蓝色超小赖氨酸-京尼平纳米粒制备及肿瘤细胞染色

Preparation of Ultrasmall Blue Lysine-genipin Nanoparticles for Glioma Cell Staining

关键词:  蓝色超小纳米粒  京尼平  赖氨酸  反相微乳液法

Key words:  ultrasmall blue nanoparticles  genipin  lysine  reversed microemulsion

摘要:

目的 利用京尼平交联制备蓝色超小纳米粒，用于肿瘤细胞染色。方法 将赖氨酸（lysine，Lys）等伯氨基化合物分别置于反相微乳液中和京尼平反应，观察颜色变化，测定产物OD_{585 nm}、粒径大小和分布，筛选伯氨基化合物。考察其浓度、摩尔比及预交联时间对粒径大小和分布的影响，确定最佳制备条件。制备所得纳米粒进行透射电子显微镜观察和表征，考察其在不同pH值和温度下的颜色稳定性、染色胶质瘤细胞U87的能力和对CHO细胞毒性。结果 Lys交联产物颜色最深、粒径最小且分布最窄。Lys浓度和预交联时间对粒径的影响较大，在Lys为30 mg·mL^-1、–NH₂与京尼平摩尔比为1.5∶1和预交联时间2 min下，得到粒径为3.8~6.8 nm的超小球形纳米粒Lys-g NPs。Lys-g NPs溶液在不同pH值和温度下呈蓝色，颜色稳定，能有效染色肿瘤细胞，使细胞团（10⁵）和单个细胞呈肉眼可见的蓝色，没有明显的细胞毒性（≤2.5 mg·mL^-1）。结论 Lys-g NPs是一种蓝色稳定的超小纳米粒，能有效染色肿瘤细胞，具有体内肿瘤染色显像裸眼可视的潜能。

Abstract:

OBJECTIVE To stain glioma cells by ultrasmall blue nanoparticles which were prepared through crosslinking with genipin.METHODS Various amino compounds including lysine (Lys) were cross-linked by genipin in reversed microemulsion.Color change, particle size and distribution of the products were characterized to screen out the specific amino compound.Next, the factors such as concentration, molar ratio between –NH₂ and genipin, and crosslinking time in preparation were optimized.To investigate the stability, the nanoparticles were placed different pH and temperature solutions.Finally, the nanoparticles were used to stain U87 cells and cytotoxicity to CHO cells was tested.RESULTS Lys-g NPs were the smallest, with the deepest color and good polydispersity.The optimal conditions for Lys-g NPs preparation were 30 mg·mL^-1 of Lys, 1.5∶1 of –NH₂ to genipin, and 2 min of crosslinking time.At the condition, 3.8-6.8 nm of Lys-g NPs were observed under TEM.Lys-g NPs maintained dark blue and stable in different pH solutions (3.0–12.0) and temperatures (4–60 ℃).More importantly, both the cell pellets(10⁵) and the single cells showed visible blue after incubated with the Lys-g NPs.Moreover, the Lys-g NPs were non-toxicity to cells even at very high dosages(≤2.5 mg·mL^-1).CONCLUSION Ultrasmall dark blue, color stable, and non-toxicity Lys-g NPs are prepared successfully and hold great potential for tumor staining in vivo.