Preparation method of phospholipid membranization aggregate containing cholesterol

Abstract

The invention discloses a preparation method of a phospholipid membranization aggregate containing cholesterol, and the phospholipid membranization aggregate prepared by the method comprises an aggregate and a phospholipid membrane, the coacervate is formed by carboxylated amylose with negative electricity and poly (dimethylaminoethyl methacrylate) bromoethane-cholesterol with positive electricity through electrostatic interaction, an ethanol solution of dimyristoyl phosphatidylcholine is added into a coacervate solution, and due to the anchoring effect of cholesterol, the coacervate with negative electricity can be formed. The phospholipid can be more efficiently and accurately assembled on the surface of the aggregate, and the formed phospholipid membrane is relatively denser. The phospholipid membranization condensation body prepared by the invention can reversibly form cavitation bubbles in the interior in a hypotonic state to balance environmental changes, and in the process, an entrapment substance in the interior is subjected to a capture-release-capture process; and in a hypertonic state, due to the existence of a hypertonic agent polyethylene glycol, the phospholipid membranization condensation body tends to generate dispersive deformation and then gradually and completely recovers, so that the change of osmotic pressure is responded.

Description

Technical Field

[0001] The invention belongs to the technical field of micro-compartments and relates to a method for efficiently enriching phospholipid membranes on the surface of condensates. Background Art

[0002] Cells with autonomous life activities have strong vitality in nature. When the internal and external environment of the organism changes, they will produce adaptive changes to respond to these stimuli. This adaptive change is a series of reactions and adjustments made by cells in order to maintain homeostasis and adapt to environmental changes. Cell adaptive changes include morphological transformation, adjustment of physiological functions, and changes in metabolic activities.

[0003] In order to explore the morphological response behavior of cells under stimulation, researchers turned their attention to artificial cells. As a new bionic system, artificial cells can be used to understand and study the behavior and characteristics of biological cells, and therefore have made great progress. Artificial cells can be divided into two categories: membrane-containing and membrane-free. Membrane-containing artificial cells include liposomes, polymer vesicles, colloidal vesicles, protein vesicles, etc., while membrane-free artificial cells mainly refer to condensates. Condensates are microstructures with droplet properties formed by phase separation in a molecularly crowded environment, which can dynamically allow the transfer and exchange of biological molecules. However, traditional condensates have poor stability due to the lack of membrane structure, and are prone to fusion and dissociation, losing their specific functions.

[0004] At present, the strategies for the membraneization of aggregates include surface self-assembly mediated by phospholipids, protein-polymer conjugates, cell walls, block copolymers and heteropolyacids, or the spontaneous morphological transformation of aggregates under external stimuli to lead to membraneization. Signal transmission, membrane fusion, membrane deformation and other processes can occur on these membrane structures. However, the structural stability of these membranes is poor, and the binding force with the aggregates is weak, which makes them at risk of falling off under external stimuli.

[0005] In order to improve the stability of the aggregates and enable them to return to a steady state under external stimulation, it is urgently necessary to develop a new method for surface film formation of aggregates so that they can simulate the morphological response behavior of real cells under osmotic pressure stimulation. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for preparing a phospholipid membrane coacervate containing cholesterol. The phospholipid membrane coacervate prepared by the method includes two parts: coacervate and phospholipid membrane, wherein the coacervate is formed by electrostatic interaction between negatively charged carboxylated amylose (Su-Am) and positively charged poly(dimethylaminoethyl methacrylate) bromide-cholesterol (PMEDAB-Chol), and then an ethanol solution of dimyristoylphosphatidylcholine (DMPC) is added to the coacervate solution. Due to the anchoring effect of cholesterol, phospholipids will be more efficiently and accurately assembled on the coacervate surface, and the formed phospholipid membrane will also be relatively denser. The phospholipid membrane coacervate prepared by the present invention will reversibly form cavitation inside to balance environmental changes under hypotonic conditions, and the internal encapsulated material will undergo a capture-release-capture process during this process; and under hypertonic conditions, due to the presence of the hypertonic agent polyethylene glycol (PEG), the phospholipid membrane coacervate will tend to undergo diffuse deformation and then gradually recover completely, thereby responding to changes in osmotic pressure.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] A method for preparing a phospholipid membrane aggregate containing cholesterol comprises the following steps:

