CN106177949A - 一种荧光‑磁性双模态纳米载体及制备方法 - Google Patents
一种荧光‑磁性双模态纳米载体及制备方法 Download PDFInfo
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Abstract
本发明涉及荧光‑磁性双模态纳米载体及制备方法,其特征在于,包括有机荧光剂和磁性纳米颗粒,其中有机荧光剂通过修饰物葡聚糖包裹在磁性纳米颗粒表面,形成荧光‑磁性双模态纳米载体;所述有机荧光剂为异硫氰酸荧光素、四甲基异硫氰酸罗丹明、菁类染料、藻红蛋白或氨甲基香豆素醋酸酯;所述磁性纳米颗粒为Fe3O4磁性纳米颗粒,粒径为10nm~50nm。本发明的有益效果是:载体具有良好、稳定的光学显像能力,易于对载体进行实时、动态观察;所用载体粒径和有机荧光材料可根据不同需要或者应用场景进行选择,适用性强;载体具有良好的磁响应和磁成像能力,可在磁场引导下实现对目标的靶向运输,亦能在磁共振下观察载体在活体内的位置和运动路径。
Description
技术领域
本发明属于生物医学纳米荧光技术和功能化材料领域,具体涉及一种携带有机荧光标记的磁性纳米载体及其制备的方法。
背景技术
在生物学和医学领域中,常常需要用到病毒和非病毒等,作为运输DNA、RNA、蛋白、脂类等生物大分子的载体。但是病毒载体存在着潜在的致病可能,存在生物安全隐患,而非病毒载体虽然安全性高于病毒载体,但是存在缺乏靶向性、转运效率不高等缺点。
随着纳米技术的发展,对于纳米级非病毒载体的探索方兴未艾。对于Fe3O4纳米颗粒而言,由于粒径达到纳米级别还具备了超顺磁性,即在外加磁场中有较强磁性,磁场撤除时磁性又很快消失,故可在外加磁场的引导下,实现在局部浓聚富集,可作为各种大分子物质的载体,起到靶向运输的作用。但是单纯Fe3O4纳米颗粒的水溶性、分散度等物理特性并不理想;缺少共价键等大分子结合位点,加载大分子物质的量受到限制;且容易被生物体内免疫细胞吞噬;因此需要对其进行改性,在其外包裹大分子物质,增加其亲水性、降低清除率,从而延长其在血液系统中的循环时间,并将大分子物质递送到目标部位。
有机荧光标记技术作为一种实时、非同位素可视标记法,被广泛应用于生物学医学等研究中。将目的物标记后,通过光学检测设备,可动态、实时、精确地对目的物进行示踪。但传统的荧光试剂存在着一些缺陷:如多数荧光试剂存在光漂白致使荧光信号不稳定、发出的荧光光子平均数量少,荧光试剂及其光解产物对活体细胞有一定毒副作用等。此外,既往的荧光标记方法连接的有机荧光分子数量较少,可视化效果不够理想;且荧光基团对目标物进行标记的方法较复杂,所需成本较高;连接荧光基团所使用的修饰材料容易对荧光的观察产生干扰,无法满足研究和应用的需求。
发明内容
本发明要解决的技术问题是提供一种制备方法简便,荧光稳定性高,具备顺磁性,易于用光学方法或磁共振方法观察、便于用外加磁场控制的荧光-磁性双模态纳米载体及其制备方法。
为了实现上述技术目的,本发明采用了以下技术方案:
这种荧光-磁性双模态纳米载体,其特征在于,包括有机荧光剂和磁性纳米颗粒,其中有机荧光剂通过修饰物葡聚糖包裹在磁性纳米颗粒表面,形成荧光-磁性双模态纳米载体;所述有机荧光剂为异硫氰酸荧光素、四甲基异硫氰酸罗丹明、菁类染料、藻红蛋白或氨甲基香豆素醋酸酯;所述磁性纳米颗粒为Fe3O4磁性纳米颗粒,粒径为10nm~50nm。
作为优选:所述葡聚糖的分子量为6kD~40kD。
荧光-磁性双模态纳米载体的制备方法,包括如下步骤:
1)将柠檬酸盐溶于去离子水,制成浓度为0.05~0.1mol/L的柠檬酸盐溶液;
2)将Fe3O4磁性纳米颗粒加入柠檬酸盐溶液中,超声乳化后,加入含葡聚糖的柠檬酸盐溶液,继续超声乳化;葡聚糖、Fe3O4磁性纳米颗粒和柠檬酸盐的摩尔比为1:1:50~1:1.5:200;
3)步骤2中,超声乳化工作频率为30kHZ~50kHZ,乳化时间为20~40min;
4)将含有Fe3O4磁性纳米颗粒和葡聚糖的柠檬酸盐溶液置于水浴中反应,得到葡聚糖包裹的Fe3O4磁性纳米颗粒;
5)步骤4中,水浴温度为80~85℃,反应时间为2~4h;
