A method for preparing macrophage membrane-coated artificial cells

Abstract

The application discloses a preparation method of macrophage membrane coated artificial cells and belongs to the technical field of artificial cell preparation. Polydiallyldimethylammonium chloride and sulfobutyl-beta-cyclodextrin are dissolved in ultrapure water, and the aqueous solutions of the two are mixed to obtain a PDDA / SBE-beta-CD condensate droplet solution; equal-mass-ratio macrophage membrane suspension liquid and the PDDA / SBE-beta-CD condensate droplet solution are mixed in equal ratio, in-situ coating of the macrophage membrane is realized by using ultrasonic coating technology, and macrophage membrane coated artificial cells are obtained. The PDDA / SBE-beta-CD condensate droplet constructed by the application has good stability under physiological salt concentration, pH conditions and 37 DEG C; the macrophage membrane coated artificial cells are prepared by mixing the macrophage membrane suspension liquid, and the macrophage membrane as the shell can not only prevent the condensate from gathering, but also increase the structural stability and biocompatibility.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of artificial cell preparation, and particularly relates to a preparation method of macrophage membrane-coated artificial cells. BACKGROUND

[0002] Phase separation technology is a technology for separating different components by regulating the solubility difference of substances in the system. Liquid-liquid phase separation (LLPS) is a common type. The principle is that under the conditions of temperature, pressure or component change, the uniform solution spontaneously splits into two or more immiscible liquid phases due to the imbalance of intermolecular forces. The solute is differentially distributed between the phases, and finally forms liquid droplet-like compartments with clear interfaces. In the field of chemistry, it is used to realize organic synthesis product purification, metal ion separation and other processes. In the field of materials, it can guide the preparation of functional materials such as porous membranes and microspheres, regulate the microstructure of materials, and optimize their filtration and adsorption performance. In the field of biomedicine, it is widely used, which can be used to drive the formation of membraneless organelles in cells, regulate gene expression, and also be used to construct artificial cells, efficiently enrich biological macromolecules to improve enzymatic reaction efficiency, or develop intelligent drug delivery systems.

[0003] Artificial cells are artificial microcavity structures that simulate the structure and function of biological cells, aiming to understand the origin of life and develop new biological technologies. Among them, the design of coacervate based on the bottom-up construction mode is becoming a frontier hotspot. The design of this kind of coacervate artificial cell is based on the formation principle of biological coacervate. By using phase separation mediated biological macromolecular coacervate formation, through the design of peptide materials with specific sequences, the controllable formation and function regulation of coacervate are realized. Compared with liposomes and nanoparticles, coacervate artificial cells show unique advantages. The core advantage lies in its extraordinary enrichment ability and dynamic selectivity. It not only can efficiently concentrate reactants and accelerate reactions, but also can intelligently recruit or exclude molecules like natural organelles, realizing the dynamic distribution of substances. Its application in the fields of artificial cell model construction, drug delivery, micro-nano reaction and wastewater treatment shows great potential, and provides a new platform for constructing more intelligent and efficient life-like systems.

[0004] Membrane modification technology is a cutting-edge biomaterial technology that coats natural cell membranes on the surface of synthetic materials to endow them with specific biological functions. In the field of materials science, this technology effectively combines the designability of artificial materials with the biological activity of cell membranes by constructing biomimetic materials, significantly improving the in vivo compatibility, stability, and functional diversity of materials. Its core advantages include: achieving active targeted delivery by using membrane surface targeting molecules to accurately identify diseased tissues or cells; conferring immune evasion ability by simulating the surface characteristics of autologous cells to avoid recognition and clearance by the immune system, thereby prolonging systemic circulation time; and providing an enzymatic catalytic microenvironment to promote specific biochemical reactions. Taking macrophage membrane modification as an example, this technology utilizes the naturally expressed chemokine receptors and immune regulatory proteins of macrophages to enable the carrier to efficiently accumulate in inflammatory or tumor regions and cross biological barriers, demonstrating important application potential in targeted therapy, inflammation regulation, and tissue repair.

