A method for preparing cholesterol-containing phospholipid membrane aggregates

Abstract

This invention discloses a method for preparing cholesterol-containing phospholipid membrane-like aggregates. The phospholipid membrane-like aggregates prepared by this method comprise two parts: an aggregate and a phospholipid membrane. The aggregate is formed by electrostatic interaction between negatively charged carboxylated linear starch and positively charged poly(dimethylaminoethyl methacrylate) bromide-cholesterol. An ethanol solution of myristoyl phosphatidylcholine is added to the aggregate solution. Due to the anchoring effect of cholesterol, the phospholipids assemble more efficiently and accurately on the aggregate surface, resulting in a relatively denser phospholipid membrane. The phospholipid membrane-like aggregates prepared by this invention reversibly form vacuoles internally to balance environmental changes under hypotonic conditions. During this process, the internal inclusions undergo a capture-release-capture process. Under hypertonic conditions, due to the presence of the hypertonic agent polyethylene glycol, the phospholipid membrane-like aggregates tend to undergo diffuse deformation and then gradually recover completely, thus responding to changes in osmotic pressure.

Description

Technical Field

[0001] This invention belongs to the field of microcompartment technology and relates to a method for efficiently enriching phospholipid films on the surface of aggregates. Background Technology

[0002] Cells with autonomous life activities possess remarkable vitality in nature, adapting to changes in both the internal and external environments of organisms. These adaptive changes constitute a series of responses and regulations undertaken by cells to maintain homeostasis and adapt to environmental changes. Cellular adaptive changes include morphological transformation, adjustment of physiological functions, and alterations in metabolic activity.

[0003] To investigate the morphological response of cells to stimuli, researchers have turned their attention to artificial cells. As a novel biomimetic system, artificial cells have seen significant development in understanding and studying the behavior and characteristics of biological cells. Artificial cells can be broadly classified into membrane-bound and non-membrane-bound categories. Membrane-bound artificial cells include liposomes, polymer vesicles, colloidal vesicles, and protein vesicles, while non-membrane-bound artificial cells mainly refer to condensates. Condensates are microstructures with droplet-like properties formed by phase separation in a molecularly crowded environment, dynamically allowing the transfer and exchange of biomolecules. However, traditional condensates lack membrane structures, resulting in poor stability and a tendency to fuse and dissociate, thus losing their specific functions.

[0004] Current strategies for condensate surface membrane formation include surface self-assembly mediated by phospholipids, protein-polymer conjugates, cell walls, block copolymers, and heteropolyacids, or spontaneous morphological transformation of condensates under external stimuli leading to membrane formation. Signal transmission, membrane fusion, and membrane deformation can occur on these membrane structures. However, these membranes have poor structural stability and weak binding forces with condensates, making them susceptible to detachment under external stimuli.

[0005] In order to improve the stability of aggregates and enable them to recover to a steady state under external stimuli, it is urgent to develop a novel method for surface film formation of aggregates so that it can simulate the morphological response behavior of real cells under osmotic pressure stimulation. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for preparing cholesterol-containing phospholipid membrane-like aggregates. The phospholipid membrane-like aggregates prepared by this method comprise two parts: an aggregate and a phospholipid membrane. The aggregate is formed by the electrostatic interaction of negatively charged carboxylated linear starch (Su-Am) and positively charged poly(dimethylaminoethyl methacrylate) bromide-cholesterol (PMEDAB-Chol). Then, an ethanol solution of myristoyl phosphatidylcholine (DMPC) is added to the aggregate solution. Due to the anchoring effect of cholesterol, the phospholipids assemble more efficiently and accurately on the aggregate surface, resulting in a relatively denser phospholipid membrane. Under hypotonic conditions, the phospholipid membrane-like aggregates prepared by this invention reversibly form vacuoles internally to balance environmental changes. During this process, the internal loading undergoes a capture-release-capture process. Under hypertonic conditions, due to the presence of the hypertonic agent polyethylene glycol (PEG), the phospholipid membrane-like aggregates tend to undergo diffuse deformation and then gradually recover completely, thus responding to changes in osmotic pressure.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A method for preparing cholesterol-containing phospholipid membrane condensates includes the following steps:

[0009] Step S1: Cholesterol formyl chloride, ethyl hydroxyacrylate and triethylamine are mixed in a molar ratio of 2.5:6:2.7 and reacted under the catalysis of triethylamine. Cholesterol monomers with terminal double bonds are obtained by removing reactants and byproducts for subsequent copolymerization reaction. The reaction time is 4-8 hours.

