CN105860960A - 一种基于金纳米颗粒的细胞膜荧光探针及其制备方法和应用 - Google Patents
一种基于金纳米颗粒的细胞膜荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明属于检测技术领域，具体涉及一种基于金纳米颗粒的细胞膜荧光探针及其制备方法和应用。该基于金纳米颗粒的细胞膜荧光探针具有如下通式：
Description
技术领域
本发明属于探针检测技术领域，具体涉及一种基于金纳米颗粒的细胞膜荧光探针及其制备方法和应用。
背景技术
细胞膜具有重要的生理功能，它既是细胞维持稳定代谢的胞内环境，又能调节和选择物质进出细胞。细胞膜通过胞饮作用、吞噬作用或胞吐作用吸收、消化和外排细胞膜外、内的物质。在细胞识别、信号传递、纤维素合成和微纤丝的组装等方面，质膜也发挥重要作用。天然产物的结构修饰及生理活性研究一直是新药研发的重要途径。由于结构上的特殊性以及本身具有的生物活性，以氨基酸为基础的化合物合成以及研发比较活跃。氨基酸是生物功能大分子蛋白质的基本组成单位，是构成动物营养所需蛋白质的基本物质。蛋白质是生物体内重要的活性分子，包括催化新陈代谢的酶。例如谷氨酸、精氨酸、天门冬氨酸、胱氨酸等氨基酸可以单独作用治疗一些疾病，主要用于治疗肝病疾病、消化道疾病、脑病、心血管病、呼吸道疾病以及用于提高肌肉活力、儿科营养和解毒等。此外，氨基酸衍生物在检测细胞膜上也表现出比较好的功能。因此，在细胞膜荧光探针的结构中引入氨基酸结构，设计合成新型结构的氨基酸衍生物，利用这些结构新颖的氨基酸来实现细胞膜荧光探针的靶向作用。
此外，用于生物分子和细胞上的一种重要的标志物——金纳米颗粒，也在荧光探针研究中有广泛的应用。基于纳米颗粒NPs的实验方法已被广泛应用于化学及生物探测。因为纳米颗粒NPs具有大的表面积及球形形状，纳米颗粒能用作许多染料分子封装载体，已被主要用于荧光免疫分析的信号记录以提高检测灵敏度。金纳米颗粒作为一种新型的纳米材料，最近，科学家的注意力转向金纳米颗粒用于医疗的可能性。鉴于金纳米颗粒光电特性易被修饰等特点，其可用于细胞检测、基因调节药物合成、药物运输、光化学治疗等方面。金纳米颗粒可以使一些特殊物质进入细胞，如多价药物和反义因子。这些物质可以控制细胞功能，调节基因表达，也可以检测细胞内分析物。金纳米颗粒的这些特点，符合目前荧光探针研究的需要。因此，功能化金纳米颗粒的改进成为目前荧光探针研究的重要趋势之一。
发明内容
本发明目的在于克服现有技术缺陷，提供一种基于金纳米颗粒的细胞膜荧光探针及其制备方法和应用。
为实现上述目的，本发明采用如下技术方案：
一种基于金纳米颗粒的细胞膜荧光探针，其具有如下通式：
；
其中，R为氨基酸残基，X为荧光基团。
具体的，所述R为L-色氨酸残基、L-半胱氨酸残基、L-赖氨酸残基或L-谷氨酸残基；所述X为绿色荧光基团或红色荧光基团。
上述基于金纳米颗粒的细胞膜荧光探针的制备方法，其合成路线如下所示：
；
具体步骤如下：制备含化合物b（即巯基丙酸修饰的金纳米颗粒b）的溶液，MES调节pH值为6－7，加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐（EDC）和N-羟基琥珀酰亚胺（NHS），反应30min，再加入氨基酸-多胺衍生物a，然后于20－45℃反应24 h，固液分离获得化合物c；将化合物c溶于三蒸水中，与FITC（异硫氰酸荧光素）或BODIPY（氟硼荧染料）的二甲基甲酰胺（DMF）溶液混合后，于20－45℃反应12－24 h后，离心得到氨基酸-多胺衍生物修饰的金纳米颗粒d或e。
其中，FITC的结构为，BODIPY的结构为，
