Preparation method and application of a phospholipid carrier coated with annexin

Abstract

This invention relates to a method for preparing annexin-coated phospholipid carriers and its application. The preparation method includes adding annexin and a phospholipid carrier to a buffer solution, mixing and incubating, and then preparing the carrier. The incubation temperature is 4℃-37℃, and the time is 1-10 min. This invention achieves rapid coating of annexin at a suitable temperature and within a short time, solving problems such as decreased protein activity, damage to the physicochemical properties of nanocarriers, and difficulty in rapid on-site application of formulations caused by high-temperature and long-term incubation in traditional preparation processes.

Description

Technical Field

[0001] This invention relates to the field of drug delivery system technology, and in particular to a method for preparing an annexin-coated phospholipid carrier and its application. Background Technology

[0002] Phospholipid nanocarriers, as important drug delivery systems, have broad application potential in cancer treatment and other disease treatments. In recent years, with the gradual elucidation of transvascular barrier transport mechanisms (such as enhanced transcytosis (ETR) mechanisms), annexin has been used to improve the tissue entry efficiency and therapeutic efficacy of phospholipid nanocarriers by mediating transcytosis and transendothelial tumor entry. Previous studies have shown that coating phospholipid nanocarriers with annexin can significantly enhance their tumor entry efficiency and promote drug accumulation at the lesion site, thereby improving therapeutic effects.

[0003] However, existing methods for preparing annexin-coated nanocarriers rely on high incubation temperatures (37°C) and long incubation times (1 hour). This not only easily leads to structural instability and decreased functional activity of annexin, but may also affect the physicochemical properties of the nanocarriers and drugs. Furthermore, the complex and time-consuming procedures cannot meet the clinical application requirements for rapid preparation, immediate use, and compatibility with temperature-sensitive drugs, thus limiting the application of annexin coating technology in real-world medical scenarios.

[0004] Therefore, there is an urgent need to develop a simplified preparation process for coating annexin under milder and shorter conditions to ensure the stability of the protein structure and nanomedicine, while significantly improving ease of operation, time efficiency, and clinical applicability. This invention proposes an innovative solution based on these needs to overcome the limitations of existing technologies. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for preparing annexin-coated phospholipid carriers and its application. This invention achieves rapid annexin coating at a suitable temperature and within a short time, solving problems such as decreased protein activity, damaged physicochemical properties of nanocarriers, and difficulty in rapid on-site application of formulations caused by high-temperature and prolonged incubation in traditional preparation processes. This invention aims to significantly simplify the preparation process and improve operational convenience while ensuring annexin coating efficiency, making annexin-coated phospholipid nanocarriers more suitable for on-the-go preparation and bedside loading needs in clinical settings, thereby enhancing its application feasibility and clinical promotion value.

[0006] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing an annexin-coated phospholipid carrier, the method comprising adding annexin and a phospholipid carrier to a buffer solution, mixing and incubating, and then obtaining the coating. The incubation temperature is 4℃-37℃, and the time is 1-10 min. The 4℃-37℃ can be, for example, 4℃, 5℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, or 37℃. The 1-10 min can be, for example, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, or 10 min.

[0007] The core innovation of this invention lies in the discovery that the adsorption process of annexin on the surface of nanocarriers is temperature-insensitive and exhibits extremely rapid kinetics, thus overcoming the technical bias of traditional methods that require long-term incubation at 37°C. An unexpected technical phenomenon is that the adsorption process of annexin A2 on the surface of liposomes exhibits temperature insensitivity and rapid kinetics. Specifically, within a wide temperature range of 4°C to 37°C, effective adsorption equilibrium can be reached with only a short incubation of 1-10 minutes, which contradicts the traditional understanding held by those skilled in the art that "protein-liposome nanoparticle adsorption requires long periods and thermodynamic driving."

