CN105381474A - 一种叶酸修饰的四氧化三铁/金星形纳米颗粒的制备方法 - Google Patents
一种叶酸修饰的四氧化三铁/金星形纳米颗粒的制备方法 Download PDFInfo
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- CN105381474A CN105381474A CN201510916406.9A CN201510916406A CN105381474A CN 105381474 A CN105381474 A CN 105381474A CN 201510916406 A CN201510916406 A CN 201510916406A CN 105381474 A CN105381474 A CN 105381474A
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Abstract
本发明涉及一种叶酸修饰的四氧化三铁/金星形纳米颗粒的制备方法，包括：温和“还原法”合成氧化铁纳米颗粒；制备柠檬酸稳定的带有银种子的四氧化三铁纳米颗粒；在金生长溶液中制备星形复合纳米颗粒；PEI的巯基化修饰；星形复合纳米颗粒的表面聚乙烯亚胺PEI和聚乙二醇-叶酸COOH-PEG-FA修饰。本发明反应条件温和，工艺简单，易于操作，制备的氧化铁金星形核壳结构纳米颗粒具有很好的生物医学成像性能和肿瘤的治疗效果，在体内肿瘤成像诊断和光热治疗领域具有潜在的应用价值。
Description
技术领域
本发明属于纳米颗粒的制备及应用领域，特别涉及一种叶酸修饰的四氧化三铁/金星形纳米颗粒的制备方法。
背景技术
近年来，四氧化三铁/金复合纳米颗粒在生物医学领域中受到越来越多的关注，特别是用作MR和CT双模态成像的造影剂和肿瘤的光热治疗，能有效地克服单模态成像的局限性和缺点，提高疾病诊断的准确度并实现其有效的治疗。已有研究在水热法合成四氧化三铁的基础上(专利公开号：201210277624.9；Caietal.，ACSAppl.Mater.Interfaces2013，5(5),1722–1731)，诱导纳米金在四氧化三铁纳米颗粒表面长成星形结构，并探索了其作为肿瘤MR/CT双模态成像和用于光热治疗的可行性(专利申请号：201410853396.4；Biomaterials，2015,38,10-21.)。
然而，上述方法的制备过程复杂，产物制备周期长，产量低，r2弛豫率低，未实现肿瘤的光声(PA)成像。为了减少造影剂的使用剂量并达到对肿瘤灵敏检测和光热治疗效果，制备具有更高弛豫率和更多功能的四氧化三铁/金复合纳米探针就显得尤为有意义。
检索国内外文献，尚没有发现关于温和“还原法”制备的氧化铁/银复合纳米颗粒的种子，以及在此基础上合成叶酸修饰的多功能星形纳米颗粒及其用于体内肿瘤模型MR，CT，PA成像和光热治疗研究的相关报道。
发明内容
本发明所要解决的技术问题是提供一种叶酸修饰的四氧化三铁/金星形纳米颗粒的制备方法，该方法工艺简单，反应条件温和，易于操作，成本较低。该方法制备的复合型星形核壳结构纳米颗粒具有良好的水溶性和胶体稳定性；所用的合成原料为廉价和环境友好材料，具有产业化实施的前景。
本发明的一种叶酸修饰的四氧化三铁/金星形纳米颗粒的制备方法，包括：
(1)将三价铁盐溶于超纯水中，氮气鼓吹后加入亚硫酸钠溶液，反应30～40分钟；然后加入NH3·H2O，60～70℃下恒温搅拌反应30～40分钟，加入柠檬酸，继续恒温搅拌，反应1.5～3小时；将反应后的产物磁分离洗涤，纯化，得到柠檬酸CA包裹的超小型超顺磁四氧化三铁纳米颗粒Fe3O4-CA水溶液；其中，三价铁盐、超纯水和NH3·H2O的配比为1.2～1.4g：20～25mL：2～3mL，三价铁盐、亚硫酸钠和柠檬酸的质量比为1.2～1.4：0.21～0.25：0.50～0.53；
(2)将AgNO3加入到步骤(1)中得到的Fe3O4-CA纳米颗粒溶液中，搅拌，加入冰浴处理的NaBH4的乙醇和水的混合溶液，搅拌2～4h，磁分离洗涤，得到Fe3O4-CA-Ag纳米颗粒水溶液；其中，AgNO3与NaBH4的摩尔比为1：5；
(3)将十六烷基三甲基溴化铵CTAB溶于水中，加入HAuCl4溶液，搅拌，加入AgNO3，搅拌2～3分钟，加入抗环血酸AA，溶液变成无色时加入步骤(2)中得到的Fe3O4-CA-Ag水溶液，反应2～3，离心洗涤除去CTAB，得到核壳结构的复合纳米星形颗粒Fe3O4/AuNSs水溶液；其中，CTAB、HAuCl4、AA、AgNO3和Fe3O4-CA-Ag的投料比为547mg：19.7mg：21mg：0.2mg：220μg；