[0009] Step S1: Cholesterol acetyl chloride, hydroxyethyl acrylate and triethylamine are mixed in a molar ratio of 2.5:6:2.7, reacted under the catalytic action of triethylamine, and cholesterol monomers with double bonds at the ends are obtained by removing reactants and by-products for subsequent copolymerization, wherein: the reaction time is 4 to 8 hours;

[0010] Step S2: dimethylaminoethyl methacrylate and ethyl bromide are mixed in a molar ratio of 3:2, and reacted with acetone as a solvent to obtain positively charged dimethylaminoethyl methacrylate ethyl bromide (MEDAB) by a precipitation method, wherein: the reaction temperature is 60-80° C., and the reaction time is 4-6 hours;

[0011] Step S3: Cholesterol monomer and MEDAB are mixed in a molar ratio of 3:23, and a polymerization reaction is carried out by a reversible addition-fragmentation chain transfer polymerization (RAFT) method to obtain a positively charged block copolymer containing cholesterol, PMEDAB-Chol, by a precipitation method, wherein: the polymerization reaction temperature is 60-80° C., and the time is 8-10 hours;

[0012] Step S4: reacting amylose and succinic anhydride at a molar ratio of 1:233 to obtain amylose (Su-Am) containing a carboxyl group at the end, the structure of which has a strong negative charge, wherein: the reaction temperature is 60-80° C., and the reaction time is 10-18 hours;

[0013] Step S5: dissolving the positively charged PMEDAB-Chol and the negatively charged Su-Am in a buffer solution to form a positively charged PMEDAB-Chol solution and a negatively charged Su-Am solution, wherein: the concentrations of the positively charged PMEDAB-Chol solution and the negatively charged Su-Am solution are both 1 mg / mL; the buffer solution is a phosphate buffer solution (PBS) with a pH of 7.4 and a concentration of 10 mM;

[0014] Step S6: mixing the PMEDAB-Chol solution and the Su-Am solution in a volume ratio of 1 to 2:1, and immediately causing liquid-liquid phase separation to form aggregates that are positively charged as a whole and contain cholesterol;

[0015] Step S7: dissolving dimyristoylphosphatidylcholine (DMPC) in EP tube with chloroform, then drying with argon gas, and dissolving again with anhydrous ethanol to form a phospholipid stock solution with a final concentration of 15-20 mg / mL;

[0016] Step S8: adding the phospholipid stock solution obtained in step S7 to the cholesterol-containing aggregates; DMPC can be quickly assembled on the surface of the phospholipid membrane aggregates to form a membrane structure due to the anchoring effect of cholesterol, thereby obtaining phospholipid membrane aggregates containing cholesterol, wherein the volume ratio of the cholesterol-containing aggregates to the phospholipid stock solution is 12:1.

[0017] A method for controlling the morphology of the above-mentioned cholesterol-containing phospholipid membrane aggregates under osmotic pressure changes comprises the following steps:

[0018] Step S1: placing the phospholipid membrane coacervate containing cholesterol on a glass slide, adding H2O, and observing the morphological transformation of the coacervate under a microscope, wherein the volume ratio of the phospholipid membrane coacervate containing cholesterol to deionized water is 2 to 5:1;

[0019] Step S2: placing the phospholipid membrane aggregates containing cholesterol on a glass slide, adding PEG, and observing the morphological transformation of the aggregates under a microscope, wherein: the volume ratio of the phospholipid membrane aggregates containing cholesterol and PEG is 1:1-4, and the molecular weight of PEG is 800-1200Da.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] 1. The present invention provides a method for efficiently forming a phospholipid membrane on the surface of a coacervate. The anchoring of cholesterol in the coacervate to phospholipids makes the phospholipid semipermeable membrane on the surface of the coacervate more continuous and effectively blocks the entry of large molecules. The greatly improved structural stability becomes the basis for the subsequent reversible transformation of the morphology of the membrane coacervate, solving the problem that the traditional membrane coacervate is still not stable and will detach under external stimulation.