[0008] This invention creatively discovers that, over a wide temperature range, especially under conditions without artificial heat sources, the preparation time for coating annexin with phospholipid carriers can be reduced from 1 hour to less than 10 minutes through simple physical mixing. This reduces the complexity of clinical procedures, significantly shortens patient drug administration preparation time, and expands the application of this technology in clinical scenarios requiring rapid drug delivery. The short incubation time at lower temperatures reduces the risk of annexin denaturation, and the applicable temperature range is wider. Compared to the constant temperature of 37°C in existing technologies, this invention eliminates the need for specialized temperature control equipment, enhancing the clinical applicability of this technology.

[0009] Preferably, the incubation temperature is 4-25℃. For example, it can be 4℃, 5℃, 10℃, 15℃, 20℃, or 25℃, etc.

[0010] Preferably, the annexin comprises any one or a combination of at least two of recombinant human Annexin A2 protein, annexin A1, annexin A10, annexin A11, annexin A13, annexin A3, annexin A4, annexin A5, annexin A6, annexin A7, annexin A8, annexin A8L1, annexin A8L2, or annexin A9.

[0011] The biological basis of this invention lies in the fact that A2 protein, as a phospholipid-binding protein, binds to the liposome membrane primarily through interactions of specific structural domains, rather than through a temperature-driven diffusion process. This unique binding mechanism allows the protein coating process to be completed rapidly at ambient temperatures, while maintaining the protein's structural integrity and biological activity.

[0012] Based on the above findings, this invention establishes a novel "short-duration, wide-temperature-range" technology, overcoming the disadvantages of existing technologies that require heating at 37°C and long incubation periods of one hour. It demonstrates that effective coating of A2 protein does not require active heating at 37°C and can be efficiently completed at room temperature (10-30°C) or even low temperatures (4°C). The incubation time is drastically reduced from "hours" to "minutes" in existing technologies. Experiments have confirmed that there is no significant difference in protein adsorption within a wide temperature range of 33°C from 4°C to 37°C. This discovery gives this method extremely strong clinical applicability.

[0013] Preferably, the phospholipid carrier comprises liposomes and / or lipid nanoparticles.

[0014] Preferably, the liposomes include any one or a combination of at least two of the following: empty liposomes, drug-loaded liposomes, PEGylated liposomes, or special functional liposomes.

[0015] Preferably, the drug-loaded liposomes include any one or a combination of at least two of doxorubicin hydrochloride liposomes, irinotecan liposomes, paclitaxel liposomes, cisplatin liposomes, or vincristine liposomes.

[0016] Preferably, the special functional liposomes include any one or a combination of at least two of thermosensitive liposomes, pH-sensitive liposomes, or magnetically responsive liposomes.

[0017] Preferably, the structure of the lipid nanoparticles includes phospholipid components, cholesterol components, PEGylated lipids, and ionizable lipids.

[0018] Preferably, the phospholipid components include any one or a combination of at least two of the following: lecithin, soybean phospholipid, phosphatidylserine, phosphatidylethanolamine, dioleoylphosphatidylethanolamine, 1,2-dimyristoyl-sn-glycerol-3-phosphate glyceride, phosphatidylinositol, phosphatidic acid, dipalmitoylphosphatidylcholine, distearylphosphatidylcholine, dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, or hydrogenated soybean phosphatidylcholine.

[0019] Preferably, the cholesterol component includes any one or a combination of at least two of cholesterol, cholesterol hemisuccinate, cholesterol oleate, or phytosterols.

[0020] Preferably, the PEGylated lipids include any one or a combination of at least two of DSPE-PEG2000, DOPE-PEG2000, or DMPE-PEG2000.

[0021] Preferably, the ionizable lipids include any one or a combination of at least two of the following: ionic and cationic lipids, permanent cationic lipids, anionic and amphoteric lipids, or targeted and functionalized lipids.

[0022] Preferably, the ionic and cationic lipids include any one or a combination of at least two of ionizable lipids, DLin-MC3-DMA, SM-102, ALC-0315, DODMA, or Dlin-DMA.

