Method for changing phospholipid membrane structure through light regulation and control and application

Abstract

The invention discloses a method for changing a phospholipid membrane structure through light regulation and application, and belongs to the technical field of biology. The method comprises the following step: irradiating a component containing a phospholipid membrane structure by using terahertz waves to obtain a phenomenon that the phospholipid membrane structure is loosened. And the light frequency of the terahertz wave is 0.1 THz. According to the invention, the liposome irradiated by terahertz waves is observed by using a cryoelectron microscope, and the particle size of the liposome is increased. Under the irradiation condition, the structure of the liposome is changed. The invention provides a novel technical method for changing a phospholipid membrane structure by utilizing light regulation and control.

Description

Technical Field

[0001] The invention belongs to the field of biological technology, and in particular relates to a method for changing the structure of phospholipid membrane by light regulation. Background Art

[0002] Terahertz waves refer to electromagnetic waves with a frequency range of 0.1 to 10 THz, which belongs to mid- and far-infrared light. With the rapid development of modern physics since the 1980s, the terahertz gap has begun to attract attention and exploration, and has gradually been popularized in fields such as biomedicine.

[0003] Phospholipids are important lipid components in organisms, and the membrane structure they form is very important in organisms. Studies have shown that phospholipid membrane structures are involved in many physiological processes, such as material transport, cell communication, energy conversion, and morphology maintenance. First, the membrane structure maintains the cell structure and morphology from a physical level, and on this basis, isolates and compartmentalizes the cells so that the organelles in the cells perform their respective functions; secondly, as a semipermeable membrane, it selectively controls the entry and exit of substances in the cell and maintains the stability of the internal environment of the cell; at the same time, the various proteins present in the membrane structure are responsible for different functions, such as transport proteins for selective transport of substances, and receptor proteins for signal molecule transmission and conduction. The normal operation of its functions is closely related to the functions of cells and even living organisms, but in specific biological processes, the destruction of the membrane structure is a necessary physiological function. For example, the process of apoptosis, the process of cell division, the process of endocytosis and exocytosis, etc. Therefore, it is crucial to maintain the orderliness of the biological membrane structure.

[0004] At present, most of the methods for studying membrane structure are to change the properties of phospholipid membrane structure through drugs or mechanical means. The former will not only cause changes in the membrane structure of the targeted part, but also cause changes in other parts, while the latter will produce more drastic changes and is difficult to be applied in practice. There is no technical method for light-regulated phospholipid membrane structure. At the same time, the present invention deeply studies the mechanism of light-regulated changes in phospholipid membrane structure, which is conducive to providing new mechanisms and perspectives for studying diseases related to changes in phospholipid membrane structure. Summary of the invention

[0005] In view of the problems existing in the above-mentioned prior art, the purpose of the present invention is to design and provide a method for changing the structure of phospholipid membranes by means of light regulation. The present invention applies light conditions with a light frequency of 0.1THz through molecular dynamics simulation to obtain the phenomenon that the phospholipid membrane structure becomes loose. Subsequently, cryo-electron microscopy was used to observe that the liposomes were subjected to light with a light frequency of 0.1THz, and it was found that the liposome particle size increased. That is, under this irradiation condition, the liposome structure changes. For the simulated structure and the liposome structure, in vitro light stimulation is performed respectively, and one or more of the two technical means of molecular dynamics simulation and cryo-electron microscopy are used for detection to achieve the change of the phospholipid membrane structure.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In one aspect, the present invention provides a method for changing the structure of a phospholipid membrane using light regulation, comprising irradiating the biological membrane structure with terahertz waves.

[0008] In the method of using light regulation to change the structure of a phospholipid membrane, the light frequency of the terahertz wave is 0.1 THz.

[0009] In the method for changing the structure of a phospholipid membrane by light regulation, the electric field intensity of the terahertz wave frequency is 0.6-0.8 V / nm.

[0010] In the method of using light regulation to change the structure of a phospholipid membrane, the electric field direction of the terahertz wave is perpendicular to the plane of the phospholipid membrane structure.

[0011] In the method for changing the structure of phospholipid membrane by light regulation, the irradiation time is 20 to 30 minutes.

[0012] In the method for changing the structure of phospholipid membrane by light regulation, the biological membrane structure is a liposome.

[0013] In a second aspect, the present invention provides use of any of the methods described above in changing the structure of phospholipid membranes.

[0014] In a third aspect, the present invention provides a liposome with a changed phospholipid membrane structure, which is prepared by any of the methods described above.

[0015] In a fourth aspect, the present invention provides a drug delivery system comprising the liposome with altered phospholipid membrane structure.

[0016] In a fifth aspect, the present invention provides the use of the liposomes with altered phospholipid membrane structure in the preparation of treatments for diseases associated with altered phospholipid membrane structure.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The method of changing the structure of phospholipid membranes by light regulation in the present invention confirms that terahertz waves with a light frequency of 0.1 THz can change the structure of phospholipid membranes, and may further change the structure of biological membranes, change the overall cell and related functions of the body, and has potential value in the field of studying light-regulated liposomes for drug delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The molecular dynamics simulation shows the changes in the phospholipid membrane structure at the end of the simulation under different electric field conditions, where A is the initial model constructed, B is the applied electric field waveform, and C is the damage process of the phospholipid membrane structure under 0.4-0.8V / nm. The blue part in the figure is phospholipids.

