金纳米颗粒介导的细胞光穿孔表征方法

Cell Optoporation Characterization Method for Gold-Nanoparticle Mediation

提出了一种新的表征方法--膜电位测量。膜电位恢复时间与膜穿孔尺寸相对应,因此建立起细胞膜穿孔尺寸的大小与激光能量阈值之间的关系,可为准确地向细胞内递送不同分子量的外源物质提供理论支持。将金纳米颗粒与胃癌细胞共同培养,在保证细胞不受金纳米颗粒毒性影响的前提下,选择不同能量纳秒脉冲激光照射共孵育后的胃癌细胞,并采用碘化丙啶(PI)和钙黄绿素乙酰甲酯(Calcein-AM)对穿孔后的细胞进行染色验证。结果发现:当加入直径为100 nm的金纳米颗粒,其数量与细胞数比为400\:1,激光能量密度在20 mJ/cm^2时,可以在保证细胞活性的前提下成功实现532 nm脉冲激光的细胞膜穿孔;在穿孔条件下,采用光标测技术测量细胞膜电位,发现细胞膜电位先增加后复原,最大增量为50 mV,恢复时间为250 s。膜电位结果再次验证,光穿孔造成的细胞膜损伤是可以恢复的,而且可以用膜电位变化来表征。

A new characterization method,namely membrane voltage measurement method,is proposed.Because the recovery time of membrane potential is dependent on the size of optoporation,establishing the relationship between the size of cytomembrane optoporation and the laser energy threshold can provide theoretical support for importing different exogenous substances into cells.In this study,gastric cancer cells are cultured with gold nanoparticles.On the premise that cells are not affected by the toxicity of gold nanoparticles,gastric cancer cells incubated with gold nanoparticles are irradiated using a nanosecond-pulsed laser with different energies.Propidium iodide and calcein-AM are used for staining verification of perforated cells.The results indicate that optoporation of cell membrane can be successfully achieved with 400 nanoparticles(diameter of 100 nm)per cell and 20 mJ/cm^2 energy density by 532 nm pulsed laser,without obvious dead cells.During the optoporation,the membrane potential is measured using optical mapping technique.It is found that the membrane potential firstly increases and then restores with 50 mV maximum increment and 250 s recovery time.These results confirm that the cell membrane damage can be recovered by optoporation and characteristiced by membrane potential change.