[0022] 2. Since membrane aggregates are extremely stable, they can be used to simulate the morphological regulation behavior of cells under changes in osmotic pressure. In addition, membrane aggregates have recoverable properties during morphological transformation and exhibit response behaviors similar to real cells, which solves the problem that traditional artificial cells directly rupture when faced with changes in osmotic pressure, making it difficult to further explore the cell behavior mechanism.

[0023] 3. Compared with the traditional method, the present invention first introduces cholesterol in the design of the building blocks of the condensate. When phospholipids are added, the anchoring effect of cholesterol on phospholipids greatly improves the assembly efficiency and accuracy. This assembly method is relatively simple and does not introduce the formation of new chemical bonds. Compared with the assembly without cholesterol, the stability is improved from 6h to 6days. The present invention achieves the most dense assembly, effectively improving the problem of easy fusion and dissociation of traditional condensates, which is conducive to its application in more scenarios or simulation of more complex cell behaviors.

[0024] 4. Due to its strong stability, phospholipid membrane aggregates can respond and balance under different osmotic pressure environments while ensuring that their own structure can be restored, showing morphological transformation behaviors similar to real cells, which is conducive to revealing the response mechanism of cells under environmental changes, which is difficult to achieve in previous studies. In this process, the position of the encapsulated object is also monitored, and the directional occurrence of the transport behavior is found. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the preparation of the phospholipid membrane aggregates of the present invention and their response behavior under osmotic pressure;

[0026] Figure 2 Characterization of the phospholipid membrane coacervate of the present invention, (a) is a schematic diagram of the structure, (b) is a laser confocal microscopy image of the phospholipid membrane coacervate, scale bar: 5μm, (c) is a 3D image of the phospholipid membrane coacervate and a fluorescence intensity map of phospholipids, scale bar: 3μm, (d) is a laser confocal microscopy image of the cholesterol-free phospholipid membrane coacervate and a fluorescence intensity map of each component, scale bar: 3μm, (e) is a microscopic image of the cholesterol-free phospholipid membrane coacervate after being placed for 6 hours, scale bar: 25μm, (f) a statistical graph of the turbidity of the phospholipid membrane coacervate at different days;

[0027] Figure 3 This is a laser confocal microscope image of the phospholipid membrane aggregate of the present invention after adding cyclodextrin (β-CD), scale bar: 10 μm;

[0028] Figure 4 It is a fluorescence statistical diagram of the seizure of different biomacromolecules by phospholipid membrane aggregates without phospholipids, phospholipid membrane aggregates without cholesterol, and phospholipid membrane aggregates with cholesterol;

[0029] Figure 5 Characterization of the phospholipid membrane aggregates of the present invention under a hypotonic environment, (a) is a schematic diagram of morphological changes under a hypotonic environment, (b) an in situ observation microscope image of the phospholipid membrane aggregates under a hypotonic environment, scale bar: 5 μm, (c) a laser confocal microscope image of the changes in the phospholipid membrane during the temporal transition of the phospholipid membrane aggregates under a hypotonic environment, scale bar: 5 μm, (d) a laser confocal microscope image of the positioning of the hydrophilic and hydrophobic regions inside the phospholipid membrane aggregates under hypotonic conditions, scale bar: 5 μm;

[0030] Figure 6 The present invention characterizes the spatiotemporal transport of the phospholipid membrane aggregates in a hypotonic environment. (a) is a schematic diagram of the spatiotemporal transport of the entrapped object in a hypotonic environment. (b) is a laser confocal microscope image of in situ observation of the spatiotemporal transport of the phospholipid membrane aggregates in a hypotonic environment. Scale bar: 2 μm. (c) is a fluorescence intensity image of the spatiotemporal transport of the entrapped object in a hypotonic environment at different times.

