涉及一种纳米载体，更具体地，涉及一种PEG/PEI修饰的磁性纳米颗粒的制备方法及其应用。
背景技术
磁性纳米颗粒，由于纳米尺寸效应，展现出与宏观磁性材料截然不同的物理化学特性，比如：具有大的比表面积、超顺磁特性及具有多样化的拓扑磁结构等，再结合其表面易功能化等特点，可在恒定磁场下聚集和定位，使其在靶向药物载体及磁控药物释放、在交变磁场下吸收电磁波产热进行肿瘤热疗等生物医学领域被广泛应用。
现有技术中，利用肝癌特异性启动子和乏氧反应序列引导下游抑癌基因的表达构建肝癌特异性的治疗基因，并用磁性纳米颗粒PEI-Fe3O4作为基因载体运送治疗基因，从而将肿瘤靶向基因治疗与磁流体热疗结合起来，极大地提高治疗的安全性、特异性和有效性。
此外，Yang等发表了通过对超顺磁纳米颗粒表面包覆一层生物兼容性蛋白体等有机物作为药物载体，成功地应用于肿瘤靶向治疗，结果表明超顺磁纳米颗粒临床应用的可行性。
研究发现，磁性纳米Fe3O4粒子具有极好的生物相容性、无毒、靶向性强等特点，故可作为缓释靶向药物载体,靶向药物可通过体外磁场的导向作用直接定位作用于病变部位，达到减少药剂用量、降低药物毒副作用、提高药物治疗指数的目的。
Vivek等人利用超顺磁纳米铁构建了包裹抗癌药物的乳腺癌HER2靶向的纳米颗粒，结果表明该纳米颗粒具有良好的乳腺癌靶向性以及抗乳腺癌细胞增殖作用。
然而目前这种载体***存在如下缺陷：
1.载体***主动靶向性不足,磁性纳米颗粒在血液中的运动会受血流速度和磁场强度的共同影响，这意味着需要建立一个足够大的磁场强度对抗组织中的血液线性流速，使药物载体到达并维持在治疗区。
2.超顺磁颗粒过小的尺寸，容易被内质网吞噬不易在肿瘤组织定位和滞留。
3.超顺磁颗粒容易出现团聚。
因此，迫切需要寻找一种新型低毒、高效的生物医用磁性纳米颗粒来弥补超顺磁颗粒的不足，进而实现更佳的生物医学应用性能。
发明内容
为了解决上述问题，本发明通过调制纳米颗粒的尺寸及几何结构，发现了一种磁化闭合分布的独特磁结构—涡旋磁畴。具有磁涡旋结构的涡旋磁纳米颗粒，由于磁矩闭合分布，降低了杂散场，能有效削弱颗粒间的磁相互作用，从而避免颗粒团聚现象的发生。同时，涡旋磁纳米颗粒由于较大的颗粒尺寸，呈现出比超顺磁颗粒更高的磁化率和饱和磁化强度。此外，涡旋磁纳米颗粒较大的粒径尺寸，既可以在颗粒外包裹更多的生物分子(药物分子、荧光剂等)形成医用纳米团簇颗粒，又可以实现颗粒在肿瘤部位具有选择高通透性和滞留性，有利于其在肿瘤组织中富集和肿瘤治疗效果的提升。
本发明提供了一种磁性纳米颗粒的制备方法，其特征在于，由如下步骤制备而成:
S1.向涡旋铁颗粒中加入醇后，超声使其分散；
S2.经磁吸附分离，去除醇；
S3.添加PEG和/或PEI、水后，超声乳化；
S4.弃上清液后，用水和醇分别洗涤，收集沉淀干燥后即得到PEG和/或PEI 修饰的磁性纳米颗粒。
进一步地，本发明提供的的一种磁性纳米颗粒的制备方法，其特征还在于:
S1中涡旋铁颗粒与醇的配料比例为：每1mg涡旋铁颗粒添加0.1-1ml醇。
进一步地，本发明提供的的一种磁性纳米颗粒的制备方法，其特征还在于:
所述涡旋铁颗粒的制备方法为：
进一步地，本发明提供的的一种磁性纳米颗粒的制备方法，其特征还在于:
重复S1-S2至少两次。
进一步地，本发明提供的的一种磁性纳米颗粒的制备方法，其特征还在于:
在S1中，超声的时间为10-30min。
进一步地，本发明提供的的一种磁性纳米颗粒的制备方法，其特征还在于:
在S3中，PEG的用量为每1mg涡旋铁颗粒10mg以上的PEG：
PEI的用量为每1mg涡旋铁颗粒10ul以上的PEI；
水的用量为：每1mg涡旋铁颗粒添加0.1-1ml水；
所述PEI为PEI的水溶液，其质量百分比浓度为40-90％。
进一步地，本发明提供的的一种磁性纳米颗粒的制备方法，其特征还在于:
所述PEG选自分子量为1000以上的PEG中的一种或几种；
所述PEI选自分子量为800以上的PEI中的一种或几种。
进一步地，本发明提供的的一种磁性纳米颗粒的制备方法，其特征还在于:
在S3中，超声次数为两次以上。
进一步地，本发明提供的的一种磁性纳米颗粒的制备方法，其特征还在于:
在S3中，每次超声的时间为1-5min。
另外，本发明研究表明，采用上述方法制备的一种磁性纳米颗粒，其特征还在于，具有如下任一或任几种的用途：
作为药物载体；
作为具有缓释效果的药物载体；
作为具有靶向作用的药物载体。
另外，本发明还提供了一种载药磁性纳米颗粒，其特征在于:
将药物I/因子I通过PEG与上述方法制备的一种磁性纳米颗粒偶联；
和/或
将药物II/因子II通过PEI静电吸附在如上述方法制备的一种磁性纳米颗粒上。
上述药物和因子均可选用任何具有靶向或治疗作用的药物或银子。
该载药载体的制备方法为：将干燥后经PEG和/或PEI修饰的磁性纳米颗粒中加入指定量的药物/因子，进行超声乳化实现。
纳米颗粒和药物/因子的质量比例为5：0.1-10。
另外，本发明还提供了一种载药磁性纳米颗粒，其特征在于:通过如下制备方法获得：
S1.将药物I/因子I通过PEG与上述方法制备的一种磁性纳米颗粒偶联；
和/或
将药物II/因子II通过PEI静电吸附在上述方法制备的一种磁性纳米颗粒上；
S2.通过超声将仿生用的细胞膜包裹在S1的复合纳米材料上。
该载药薄膜载体的制备方法为：将干燥后经PEG和/或PEI修饰的磁性纳米颗粒中加入指定量的药物/因子，进行超声乳化后，加入细胞膜溶液，经超声、离心、洗涤后，用去离子水重悬得到细胞膜包裹的携载DOX的PEG和/或PEI修饰的磁性纳米颗粒。
纳米颗粒和药物/因子的质量比例为5：0.1-10。
细胞膜溶液的添加量为每mg纳米颗粒添加1-20ul细胞膜溶液。
附图说明
图1为磁性纳米颗粒TEM形态图；
其中，图1a为磁性纳米环TEM形态图；
图1b为磁性纳米环TEM形态图；
图1c为磁性能测试结果；
图2为不同用量的PEG修饰下的纳米环红外谱图；
图3为不同用量的PEG修饰下的纳米棒红外谱图；
图4为PEG修饰的纳米环的稳定性比对图；
其中，图4a为初始状态图；
图4b为20min后的状态图；
图4c为40min后的状态图；
图5a为不同用量的PEG修饰下的纳米棒环载DOX后的红外谱图；
图5b.PEG-Fe3O4携载DOX的紫外图；
图6为不同用量的PEI修饰下的纳米环红外谱图；
图7为PEI修饰的纳米棒的电位图；
其中，图7a为裸棒电位图；
图7b为500ulPEI的电位图；
图7c为1000ulPEI的电位图。
图8.PEG、PEI包覆棒状Fe3O4红外图谱；
图9.PEG、PEI包覆棒状Fe3O4电位图；
图10.PEG/PEI-Fe3O4携载DOX载药率；
图11.PEG/PEI-Fe3O4携载DOX包封率；
图12.环状、棒状PEG/PEI-Fe3O4包膜后携载2mgDOX的包封率和载药率；
