CN115177741A - 一种金属-有机复合纳米药物及其制备方法和应用 - Google Patents
一种金属-有机复合纳米药物及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开一种金属‑有机复合纳米药物,包含依次经第一表面改性剂和交联剂处理的异质结构金属基纳米颗粒以及经第二表面改性剂处理的药物成分化合物,所述纳米颗粒通过交联剂与所述药物成分化合物偶联,所述药物成分化合物为人参皂苷,所述纳米颗粒为Au@CoFeB。本发明还公开纳米药物的制备和应用。本发明提供的纳米药物可良好抑制肝癌的发展,并且同时具有离体和活体分子影像示踪功能。
Description
技术领域
本发明涉及纳米药物技术领域,具体涉及一种金属-有机复合纳米药物及其制备方法和应用。
背景技术
纳米药物具有尺寸可控、大的质量比和独特的物理化学特性,因此可在分子水平上用于生物***的监测、控制、诊断和治疗以及组织修复(Tekade,R.;Maheshwari,R.;Soni,N.;Tekade,M.;Chougule,M.,Nanotechnology-Based Approaches for Targetingand Delivery of Drugs and Genes.Elsevier,Amsterdam,2017,pp 3-61.)。近年来,合成纳米药物的主要方法集中在以下几个方面:1)通过提高分析方法的敏感性和综合性来获得连贯结果的诊断(Alhareth,K.;Sancey,L.;Tsapis,N.;Mignet,N.,How should we planthe future of nanomedicine for cancer diagnosis and therapy?InternationalJournal of pHarmaceutics 2017,532,(2),657-659);2)携带生物活性剂的药物输送(Peng,F.;Zhang,W.;Qiu,F.,Self-assembling Peptides in Current Nanomedicine:Versatile Nanomaterials for Drug Delivery.Cur-rent Medicinal Chemistry 2019,26,(1),1-26.);3)通过组织工程和植入物以克服与血管移植物有关的局限性(Hu,E.;Bayindir-Buchhalter,I.;Goebel,U.,Nanomedicine,Biofab-rication,TissueEngineering and Much More-Advanced Healthcare Materials Welcomes2019.Advanced Healthcare Mate-rials 2019,8,(1),1801576.);和4)生物利用度提高和医疗设备。此外,已经使用了广泛的纳米材料来制备纳米药物,包括纳米悬浮液、聚合物纳米颗粒、氧化铁、金属纳米颗粒、树枝状聚合物和脂质体等。因此,纳米药物改善并扩展了一系列药物分子的药代动力学、溶解性和稳定性,这些分子已广泛用于各种生物医学应用中,包括特定的药物输送、治疗、成像和诊断。然而,纳米药物的使用仍然具有对人类健康的众多不清楚的影响以及需要克服的各种生物障碍的特征,包括稳定性、表面修饰和功能化、多模式功能,有效的药物递送以及两者之间的平衡和副作用(Cencini,E.;Sicuranza,A.;Fabbri,A.;Ferrigno,I.;Rigacci,L.;Cox,M.C.;Raspadori,D.;Bocchia,M.,Study ofgene polymorpHisms as predictors of treatment efficacy and toxicity inpatients with indo-lent non-hodgkin lympHomas and mantle cell lympHomareceiv-ing bendamustine and rituximab.British Journal of Haematology 2019,184,(2),223-231.)。
由贵金属和磁性成分组成的纳米杂化物由于其独特的理化性质、高稳定性和优异的应用前景,有望在包括能量转换(Wang,S.;Niu,S.Y.;Li,H.S.;Lam,K.K.;Wang,Z.R.;Du,P.Y.;Leung,C.W.;Qu,S.X.,Synthesis and controlled morpHology of Ni@Ag coreshell nanowires with excellent catalytic efficiency andrecyclability.Nanotechnology 2019,30,(38),9.)、医学成像(S.D.;Gwenin,V.V.;Gwenin,C.D.,Magnetic Functional-ized Nanoparticles for Biomedical,DrugDelivery and Imaging Applications.Nanoscale Research Letters.2019,14,(1),188.)和疾病检测在内的广泛领域中应用。此外,对这些纳米颗粒的表面进行适当的修饰和/或与某些药物或有机成分的结合可导致宽广的治疗窗和临床应用,而对健康细胞和/或组织的副作用则没有或明显减少。这些改变使某些混合纳米颗粒具有诊断,成像,药物输送,治疗甚至合成疫苗开发等吸引人的特征,因而可作为多种疾病的有效化学治疗药物(Chen,H.;Liu,F.;Lei,Z.;Ma,L.;Wang,Z.,Fe2O3@Au Core@Shell Nanoparticle-GrapHene Nanocomposites as Theranostic Agents for Bioimaging and Chemo-pHotothermal Synergistic Therapy.RSC Advances 2015,5,(103),84980-84987.)。因此,多模式纳米药物的可控制备和功能化将其跨学科应用扩展到基础研究和临床应用(例如,超灵敏生物探针,高效纳米药物和纳米酶,超灵敏的生物医学分子成像和某些疾病的诊断等)。