[0020] Figure 2 The changes in the number of hydrogen bonds between phospholipid membrane structures during the molecular dynamics simulation process; A is the change in the number of hydrogen bonds over time during the whole simulation process, B is the T-test analysis of the average number of hydrogen bonds during the simulation process, and C is the thickness of the simulation system at the end of the simulation. The results are expressed as mean ± standard deviation, and *** indicates p < 0.001 compared with the control group;

[0021] Figure 3 The results of changes in liposome structure detected by cryo-electron microscopy; A is a schematic diagram of different types of liposome morphology; B is the liposome structure photographed by cryo-electron microscopy; C is the statistical result of the average particle size of liposomes in the control group and the irradiated group; D is the normal distribution diagram of the liposome particle size in the control group and the irradiated group. The number of liposomes counted in C and D are both greater than 1000. The results are expressed as mean ± standard deviation, and *** indicates p < 0.001 compared with the control group. DETAILED DESCRIPTION

[0022] The specific implementation of the present invention will be described below in conjunction with the accompanying drawings so that those skilled in the art can better understand the present invention. It should be noted that in the following description, when the detailed description of known functions and designs may dilute the main content of the present invention, these descriptions will be omitted here.

[0023] The method for changing the structure of the phospholipid membrane of the present invention is: using a terahertz wave with a light frequency of 0.1THz to stimulate and intervene the phospholipid membrane structure, realize light regulation, and then change the phospholipid membrane structure. The present invention first constructs a phospholipid bilayer by molecular dynamics simulation modeling, and simulates the irradiation process of the terahertz light wave by applying a light frequency of 0.1THz, an electric field direction perpendicular to the plane of the phospholipid layer, and an electric field strength of 0.4, 0.5, 0.6, 0.7, and 0.8V / nm. The simulation time is 10ns, and it is found that the applied electric field strength will destroy the phospholipid membrane structure when it is large enough. Subsequently, an electric field strength of 0.5V / nm that fails to destroy the phospholipid membrane structure is used for subsequent simulation. By comparing the parameters such as the number of hydrogen bonds after the simulation, it is found that the phospholipid membrane structure system becomes loose, that is, the membrane structure is disturbed and the stability changes. Subsequently, the constructed liposomes are irradiated with a 0.1THz terahertz source, and the irradiated liposomes are observed using a cryo-electron microscope, and it is found that the average particle size of the liposomes increases, that is, the membrane structure has changed.

[0024] Embodiment 1:

[0025] In order to evaluate the effect of terahertz waves with an optical frequency of 0.1 THz on the structure of phospholipid membranes, molecular dynamics simulations were used to conduct a preliminary assessment of the terahertz frequency intensity used.

[0026] Considering that the means of observing the microstructure of phospholipid membranes are relatively limited, the stability of the membrane structure was first simulated with the help of molecular dynamics simulation. The most important component of the phospholipid membrane structure is phosphatidylcholine, so one of the phosphatidylcholines was used to build the model. The model was built with the help of the Charmm-GUI online website. The model is a cuboid with an initial size of 10nm×10nm×8.5nm. The simulation system contains 294 phospholipid molecules, 13148 water molecules, 34 potassium ions and 34 chloride ions.

[0027] The simulation duration is 100ns, and the terahertz condition with a light frequency of 0.1THz is applied with the help of an external electric field. The reason for applying the electric field here is that the magnetic field component is small enough compared to the electric field, so only the electric field is applied in the simulation to simulate the terahertz light wave irradiation. The direction of the electric field is perpendicular to the surface of the phospholipid membrane and downward, which is defined as the Z direction. The electric field strength is 0.4, 0.5, 0.6, 0.7, and 0.8V / nm. With the help of the trajectory extraction command of GROMACS, the last frame of the simulation is extracted. After graphical processing using VMD, it is found that when the electric field condition is lower than 0.6V / nm, the phospholipid membrane structure will not rupture, and when it is higher than this value, the phospholipid membrane structure will rupture rapidly. The mechanism of this rupture process is electroporation. The initial model constructed is as follows: Figure 1 A, the applied electric field waveform is shown in 1B, and the damage process of the phospholipid membrane structure at 0.4-0.8V / nm is shown in Figure 1 C. The simulation results preliminarily show that the terahertz condition with a light frequency of 0.1 THz will destroy the membrane structure under the electric field condition of 0.6 V / nm and above.

[0028] Embodiment 2:

[0029] In order to explore whether the effect of terahertz waves with an optical frequency of 0.1 THz on the phospholipid membrane structure only causes damage to the phospholipid membrane structure, subsequent simulations were performed using the terahertz frequency intensity obtained in Example 1 that would not damage the phospholipid membrane structure.

