CN111420072A - 一种mri-sers双模式造影剂的制备方法 - Google Patents
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Abstract
本发明公开了一种MRI‑SERS双模式造影剂的制备方法,包括以下步骤,S1:Fe3O4/Au种子样品的制备;S2:Fe3O4/Au纳米复合材料的制备;S3:改性Fe3O4/Au纳米复合材料的制备。本发明方法采用SiO2代替现有方法中常用试剂PEI对Fe3O4进行包覆,利用SiO2层保护磁性纳米粒子,由于SiO2层的覆盖,其SERS信号检测高于PEI修饰合成的Fe3O4/Au复合纳米材料;在材料的MRI和SERS性能研究中,SiO2修饰合成的Fe3O4/Au复合纳米材料也显示出很好的生物相容性,可作为MRI‑SERS双模式造影剂应用于医学成像领域。
Description
技术领域
本发明属于纳米催化复合材料技术领域,尤其涉及一种MRI-SERS双模式造影剂的制备方法。
背景技术
近几年来,多模式医学造影剂在生物检测领域受到广泛关注。在众多的多模式造影剂中,将MRI和SERS成像技术结合,大大提高对肿瘤检测的灵敏度和准确性。目前在MRI-SERS双模式造影剂的研究中,常用四氧化三铁纳米粒子与金纳米粒子通过PEI结合,由于Fe3O4易被酸性环境腐蚀、溶解,因此,不利于长时间的监测。
发明内容
本发明为解决现有技术中的上述问题,提出了一种MRI-SERS双模式造影剂的制备方法,采用SiO2进行Fe3O4的包覆代替常用试剂PEI,利用SiO2层保护磁性纳米粒子,其SERS信号检测高于PEI修饰合成的Fe3O4/Au复合纳米材料;同时,该材料也显示很好的生物相容性,可作为MRI和SERS双模式成像试剂在医学成像领域使用。
为实现上述目的,本发明采用以下技术方案:
本发明提供一种MRI-SERS双模式造影剂的制备方法,其特征在于,包括以下步骤,
S1:Fe3O4/Au种子样品的制备;
将4-8mgFe3O4/SiO2微球样品分散于盛有20-40mL水的烧瓶中,超声处理后,加入由1.5-3mL HAuCl4和1.5-3mL NaOH组成的混合水溶液,搅拌均匀,最后,在剧烈搅拌下向烧瓶中注入新鲜制备的1-2mL还原剂水溶液;
S2:Fe3O4/Au纳米复合材料的制备;
将经步骤S1制得的Fe3O4/Au种子样品分散于盛有60-120mL超纯水的烧瓶中,加入1-2mL NaAuCl4和1-2mL SiO2水溶液,搅拌均匀,最后,在剧烈搅拌下将1-2mL抗坏血酸水溶液注射入烧瓶,该过程重复两次,最终得到Fe3O4/Au纳米复合材料;
S3:改性Fe3O4/Au纳米复合材料的制备;
经磁分离收集新鲜经步骤S2制得的Fe3O4/Au样品分散于30-60mL 4-ATP的乙醇溶液中,搅拌均匀,所得产品经乙醇和牛血清白蛋白水溶液洗涤数次后,分散于10-20mL磷酸盐缓冲盐水,并通过叶酸进行修饰,制得以0.5-1mL4-ATP和叶酸的混合液改性的Fe3O4/Au纳米复合材料。
优选的,步骤S1中,超声时间为10-15min。
优选的,步骤S1中,HAuCl4的浓度为10mmol/L;所述NaOH的浓度为10mmol/L;所述还原剂水溶液的浓度为60mmol/L。
优选的,所述还原剂为硼氢化钠、柠檬酸三钠和葡萄糖中的一种。
优选的,步骤S1中,所述搅拌的时间为30min。
优选的,步骤S2中,所述NaAuCl4的浓度为5mmol/L;所述SiO2的浓度为2mg·mL-1;所述抗坏血酸水溶液的浓度为15mmol/L。
优选的,步骤S2中,所述搅拌的时间为30-45min。
优选的,所述4-ATP乙醇溶液的浓度为0.1mmol/L;所述牛血清白蛋白水溶液的浓度为1mg·mL-1;所述叶酸溶液的浓度为1mmol/L。
