Molecular dynamics simulation method for ultrasonic cavitation effect of cell membrane

Abstract

The invention discloses a molecular dynamics simulation method of a cell membrane ultrasonic cavitation effect, and belongs to the field of ultrasonic medical treatment and molecular dynamics simulation. Through a molecular dynamics method, ultrasonic waves are generated by periodically applying speeds in different directions to particles in simulation and act on a constructed coarse-grained cell membrane system; and the generation of the cavitation effect is confirmed according to the visualization result of the cavitation bubble forming process and the pressure and density parameter calculation of the generation process, which indicates that the ultrasonic wave successfully acts on the system. According to the method, the cavitation effect and influence of ultrasonic waves on a cell membrane system are simulated by using an unbalanced dynamic simulation method of the ultrasonic wave action model under the NVE ensemble for the first time, a method for generating the cavitation effect by the ultrasonic waves under a microscopic view angle is provided, and certain theoretical support is provided for ultrasonic-assisted medical treatment; and the development of the future ultrasonic medical field is facilitated.

Description

Technical Field

[0001] The invention belongs to the field of molecular dynamics simulation and ultrasonic therapy, and in particular relates to a molecular dynamics simulation method for ultrasonic cavitation effect of cell membrane. Background Art

[0002] Ultrasound is a sound wave with a frequency higher than the human hearing range, usually between 20KHz and hundreds of MHz. It can propagate in media such as gas, liquid and solid, and the propagation speed is different in different media. The wavelength of ultrasound is inversely proportional to its frequency. The higher the frequency, the shorter the wavelength, and reflection and refraction will occur when encountering the interface of different media. In addition, ultrasound will also produce cavitation effect: under the action of ultrasound, the pressure inside the liquid will change dramatically. When the local pressure in the liquid is lower than the vapor pressure of the liquid, bubbles will form, and the negative pressure at the gas-liquid interface will also erode and damage the surrounding medium. When the bubble reaches a certain size, it will collapse due to the increase of surrounding pressure or the change of ultrasound. When the bubble collapses, a large amount of energy will be released, generating a small shock wave of high temperature and high pressure. Therefore, cavitation effect ultrasound has many applications in treatment, such as promoting blood circulation, relieving pain, accelerating tissue healing, and is often used for the rehabilitation of muscle injuries, arthritis and other diseases.

[0003] Although the effect of ultrasound on the macroscopic level is very significant, it is currently difficult to observe the process and influence of ultrasound on tissue cells at the molecular level. Therefore, theoretical methods are needed to study the effects and mechanisms of ultrasound at the microscopic level. Molecular dynamics simulation, as a method to study the mechanism of intracellular molecular reactions, can simulate the chemical and physical properties of macromolecules such as proteins, lipids, DNA and various small molecules on cells at the atomic level, thereby revealing the interaction, conformational changes and dynamic processes of the ultrasonic cavitation effect on molecules at the molecular level. Molecular dynamics simulation can also provide an in-depth understanding of experimental research at the microscopic level and predict phenomena that are difficult to observe in experiments.

[0004] Through the analysis of relevant literature, most of the molecular dynamics simulation methods for studying cavitation collapse under ultrasound usually first preset an artificial cavitation [1-3] , bubbles are formed by manually removing the spherical part of the water molecules. The advantage of this method is that the position of the cavitation can be adjusted freely, but it only simulates the impact of the collapse after the cavitation is generated, and cannot restore the pressure and density state of water molecules at the gas-liquid interface when the cavitation is generated, and cannot simulate the erosion of the cell membrane when the cavitation is generated. Therefore, it is not comprehensive enough to only simulate the jet generated by the collapse of bubbles in this state. For the generation of cavitation, the existing simulation method is to apply a huge negative pressure to the liquid to make it reach a critical state to generate cavitation. [4]. However, this method also cannot accurately simulate the effect of ultrasound on liquid pressure and density. At present, the simulation research on the cavitation formation process under ultrasound is not comprehensive. The present invention uses an ultrasound action model combined with NVE non-equilibrium molecular dynamics simulation (NEMD) to apply ultrasound to the generation of cavitation effect in the cell membrane system for the formation process of cavitation under ultrasound. Different from the previous method of simulating preset cavitations, the present invention uses an ultrasound action model to act on the cell membrane system to naturally form cavitations. The cavitations formed by this method can more completely reflect the generation of cavitation effect under ultrasound. At the same time, the present invention also uses the NEMD simulation method to simulate the dynamic response of the system under external disturbances or gradients, and to show the changes produced at the molecular level under ultrasound, providing new insights for future medical research such as ultrasound therapy and ultrasonic drug delivery.

[0005] References

[0006] [1]Dandan Sun,Xubo Lin,Zuoheng Zhang,Ning Gu.Impact ofShock-InducedLipid Nanobubble Collapse on a PhospholipidMembrane.J.Phys.Chem.C 2016,DOI:10.1021.