6)加入有机荧光剂,均匀震荡搅拌,得到的反应产物需用去离子水洗涤6~8次,得到荧光-磁性双模态纳米载体;
7)步骤6中,葡聚糖和有机荧光剂的摩尔比为1:10~1:20,反应需在4℃避光条件下进行,时间为6~8h。
本发明所公开的一种荧光-磁性双模态纳米载体及制备方法,其优点表现在:1)载体具有良好、稳定的光学显像能力,易于对载体进行实时、动态观察;2)所用载体粒径和有机荧光材料可根据不同需要或者应用场景进行选择,适用性强;3)载体具有良好的磁响应和磁成像能力,可在磁场引导下实现对目标的靶向运输,亦能在磁共振下观察载体在活体内的位置和运动路径;4)载体制备方法简单,具有良好的生物安全性,不会对生理活动造成影响;5)所用修饰物葡聚糖在载体表面形成正电荷界面,使其易被表面带负电荷的细胞摄取,提高了载体的转运效率;6)葡聚糖侧枝基团大大增加有机荧光材料和生物大分子的结合位点,且其在可见光到近红外光范围内又是透明的,能避免对荧光信号形成干扰。
附图说明
图1制备完成后,在透射电镜下所见荧光-磁性双模态纳米载体;
图2制备完成后,紫外光下携带荧光标记的荧光-磁性双模态纳米载体;
图3制备完成后,自然光下携带荧光标记的荧光-磁性双模态纳米载体;
图4无外加磁场下,携带荧光标记的荧光-磁性双模态纳米载体在细胞内的分布情况(黑箭头所指为进入细胞内部的纳米载体);
图5给予外加磁场后,携带荧光标记的荧光-磁性双模态纳米载体在细胞内的分布情况(黑箭头所指为进入细胞内部的纳米载体)。
具体实施方式
下面结合实施例对本发明做进一步描述。下述实施例的说明只是用于帮助理解本发明。应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以对本发明进行若干改进和修饰,这些改进和修饰也落入本发明权利要求的保护范围内。
这种荧光-磁性双模态纳米载体及制备方法,它包括有机荧光剂和磁性纳米颗粒,其中有机荧光剂通过连接物葡聚糖包裹在磁性纳米颗粒表面,形成荧光-磁性双模态纳米载体。其具体制备步骤如下:将葡聚糖和Fe3O4磁性纳米颗粒分别溶于柠檬酸盐溶液中,将含有Fe3O4磁性纳米颗粒的柠檬酸盐溶液超声乳化后,加入含有葡聚糖的柠檬酸盐溶液继续超声乳化,然后将溶液转移到水浴中,用搅拌器搅拌反应,并加入用DMSO溶解的有机荧光剂,得到荧光-磁性双模态纳米载体。
所用葡聚糖分子量为6kD~40kD,Fe3O4磁性纳米颗粒粒径为10nm~50nm,有机荧光剂为异硫氰酸荧光素、四甲基异硫氰酸罗丹明、菁类染料、藻红蛋白或者氨甲基香豆素醋酸酯。
所用柠檬酸盐溶液为柠檬酸盐溶于去离子水而成,其浓度为0.05~0.1mol/L。
葡聚糖、Fe3O4磁性纳米颗粒和有机荧光剂的摩尔比为1:1:10~1:1.5:20,葡聚糖与柠檬酸盐的摩尔比为1:50~1:200。
超声乳化工作频率为30kHZ~50kHZ,乳化时间为20~40min。
含有Fe3O4磁性纳米颗粒和葡聚糖的柠檬酸盐溶液需要在80~85℃水浴中反应2~4h。
有机荧光剂溶于DMSO溶液,加入后需在4℃避光条件下均匀震荡搅拌,反应6~8h。
得到的反应产物需用去离子水洗涤6~8次,去除未参与反应的单体,从而得到荧光-磁性双模态纳米载体溶液。
实施例1
1)将0.05mol柠檬酸钠溶于1L去离子水中,得到0.05mol/L的溶液。
2)将粒径为20nm的Fe3O4磁性纳米颗粒加入柠檬酸钠溶液中,工作频率为30kHZ的超声乳化20min后,加入含葡聚糖(分子量为20kD)的柠檬酸钠溶液,继续超声乳化20min,其中葡聚糖、Fe3O4磁性纳米颗粒和柠檬酸钠的摩尔比为1:1:50。
3)将步骤(2)中所得混合液置于温度为80℃的水浴中,反应4h。
4)将步骤(2)中所得混合液降温至4℃,避光条件下加入溶于DMSO的异硫氰酸荧光素,均匀震荡搅拌6h。
5)将步骤(4)中所得混合液用去离子水洗涤6~8次,得到荧光-磁性双模态纳米载体。
实施例2
1)将0.075mol柠檬酸钠溶于1L去离子水中,得到0.075mol/L的溶液。
