Functionalized artificial cell based on complex coacervate, preparation and application thereof

Abstract

The present invention relates to a functionalized artificial cell based on a composite coacervate, preparation and application thereof, and relates to the field of biomedicine technology. The preparation method is as follows: a positively charged solution and a negatively charged solution are mixed, the positively charged solution being a polylysine solution or an ε-polylysine hydrochloride solution, and the negatively charged solution being a deoxyribonucleic acid solution; a coacervate droplet suspension containing functional components is obtained by liquid-liquid phase separation; phospholipids and PEGylated phospholipids are dissolved in an organic solvent and then subjected to vacuum rotary evaporation until the organic solvent evaporates to obtain a lipid membrane; the coacervate droplet suspension is added to the lipid membrane for hydration until the lipid membrane is completely dissolved, thereby obtaining a functionalized artificial cell; at least one of the positively charged solution, the negatively charged solution and the phospholipid solution contains a functional component. The functionalized artificial cell based on the composite coacervate constructed by the present invention has a cell membrane-like and cytoplasm-like structure, and has good physiological stability and biocompatibility.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more particularly to a functionalized artificial cell based on complex coacervates, and its preparation and application. Background Art

[0002] Liquid-liquid phase separation (LLPS) is the phenomenon in which a homogeneous liquid phase separates into two or more distinct, immiscible phases through interactions such as charge-charge, hydrogen bonding, or π-π stacking. Multiple independent studies have confirmed that LLPS underlies the formation of membraneless organelles in cells. These condensed droplets, with their internal molecular crowding, lower dielectric constant than the surrounding aqueous continuous phase, and at least 50% water content, can enrich external solutes, serve as sites for specific catalytic reactions, and act as compartments for storing specific enzymes during stress. They are a natural choice for compartmentalizing and protecting active components within cells. Therefore, constructing condensed droplets in vitro to mimic the characteristics of membraneless organelles has become a simple and efficient strategy for encapsulating therapeutic drugs. First, condensed droplets offer universal and efficient loading for therapeutic drugs. Therapeutic drugs are concentrated in condensed droplets through non-covalent interactions, enabling efficient loading of small molecules, biomacromolecules, and even nanoparticles of varying charge and hydrophilicity without the need for any chemical modification. Secondly, the composition of condensate droplets is simple and can be prepared quickly. Compared with conventional delivery systems such as large lipid vesicles, polymer microspheres, and gels, condensate droplets are usually formed in an instant with 1-2 materials. When preparing and loading drugs, no organic solvents are required, and the activity of biological drugs such as peptides, proteins, nucleic acids and vesicles can be effectively retained. This simple composition and easy preparation are of great significance to the process flow and quality control in large-scale production. Therefore, the discovery and development of condensate-based delivery systems with physiological stability and biocompatibility is expected to promote the development of synthetic biology and pharmaceutical fields.

