CN110123765B - 一种千金子甾醇磁性靶向微球制剂的制备方法和应用 - Google Patents
一种千金子甾醇磁性靶向微球制剂的制备方法和应用 Download PDFInfo
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Abstract
本发明涉及医药科技领域,具体涉及一种千金子甾醇磁性靶向微球制剂的制备方法和应用。所述千金子甾醇磁性靶向微球制剂,包含以下组分:千金子甾醇为4‑10wt%,DIB‑PEG‑NH2修饰的磁性四氧化三铁为40‑80wt%,叶酸修饰的聚乳酸‑羟基乙酸共聚物为10‑50wt%。本发明是基于生物可降解载体聚乳酸‑羟基乙酸共聚物构建难溶性药物多功能靶向递送系统,具有延长药物半衰期、减少毒副作用、同时具有肿瘤靶向功能等特点。本发明制备的为微球制剂进入体内后,在体外外加磁场的作用下及主动靶向作用下定向移动,定位在癌靶器官病变组织浓集,达到高效治疗的目的。
Description
技术领域
本发明涉及医药科技领域,具体涉及一种千金子甾醇磁性靶向微球制剂的制备方法和应用。
背景技术
本发明应用的千金子甾醇(Euphorbiasteroid),又名千金子素L1(Euphorbiafactor L1)、大戟因子L1,为大戟科植物续随子 Euphorbia lathyris L.干燥成熟种子千金子中提取出的有效活性成分,因此又名续随二萜酯,也是2015版《中国药典》的指标成分。千金子甾醇粉末为无色针晶,化合物极性小,难溶于水,易溶于一些有机溶剂,如乙醚、乙酸乙酯、氯仿等溶剂中。现代研究表明,千金子甾醇具有显著的抗肿瘤活性,可用于治疗肺癌、宫颈癌、乳腺癌、肝癌、慢性粒细胞性白血病以及急性单核细胞性白血病等。
磁性靶向药物之所以能够应用于靶向治疗,主要是通过磁性靶向给药系统对病变部位进行治疗。将磁靶向药物用于磁靶向给药系统的基本原理是:将磁性载药粒子注射到体内,在外部加以磁场,通过外加磁场的诱导性以及磁性粒子在体内的流动性,能够将磁性载药微粒逐渐转移到病变区,然后载药微粒以特定的受控方式(酶的活性或者其他生理条件的改变,例如pH值、温度改变或者渗透压等)进行缓慢释放,在病变区集中并发挥药物的作用。在本质上,磁靶向制剂的靶向过程是体内血流对载药微粒的推动作用以及外加磁场对磁性载药微粒的磁力综合作用的结果。
实现诊疗一体化及成像介导的治疗,诊疗一体化实现肿瘤的精准诊断和高效治疗一直是现代医学关注和追求的重要目标,目前还未见千金子甾醇磁性靶向微球制剂方面的报导。
发明内容
本发明的目的是提供一种千金子甾醇磁性靶向微球制剂及制备方法和用途,该微球制剂能够有效的治疗肺癌、宫颈癌、乳腺癌、肝癌等癌症,并能增强癌症组织的核磁共振(MRI)成像。
本发明提供了一种千金子甾醇磁性靶向微球制剂及制备方法,它包含以下组分(按重量百分比计):
千金子甾醇(EFL1)为 4-10%,DIB-PEG-NH2修饰的磁性四氧化三铁Fe3O4(4-28 nm)为40-80%,叶酸(FA)修饰的聚乳酸-羟基乙酸共聚物(poly(lactic-co-glycolic acid),PLGA)为10-50%。
本发明还提供千金子甾醇磁性靶向微球制剂Fe3O4纳米颗粒制备方法,采用以下步骤:
(1)磁性Fe3O4纳米颗粒的制备
金属前驱体-油酸铁的制备:称取六水合三氯化铁和油酸钠形成的混合溶液加热,冷凝回流,旋转蒸发除去溶剂,得到金属前驱体-油酸铁;
单分散Fe3O4纳米颗粒的制备:将金属前驱体-油酸铁、油酸和1-十八烯,混合均匀,离心收集反应产物,将反应产物用环己烷分散后,清洗,得到分散在环己胺中的Fe3O4纳米颗粒;