2)将粒径为30nm的Fe3O4磁性纳米颗粒加入柠檬酸钠溶液中,以工作频率为40kHZ的超声乳化30min后,加入含葡聚糖(分子量为10kD)的柠檬酸钠溶液,继续超声乳化30min,其中葡聚糖、Fe3O4磁性纳米颗粒和柠檬酸钠的摩尔比为1:1:100。
3)将步骤(2)中所得混合液置于温度为85℃的水浴中,反应2h。
4)将步骤(2)中所得混合液降温至4℃,避光条件下加入溶于DMSO的四甲基异硫氰酸罗丹明,均匀震荡搅拌7h。
5)将步骤(4)中所得混合液用去离子水洗涤6~8次,得到荧光-磁性双模态纳米载体。
实施例3
1)将0.1mol柠檬酸钠溶于1L去离子水中,得到0.1mol/L的溶液。
2)将粒径为50nm的Fe3O4磁性纳米颗粒加入柠檬酸钠溶液中,以工作频率为50kHZ的超声乳化40min后,加入含葡聚糖(分子量为6kD)的柠檬酸钠溶液,继续超声乳化40min,其中葡聚糖、Fe3O4磁性纳米颗粒和柠檬酸钠的摩尔比为1:1:200。
3)将步骤(2)中所得混合液置于温度为85℃的水浴中,反应4h。
4)将步骤(2)中所得混合液降温至4℃,避光条件下加入溶于DMSO的四甲基异硫氰酸罗丹明,均匀震荡搅拌8h。
5)将步骤(4)中所得混合液用去离子水洗涤6~8次,得到荧光-磁性双模态纳米载体。
荧光-磁性双模态纳米载体的性能检测
荧光-磁性双模态纳米载体的电镜观测:将纳米复合物溶液滴于铜网上,干燥后置于透射电子显微镜下观测,其颗粒尺寸小于20纳米(如图1)。
荧光-磁性双模态纳米载体的磁响应及荧光特性:将双模态纳米载体及单纯磁性纳米载体水溶液分别置于石英比色器中,一侧置永久磁铁,可见顺磁性载体聚集于靠近磁铁的器壁;在紫外灯照射下溶双模态纳米载体呈现荧光,而单纯磁性纳米载体不发光(如图2和图3)。
荧光-磁性双模态纳米载体在磁场引导下进入细胞的能力以及实时观察:取2个含有贴壁细胞的培养皿(皿中含4毫升培养液),分别加入双模态纳米载体溶液15微升,一个皿不给予磁场,另一个皿底给予200mT的磁场,37℃培养1小时,置于紫外荧光显微镜下数码成像(如图4和图5)。
Claims (3)
1.一种荧光-磁性双模态纳米载体,其特征在于,包括有机荧光剂和磁性纳米颗粒,其中有机荧光剂通过修饰物葡聚糖包裹在磁性纳米颗粒表面,形成荧光-磁性双模态纳米载体;所述有机荧光剂为异硫氰酸荧光素、四甲基异硫氰酸罗丹明、菁类染料、藻红蛋白或氨甲基香豆素醋酸酯;所述磁性纳米颗粒为Fe3O4磁性纳米颗粒,粒径为10nm~50nm。
2.根据权利要求书1所述的荧光-磁性双模态纳米载体,其特征在于:所述葡聚糖的分子量为6kD~40kD。
3.如权利要求书1所述荧光-磁性双模态纳米载体的制备方法,其特征在于,包括如下步骤:
1)将柠檬酸盐溶于去离子水,制成浓度为0.05~0.1mol/L的柠檬酸盐溶液;
2)将Fe3O4磁性纳米颗粒加入柠檬酸盐溶液中,超声乳化后,加入含葡聚糖的柠檬酸盐溶液,继续超声乳化;葡聚糖、Fe3O4磁性纳米颗粒和柠檬酸盐的摩尔比为1:1:50~1:1.5:200;
3)步骤2中,超声乳化工作频率为30kHZ~50kHZ,乳化时间为20~40min;
4)将含有Fe3O4磁性纳米颗粒和葡聚糖的柠檬酸盐溶液置于水浴中反应,得到葡聚糖包裹的Fe3O4磁性纳米颗粒;
5)步骤4中,水浴温度为80~85℃,反应时间为2~4h;
6)加入有机荧光剂,均匀震荡搅拌,得到的反应产物需用去离子水洗涤6~8次,得到荧光-磁性双模态纳米载体;
7)步骤6中,葡聚糖和有机荧光剂的摩尔比为1:10~1:20,反应需在4℃避光条件下进行,时间为6~8h。
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CN110292635A (zh) * | 2019-08-08 | 2019-10-01 | 鲁东大学 | 一种基于天然硅藻外壳和藻胆蛋白的新型材料及应用 |
CN110292635B (zh) * | 2019-08-08 | 2021-07-16 | 鲁东大学 | 一种基于天然硅藻外壳和藻胆蛋白的材料及应用 |
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