[0031] Figure 7 Characterization of the phospholipid membrane aggregates of the present invention under a hypertonic environment, (a) is a schematic diagram of morphological changes under a hypertonic environment, (b) an in situ observation microscope image of the morphological transformation of the phospholipid membrane aggregates under a hypertonic environment, scale: 5 μm, (c) a time-correlated binary image of the morphological transformation of the phospholipid membrane aggregates under a hypertonic environment, scale: 3 μm, (d) a trajectory tracking image of the phospholipid membrane aggregates during diffuse deformation under a hypertonic environment, scale: 3 μm, (e) a trajectory tracking image of the phospholipid membrane aggregates during recovery under a hypertonic environment, scale: 3 μm;

[0032] Figure 8 The characterization of the spatiotemporal transport of the phospholipid membrane aggregates in the hypertonic environment of the present invention, (a) is a schematic diagram of the spatiotemporal transport of the entrapped material in the hypertonic environment, (b) is a laser confocal microscope image of the in situ observation of the spatiotemporal transport of the phospholipid membrane aggregates in the hypertonic environment, scale bar: 25 μm;

[0033] Fig. 9 Characterization of the phospholipid membrane of the phospholipid membrane aggregates under a hypertonic environment of the present invention, (a) is a schematic diagram of the changes of the phospholipid membrane under a hypertonic environment, (b) is a laser confocal microscope image of the in situ observation of the changes of the phospholipid membrane of the phospholipid membrane aggregates under a hypertonic environment, scale bar: 25μm. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described below in conjunction with the embodiments, but is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be included in the protection scope of the present invention.

[0035] Example 1: Preparation and characterization of phospholipid membrane aggregates:

[0036] like Figure 1 As shown, the preparation process of phospholipid membrane coacervate is as follows:

[0037] (1) Preparation of double bond cholesterol monomer:

[0038] 1.125g cholesterol formoyl chloride (2.5mmol) is added to a 100mL three-bottom round-bottom flask and dissolved with 10mL dichloromethane, followed by 0.2777g triethylamine (2.74mmol) and placed in an ice bath. Then, 0.4375g hydroxyethyl acrylate (6.07mmol) is dissolved in 5mL dichloromethane, slowly added dropwise to the reaction mixture, and the reaction is allowed to proceed at room temperature for 6h. After the reaction is completed, the reaction solution is extracted stepwise with 20mL of 0.5M hydrochloric acid, 20mL of NaHCO3 solution (pH 8.5), 20mL of H2O and 20mL of saturated NaCl solution. After adding anhydrous Na2SO4 and drying, filter and rotary evaporation, vacuum drying obtains the product.

[0039] (2) Preparation of dimethylaminoethyl methacrylate ethyl bromide (MEDAB):

[0040] Dimethylaminoethyl methacrylate (DMAEMA, 10 g, 0.06 mol) and ethyl bromide (4.6 g, 0.04 mol) were dissolved in 30 mL of acetone at a molar ratio of 3:2 and reacted in a round-bottom flask. The reaction was carried out at 50 °C under N2 protection for 5 h. After the reaction was stopped, the excess solvent was removed by vacuum distillation at 50 °C. The yellow viscous crude product was then purified three times by precipitation in anhydrous diethyl ether. The crude product was dissolved in 200 mL of diethyl ether during precipitation. Finally, it was dried in vacuum at room temperature to obtain a white powder as the final product.

[0041] (3) Preparation of block copolymer—PMEDAB-Chol:

[0042] Thiothiazolinyl activated trisulfide-RAFT initiator (6.8 mg, 14.7 μmol), AIBN (1 mg, 6.0 μmol), double bond cholesterol monomer (80 mg, 0.15 mmol), MEDAB (220 mg, 1.15 mmol) and DMF (4 mL) were added to a 10 mL round bottom flask. The polymerization reaction was carried out at 65 ° C for 9 h, and then purified by precipitation in diethyl ether / hexane (1:2 volume ratio) twice, and finally precipitated in chloroform once to obtain the product.