(2)聚乙二醇二胺修饰DIB-PEG-NH2的制备
称取聚乙二醇二胺溶于二氯甲烷与无水乙醇的混合溶液中;冰浴条件下3,4二羟基苯甲醛(DIB)溶液缓慢加入聚乙二醇二胺溶液中,搅拌反应加入硼氢化钠,过滤,旋蒸析出干燥得到DIB-PEG-NH2;
(3)水溶性Fe3O4纳米颗粒(Fe3O4-DIB-PEG-NH2)的制备
将步骤(1)制备的Fe3O4纳米颗粒,干燥后使用三氯甲烷分散,缓慢加入含有DIB-PEG-NH2的三氯甲烷溶液,反应,得到Fe3O4-DIB-PEG-NH2纳米颗粒;
本发明还提供千金子甾醇磁性靶向微球制剂叶酸修饰的聚乳酸-羟基乙酸共聚物FA-PEG-PLGA制备方法,采用以下步骤:
(1)叶酸修饰的聚乙二醇胺FA-PEG-NH2的合成
取叶酸(FA)溶于N,N-二甲基甲酰胺(DMF)中,加入N, N’-二环己基碳二亚胺(DCC)和N-羟基丁二酰亚胺(NHS),于室温下氮气流活化,利用低温冰浴以及加入低极性的乙醚进行沉淀析出,无水乙醇重新分散沉淀产物,对沉淀出的FA-PEG-NH2进行过滤和真空干燥,得到叶酸修饰的聚乙二醇胺(FA-PEG-NH2);
(2)叶酸修饰的聚乳酸-羟基乙酸共聚物(FA-PEG-PLGA)的合成
取聚乳酸-羟基乙酸共聚物(PLGA)溶于二氯甲烷中,加入二环己基碳二亚胺和N-羟基丁二酰亚胺,于室温下氮气流活化,将步骤(1)合成好的FA-PEG-NH2加入上述活化液中,氮气流下搅拌继续反应,得到FA-PEG-PLGA。
本发明还提供千金子甾醇磁性靶向微球制剂叶酸修饰的聚乳酸-羟基乙酸共聚物FA-PEG-PLGA制备方法,采用以下步骤:
采用W1/O/W2法制备初乳,W1相逐滴加入到油相(O)O相中,混匀,后将混合溶液超声加入到W2溶液中混匀,形成初乳液后过膜,后转入等体积生理盐水中固化,旋转蒸发去除有机溶剂,去离子水清洗,冷冻干燥即得;其中W1为制备的Fe3O4-DIB-PEG-NH2水溶液,W2为聚乙烯醇(PVA)PVA水溶液,油相(O)为制备的FA-PEG-PLGA与EFL1的二氯甲烷溶液。
一种上述千金子甾醇磁性靶向微球制剂的应用,所述制剂用于制备治疗、预防或缓解肿瘤的药物。所述肿瘤为肺癌、宫颈癌、乳腺癌、肝癌、慢性粒细胞性白血病以及急性单核细胞性白血病、卵巢癌、肾癌、脑癌、结肠癌或胃癌。
本发明生产工艺是将千金子甾醇与修饰后的纳米级四氧化三铁混合被修饰后的医用高分子材料包裹制成微球,应用时用溶剂将其溶解,静脉注射给药。
有益效果
(1)本发明将PLGA磁性微球将磁性纳米颗粒与PLGA等有机高分子材料、生物大分子以及其他无机物等非磁性材料以一定的结构相复合,进一步采用化学修饰手段经表面改性、共聚、吸附、键合等途径使微球表面带有功能基团或靶分子,从而构建形成多功能和磁响应的多功能微球。
(2)本发明制备包载抗癌药物千金子甾醇及磁性四氧化三铁纳米颗粒的叶酸介导PLGA空心微球,将治疗和诊断相结合,构建主动靶向、磁靶向、MRI成像的多功能诊疗一体化靶向给药系统用于癌症的靶向治疗。
(3)本发明的微球制剂注入人体后会有双重靶向作用,首先是主动靶向,制剂在叶酸的作用下,靶向至细胞表面高表达叶酸受体的肿瘤细胞。其次,在外加磁场的作用下,制剂中的四氧化三铁能够选择性的定位于肿瘤靶区,对正常组织无太大影响。
(4)本发明所用的纳米磁性四氧化三铁是经过化学修饰后所得,能够溶于水溶液;所用的PLGA为经修饰后载叶酸的高分子材料;所应用的微球载药系统作为一种载体,可装载药物,也可装载造影剂或自身具有光热特性,其独特优势将多种功能(包括各种诊断和治疗方法)整合到一个体系中,实现诊疗一体化及成像介导的治疗,诊疗一体化实现肿瘤的精准诊断和高效治疗一直是现代医学关注和追求的重要目标。