(4)将超支化聚乙烯亚胺PEI溶于水中，加入巯基乙酸甲酯，60～80℃水浴反应20～30小时，透析，真空冷冻干燥，得到巯基化的PEI，即PEI-SH；其中，PEI与水的比例为1.0g:10ml，PEI和巯基乙酸甲酯的摩尔比为1：30；
(5)向步骤(3)中得到的Fe3O4/AuNSs水溶液中加入步骤(4)中制备得到的PEI-SH水溶液，超声，搅拌反应20～30小时，得到PEI包裹的Fe3O4/AuNSs，即Fe3O4Au-PEINSs；其中，Fe3O4/AuNSs水溶液与PEI-SH水溶液的质量比1：15；
(6)将FA溶解于DMSO中，用EDC和NHS活化2～4小时，加入到NH2-PEG-COOH的DMSO溶液中，搅拌反应2～4天，透析，冷冻干燥，得到COOH-PEG-FA；其中FA和NH2-PEG-COOH的质量比为2：1；
(7)将步骤(6)中得到的COOH-PEG-FA、EDC和NHS溶解于DMSO中，搅拌活化2～4h；将步骤(5)中制备得到的Fe3O4Au-PEINSs分散于DMSO中，加入活化的COOH-PEG-FA溶液，搅拌反应2～4天，离心洗涤，分散于水中，得到Fe3O4Au-PEI-PEG-FANSs水溶液；其中，COOH-PEG-FA与Fe3O4Au-PEINS上PEI的摩尔比30：1；
(8)向步骤(7)中得到的Fe3O4Au-PEI-PEG-FANSs水溶液中加入三乙胺，搅拌20～40分钟；然后加入乙酸酐，反应20～30小时，离心洗涤，分散在超纯水中，得到FA修饰的星形纳米颗粒Fe3O4Au-PEI.Ac-PEG-FA；其中，三乙胺、乙酸酐与Fe3O4Au-PEI-PEG-FANSs上氨基的摩尔比是10：10：1。
所述步骤(1)中三价铁盐为FeCl3·6H2O。
所述步骤(1)中氮气鼓吹的时间为10～15分钟。
所述步骤(1)中NH3·H2O的质量百分比浓度为25～28％。
所述步骤(1)中恒温搅拌的时间为30～40分钟。
所述步骤(2)中搅拌的时间为10～15分钟。
所述步骤(3)中HAuCl4溶液的浓度为30mg/mL。
所述步骤(4)中PEI的分子量Mw＝25000。
所述步骤(4)中巯基乙酸甲酯的纯度为95％。
所述步骤(4)中透析为用截留分子量8000～14000的透析袋对蒸馏水透析2～3天。
所述步骤(6)中EDC、NHS和FA的摩尔比为0.9：0.9：1.0。
所述步骤(7)中COOH-PEG-FA、EDC和NHS的摩尔比为1：10：10。
本发明采用温和“还原法”制备高弛豫率的氧化铁(Fe3O4)纳米颗粒(专利申请号：201410604468.1；Biomater.Sci.,2015,3,721)，在此基础上进一步合成氧化铁/银(Fe3O4/Ag)复合纳米颗粒作为种子，然后在金生长溶液中控制星形纳米颗粒(NSs)的生长，紧接着在其表面先后修饰聚乙烯亚胺(PEI)和聚乙二醇-叶酸分子(COOH-PEG-FA)，乙酰化，最终制备出叶酸修饰的星形纳米颗粒(Fe3O4Au-PEI.Ac-PEG-FANSs)。实验数据表明，制备的Fe3O4Au-PEI.Ac-PEG-FANSs不仅具有良好的水溶性和胶体稳定性，而且同时表现出优良的T2加权成像效果(r2的弛豫率高达549.07mM-1s-1)，X射线衰减性能，光声成像能力和快速升温的特点。此外，COOH-PEG-FA的修饰赋予了复合星形纳米颗粒更好的生物相容性及用于生物体内对特定肿瘤部位的成像检测的能力。以叶酸受体高表达的HeLa细胞(一种人宫颈瘤癌细胞)为模型细胞和肿瘤模型对制备的多功能星形纳米颗粒的性质进行一一评价。本研究制备的Fe3O4Au-PEI.Ac-PEG-FANSs纳米探针具备的良好特性，特别是高的弛豫率，优良的CT和光声成像效果，快速升温杀死癌细胞和肿瘤的能力等，使得其有望实现不同疾病的灵敏诊断和治疗。因此，本发明制备的纳米探针有望用于临床“诊疗一体化”。
本发明操作简便易行，原材料成本低。制备的复合型纳米颗粒具有良好的水溶性、胶体稳定性和生物相容性。体外和动物体内实验结果显示该星形复合纳米颗粒不仅具有很好的MR，CT和PA成像效果，同时还能对癌细胞和肿瘤达到光热治疗的效果。因此，该方法制备的氧化铁金星形核壳结构纳米颗粒在MR，CT和PA三模态成像诊断和癌症的治疗领域有着潜在的应用。