氨基酸-多胺衍生物修饰的金纳米颗粒d或e在制备荧光探针上的应用。
氨基酸-多胺衍生物a的制备可采用本领域常规方法合成即可，也可以参照如下步骤进行制备：以制备色氨酸-多胺衍生物为例，圆底烧瓶中先加入0.45 moL 1,4-二溴丁烷和100 moL丙酮，搅拌下分批加入27.7 g邻苯二甲酰亚胺钾盐，搅拌回流反应12 h，冷却，过滤除去反应生成的KBr，减压蒸去丙酮和过量的1,4-二溴丁烷，剩余物用无水乙醇重结晶，然后溶于60 mL乙腈中，加入10 mmoL色氨酸、20 mmoL K2CO3，在室温下搅拌15 min，随后升温至70 ℃，反应过夜后冷却，溶液中有大量白色不溶物，过滤除去，减压蒸除乙腈，得淡黄色油状物，通过层析柱分离提纯得到黄色化合物；把该黄色化合物用少量乙醇溶解置于圆底烧瓶中，缓慢滴加10当量的水合肼，室温搅拌过夜，有大量白色固体析出，用滤纸过滤除去白色固体，得淡黄色滤液，减压蒸除乙醇，得黄色油状物，通过葡聚糖层析柱分离提纯的化合物a。制备其它氨基酸-多胺衍生物时，只需要将色氨酸替换为相应的氨基酸即可。
具体的，制备过程中，巯基丙酸修饰的金纳米颗粒b与氨基酸-多胺衍生物a的摩尔比优选为1：15。
上述基于金纳米颗粒的细胞膜荧光探针在标识癌细胞细胞膜荧光探针上的应用。
上述基于金纳米颗粒的细胞膜荧光探针在标识正常细胞细胞膜荧光探针上的应用。
和现有技术相比，本发明的有益效果：
1）本发明把氨基酸和多胺结合在一起，可以改善多胺类化合物的水溶性和生物性能，制成具有生物靶向性的纳米材料，从而实现荧光探针的靶向作用；
2）本发明通过有效的方法将氨基酸-多胺衍生物修饰到金纳米颗粒表面，使得这些荧光金纳米颗粒可以特异性地识别细胞，制成具有生物靶向性和特异性高的纳米材料，实现荧光探针的靶向作用，其标记的细胞则可以通过多种检测方法进行检测；
3）本发明基于金纳米颗粒的细胞膜荧光探针的制备方法简单易行，成本较低。
附图说明
图1为实施例1中巯基丙酸修饰的金纳米颗粒b的透射电镜图谱；图中可以看出巯基丙酸修饰的金纳米颗粒b为粒径约4 nm的均匀分散的球体；
图2为实施例1制备所得赖氨酸-多胺衍生物修饰的金纳米颗粒d 的透射电镜图谱；图中可以看出赖氨酸-多胺衍生物修饰的金纳米颗粒d为粒径约8 nm的球体；
图3为实施例1制备所得赖氨酸-多胺衍生物修饰的金纳米颗粒d 的紫外吸收图谱；
图4为实施例1制备所得赖氨酸-多胺衍生物修饰的金纳米颗粒d 的荧光图谱；
图5为实施例1制备所得赖氨酸-多胺衍生物修饰的金纳米颗粒d 的共聚焦图谱；
图6为实施例1制备所得赖氨酸-多胺衍生物修饰的金纳米颗粒 e 的共聚焦图谱。
具体实施方式
以下结合实施例对本发明的技术方案作进一步地详细介绍，但本发明的保护范围并不局限于此。
实验仪器名称与型号：
德国Bruker AV-400型核磁共振仪；
美国Perkin-Elmer Lambda-850紫外分光光度计；
美国Perkin-Elmer Ls55荧光分光光度计；
日本JEOL JEM-200CX透射电子显微镜；
德国Leica SP5共焦焦荧光显微镜；
实施例
1
R选为L-赖氨酸残基，制备基于金纳米颗粒的细胞膜荧光探针时的合成步骤如下：
1）制备化合物b：依据参考文献（Yan-Juan
Gu , Jinping Cheng , Chun-Chi Lin. Nuclear penetration of surface
functionalized gold nanoparticles. Toxicology and Applied Pharmacology 237