[0023] Preferably, the permanent cationic lipid includes any one or a combination of at least two of DOTAP, DOTMA, DDAB, or CTAB.

[0024] Preferably, the anionic and amphoteric lipids include cardiolipin and / or sphingomyelin.

[0025] Preferably, the targeted and functionalized lipids include DSPE-PEG-maleimide, DSPE-PEG-folic acid, DSPE-PEG-RGD, GM1, GM3, glyceryl monolaurate and / or squalene.

[0026] Preferably, the concentration of the phospholipid carrier is 1-5 mg / mL; more preferably, it is 2-3 mg / mL. The 1-5 mg / mL concentration can be, for example, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, or 5 mg / mL. The 2-3 mg / mL concentration can be, for example, 2 mg / mL, 2.2 mg / mL, 2.4 mg / mL, 2.6 mg / mL, 2.8 mg / mL, or 3 mg / mL.

[0027] Preferably, the mass ratio of annexin to phospholipid carrier is 50-1000 μg / mg; more preferably, it is 250-300 μg / mg. The 50-1000 μg / mg can be, for example, 50 μg / mg, 100 μg / mg, 200 μg / mg, 300 μg / mg, 400 μg / mg, 500 μg / mg, 600 μg / mg, 700 μg / mg, 800 μg / mg, 900 μg / mg, or 1000 μg / mg. The 250-300 μg / mg can be, for example, 250 μg / mg, 260 μg / mg, 270 μg / mg, 280 μg / mg, 290 μg / mg, or 300 μg / mg.

[0028] Preferably, the mixing and incubation method includes any one or a combination of at least two of the following: oscillating mixing, blowing mixing, stirring mixing, vortex mixing, microfluidic mixing, or dropwise mixing.

[0029] Preferably, the buffer solution includes any one or a combination of at least two of PBS buffer, sodium chloride buffer, calcium chloride buffer, or HEPES buffer.

[0030] Preferably, the buffer solution also includes a growth promoter.

[0031] Preferably, the growth promoter comprises any one or a combination of at least two of serum protein, insulin, transferrin, growth factor, ethanolamine, or sodium selenite.

[0032] Preferably, the preparation method further includes centrifugation and ultrafiltration after mixing and incubation.

[0033] Preferably, the ultrafiltration tube in the ultrafiltration step has a molecular weight cutoff of ≥100 kDa. In one specific embodiment of the present invention, the molecular weight cutoff of the ultrafiltration tube is 100 kDa.

[0034] Preferably, the centrifugal force is 6000-10000 g, the time is 20-40 min, and the temperature is 0-4℃. The 6000-10000 g can be, for example, 6000 g, 7000 g, 8000 g, 9000 g, or 10000 g. The 20-40 min can be, for example, 20 min, 25 min, 30 min, 35 min, or 40 min. The 0-4℃ can be, for example, 0℃, 1℃, 2℃, 3℃, or 4℃.

[0035] In a second aspect, the present invention provides a protein-coated liposome, wherein the protein-coated liposome is prepared by the method described in the first aspect for preparing annexin-coated phospholipid carriers.

[0036] Thirdly, the present invention provides the use of protein-coated liposomes according to the third aspect in the preparation of a drug delivery system.

[0037] Preferably, the drug delivery system is used for tumor-like lesions and / or non-tumor-like lesions.

[0038] Preferably, the tumor lesion sites include any one or a combination of at least two of the following: breast cancer, pancreatic cancer, liver cancer, ovarian cancer, osteosarcoma, prostate cancer, glioma, melanoma, myxofibrosarcoma, skin cancer, lung cancer, or gastric cancer.

[0039] Preferably, the non-tumor lesion sites include psoriasis and / or diabetes.