[0010] Step S2: Dimethylaminoethyl methacrylate and bromoethane are mixed in a molar ratio of 3:2 and reacted with acetone as solvent to obtain positively charged dimethylaminoethyl methacrylate bromoethane (MEDAB) by precipitation. The reaction temperature is 60-80℃ and the reaction time is 4-6h.

[0011] Step S3: Cholesterol monomer and MEDAB are mixed at a molar ratio of 3:23 and polymerized by reversible addition-fragmentation chain transfer polymerization (RAFT). A positively charged block copolymer containing cholesterol, PMEDAB-Chol, is obtained by precipitation. The polymerization temperature is 60-80℃ and the time is 8-10h.

[0012] Step S4: Reaction of amylose and succinic anhydride at a molar ratio of 1:233 yields amylose (Su-Am) with terminal carboxyl groups, which has a strongly negatively charged structure. The reaction temperature is 60–80 °C and the reaction time is 10–18 h.

[0013] Step S5: Dissolve the positively charged PMEDAB-Chol and the negatively charged Su-Am in buffer solution to form a positively charged PMEDAB-Chol solution and a negatively charged Su-Am solution, respectively. The concentration of both the positively charged PMEDAB-Chol solution and the negatively charged Su-Am solution is 1 mg / mL. The buffer solution is phosphate-buffered saline (PBS) buffer with a pH of 7.4 and a concentration of 10 mM.

[0014] Step S6: After mixing PMEDAB-Chol solution and Su-Am solution at a volume ratio of 1 to 2:1, liquid-liquid phase separation occurs immediately, forming a positively charged aggregate containing cholesterol.

[0015] Step S7: Dissolve DMPC in chloroform in an EP tube, then dry it with argon gas, and dissolve it again with anhydrous ethanol to form a phospholipid stock solution with a final concentration of 15-20 mg / mL.

[0016] Step S8: Add the phospholipid reserve solution obtained in step S7 to the cholesterol-containing aggregate. Due to the anchoring effect of cholesterol, DMPC can quickly assemble on the surface of the phospholipid membrane aggregate to form a membrane structure, thereby obtaining a cholesterol-containing phospholipid membrane aggregate. The volume ratio of the cholesterol-containing aggregate to the phospholipid reserve solution is 12:1.

[0017] A method for regulating the morphology of the above-mentioned cholesterol-containing phospholipid membrane condensates under osmotic pressure changes includes the following steps:

[0018] Step S1: Place the cholesterol-containing phospholipid membrane condensate on a glass slide, add H2O, and observe the morphological transformation of the condensate under a microscope, wherein the volume ratio of the cholesterol-containing phospholipid membrane condensate to deionized water is 2-5:1.

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

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

[0021] 1. This invention provides a method for efficiently forming a phospholipid membrane on the surface of agglomerates. The anchoring of cholesterol to phospholipids within the agglomerates makes the phospholipid semipermeable membrane on the surface of the agglomerates more continuous and effectively blocks the entry of macromolecules. The significant improvement in structural stability forms the basis for the subsequent reversible transformation of the membrane-formed agglomerates, solving the problem that traditional membrane-formed agglomerates are still not very stable and will detach under external stimuli.