图13.环状、棒状PEG/PEI-Fe3O4包膜后携载2mgDOX的的释药率。
图14.环状、棒状PEG/PEI-Fe3O4包膜后携载2mgDOX的的释药率折线图。
具体实施方式
实施例1.涡旋铁颗粒的制备：
实施例1-1环状涡旋铁颗粒的制备：
用水热法合成α-Fe2O3纳米环，反应物是FeCl3·6H2O，引入的表面活性剂是NH4H2PO4和Na2SO4。称取0.43g的FeCl3·6H2O，0.00092g的NH4H2PO4和 0.00625g的Na2SO4，将其溶解于蒸馏水中，将混合物放在磁力搅拌器上剧烈搅拌至混合均匀后，转移到不锈钢高压釜中，放到220℃的烘箱中，恒温反应48小时,冷却至室温后，洗涤，离心，得到所需沉淀物。最后干燥，获得所需α-Fe2O3纳米环颗粒。再将制备的α-Fe2O3作为原料，通过氢还原法得到Fe3O4纳米环，得最终产物环状Fe3O4纳米颗粒。TEM如图1a所示。
实施例1-2棒状涡旋铁颗粒的制备：
称0.432g的FeCl3·6H2O和0.0046gNH4H2PO4，加入少量的蒸馏水溶解并剧烈搅拌，当溶质完全溶解后，将混合物转移到不锈钢高压釜中，在205℃下水热处理48小时，冷却至室温后，洗涤，在60℃下干燥24小时，即获得所需的棒状α-Fe2O3纳米颗粒。再使用制备好的α-Fe2O3作为原料，通过氢还原法制备 Fe3O4纳米棒。将干燥的α-Fe2O3粉末在连续氢气/氩气流中在500℃下退火，然后冷却至室温，同时保持连续的氢气流，即得最终产物棒状Fe3O4纳米颗粒。TEM 如图1b所示。
实施例1-3球状涡旋铁颗粒。
如图1c所示，各种形态的磁性能测试结果表明，环和棒的磁性能较为
实施例2.PEG修饰的磁性纳米颗粒的制备：
称量实施例1中制备好的涡旋铁颗粒20mg，加入4ml无水乙醇，超声20分钟(根据不同物料量的要求，该超声时间可调控范围为10-30min)使其分散于无水乙醇。后磁吸附分离，弃去无水乙醇，重复两次。
称取不同量的PEG4000进行实验(在本实施例中，选用了200mg、500mg、 1000mg、2000mg进行实验例的制备，PEG可选用1000以上分子量的PEG，优选为PEG2000和PEG4000)，加去离子水10ml(根据不同物料量的要求，该水的用量可调控范围为5-20ml)，超声20分钟(根据不同物料量的要求，该超声时间可调控范围为10-30min)，再将其超声乳化数次，每次3分钟(根据不同物料量的要求，该超声时间可调控范围为1-5min)。离心弃去上清，用去离子水洗三遍，乙醇洗三遍。收集沉淀干燥，即得到PEG修饰的磁性纳米颗粒。
实验例2-1.红外分析结果：
如图2所示，与裸铁相比。PEG修饰的铁在580cm-1附近显示出较强的特征吸收峰，在1104cm-1处出现了比较强的C-O-C吸收峰，在1623cm-1附近也有 C＝O弯曲振动峰，在2884cm-出现了C-H振动峰，在3385cm-1附近也出现了羟基 O-H的伸缩振动峰，这些明显的基团特征吸收峰的出现表明PEG成功的修饰在粒子中。
如图3所示，同样，当纳米棒包覆PEG后，其铁的特征峰减小，并且有出现PEG中基团的吸收峰，说明PEG成功地修饰在铁表面。
实验例2-2.稳定性实验：
如图4a，4b，4c所示，当PEG的添加量为200mg时，沉静20min即出现了明显的固液分离现象；
当PEG的添加量为500mg时，沉静40min即出现了明显的固液分离现象；