[0040] Compared with the prior art, the present invention has at least the following beneficial effects: 1. The preparation method of this invention significantly improves clinical operational efficiency: the incubation time for the protein coating step is drastically reduced from 1 hour in the prior art to 5 minutes, greatly shortening the preparation time before drug administration. This allows the technology to better meet the needs of clinical point-of-care drug administration, improve diagnostic and treatment efficiency, and enhance the patient's medical experience.

[0041] 2. This invention verifies the feasibility of lower incubation temperatures (4-25℃), effectively avoiding the risk of denaturation or inactivation that may occur when proteins are processed under high temperatures for extended periods, thus improving the safety of clinical drug use. It better maintains the native conformation and biological activity of the A2 protein, thereby ensuring the targeting performance and therapeutic efficacy of the final product. No specialized temperature control equipment is required, significantly reducing the complexity of clinical procedures and equipment requirements.

[0042] 3. The preparation method provided by this invention is simple and convenient to operate, making it very suitable for direct implementation in clinical pharmacies or bedside environments. The entire preparation process requires no special equipment; it only requires simple mixing followed by short-term standing within a wide temperature window, greatly promoting the clinical application and dissemination of this technology.

[0043] 4. The protein-coated liposomes prepared by the method of this invention show no statistically significant difference in protein adsorption capacity compared to existing technologies, ensuring that the core performance is not compromised. This method works effectively over a wide temperature range, indicating that the process is insensitive to changes in clinical environmental conditions, possesses excellent robustness and reproducibility, and is more suitable for stable application in changing clinical environments. Attached Figure Description

[0044] Figure 1 This is a technical roadmap for Example 1.

[0045] Figure 2 This is a flowchart illustrating the process of coating annexin onto a phospholipid carrier.

[0046] Figure 3 Electrophoresis results of proteins in the initial filtrate of each group in the experiment with optimal coating parameters.

[0047] Figure 4 The response surface plot shows the protein coating efficiency during co-incubation for 5 min as a function of protein input and temperature.

[0048] Figure 5 The response surface plot shows the protein coating efficiency during 30 min of co-incubation as a function of protein input and temperature.

[0049] Figure 6The response surface plot shows the protein coating efficiency during co-incubation for 60 min as a function of protein input and temperature.

[0050] Figure 7 The graph shows the protein electrophoresis results in the initial filtrate of each group in the temperature insensitivity test.

[0051] Figure 8 This is a graph showing the results of the temperature insensitivity test for protein adsorption efficiency in each group. Detailed Implementation

[0052] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0053] Example 1 This embodiment determines the optimal coating parameters through system condition filtering. This embodiment systematically investigated the effects of three key parameters—protein dosage, incubation temperature, and incubation time—on the coating efficiency of A2 protein on liposome surfaces through orthogonal experiments. The specific experimental procedure is as follows: Figure 1 As shown. The liposomes used were doxorubicin hydrochloride liposomes (Doxorubicin). ® The protein used was recombinant human Annexin A2 protein, purchased from Wuhan Kaituo Saisi Co., Ltd. (CSPC Pharmaceutical Group Co.), at a concentration of 2 mg / mL. A Box-Behnken response surface methodology was employed, with 15 experimental groups. Specific experimental conditions are shown in Table 1.

[0054] Table 1 The specific implementation process is as follows: Thaw the Annexin A2 protein solution at -80°C, centrifuge at 13,000 rpm for 10 min at 4°C, and collect the supernatant. This centrifugation step is to prevent protein precipitation interference. After diluting 10 times, the protein concentration was measured to be 1.717 mg / mL using the BCA method. Add 5.823 μL, 14.558 μL, and 29.116 μL of protein solution to Protein LowBind EP tubes with corresponding protein input volumes of 10, 25, and 50 μg. Adjust the volume of each protein solution to 50 μL using 1×PBS buffer pre-chilled at 4°C.