现在的纳米药物要么是将原有药物简单纳米化,特别是对以一些有机物或高分子类药物,纳米化除了提高其分散性外,其他性能,尤其是疗效提高微乎其微,而毒副作用反而会变得更大,这些简单纳米化的药物不存在像金属基纳米材料在纳米尺度下的对疗效起重要作用的量子效应和表面活化效应。而纯的无机纳米药物,如无机非金属类,像二氧化硅等,其基本充当载体作用,而对纯金属,如金等可能还是癌细胞逃逸转移的很好的运载工具,同时也具有很大的副作用。另外,无论是无机非类、有机聚合物类还是金属类的纳米颗粒,目前的粒径都很大,各个组份基本都大于10nm以上,其进入人体后,很难被人体各器官或组织的表皮网络细胞以及肾小管等给清除掉,存在永久滞留人体的潜在危害。对于无机非金属和金属类纳米颗粒,其组成部分尺寸最好都小于6nm,但目前基本没有此类纳米材料的可量产方法,一次价格非常昂贵,病人一时无法接受。而有机聚合物类纳米药物,如果持续分解,还存在分解物(特别是产生的活性官能团)对正常组织和器官的损害以及癌变作用。另外,对于疗效的机理研究,需要能够进行离体和活体密切对照的具有示踪影像功能的纳米药物,目前基本全靠具有寿命短、易光敏化分解的荧光剂标记。特别是对有机类纳米药物,基本无法长时间稳定地进行分子影像学研究其药代动力学和药效机理。因此迫切需要能够同时具有离体和活体分子影像示踪功能的纳米药物。
发明内容
为解决现有技术中存在的问题,本发明的一个目的在于提供一种金属-有机复合纳米药物,包含依次经第一表面改性剂和活化剂处理的纳米颗粒以及经第二表面改性剂处理的药物成分化合物;所述纳米颗粒通过交联剂与所述药物成分化合物偶联,其中所述药物成分化合物为人参皂苷,所述纳米颗粒为Au@CoFeB。
根据本发明的纳米药物,其中所述第一表面改性剂选自3-氨丙基三甲氧基硅氧烷、乙烯基三甲氧基硅烷、磷酸酯双钛酸酯偶联剂和二异丙氧基二乙酰丙酮钛酸酯中的至少一种;优选为3-氨丙基三甲氧基硅氧烷。
根据本发明的纳米药物,所述第二表面改性剂选自3-氨丙基三甲氧基硅氧烷、3-巯丙基-三乙氧基硅烷偶联剂、十七氟癸基三甲基氧基硅烷和异丙基三(二辛基焦磷酸酰氧基)钛酸酯中的至少一种;优选为3-氨丙基三甲氧基硅氧烷、3-巯丙基-三乙氧基硅烷偶联剂和异丙基三(二辛基焦磷酸酰氧基)钛酸酯中的至少一种。
根据本发明的纳米药物,其中所述交联剂选自辛二酸(N-羟基琥珀酰亚胺酯)、乙二醇双(丁二酸N-羟基琥珀酰亚胺酯)、聚乙二醇二琥珀酰亚胺琥珀酸酯、琥珀酰-亚胺丁二酸酯聚、乙二醇琥珀酰-亚胺丁二酸酯和氮丙啶交联剂XR-100中的至少一种;优选为辛二酸(N-羟基琥珀酰亚胺酯)、乙二醇双(丁二酸N-羟基琥珀酰亚胺酯)、聚乙二醇二琥珀酰亚胺琥珀酸酯、聚乙二醇琥珀酰-亚胺丁二酸酯和氮丙啶交联剂XR-100中的至少一种。
根据本发明的纳米药物,在一种具体实施方案中,所述纳米药物采用包含以下步骤的方法制备:
(1)制备Au@CoFeB纳米颗粒;
(2)纳米颗粒的表面改性:
a)将(1)中的纳米颗粒加入到含有第一表面改性剂的第一有机溶液中进行搅拌,
b)将a)中的加有纳米颗粒的第一有机溶剂溶液经超声清洗、离心和干燥,得到表面改性的纳米颗粒;
(3)纳米颗粒的偶联活化:
a)将(2)中得到的表面改性的纳米颗粒和交联剂分别分散到第二有机溶剂中,搅拌后进行孵化,
b)将a)中加有表面改性的纳米颗粒和交联剂的溶液进行离心和干燥,得到活化后的纳米颗粒;
(4)纳米颗粒的pH调控:
将(3)中所得的活化后的纳米颗粒分散到缓冲液中调节pH值,随后经离心-超声清洗,得到经表面改性和活化的纳米颗粒;
(5)人参皂苷的表面改性:
a)将人参皂苷加入到含有第二表面改性剂的第三有机溶液中进行搅拌;
b)将a)中加有人参皂苷的第三有机溶剂溶液经超声清洗、离心和干燥,得到表面改性的人参皂苷;
(6)纳米颗粒和人参皂苷的偶联:
a)将步骤(3)得到的经表面改性和活化的纳米颗粒和步骤(5)得到的表面改性的人参皂苷放入第四有机溶剂中孵化;
b)将a)中的加有经表面改性和活化的纳米颗粒及表面改性的人参皂苷的第四有机溶剂溶液经过离心-超声清洗-离心后进行干燥处理,得到复合纳米药物。
根据本发明的纳米药物,其中所述纳米颗粒为核壳结构,核壳结构的金属内核为面心立方晶体结构的Au;核壳结构的壳层为面心立方晶体结构的CoFeB;
所述纳米药物的整体结构为6-7.2nm的超小纳米药物单元偶联在一起构建的尺寸为250-350nm的纳米药物聚集体;纳米颗粒的动力学半径约100-200nm;
所述纳米颗粒表面带有+7-12mV;所述纳米药物表面带有+25-30mV的正电势。
根据本发明的纳米药物,其中第一有机溶剂选自苯、甲苯、对二甲苯和间二甲苯中的至少一种;优选为甲苯、对二甲苯和间二甲苯中的至少一种。
第二有机溶剂选自对二甲基亚砜、N-甲基甲酰胺、N-甲基-2-吡咯烷酮、二甲苯和邻二甲苯中的至少一种;优选为二甲基亚砜、N-甲基甲酰胺和N-甲基-2-吡咯烷酮中的至少一种。
第三有机溶剂选自对二甲苯、邻二甲苯和甲苯中的至少一种;优选为甲苯。
