Shigematsu Taiki, Shinoda Yuya, Takagi Reiya, Ujihara Yoshihiro, Sugita Shukei, Nakamura Masanori
Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Yamaguchi 755-8611, Japan.
Department of Electrical and Mechanical Engineering, Graduate School of Engineering, Nagoya Institute of Technology, Nagoya, Aichi 466-8555, Japan.
Langmuir. 2025 Feb 11;41(5):3209-3219. doi: 10.1021/acs.langmuir.4c03943. Epub 2025 Jan 28.
Second harmonic generation (SHG) measurements using SHG-active dye molecules have recently attracted attention as a method to detect the formation of pores in phospholipid bilayers. The bilayers, in which the dye molecules are embedded in the outer leaflet, exhibit a noncentrosymmetric structure, generating SHG signals. However, when pores form, these dye molecules translocate through the pores into the inner leaflet, leading to a more centrosymmetric structure and the subsequent loss of the SHG signals. A decrease in the SHG signals has been experimentally observed in membranes subjected to electrical stimuli. However, the characteristics of the interleaflet translocation of SHG-active dye molecules through pores remain unclear, hindering quantitative estimation of the membrane conditions, such as the pore size and density, based on the SHG signal reduction. In this study, we investigated the interleaflet translocation characteristics of Ap3, an SHG-active dye molecule, using molecular dynamics (MD) simulations and two-dimensional random-walk (RW) simulations. The MD simulations revealed that Ap3 molecules only translocate between the leaflets along the pore sidewalls. We determined the lateral diffusion coefficient of Ap3 within the membrane plane and its propensity for interleaflet movement at the pore wall. Based on these movement characteristics, the RW model successfully reproduced the characteristic time scale of the interleaflet translocation observed in the MD simulations. By varying the pore size and density in the RW simulations, we estimated that the characteristic time scale of interleaflet translocation depends on the -0.31 power of the pore radius and the -1.13 power of the pore density. Using these findings, we estimated the number of pores that probably formed in membranes during previous electroporation experiments. These results indicate the potential of optical measurement of the dye molecule movement for the indirect quantitative estimation of the pore size and number, which are challenging to measure optically.
使用具有二次谐波产生（SHG）活性的染料分子进行二次谐波产生测量，作为一种检测磷脂双层中孔形成的方法，最近受到了关注。染料分子嵌入外层小叶的双层膜呈现非中心对称结构，产生SHG信号。然而，当孔形成时，这些染料分子通过孔转移到内层小叶，导致结构更加中心对称，随后SHG信号消失。在受到电刺激的膜中，实验观察到SHG信号减弱。然而，具有SHG活性的染料分子通过孔进行小叶间转移的特性仍不清楚，这阻碍了基于SHG信号减弱对膜条件（如孔径和密度）进行定量估计。在本研究中，我们使用分子动力学（MD）模拟和二维随机游走（RW）模拟，研究了具有SHG活性的染料分子Ap3的小叶间转移特性。MD模拟表明，Ap3分子仅沿着孔侧壁在小叶之间转移。我们确定了Ap3在膜平面内的横向扩散系数及其在孔壁处小叶间移动的倾向。基于这些移动特性，RW模型成功再现了MD模拟中观察到的小叶间转移的特征时间尺度。通过在RW模拟中改变孔径和密度，我们估计小叶间转移的特征时间尺度取决于孔半径的-0.31次方和孔密度的-1.13次方。利用这些发现，我们估计了先前电穿孔实验中膜中可能形成的孔的数量。这些结果表明，通过光学测量染料分子的运动来间接定量估计孔径和数量具有潜力，而这些参数通过光学测量具有挑战性。