本发明使用核磁共振氢谱分析(1HNMR)、紫外可见吸收光谱(UV-Vis)、热重分析(TGA)、电感耦合等离子体原子发射光谱法(ICP-OES)、Zeta电势及动态光散射(DLS)和透射电子显微镜(TEM)等方法表征制备的星形核壳结构性纳米颗粒，并通过MR成像仪，CT成像系统和PA成像系统测定纳米颗粒的T2弛豫性能，X射线衰减性能和PA信号增强性能，并评价了纳米颗粒作为热疗试剂在近红外激光照射下的升温效率。然后利用MTT法评价纳米颗粒的细胞毒性和纳米颗粒在808nm的激光照射下对细胞的杀伤效果，最后通过负荷肿瘤的裸鼠，对纳米颗粒的体内成像和治疗性能进行评价。
有益效果
(1)本发明先利用温和“还原法”合成带有银种子的氧化铁纳米颗粒；再在金生长溶液中制备星形复合纳米颗粒；然后对制备的星形复合纳米颗粒的进行表面PEI和COOH-PEG-FA修饰；最后乙酰化。制备方法操作工艺简单，反应条件温和，易于操作纯化，所用均为廉价的和环境友好的材料；
(2)本发明制备的复合型纳米颗粒具有良好的水溶性、胶体稳定性和生物相容性。体外和动物体内实验结果显示该星形复合纳米颗粒不仅具有很好的MR，CT和PA成像效果，同时能对癌细胞和肿瘤达到光热治疗的效果。该方法制备的氧化铁金星形核壳结构纳米颗粒在多模态成像诊断和癌症的治疗领域有着潜在的应用。
附图说明
图1为实施例2中PEI-SH的1HNMR图谱；
图2为实施例4中COOH-PEG-FA进行1HNMR图谱；
图3为实施例5中Fe3O4Au(a)，Fe3O4Au-PEI(b)和Fe3O4Au-PEI-PEG-FA(c)的热重分析图；
图4为实施例5中Fe3O4Au-PEI.Ac-PEG-FANSs激光照射前(a)后(b)的紫外吸收图谱；
图5为实施例6中Fe3O4Au-PEI.Ac-PEG-FANSs的透射电镜图片(a)和高分辨透射电镜图片(b)；
图6为实施例7中Fe3O4Au-PEI.Ac-PEG-FANSs在不同Fe浓度下(0.00125-0.02mM)的T2加权成像(a)和T2弛豫时间倒数与Fe浓度的线性关系图(b)；在不同Au浓度下(4-64mM)的CT成像图片(c)和CT值随金浓度变化的线性关系图(d)；
图7为实施例8中Fe3O4Au-PEI.Ac-PEG-FANSs在不同Au浓度下(0-2.0mM)经过808nm激光照射后的PA成像图(a)和PA值随金浓度变化的线性关系图(b)；
图8为实施例9中Fe3O4Au-PEI.Ac-PEG-FANSs在不同Au浓度下(0-20mM)经过808nm激光照射后的升温曲线图(a)和在相同Au浓度下(10mM)经过不同功率的激光照射后升温曲线图(b)；
图9为实施例10中MTT法测试的HeLa细胞经过PBS缓冲液(对照)和本发明制备的Fe3O4Au-PEI.Ac-PEG-FANSs(Au的浓度范围在0.2-2.0mM)处理24小时后的细胞活力；
图10为实施例11中MTT法测试的HeLa细胞在与本发明制备的Fe3O4Au-PEI.Ac-PEG-FANSs(Au浓度0.1-0.8mM)共培养6小时后，不经过和经过的激光照射后细胞的存活率；
图11为实施例12中叶酸受体低表达(被叶酸阻断)和叶酸受体高表达(未被叶酸阻断)的HeLa细胞与经过PBS缓冲液(对照)和本发明制备的Fe3O4Au-PEI.Ac-PEG-FANSs的PBS溶液(金的浓度为0.5、1.0和2.0mM)共培养4小时后，ICP测出的细胞中金的吞噬量；
图12为实施例13中瘤内注射本发明制备的Fe3O4Au-PEI.Ac-PEG-FANSs([Fe]＝1.31mM,[Au]＝70mM，0.1mL)到裸鼠肿瘤部位后的MR(a)和CT(c)成像以及相应的MR(b)和CT(d)信号强度的变化；
图13是实施例14中瘤内注射本发明制备的Fe3O4Au-PEI.Ac-PEG-FANSs([Au]＝100mM，40μL)到裸鼠肿瘤部位前后经808nm激光照射后的PA成像(a)和PA信号值(b)的变化；
图14为实施例15中瘤内注射本发明制备的Fe3O4Au-PEI.Ac-PEG-FANSs([Au]＝20mM，0.1mL)(Ar1)和PBS(0.1mL)(Ar2)到裸鼠肿瘤部位后的肿瘤部位的热敏成像图片(a)和温度变化曲线(b)；
图15是实施例16中经过不同方式处理后裸鼠肿瘤的体积变化(a)和生存率(b)。荷瘤鼠分为四组：不做任何处理，瘤内注射PBS(0.1mL)且经过激光照射，瘤内注射本发明制备的Fe3O4Au-PEI.Ac-PEG-FANSs([Au]＝20mM，0.1mL)但不经过激光照射，瘤内注射本发明制备的Fe3O4Au-PEI.Ac-PEG-FANSs([Au]＝20mM，0.1mL)且经过激光照射。