(2009) 196–204）制备巯基丙酸修饰的金纳米颗粒b，即将2 ml 15 mmol/L HAuCl4与773 μL 38.8 mM柠檬酸三钠溶于30 mL三蒸水中，缓慢滴入新鲜配制的0.1 mol/L的NaBH4水溶液3 ml，反应2 h，溶液从无色变为橙色。调节pH至11，加入3 ml 0.01 M的巯基丙酸（MPA）乙醇溶液，反应2 h；
2）制备化合物c：MES调节pH至6.5，加入7 mg (0.035 mmol) 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐（EDC）、10.5 mg (0.0875
mmol) N-羟基琥珀酰亚胺（NHS），反应30
min，再加入15.7 mg(0.045 mmol)赖氨酸-多胺衍生物a ()，室温搅拌24h，离心得到化合物c；
3）制备化合物d或e：将化合物c溶于三蒸水中，加入200 μL含1 mg FITC或9 mg BODIPY的DMF溶液，室温搅拌24 h，离心得到赖氨酸-多胺衍生物修饰的金纳米颗粒d或e。
利用紫外光谱和荧光光谱对赖氨酸-多胺衍生物修饰的金纳米颗粒d进行表征，结果见图3和4。图3可以看出：其吸收峰的位置为320 nm；图4可以看出金纳米颗粒c与FITC连接成功。利用激光共聚焦荧光显微镜对金纳米颗粒d及e进行表征，结果见图5和6。图5可以看出：此探针在488 nm处激发光为绿色；图6可以看出：此探针在633 nm处激发光为红色。
实施例
2
R选为L-色氨酸残基，制备基于金纳米颗粒的细胞膜荧光探针时的合成步骤如下：
1）制备化合物b：同实施例1；
2）制备化合物c：MES调节pH至6.5，加入7 mg (0.035 mmol) 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐（EDC）、10.5 mg (0.0875
mmol) N-羟基琥珀酰亚胺（NHS），反应30
min，再加入12.3 mg(0.045 mmol)色氨酸-多胺衍生物a()，室温搅拌24 h，离心得到化合物c；
3）制备化合物d或e：将化合物c溶于三蒸水中，加入200 μL 含1 mg FITC或9 mg BODIPY的DMF溶液，室温搅拌24 h，离心得到色氨酸-多胺衍生物修饰的金纳米颗粒d或e。
实施例
3
R选为L-半胱氨酸残基，制备基于金纳米颗粒的细胞膜荧光探针时的合成步骤如下：
1）制备化合物b：同实施例1；
2）制备化合物c：MES调节pH至6.5，加入7 mg (0.035 mmol) 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐（EDC）、10.5 mg (0.0875
mmol) N-羟基琥珀酰亚胺（NHS），反应30
min，再加入10.5 mg (0.045 mmol)半胱氨酸-多胺衍生物a ()，室温搅拌24 h，离心得到化合物c；
3）制备化合物d或e：将化合物c溶于三蒸水中，加入200 μL 含1 mg FITC或9 mg BODIPY的DMF溶液，室温搅拌24 h，离心得到半胱氨酸-多胺衍生物修饰的金纳米颗粒d或e。
实施例
4
R选为L-谷氨酸残基，制备基于金纳米颗粒的细胞膜荧光探针时的合成步骤如下：
1）制备化合物b：同实施例1；
2）制备化合物c：MES调节pH至6.5，加入7 mg (0.035 mmol) 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐（EDC）、10.5 mg (0.0875
mmol) N-羟基琥珀酰亚胺（NHS），反应30
min，再加入9.7 mg (0.045 mmol)谷氨酸-多胺衍生物a ()，室温搅拌24 h，离心得到化合物c；