第四有机溶剂选自二甲亚砜、N-甲基吡咯烷酮、N-乙基乙酰胺和N-甲基甲酰胺中的至少一种;优选为二甲亚砜、N-甲基吡咯烷酮和N-乙基乙酰胺中的至少一种。
根据本发明的纳米药物,步骤(4)中所用缓冲液可以为基于本领域技术人员所公知的缓冲液,例如所述缓冲液可以为磷酸盐PBS缓冲液、柠檬酸–Na2HPO4缓冲液、Trizma缓冲液、胎牛血清FBS缓冲液、碳酸钠-碳酸氢钠缓冲溶液、和醋酸钠-醋酸缓冲液中的至少一种,优选为磷酸盐缓冲液、胎牛血清FBS缓冲液和柠檬酸-Na2HPO4缓冲液中的至少一种,进一步优选为磷酸盐PBS缓冲液。
本发明的另一个目的在于提供一种复合纳米药物的制备方法,包括以下步骤:
(1)制备Au@CoFeB纳米颗粒;
(2)纳米颗粒的表面改性:
a)将(1)中的纳米颗粒加入到含有第一表面改性剂的第一有机溶液中进行搅拌,
b)将a)中的加有纳米颗粒的第一有机溶剂溶液经超声清洗、离心和干燥,得到表面改性的纳米颗粒;
(3)纳米颗粒的偶联活化:
a)将(2)中得到的表面改性的纳米颗粒和交联剂分别分散到第二有机溶剂中,搅拌后进行孵化,
b)将a)中加有表面改性的纳米颗粒和交联剂的溶液进行离心和干燥,得到活化后的纳米颗粒;
(4)纳米颗粒的pH调控:
将(3)中所得的活化后的纳米颗粒分散到缓冲液中调节pH值,随后经离心-超声清洗,得到经表面改性和活化的纳米颗粒;
(5)人参皂苷的表面改性:
a)将人参皂苷加入到含有第二表面改性剂的第三有机溶液中进行搅拌;
b)将a)中加有人参皂苷的第三有机溶剂溶液经超声清洗、离心和干燥,得到表面改性的人参皂苷;
(6)纳米颗粒和人参皂苷的偶联:
a)将步骤(3)得到的经表面改性和活化的纳米颗粒和步骤(5)得到的表面改性的人参皂苷放入第四有机溶剂中孵化;
b)将a)中的加有经表面改性和活化的纳米颗粒及表面改性的人参皂苷的第四有机溶剂溶液经过离心-超声清洗-离心后进行干燥处理,得到复合纳米药物。
作为一种优选方案,步骤(1)中,Au@CoFeB的纳米颗粒采用微流控法、水热法、磁控溅射法、电沉积法制备;进一步优选为微流控法。
具体而言,所述微流控法就是使用微米尺度(亚毫米)构建的连续流反应工艺,将合成纳米材料或药物时的反应物的混合、反应成核、纳米颗粒或药物生长和生长终止过程控制在μL到pL甚至更小的反应体积内。该方法和传统的釜式反应器相比,具有精确设计和调控反应不同阶段的动力学参数、快速的物质和能量交换、混合和反应均匀、可以平行放大作业。无釜式反应器不可避免的放大效应、环境友好、安全和废物最小化、可充分利用微流道的高比表面积效应对反应产物进行调控的特点。微流控法制备纳米颗粒的方法可以参考现有技术,在此不再赘述。
作为一种优选方案,步骤(2)-a)中,搅拌时间优选为20-28小时;步骤(2)-b)中,优选离心的条件包括:离心转速为10000-20000rpm;离心时间为5-40分钟;优选为12000-16000rpm;离心时间为10-30分钟。
作为一种优选方案,步骤(3)-a)中,搅拌时间优选为1-3小时;步骤(3)-b)中,优选离心的条件包括;离心转速为10000-20000rpm;离心时间为5-15分钟。
作为一种优选方案,步骤(4)中,将pH值调节为7-7.8;优选离心的条件包括;离心转速为10000-14000rpm;离心时间为5-15分钟。
作为一种优选方案,步骤(5)-a)中,搅拌时间优选为20-28小时;优选离心的条件包括;离心转速为10000-14000rpm;离心时间为5-15分钟。
作为一种优选方案,步骤(6)-a)中,孵化时间优选为1.5-2.0小时;步骤(6)-a)中,所述第四有机溶剂优选为二甲基亚砜。
本发明的另一个目的在于提供本发明的复合纳米药物在制备治疗肝癌药物中的应用。
根据本发明的应用,其中复合纳米药物中的Au@CoFeB的浓度为0.00001-100000μg/mL。
本发明提供的复合纳米药物可以充分利用无机,特别是金属基纳米颗粒的多模态和表面数层原子层的活性和量子效应产生的疗效,以及有机药物的疗效和对金属内层及生命有机体的保护作用,构建无机-有机复合纳米药物,充分发挥有机药物-无机纳米颗粒的协同效果,获得高疗效、低或无毒副作用的原创型纳米药物,该药物同时具有离体和活体分子影像示踪功能。
附图说明
图1.合成纳米颗粒的微流控装置结构示意图。
图2A是使用实施例1工艺制备的Au@CoFeB-Rg3纳米药物的广角透射电镜照片,图中右上是尺寸分布图,其下是单个颗粒图像的放大图。图2B-8是是使用实施例1工艺制备的纳米颗粒Au@CoFeB的XRD谱。图2B-9是是使用实施例1工艺制备的纳米颗粒Au@CoFeB-Rg3的XRD谱.图2C-10是是使用实施例1工艺制备的纳米颗粒Au@CoFeB的XPS谱图。图2C-11是纳米药物Au@CoFeB-Rg3的XPS谱图。图2D-12是纳米颗粒Au@CoFeB的FT-IR谱图。图2D-13是纳米药物Au@CoFeB-Rg3的FT-IR谱图。图2E-14是是使用实施例1工艺制备的纳米颗粒Au@CoFeB的水动力学直径分布图。图2E-15是是使用实施例1工艺制备的纳米药物Au@CoFeB-Rg3的水动力学直径分布图。图2F-16是使用实施例1工艺制备的纳米颗粒Au@CoFeB的表面Zeta电势。图2F-17是是使用实施例1工艺制备的纳米药物Au@CoFeB-Rg3的表面Zeta电势。