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。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1
将1.3g三价铁盐溶解于超纯水中，移入250mL三口瓶中搅拌；氮气鼓吹10～15分钟后，将0.2g亚硫酸钠溶液加入到三口瓶中，反应30～40分钟；接着加入2mLNH3·H2O，60～70℃下恒温搅拌反应30～40分钟；然后加入0.5g柠檬酸，继续在60～70℃下恒温搅拌反应30～40分钟，接着在室温下反应1.5～3小时；反应结束后，将产物磁分离洗涤纯化，即得柠檬酸CA包裹的超小型超顺磁氧化铁纳米颗粒(Fe3O4-CA)水溶液(浓度为1mg/mL)，储存在4℃备用。将3.4mgAgNO3溶液逐滴加入到制备的Fe3O4-CA纳米颗粒溶液中，搅拌10～15min，然后加入3.8mgNaBH4的乙醇和水混合溶液(v/v＝1:2)，搅拌2-4h，磁分离洗涤，得到Fe3O4-CA-Ag纳米颗粒，分散于15mL水中，储存在4℃备用。称547mg十六烷基三甲基溴化铵(CTAB)溶解于15mL水中，加入658μLHAuCl4溶液(30mg/mL)，搅拌2～3分钟后加入0.2mgAgNO3，再搅拌2～3分钟后加入21mg抗坏血酸(AA)，待溶液变成无色时加入制备的Fe3O4-AC-Ag水溶液(220μL)。反应结束后，离心洗涤除去CTAB，即得到复合纳米星形颗粒(Fe3O4/AuNSs)，储存在4℃备用。
实施例2
称1.0gPEI溶于10mL水中并向其中加入108μL巯基乙酸甲酯(Methylthioglycolate，95％，Mw＝106.14，ρ＝1.187)在60～80℃水浴反应2天，透析除去副产物和杂质(用截留分子量为8000-14000的透析袋对蒸馏水透析三天(6次，2L/次))，然后真空冷冻干燥，即得PEI-SH，储存在-20℃备用。取少量的PEI-SH进行1HNMR图谱分析，如图1所示，结果表明PEI-SH在3.4ppm处有一个明显的谱峰，该峰是由PEI上的氨基和巯基乙酸甲酯发生水解反应后形成的-CO-CH2-S-上的氢质子导致的，从而说明PEI成功巯基化修饰。
实施例3
向实施例1制备的Fe3O4AuNSs水溶液中加入实施例2中制备的PEI-SH水溶液(0.1g)，超声30分钟后搅拌反应1天，得到PEI包裹的Fe3O4AuNSs，即Fe3O4/Au-PEINSs，储存在4℃备用。
实施例4
将1.8mgFA溶解于DMSO中后，先用0.7mgEDC和0.4mgNHS活化2～4h，然后逐滴加入到4.0mgNH2-PEG-COOH的DMSO溶液中，搅拌反应2～4天，透析除去副产物和杂质(用截留分子量为1000的透析袋对蒸馏水透析三天(6次，2L/次))，然后真空冷冻干燥，即得COOH-PEG-FA，储存在-20℃备用。取少量的COOH-PEG-FA进行1HNMR图谱分析，如图2所示，结果表明PEG-FA在6.62，7.70和8.64ppm处有明显的谱峰，该峰是由FA苯环上的氢质子导致的，从而说明成功制备COOH-PEG-FA。
实施例5
将实施例4中制备的1.13mgCOOH-PEG-FA以及0.89mgEDC和0.53mgNHS溶解于DMSO后，搅拌活化2～4h；将实施例3中制备的8.0mgFe3O4Au-PEINSs分散于DMSO中，然后将活化的COOH-PEG-FA溶液加入其中，搅拌反应2～4天，再离心洗涤，并分散于水中，得到Fe3O4Au-PEI-PEG-FANSs，储存在4℃备用。向制备的Fe3O4Au-PEI-PEG-FANSs水溶液中加入12.6μL三乙胺，并搅拌20～40分钟；然后向上述溶液中加入10.3μL乙酸酐，继续搅拌反应20～30小时；离心洗涤并分散于超纯水中，即得最终产物FA修饰的复合星形纳米颗粒(Fe3O4Au-PEI.Ac-PEG-FANSs)，储存在4℃备用。
取Fe3O4Au-PEINSs(实施例3)，Fe3O4Au-PEI-PEG-FANSs(实施例5)和Fe3O4Au-PEI.Ac-PEG-FANSs(实施例5)用超纯水分别配制成1.5mL的水溶液用于测表面电势和水动力直径(见表1)。电势结果显示PEI修饰后星形纳米颗粒的表面电势是+31.4mV，而经过COOH-PEG-FA修饰和乙酰化处理后电势降低到+28.3mV和+14.4mV。水动力粒径测试结果显示Fe3O4Au-PEINSs的水动力粒径是211.3nm，而经过COOH-PEG-FA修饰和乙酰化处理后的星形纳米颗粒的水动力粒径分别是226.1nm和244.2nm。