附图说明
图1为实施例1中FA-PEG-PLGA-EFL1-Fe3O4扫描电镜(A)与透射电镜图(B);
图2为实施例1中FA-PEG-PLGA-EFL1-Fe3O4在裸鼠KB肿瘤模型的MRI磁共振横断位成像图;
图3为实施例1中FA-PEG-PLGA-Fe3O4在裸鼠KB肿瘤模型的MRI磁共振冠状位影像图;
图4为实施例1中FA-PEG-PLGA-Fe3O4在裸鼠Hela肿瘤模型的MRI磁共振横轴位影像图;
图5为实施例1中主FA-PEG-PLGA-Fe3O4在裸鼠Hela肿瘤模型的MRI磁共振横轴位影像图。
具体实施方式以下实施例中所用FA-PEG-PLGA的制备方法为:取叶酸(FA)溶于N,N-二甲基甲酰胺(DMF)中,加入N, N’-二环己基碳二亚胺(DCC)和N-羟基丁二酰亚胺(NHS),于室温下氮气流活化,利用低温冰浴以及加入低极性的乙醚进行沉淀析出,无水乙醇重新分散沉淀产物,对沉淀出的FA-PEG-NH2进行过滤和真空干燥,得到叶酸修饰的聚乙二醇胺(FA-PEG-NH2);
所用Fe3O4-DIB-PEG-NH2的制备方法为:(1)金属前驱体-油酸铁的制备:称取六水合三氯化铁和油酸钠形成的混合溶液加热,冷凝回流,旋转蒸发除去溶剂,得到金属前驱体-油酸铁;单分散Fe3O4纳米颗粒的制备:将金属前驱体-油酸铁、油酸和1-十八烯,混合均匀,离心收集反应产物,将反应产物用环己烷分散后,清洗,得到分散在环己胺中的Fe3O4纳米颗粒;
(2)聚乙二醇二胺修饰DIB-PEG-NH2的制备,包括:
称取聚乙二醇二胺溶于二氯甲烷与无水乙醇的混合溶液中;冰浴条件下3,4二羟基苯甲醛(DIB)溶液缓慢加入聚乙二醇二胺溶液中,搅拌反应加入硼氢化钠,过滤,旋蒸析出干燥得到DIB-PEG-NH2;
(3)水溶性Fe3O4纳米颗粒(Fe3O4-DIB-PEG-NH2)的制备
将步骤(1)制备的Fe3O4纳米颗粒,干燥后使用三氯甲烷分散,缓慢加入含有步骤(2)制备的DIB-PEG-NH2的三氯甲烷溶液,反应,得到Fe3O4-DIB-PEG-NH2纳米颗粒。
实施例1. 千金子甾醇磁性靶向微球的制备
采用溶剂挥发法,取FA-PEG-PLGA0.1g先溶解于少量DMSO溶液中以保证材料完全溶解,后加二氯甲烷稀释至10ml,取千金子甾醇5mg溶于其中,作为油相O;取0.04g Fe3O4-DIB-PEG-NH2溶于水中作为水相W1;取0.8g聚乙烯醇PVA溶于40ml水中作为水相W2。W1相逐滴加入到O相中,涡旋混匀5 min,后将混合溶液超声15s,加入到W2溶液中涡旋混匀5 min,形成初乳液后通过滤膜,后转入等体积生理盐水中固化4 h,旋转蒸发去除有机溶剂,去离子水清洗3次, 分散于适量去离子水中,冷冻干燥即得FA-PEG-PLGA-EFL1-Fe3O4。
实施例2. 千金子甾醇磁性靶向微球的制备
采用溶剂挥发法,取FA-PEG-PLGA0.2g先溶解于少量DMSO溶液中以保证材料完全溶解,后加二氯甲烷稀释至10ml,取千金子甾醇10mg溶于其中,作为油相O;取0.03g Fe3O4-DIB-PEG-NH2溶于水中作为水相W1;取0.6g聚乙烯醇PVA溶于40ml水中作为水相W2。W1相逐滴加入到O相中,涡旋混匀5 min,后将混合溶液超声15s,加入到W2溶液中涡旋混匀5 min,形成初乳液后通过滤膜,后转入等体积生理盐水中固化4 h,旋转蒸发去除有机溶剂,去离子水清洗3次, 分散于适量去离子水中,冷冻干燥即得FA-PEG-PLGA-EFL1-Fe3O4。