[0043] (4) Preparation of carboxylated amylose (Su-Am):

[0044] 100 mg of amylose (8.3 μmol) and 185 mg of succinic anhydride (1.85 mmol) were dissolved in 15 mL of DMSO and heated at 60°C. When the starch was completely dissolved, 5 mg of DMAP (0.04 mmol) was added and the reaction mixture was stirred for 16 h. After the reaction was completed, the mixture was diluted with 30 mL of H2O and dialyzed extensively using a dialysis tubing with a molecular weight cutoff of 3.5 kDa to remove impurities.

[0045] (5) Preparation of phospholipid membrane aggregates:

[0046] Dilute 20 mg / mL Su-Am stock solution to 1 mg / mL, and 5 mg / mL PMEDAB-Chol stock solution to 1 mg / mL. Su-Am and PMEDAB-Chol were mixed at a volume ratio of 1:2 to prepare coacervate droplets, and the final solution volume was 60 μL. After 20 minutes, 5 μL of phospholipid ethanol solution (20 mg / mL) was added. Since the cholesterol on the coacervate can anchor the phospholipid, a more dense phospholipid membrane mixed structure - phospholipid membrane coacervate (65 μL) was finally obtained.

[0047] (6) Research results:

[0048] Figure 2 a is the binding mechanism of cholesterol and phospholipids in phospholipid membrane aggregates. The hydrophobic structure of cholesterol can spontaneously insert into the hydrophobic region of the phospholipid membrane, effectively reducing the lateral flow of phospholipids and positioning them on the surface of the aggregate. The phospholipid membrane and aggregates were fluorescently labeled to confirm the position of each component, and observed under a laser confocal microscope. It was found that the phospholipids were continuously assembled on the surface of the aggregate and were uniformly dispersed as a whole ( Figure 2 b) and presents a globe-shaped structure with clear edges ( Figure 2 c).

[0049] In the control experiment, when other experimental conditions remained unchanged, the assembly of phospholipids was further observed after cholesterol was not added to the construction of the aggregates. The results showed that phospholipids could not be continuously and densely coated on the surface of the aggregates, and therefore could not effectively improve their stability ( Figure 2 d), after 6 hours, it completely dissociated and could not be observed under a microscope ( Figure 2 e). However, the phospholipid membrane aggregates containing cholesterol will not show a large decrease in turbidity until they are placed stably for 6 days, which means they begin to dissociate ( Figure 2 f).

[0050] Example 2: Analysis of the effect of cholesterol on phospholipid anchoring:

[0051] The key to assembly mentioned in the present invention is the anchoring effect of cholesterol on phospholipids. Therefore, in theory, as long as the anchoring of cholesterol on phospholipids is released, the phospholipids will fall off the surface of the coacervate, and then the coacervate will lose the protection of phospholipids and lose stability. In order to verify from the opposite side that phospholipids improve the stability of the coacervate, it was found from the literature that cyclodextrin has an affinity for non-polar molecules (such as cholesterol). And through experimental verification, it was concluded that the interaction between cyclodextrin and cholesterol is greater than the anchoring effect of phospholipids and cholesterol. Therefore, β-cyclodextrin (β-CD) was added to the phospholipid membrane coacervate and its structural changes were observed under a laser confocal microscope. From Figure 3 It can be clearly seen that when β-CD is not added, the fluorescence of the phospholipids on the surface of the coacervate is very clear. However, as β-CD is added and gradually diffused, the phospholipids on the membrane are unloaded and the fluorescence gradually disperses into the solution. The shedding of phospholipids reduces the stability of the coacervate and gradually collapses on the glass substrate. This phenomenon not only confirms that the anchoring effect of cholesterol on phospholipids is necessary for the formation of phospholipid membrane coacervates, but also illustrates the important role of phospholipid coating in improving the stability of coacervates.