[0003] However, the biological application of coacervates still faces many challenges. (1) It is difficult to screen suitable coacervates: For simple coacervates formed by using only one coacervate material, there is no clear design rule to guide their synthesis; for complex coacervates formed by two or more coacervates, the inherent properties of the materials such as charge density, charge length, molecular weight and hydrophilicity may affect their interactions, so that they do not undergo phase separation (maintaining a homogeneous solution) or undergo liquid-solid phase separation (precipitation), which brings great challenges to screening coacervates that only undergo LLPS. (2) The preparation conditions of physiologically stable coacervate droplets are harsh: When preparing coacervates, external environmental factors including pH, temperature and ionic strength, especially ionic strength, will affect the formation of coacervates. When the ionic strength gradually increases, the material may transform from a solid aggregate to a coacervate droplet and eventually form a homogeneous solution. Once the ionic strength is higher than the critical salt concentration of the coacervate, the coacervate will disaggregate and transform into a homogeneous solution. Although some coacervates are stable at low salt concentrations, they will still disaggregate when transferred to a solution above the critical salt concentration. At present, the critical salt concentration of some common coacervate droplets is lower than the physiological salt condition. (3) Self-fusion characteristics of coacervates: Due to the inherent membrane-less nature of coacervates, they undergo self-fusion. Suspended coacervate droplets aggregate into large coacervate phases within minutes to hours, which contributes to the instability of coacervates and limits their drug delivery routes. Therefore, in order for coacervates to better realize their potential in biological applications, it is necessary to conduct more in-depth research on the above issues and explore solutions. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for preparing functionalized artificial cells based on complex coacervates, so as to overcome the technical problem of poor stability of coacervates in the prior art and realize their application in living organisms. The complex coacervates formed by polylysine or ε-polylysine hydrochloride / deoxyribonucleic acid through LLPS of the present invention have good stability at physiological salt concentration, pH and temperature; and can quickly and efficiently encapsulate therapeutic drugs with diverse physicochemical properties, including small molecules, biomacromolecules and nanoparticles, without the use of organic solvents. Through a modified thin film hydration method, a phospholipid membrane is coated on the surface of the complex coacervate droplets to prepare artificial cells with cell membrane-like and cytoplasm-like structures. The phospholipid membrane not only prevents the aggregation of the complex coacervate, increasing its structural stability and biocompatibility, but also reduces the recognition of the endothelial reticular system in the body, thereby prolonging the circulation time of the artificial cell body and effectively protecting the activity of the drug.

[0005] According to a first aspect of the present invention, there is provided a method for preparing a functionalized artificial cell based on a complex coacervate, comprising the following steps:

[0006] (1) mixing a positively charged solution with a negatively charged solution, wherein the positively charged solution is a polylysine solution or an ε-polylysine hydrochloride solution, and the negatively charged solution is a deoxyribonucleic acid solution; and obtaining a coacervate droplet suspension by liquid-liquid phase separation;

[0007] (2) dissolving the phospholipid and the PEGylated phospholipid in an organic solvent and then performing vacuum rotary evaporation until the organic solvent evaporates to obtain a lipid film;

[0008] (3) adding the coacervate droplet suspension obtained in step (1) to the lipid membrane obtained in step (2) for hydration until the lipid membrane is completely dissolved, thereby obtaining the functionalized artificial cell based on the complex coacervate;

[0009] Among them, at least one of the positively charged solution in step (1), the negatively charged solution in step (2), and the solution obtained by dissolving the phospholipid and the PEGylated phospholipid in the organic solvent in step (2) contains a functional component.

[0010] Preferably, the functional components contained in the positively charged solution and the negatively charged solution are at least one of enzymes, small molecule drugs, polypeptide drugs, protein drugs, nucleic acid drugs, nanoparticles and biological vesicles.

[0011] Preferably, the phospholipid is linked to a drug or active ingredient;

[0012] Preferably, in step (2), after the phospholipids and PEGylated phospholipids are dissolved in an organic solvent, the method further comprises adding at least one of fat-soluble enzymes, small molecule drugs, polypeptide drugs and protein drugs to the resulting solution.

[0013] Preferably, the mass ratio of the solute in the positively charged solution to the solute in the negatively charged solution is (0.05-20):1.

[0014] Preferably, the mass ratio of the solute in the positively charged solution to the solute in the negatively charged solution is (0.2-10):1;

[0015] Preferably, the mass ratio of the solute in the positively charged solution to the solute in the negatively charged solution is (2.6-10):1.

[0016] Preferably, the concentration of the solute in the positively charged solution is 1 mg / mL-6 mg / mL, and the concentration of the solute in the negatively charged solution is 1 mg / mL-6 mg / mL.

[0017] Preferably, the enzymes are uricase and catalase.

[0018] According to another aspect of the present invention, a functionalized artificial cell based on complex coacervates prepared by any one of the methods is provided.

[0019] According to another aspect of the present invention, there is provided a use of the functionalized artificial cells based on complex coacervates for preparing a drug delivery system.