[0055] 50 μL of doxorubicin hydrochloride liposomes (concentration 2 mg / mL) and 50 μL of the corresponding mass of the prepared A2 protein solution were mixed in a Protein LowBind EP tube. Each group was then incubated with shaking at 200 rpm under the conditions shown in Table 1 to prepare protein-coated liposomes. The specific procedure is as follows: Figure 2 As shown.

[0056] After incubation, each group of solutions was brought to a final volume of 200 μL using 1×PBS buffer pre-chilled at 4°C. The solution was then added to an ultrafiltration tube with a molecular weight cutoff of 100 kDa, and centrifuged at 4°C and 8000g for 30 min. The supernatant from the first ultrafiltration was collected. Subsequently, the chopped liposomes were resuspended in 200 μL of 1×PBS buffer pre-chilled at 4°C by pipetting. This ultrafiltration and resuspension process was repeated three times to completely remove free protein. The chopped liposomes in the ultrafiltration tube were then collected, thus obtaining A2 protein-coated liposomes.

[0057] Qualitative and semi-quantitative analysis of the A2 protein-coated liposomes was performed using SDS-PAGE. The ultrafiltered liposome solution was brought to a final volume of 70 μL. 20 μL of 10% SDS and 10 μL of Tris-HCl (pH = 8.8) were added to each tube. The tubes were boiled in a metal bath at 100°C for 5 min, incubated at room temperature for 5 min, and then 33.33 μL of 4× Loading Buffer was added. The tubes were incubated at room temperature for 5 min, and then boiled in a metal bath for another 10 min. Gel electrophoresis was then performed at 140 V for 40 min, and the protein was stained using a rapid silver staining kit. The staining results are shown below. Figure 3 As shown.

[0058] Precise protein quantification was performed using the BCA method. The concentration of free protein in the initial ultrafiltration supernatant was determined using a micro-BCA protein assay kit. The free protein mass was calculated as: free protein concentration × supernatant volume; coated protein mass = protein input mass - free protein mass; protein coating efficiency = coated protein mass / protein input mass. The protein coating efficiencies of each group were fitted into a Box-Behnken design to obtain the response surface curves of protein coating efficiency as a function of protein input mass, temperature, and time. Figure 4 , Figure 5 and Figure 6 As shown.

[0059] The results above show that the protein coating efficiency is not sensitive to temperature in the temperature range of 4°C to 37°C, and the group with an input protein amount of 25 μg is closest to the peak value shown in the coating efficiency fitting curve.

[0060] These results demonstrate that the adsorption of Annexin A2 protein onto the surface of liposomes is a temperature-insensitive and extremely rapid process. Protein coating of liposomes does not require the traditional long incubation at 37°C; it can be completed in just 5 minutes at room temperature or even 4°C. This provides experimental evidence to support the "short-duration" conditions for protein adsorption onto liposomes as claimed in this invention.

[0061] Example 2 This embodiment directly verifies the coating temperature insensitivity. Using the method described in Example 1, the effects of 4℃, 25℃, and 37℃ on the coating amount of A2 protein were directly compared under fixed protein dosage and incubation time to verify the effectiveness of wide-temperature-range coating (4-37℃). The liposomes used were doxorubicin hydrochloride liposomes (Doxorubicin). ® The protein was recombinant human Annexin A2 protein, purchased from Wuhan Kaituo Sais Biotechnology Co., Ltd. (CSPC Pharmaceutical Group Co.). The concentration was 2 mg / mL.

[0062] The preparation method of Example 1 was used, with a protein input of 25 μg; an incubation time of 5 min; and incubation temperatures of 4°C, 25°C, and 37°C. To ensure the reliability of the evidence, three parallel replicates were set for each temperature condition.