取Fe3O4Au(实施例1)，Fe3O4Au-PEINSs(实施例3)，Fe3O4Au-PEI-PEG-FANSs(实施例5)的水溶液，真空冷冻干燥，然后进行热重分析(见附图3)。结果表明，Fe3O4Au在温度升至700℃时，几乎不失重(见附图3a)；Fe3O4Au-PEI和Fe3O4Au-PEI-PEG-FA在温度升至700℃时，重量为原来的95.2％和87.6％，经过计算，PEI上载率为4.8％(见附图3b)，COOH-PEG-FA上载率7.6％(见附图3c)，由此表明PEI和COOH-PEG-FA已成功连接到Fe3O4Au纳米颗粒的表面。
别取25μLFe3O4Au-PEI.Ac-PEG-FANSs(实施例5)的水溶液于2mL离心管中，再向其中加700μL超纯水，超声均匀，测紫外吸收(见附图4)。从图4a中我们可以看出，星形纳米颗粒在810nm附近有一个很强的紫外吸收峰，从而为近红外的成像和光热治疗提供了可行性。此外，在808nm激光(2.0W/cm2，20min)连续照射后，该纳米颗粒的吸收峰没有明显的变化，表明本发明制备的纳米颗粒具有较好的光热稳定性，能够长期暴露于激光下进行疾病的光热治疗(图4b)。
表1星形纳米颗粒分散于水中的电势和水动力粒径
实施例6
取Fe3O4Au-PEI.Ac-PEG-FANSs(实施例5)纳米颗粒水溶液5μL，然后用超纯水配制成100μL的纳米颗粒悬浮液。并将纳米颗粒悬浮液5μL滴在铜网表面，在空气中晾干后用于TEM测试(见附图5)。TEM结果显示Fe3O4Au-PEI.Ac-PEG-FANSs的形貌均为星形核壳结构。通过分别随机测量300个纳米颗粒的直径计算得到Fe3O4Au-PEI.Ac-PEG-FA纳米颗粒的直径是149.5±21.6nm(图5a)。高分辨TEM显示纳米颗粒的表面包覆一层有机层(图5b)。
实施例7
弛豫率(r2)是评价Fe3O4纳米材料用作核磁共振(MR)成像阴性造影剂的重要指标，弛豫率可通过不同铁浓度下的弛豫时间(T2)的倒数拟合计算得到。将制备的Fe3O4Au-PEI.Ac-PEG-FANSs(实施例5)通过ICP-OES测试法测定溶液中Fe元素的浓度，接着在EP管中用超纯水配制Fe浓度依次为0.00125、0.0025、0.005、0.01和0.02mM的水溶液2mL，利用磁共振成像仪测定材料在不同的Fe浓度下的成像效果和T2弛豫效应(见附图6)。T2加权成像结果表明本发明制备的Fe3O4Au-PEI.Ac-PEG-FANSs随着铁浓度的增加，MR信号强度随之减弱(见附图6a)。弛豫率测试结果表明Fe3O4Au-PEI.Ac-PEG-FANSs的弛豫时间倒数随着铁浓度的增加(在0.0025-0.02mM浓度范围内)具有很好的线性关系。并且通过计算可得本发明制备的Fe3O4Au-PEI.Ac-PEG-FANSs的r2弛豫率是549.07mM-1s-1(见附图6b)。因此，本发明所制备的Fe3O4Au-PEI.Ac-PEG-FANSs可作为MRI分子影像学诊断中的优良T2信号衰减造影剂。
类似的，通过ICP-OES测试法测定溶液中Au元素的浓度，接着在EP管中用超纯水配制Au浓度依次为4、8、16、32和64mM的水溶液2mL，使用临床医用CT成像系统测定星形纳米颗粒的CT效应。结果表明随着金浓度的增加(在4-64mM浓度范围内)，星形纳米颗粒的CT信号强度也随之增强(见附图6c)。而且，星形纳米颗粒的CT值随着金浓度的增加具有很好的线性关系(见附图6d)。由此说明本发明的Fe3O4Au-PEI.Ac-PEG-FANSs有望用作CT成像的造影剂。
实施例8
由于Fe3O4Au-PEI.Ac-PEG-FANSs(实施例5)在800nm左右有明显的近红外吸收峰，据此可一步探究本发明制备的的PA成像效应，测量了该星形纳米颗粒的808nm激光照射下的光声信号增强特性(如图7所示)。依据实施例(实施例7)测定的Au元素的浓度，配制成Au浓度依次为0、0.25、0.5、1.0和2.0mM的Fe3O4Au-PEI.Ac-PEG-FANSs水溶液，各取15μL注射到自制琼脂糖板孔洞里，利用光声成像系统在808nm波长激光下测定星形纳米颗粒的光声效应(见附图7)。结果表明，随着金浓度的增加(0-2.0mM)，星形纳米颗粒的PA信号强度也随之增强(见附图7a)。而且，复合纳米颗粒的PA值随着金浓度的变化具有很好的线性关系(见附图7b)。说明了本发明的Fe3O4Au-PEI.Ac-PEG-FANSs有望用作PA成像的造影剂。