[0020] Preferably, the drug delivery system is a hyperuricemia drug delivery system.

[0021] In general, the above technical solutions conceived by the present invention have the following technical advantages compared with the existing technology:

[0022] (1) The present invention successfully overcomes the challenges of traditional condensate droplets in drug delivery. The screened positively charged solutions and negatively charged solutions have suitable charge properties and can be used to prepare condensate droplets with physiological stability, effectively solving the limitation that existing conventional condensates cannot be used in vivo.

[0023] (2) The condensate droplets provided by the present invention can quickly and efficiently concentrate various types of bioactive ingredients, including small molecule drugs, biomacromolecule drugs and nanoparticles, significantly improving the drug loading success rate and encapsulation efficiency, and providing a broader prospect for the combined treatment strategy of various diseases.

[0024] (3) The condensate droplets provided by the present invention do not require the use of additional organic solvents and surfactants when encapsulating various active ingredients, thus avoiding the introduction of toxic substances, reducing potential harm to organisms, and providing convenience for the supervision and quality control of the preparation process.

[0025] (4) By introducing a PEGylated phospholipid membrane onto the surface of the coacervate, the present invention successfully improves the biocompatibility and stability of the coacervate, resolving the key issues of unstable physiological salt concentration and hemolysis in commonly used complex coacervates. The constructed artificial cells have cell membrane- and cytoplasm-like structures, which are closer to the configuration of real biological cells. Both the cell membrane- and cytoplasm-like structures can be functionally modified, adapting to diverse research and application needs.

[0026] (5) The preparation method of the condensate droplets provided by the present invention is simple, the operation process is simple and clear, the preparation process is time-saving, and the conditions are controllable, thus having the potential for efficient production.

[0027] (6) Preferably, the functionalized artificial cells of the present invention can easily confine the circulating catalytic system of uricase and catalase within the artificial cells, and can simultaneously remove hydrogen peroxide while effectively degrading uric acid, thereby reducing the damage of hydrogen peroxide to the body, showing obvious advantages in treating hyperuricemia and reducing kidney damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1This is a turbidity density diagram of poly-lysine / deoxyribonucleic acid aggregates at different ratios and concentrations prepared in Example 1.

[0029] Figure 2 The surface charge properties and particle size of the poly-lysine / deoxyribonucleic acid aggregates after optimizing the composite ratio in Example 2.

[0030] Figure 3 These are the morphological characteristics of the polylysine / deoxyribonucleic acid condensate in Example 3.

[0031] Figure 4 The stability of the polylysine / deoxyribonucleic acid condensate in Example 3 at different sodium chloride concentrations, pH values ​​and temperatures.

[0032] Figure 5 This is to confirm the fluidity inside the polylysine / deoxyribonucleic acid condensate in Example 3.

[0033] Figure 6 This is a fluorescent image of various molecules concentrated in the poly-lysine / deoxyribonucleic acid condensate in Example 6.

[0034] Figure 7 This is the fluorescence image of the artificial cell in Example 7.

[0035] Figure 8-9 For the study of biocompatibility of aggregates and artificial cells; Figure 8 Comparison of hemolytic properties between aggregates and artificial cells, Figure 9 Comparison of the hemolytic properties of aggregates and artificial cells.

[0036] Figure 10 The spatial confinement provided by the functionalized artificial cells prepared in Example 10 for the cyclic catalytic system protects cells from being killed by hydrogen peroxide in the presence of uric acid.