[0022] 2. Due to the extremely high stability of membrane-bound condensates, they can be used to simulate the morphological regulation behavior of cells under changes in osmotic pressure. Furthermore, membrane-bound condensates have recoverable characteristics during morphological transitions, exhibiting response behaviors similar to real cells. This solves the problem that traditional artificial cells rupture directly when faced with changes in osmotic pressure, making it difficult to further explore the mechanisms of cell behavior.

[0023] 3. Compared to traditional methods, this invention first introduces cholesterol into the design of the building blocks of the condensate. The addition of phospholipids significantly improves assembly efficiency and accuracy through cholesterol's anchoring effect. This assembly method is relatively simple and does not introduce the formation of new chemical bonds. Compared to assemblies without cholesterol, stability is improved from 6 hours to 6 days. This invention achieves maximally dense assembly, effectively improving the problem of easy fusion and dissociation in traditional condensates, making it suitable for application in more scenarios or simulating more complex cell behaviors.

[0024] 4. Phospholipid membrane condensates, due to their strong stability, can maintain their structural integrity and reach equilibrium under different osmotic pressures, exhibiting morphological transformation behavior similar to real cells. This is beneficial for revealing the response mechanisms of cells to environmental changes, something that has been difficult to achieve in previous studies. Furthermore, monitoring the location of the encapsulated material revealed the directional nature of transport behavior. Attached Figure Description

[0025] Figure 1 This is a schematic diagram illustrating the preparation of phospholipid membrane aggregates and their response behavior under osmotic pressure according to the present invention;

[0026] Figure 2 For characterization of the phospholipid membrane condensate of the present invention, (a) is a schematic diagram of the structure, (b) is a laser confocal microscope image of the phospholipid membrane condensate, scale bar: 5 μm, (c) is a 3D image of the phospholipid membrane condensate and a fluorescence intensity diagram of the phospholipid, scale bar: 3 μm, (d) is a laser confocal microscope image of the cholesterol-free phospholipid membrane condensate and a fluorescence intensity diagram of each component, scale bar: 3 μm, (e) is a microscope image of the cholesterol-free phospholipid membrane condensate after 6 h of storage, scale bar: 25 μm, and (f) is a statistical graph of the turbidity of the phospholipid membrane condensate at different days.

[0027] Figure 3 Laser confocal microscopy image of the phospholipid membrane aggregates of the present invention after the addition of cyclodextrin (β-CD), scale bar: 10 μm;

[0028] Figure 4 Fluorescence statistics of the sequestration of different biomacromolecules by phospholipid-free membrane aggregates, cholesterol-free phospholipid membrane aggregates, and cholesterol-containing phospholipid membrane aggregates.

[0029] Figure 5 For the 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) is an in-situ microscopic image of the phospholipid membrane aggregates under a hypotonic environment, scale bar: 5 μm, (c) is a laser confocal microscopic image of the phospholipid membrane changes during temporal transitions of the phospholipid membrane aggregates under a hypotonic environment, scale bar: 5 μm, and (d) is a laser confocal microscopic image of the localization of hydrophilic and hydrophobic regions inside the phospholipid membrane aggregates under hypotonic conditions, scale bar: 5 μm;

[0030] Figure 6 To characterize the spatiotemporal transport of the phospholipid membrane aggregates of the present invention under hypotonic conditions, (a) is a schematic diagram of the spatiotemporal transport of the loads under hypotonic conditions, (b) is a laser confocal microscope image of the phospholipid membrane aggregates during the spatiotemporal transport of the loads under hypotonic conditions, scale bar: 2μm, and (c) is a fluorescence intensity diagram of the phospholipid membrane aggregates during the spatiotemporal transport of the loads under hypotonic conditions at different times.