图3A-18是使用实施例2工艺制备的由暗场显微镜拍摄的Au@CoFeB纳米颗粒的表面等离子体散射光图形。图3A-19是使用实施例2工艺制备的由暗场显微光波谱仪表征的Au@CoFeB纳米颗粒表面等离子体散射光波谱。图3B-20是使用实施例2工艺制备的由暗场显微镜拍摄的Au@CoFeB-Rg3纳米药物的表面等离子体散射光图形。图3B-21是使用实施例2工艺制备的由暗场显微光波谱仪表征的Au@CoFeB-Rg3纳米药物的表面等离子体散射光波谱。图3C是使用实施例2工艺制备的Au@CoFeB纳米颗粒的T2WI浓度(0μg/mL、7.5μg/mL、15.1μg/mL、31.3μg/mL、93.5μg/mL、187.0μg/mL)依赖性的磁共振图像(图上部图像)和磁共振弛豫速率(T2 -1,单位为s-1)的浓度依赖性曲线。图3D是使用实施例2工艺制备的Au@CoFeB-Rg3纳米药物的T2WI浓度(0μg/mL、4.1μg/mL、12.4μg/mL、37.3μg/mL、112.0μg/mL)依赖性的磁共振图像(图上部图像)和磁共振弛豫速率(T2 -1,单位为s-1)的浓度依赖性曲线。图3E是使用实施例2工艺制备的Au@CoFe(B)-Rg3纳米药物浓度(400μg/mL、800μg/mL、1100μg/mL、1500μg/mL、4000μg/mL)依赖性的计算机辅助X光断层扫描(CT)图像(图上部图像)和三点平均的信号(HU)的浓度依赖曲线。
图4A是使用实施例1工艺制备的Au@CoFeB纳米颗粒浓度依赖的Jurkat-CT(4A-22)、3T3(4A-23)、K562-CT(4A-24)和HEP-G2/C3A(4A-25)细胞24小时存活率。图4B是使用实施例1工艺制备的Au@CoFeB-Rg3纳米药物浓度依赖的Jurkat-CT(4B-26)、3T3(4B-27)、K562-CT(4B-28)和HEP-G2/C3A(4B-29)细胞24小时存活率。
图5A是使用实施例3工艺制备的Au@CoFeB纳米颗粒浓度依赖的Jurkat-CT(5A-30)、3T3(5A-31)、K562-CT(5A-32)和HEP-G2/C3A(5A-33)细胞增殖存活率。图5B是使用实施例3工艺制备的Au@CoFeB-Rg3纳米药物浓度依赖的Jurkat-CT(5B-34)、3T3(5B-35)、K562-CT(5B-36)和HEP-G2/C3A(5B-37)细胞增殖存活率。
图6使用实施例4工艺制备的制备的Au@CoFeB纳米颗粒和Au@CoFeB-Rg3纳米药物和PBS缓冲液以及其它纳米药物(Fe@Fe3O4-Rg3、FePt@Fe3O4-Rg3)在两种浓度下(斜纹柱,95-190μg/mL;网格柱,474-947μg/mL)对人慢性髓系白血病细胞K562癌细胞孵化24小时的存活率实验结果。
图7A是使用实施例5工艺制备的Au@CoFeB-Rg3和Au@CoFeB纳米颗粒抗肝癌疗效的活体动物实验在不同用药方案后将小鼠牺牲后的肝部肿瘤图像。图7B是使用实施例5工艺制备的Au@CoFeB-Rg3和Au@CoFeB纳米颗粒抗肝癌疗效的活体动物实验在不同用药方案的小鼠在不同阶段处通过生物荧光标记的肝部肿瘤大小荧光图。图7C是使用实施例5工艺制备的Au@CoFeB(7C-40)和Au@CoFeB-Rg3(7C-41)和对照组生理盐水(7C-38)及Rg3(7C-39)抗肝癌疗效的活体动物实验在21天内不同组小鼠的绝对(上部折线)和相对体重变化(下部折线)。图7D是使用实施例5工艺制备的Au@CoFeB纳米颗粒(7D-44)和Au@CoFeB-Rg3(7D-45)和对照组生理盐水(7D-42)及Rg3(7D-43)抗肝癌疗效的活体动物实验在不同药物处理后的肿瘤部位生物荧光强度的定量值。
附图标记说明
1、第一注射泵;2、第二注射泵;3、第一微流道管;4、第二微流道管;5、Y字形反应物料液混合器;6、第三微流道管;7、收集器。
具体实施方式
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,或按照制造厂商所建议的条件。
纳米颗粒和纳米药物的合成
实施例1:
(1)制备Au@CoFeB纳米颗粒:
如图1所示,将0.42g聚乙烯吡咯烷酮(PVP,K-30,重均分子量30,000),0.07g(0.35mmol)的FeCl2·4H2O和0.0833g(0.35mmol)的CoCl2·6H2O在氮气保护下溶解在50毫升N-甲基-2-吡咯烷酮(NMP)中,形成金属盐溶液。接着,在氮气保护下将0.4g(10.5mmol)的NaBH4溶解在50ml的NMP中以形成还原溶液。
接下来,使用图1所示过程在氮气保护下于120℃进行微流体合成:将50ml带PVP的金属盐溶液和50ml还原溶液分别引入每个注射器中,并将注射器固定在第一注射泵1和第二注射泵2上。随后将其通过第一微流道管3和第二微流道管4引入Y字形反应物料液混合器5完成还原反应,第一注射泵1和第二注射泵2的流速均为3ml/min。接下来,溶液进入第三微通道管6以完成快速成核并完成纳米颗粒的生长。根据以下反应路线形成CoFeB纳米颗粒。当反应完成时,将获得的新鲜纳米颗粒分散溶液收集在收集器7中。然后,使用离心机以15,000rpm的速度沉淀溶液30分钟,并倾析出顶部上清液。使用NMP将获得的颗粒洗涤两次,得到预合成的CoFeB纳米颗粒。
将2.5ml(0.36mmol)的HAuCl4溶液溶解在50ml的NMP中。然后使用图1所示装置在氮气保护条件下在室温下进行微流体合成:分别使用第一注射泵1和第二注射泵2将50ml稀释的HAuCl4溶液和50ml预合成的CoFeB纳米颗粒的NMP溶液泵送入第一微流道管3和第二微流道管4,一起进入Y字形反应物料液混合器5完成还原反应和快速成核,第一注射泵1和第二注射泵2的流速均为3ml/min。在该步骤中,将0.2g的NaBH4溶解在20ml的NMP中,并预先放入收集器7中。当反应完成时,将收集器7彻底摇动并静置30分钟,以完成CoFeB纳米颗粒的置换,还原和表面重排。随后向收集器中加入2ml乙醇以破坏溶液的平衡。沉淀的纳米颗粒重新溶解在相同体积的NMP中。重复清洗过程两次,以去除大部分表面活性剂。最后将瓶中的最终黑色浆料在真空条件下干燥成黑色粉末,并保存在干燥器中以备将来使用。