[0037] Figure 11 The artificial cell ArtPC group without active ingredients was prepared according to Example 7, and the artificial cell Uri / Cat@ArtPC group containing uricase and catalase was prepared according to Example 10. The uric acid-lowering effect and renal damage alleviation effect of the free uricase and catalase Uri / Cat group and the normal saline Saline group in hyperuricemia mice were evaluated. DETAILED DESCRIPTION

[0038] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely for the purpose of explaining the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0039] The present invention provides a method for preparing a functionalized artificial cell based on a complex coacervate, comprising the following steps:

[0040] (1) mixing a positively charged solution with a negatively charged solution, wherein the positively charged solution is a polylysine solution or an ε-polylysine hydrochloride solution, and the negatively charged solution is a deoxyribonucleic acid solution; obtaining a suspension of aggregate droplets containing functional components by liquid-liquid phase separation; the main driving force of the liquid-liquid phase separation is electrostatic interaction;

[0041] (2) dissolving the phospholipid in an organic solvent to obtain a phospholipid solution, and then performing vacuum rotary evaporation until the organic solvent evaporates to obtain a lipid film;

[0042] (3) adding the coacervate droplet suspension obtained in step (1) to the lipid membrane obtained in step (2) for hydration until the lipid membrane is completely dissolved, thereby obtaining the functionalized artificial cell based on the complex coacervate;

[0043] At least one of the positively charged solution, the negatively charged solution and the phospholipid solution contains a functional component.

[0044] In some embodiments, the molecular weight of ε-polylysine hydrochloride and polylysine is 1000-20000 Da.

[0045] In some embodiments, the solution for preparing polylysine and deoxyribonucleic acid includes ultrapure water, sodium chloride solution, glucose solution, phosphate buffer, acetate buffer, Hank's balanced salt solution and Tris-HCl buffer.

[0046] In some embodiments, the organic solvent is chloroform.

[0047] In some embodiments, the concentration of the condensate droplets is 1 mg / mL-10 mg / mL, and the mass ratio of polylysine to deoxyribonucleic acid is 20:1-1:20.

[0048] In some embodiments, in step (2), the phospholipids further include dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidic acid (DOPA), dioleoylphosphatidylcholine (DOPC), dioctadecyldimethylammonium bromide (DDAB), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylglycerol (DOPG) and distearoylphosphatidylglycerol (DSPG).

[0049] In some embodiments, in step (2), the PEGylated phospholipid further comprises PEGylated distearoylphosphatidylethanolamine (DSPE-PEG), PEGylated dioleoylphosphatidylethanolamine (DOPE-PEG) and PEGylated tetradecanoylphosphatidylethanolamine (DMPE-PEG).

[0050] In some embodiments, in step (2), a functionalized phospholipid is used.

[0051] In some embodiments, the hydration in step (3) is performed by rotary hydration using a rotary evaporator or by stirring hydration on a magnetic stirrer.

[0052] In some embodiments, in step (2), the surface of the condensate droplets formed by polylysine and deoxyribonucleic acid is positively charged, that is, the mass ratio of polylysine to deoxyribonucleic acid is greater than 2.4.

[0053] The complex coacervates of the present invention and the functionalized artificial cells based on the complex coacervates are used to prepare drug delivery systems.

[0054] In some embodiments, the present invention further incorporates uricase and catalase into the artificial cells, enabling them to participate in simple metabolic reactions in vivo, effectively reducing blood uric acid levels and improving kidney damage in mice with hyperuricemia. These functionalized artificial cells based on complex aggregates exhibit excellent physiological stability and biocompatibility, and are easily functionalized within the cytoplasm-like aggregate lumen and cell membrane-like phospholipid outer membrane, paving the way for a wide range of applications in artificial cells and drug delivery.

[0055] The following are specific embodiments

[0056] Example 1

[0057] In this example, the concentration and ratio of polylysine and deoxyribonucleic acid were screened. The specific process included: dissolving polylysine and deoxyribonucleic acid at equal mass concentrations in phosphate buffer at pH 7.4, and the concentrations were 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL and 6 mg / mL, respectively. Figure 1Mix the polylysine solution and deoxyribonucleic acid solution at the same mass ratio, observe that the solution immediately becomes turbid and use a fully automatic multifunctional microplate reader to detect the turbidity of the solution to preliminarily determine whether coacervates are formed. Figure 1 The turbidity contour plots show that the concentration and ratio of the coagulated materials regulate the occurrence of LLPS. When the concentration of the polylysine solution and the DNA solution range from 2 to 6 mg / mL and the mass ratio of polylysine to DNA ranges from 10:1 to 1:5, the solution turbidity is greater than 50%, indicating that coagulation is fully formed.