[0005] However, current research on applying membrane modification technology to artificial cells is still in its infancy. Given the significant advantages of membrane modification in targeted delivery, immune evasion, and biocompatibility, as well as the unique potential of artificial cells in biomimetic construction and efficient catalysis, the integration of the two is expected to break through existing technical bottlenecks and construct intelligent life-like systems with greater biological authenticity, functional integration, and environmental responsiveness. Current research should focus on exploring the effects of membrane modification on the structural stability, material exchange behavior, and dynamic functional regulation of artificial cells, and promoting the in-depth application of this composite system in drug delivery, synthetic biology, and intelligent biomaterials. SUMMARY

[0006] The purpose of the present application is to provide a preparation method of macrophage membrane coated artificial cells. The present application is based on the principle of liquid-liquid phase separation (LLPS), and uses the electrostatic interaction of two components to construct PDDA / SBE-β-CD condensate droplets. Compared with the co-extrusion membrane coating method of rigid nano-particles, the mechanical stability of micrometer-sized condensate droplets is relatively poor, and they cannot withstand the large shear force during the extrusion process, which can cause structural damage, leakage of contents, or uncontrollable fusion with the membrane material. Therefore, on the basis of condensate droplets, macrophage membrane coated artificial cells are prepared by optimizing the 4℃ low temperature, intermittent ultrasonic membrane coating scheme to coat the macrophage membrane on the outside of the condensate droplets.

[0007] The present application is realized by the following technical scheme. A preparation method of macrophage membrane coated artificial cells, comprising the following steps:

[0008] Step 1: polydiallyldimethylammonium chloride (PDDA) and sulfobutyl-β-cyclodextrin (SBE-β-CD) are dissolved in ultrapure water, and the aqueous solutions of the two are mixed to obtain a PDDA / SBE-β-CD condensate droplet solution;

[0009] Step 2: equal mass ratio of macrophage membrane suspension and PDDA / SBE-β-CD condensate droplets are mixed in equal ratio, and in-situ coating of macrophage membrane is realized by ultrasonic coating technology to obtain macrophage membrane coated artificial cells.

[0010] Further preferably, the mass ratio of polydiallyldimethylammonium chloride to sulfobutyl-β-cyclodextrin is 1:1.4~1.6.

[0011] Further preferably, polydiallyldimethylammonium chloride is dissolved in ultrapure water to obtain a polydiallyldimethylammonium chloride aqueous solution with a concentration of 2.5~20mg / mL; sulfobutyl-β-cyclodextrin is dissolved in ultrapure water to obtain a sulfobutyl-β-cyclodextrin aqueous solution with a concentration of 2.5~20mg / mL.

[0012] Further preferably, the preparation process of the macrophage membrane suspension is as follows: the macrophage membrane is dissolved in water at 10mg / ml to prepare the macrophage membrane suspension, and is dispersed by ultrasonic at 200W for 15~20min at 4℃.

[0013] Further preferably, the preparation process of the macrophage membrane is as follows: the macrophage cells are cultured in culture medium, and when the cells grow to a certain density, the cell suspension is collected and centrifuged to obtain cell precipitate; the Tris-HCl buffer containing protease inhibitor, PMSF and MgCl2 is added to the cell precipitate, and is lysed on ice; after the lysis is completed, the cells are broken by using a cell disrupter; then, the supernatant is obtained by low-speed centrifugation, and the precipitate is obtained by high-speed centrifugation, and the precipitate is resuspended in ultrapure water and freeze-dried to obtain the macrophage membrane.

[0014] Further preferably, in the Tris-HCl buffer containing protease inhibitor, PMSF and MgCl2, the final concentration of protease inhibitor is 1×, the final concentration of PMSF is 1mM, the final concentration of MgCl2 is 10mM, and the pH is 7.0-7.4; the lysis time on ice is 30~60min.