实施例9
依据实施例(实施例7)测得的Au元素的浓度，在EP管中用超纯水配制Au浓度依次为1、2、4、10和20mM的水溶液100μL，以等体积的水做对照，用808nm激光照射(输出功率1.0W/cm2，照射5分钟)，观察温度变化情况(见附图8a)。结果表明，水在照射5分钟后，温度仅稍微的升高。而对于Fe3O4Au-PEI.Ac-PEG-FANSs，随着照射时间的延长，纳米颗粒水溶液的温度明显升高。而且随着Au浓度的增加，温度的升高更加明显。在相同浓度下(10mM)，分别用输出功率为0.25，0.5，1.0和1.5W/cm2的808nm的激光照射，观察温度变化情况(见附图8b)。结果表明，随着对Fe3O4Au-PEI.Ac-PEG-FANSs(实施例5)水溶液照射时间的延长，水溶液的温度明显升高。而且随着输出功率的增加，温度的升高更加明显。
实施例10
通过MTT比色法测定HeLa细胞的活力来检测Fe3O4Au-PEI.Ac-PEG-FANSs的细胞相容性。以HeLa细胞为模型细胞评价制备的Fe3O4Au-PEI.Ac-PEG-FANSs(实施例5)对细胞增殖的影响。用无菌PBS配置不同浓度的Fe3O4Au-PEI.Ac-PEG-FANSs的PBS溶液，并用紫外照射杀菌过夜。HeLa细胞种植于96孔板(1×104细胞/孔)，在37℃和5％CO2条件下过夜培养，贴壁后弃去培养基，再用200μL含Fe3O4Au-PEI.Ac-PEG-FANSs(实施例5)(Au浓度为0.2、0.4、0.8、1.5和2.0mM)的DMEM培养基在37℃和5％CO2条件下培养24小时。然后，向培养板孔中加入20μLMTT，继续在37℃下培养4小时后，弃去培养液，并加入200μLDMSO，振荡15分钟后在570nm处测量吸光值，并根据此值计算细胞的活力(见附图9)。不同浓度的材料对细胞增殖的影响以缓冲液PBS为对照进行比较。与PBS对照组相比，即使Au的实验浓度高达2.0mM，细胞活力也仍然保持在80％以上，这充分说明本发明制备的Fe3O4Au-PEI.Ac-PEG-FANSs具有良好的生物相容性。
实施例11
以HeLa细胞为模型细胞，在808nm激光照射下评价Fe3O4Au-PEI.Ac-PEG-FANSs(实施例5)对肿瘤细胞的杀伤作用。用无菌PBS配置不同浓度的Fe3O4Au-PEI.Ac-PEG-FANSs的PBS溶液，并用紫外照射杀菌过夜。将HeLa细胞种植于96孔板(1×104细胞/孔)，在37℃和5％CO2条件下过夜培养，贴壁后弃去培养基，再用200μL含本发明制备的Fe3O4Au-PEI.Ac-PEG-FANSs(实施例5)(Au浓度为0.1、0.2、0.4和0.8mM)的DMEM培养基培养6小时，用PBS洗涤3次，用808nm激光器照射(输出功率1.0W/cm2，照射5分钟)，未照射的细胞用作对照组。然后，向培养板孔中加入20μLMTT，继续在37℃下培养4小时后，弃去培养液，并加入200μLDMSO，振荡15分钟后在570nm处测量吸光值，并根据此值计算细胞的活力(见附图10)。结果表明随着浓度的增加，未照射的细胞仍然保持很高的细胞存活率；而经过激光照射5分钟，细胞表现出死亡趋势，并且随着金浓度的增加，细胞的存活率越低，并且在金浓度为0.8mM时，仅有24.7％的细胞存活。该实验结果说明本发明制备的Fe3O4Au-PEI.Ac-PEG-FANSs在近红外激光照射下能引起肿瘤细胞的死亡。
实施例12
通过定量分析HeLa细胞对本发明制备的Fe3O4Au-PEI.Ac-PEG-FANSs(实施例5)在不同Au浓度下的吞噬量来检测叶酸的靶向性效果。将叶酸受体高低表达的HeLa细胞以2×105每孔的密度种植于12孔板中，在37℃和5％CO2条件下培养过夜，贴壁后弃去培养基，再与含的Fe3O4Au-PEI.Ac-PEG-FANSs(实施例5)(Au浓度为0.5、1.0和2.0mM)的DMEM培养基(1mL)共培养4小时。培养结束后用PBS清洗3次，胰酶消化，离心，王水(硝酸与盐酸体积比为1:3)消化，ICP-OES检测HeLa细胞中金的含量，以此评价其靶向性效果(见附图11)。结果表明，在Au浓度0.5-2.0mM范围内，Fe3O4Au-PEI.Ac-PEG-FA纳米探针培养的叶酸受体高表达(未被叶酸阻断)的HeLa细胞对Au元素的吞噬量明显高于低叶酸受体表达(被叶酸阻断)的HeLa细胞。因此，叶酸的修饰使得Fe3O4Au-PEI.Ac-PEG-FANSs对叶酸受体高表达的HeLa细胞有特异靶向能力。