[0058] Example 2

[0059] Following the method in Example 1, 2 mg / mL polylysine solution and deoxyribonucleic acid solution were selected to further optimize the mass ratio of polylysine to deoxyribonucleic acid. The particle size and potential of the coacervate droplets were measured using a dynamic light scattering particle size analyzer within the mass ratio range of 1:1-3:1. Figure 2 The results show that isoelectric aggregates form within a polylysine to deoxyribonucleic acid mass ratio of 2.2:1 to 2.4:1. When the polylysine to deoxyribonucleic acid mass ratio is greater than or equal to 2.6, stable positively charged aggregate droplets can be prepared. These positively charged aggregates are more suitable for subsequent phospholipid membrane coating.

[0060] Example 3

[0061] According to the method in Example 2, a positively charged aggregate closest to the isoelectric point was prepared, i.e., the mass ratio of polylysine to deoxyribonucleic acid was 2.6:1. The positively charged aggregate was placed in a 24-well cell culture plate and the morphology was observed under a white light microscope. Figure 3 As shown, positively charged aggregates form bright round bubble-like structures.

[0062] Example 4

[0063] According to the method in Example 3, positively charged aggregates closest to the isoelectric point were prepared, and the turbidity changes under different ionic strength, temperature and pH conditions were investigated using a fully automatic multifunctional microplate reader to evaluate the structural stability of the polylysine / deoxyribonucleic acid aggregates. Figure 4 As shown, the turbidity of the coacervate does not change significantly under physiological salt concentration, temperature and pH, indicating that the coacervate droplets exist stably under these conditions.

[0064] Example 5

[0065] According to the method in Example 3, positively charged aggregates closest to the isoelectric point were prepared, and the aggregates were stained with 4',6-diamidino-2-phenylindole (DAPI) to make them have blue fluorescence under a laser confocal microscope. Fluorescence recovery after photobleaching was further detected using a confocal microscope to investigate the liquid flow properties inside the aggregates. Figure 5 As shown, before photobleaching, the two aggregates shown under the microscope have uniformly distributed fluorescence intensity. After photobleaching, the fluorescence in the upper portion of the right droplet is quenched. The recovery half-life of this positively charged aggregate is 4.453 ± 0.294 seconds. This confirms that the aggregates have liquid-like flow properties rather than solid state, allowing the fluorescent molecules to diffuse within the droplet, restoring fluorescence intensity.

[0066] Example 6

[0067] Doxorubicin, fluorescein isothiocyanate, methylene blue, fluorescein isothiocyanate-labeled bovine serum albumin, sulfocyanine 5-labeled small interfering RNA, and fluorescein isothiocyanate-labeled polylactic acid nanoparticles were selected as representative fluorescent materials to evaluate the loading capacity of droplets for various substances. Prepare the mother solutions of doxorubicin (368nM), fluorescein isothiocyanate (514nM), methylene blue (625nM), fluorescein isothiocyanate-labeled bovine serum albumin (75nM), Cy5-siRNA (2μM), and fluorescein isothiocyanate-labeled polylactic acid nanoparticles (1mg / mL). Mix 70μL of negatively charged or neutrally charged fluorescent substance mother solution with 1011μL of polylysine solution, and then add 389μL of deoxyribonucleic acid solution (2mg / mL) to prepare coacervates. On the contrary, after mixing the positively charged fluorescent substance mother solution with the deoxyribonucleic acid solution, polylysine solution was added to prepare coacervates. As Figure 6 As shown, the slightly water-soluble and positively charged anti-tumor drug doxorubicin, the slightly water-soluble and negatively charged fluorescein isothiocyanate, the water-soluble and positively charged methylene blue, the water-soluble and negatively charged biomacromolecule bovine serum albumin, the water-soluble and negatively charged biomacromolecule small interfering RNA, and nanoparticles can all be enriched in polylysine / deoxyribonucleic acid aggregates. Therefore, these representative small molecules, biomacromolecules, and nanoparticles with representative physical and chemical properties can all be loaded into aggregates.