[0031] Figure 7 For the characterization of the phospholipid membrane aggregates of the present invention under hyperosmotic conditions, (a) is a schematic diagram of morphological changes under hyperosmotic conditions, (b) is an in-situ microscopic image of the morphological transformation of the phospholipid membrane aggregates under hyperosmotic conditions, scale bar: 5 μm, (c) is a time-correlated binary image of the morphological transformation of the phospholipid membrane aggregates under hyperosmotic conditions, scale bar: 3 μm, (d) is a trajectory tracking diagram of the diffuse deformation of the phospholipid membrane aggregates under hyperosmotic conditions, scale bar: 3 μm, and (e) is a trajectory tracking diagram of the recovery of the phospholipid membrane aggregates under hyperosmotic conditions, scale bar: 3 μm;

[0032] Figure 8 To characterize the spatiotemporal transport of the phospholipid membrane aggregates of the present invention under a hyperosmotic environment, (a) is a schematic diagram of the spatiotemporal transport of the loads under a hyperosmotic environment, and (b) is an in-situ observation of the spatiotemporal transport of the phospholipid membrane aggregates under a hyperosmotic environment using a laser confocal microscope. Scale bar: 25 μm.

[0033] Figure 9 The following is a characterization of the phospholipid membrane aggregates of the present invention under a hyperosmotic environment: (a) is a schematic diagram of the changes in the phospholipid membrane under a hyperosmotic environment, and (b) is an in-situ observation of the changes in the phospholipid membrane aggregates under a hyperosmotic environment using a laser confocal microscope. Scale bar: 25 μm. Detailed Implementation

[0034] The technical solution of the present invention will be further described below with reference to the embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention.

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

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

[0037] (1) Preparation of double-bonded cholesterol monomers:

[0038] 1.125 g of cholesterol formyl chloride (2.5 mmol) was added to a 100 mL round-bottom flask and dissolved in 10 mL of dichloromethane. Then, 0.2777 g of triethylamine (2.74 mmol) was added and the flask was placed in an ice bath. Next, 0.4375 g of ethyl hydroxyacrylate (6.07 mmol) was dissolved in 5 mL of dichloromethane and slowly added dropwise to the reaction mixture. The reaction was allowed to proceed at room temperature for 6 h. After the reaction was complete, the reaction solution was extracted stepwise with 20 mL of 0.5 M hydrochloric acid, 20 mL of NaHCO3 solution (pH 8.5), 20 mL of H2O, and 20 mL of saturated NaCl solution. After drying with anhydrous Na2SO4, the solution was filtered, rotary evaporated, and dried under vacuum to obtain the product.

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

[0040] Dimethylaminoethyl methacrylate (DMAEMA, 10 g, 0.06 mol) and bromoethane (4.6 g, 0.04 mol) were dissolved in 30 mL of acetone at a molar ratio of 3:2, and the reaction was carried out in a round-bottom flask. The reaction was conducted at 50 °C under N2 protection for 5 h. After the reaction was stopped, 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. During precipitation, the crude product was dissolved in 200 mL of diethyl ether. Finally, the product was dried under vacuum at room temperature to obtain a white powder as the final product.

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

[0042] A 10 mL round-bottom flask was filled with a trisulfide-RAFT initiator activated with thiothiazoline (6.8 mg, 14.7 μmol), AIBN (1 mg, 6.0 μmol), double-bonded cholesterol monomer (80 mg, 0.15 mmol), MEDAB (220 mg, 1.15 mmol), and DMF (4 mL). The polymerization reaction was carried out at 65 °C for 9 h. The product was then purified twice by precipitation in diethyl ether / hexane (1:2 volume ratio) and finally precipitated once in chloroform 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. Once 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 complete, the mixture was diluted with 30 mL of H₂O and subjected to extensive dialysis using a dialysis tube with a molecular weight cutoff of 3.5 kDa to remove impurities.

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

[0046] A 20 mg / mL Su-Am stock solution was diluted to 1 mg / mL, and a 5 mg / mL PMEDAB-Chol stock solution was diluted to 1 mg / mL. Su-Am and PMEDAB-Chol were mixed at a volume ratio of 1:2 to prepare aggregate droplets, with a final solution volume of 60 μL. After 20 min, 5 μL of an ethanol solution of phospholipids (20 mg / mL) was added. Since cholesterol on the aggregates can anchor phospholipids, a more compact hybrid structure of phospholipid membranes—phospholipid membrane aggregates (65 μL)—was finally obtained.