(2)纳米颗粒的表面改性:
首先将10mg步骤(1)制备的Au@CoFeB纳米颗粒溶解到50ml含有1wt%3-氨丙基三甲氧基硅氧烷(APTMS)无水甲苯溶液中。然后,将混合溶液在室温下搅拌24小时。搅拌完成后,使用离心机以12000rpm的速度沉离心10分钟。随后,倾析出顶部上清液,并将沉淀的纳米颗粒用乙醇洗涤一次。最后获得Au@CoFeB-APTMS纳米颗粒。
(3)纳米颗粒的偶联活化:
首先,将5mg制备的Au@CoFeB-APTMS纳米颗粒溶解在5ml的二甲基亚砜中。然后,将5mg辛二酸(N-羟基琥珀酰亚胺酯)偶联剂溶液缓慢加入到溶液中。在室温下孵育1小时以上后,将混合溶液以12,000rpm的速度离心10分钟。然后倒出顶部上清液,并将瓶中最终沉淀的纳米颗粒在真空下干燥,即可以得到Au@CoFeB-APTMS-DSS纳米颗粒。
(4)纳米颗粒的pH调控:
将干燥后的样品溶于10mL磷酸盐缓冲溶液(pH=7.4)中,调节纳米颗粒的pH值。然后以12,000rpm的速度离心10分钟后,将瓶中的最终浆液用去离子水洗涤并在真空下干燥。
(5)人参皂苷的表面改性:
首先将200mg人参皂苷Rg3溶解在50mL含有1w%APTMS的无水甲苯溶液中。在环境温度下搅拌24小时后,将混合溶液以1200rpm的速度离心10分钟。然后,将沉淀的浆液用乙醇洗涤3次,获得改性后的人参皂苷Rg3。
(6)纳米颗粒和人参皂苷的偶联:
在真空中干燥后,将5mg表面改性后的人参皂苷Rg3和5mg经过表面改性和活化的Au@CoFeB纳米颗粒分散在5ml的二甲基亚砜中。在室温下孵育2小时以实现Au@CoFeB纳米颗粒与人参皂苷Rg3之间的偶联。随后将溶液离心并用去离子水洗涤两次。随后将沉淀的浆液在真空中干燥,获得Au@CoFeB-Rg3纳米药物。
实施例2-5都利用了实施例1的工艺和装置制备纳米颗粒,但改变了部分的原料、原料用量以及反应条件,如表1所示。
表1 实施例2-5中各项工艺参数一览表
对通过实施例1中制备的典型的纳米颗粒和纳米药物的微观结构以及多模成像特性进行表征。根据高分辨TEM图像(图2A)和XRD谱图(图2B),纳米颗粒偶联药物前后,其金属内核仍保持核心为面心立方晶体的Au和面心立方晶体结构的CoFeB合金壳层的核壳结构;XPS谱图(图2C:10、11)则表明该纳米药物含有Au、Co、Fe、B、Si(Ti)、C、N(P)和O元素,其中Si主要来自纳米颗粒的表面改性和交联剂,C、N(P)和O来自有机的药物和偶联剂,如Rg3和辛二酸(N-羟基琥珀酰亚胺酯),而硼(B)则来自使用的还原剂硼氢化钠;当使用磷酸酯双钛酸酯类偶联剂时,药物的Si会变成Ti,同时增加一个元素P。FT-IR谱图(图2D)则证实了使用该方法的确获得了由异质结构金属Au@CoFeB(图2D:12)和药物Rg3共轭构建的金属-有机复合化的纳米药物Au@CoFeB-Rg3(图2D:13);结合TEM图像(图2A)和水动力学直径(图2E)检测结果则表明纳米颗粒和药物偶联成纳米药物后的纳米药物(图2E:15)整体结构的确是由约6.6nm的超小纳米药物单元偶联在一起构建的尺寸约300nm的纳米药物聚集体;而单纯的金属纳米颗粒(图2E:14)的动力学半径约150nm,主要是由于该纳米药物的无机部分的壳层CoFeB磁性材料,其产生的磁偶极作用具有将其聚集在一起的功能,在水溶液中也会以聚集体存在,这些小于1微米的聚集体非常有利于提高药物在体内停留时间和输运到病灶的效率,而在病灶处微酸性的细胞微环境,则又很快解离成单个的药物,提高对病灶细胞及组织内的渗透力;而Zeta电势(图2F)表征结果表明,该类纳米颗粒(图2F:16)及纳米药物(图2F:17)表面分别带有+10mV和+27mV的正电势,这些正电势特别有利于提高药物递送速率和提高药物利用率起关键作用的细胞内化过程,药物的这个特点和药物以聚集体形式的集团输运模式一起,共同提高药物进入细胞内的比率,即药物的利用率,克服纳米药物在癌症治疗上的关键难题:药物利用率低下,平均不到1.0%。
图3是对通过实施例2制备的纳米颗粒和纳米药物的多模态影像功能表征。如图3A所示,对于含有贵金属组份的磁性异质结构纳米颗粒,其具有很强的表面等离子体共振(LSPR)散射光特性,虽然其只有5nm左右,但由于其可以散射光具有400-600纳米的光学尺寸,仍然可以通过光学显微镜观测到单个纳米颗粒图像(图3A:18、20),并可以用表面等离子体波谱仪对单个纳米颗粒的光学散射特性表征(图3A:19、21)。而经过表面改性并偶联上药物后,虽然表面的介电常数有所改变,但其LSPR散射光强度仍然保持很强(图3B-20),仍可以使用暗场显微镜和表面等离子体波谱仪对其成像示踪。制备的Au@CoFe(3A-18)和偶联有Rg3的Au@CoFe-Rg3纳米药物(3B-20)的暗场显微LSPR光学图像和其中一个颗粒(3A-19)和药物的LSPR波谱(3B-21),经过药物共轭后,由于表面介电常数改变,整体颗粒的散射光发生了红移。