[0052] Example 3: Analysis of the blocking effect of phospholipid membrane aggregates on macromolecules:

[0053] The phospholipid coating effectively prevents the capture of macromolecules by aggregates. Figure 4 As shown in the figure, membraneless coacervates can effectively capture various biomacromolecules, which is manifested by the fact that when the corresponding fluorescently labeled biomacromolecules are added to the coacervates, a strong fluorescence intensity can be detected inside them. However, the capture degree of macromolecules by cholesterol-free phospholipid membrane coacervates is slightly reduced but still at a high level. Only when cholesterol is present can phospholipids form a dense membrane structure on the surface of the coacervate, thereby blocking the capture of macromolecules, and almost no fluorescence can be detected inside the coacervate.

[0054] Example 4: Morphological Control of Phospholipid Membrane Aggregates in Hypotonic Environment:

[0055] Take 10 μL of phospholipid membrane aggregates under the slide, add 3 μL of H2O, and observe the morphological changes under a microscope ( Figure 5 ab). After adding H2O, cavitation bubbles immediately appeared inside the aggregates. As time went by, the size of the cavitation bubbles gradually decreased and then disappeared, returning to the initial state. The instantaneous addition of H2O caused the system to be in an unbalanced state with a high total potential energy, so the equilibrium state with the minimum total potential energy had to be reached through the migration of solvent molecules. No obvious movement of the phospholipid position was observed under the microscope during the process of H2O passively penetrating the phospholipid membrane into the aggregates, indicating that the phospholipid membrane still maintained good continuity, and no phospholipid assembly was observed on the cavitation bubbles, indicating that the assembly of phospholipids on the surface of the aggregates maintained good stability due to the anchoring of cholesterol, so that it would not disassemble under hypotonic conditions ( Figure 5 c). At the same time, the hydrophobic dye Rhodamine B and the hydrophilic dye calcein were added to locate the hydrophilic and hydrophobic positions. It was found that the phospholipid membrane condensate phase still maintained strong hydrophobicity, while the vacuoles were hydrophilic, which confirmed that the vacuoles came from the influx of external H2O in a hypotonic environment ( Figure 5 d).

[0056] Example 5: Spatiotemporal transport of phospholipid membrane aggregates in a hypotonic environment:

[0057] In the previous analysis, it has been proved that in a hypotonic environment, external H2O will flow into the phospholipid membrane to produce cavitation. Considering its inability to capture biological macromolecules, fluorescently labeled dextran (FITC-Dextran, 50kPa) was added to the solution of phospholipid membrane aggregates, and then by creating a hypotonic state, external H2O carrying FITC-Dextran (50kPa) flowed into the aggregates to capture substances that could not be captured. Figure 6 As shown, under the microscope, it was observed that in the initial state, FITC-Dextran was evenly distributed outside the phospholipid membrane aggregates and was not captured. When H2O was added, the hypotonic environment formed caused H2O to flow into the phospholipid membrane aggregates to form vacuoles. Along with this process, obvious green fluorescence was observed in the vacuoles, indicating that FITC-Dextran was successfully captured, and then it was released again with the disappearance of the vacuoles. This proves that the encapsulated material experienced a release-capture-release process inside the phospholipid membrane aggregates, showing spatiotemporal transmission behavior.

[0058] Example 6: Morphological Control of Phospholipid Membrane Aggregates in Hypertonic Environment:

[0059] Polyethylene glycol (PEG) was selected as a hypertonic inducer to form a hypertonic environment to explore the effect on phospholipid membrane aggregates. The volume ratio of phospholipid membrane aggregates and PEG was 1:2. At the moment of adding PEG, an osmotic pressure difference (36.6 kPa) was formed inside and outside the phospholipid membrane, so the phospholipid membrane aggregates would undergo diffuse deformation and then gradually recover completely ( Figure 7 ab). The reason why phospholipid membrane aggregates can maintain extremely high stability and recover after such a large deformation is that cholesterol has a strong anchoring effect on phospholipids. When phospholipid membrane aggregates are subjected to a hypertonic environment and tend to dissociate, they can still firmly grasp the phospholipids so that their internal components do not disperse.