[0068] Example 7

[0069] According to the method in Example 2, positively charged coacervates closest to the isoelectric point were prepared for use. Phospholipids (80 mol% DPPC and 20 mol% DSPE-PEG2000) were dissolved in chloroform, poured into a round-bottom flask and vacuum rotary evaporated for 30 minutes to prepare a phospholipid film. The coacervates were then slowly added to the bottom of the flask and hydrated for 1 hour. The mass ratio of phospholipids to coacervates was 1:10. Use a pipette to gently transfer the solution in the round-bottom flask to a centrifuge tube and let it stand for 4 hours. After 4 hours, the centrifuge tube was placed in a centrifuge, centrifuged at 3000 rpm / min for 10 minutes, and then the bottom precipitate was collected and resuspended in phosphate buffer to construct an artificial cell. The coacervates were fluorescently labeled with DAPI, and the phospholipid membrane was fluorescently labeled with a lipid red fluorescent probe (DiI). As Figure 7 As shown, the red ring of fluorescence is completely enclosed within the blue circular fluorescence. By scanning a single droplet layer by layer, a 3D reconstruction of the artificial cell was obtained. This image demonstrates that the phospholipid membrane has effectively coated the surface of the aggregate droplet, resulting in the creation of an artificial cell with cytoplasm-like and cell membrane-like structures.

[0070] Example 8

[0071] Prepare the aggregates and artificial cells according to Example 1, Example 3 and Example 7 for later use. Take 500 μL of whole blood from healthy Kunming mice, place it in a centrifuge tube moistened with sodium heparin and shake it evenly. Centrifuge at 3000 rpm / min for 10 minutes at 4°C to separate the plasma and red blood cells, and collect the red blood cells from the bottom of the centrifuge tube to avoid the white blood cells and platelets in the upper layer. After washing 3 times with pre-cooled phosphate buffer, the red blood cells were added to a mixture containing ultrapure water, physiological saline, aggregates and artificial cells of different concentrations, so that each sample contained 2% (w / v) red blood cells. After incubating the sample in a constant temperature shaker at 37°C for 4 hours, centrifuge it at 3000 rpm / min for 10 minutes. Aspirate the supernatant in the centrifuge tube into a 96-well plate, use a fully automatic multifunctional enzyme marker to measure the absorbance at 540 nm, and calculate the hemolysis rate of each sample. As Figure 8 As shown in the figure, at a concentration of 5 mg / mL, the hemolysis rate of the aggregates was as high as 39.7±0.7%. Compared with the membrane-free aggregates, the hemolysis rate of the artificial cells was significantly improved.

[0072] Example 9

[0073] The aggregates and artificial cells were prepared according to Example 1, Example 3 and Example 7. The effects of the aggregates and artificial cells on the viability of dendritic cells and venous endothelial cells were detected by MTT assay, and the safety of the aggregates and artificial cells was evaluated at the cellular level. 4The cells were seeded at a density of 100 μL in a 96-well plate, and 100 μL of complete culture medium was added to each well. After culturing for 12 hours, the culture medium was replaced with blank culture medium containing different concentrations of aggregates or artificial cells. After culturing for 24 hours, 10 μL of MTT (5 mg / mL) was added to each well and cultured in the incubator for another 4 hours. The culture medium was slowly aspirated from each well, and then 150 μL of dimethyl sulfoxide was added. After the purple crystals at the bottom were completely dissolved, the absorbance of each well was detected at 490 nm. Figure 9 As shown, at a concentration of 500 μg / mL, the aggregates killed both dendritic cells and venous endothelial cells, with cell survival rates below 85%. When the concentration was increased to 1000 μg / mL, the artificial cells did not cause a decrease in the survival rate of either cell type, while the aggregates killed nearly half of the cells. Therefore, the membrane-bound aggregates, or artificial cells, exhibited improved biocompatibility.