图3C是通过实施例2制备的纯Au@CoFeB纳米颗粒在T2WI实验条件的自选-回波序列为回波(TE)持续时间是33ms、脉冲重复时间间隔(TR)是2500ms下的磁共振成像结果,其具有很好的T2WI增强效果,其弛豫速率(T2 -1,s-1)和纳米颗粒浓度具有很好的线性关系。图3D是合成的纯Au@CoFeB-Rg3纳米药物在T2WI实验条件的自选-回声序列为回声(TE)时间是33ms、脉冲重复时间间隔(TR)是2500ms下的磁共振成像结果,和纳米颗粒一样,其具有很好的T2WI增强效果,其弛豫速率(T2 -1,s-1)和纳米颗粒浓度具有很好的线性关系,同时可以看出,纳米药物的弛豫速率比纳米颗粒更高,说明金属-有机复合有利于进一步增强其分子影像增强效果。
图3E上部图像是是Au@CoFeB-Rg3纳米药物不同浓度下(400μg/mL、800μg/mL、1100μg/mL、1500μg/mL、4000μg/mL)的计算机辅助X光断层扫描(CT)图像和三点平均的信号(亨氏单位:HU)的浓度依赖曲线(图中曲线)。可见,其具有很优异的CT影像效果;同时在很大浓度范围内,其CT值(HU是测定人体某一局部组织或器官密度大小的一种计量单位:空气为-1000,致密骨为1000)和浓度具有优异的线性关系,可以在临床中通过分子影像示踪法高时空分辨在线研究病理和药理。
应用实施例
纳米药物Au@CoFeB和Au@CoFeB-Rg3抗肿瘤效果及其药理研究
应用实施例1
通过细胞生物学方法以人慢性髓性白血病K562细胞系和肝癌细胞系(HEP-G2/C3A)为癌细胞病理模型、以人上皮成纤细胞系3T3和人免疫细胞JurkatT细胞系为正常健康细胞模型,对合成的纳米药物的抗肿瘤效果和对正常细胞的毒副作用进行了研究。
配制不同Au@CoFeB和Au@CoFeB-Rg3浓度的细胞培养用磷酸盐PBS缓冲液,通过BDLSRFortessaTM细胞分析仪(BD biosciences)使用光散射法检测各种细胞在不同Au@CoFeB和Au@CoFeB-Rg3浓度下,孵育24小后,各种细胞的24小时存活率。
如图4A所示为实施例1制备的Au@CoFeB(浓度从0.00001μg/mL到500μg/mL)和Au@CoFeB-Rg3(浓度从0.00001μg/mL到1000μg/mL)对正常细胞(3T3:4A-23、4B-27)、T细胞(Jurkat:4A-22、4B-26)、白血病细胞K562(4A-24、4B-28)和实体瘤肝癌细胞(HEP-G2/C3A)(4A-25、4B-29)毒性的影响。24小时细胞毒性检测表明(4A和4B),实施例1制备的Au@CoFeB-Rg3纳米药物(图4B)对K562细胞(4B-28)和HEP-G2/C3A(4B-29)细胞的毒性明显高于Au@CoFeB(4A:24、25),也都高于纯Rg3对肝癌细胞和血癌细胞的致死率,说明异质结构金属纳米颗粒和有机的药物之间有明显的协同抗肿瘤效果。Au@CoFeB对免疫细胞和血癌细胞的24小时毒性明显低于对3T3和肝癌细胞,说明Au@CoFeB对可以成实体组织的细胞比自由悬浮细胞具有高毒性,同时可以看出,Au@CoFeB在低浓度(9.5μg/mL)。当Au@CoFeB浓度低于250μg/mL时,对所考察细胞毒性很低,说明该浓度下可以作为光学生物分子纳米探针使用。当Au@CoFeB-Rg3浓度为95μg/mL,对K562细胞已具有强致死率;不超过该浓度对其他细胞无明显毒性(细胞存活>70%)。当Au@CoFeB-Rg3浓度超过200μg/mL,其对肝癌细胞和血癌细胞致死率大幅度提高;虽然该浓度对Jurkat细胞也有明显毒性,但对3T3的毒性明显比对癌细胞的小很多。
应用实施例2
通过细胞生物学方法以人慢性髓性白血病K562细胞系和肝癌细胞系(HEP-G2/C3A)为癌细胞病理模型、以人上皮成纤细胞系3T3和人免疫细胞JurkatT细胞系为正常健康细胞模型,对合成的纳米药物的抗肿瘤效果和对正常细胞的毒副作用进行了研究。配制不同Au@CoFeB和Au@CoFeB-Rg3浓度的细胞培养用PBS缓冲液,通过BD LSRFortessaTM细胞分析仪(BD biosciences购自BD biosciences公司,)使用光散射法检测各种细胞在不同Au@CoFeB和Au@CoFeB-Rg3浓度下,孵育24小后,各种细胞的24小时存活率。
这次使用实施例1制备的Au@CoFeB浓度分别是2500μg/mL、10000μg/mL、100000μg/mL;Au@CoFeB-Rg3浓度为2500μg/mL、20000μg/mL、100000μg/mL。检测它们对白血病细胞K562细胞和肝癌细胞HEP-G2/C3A的毒性,其24小时的细胞存活率均为零,100%被杀死。
应用实施例3
使用96孔板通过基于ATP细胞活力检测的细胞效价-glo荧光细胞活力测定仪(Promega,G7570)对通过实施例3制备的不同浓度下纳米颗粒和纳米药物的K562,HepG2/C3A,和Jurkat细胞的细胞增殖率进行检测。其中缓冲液使用10%的胎牛血清FBS缓冲液,缓冲液中Au@CoFeB的浓度选取为0.00001μg/ml,9.5μg/ml,47.0μg/ml,95.0μg/ml和474.0μg/ml;Au@CoFe-Rg3选取为0.00001μg/ml,19μg/ml,95.0μg/ml,190.0μg/ml和950.0μg/ml。一起在细胞培养箱(3%CO2,310K)中培育4天后检测其增值。
纳米颗粒和纳米药物对细胞增殖的效果表明:当Au@CoFeB浓度超过50μg/mL(图5A)、Au@CoFeB-Rg3浓度超过100μg/mL(图5B)后,不论是实体瘤肝癌细胞(HEP-G2/C3A:5A-33、5B-37)还是可以自由流动的白血病癌细胞(K562:5A-32、5B-36),纳米药物对癌细胞增殖具有明显抑制作用,但对正常细胞3T3(5A-31、5B-35)无明显毒性;特别是当Au@CoFeB-Rg3浓度达到950μg/mL时,增殖培养4天后,癌细胞基本全部死亡。该增殖抑制效果研究表明,Au@CoFeB自身对癌细胞的增殖具有明显抑制作用,特别是当浓度达到474μg/mL时,癌细胞增殖基本全部被抑制。一个特别的现象是,当使用低的Au@CoFeB浓度(如19μg/mL)时,其可以提高免疫细胞Jurkat的增殖速率(5A-30),但是纳米药物对其有一定毒性(5B-34),说明如何适当设计好纳米颗粒的尺寸、组成和结构,它们可以具有提高免疫细胞活性的作用,将来特定浓度和成分的纳米颗粒可以和免疫疗法联合使用,提高癌症治疗效果。