[0047] (6) Research results:

[0048] Figure 2 A represents the binding mechanism of cholesterol and phospholipids in phospholipid membrane condensates. The hydrophobic structure of cholesterol can spontaneously insert into the hydrophobic region of the phospholipid membrane, effectively reducing the lateral flow of phospholipids and causing them to be positioned and assembled on the condensate surface. Fluorescent labeling of the phospholipid membrane and condensates was used to confirm the location of each component, and observation under a laser confocal microscope revealed that phospholipids were continuously assembled on the condensate surface and uniformly dispersed overall. Figure 2 b), and exhibits a clearly defined Earth-shaped structure ( Figure 2 c).

[0049] In the control experiment, with other experimental conditions kept constant, the assembly of phospholipids was further observed after cholesterol was not added during the construction of the aggregates. The results showed that phospholipids could not continuously and densely coat the aggregate surface, thus failing to effectively improve its stability. Figure 2 d), completely dissociated after 6 hours and could not be observed under a microscope. Figure 2 e). Phospholipid membrane aggregates containing cholesterol only show a significant decrease in turbidity after being stably stored for 6 days, indicating the beginning of dissociation. Figure 2 f).

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

[0051] The key to assembly, consistently mentioned in this invention, is the anchoring effect of cholesterol on phospholipids. Therefore, theoretically, if the anchoring of cholesterol to phospholipids is broken, the phospholipids will detach from the surface of the aggregates, thus causing the aggregates to lose their phospholipid protection and stability. To verify from the opposite perspective that phospholipids improve aggregate stability, literature review revealed that cyclodextrins have an affinity for nonpolar molecules (such as cholesterol). Furthermore, experimental verification showed that the interaction between cyclodextrin and cholesterol is greater than the anchoring effect of phospholipids on cholesterol. Therefore, β-cyclodextrin (β-CD) was added to phospholipid membrane aggregates, and structural changes were observed under a laser confocal microscope. Figure 3 As can be clearly seen, without the addition of β-CD, the fluorescence of phospholipids on the surface of the aggregates is very clear. However, with the addition of β-CD and its gradual diffusion, the phospholipids on the membrane are unloaded, and the fluorescence gradually disperses into the solution. The detachment of phospholipids reduces the stability of the aggregates, causing them to gradually collapse onto the glass substrate. This phenomenon confirms that the anchoring effect of cholesterol on phospholipids is necessary for the formation of phospholipid membrane aggregates, and also illustrates the important role of phospholipid coating in improving aggregate stability.

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

[0053] Phospholipid coating effectively prevents aggregates from capturing large molecules. For example... Figure 4 As shown, membrane-free condensates can effectively capture various biomolecules; when fluorescently labeled biomolecules are added to the condensates, strong fluorescence intensity can be detected inside. Cholesterol-free phospholipid membrane-bound condensates show a slightly reduced but still high level of macromolecule capture. Only in the presence of cholesterol can phospholipids form a dense membrane structure on the condensate surface, thus blocking macromolecule capture; fluorescence is almost undetectable inside the condensate.

[0054] Example 4: Morphology regulation of phospholipid membrane aggregates in a hypotonic environment:

[0055] Take 10 μL of phospholipid membrane condensate under a glass slide, add 3 μL of H2O, and observe the morphological changes under a microscope. Figure 5 (ab) Upon addition of H2O, vacuoles immediately appeared inside the condensate. Over time, the size of these vacuoles gradually decreased and eventually disappeared, returning to the initial state. The instantaneous addition of H2O resulted in a non-equilibrium state with a high total potential energy, thus requiring the migration of solvent molecules to reach an equilibrium state with the minimum total potential energy. During the passive permeation of H2O through the phospholipid membrane into the condensate, no significant movement of phospholipid positions was observed under the microscope, indicating that the phospholipid membrane maintained good continuity. Furthermore, no phospholipid assembly was observed on the vacuoles, suggesting that the phospholipid assembly on the condensate surface maintained good stability due to cholesterol anchoring, preventing disassembly under hypotonic conditions. Figure 5 c). Simultaneous addition of the hydrophobic dye Rhodamine B and the hydrophilic dye Calcein to pinpoint the hydrophobic and hydrophilic locations revealed that the phospholipid membrane aggregate phase maintained strong hydrophobicity, while the cavitation bubbles were hydrophilic. This confirms that the cavitation bubbles originated from the inflow of external H2O under a hypotonic environment. Figure 5 d).