[0060] In the right Figure 7 After the image in b is binarized, the microstructural changes of the phospholipid membrane aggregates during the diffusion deformation and recovery process can be observed more directly ( Figure 7 c). Automatically tracking the trajectory revealed that the diffusion deformation of the phospholipid membrane aggregates started from the edge and expanded outwards ( Figure 7 d), and shrinks inwards to the edge during recovery ( Figure 7 e).

[0061] Example 7: Spatiotemporal transport of phospholipid membrane aggregates in a hypotonic environment:

[0062] During the process of PEG-induced deformation of phospholipid membrane aggregates, the internal encapsulated substances will be affected. Fluorescein was added to the phospholipid membrane aggregates, and the positional changes of the encapsulated substances were studied by inducing deformation. Before PEG was added, the encapsulated fluorescein produced stable and uniform green fluorescence in the phospholipid membrane aggregates. After the addition of PEG, during the gradual deformation of the phospholipid membrane aggregates, it was found that the encapsulated substances were leaking out. When the deformation was complete, there was no fluorescence inside the aggregates, which indicated that the internal fluorescein was almost completely leaked. This is because it is difficult for the aggregates to maintain their initial capture performance under large deformation, so the encapsulated substances will be released. In the process of deformation recovery, the phospholipid membrane aggregates gradually recovered and thus could recapture the fluorescein, and obvious green fluorescence could be observed inside ( Figure 8 ). The whole process involves capture-release-capture.

[0063] Example 8: Analysis of changes in phospholipid membranes of phospholipid membrane aggregates under hypotonic conditions:

[0064] Since the scale of phospholipid membrane aggregates changes greatly when undergoing diffuse deformation, the phospholipids on the membrane are bound to be greatly affected. Fig. 9 As shown in the figure, when PEG is not added, the phospholipids on the membrane are stable and continuous. When PEG is added to form a hypertonic environment, causing the phospholipid membrane aggregates to undergo diffuse deformation, it can be clearly seen that a large number of surface phospholipids are disassembled and gradually assembled into large-scale phospholipid micelles in the solution. This is because the deformation causes the phospholipid membrane aggregates to have a large tendency to disperse, so the anchoring effect of cholesterol on phospholipids is insufficient to stabilize them on the surface. With the gradual recovery of the diffuse phase transition, some free phospholipids that have not formed micelles will reassemble on the surface of the aggregates, but the phospholipids that have formed micelles no longer have the ability to move freely, so they do not have the possibility of reassembly.

Claims

1. A method for preparing a phospholipid membrane aggregate containing cholesterol, characterized in that The method comprises the following steps: Step S1: Cholesterol acetyl chloride, hydroxyethyl acrylate and triethylamine are mixed, reacted under the catalytic action of triethylamine, and cholesterol monomers with double bonds at the ends are obtained by removing reactants and by-products for subsequent copolymerization, wherein the molar ratio of cholesterol acetyl chloride, hydroxyethyl acrylate and triethylamine is 2.5:6:2.7, and the reaction time is 4 to 8 hours; Step S2: dimethylaminoethyl methacrylate and ethyl bromide are mixed, and reacted with acetone as solvent to obtain positively charged dimethylaminoethyl methacrylate ethyl bromide MEDAB by precipitation method, wherein: the molar ratio of dimethylaminoethyl methacrylate to ethyl bromide is 3:2, the reaction temperature is 60-80° C., and the reaction time is 4-6 hours; Step S3: mixing cholesterol monomer and MEDAB, performing polymerization reaction by reversible addition-fragmentation chain transfer polymerization, and obtaining a positively charged block copolymer containing cholesterol, PMEDAB-Chol, by precipitation method, wherein: the molar ratio of cholesterol monomer to MEDAB is 3:23, the polymerization reaction temperature is 60-80° C., and the time is 8-10 h; Step S4: reacting amylose with succinic anhydride to obtain amylose Su-Am containing a carboxyl group at the end, whose structure has a strong negative charge, wherein: the molar ratio of amylose to succinic anhydride is 1:233, the reaction temperature is 60-80°C, and the reaction time is 10-18h; Step S5: dissolving PMEDAB-Chol and Su-Am in a buffer solution to form a positively charged PMEDAB-Chol solution and a negatively charged Su-Am solution; Step S6: After the PMEDAB-Chol solution and the Su-Am solution are mixed, liquid-liquid phase separation occurs immediately to form aggregates that are positively charged as a whole and contain cholesterol; Step S7: dissolving dimyristoylphosphatidylcholine in EP tube with chloroform, then drying with argon gas, and dissolving again with anhydrous ethanol to form a phospholipid stock solution; Step S8: adding a phospholipid stock solution to the cholesterol-containing aggregates, DMPC can be quickly assembled on the surface of the phospholipid membrane aggregates to form a membrane structure due to the anchoring effect of cholesterol, thereby obtaining phospholipid membrane aggregates containing cholesterol.