[0015] Further preferably, the cell disrupter is set to 50W power, with 3s on and 6s off, and the cells are broken in ice water bath for 5~10min.

[0016] Further preferably, the low-speed centrifugation to obtain the supernatant is set to 1000~2000rpm for 10min, and the high-speed centrifugation to obtain the precipitate is set to 12000rpm for 30min.

[0017] Further preferably, the ultrasonic membrane coating technique is 200W ultrasonic for 15-20min at 4℃.

[0018] The present application has the following advantages: the present application uses the double-component electrostatic effect to construct PDDA / SBE-β-CD coacervate droplets, and on this basis, the membrane modification technology is used to coat the macrophage membrane outside the coacervate droplets to prepare the macrophage membrane coated artificial cells; the PDDA / SBE-β-CD coacervate droplets constructed by the present application have good stability under physiological salt concentration, pH conditions and 37℃; and the coacervate droplets can quickly and efficiently enrich dyes with various physical and chemical properties without using organic solvents. The macrophage membrane coated artificial cells are prepared by mixing the coacervate droplets with the macrophage membrane suspension. The macrophage membrane as the shell not only prevents the coacervate from gathering, but also increases the structural stability and biocompatibility of the coacervate. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The workflow diagram for constructing the macrophage membrane coated artificial cells.

[0020] Figure 2 The bright field image of the PDDA and four negative substances forming the coacervate droplets in Example 1.

[0021] Figure 3 The partial turbidity contour map of the PDDA / SBE-β-CD coacervate droplets formed in Example 1.

[0022] Figure 4 The size measurement of the PDDA / SBE-β-CD coacervate droplets in Example 1.

[0023] Figure 5 The confocal image (FITC dye) of the PDDA / SBE-β-CD coacervate droplets in Example 1.

[0024] Figure 6 The stability of the PDDA / SBE-β-CD coacervate droplets in Example 1 under different sodium chloride concentrations, different pH and different temperatures.

[0025] Figure 7 The stability of the PDDA / SBE-β-CD coacervate droplets in Example 1 in physiological saline.

[0026] Figure 8 The confocal image (Dil dye) of the macrophage membrane coated artificial cells in Example 2.

[0027] Figure 9 The surface protein Western Blot result map of the macrophage membrane coated artificial cells in Example 2.

[0028] Figure 10 Hemolysis rate of PDDA / SBE-β-CD coacervate droplets with macrophage membrane-coated artificial cells in Example 2.

[0029] Figure 11 Endothelial cell viability after treatment of PDDA / SBE-β-CD coacervate droplets with macrophage membrane-coated artificial cells in Example 2. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. The specific embodiments described herein are only used to explain the present application, and are not used to limit the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0031] The present application relates to the preparation and characterization of PDDA / SBE-β-CD coacervate droplets, and the preparation and characterization of macrophage membrane-coated artificial cells. The workflow is as shown below. Figure 1 The following are specific embodiments.

[0032] Example 1

[0033] This embodiment aims to screen the suitable concentration and ratio of SBE-β-CD and PDDA for liquid-liquid phase separation to form coacervate, and to systematically evaluate its stability. The specific implementation process is as follows:

[0034] Prepare a 10 mg / ml solution of the cationic polymer polydiallyldimethylammonium chloride (PDDA) as the positive component of the coacervate droplet formation. Prepare a 10 mg / ml solution of the anionic compound sulfobutyl-β-cyclodextrin (SBE-β-CD), and also prepare 10 mg / ml of commonly used anionic polymers heparin, polyglutamic acid, and polyaspartic acid as controls. Figure 2 A- Figure 2 D are the bright field morphologies of coacervate droplets formed by mixing PDDA and SBE-β-CD, heparin, polyglutamic acid, and polyaspartic acid, etc. in different mass ratios, respectively. The results show that PDDA and SBE-β-CD form the most uniform coacervate droplet particle size, so SBE-β-CD is selected as the negative component of the subsequent coacervate droplets.