应用实施例4
使用96孔板通过基于ATP细胞活力检测的细胞效价-glo荧光细胞活力测定仪(Promega,G7570)对通过实施例3制备的不同纳米药物在其不同浓度下的K562,HepG2/C3A,和Jurkat细胞的细胞增殖率进行检测。其中缓冲液使用10%的胎牛血清FBS缓冲液.这次使用的Au@CoFeB浓度分别是2500μg/mL、10000、100000μg/mL;Au@CoFeB-Rg3浓度为5000μg/mL、20000μg/mL、100000μg/mL。一起在细胞培养箱(3%CO2,310K)中培育4天后检测其增值。实验结果表明,当Au@CoFeB浓度超过2500μg/mL、Au@CoFeB-Rg3浓度超过5000μg/mL后,血癌细胞K562和实体瘤肝癌HepG2/C3A细胞均全部死亡。而Jurkat细胞和3T3细胞仍然分别有30%和20%以上的4天增值培养存活率。说明,高浓度下,该类药物对癌细胞具有高致死性,而对正常细胞也有一定毒性,因此,临床应用必须控制用药量。
应用实施例5
考察了使用实施例4工艺制备的Au@CoFeB纳米颗粒和Au@CoFeB-Rg3纳米药物与其它纳米药物(Fe@Fe3O4-Rg3、FePt@Fe3O4-Rg3)在两种浓度下)对人慢性髓系白血病细胞K562癌细胞孵化24小时的存活率实验结果。如图6所示:斜纹柱,药物浓度为95-190μg/mL;网格柱,药物浓度为474-947μg/mL。结果表明,Au@CoFeB-Rg3纳米药物对该类白血病细胞的杀死率比前两者在低浓度下(95-190μg/mL)均提高了近7倍;在高浓度下(474-947μg/mL)提高了40多倍。而Au@CoFeB纳米颗粒在高浓度下则比Fe@Fe3O4-Rg3和FePt@Fe3O4-Rg3纳米药物也分别提高了27倍和23倍;在低浓度下分别比Fe@Fe3O4-Rg3和FePt@Fe3O4-Rg3纳米药物提高1.4倍和1.5倍。
应用实施例6
使用裸鼠并通过二甲基亚硝胺在其肝部建立原位肝癌模型。当肿瘤长大约4周后开始给药,每两天给一次,每次约70mg/kg,连续5次后停药,然后饲养老鼠,每7天对老鼠称体重,并通过肿瘤处的荧光分析检测肿瘤相对大小。共分四组,一组为使用生理盐水组作为对照,另三组分别为纯Rg3组、通过实施例5制备的纯Au@CoFeB和纳米药物Au@CoFeB-Rg3组。到第21天后,将所有老鼠牺牲掉并进行解剖,对肝部及其肿瘤进行解剖并作各种生化和病理切片分析。图7A是不同药物和对照组处理下的最后的小鼠的肿瘤图像,图7B是不同时间点处通过荧光法表征的小鼠肿瘤大小的荧光图像,图7C是测定的不同组小鼠在给药后不同时间点的绝对(上部折线)和相对体重变化(下部折线)(生理盐水:38;Rg3:39;Au@CoFe:40;Au@CoFeB-Rg3:41)。图7D是根据荧光值测定不同组在给药后不同时间节点处的肿瘤大小(生理盐水:42;Rg3:43;Au@CoFe:44;Au@CoFeB-Rg3:45)。可以看出,单纯Rg3组(43)比对照组(42)的肿瘤稍微小一点,而Au@CoFeB(44)则比对照组的肿瘤稍小很多,说明Au@CoFeB自身就具有很强的抗肝癌效果;对小鼠肿瘤抑制作用最强的是经过Au@CoFeB和Rg3复合成纳米药物的一组,同时可以看出给药后,随着用药及用药后时间推移,纳米药物对肝癌的发展的抑制作用越来越明显(45)。
Claims (10)
1.一种金属-有机复合纳米药物,包含依次经第一表面改性剂和交联剂处理的异质结构金属基纳米颗粒以及经第二表面改性剂处理的药物成分化合物,所述纳米颗粒通过交联剂与所述药物成分化合物偶联,其特征在于,所述药物成分化合物为人参皂苷,所述纳米颗粒为Au@CoFeB。
2.根据权利要求1所述的纳米药物,其中所述第一表面改性剂选自3-氨丙基三甲氧基硅氧烷、乙烯基三甲氧基硅烷、磷酸酯双钛酸酯偶联剂和二异丙氧基二乙酰丙酮钛酸酯中的至少一种;优选为3-氨丙基三甲氧基硅氧烷。
3.根据权利要求1所述的纳米药物,其中所述交联剂选自辛二酸(N-羟基琥珀酰亚胺酯)、乙二醇双(丁二酸N-羟基琥珀酰亚胺酯)、聚乙二醇二琥珀酰亚胺琥珀酸酯、琥珀酰-亚胺丁二酸酯、聚乙二醇琥珀酰-亚胺丁二酸酯和氮丙啶交联剂XR-100中的至少一种;优选为辛二酸(N-羟基琥珀酰亚胺酯)、乙二醇双(丁二酸N-羟基琥珀酰亚胺酯)、聚乙二醇二琥珀酰亚胺琥珀酸酯、聚乙二醇琥珀酰-亚胺丁二酸酯和氮丙啶交联剂XR-100中的至少一种。
4.根据权利要求1所述的纳米药物,其中所述第二表面改性剂选自3-氨丙基三甲氧基硅氧烷、3-巯丙基-三乙氧基硅烷偶联剂、十七氟癸基三甲基氧基硅烷和异丙基三(二辛基焦磷酸酰氧基)钛酸酯中的至少一种;优选为3-氨丙基三甲氧基硅氧烷、3-巯丙基-三乙氧基硅烷偶联剂和异丙基三(二辛基焦磷酸酰氧基)钛酸酯中的至少一种。
5.根据权利要求1所述的纳米药物,其中所述纳米药物采用包含以下步骤的方法制备:
(1)制备Au@CoFeB纳米颗粒;
(2)纳米颗粒的表面改性:
a)将(1)中的纳米颗粒加入到含有第一表面改性剂的第一有机溶液中进行搅拌,