实施例3. 千金子甾醇磁性靶向微球的制备
采用溶剂挥发法,取FA-PEG-PLGA0.1g先溶解于少量DMSO溶液中以保证材料完全溶解,后加二氯甲烷稀释至10ml,取千金子甾醇8mg溶于其中,作为油相O;取0.02g Fe3O4-DIB-PEG-NH2溶于水中作为水相W1;取0.8g聚乙烯醇PVA溶于40ml水中作为水相W2。W1相逐滴加入到O相中,涡旋混匀5 min,后将混合溶液超声15s,加入到W2溶液中涡旋混匀5 min,形成初乳液后通过滤膜,后转入等体积生理盐水中固化4 h,旋转蒸发去除有机溶剂,去离子水清洗3次,10000 r/min,10 min,分散于适量去离子水中,冷冻干燥即得FA-PEG-PLGA-EFL1-Fe3O4。
实施例4.千金子甾醇磁性靶向微球的形态观察。
扫描电镜(SEM):将导电胶粘在样品台上,取少量微球平铺于导电胶布上,将样品置于扫描电镜样品室中,抽真空,观察其形貌。透射电镜(TEM):取出少量实施例1中制备的超纯水分散的千金子甾醇磁性靶向微球,挥干溶剂,制得干燥透射电镜样品,在Tecnai T20下观察其形貌。
扫描电镜图显示工艺优化后制备的微球表面光滑、多孔,无黏连情况;透射电镜显示其为中空的空腔结构,Fe3O4被包载于空腔中,散落分布。
实施例5. 千金子甾醇磁性靶向微球的马尔文粒度测定
取实施例1中FA-PEG-PLGA-EFL1-Fe3O4,置于马尔文粒度测定仪配套的比色皿中,在粒度分析项下选择对应的溶剂、比色皿型号,设置参数后测定,应用Malvern Nano-ZS90激光粒度分析仪进行粒径的测定,应用动态光散射软件进行数据处理,记录待测样品的平均粒径。
表1 PLGA微球制剂粒径与Zeta电位(n=3)
实施例6. 千金子甾醇磁性靶向微球体外释放度的测定
取为实施例1中冷冻干燥后的FA-PEG-PLGA-EFL1-Fe3O4微球,分别置于具塞密封离心管中,采用恒温水浴振荡法测定,以pH6.8缓冲液(含有0.5%吐温-80)为释放介质,恒温振荡,分别于12、24 h以及3、6、9、12、15 d吸取溶液5mL,补充相应pH缓冲液5 mL,将微球重新悬浮进行进一步的药物释放。每个时间点离心上清液用微孔滤膜过滤,取续滤液,注入HPLC,计算出PLGA微球在不同时间的累积释放量,并绘制体外释放曲线。
表2 千金子甾醇磁性微球释药模型拟合结果
微球体外释放模型拟合程度为Ritger-Peppas模型> Higuchi 模型>First-order模型> Zero-order 模型。所制备的微球释放度符合Ritger-Peppas模型,Qt=ktn 即 lnQt=nlnt+lnk,n=0.31<0.45,药物释放机制为Fickian 扩散。
实施例7. 千金子甾醇磁性靶向给药系统小鼠组织分布研究
取实施例1中FA-PEG-PLGA-EFL1-Fe3O4微球小鼠尾静脉注射,取组织。精密移取组织匀浆液置于具塞EP管,加入汉黄芩素内标溶液,离心,取上清液用 UPLC-MS/MS进行检测分析。
结果显示在相同给药剂量下,各个微球制剂组大鼠肝脏与肺组织的药物浓度均明显高于肾脏组织,说明微球制剂在血液系统中被巨噬细胞所识别吞噬,因而主要分布在网状内皮系统丰富的肝脏中,即体现了对肝脏组织的被动靶向性,同时磁场的加入减弱了制剂对非靶区肝脏组织的被动靶向,增强了对靶区肺组织的磁靶向作用。组织分布实验结果初步表明微球制剂可以提高药物在肺、肝2个组织的靶向性并且延长药物在组织中的滞留时间。
实施例8. 千金子甾醇磁性靶向给药系统荷瘤裸鼠模型MRI成像