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

[0057] Previous analyses have demonstrated that under hypotonic conditions, external H2O flows into the phospholipid membrane, creating cavitation. Given its inability to capture biomolecules, fluorescently labeled dextran (FITC-Dextran, 50 kPa) is added to the solution of the phospholipid membrane aggregates. Subsequently, by creating a hypotonic state, external H2O carrying FITC-Dextran (50 kPa) flows into the aggregates, thus capturing substances that would otherwise be impossible to trap. Figure 6 As shown in the microscopic observation, FITC-Dextran was initially uniformly distributed on the outer side of the phospholipid membrane condensate without being captured. Upon addition of H2O, the resulting hypotonic environment caused H2O to flow into the phospholipid membrane condensate, forming cavitation bubbles. During this process, a distinct green fluorescence was observed within the cavitation bubbles, indicating successful capture of FITC-Dextran. Subsequently, it was released again as the cavitation bubbles disappeared. This demonstrates that the loaded material underwent a release-capture-release process within the phospholipid membrane condensate, exhibiting spatiotemporal transport behavior.

[0058] Example 6: Morphology regulation of phospholipid membrane aggregates in a hyperosmotic environment:

[0059] Polyethylene glycol (PEG) was selected as a hyperosmotic inducer to create a hyperosmotic environment and investigate its effect on phospholipid membrane aggregates. The volume ratio of phospholipid membrane aggregates to PEG was 1:2. An osmotic pressure difference (36.6 kPa) was formed across the phospholipid membrane immediately upon the addition of PEG, causing the phospholipid membrane aggregates to undergo diffuse deformation, which then gradually and completely recovered. Figure 7 (ab). The reason why phospholipid membrane aggregates can maintain extremely high stability and recover even after such large deformation is that cholesterol has a strong anchoring effect on phospholipids. Even when phospholipid membrane aggregates are subjected to a hyperosmotic environment that tends to dissociate, cholesterol can still firmly hold onto the phospholipids, preventing their internal components from dispersing.

[0060] In the Figure 7 After binarization of the image in b, the microstructural changes of the phospholipid film condensate during diffusion deformation and recovery can be observed more directly. Figure 7 c). Automatic trajectory tracking revealed that the diffusion deformation of phospholipid membrane condensates started from the edge and expanded outwards. Figure 7 d), while during the recovery process, it contracts inward to the edge ( Figure 7 e).

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

[0062] During the deformation of phospholipid membrane-bound aggregates induced by PEG, the internal loadings are affected. Fluorescein was added to the phospholipid membrane-bound aggregates, and the positional changes of the loadings were studied by inducing deformation. Before PEG addition, the loaded fluorescein produced stable and uniform green fluorescence within the phospholipid membrane-bound aggregates. After PEG addition, during the gradual deformation of the phospholipid membrane-bound aggregates, leakage of the loadings was observed. When complete deformation occurred, no fluorescence was observed inside the aggregates, indicating that the internal fluorescein was almost completely leaked. This is because the aggregates struggle to maintain their initial trapping performance under large deformation, thus releasing the loadings. During the deformation recovery process, the phospholipid membrane-bound aggregates gradually recover and can recapture the fluorescein, resulting in the observation of obvious green fluorescence inside. Figure 8 The entire process involved the payload undergoing a capture-release-capture cycle.