优选的,步骤S3中,所述搅拌的时间为2-3h。
本发明采用上述技术方案,与现有技术相比,具有如下技术效果:
本发明MRI-SERS双模式造影剂的制备方法,采用SiO2代替现有方法中常用试剂PEI对Fe3O4进行包覆,利用SiO2层保护磁性纳米粒子,由于SiO2层的覆盖,其SERS信号检测高于PEI修饰合成的Fe3O4/Au复合纳米材料;在材料的MRI和SERS性能研究中,SiO2修饰合成的Fe3O4/Au复合纳米材料也显示出很好的生物相容性,可作为MRI-SERS双模式造影剂应用于医学成像领域。
附图说明
图1为发明制备得到的Fe3O4/SiO2和Fe3O4/SiO2/Au复合纳米材料的TEM照片;
图2为发明制备得到的Fe3O4/Au复合材料的UV-Vis和XRD图谱;
图3为发明制备得到的Fe3O4/Au纳米复合材料的生物相容性实验数据;
图4为发明制备得到的Fe3O4/Au复合材料的MRI测试结果;
图5为发明制备得到的Fe3O4/Au复合材料的Raman图谱;
图6为Fe3O4/Au纳米复合材料不同浓度下单独和与细胞孵育后分散在琼脂糖凝胶的T2加权MR图像。
具体实施方式
下面通过具体实施例对本发明进行详细和具体的介绍,以使更好的理解本发明,但是下述实施例并不限制本发明范围。
本发明提供了一种MRI-SERS双模式造影剂的制备方法,包括以下步骤,
S1:Fe3O4/Au种子样品的制备;将4-8mgFe3O4/SiO2微球样品分散于盛有20-40mL水的烧瓶中,超声处理后,加入由1.5-3mL HAuCl4和1.5-3mL NaOH组成的混合水溶液,搅拌均匀,最后,在剧烈搅拌下向烧瓶中注入新鲜制备的1-2mL还原剂水溶液;
S2:Fe3O4/Au纳米复合材料的制备;将经步骤S1制得的Fe3O4/Au种子样品分散于盛有60-120mL超纯水的烧瓶中,加入1-2mL NaAuCl4和1-2mL SiO2水溶液,搅拌均匀,最后,在剧烈搅拌下将1-2mL抗坏血酸水溶液注射入烧瓶,该过程重复两次,最终得到Fe3O4/Au纳米复合材料;
S3:改性Fe3O4/Au纳米复合材料的制备;经磁分离收集新鲜经步骤S2制得的Fe3O4/Au样品分散于30-60mL 4-ATP的乙醇溶液中,搅拌均匀,所得产品经乙醇和牛血清白蛋白水溶液洗涤数次后,分散于10-20mL磷酸盐缓冲盐水,并通过叶酸进行修饰,制得以0.5-1mL4-ATP和叶酸的混合液改性的Fe3O4/Au纳米复合材料。
作为一个优选实施例,步骤S1中,超声时间为10-15min。步骤S1中,HAuCl4的浓度为10mmol/L;所述NaOH的浓度为10mmol/L;所述还原剂水溶液的浓度为60mmol/L,所述还原剂为硼氢化钠、柠檬酸三钠和葡萄糖中的一种。步骤S1中,所述搅拌的时间为30min。步骤S2中,所述NaAuCl4的浓度为5mmol/L;所述SiO2的浓度为2mg·mL-1;所述抗坏血酸水溶液的浓度为15mmol/L。步骤S2中,所述搅拌的时间为30-45min。所述4-ATP乙醇溶液的浓度为0.1mmol/L;所述牛血清白蛋白水溶液的浓度为1mg·mL-1;所述叶酸溶液的浓度为1mmol/L。步骤S3中,所述搅拌的时间为2-3h。
应用例
一、细胞毒性试验
将人乳腺癌细胞(MCF-7)接种在含有100units mL-1青霉素,100mg mL-1链霉素和10wt%的胎牛血清(FBS)的达尔伯克改良伊格尔培养基(DMEM)中,置于37℃,5%CO2湿度环境中培养。采用MTT法评估了纳米粒子对MCF-7的细胞毒性。所有实验重复三次。
