叶酸-聚乙烯亚胺复合碳点的跨膜机制和胞内分布

Transmembrane mechanism and intracellular trafficking of folic acid-polyethyleneimine carbon dots

目的:探讨以叶酸和聚乙烯亚胺(PEI)为原料合成的荧光碳点跨MC3T3-E1细胞膜转运途径、胞内分布其对细胞的影响,并阐明其机制。方法:以叶酸和PEI为原料,通过水热法合成具有细胞成像功能的荧光碳点,采用MTT法筛选碳点的最佳使用浓度。将MC3T3-E1细胞分为空白对照组、叶酸组和碳点组,通过细胞周期、细胞凋亡和细胞活性氧(ROS)水平检测评估碳点的生物相容性;通过胞膜窖途径抑制剂制霉菌素、巨胞饮途径抑制剂诺考达唑的使用,探讨细胞摄取碳点的途径;利用碳点在紫外光激发下发射蓝色荧光的特点,通过各种细胞器探针进行荧光共定位以观察碳点在细胞内的分布。结果:与空白对照组比较,24h时100~450mg·L-1碳点组细胞增殖率明显升高(P<0.05);在48h时,当碳点浓度达到350mg·L-1时,细胞增殖率仅有空白对照组的68.4%(P<0.05)。与空白对照组比较,碳点组G0期和G1期细胞比例明显下降(P<0.05),S期细胞比例明显升高(P<0.05);G2期和M期细胞比例亦升高,但差异无统计学意义(P>0.05);与空白对照组比较,叶酸组和碳点组细胞凋亡率无明显改变(P>0.05),但细胞内ROS水平明显降低(P<0.05)。与空白对照组比较,制霉菌素组细胞摄取碳点量减少(P<0.05)。倒置荧光显微镜观察,碳点的蓝色荧光与线粒体的红色荧光较好重叠,与溶酶体的红色荧光较好重叠,与内质网的红色荧光不完全重叠,与高尔基体的红色荧光重叠得较差。结论:碳点生物相容性较好,被细胞摄取后可以分布至细胞质的主要细胞器上,可作为一种非病毒载体应用到转基因治疗中。

Objective:To investigate the transport pathway and intracellular distribution of the of fluorescent carbon dots(CDs)synthesized by folic acid and polyethyleneimine(PEI)through the membrane of MC3T3-E1 cells and its effect on the cells,and to clarify the mechanism.Methods:The fluorescent CDs with the function of cell imaging were synthesized by hydrothermal method using folic acid and PEI as the raw materials;MTT assay was applied to screen the best concentration of CDs.The MC3T3-E1 cells were divided into blank control group,folic acid group and CDs group.The biocompatibility of CDs was evaluated by the detection of cell cycle,apoptosis and cellular reactive oxygen species(ROS)level.Nystatin as a kind of caveolae inhibitor and nocodazole as a kind of macropinocytosis inhibitor were used to find out the pathway through which the cells took in the CDs.Using the charcteristic of CDs with blue fluorescence stimulated by ultraviolet ray,the organelle probes were used to observe the distribution of CDs.Results:Compared with blank control group,the cells in different concentrations(100-450 mg·L-1)of CDs groups showed no cytotoxicity at 24 h(P>0.05);at 48 h,the cell proliferation rate was reduced to 68.4%of blank control group when the concentration of CDs reached 350 mg·L-1(P<0.05).Compared with blank control group,the percentages of cells in G 0 phase and G 1 phase in CDs group were decreased(P<0.05),and the percentage of cells in S phase was increased(P<0.05);the percentages of cells in G 2 phase and M phase were increased,but there no was significant differences(P>0.05).Compared with blank control group,the apoptotic rates of the cells in folic acid group and CDs group had no significant differences(P>0.05).Compared with blank control group,the intracellular ROS levels in folic acid group and CDs group were significantly decreased(P<0.05).Compared with blank control group,the uptake amount of CDs in the cells was decreased in nystatin group(P<0.05).The blue fluorescence of CDs overlapped with the red fluorescence of mitochondria under an inverted fluorescence microscope,the blue fluorescence of CDs overlapped with the red fluorescence of lysosomes;they didn’t overlap completely with the red fluorescence of the endoplasmic reticulum;the blue fluorescence of CDs overlapped poorly with the red fluorescence of Golgi apparatus.Conclusion:CDs perform well in biocompatibility and they can be distributed to different organelles after taken in by the cells.They can be used as a kind of gene carrier in transgenic therapy.