[0063] Example 8: Analysis of changes in phospholipid membranes in hypotonic environments:

[0064] Because phospholipid membrane condensates undergo large-scale changes during diffuse deformation, the phospholipids on the membrane are inevitably significantly affected. Figure 9 As shown, the phospholipids on the membrane are stable and continuous without the addition of PEG. When the addition of PEG creates a hyperosmolar environment that causes diffuse deformation of the phospholipid membrane aggregates, a large amount of surface phospholipids can be clearly seen disassembling and gradually assembling 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. As the diffuse phase transition gradually recovers, some free phospholipids that have not formed micelles will reassemble on the surface of the aggregates. However, the phospholipids that have already formed micelles no longer have the ability to move freely and therefore do not have the possibility of reassembly.

Claims

1. A method for preparing cholesterol-containing phospholipid membrane aggregates, characterized in that... The method includes the following steps: Step S1: Cholesterol formyl chloride, ethyl hydroxyacrylate and triethylamine are mixed and reacted under the catalysis of triethylamine. Cholesterol monomers with terminal double bonds are obtained by removing reactants and byproducts for subsequent copolymerization reaction. The molar ratio of cholesterol formyl chloride, ethyl hydroxyacrylate and triethylamine is 2.5:6:2.7, and the reaction time is 4~8 h. Step S2: Dimethylaminoethyl methacrylate and bromoethane are mixed and reacted with acetone as solvent. Positively charged dimethylaminoethyl methacrylate bromoethane MEDAB is obtained by precipitation. The molar ratio of dimethylaminoethyl methacrylate to bromoethane is 3:2, the reaction temperature is 60~80℃, and the reaction time is 4~6 h. Step S3: Cholesterol monomer and MEDAB are mixed and polymerized by reversible addition-fragmentation chain transfer polymerization. A positively charged block copolymer containing cholesterol, PMEDAB-Chol, is obtained by precipitation. The molar ratio of cholesterol monomer to MEDAB is 3:

23. The polymerization temperature is 60~80℃ and the time is 8~10 h. Step S4: Reaction of amylose and succinic anhydride yields amylose Su-Am with carboxyl groups at the ends, which has a strongly negatively charged structure. The molar ratio of amylose to succinic anhydride is 8.3 μmol: 1.85 mmol. The reaction temperature is 60-80℃ and the reaction time is 10-18 h. Step S5: Dissolve PMEDAB-Chol and Su-Am in buffer solution to form a positively charged PMEDAB-Chol solution and a negatively charged Su-Am solution, respectively; Step S6: After mixing PMEDAB-Chol solution and Su-Am solution, liquid-liquid phase separation occurs immediately, forming a positively charged aggregate containing cholesterol. Step S7: Dissolve DMPC (dimyristic phosphatidylcholine) in EP tube with chloroform, then dry with argon gas, and dissolve again with anhydrous ethanol to form a phospholipid stock solution. Step S8: Add a phospholipid reserve solution to the cholesterol-containing aggregate. Due to the cholesterol anchoring effect, DMPC can quickly assemble on the surface of the phospholipid membrane aggregate to form a membrane structure, thus obtaining a cholesterol-containing phospholipid membrane aggregate.

2. 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 phosphate buffer with a pH of 7.4 and a concentration of 10 mM.

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

1.

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

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

1.

6. A method for morphological regulation of cholesterol-containing phospholipid membrane aggregates prepared by the method according to any one of claims 1-5 under osmotic pressure changes, characterized in that... The method includes the following steps: Step S1: Place the cholesterol-containing phospholipid membrane condensate on a glass slide, add H2O, and observe the morphological transformation of the condensate under a microscope, wherein the volume ratio of the cholesterol-containing phospholipid membrane condensate to H2O is 2~5:

1. Step S2: Place the cholesterol-containing phospholipid membrane aggregates on a glass slide, add PEG, and observe the morphological transformation of the aggregates under a microscope. The volume ratio of the cholesterol-containing phospholipid membrane aggregates to PEG is 1:1~4, and the molecular weight of PEG is 800~1200 Da.

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