实施例13
ICP测试结果显示Fe3O4Au-PEI.Ac-PEG-FANSs中Fe元素和Au元素的摩尔比例是1:53.4。为了探索本发明制备的Fe3O4Au-PEI.Ac-PEG-FANSs(实施例5)用作MR成像造影剂的潜力，将HeLa荷瘤裸鼠麻醉后，直接瘤内注射本发明制备的Fe3O4Au-PEI.Ac-PEG-FANSsPBS溶液([Fe]＝1.31mM,[Au]＝70mM,0.1mL)，再将裸鼠置于动物线圈中，注射30min后用临床核磁共振成像体系评价肿瘤部位(圆圈)的MR成像效果。结果表明，注射星形纳米颗粒30分钟后，裸鼠肿瘤较注射前(0分钟)明显的变暗(见附图12a)，其肿瘤部位的MR信号强度从注射前的364.9降低到21.3(见附图12b)。在探索其用作CT成像造影剂的潜力时，用临床医用CT成像系统评价星形纳米颗粒注射后肿瘤部位(箭头)的CT效果。结果表明，注射星形纳米颗粒30分钟后，肿瘤部位的CT信号强度较注射前(0分钟)明显变亮(见附图12c)，裸鼠肿瘤部位的亨氏单位从30.7HU增加到270.9HU(见附图12d)。该组实验结果说明，制备的Fe3O4Au-PEI.Ac-PEG-FANSs具有很好的肿瘤MR和CT成像性能。
实施例14
为了探索本发明制备的Fe3O4Au-PEI.Ac-PEG-FANSs(实施例5)用作PA成像造影剂的潜力，将HeLa荷瘤裸鼠麻醉后，瘤内注射Fe3O4Au-PEI.Ac-PEG-FANSsPBS溶液([Au]＝100mM，40μL)，然后用光声成像系统在808nm波长激光下观察星形纳米颗粒注射后肿瘤的光声效果(见附图13)。结果表明，注射纳米颗粒30分钟后，裸鼠肿瘤较注射前(0分钟)PA信号强度明显增强(见附图13a)。对信号强度定量测量可知，肿瘤部位的PA值从注射前的0.14升高到2.82(见附图13b)。结果说明，本发明制备的Fe3O4Au-PEI.Ac-PEG-FANSs具有很好的PA成像性能。
实施例15
为了探索本发明制备的Fe3O4Au-PEI.Ac-PEG-FANSs(实施例5)在体内光热升温能力，将HeLa荷瘤裸鼠麻醉后，直接瘤内注射本发明制备的Fe3O4Au-PEI.Ac-PEG-FANSsPBS溶液([Au]＝20mM,0.1mL)。接着，使用808nm激光照射(输出功率1.0W/cm2，照射5分钟)，并通过红外摄像机来评价体内肿瘤部位的热成像效果，同时以注射相同体积PBS的裸鼠作为对照(见附图14)。结果表明，注射后2.5分钟，注射Fe3O4Au-PEI.Ac-PEG-FANSs的裸鼠肿瘤部位的温度明显升高，达到了51.5℃，而此时注射PBS的裸鼠的肿瘤部位的温度仅仅达到30.5℃。在随后的一段时间内，注射Fe3O4Au-PEI.Ac-PEG-FANSs的裸鼠肿瘤部位的温度一直保持在50℃以上。注射5分钟后，注射星形纳米颗粒的裸鼠肿瘤部位温度高达57.6℃，而对照裸鼠肿瘤部位的温度仅仅达到在31.3℃左右。实验结果说明本发明制备的Fe3O4Au-PEI.Ac-PEG-FANSs能使肿瘤部位的温度升高，从而达到杀死肿瘤细胞的作用。
实施例16
将负荷肿瘤的裸鼠分为四组，分别经过不同方式处理：不经过任何处理，瘤内注射PBS(0.1mL)且经过808nm激光照射(输出功率1.0W/cm2，照射5分钟)，瘤内注射本发明制备的Fe3O4Au-PEI.Ac-PEG-FANSs(实施例5)PBS溶液([Au]＝20mM，0.1mL)但不经过激光照射，瘤内注射本发明制备的Fe3O4Au-PEI.Ac-PEG-FANSsPBS溶液([Au]＝20mM，0.1mL)且经过808nm激光照射(输出功率1.0W/cm2，照射5分钟)。各组荷瘤裸鼠均在第0，2和4天各处理一次(见附图15)。结果表明，只有瘤内注射星形纳米颗粒且经过激光照射的裸鼠肿瘤才完全消失(处理后第七天)，其余三组裸鼠肿瘤均继续快速生长(见附图15a)。而且，只有瘤内注射星形纳米颗粒且经过激光照射的裸鼠未出现死亡现象，其余三组在60天内均完全死亡(见附图15b)。该实验结果说明本发明制备的Fe3O4Au-PEI.Ac-PEG-FANSs在近红外激光的照射下能引起肿瘤的消融，从而达到治疗肿瘤的目的。
Claims (10)
1.一种叶酸修饰的四氧化三铁/金星形纳米颗粒的制备方法，包括：