3）制备化合物d或e：将化合物c溶于三蒸水中，加入200 μL 含1 mg FITC或9 mg BODIPY的DMF溶液，室温搅拌24 h，离心得到谷氨酸-多胺衍生物修饰的金纳米颗粒d或e。
活性实验
细胞培养：HepG2（肝癌细胞），MCF-7（人乳腺癌细胞）和QSG-7701（人胚肝细胞），细胞用含10%(v/v)胎牛血清且含1% (v/v)青链霉素混合液的1640培养基于培养瓶中培养。培养瓶置于37 ℃，含5% CO2且湿度为90%的培养箱中进行培养。
细胞毒性测试：实施例1至4制备得到的氨基酸-多胺衍生物修饰的金纳米颗粒d或e在HepG2，QSG-7701和MCF-7细胞中的细胞毒性采用MTT法测定：细胞传代培养3、4次后，当细胞生长到对数期时，将细胞用0.25%胰蛋白酶消化成单细胞悬液，采用血球计数板进行活细胞计数，调整活细胞浓度为5×104/mL接种于96孔培养板中，每孔100 μL，于37℃、含5% CO2且湿度为90%的培养箱中培养24 h后，吸出旧培养基，再分别加入用培养基稀释的不同浓度（1-50 μM）的实施例1至4制备得到的产物d或e样品。加入样品后的96孔板置于37℃、含5% CO2的培养箱中孵育48 h，然后加入MTT 20 μL/孔（2.5 mg/mL），4h后弃上清液，加入DMSO 100 μL/孔，振荡10min左右，用M200酶标仪测定OD值，波长设置为570 nm和690 nm双波长。没有加入样品的孔的细胞存活率作为对照，设为100%，计算细胞存活率，评价样品的细胞毒性。结果见下表1和2。
表1 实施例1至4制备得到的产物d的细胞毒性
表2 实施例1至4制备得到的产物e的细胞毒性
表 1 及表 2 测试了实施例1至4制备得到的氨基酸-多胺衍生物修饰的金纳米颗粒d或e对HepG2、MCF-7和QGY-7701细胞的体外生长抑制活性。IC50值为能使正常分裂生长的细胞数抑制到50%水平时的样品浓度值（mg/ml），IC50值越大表明样品的细胞毒性越弱。实验结果表明：本发明基于金纳米颗粒的细胞膜荧光探针，即氨基酸-多胺衍生物修饰的金纳米颗粒d或e对细胞的毒性比较小。
综上说明：本发明氨基酸-多胺衍生物修饰的金纳米颗粒d或e可以作为靶向性细胞膜荧光探针使用。
Claims (6)
1.一种基于金纳米颗粒的细胞膜荧光探针，其特征在于，具有如下通式：
；
其中，R为氨基酸残基，X为荧光基团。
2.如权利要求1所述基于金纳米颗粒的细胞膜荧光探针，其特征在于，所述R为L-色氨酸残基、L-半胱氨酸残基、L-赖氨酸残基或L-谷氨酸残基；所述X为绿色荧光基团或红色荧光基团。
3.权利要求1所述基于金纳米颗粒的细胞膜荧光探针的制备方法，其特征在于，合成路线如下所示：
；
具体步骤如下：制备含化合物b的溶液，MES调节pH值为6－7，加入EDC和NHS，反应30min，再加入氨基酸-多胺衍生物a，然后于20－45℃反应24 h，固液分离获得化合物c；将化合物c溶于三蒸水中，与含FITC或BODIPY的DMF溶液混合后，于20－45℃反应12－24 h后，离心得到氨基酸-多胺衍生物修饰的金纳米颗粒d或e。
4.如权利要求3所述基于金纳米颗粒的细胞膜荧光探针的制备方法，其特征在于，巯基丙酸修饰的金纳米颗粒b与氨基酸-多胺衍生物a的摩尔比为1：15。
5.权利要求1所述基于金纳米颗粒的细胞膜荧光探针在标识癌细胞细胞膜荧光探针上的应用。
6.权利要求1所述基于金纳米颗粒的细胞膜荧光探针在标识正常细胞细胞膜荧光探针上的应用。
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