[0074] Example 10

[0075] Artificial cells (named ArtPC) were prepared according to the method of Example 7. Uricase (Uri) and catalase (Cat) were added to a polylysine solution and then mixed with a deoxyribose solution to form aggregates. Droplets of aggregates enriched with uricase and catalase were then used according to the method of Example 7 to prepare artificial cells capable of decomposing uric acid and scavenging hydrogen peroxide, resulting in functionalized artificial cells based on the composite aggregates (named Uri / Cat@ArtPC).

[0076] Example 11

[0077] According to the method of Example 7, functionalized artificial cells Uri / Cat@ArtPC based on complex coacervates were prepared. The effect of hydrogen peroxide, a byproduct of uric acid decomposition, on the viability of dendritic cells and venous endothelial cells was detected by MTT assay. 4 The two cells were seeded in a 96-well plate at a density of 100 μL, and 100 μL of complete culture medium was added to each well. The culture was continued for 12 hours. After adding culture medium containing uric acid or not containing uric acid to the two cells, the following five groups of treatments were performed: culture medium (negative control group Saline), hydrogen peroxide (positive control group H2O2), Uri, free Uri and Cat (named Uri / Cat) and Uri / Cat@ArtPC. After continuing to culture for 24 hours, 10 μL of MTT (5 mg / mL) was added to each well, and the culture was continued in the incubator for 4 hours. The culture medium was slowly aspirated from each well, and then 150 μL of dimethyl sulfoxide was added. After the purple crystals at the bottom were completely dissolved, the absorbance of each well at 490 nm was detected to calculate the cell viability of each group. Figure 10As shown, under uric acid-free conditions, the cell viability of the free Uri group and the Uri / Cat group decreased slightly compared with the Saline group and the Uri / Cat@ArtPC group, which may be due to the toxicity of the heterologous proteins Uri and Cat at these doses. The encapsulation of Uri and Cat by ArtPC can reduce their toxicity. In the presence of uric acid, the cytotoxicity of the Uri group was similar to that of the positive control group H2O2 group. While the free Uri / Cat group only slightly improved cell viability, the Uri / Cat@ArtPC group effectively protected cells from hydrogen peroxide damage. Although Cat can decompose hydrogen peroxide, a byproduct of uric acid degradation, the Uri / Cat group did not achieve the expected protective effect compared with the Uri / Cat@ArtPC group. Therefore, using ArtPC as a Uri carrier and further loading Cat can effectively provide spatial confinement and improve the safety of treatment.

[0078] Example 12 (Treatment of Hyperuricemia)