[0063] The specific experimental procedure is as follows: 14.558 μL of Annexin A2 protein solution with a concentration of 1.717 mg / mL (corresponding to a protein mass of 25 μg) was added to a Protein LowBind EP tube. The protein solutions of each group were then brought to a final volume of 50 μL using 1× PBS buffer pre-cooled at 4°C. 50 μL of doxorubicin hydrochloride liposomes (concentration of 2 mg / mL) was mixed with 50 μL of the above A2 protein solution in a Protein LowBind EP tube. Subsequently, each group was incubated with shaking at 200 rpm under the conditions shown in Table 2. Ultrafiltration was then performed using the method in Example 1 to prepare protein-coated liposomes.

[0064] Table 2 The method described in Example 1 was used, and SDS-PAGE was employed for detection. Specific results are as follows: Figure 7 As shown. Precise protein quantification was performed using the BCA method. The concentration of free protein in the initial ultrafiltration supernatant was determined using a micro-BCA protein assay kit. The free protein mass was calculated as: Free protein mass = Free protein concentration × Supernatant volume; Coated protein mass = Protein input mass - Free protein mass. The protein adsorption efficiencies for each group are shown in the figure. Figure 8 As shown.

[0065] The hydrodynamic particle size distribution of the obtained protein-coated liposomes was uniform, as determined by a particle size analyzer, with a polydispersity index (PDI) of less than 0.2, indicating that this preparation method can form a well-monodispersed nanoparticle system. Zeta potential measurements showed that the surface potential of the coated liposomes did not change significantly compared to the uncoated liposomes, suggesting that the coating process of A2 protein on the liposome surface may have achieved tight adsorption through specific orientation or conformation, without significantly altering the overall charge characteristics of the liposome surface. This helps maintain the stability of the formulation in the physiological environment. The results are shown in Table 3.

[0066] Table 3 Quantitative analysis using the BCA method showed that the protein adsorption reached over 70% of the saturation value at three temperatures: 4℃, 25℃, and 37℃, with good batch-to-batch repeatability (relative standard deviation less than 5%). This directly confirms that the adsorption of A2 protein on liposomes is a temperature-insensitive process, providing experimental evidence for supporting the "wide temperature range" conditions for protein adsorption on liposomes as claimed in this invention.

[0067] In summary, this invention addresses the problems of decreased protein activity, impaired physicochemical properties of nanocarriers, and difficulty in rapid on-site application of formulations caused by high-temperature and prolonged incubation in traditional preparation processes, by achieving rapid coating of annexin at suitable temperatures and within a short time. This invention aims to significantly simplify the preparation process and improve operational convenience while ensuring annexin-coated phospholipid nanocarriers, making them more suitable for point-of-care preparation and bedside loading in clinical settings, thereby enhancing their application feasibility and clinical promotion value.

[0068] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for preparing a phospholipid carrier coated with annexin, characterized in that, The preparation method includes adding annexin and phospholipid carrier to a buffer solution, mixing and incubating, and then obtaining the product. The incubation temperature is 4℃-37℃, and the time is 1-10 min.

2. The method for preparing annexin-coated phospholipid carriers according to claim 1, characterized in that, The annexins include any one or a combination of at least two of the following: recombinant human Annexin A2 protein, annexin A1, annexin A10, annexin A11, annexin A13, annexin A3, annexin A4, annexin A5, annexin A6, annexin A7, annexin A8, annexin A8L1, annexin A8L2, or annexin A9.