b)将a)中加有纳米颗粒的第一有机溶剂溶液经超声清洗、离心和干燥,得到表面改性的纳米颗粒;
(3)纳米颗粒的偶联活化:
a)将(2)中得到的表面改性的纳米颗粒和交联剂分别分散到第二有机溶剂中,搅拌后进行孵化,
b)将a)中加有表面改性的纳米颗粒和交联剂的溶液进行离心和干燥,得到活化后的纳米颗粒;
(4)纳米颗粒的pH调控:
将(3)中所得的活化后的纳米颗粒分散到缓冲液中调节pH值,随后经离心-超声清洗,得到经表面改性和活化的纳米颗粒;
(5)人参皂苷的表面改性:
a)将人参皂苷加入到含有第二表面改性剂的第三有机溶液中进行搅拌,
b)将a)中加有人参皂苷的第三有机溶剂溶液经超声清洗、离心和干燥,得到表面改性的人参皂苷;
(6)纳米颗粒和人参皂苷的偶联:
a)将步骤(3)得到的经表面改性和活化的纳米颗粒和步骤(5)得到的表面改性的人参皂苷放入第四有机溶剂中孵化;
b)将a)中加有经表面改性和活化的纳米颗粒及表面改性的人参皂苷的第四有机溶剂溶液经过离心-超声清洗-离心后进行干燥处理,得到复合纳米药物。
6.根据权利要求5所述的纳米药物,其中步骤(1)中的Au@CoFeB纳米颗粒采用微流控法、水热法、磁控溅射法或电沉积法制备。
7.根据权利要求5所述的纳米药物,其中所述纳米颗粒为核壳结构,核壳结构的金属内核为面心立方晶体结构的Au;核壳结构的壳层为面心立方晶体结构的CoFeB;
所述纳米药物的整体结构为6-7.2nm的超小纳米药物单元偶联在一起构建的尺寸为250-350nm的纳米药物聚集体;纳米颗粒的动力学半径约100-200nm;
所述纳米颗粒表面带有+7-12mV;所述纳米药物表面带有+25-30mV的正电势。
8.权利要求1-7中任一项纳米药物的制备方法,包括以下步骤:
(1)制备Au@CoFeB纳米颗粒;
(2)纳米颗粒的表面改性:
a)将(1)中的纳米颗粒加入到含有第一表面改性剂的第一有机溶液中进行搅拌,
b)将a)中的加有纳米颗粒的第一有机溶剂溶液经超声清洗、离心和干燥,得到表面改性的纳米颗粒;
(3)纳米颗粒的偶联活化:
a)将(2)中得到的表面改性的纳米颗粒和交联剂分别分散到第二有机溶剂中,搅拌后进行孵化,
b)将a)中加有表面改性的纳米颗粒和交联剂的溶液进行离心和干燥,得到活化后的纳米颗粒;
(4)纳米颗粒的pH调控:
将(3)中所得的活化后的纳米颗粒分散到缓冲液中调节pH值,随后经离心-超声清洗,得到经表面改性和活化的纳米颗粒;
(5)人参皂苷的表面改性:
a)将人参皂苷加入到含有第二表面改性剂的第三有机溶液中进行搅拌;
b)将a)中加有人参皂苷的第三有机溶剂溶液经超声清洗、离心和干燥,得到表面改性的人参皂苷;
(6)纳米颗粒和人参皂苷的偶联:
a)将步骤(3)得到的经表面改性和活化的纳米颗粒和步骤(5)得到的表面改性的人参皂苷放入第四有机溶剂中孵化;
b)将a)中的加有经表面改性和活化的纳米颗粒及表面改性的人参皂苷的第四有机溶剂溶液经过离心-超声清洗-离心后进行干燥处理,得到复合纳米药物。
9.权利要求1-7中任一项所述的纳米药物在制备治疗肝癌药物中的应用。
10.根据权利要求9中的应用,其中所述纳米药物中的Au@CoFeB的浓度为0.00001-5000μg/mL。
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US20190125690A1 (en) * | 2016-01-14 | 2019-05-02 | University-Industry Cooperation Group Of Kyung Hee University | A nano complex comprising a nano drug delivery matrix; and a ginseng extract or a ginsenoside isolated therefrom |
US20190307903A1 (en) * | 2018-04-05 | 2019-10-10 | Commissariat A L'energie Atomique Et Aux Energies Alternatives | Method for manufacturing a biocompatible fluid comprising a powder of magnetic particles, biocompatible fluid comprising a powder of magnetic particles |
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KR101748079B1 (ko) * | 2016-04-08 | 2017-06-19 | 경희대학교 산학협력단 | 메조다공성 실리카 나노입자(Mesoporous silica nanoparticle)와 진세노사이드의 결합체 및 이의 용도 |
US20190307903A1 (en) * | 2018-04-05 | 2019-10-10 | Commissariat A L'energie Atomique Et Aux Energies Alternatives | Method for manufacturing a biocompatible fluid comprising a powder of magnetic particles, biocompatible fluid comprising a powder of magnetic particles |
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