[0007] [2] Nan Nan. Interaction between jet and cell membrane in sonoporation and morphological behavior of endothelial tissue[D]. Shanghai University, 2019. DOI: 10.27300.

[0008] [3] Sun Dandan. Molecular dynamics simulation study on the interaction between C60, lipid nanobubbles and cell-like membranes[D]. Southeast University, 2015.

[0009] [4] Gu Youwei. Experimental study and molecular dynamics simulation of liquid cavitation initiation[D]. Tsinghua University, 2016. Summary of the invention

[0010] The present invention proposes a molecular dynamics simulation method for ultrasonic cavitation effect of cell membrane, so as to solve the problem that the cavitation effect caused by ultrasonic wave acting on cell membrane system cannot be accurately studied at the molecular level.

[0011] In view of this, the present invention: a molecular dynamics simulation method of ultrasonic cavitation effect of cell membrane adopts a technical solution comprising the following steps:

[0012] S1: construct the ultrasonic action model and embed its code into Gromacs software;

[0013] S2: Establish a cell membrane model, evenly distribute lipid molecules according to the lipid composition of the cell membrane, and coarse-grain the system;

[0014] S3: The system is balanced using a step balance method, with a total balance time of 1 μs;

[0015] S4: Use the ultrasonic action model described in S1 to perform molecular dynamics simulation analysis under the NVE ensemble; further, in the ultrasonic model, the ultrasonic wave is generated by irradiating six shock waves from the six sides of the system box to the center of the box in sequence, and the irradiation time of each shock wave is 80 simulation steps N, which is divided into 5 irradiation cycles, each cycle is 16N, and the irradiation time interval is T int , T int =2400N.

[0016] Furthermore, the ultrasonic velocity in each pulse is defined as follows:

[0017]

[0018]

[0019] Among them, Vi represents the six ultrasonic velocities along the hexahedral box, and their directions are all from the box surface toward the box center, Vmax is the maximum speed of the ultrasonic pulse, m represents the time step, and N represents the molecular dynamics simulation step.

[0020] Furthermore, a cell membrane model was constructed based on the main phospholipid and cholesterol composition of the human cell membrane, and 4 to 6 atoms were aggregated into a coarse grain to form a coarse-grained particle. The interaction of each coarse grain was regarded as a valid interaction to simplify the interaction calculation between molecules.

[0021] Furthermore, in the model equilibrium stage, in order to ensure the stability of the system and more efficient calculation, equilibrium simulations are performed in sequence using different step sizes, ultimately allowing the system to reach an equilibrium state.

[0022] Furthermore, in the simulation process of ultrasonic wave acting on cell membrane, the non-equilibrium simulation method was adopted, and the NVE ensemble was used to simulate the system. Compared with the NPT and NVT ensembles, the system was not temperature-coupled and pressure-coupled, which eliminated the heat exchange between the system and the external environment, thereby relatively accurately reflecting the structural changes of the system and the generation of cavitation effect under the action of ultrasonic wave, as well as more reasonable pressure changes and temperature changes.

[0023] Furthermore, the simulation results of molecular dynamics were visualized and analyzed. The visualization software VMD was used to observe the structural changes of the cell membrane and the process from the generation to the expansion of vacuoles. The density module of gromacs software was used to calculate the density distribution of water molecules at different stages of ultrasound, and the pressure changes of the system during the simulation were calculated based on gmx energy.

[0024] The present invention provides a computer-readable storage medium having a computer program stored thereon, including a processor and a memory for executing the computer program stored in the memory to implement a cell membrane NVE non-equilibrium molecular dynamics simulation method under ultrasonic action as described above.

[0025] The present invention has the following advantages:

[0026] 1. The present invention adopts a coarse-grained simulation balance method of step balance to improve the calculation efficiency while ensuring the stability of the system.

[0027] 2. The present invention adopts the NVE non-equilibrium simulation method to combine the above-mentioned ultrasonic action model with molecular dynamics simulation to act on the cell membrane and simulate the generation of cavitation effect. Compared with directly removing water molecules to form cavitations and directly applying negative pressure to generate cavitations, the cavitation generation of the present invention is more consistent with the effect produced under the action of ultrasonic waves, showing the process of generating cavitation effect at the microscopic level.

[0028] 3. By simulating the cavitation effect and structural changes of cell membranes when ultrasound is applied to cells, new insights and theoretical guidance can be provided for future ultrasound therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The overall method flow chart of the present invention is

[0030] Figure 2 Coarse-grained cell membrane structure

[0031] Figure 3 The equilibrium RMSD plot

[0032] Figure 4 Ultrasonic wave action model diagram

[0033] Figure 5 The diagram of membrane structure changes under different intensities of ultrasound

[0034] Figure 6 This is a diagram of the process of cavitation effect under ultrasonic action.