[0035] Polydiallyldimethylammonium chloride (PDDA) and sulfobutyl-β-cyclodextrin (SBE-β-CD) are dissolved in water at a mass concentration of 20 mg / ml, and are diluted to a concentration gradient of 17.5, 15, 12.5, 10, 7.5, 5, and 2.5 mg / ml for subsequent use. Subsequently, according to the preparation method of the coacervate droplets in Example 1, the coacervate droplets are prepared by mixing PDDA and SBE-β-CD at different mass ratios.Figure 3 The polydiallyl dimethyl ammonium chloride (PDDA) and sulfonyl-β-cyclodextrin (SBE-β-CD) solutions were mixed at mass ratios of 10:1, 5:1, 1:1, 1:5, 1:10, 1:15, and 1:20. After mixing, the solutions quickly became turbid. The turbidity was then measured using an automated multi-functional microplate reader to preliminarily determine whether aggregate droplets had formed. Figure 3 As shown in the turbidity contour plot, when the mass ratio of polydiallyldimethylammonium chloride (PDDA) to sulfobutyl-β-cyclodextrin (SBE-β-CD) is between 1:1 and 1:20, the solution exhibits a certain degree of turbidity, indicating that aggregated droplets can form within this range. Particularly noteworthy is that when the mass ratio of PDDA to SBE-β-CD is 1:1 and the aggregated droplet concentration is 10 mg / ml, the turbidity is 98.26%, exceeding 90%, meaning that aggregated droplets can be formed under these conditions. Conversely, when the mass ratio of PDDA to SBE-β-CD is 1:5 and the aggregated droplet concentration is 10 mg / ml, the turbidity is 37.57%, below 40%, indicating that fewer aggregated droplets are formed under these conditions, and the aggregation effect is not as good as with the 1:1 mass ratio. Therefore, the range of PDDA and SBE-β-CD that can form aggregated droplets through liquid-liquid phase separation is determined to be 1:1 to 5.

[0036] Furthermore, the mass ratio range of 1:1 to 5 for polydiallyldimethylammonium chloride (PDDA) to sulfobutyl-β-cyclodextrin (SBE-β-CD) to form aggregates was investigated in detail to determine the optimal mass ratio range for aggregate droplets. Figure 4 As shown, the particle size distribution of the formed aggregate droplets was analyzed using a dynamic light scattering particle size analyzer within the PDDA to SBE-β-CD mass ratio range of 1:1 to 2. It is particularly noteworthy that when the mass ratio of polydiallyldimethylammonium chloride (PDDA) to sulfobutyl-β-cyclodextrin (SBE-β-CD) was 1:1.3, the average droplet size of the aggregate was 2.61 μm, which is not ideal for simulating real cell size. When the mass ratio was 1:1.4 to 1.6, the average droplet sizes were 4.45 μm, 3.76 μm, and 3.59 μm, respectively, which showed relatively good simulation of real cell size. However, when the mass ratio was 1:1.7, the average droplet size was 2.80 μm, which is also not ideal for simulating real cell size. Therefore, the range of PDDA to SBE-β-CD that can form good aggregate droplet sizes is determined to be 1:1.4 to 1.6.

[0037] Based on the turbidity observation of the above series of concentration gradients, it is determined that the effective working concentration range of PDDA and SBE-β-CD to form the condensate is 2.5~20mg / mL. This wide range indicates that the method of the present application has good inclusivity to the concentration of raw materials, which is convenient for flexible adjustment of the preparation process according to different application scenarios. Therefore, the subsequent examples select the concentration of 10mg / mL of polydiallyldimethylammonium chloride (PDDA) and sulfobutyl-β-cyclodextrin (SBE-β-CD) solution, and prepare the condensate according to the mass ratio of 1:1.5. Add FITC dye to the condensate so that the final concentration is 1μM. Then drop the sample into the confocal dish, and use the confocal microscope to observe the morphology in the bright field and the fluorescence field under the excitation wavelength of 495nm, respectively. As shown in Figure 5 , the PDDA / SBE-β-CD condensate droplet particle size distribution is uniform, and shows good enrichment effect on dye molecules.