显然，当PEG的添加量为2000mg时，其稳定性明显优于其他用量。
实验例2-3.毒性实验(采用CCK8试剂盒)：
A.纳米棒对231细胞24h的毒性结果如下表所示(PEG用量2000mg)：
Figure RE-GDA0003003444050000071
B.纳米环对231细胞24h的毒性结果如下表所示：
Figure RE-GDA0003003444050000072
从上述数据可以发现，无论环状还是棒状纳米材料基本无毒性。
实施例3.以实施例2的磁性纳米颗粒(PEG用量2000mg)为载体制备协载 DOX的颗粒：
将干燥后的PEG修饰的磁性纳米颗粒称取5mg，加入2mgDOX，超声乳化。
实验例3-1.红外分析结果：
如图5a所示，从图中可以发现峰波动强烈，且铁的特征峰减小，说明有DOX 在铁表面。
实验例3-2.紫外分析结果(见图5b)。
实施例4.PEI修饰的磁性纳米颗粒的制备：
称量制备好的涡旋铁颗粒20mg，加入4ml无水乙醇，超声20分钟使其分散于无水乙醇。后磁吸附分离，弃去无水乙醇，重复两次。
称取不同量的PEI(分子量1300，浓度为50％的水溶液密度是1.08g/ml浓度0.54g/ml)进行实验(在本实施例中，选用了200ul、500ul、1000ul进行实验例的制备，PEI优选采用分子量为1000以上的PEI)，加去离子水10ml，超声20分钟，再将其超声乳化数次，每次3分钟。离心弃去上清，用去离子水洗三遍，乙醇洗三遍。收集沉淀干燥，即得到PEI修饰的磁性纳米颗粒。
实验例4-1.红外分析结果：(从下至上200，500，1000)
如图6所示，有明显的PEI特征峰，说明PEI成功的修饰在铁表面。
实验例4-2.电位结果：
如图7所示，棒包PEI 500ul的电位：8.7mV
棒包PEI 1000ul的电位：35.6mV
由此可见，当负载PEI后电位均优于裸棒状态，当PEI的用量为1000ul时，显然电位能达到最佳预期值。
实施例5.PEG/PEI共同修饰的磁性纳米颗粒的制备：
称量制备好的涡旋铁颗粒20mg，加入4ml无水乙醇，超声20分钟使其分散于无水乙醇。后磁吸附分离，弃去无水乙醇，重复两次。称取适量2000mgPEG4000， 1000ul PEI(分子量1300，浓度为50％的水溶液密度是1.08g/ml，浓度0.54g/m) 加去离子水10ml，超声20分钟，再将其超声乳化数次，每次3分钟。离心弃去上清，用去离子水洗三遍，乙醇洗三遍。收集沉淀干燥，即得到PEG/PEI修饰的磁性纳米颗粒。
实验例5-1.红外分析结果(棒状)：
如图8所示，与裸铁相比，PEG，PEI修饰的纳米颗粒除了在1045cm-1、1408cm-1附近显示出PEG特征吸收峰之外，在1623cm-1处出现N-H弯曲振动吸收峰，在 2857cm-1、2922cm-1出现了C-H的伸缩振动峰，在3445cm-1附近出现了N-H的伸缩振动峰，这些明显的基团特征吸收峰的出现表明PEG和PEI成功的修饰在粒子中。
实验例5-2.电位结果(棒状)：
如图9所示，PEI-PEG-Fe3O4棒电位为：50.8mV。
实施例6.以实施例5的磁性纳米颗粒为载体制备协载DOX的颗粒：
将干燥后的实施例5所制备的PEG/PEI修饰的磁性纳米颗粒称取5mg，加入特定量的DOX(根据靶向药物的不同，该药物的添加量可进行调整)，超声乳化。
实验例6-1.载药率结果：