(1)将三价铁盐溶于超纯水中，氮气鼓吹后加入亚硫酸钠溶液，反应30～40分钟；然后加入NH3·H2O，60～70℃下恒温搅拌反应30～40分钟，加入柠檬酸，继续恒温搅拌，反应1.5～3小时；将反应后的产物磁分离洗涤，纯化，得到柠檬酸CA包裹的超小型超顺磁四氧化三铁纳米颗粒Fe3O4-CA水溶液；其中，三价铁盐、超纯水和NH3·H2O的配比为1.2～1.4g：20～25mL：2～3mL，三价铁盐、亚硫酸钠和柠檬酸的质量比为1.2～1.4：0.21～0.25：0.50～0.53；
(2)将AgNO3加入到步骤(1)中得到的Fe3O4-CA纳米颗粒水溶液中，搅拌，加入冰浴处理的NaBH4的乙醇和水的混合溶液，搅拌2～4h，磁分离洗涤，得到Fe3O4-CA-Ag纳米颗粒水溶液；其中，AgNO3与NaBH4的摩尔比为1:5；
(3)将十六烷基三甲基溴化铵CTAB溶于水中，加入HAuCl4溶液，搅拌，加入AgNO3，搅拌2～3分钟，加入抗环血酸AA，溶液变成无色时加入步骤(2)中得到的Fe3O4-CA-Ag水溶液，反应2～3分钟，离心洗涤除去CTAB，得到核壳结构的复合纳米星形颗粒Fe3O4/AuNSs水溶液；其中，CTAB、HAuCl4、AA、AgNO3和Fe3O4-CA-Ag的投料比为547mg：19.7mg：21mg：0.2mg：220μg；
(4)将超支化聚乙烯亚胺PEI溶于水中，加入巯基乙酸甲酯，60～80℃水浴反应20～30小时，透析，真空冷冻干燥，得到巯基化的PEI，即PEI-SH；其中，PEI与水的比例为1.0g：10ml，PEI和巯基乙酸甲酯的摩尔比为1：30；
(5)向步骤(3)中得到的Fe3O4/AuNSs水溶液中加入步骤(4)中制备得到的PEI-SH水溶液，超声30分钟，搅拌反应20～30小时，得到PEI包裹的Fe3O4/AuNSs，即Fe3O4Au-PEINSs；其中，Fe3O4/AuNSs水溶液与PEI-SH水溶液的质量比为1：15；
(6)将FA溶解于DMSO中，用EDC和NHS活化2～4小时，加入到NH2-PEG-COOH的DMSO溶液中，搅拌反应2～4天，透析，冷冻干燥，得到COOH-PEG-FA；其中FA和NH2-PEG-COOH的摩尔比为2：1；
(7)将步骤(6)中得到的COOH-PEG-FA、EDC和NHS溶解于DMSO中，搅拌活化2～4h；将步骤(5)中制备得到的Fe3O4Au-PEINSs分散于DMSO中，加入活化的COOH-PEG-FA溶液，搅拌反应2～4天，离心洗涤，分散于水中，得到Fe3O4Au-PEI-PEG-FANSs水溶液；其中，COOH-PEG-FA与Fe3O4Au-PEINSs上PEI的摩尔比是30：1；
(8)向步骤(7)中得到的Fe3O4Au-PEI-PEG-FANSs水溶液中加入三乙胺，搅拌20～40分钟；然后加入乙酸酐，反应20～30小时，离心洗涤，分散在超纯水中，得到FA修饰的星形纳米颗粒Fe3O4Au-PEI.Ac-PEG-FA；其中，三乙胺、乙酸酐与Fe3O4Au-PEI-PEG-FANSs上氨基的摩尔比是10：10：1。
2.根据权利要求1所述的一种叶酸修饰的四氧化三铁/金星形纳米颗粒的制备方法，其特征在于，所述步骤(1)中三价铁盐为FeCl3·6H2O。
3.根据权利要求1所述的一种叶酸修饰的四氧化三铁/金星形纳米颗粒的制备方法，其特征在于，所述步骤(1)中氮气鼓吹的时间为10～15分钟。
4.根据权利要求1所述的一种叶酸修饰的四氧化三铁/金星形纳米颗粒的制备方法，其特征在于，所述步骤(1)中NH3·H2O的质量百分比浓度为25～28％。
5.根据权利要求1所述的一种叶酸修饰的四氧化三铁/金星形纳米颗粒的制备方法，其特征在于，所述步骤(1)中恒温搅拌的时间为30～40分钟。
6.根据权利要求1所述的一种叶酸修饰的四氧化三铁/金星形纳米颗粒的制备方法，其特征在于，所述步骤(2)中搅拌的时间为10～15分钟。
7.根据权利要求1所述的一种叶酸修饰的四氧化三铁/金星形纳米颗粒的制备方法，其特征在于，所述步骤(3)中HAuCl4溶液的浓度为30mg/mL。
8.根据权利要求1所述的一种叶酸修饰的四氧化三铁/金星形纳米颗粒的制备方法，其特征在于，所述步骤(4)中透析为用截留分子量8000～14000的透析袋对蒸馏水透析2～3天。
9.根据权利要求1所述的一种叶酸修饰的四氧化三铁/金星形纳米颗粒的制备方法，其特征在于，所述步骤(6)中EDC、NHS和FA的摩尔比为0.9：0.9：1.0。
10.根据权利要求1所述的一种叶酸修饰的四氧化三铁/金星形纳米颗粒的制备方法，其特征在于，所述步骤(7)中COOH-PEG-FA、EDC和NHS的摩尔比为1：10：10。
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