[0030] The electric field intensity of the terahertz frequency used in this embodiment is 0.5V / nm. The direction of the electric field is applied in the same manner as in Example 1. The simulation result of 0.5V / nm in Example 1 was continued for 10ns as the irradiation group, and the simulation result of 100ns without applying an external electric field was continued for 10ns as the control group. The irradiation group n=9, the control group n=5. First, the number of hydrogen bonds between the simulated phospholipid layer structure and the water molecules was statistically analyzed.

[0031] like Figure 2 A is the change in the number of hydrogen bonds between the phospholipid membrane structure and water molecules with simulation time. Figure 2B is the change in the average number of hydrogen bonds during the whole simulation process. The results show that after the simulation reaches a certain degree of stability, the number of hydrogen bonds between the phospholipid membrane structure and water molecules in the irradiated group increases significantly. Secondly, the thickness of the simulation system is tested. The results are as follows Figure 2 C is the thickness of the simulation system at the end of the simulation, indicating that the simulation system as a whole has been compressed. Combined with the change in the number of hydrogen bonds, it can be concluded that the degree of water molecules invading the phospholipid membrane structure has deepened, the membrane structure has changed, and the instability has increased. That is, light waves with a frequency of 0.1 THz will change the stability and order of the phospholipid membrane structure.

[0032] Embodiment 3:

[0033] Liposomes are a commonly used in vitro phospholipid layer model. After terahertz light wave irradiation, cryo-electron microscopy can be used to further verify whether light with a frequency of 0.1 THz can change the structure of the phospholipid layer. The preparation of liposomes and cryo-electron microscopy observation methods are as follows:

[0034] 1. First, re-prepare the purchased liposomes. Dissolve the liposomes in chloroform to make a lipid solution. Transfer the prepared liposome solution to a glass test tube and dry it overnight in a vacuum drying pump to dehydrate the lipid solution into powder. Use PBS buffer to re-dissolve the liposome powder, transfer the dissolved solution to a 1.5mL centrifuge tube, and use an ultrasonic disruptor to fully break it up. Then, freeze and thaw the liposomes in liquid nitrogen and a 95°C metal bath through repeated freeze-thaw cycles, and store the prepared samples at -80°C for later use.

[0035] 2. Then, prepare and observe the samples for cryo-electron microscopy. Thaw the prepared samples in an ice bath, add PBS for dilution, and then divide them into groups and irradiate them with terahertz light waves with a frequency of 0.1 THz for 30 minutes. After irradiation, use a Vitrobot suspension cryo-electron microscopy sample preparation machine to prepare the samples. The sample preparation parameters are 100% humidity, 4°C temperature, 2s adsorption time, 4 levels of adsorption force, and 1 adsorption. The prepared copper mesh is quickly immersed in liquid ethane for freezing and stored in liquid nitrogen. The samples are observed with the aid of a FEI Talos F200C cryo-electron microscope with a magnification of 22000×.

[0036] 3. Finally, the collected sample images were analyzed with the help of ImageJ, and the particle size statistics were performed with the help of GraphpadPrism.

[0037] According to the observation results of liposomes, there are three types of classification, such as Figure 3 A shows schematic diagrams of unilamellar liposomes, multilamellar liposomes and multivesicular liposomes. Figure 3 B is the result of liposome photography. Figure 3 C is the statistical result of the average particle size of liposomes, Figure 3 D is the normal distribution diagram of liposome particle size. The results show that the particle size of liposomes in the irradiated group increased significantly compared with that in the control group.

[0038] The statistical results of the number of liposome types are shown in Table 1 below. No obvious changes were observed, indicating that the method is more gentle. The above results show that after the terahertz light wave with a light frequency of 0.1THz acts, the liposome structure changes and tends to membrane fusion, that is, the light with a light frequency of 0.1THz will change the phospholipid membrane structure.

[0039] Table 1 Statistical results of the number of liposome types

[0040]

Claims

1. A method for changing the structure of phospholipid membrane by light regulation, characterized in that: This involves irradiating biological membrane structures with terahertz waves.

2. A method for changing the structure of phospholipid membrane by light regulation as claimed in claim 1, characterized in that: The optical frequency of the terahertz wave is 0.1 THz.

3. The method for changing the structure of phospholipid membrane by light regulation according to claim 1, characterized in that: The electric field intensity of the terahertz wave frequency is 0.6-0.8 V / nm.

4. The method for changing the structure of phospholipid membrane by light regulation according to claim 1, characterized in that: The electric field direction of the terahertz wave is perpendicular to the structural plane of the phospholipid membrane.

5. The method for changing the structure of phospholipid membrane by light regulation according to claim 1, characterized in that: The irradiation time is 20 to 30 minutes.

6. The method for changing the structure of phospholipid membrane by light regulation according to claim 1, characterized in that: The biological membrane structure is a liposome.

7. Use of the method described in any one of claims 1 to 6 in changing the structure of phospholipid membranes.

8. A liposome with a modified phospholipid membrane structure, characterized in that: It is prepared by the method according to any one of claims 1 to 6.

9. A drug delivery system, characterized in that: A liposome comprising the phospholipid membrane structure changed as claimed in claim 8.

10. Use of the liposome with altered phospholipid membrane structure as claimed in claim 8 in preparing a treatment for diseases related to altered phospholipid membrane structure.

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