[0035] Figure 7 The water density distribution diagram at different stages of ultrasonic action

[0036] Figure 8The pressure of the system changes with the simulation time when ultrasonic waves act on it DETAILED DESCRIPTION

[0037] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] like Figure 1 As shown, a method for simulating cell membrane molecular dynamics under ultrasonic action of the present invention comprises the following steps:

[0039] S1: Use CHARMM-GUI cell membrane construction tool to build a human cell membrane model and fill it with water molecules

[0040] S2: Use the Martini Maker tool to coarse-grain the cell membrane model constructed in S1 and generate a coarse-grained force field.

[0041] S3: Energy minimization and piecewise equilibrium simulation were performed using Gromacs software to bring the system to equilibrium.

[0042] S4: The ultrasonic action model was embedded in the Gromacs software, and the NVE non-equilibrium molecular dynamics simulation method was used to perform ultrasonic simulation of the above-mentioned balanced cell membrane model for 100 ns.

[0043] S5: Use the orthogonal perspective of VMD visualization software to observe the morphological changes of the cell membrane and calculate the density and pressure changes of the analysis system.

[0044] Further, the step 1: using the CHARMM-GUI molecular dynamics modeling tool to establish a 25×25×35 nm 3 box, and build a 25×25nm 2 The cell membrane of the outer leaflet is composed of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) 55%, sphingomyelin (DPSM) 5%, 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) 20%, cholesterol (CHOL) 20%, and the inner leaflet is composed of POPC 50%, POPE 20%, 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS) 5%, CHOL 20%, phosphatidylinositol 4,5-bisphosphate (POP5) 5%. Then water molecules are filled into the system, and 150nM NaCl is added to keep the system electrically neutral.

[0045] Furthermore, step 2: use the Martini Maker tool to coarse-grain the cell membrane model constructed in S1, use martini22 as the coarse-grain force field, and coarse-grain the water molecules into non-polar water molecules. In this model, water molecules are regarded as particles without charge, and the van der Waals force and hydrophobic interaction are mainly considered. The number of system particles is reduced while the computational efficiency is enhanced. Finally, a cell membrane system containing 224,961 coarse-grained particles is constructed, such as Figure 2 shown.

[0046] Furthermore, in step 3, the energy minimization and piecewise equilibrium simulation of the coarse-grained model are performed using Gromacs software. First, the step size is set to 1 fs for 20 ns simulation, then the step size is set to 2 fs for 20 ns equilibrium simulation, and the step size is gradually increased, and finally the step size of 20 fs is used for 1 μs equilibrium simulation. The results are shown in Fig. Figure 3 As shown, according to the calculated RMSD, the system has reached the expected equilibrium state.

[0047] Furthermore, the step 4: embed the ultrasonic action model into the Gromcs software, and use the NVE non-equilibrium molecular dynamics simulation method to perform ultrasonic simulation on the above-mentioned balanced cell membrane model for 100ns: the ultrasonic model is established by irradiating six shock waves from the six sides of the system box to the center of the box in sequence, and the irradiation time of each shock wave is 80 simulation steps N, which is divided into 5 irradiation cycles, each cycle is 16N, and the irradiation time interval is T int , T int =2400N, such as Figure 4 shown.

[0048] Furthermore, the ultrasonic velocity in each pulse is defined as follows:

[0049]

[0050]

[0051] Among them, Vi represents the six ultrasonic velocities along the hexahedral box, and their directions are all from the box surface toward the box center, Vmax is the maximum speed of the ultrasonic pulse, m represents the time step, and N represents the molecular dynamics simulation step.

[0052] Furthermore, the ultrasonic model described in step 4 is applied to the cell membrane coarse-grained model after equilibrium in step 3. During the simulation, the temperature coupling and pressure coupling parameters are set to NO, the simulation ensemble is the NVE ensemble, and a non-equilibrium simulation is performed. The ultrasonic intensity is set to 10Mhz, 20Mhz, 30Mhz, and 70Mhz according to the ultrasonic parameters. The simulation is performed for 100ns respectively.

[0053] Furthermore, in step 5, the simulated structure is displayed using a VMD visualization tool, and the morphological changes of the cell membrane are observed using an orthogonal perspective. Figure 5 As shown in the figure, under the action of ultrasound of different intensities, the cell membrane exhibits different degrees of vibration stretching, and the degree of stretching increases with the increase of ultrasound intensity. Finally, at 70Mhz, the cell membrane cannot withstand the excessive vibration, and some membrane molecules detach from the cell membrane. At the same time, the cavitation effect occurs in the simulation process of 70Mhz ultrasound intensity, as shown in the figure. Figure 6 As shown, when the liquid pressure reaches a critical point, cavitation occurs and further expands, eventually destroying the cell membrane.