为研究Fe3O4/Au纳米复合材料在生物应用中的可行性,确定复合材料的毒性,本实验使用人乳腺癌细胞系MCF-7的MTT活力测定法评估Fe3O4/Au纳米复合材料的细胞毒性。假设未处理细胞的存活率为100%,如图3所示,MCF-7细胞用不同浓度的Fe3O4/Au纳米复合物处理24h。浓度范围为20-120g/mL(对应于Fe3O4/Au复合材料的重量浓度),与未处理细胞相比,细胞存活率高于80%。即使浓度增加到120ug/mL,也不会记录到明显的细胞死亡或增殖缺陷。该结果表明,Fe3O4/Au纳米复合材料对细胞没有任何相当大的毒性问题。
二、体外MRI和SERS检测应用
MCF-7细胞保持在含有10%FBS的DMEM培养基中。将乙醇处理的载玻片放置于6孔板中。将含2mLMCF-7细胞的DMEM培养基接种于六孔板中,初始密度为6×105细胞/孔。细胞在37℃下培养24h。将不同量(0.115、0.229、0.343μmol Fe)的纳米颗粒置于DMEM培养液中。培养4h后,用PBS洗涤细胞两次,以除去未吸附的纳米颗粒和DMEM培养液。对于SERS实验,将细胞与4%甲醛混合30分钟,用PBS洗涤三次。对于MRI实验,从孔中取出细胞,用PBS洗涤两次,用胰蛋白酶-EDTA分离。将细胞离心两次,将其以5×106个细胞/mL的密度分散在1%低熔点琼脂糖凝胶中。
为了评估磁性样品Fe3O4/Au纳米结构作为MRI造影剂的功效,研究了水分散的Fe3O4/Au纳米复合材料的MRI弛豫性。图4为不同浓度Fe3O4/Au下的1/T2的反弛豫时间关系图。可以看出,松弛率与纳米复合材料的浓度呈良好的线性关系。在Fe3O4/Au的MRI测试中,Fe是MRI信号的主要来源,因此r值(1/T2)是根据Fe原子的浓度计算的。对于r2和r1,Fe3O4/Au的斜率分别为64和1mM-1s-1。该结果表明,该材料可用作MRI成像应用的T2剂。
如图3所示,Fe3O4/Au纳米复合材料的MRI-SERS预期特性及其细胞相容性表明,新材料应具有优异的生物检测性能。本发明采用细胞内成像研究了基于MCF-7细胞的Fe3O4/Au纳米复合材料的体外MRI和SERS检测能力。
对于SERS体外实验,将0.229mol(以Fe浓度计算)Fe3O4/Au纳米复合材料分散在2mL的DMEM中,37℃下孵育4小时。样品用PBS洗涤并用4%多聚甲醛固定。用633nm(8mW)收集MCF-7细胞的拉曼数据2s。如图5所示,MCF-7细胞样品的拉曼光谱完全体现了靶向分子4-ATP的峰值,且细胞样品没有观察到其他峰。说明Fe3O4/Au纳米复合材料被MCF-7细胞均匀吸收。该结果进一步证实,Fe3O4/Au纳米复合材料可用作基于SERS的癌症检测。
为了研究作为MRI造影剂的可行性,使用1.5T临床MRI系统研究了Fe3O4/Au纳米复合材料与MCF-7细胞孵育的Fe3O4/Au纳米复合材料的对比成像。为了模拟人体活体组织,制备了1%低熔点琼脂糖凝胶作为分散体。图6中的左侧图片显示了分散在1mL琼脂糖凝胶中的各种Fe浓度(0.046、0.115、0.229、0.334和0.458mM)的Fe3O4/Au纳米复合材料的T2加权MR图像。观察到明显的黑色对比度,其随Fe浓度增高而增加。当Fe浓度为0.229mM时,对比度图像强度较高。因此,对于MCF-7细胞的MR成像,研究了Fe浓度为0.115、0.229和0.343mM时的T2加权图像。将MCF-7细胞与不同浓度的Fe3O4/Au纳米复合物孵育4小时,然后分散在1mL琼脂糖凝胶中。作为对比,未添加Fe3O4/Au纳米复合材料的细胞也分散在1mL琼脂糖凝胶中。图6中的右侧MR图像显示了孵育于Fe3O4/Au纳米复合材料的MCF-7细胞的T2加权图像,获得了明显的暗对比度,且其随着Fe浓度的增加而增加。所有图像均以相同的序列在相同的临床磁共振成像仪上获得。松弛率和MR成像结果表明,Fe3O4/Au纳米复合材料应用于T2加权MRI造影剂,具有非常好的应用前景。