2. The method for preparing the cholesterol-containing phospholipid membrane aggregate according to claim 1, characterized in that In the step S1, the molar ratio of cholesterol methyl chloride, hydroxyethyl acrylate and triethylamine is 2.5:6:2.7, and the reaction time is 4 to 8 hours.

3. The method for preparing the cholesterol-containing phospholipid membrane aggregate according to claim 1, characterized in that In the step S2, the molar ratio of dimethylaminoethyl methacrylate to ethyl bromide is 3:2, the reaction temperature is 60-80° C., and the reaction time is 4-6 hours.

4. The method for preparing the cholesterol-containing phospholipid membrane aggregate according to claim 1, characterized in that In the step S3, the molar ratio of cholesterol monomer to MEDAB is 3:23, the polymerization temperature is 60-80° C., and the polymerization time is 8-10 hours.

5. The method for preparing the cholesterol-containing phospholipid membrane aggregate according to claim 1, characterized in that In step S4, the molar ratio of amylose to succinic anhydride is 1:233, the reaction temperature is 60-80° C., and the reaction time is 10-18 hours.

6. The method for preparing cholesterol-containing phospholipid membrane aggregates according to claim 1, characterized in that In step S5, the concentrations of the positively charged PMEDAB-Chol solution and the negatively charged Su-Am solution are both 1 mg / mL, and the buffer solution is a phosphate buffer solution with a pH of 7.4 and a concentration of 10 mM.

7. The method for preparing cholesterol-containing phospholipid membrane aggregates according to claim 1, characterized in that The method for preparing phospholipid membrane aggregates containing cholesterol according to claim 1 is characterized in that in step S6, the volume ratio of PMEDAB-Chol solution to Su-Am solution is 1 to 2:

1.

8. The method for preparing cholesterol-containing phospholipid membrane aggregates according to claim 1, characterized in that The method for preparing phospholipid membrane aggregates containing cholesterol according to claim 1 is characterized in that in step S7, the concentration of the phospholipid stock solution is 15-20 mg / mL.

9. The method for preparing cholesterol-containing phospholipid membrane aggregates according to claim 1, characterized in that The method for preparing cholesterol-containing phospholipid membrane coacervates according to claim 1 is characterized in that in step S8, the volume ratio of the cholesterol-containing coacervates to the phospholipid stock solution is 12:

1.

10. A method for controlling the morphology of phospholipid membrane aggregates containing cholesterol prepared by the method according to any one of claims 1 to 9 under osmotic pressure changes, characterized in that The method comprises the following steps: Step S1: placing the phospholipid membrane coacervate containing cholesterol on a glass slide, adding H2O, and observing the morphological transformation of the coacervate under a microscope, wherein the volume ratio of the phospholipid membrane coacervate containing cholesterol to deionized water is 2 to 5:1; Step S2: placing the phospholipid membrane aggregates containing cholesterol on a glass slide, adding PEG, and observing the morphological transformation of the aggregates under a microscope, wherein: the volume ratio of the phospholipid membrane aggregates containing cholesterol and PEG is 1:1-4, and the molecular weight of PEG is 800-1200Da.

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