通过构建BALB/c-nu 裸鼠KB和Hela肿瘤模型,MRI核磁共振成像实验验证实施例1中FA-PEG-PLGA-EFL1-Fe3O4微球可以在磁场作用下靶向到肿瘤组织并在肿瘤细胞内富集,实施例1中FA-PEG-PLGA-EFL1-Fe3O4微球可以在叶酸介导的主动靶向作用下同样靶向到肿瘤组织,两者均能提高MRI影像图像的清晰度从而便于肿瘤部位的诊断治疗,且存在一定的协同双重靶向于肿瘤部位的特性,初步表明本课题构建的磁靶向、主动靶向双重靶向给药系统在实现肿瘤诊疗一体化方面具有较大的潜力。
Claims (3)
1.一种千金子甾醇磁性靶向微球制剂,其特征在于,所述制剂包含以下组分:千金子甾醇(EFL1)为4-10wt%,DIB-PEG-NH2修饰的磁性四氧化三铁(Fe3O4)为40-80wt%,叶酸(FA)修饰的聚乳酸-羟基乙酸共聚物(PLGA)为10-50wt%;
所述的千金子甾醇磁性靶向微球制剂的制备方法,采用以下步骤:
(1)磁性Fe3O4纳米颗粒的制备,包括:
金属前驱体-油酸铁的制备:称取六水合三氯化铁和油酸钠形成的混合溶液加热,冷凝回流,旋转蒸发除去溶剂,得到金属前驱体-油酸铁;单分散Fe3O4纳米颗粒的制备:将金属前驱体-油酸铁、油酸和1-十八烯,混合均匀,离心收集反应产物,将反应产物用环己烷分散后,清洗,得到分散在环己胺中的Fe3O4纳米颗粒;
(2)DIB-PEG-NH2的制备,包括:
称取聚乙二醇二胺溶于二氯甲烷与无水乙醇的混合溶液中;冰浴条件下3,4二羟基苯甲醛(DIB)溶液缓慢加入聚乙二醇二胺溶液中,搅拌反应加入硼氢化钠,过滤,旋蒸析出干燥得到DIB-PEG-NH2;
(3)水溶性Fe3O4纳米颗粒Fe3O4-DIB-PEG-NH2的制备,
将步骤(1)制备的Fe3O4纳米颗粒,干燥后使用三氯甲烷分散,缓慢加入含有DIB-PEG-NH2的三氯甲烷溶液,反应,得到Fe3O4-DIB-PEG-NH2纳米颗粒;
(4)叶酸修饰的聚乙二醇胺FA-PEG-NH2的合成
取FA溶于N,N-二甲基甲酰胺(DMF)中,加入N,N’-二环己基碳二亚胺(DCC)和N-羟基丁二酰亚胺(NHS),于室温下氮气流活化,利用低温冰浴以及加入低极性的乙醚进行沉淀析出,无水乙醇重新分散沉淀产物,对沉淀出的FA-PEG-NH2进行过滤和真空干燥,得到叶酸修饰的聚乙二醇胺(FA-PEG-NH2);
(5)叶酸修饰的聚乳酸-羟基乙酸共聚物(FA-PEG-PLGA)的合成
取PLGA溶于二氯甲烷中,加入二环己基碳二亚胺和N-羟基丁二酰亚胺,于室温下氮气流活化,将步骤(4)合成好的FA-PEG-NH2加入上述活化液中,氮气流下搅拌继续反应,对沉淀出的FA-PEG-PLGA进行过滤和干燥,得到FA-PEG-PLGA;
(6)千金子甾醇磁性靶向微球的制备
采用W1/O/W2法制备初乳,W1相逐滴加入到油相(O)相中,混匀,后将混合溶液超声加入到W2溶液中混匀,形成初乳液后过膜,后转入等体积生理盐水中固化,旋转蒸发去除有机溶剂,去离子水清洗,冷冻干燥即得;其中W1为Fe3O4-DIB-PEG-NH2水溶液,W2为聚乙烯醇(PVA)水溶液,油相(O)为制备的FA-PEG-PLGA与EFL1的二氯甲烷溶液。
2.根据权利要求1所述的微球制剂,其特征在于,所述DIB-PEG-NH2修饰的磁性四氧化三铁Fe3O4粒径为4-28nm。
3.根据权利要求1所述的微球制剂,其特征在于,所述千金子甾醇磁性靶向微球粒径为550nm-850nm,Zeta电位绝对值为15-22mv。
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