Simulation And Experimental Study Of The Effect Of Nuclear Lamina On The Mechanical Properties Of Cell Nuclear Membrane Piercing

Gene editing is an important biotechnology that can be used to treat genetic diseases,improve crops,and produce biopharmaceuticals.One crucial step in gene editing is to deliver the target gene into the nucleus of the cell.The current precise and controllable method is to use a nanoneedle to carry the desired gene into the nucleus,which requires breaking through the cell membrane,cytoplasm,and nuclear membrane in sequence while ensuring the cell remains intact.However,the complexity of the nuclear structure,especially its high self-protection,makes it difficult to penetrate the nuclear membrane.Therefore,how to improve the delivery efficiency of exogenous molecules into the nucleus is a major challenge.The nuclear lamina,one of the main structures in the nucleus,plays an important role in maintaining the shape of the nucleus.This study analyzed how the nuclear lamina affects the mechanical properties of the nuclear membrane during the process of penetration and explored methods to improve the penetration rate of the nuclear membrane.This study used a combined approach of simulation and experiment to construct a finite element simulation model of nanoneedle penetration into the nucleus and analyzed the mechanical properties of the nuclear membrane during the process.Firstly,a nuclear penetration model was constructed using Abaqus and Python secondary development,including the nuclear membrane,nuclear lamina A and B,nucleoplasm,and glass slide with the nanoneedle.Then,by analyzing the stress distribution of the nuclear membrane under different loading speeds and diameters of the nanoneedle,the parameters of the nanoneedle were optimized.The results showed that the optimal penetration speed of the nanoneedle should be smaller than 20 μm/s,and a nanoneedle diameter less than 200 nm.Finally,by comparing the simulation results of models with different nuclear lamina structures,it was demonstrated that nuclear lamina A is the main structure maintaining the stiffness of the nucleus.Moreover,by comparing models with different nuclear lamina densities,it was shown that increasing the nuclear lamina density can improve the stress concentration on the nuclear membrane and promote nuclear membrane penetration,and the influence of the nuclear lamina A density was significant.To verify the accuracy of the simulation model,AFM was used for experimental analysis of nanoneedle penetration into the nucleus.Firstly,the effects of extracting and removing nuclear components from isolated nuclei were compared,and the isolated nucleus could increase the penetration rate of the nuclear membrane by 30%.Then,by changing the nanoneedle penetration speed and position,the influence of these parameters on nuclear membrane penetration was analyzed.It was demonstrated that higher penetration speed and higher penetration height were more conducive to increasing the penetration rate of the nuclear membrane.Finally,by treating cells with OPN,it was shown that as the density of nuclear lamina A decreased in isolated nuclei,the penetration rate of the nuclear membrane decreased by 26.7%,indicating that the density of nuclear lamina A is related to the penetration characteristics of the nuclear membrane.This study revealed the mechanism of nuclear membrane penetration at the nanoscale,demonstrated that increasing the density of the nuclear lamina can improve the stress concentration on the nuclear membrane and increase the penetration rate of the nanoneedle into the nucleus.The study also optimized the parameters of nanoneedle penetration and proposed selecting the appropriate nanoneedle diameter,penetration speed,and position to increase the penetration rate,providing theoretical guidance for applications such as gene editing.