[0038] The effects of different ion strengths, pH environments and temperature conditions on the turbidity of the solution were investigated by the full-automatic multifunctional enzyme marker, so as to evaluate the structural stability of the PDDA / SBE-β-CD condensate droplet. As shown in Figure 6 , under the conditions of physiological salt concentration, physiological pH value and normal body temperature, the turbidity of the condensate droplet is higher than 90%, indicating that the PDDA / SBE-β-CD condensate droplet can exist stably in the above environment.

[0039] The prepared condensate was stored in physiological saline, and the sample was dropped into the confocal dish at different time points, and the morphology was observed by the bright field of the confocal microscope, and the hydrodynamic diameter and polydispersity index (PDI) were counted. As shown in Figure 7 , the particle size and PDI of the PDDA / SBE-β-CD condensate droplet only change slightly, and the particle size is stably maintained at 3μm.

[0040] Example 2

[0041] This example demonstrates the effect of macrophage membrane modification on the surface of PDDA / SBE-β-CD condensate droplets, and verifies the integrity of the coated membrane and its biocompatibility, and the specific process includes:

[0042] According to the preferred optimal concentration of the condensate droplet in Example 1, the condensate is prepared by using polydiallyldimethylammonium chloride (PDDA) and sulfobutyl-β-cyclodextrin (SBE-β-CD) with a concentration of 10 mg / mL, and a mass ratio of 1:1.5. Then, the same mass of 10 mg / mL macrophage membrane suspension is slowly added to the condensate, and the film coating operation is completed by low-temperature ultrasonic treatment. Dil dye is added to a final concentration of 5 μM, and the sample is dropped into a confocal dish. The morphology and structure are observed under bright field and 510 nm fluorescence excitation using a confocal microscope. As shown in FIG. 9, the obtained macrophage membrane-coated artificial cell has uniform particle size, and the cell membrane on the outer surface is complete and continuous. Figure 8

[0043] The preparation process of the macrophage membrane suspension is as follows:

[0044] The macrophages are cultured in the culture medium, and when the cells grow to a certain density, the cell suspension is collected and centrifuged to obtain the cell precipitate. The Tris-HCl buffer containing protease inhibitors, PMSF, and MgCl2 is added to the cell precipitate, and the mixture is lysed on ice. After the lysis is completed, the cells are broken by using a cell disrupter. Then, the supernatant is obtained by low-speed centrifugation, and the precipitate is obtained by high-speed centrifugation. The precipitate is resuspended in ultrapure water and freeze-dried to obtain the macrophage membrane. In the Tris-HCl buffer containing protease inhibitors, PMSF, and MgCl2, the final concentration of the protease inhibitor is 1×, the final concentration of PMSF is 1 mM, the final concentration of MgCl2 is 10 mM, and the pH is 7.0-7.4. The lysis time on ice is 30-60 min. The cells are broken by using a cell disrupter with a power of 50 W, and the intermittent ultrasonic mode is set to 3 s on and 6 s off. The cells are broken in an ice water bath for 5-10 min. The membrane material is allowed to stabilize in the pulse interval to reduce the continuous mechanical stress. The supernatant is obtained by low-speed centrifugation at 1000-2000 rpm for 10 min, and the precipitate is obtained by high-speed centrifugation at 12000 rpm for 30 min. The ultrasonic film coating technique is performed at 4°C under 200 W ultrasonic for 15-20 min.

[0045] The macrophage membrane is dissolved in water to prepare a macrophage membrane suspension at a concentration of 10 mg / ml. The suspension is ultrasonically dispersed at 4°C under 200 W for 15-20 min to maximize the reduction of thermal damage.