[0054] Density change calculation during simulation: The result is as follows Figure 7 As shown in the figure, the density distribution of water molecules at different stages of the simulation process was calculated and plotted. It was found that when the ultrasound had not yet acted on the model, the density of water molecules was in the natural state (1000kg / m 3 , the cell membrane is in the middle and the water molecule density is 0). When ultrasound is applied to the model, the water density decreases and the density shows a periodic distribution, which is consistent with the periodic effect of ultrasound. When the cavitation effect occurs, the density distribution at the coordinates where the cavitation bubble is generated decreases significantly, and the water molecule density at the cavitation bubble boundary also shows periodicity.

[0055] According to the pressure change of the system during the simulation Figure 8 As shown in the figure, the pressure rises in a wave-like manner in the early stage of ultrasonic action, begins to drop rapidly at 30ns, and eventually becomes negative pressure, but rises cliff-like at around 90ns. This shows that water molecules produce cavitation effect under the action of ultrasound, and the internal pressure of the liquid becomes negative pressure under the action of ultrasound, reaching the conditions for cavitation. When cavitation occurs, part of the pressure is released, and the pressure of the surrounding liquid is redistributed, resulting in a sudden rise in the curve. This shows that this method can successfully load ultrasound to the system and simulate the process of cavitation effect cavitation.

Claims

1. A molecular dynamics simulation method for ultrasonic cavitation effect of cell membrane, characterized in that: The following steps are involved: S1: construct the ultrasonic action model and embed its code into Gromacs software; S2: Establish a cell membrane model, evenly distribute lipid molecules according to the lipid composition of the cell membrane, and coarse-grain the system; S3: The system is balanced using a step balance method, with a total balance time of 1 μs; S4: Molecular dynamics simulation analysis was performed under the NVE ensemble using the ultrasound action model described in S1.

2. The molecular dynamics simulation method of the ultrasonic cavitation effect of cell membrane according to claim 1, characterized in that: In this ultrasonic model, the ultrasonic wave is generated by six shock waves irradiated from the six sides of the system box to the center of the box in sequence. The irradiation time of each shock wave is 80 simulation steps N, which is divided into 5 irradiation cycles, each cycle is 16N, and the irradiation time interval is T int , T int =2400N.

3. The molecular dynamics simulation method of the ultrasonic cavitation effect of cell membrane according to claim 2, characterized in that: The ultrasonic velocity in each pulse is defined as follows: Among them, V i represents the six ultrasonic velocities along the hexahedral box, all of which are directed from the box surface toward the box center, V max is the maximum speed of the ultrasonic pulse, m represents the time step, and N represents the molecular dynamics simulation step.

4. The molecular dynamics simulation method of the ultrasonic cavitation effect of cell membrane according to claim 1, characterized in that: A cell membrane model was constructed based on the main phospholipid and cholesterol composition of human cell membrane, and 4 to 6 atoms were aggregated into a coarse grain to form a coarse-grained particle. The interaction of each coarse grain was regarded as a valid interaction to simplify the interaction calculation between molecules.

5. The molecular dynamics simulation method of the ultrasonic cavitation effect of cell membrane according to claim 1, characterized in that: In the model equilibrium stage, in order to ensure the stability of the system and more efficient calculation, equilibrium simulations are carried out in sequence using different step sizes, ultimately allowing the system to reach an equilibrium state.

6. The molecular dynamics simulation method of cell membrane ultrasonic cavitation effect according to claim 1, characterized in that: In the process of simulating the effect of ultrasound on the cell membrane, a non-equilibrium simulation method is adopted, and the NVE ensemble is used to simulate the system. Compared with the NPT and NVT ensembles commonly used in the literature, its advantage is that there is no temperature coupling and pressure coupling of the system, which eliminates the heat exchange between the system and the external environment, thereby relatively accurately reflecting the structural changes of the system under the action of ultrasound and the generation of cavitation effects, as well as more reasonable pressure changes and temperature changes.

7. The molecular dynamics simulation method of cell membrane ultrasonic cavitation effect according to claim 1, characterized in that: The simulation results of the ultrasonic cavitation effect are as follows: the visualization software VMD is used to observe the structural changes of the cell membrane and the process from the generation to the expansion of cavitation bubbles; the density module of the gromacs software is used to calculate the density distribution of water molecules at different stages of ultrasound; and the pressure changes of the system during the simulation are calculated according to the gmx energy.

8. A computer-readable storage medium having a computer program stored thereon, comprising a processor and a memory; characterized in that: The memory is used to store computer programs, and the processor is connected to the memory and is used to execute the computer programs stored in the memory to implement the molecular dynamics simulation method of ultrasonic cavitation effect of cell membrane according to any one of claims 1 to 7.

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