3. The method for preparing annexin-coated phospholipid carriers according to claim 1 or 2, characterized in that, The phospholipid carrier includes liposomes and / or lipid nanoparticles; Preferably, the liposomes include any one or a combination of at least two of the following: empty liposomes, drug-loaded liposomes, PEGylated liposomes, or special functional liposomes; Preferably, the drug-loaded liposomes include any one or a combination of at least two of doxorubicin hydrochloride liposomes, irinotecan liposomes, paclitaxel liposomes, cisplatin liposomes, or vincristine liposomes. Preferably, the special functional liposomes include any one or a combination of at least two of thermosensitive liposomes, pH-sensitive liposomes, or magnetically responsive liposomes; Preferably, the structure of the lipid nanoparticles includes phospholipid components, cholesterol components, PEGylated lipids, and ionizable lipids; Preferably, the phospholipid components include any one or a combination of at least two of the following: lecithin, soybean phospholipid, phosphatidylserine, phosphatidylethanolamine, dioleoylphosphatidylethanolamine, 1,2-dimyristoyl-sn-glycerol-3-phosphate glyceride, phosphatidylinositol, phosphatidic acid, dipalmitoylphosphatidylcholine, distearylphosphatidylcholine, dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, or hydrogenated soybean phosphatidylcholine. Preferably, the cholesterol component includes any one or a combination of at least two of cholesterol, cholesterol hemisuccinate, cholesterol oleate, or phytosterols; Preferably, the PEGylated lipids include any one or a combination of at least two of DSPE-PEG2000, DOPE-PEG2000 or DMPE-PEG2000; Preferably, the ionizable lipids include any one or a combination of at least two of the following: ionic and cationic lipids, permanent cationic lipids, anionic and amphoteric lipids, or targeted and functionalized lipids. Preferably, the ionic and cationic lipids include any one or a combination of at least two of ionizable lipids, DLin-MC3-DMA, SM-102, ALC-0315, DODMA, or Dlin-DMA. Preferably, the permanent cationic lipid includes any one or a combination of at least two of DOTAP, DOTMA, DDAB, or CTAB; Preferably, the anionic and amphoteric lipids include cardiolipin and / or sphingomyelin; Preferably, the targeted and functionalized lipids include DSPE-PEG-maleimide, DSPE-PEG-folic acid, DSPE-PEG-RGD, GM1, GM3, glyceryl monolaurate and / or squalene.

4. The method for preparing annexin-coated phospholipid carriers according to any one of claims 1-3, characterized in that, The concentration of the phospholipid carrier is 1-5 mg / mL; more preferably 2-3 mg / mL; Preferably, the mass ratio of annexin to phospholipid carrier is 50-1000 μg / mg; more preferably, it is 250-300 μg / mg.

5. The method for preparing annexin-coated phospholipid carriers according to any one of claims 1-4, characterized in that, The mixing and incubation methods include any one or a combination of at least two of the following: oscillating mixing, blowing mixing, stirring mixing, vortex mixing, microfluidic mixing, or dropwise mixing.

6. The method for preparing annexin-coated phospholipid carriers according to any one of claims 1-5, characterized in that, The buffer solution includes any one or a combination of at least two of PBS buffer, sodium chloride buffer, calcium chloride buffer, or HEPES buffer. Preferably, the buffer solution further includes a growth promoter; Preferably, the growth promoter comprises any one or a combination of at least two of serum protein, insulin, transferrin, growth factor, ethanolamine, or sodium selenite.

7. The method for preparing annexin-coated phospholipid carriers according to any one of claims 1-6, characterized in that, The preparation method further includes centrifugation and ultrafiltration after mixing and incubation; Preferably, the ultrafiltration tube in the ultrafiltration step has a molecular weight cutoff of ≥100 kDa; Preferably, the centrifugal force is 6000-10000 g, the time is 20-40 min, and the temperature is 0-4℃.

8. A protein-coated liposome, characterized in that, The protein-coated liposomes are prepared by the method described in any one of claims 1-7 for preparing annexin-coated phospholipid carriers.

9. The application of the protein-coated liposomes according to claim 8 in the preparation of drug delivery systems.

10. The application according to claim 9, characterized in that, The drug delivery system is used for tumor lesion sites and / or non-tumor lesion sites; Preferably, the tumor lesion sites include any one or a combination of at least two of the following: breast cancer, pancreatic cancer, liver cancer, ovarian cancer, osteosarcoma, prostate cancer, glioma, melanoma, myxofibrosarcoma, skin cancer, lung cancer, or gastric cancer. Preferably, the non-tumor lesion sites include psoriasis and / or diabetes.

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