[0046] ​The macrophage membrane is rich in various functional membrane proteins, and the high expression of the proteins is the key to maintaining the integrity of the cell membrane. Among them, the CD47 protein can mediate the mutual recognition with the immune system of the body, thereby helping the carrier to avoid immune clearance and realize long circulation in the body; the activated endothelial cells at the inflammation site highly express VCAM-1, and the ligands thereof include Integrin α4 and Integrin β1, which can mediate the recruitment of monocytes / macrophages to the inflammation site. By implementing the optimized low-temperature 4°C and intermittent ultrasonic membrane wrapping scheme, the functional components of the cell membrane are successfully retained without irreversible damage under the condition of a longer total ultrasonic time. Therefore, the expression of CD47, Integrin α4 and Integrin β1 in the total protein of macrophages, the separated membrane protein of macrophages and the finally constructed macrophage membrane coated artificial cells is detected by Western Blot. As shown in Figure 9 , the key membrane proteins are clearly detected on the surface of the artificial cells, which confirms the successful wrapping of the macrophage membrane and indicates that the artificial cells may have part of the biological functions derived from macrophages, such as targeting and immune escape.

[0047] Further, in vitro hemolysis experiment is used to verify whether the macrophage membrane coated artificial cells have good blood compatibility. As shown in Figure 10 , compared with the positive control, the PDDA / SBE-β-CD condensate droplets and the macrophage membrane coated artificial cells both have good blood compatibility, and the hemolysis rate is less than 2%.

[0048] To verify the toxicity difference of the artificial cells before and after the membrane modification, 5-40 μg / ml of the artificial cells are respectively incubated with endothelial cells for 8 h, and then the cell viability is detected by CCK-8 Kit. As shown in Figure 11 , with the increase of the concentration of the PDDA / SBE-β-CD condensate droplets, the cell activity gradually decreases. After the membrane modification, the macrophage membrane coated artificial cells have a higher cell survival rate, and when the concentration reaches 20 μg / ml, the cells in each group still have a survival rate of more than 80%. It is proved that the macrophage membrane coating can significantly reduce the biological toxicity of the condensate droplets.

[0049] The above is only the preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any skilled person in the technical field can make equivalent replacement or change according to the technical scheme and the inventive concept of the present application within the technical range disclosed by the present application, which should be within the protection scope of the present application.

Claims

1. A method for preparing artificial cells coated with macrophage membranes, characterized in that, Includes the following steps: Step 1: Dissolve polydiallyldimethylammonium chloride and sulfobutyl-β-cyclodextrin in ultrapure water, and mix the two aqueous solutions to obtain a PDDA / SBE-β-CD aggregate droplet solution; the mass ratio of polydiallyldimethylammonium chloride to sulfobutyl-β-cyclodextrin is 1:1.4~1.6; Step 2: Dissolve the macrophage membrane in water to prepare a macrophage membrane suspension and disperse it by ultrasound. Mix the macrophage membrane suspension with PDDA / SBE-β-CD aggregate droplets in equal mass ratio. Use ultrasound encapsulation technology to achieve in-situ coating of the macrophage membrane to obtain artificial cells coated with macrophage membrane.

2. The preparation method according to claim 1, characterized in that, Polydiallyl dimethyl ammonium chloride was dissolved in ultrapure water to obtain an aqueous solution of polydiallyl dimethyl ammonium chloride with a concentration of 2.5~20 mg / mL; sulfobutyl-β-cyclodextrin was dissolved in ultrapure water to obtain an aqueous solution of sulfobutyl-β-cyclodextrin with a concentration of 2.5~20 mg / mL.

3. The preparation method according to claim 1, characterized in that, The preparation process of macrophage membrane suspension is as follows: macrophage membrane is dissolved in water at a concentration of 10 mg / ml to prepare macrophage membrane suspension, and sonicated at 200W for 15-20 min at 4℃ to ensure full dispersion.

Images