如图10所示，5mg的PEG/PEI-Fe304，分别加入1、2、3mg的DOX。
环状：载药率为26.35％；棒状铁载药率为：28.436％。
实验例6-2.包封率结果：
如图11所示，环状铁在DOX投入1mg和2mg时相差不多，为56％左右；
棒状铁DOX投入2mg时最好，为62％左右。
实施例7.以实施例5的磁性纳米颗粒包膜后为载体制备协载DOX的颗粒：
制备方法如图12所示，关于包膜和载药过程为：将干燥后的PEG/PEI修饰的磁性纳米颗粒称取5mg，加入2mgDOX，超声乳化。加入50ul事先提取的细胞膜溶液，超声三分钟。然后离心，洗涤，后用去离子水重悬得到细胞膜包裹的携载DOX的PEG/PEI修饰的磁性纳米颗粒。
实验例7-1.载药率和包封率结果：
如图12所示，环状铁包封率70％，载药量28％；
棒状铁包封率78％，载药量31％；
较未包细胞膜的磁性纳米颗粒均要好。
实验例7-2.缓释结果：
如图13所示，环的缓释率为：65.79％；
棒的缓释率为：59.21％；
随后还会缓慢地释放，说明其作为药物载体在释药方面都可以达到要求。

Claims (10)

1.一种磁性纳米颗粒的制备方法，其特征在于，由如下步骤制备而成:
S1.向涡旋铁颗粒中加入醇后，超声使其分散；
S2.经磁吸附分离，去除醇；
S3.添加PEG和/或PEI、水后，超声乳化；
S4.弃上清液后，用水和醇分别洗涤，收集沉淀干燥后即得到PEG和/或PEI修饰的磁性纳米颗粒。
2.如权利要求1所述的一种磁性纳米颗粒的制备方法，其特征在于:
S1中涡旋铁颗粒与醇的配料比例为：每1mg涡旋铁颗粒添加0.1-1ml醇。
3.如权利要求1所述的一种磁性纳米颗粒的制备方法，其特征在于:
重复S1-S2至少两次。
4.如权利要求1所述的一种磁性纳米颗粒的制备方法，其特征在于:
在S1中，超声的时间为10-30min。
5.如权利要求1所述的一种磁性纳米颗粒的制备方法，其特征在于:
在S3中，PEG的用量为每1mg涡旋铁颗粒10mg以上的PEG：
PEI的用量为每1mg涡旋铁颗粒10ul以上的PEI；
水的用量为：每1mg涡旋铁颗粒添加0.1-1ml水；
所述PEI为PEI的水溶液，其质量百分比浓度为40-90％。
6.如权利要求1所述的一种磁性纳米颗粒的制备方法，其特征在于:
在S3中，超声次数为两次以上。
7.如权利要求1所述的一种磁性纳米颗粒的制备方法，其特征在于:
在S3中，每次超声的时间为1-5min。
8.采用如权利要求1-7任一方法制备的一种磁性纳米颗粒，其特征在于，具有如下任一或任几种的用途：
A.作为药物载体；
B.作为具有缓释效果的药物载体；
C.作为具有靶向作用的药物载体。
9.一种载药磁性纳米颗粒，其特征在于:
将药物I/因子I通过PEG与如权利要求1-7任一方法制备的一种磁性纳米颗粒偶联；
和/或
将药物II/因子II通过PEI静电吸附在如权利要求1-7任一方法制备的一种磁性纳米颗粒上。
10.一种载药磁性纳米颗粒，其特征在于，通过如下制备方法获得：
S1.将药物I/因子I通过PEG与如权利要求1-7任一方法制备的一种磁性纳米颗粒偶联；
和/或
将药物II/因子II通过PEI静电吸附在如权利要求1-7任一方法制备的一种磁性纳米颗粒上；
S2.通过超声将仿生用的细胞膜包裹在S1的复合纳米材料上。
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