综上,通过使用种子生长机制二次粒子(Au)到二次粒子(Fe 3O4)上合成独特的鳞片状Fe3O4/Au纳米结构。研究了该纳米颗粒在水溶液中的MRI和SERS性质以及在MCF-7中的进一步体外MRI和SERS活性。如图1和图2所示,复合材料由TEM、UV-Vis、XRD等进行表征。综上所述,制备的纳米复合材料在不同浓度下显示出良好的生物相容性,没有任何毒性。在用MCF-7癌细胞孵育4小时后,获得了有效的T2加权MR图像和SERS数据。这些结果清楚地表明,合成的Fe3O4/Au纳米复合材料是可用于肿瘤检测的MRI-SERS多功能剂。
以上对本发明的具体实施例进行了详细描述,但其只是作为范例,本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言,任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此,在不脱离本发明的精神和范围下所作的均等变换和修改,都应涵盖在本发明的范围内。
Claims (9)
1.一种MRI-SERS双模式造影剂的制备方法,其特征在于,包括以下步骤,
S1:Fe3O4/Au种子样品的制备;
将4-8mgFe3O4/SiO2微球样品分散于盛有20-40mL水的烧瓶中,超声处理后,加入由1.5-3mL HAuCl4和1.5-3mL NaOH组成的混合水溶液,搅拌均匀,最后,在剧烈搅拌下向烧瓶中注入新鲜制备的1-2mL还原剂水溶液;
S2:Fe3O4/Au纳米复合材料的制备;
将经步骤S1制得的Fe3O4/Au种子样品分散于盛有60-120mL超纯水的烧瓶中,加入1-2mLNaAuCl4和1-2mL SiO2水溶液,搅拌均匀,最后,在剧烈搅拌下将1-2mL抗坏血酸水溶液注射入烧瓶,该过程重复两次,最终得到Fe3O4/Au纳米复合材料;
S3:改性Fe3O4/Au纳米复合材料的制备;
经磁分离收集新鲜经步骤S2制得的Fe3O4/Au样品分散于30-60mL 4-ATP的乙醇溶液中,搅拌均匀,所得产品经乙醇和牛血清白蛋白水溶液洗涤数次后,分散于10-20mL磷酸盐缓冲盐水,并通过叶酸进行修饰,制得以0.5-1mL4-ATP和叶酸的混合液改性的Fe3O4/Au纳米复合材料。
2.根据权利要求1所述的MRI-SERS双模式造影剂的制备方法,其特征在于,步骤S1中,超声时间为10-15min。
3.根据权利要求1所述的MRI-SERS双模式造影剂的制备方法,其特征在于,步骤S1中,HAuCl4的浓度为10mmol/L;所述NaOH的浓度为10mmol/L;所述还原剂水溶液的浓度为60mmol/L。
4.根据权利要求1所述的MRI-SERS双模式造影剂的制备方法,其特征在于,所述还原剂为硼氢化钠、柠檬酸三钠和葡萄糖中的一种。
5.根据权利要求1所述的MRI-SERS双模式造影剂的制备方法,其特征在于,步骤S1中,所述搅拌的时间为30min。
6.根据权利要求1所述的MRI-SERS双模式造影剂的制备方法,其特征在于,步骤S2中,所述NaAuCl4的浓度为5mmol/L;所述SiO2的浓度为2mg·mL-1;所述抗坏血酸水溶液的浓度为15mmol/L。
7.根据权利要求1所述的MRI-SERS双模式造影剂的制备方法,其特征在于,步骤S2中,所述搅拌的时间为30-45min。
8.根据权利要求1所述的MRI-SERS双模式造影剂的制备方法,其特征在于,所述4-ATP乙醇溶液的浓度为0.1mmol/L;所述牛血清白蛋白水溶液的浓度为1mg·mL-1;所述叶酸溶液的浓度为1mmol/L。
9.根据权利要求1所述的MRI-SERS双模式造影剂的制备方法,其特征在于,步骤S3中,所述搅拌的时间为2-3h。
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