[0079] A hyperuricemia model in Kunming mice was established by daily gavage with 0.5% sodium carboxymethylcellulose solution containing 500 mg / kg hypoxanthine and 1000 mg / kg potassium oxalate. After the hyperuricemia model was successfully established, each group was given relevant drug treatment. The groups were divided into the following groups: (1) Healthy mice were treated with normal saline and named Saline group. (2) Hyperuricemia mice were treated with normal saline and named Model group. (3) Hyperuricemia mice were treated with ArtPC and named ArtPC group. (4) Hyperuricemia mice were treated with free Uri and Cat and named Uri / Cat group. (5) Hyperuricemia mice were treated with Uri / Cat@ArtPC and named Uri / Cat@ArtPC (ensuring that the Uri and Cat contents were consistent). The body weight of mice was measured throughout the experiment, and the intravenous injection volume was calculated based on the body weight of each mouse. Blood was collected from the tail tip on days 7, 9, 11, and 13, and the blood uric acid concentration was measured using an EA-11 uric acid detector (with a detection limit of 180 μM). After the experiment, the kidneys of the mice were dissected and stained with hematoxylin-eosin. Figure 11As shown, blood uric acid levels in the ArtPC and model groups remained consistently above 600 μmol / L during testing. By day 11, levels in the Uri / Cat and Uri / Cat@ArtPC groups had dropped to 481±53 μmol / L and 302±71 μmol / L, respectively. By day 13, blood uric acid levels in the Uri / Cat@ArtPC group were significantly lower than those in the Uri / Cat group and were not significantly different from those in healthy mice. Hematoxylin-eosin staining revealed normal renal parenchyma in the Saline group, with intact tubular epithelial cells and normal tubular spaces, and no signs of inflammatory cell infiltration or necrosis. In contrast, in the model group, tubular structure disappeared, with marked tubular dilation and epithelial cell atrophy, occasionally with effacement of the nucleus. The parietal cells of the renal cyst were flattened, and the spaces within the cystic lumen were enlarged. The extent of renal damage in the ArtPC group was similar to that in the Model group, with no improvement. In the Uri / Cat-treated group, tubular luminal dilation was reduced, but pink, structureless material was present within the tubular lumen. The Uri / Cat@ArtPC-treated group showed only slight tubular expansion and retained intact tubular structure. Therefore, the functionalized artificial cells based on complex coacervates in this invention have high application and promotion value, especially as a biopharmaceutical delivery platform, which can further enhance their therapeutic efficacy.

[0080] It will be easily understood by those skilled in the art that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing functionalized artificial cells based on complex coacervates, characterized in that: The following steps are involved: (1) mixing a positively charged solution with a negatively charged solution, wherein the positively charged solution is a polylysine solution or an ε-polylysine hydrochloride solution, and the negatively charged solution is a deoxyribonucleic acid solution; and obtaining a coacervate droplet suspension by liquid-liquid phase separation; (2) dissolving the phospholipid and the PEGylated phospholipid in an organic solvent and then performing vacuum rotary evaporation until the organic solvent evaporates to obtain a lipid film; (3) adding the coacervate droplet suspension obtained in step (1) to the lipid membrane obtained in step (2) for hydration until the lipid membrane is completely dissolved, thereby obtaining the functionalized artificial cell based on the complex coacervate; wherein at least one of the positively charged solution in step (1), the negatively charged solution in step (2), and the solution obtained by dissolving the phospholipid and the PEGylated phospholipid in the organic solvent in step (2) contains a functional component; The functional components contained in the positively charged solution and the negatively charged solution are enzymes, and the enzymes are uricase and catalase; The mass ratio of the solute in the positively charged solution to the solute in the negatively charged solution is (0.05-20):1; The concentration of the solute in the positively charged solution is 1 mg / mL-6 mg / mL, and the concentration of the solute in the negatively charged solution is 1 mg / mL-6 mg / mL.

2. The method for preparing a functionalized artificial cell based on complex coacervates according to claim 1, wherein: The phospholipid is connected with a drug or an active ingredient.

3. The method for preparing a functionalized artificial cell based on complex coacervates according to claim 2, wherein: In step (2), after the phospholipids and PEGylated phospholipids are dissolved in an organic solvent, the method further includes adding fat-soluble enzymes to the resulting solution.

4. The method for preparing a functionalized artificial cell based on complex coacervates according to claim 1, wherein: The mass ratio of the solute in the positively charged solution to the solute in the negatively charged solution is (0.2-10):

1.

5. The method for preparing a functionalized artificial cell based on complex coacervates according to claim 4, wherein: The mass ratio of the solute in the positively charged solution to the solute in the negatively charged solution is (2.6-10):

1.

6. A functionalized artificial cell based on complex coacervates prepared by the method according to any one of claims 1 to 5.

7. Use of the functionalized artificial cell based on complex coacervates as claimed in claim 6 for preparing a drug delivery system.

8. The use according to claim 7, characterized in that The drug delivery system is a hyperuricemia drug delivery system.

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