Phosphatidylserine-modified EGCG liposome, preparation method and application of phosphatidylserine-modified EGCG liposome in bone infection resistance

Abstract

The invention belongs to the technical field of medical materials, and particularly relates to a phosphatidylserine modified EGCG liposome, a preparation method and application of the phosphatidylserine modified EGCG liposome in bone infection resistance. The epigallocatechin gallate is entrapped in the liposome, a lipid bilayer of the epigallocatechin gallate comprises phosphatidylserine, phosphatidylcholine and cholesterol, and the molar ratio of the phosphatidylserine to the phosphatidylcholine to the cholesterol is controlled to be (1-2): 2: 1. The PS-EGCG liposome is prepared based on a film hydration method, the process is simple and convenient, and the obtained liposome is uniform in particle size, good in dispersity and stable in Zeta potential, can be effectively ingested by macrophages, plays the functions of inhibiting bacterial proliferation and regulating immune response, and is expected to be applied to treatment of infectious bone defects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a phosphatidylserine-modified EGCG liposome, its preparation method, and its application in anti-bone infection. Background Technology

[0002] Infectious bone defects refer to bone damage or defects caused by infection of bone tissue by bacteria, fungi, or other microorganisms. This infectious lesion may involve the bone marrow, cortical bone, or joints, and is usually accompanied by inflammatory response and tissue damage. Common causes include severely contaminated open fractures, postoperative infection after internal fixation of closed fractures, and hematogenous osteomyelitis.

[0003] The core of clinical treatment for infected bone defects includes two main tasks: infection control and bone defect reconstruction. The key to infection control is surgical debridement to remove infected tissue, supplemented by antibiotic treatment—which is mainly divided into two categories: local application of antibiotic-impregnated bone cement and systemic antibiotic administration.

[0004] However, current antibiotic treatments have significant limitations: even standard drugs like vancomycin for treating Staphylococcus aureus infections (including methicillin-resistant Staphylococcus aureus (MRSA)) are showing diminishing efficacy against resistant strains; and high-dose administration only provides a temporary increase in local drug concentration and often produces cytotoxic effects on host cells. Furthermore, current antibiotic treatments only aim to eliminate the pathogens, while a severe inflammatory microenvironment often exists within the defect area, leading to delayed bone loss due to prolonged inflammation.

[0005] Epigallocatechin gallate (EGCG), a natural tea extract, is widely recognized for its anti-inflammatory and antibacterial effects, thanks to its low cost and mature extraction process. It can also exert targeted anti-MRSA effects by inhibiting MRSA biofilms, demonstrating significant therapeutic potential. However, EGCG itself suffers from inherent defects such as chemical instability, rapid metabolism in vivo, and low bioavailability, hindering its clinical translation—making it difficult to achieve targeted enrichment and long-term efficacy in the complex infection microenvironment, thus preventing the full realization of its therapeutic value. Summary of the Invention

[0006] In view of this, and in order to fill the aforementioned technical gaps in the field, the present invention aims to provide a phosphatidylserine-modified EGCG liposome, its preparation method, and its application in anti-bone infection. In a first aspect, the present invention provides a phosphatidylserine-modified EGCG liposome. The liposome encapsulates epigallocatechin gallate (EGCG), and its lipid bilayer comprises phosphatidylserine (PS), phosphatidylcholine (PC), and cholesterol.

[0007] Suitablely, the molar ratio of phosphatidylserine, phosphatidylcholine, and cholesterol is (1~2):2:1. This ratio range is at least able to balance lipid membrane stability and drug encapsulation efficiency.

[0008] Preferably, the molar ratio of phosphatidylserine, phosphatidylcholine, and cholesterol is 2:2:1. Within this ratio range, liposomes exhibit a more uniform particle size distribution and enhanced phosphatidylserine PS modification effect.

[0009] Suitablely, the average hydrated particle size of the liposomes is 20–60 nm. Compared with the commonly used particle size of 100–200 nm, the liposome particle size of less than 60 nm can moderately delay its “premature” phagocytosis by macrophages, which helps the liposomes to be locally retained and release the active ingredient (EGCG) in a targeted manner.

[0010] Suitablely, the zeta potential of the liposomes is -30 mV to -50 mV. This negative potential helps to enhance the colloidal stability of the liposome dispersion system.

[0011] Phosphatidylserine (PS) on the surface of liposomes acts as an "eat-me" signal, which can be recognized by macrophages with high density infiltration at the site of bone infection, thereby enabling active targeting of liposomes.

[0012] In a second aspect, the present invention provides a method for preparing the phosphatidylserine-modified EGCG liposomes.

[0013] The method is based on thin-film hydration and homogenization treatment, and includes the following steps: Phosphatidylserine, phosphatidylcholine and cholesterol are dissolved in a suitable organic solvent, and after the solvent is removed, a uniform lipid film is formed on the inner wall of the container. The lipid film was hydrated using an aqueous solution containing EGCG to obtain a crude liposome suspension; the crude liposome suspension was then homogenized to obtain liposomes with uniform particle size.

[0014] Suitablely, the hydration process is carried out under oscillation conditions of 37-45°C and 100-300 rpm for 0.5-2 hours. These conditions are beneficial for the full hydration and recombination of the lipid film and its encapsulation of EGCG.

[0015] Suitablely, the homogenization process includes extruding the hydrated crude liposome suspension through a polycarbonate membrane with a pore size of 50-200 nm.

[0016] More preferably, the crude liposome suspension can be pretreated with short-term ultrasonication before extrusion and membrane processing.

[0017] In a third aspect, the present invention provides a drug delivery system.

[0018] To achieve controlled drug release at the site of infection, this invention combines the aforementioned liposomes with a pH-responsive hydrogel to form a composite delivery system. The system comprises: a) PS-modified EGCG liposomes as active units; b) A pH-responsive chitosan hydrogel as a carrier unit. The liposomes are encapsulated within the hydrogel.

[0019] Suitablely, the pH-responsive chitosan hydrogel is chitosan or a derivative thereof.

[0020] Under normal physiological conditions, the chitosan hydrogel maintains structural stability and can effectively retain liposomes; Considering the slightly acidic environment of MRSA infection sites, and taking into account that the inflammation induced by infection can inhibit osteoblast activity, promote osteoclast differentiation, and delay bone healing, a liposome was constructed by combining phosphatidylserine (PS), a product of programmed apoptosis that can be specifically recognized by macrophages, with EGCG. This liposome was then encapsulated in a pH-responsive chitosan hydrogel to achieve high-concentration release of EGCG to kill MRSA in the slightly acidic environment of the early stage of infection.

[0021] In a fourth aspect, the present invention provides a bone repair material with anti-infection function.

[0022] The bone repair material includes a bone repair scaffold as a structural support and the aforementioned drug delivery system as a bioactive functional unit. The drug delivery system is coated on the surface of the scaffold or loaded as a filler within the pore structure of the scaffold.

[0023] Suitablely, the material of the bone repair scaffold may be selected from any one or a combination of the following: bioinert metals, bioceramics, or biodegradable polymers.

[0024] Optionally, the bio-inert metal includes titanium and titanium alloys as well as medical stainless steel, the bioceramic includes but is not limited to hydroxyapatite or calcium phosphate, and the biodegradable polymer includes but is not limited to polylactic acid or polycaprolactone.

[0025] In a fifth aspect, the present invention provides the use of the liposomes, drug delivery system and bone repair material in the preparation of products for treating bone infections.

[0026] Suitablely, the bone infection is caused by Staphylococcus aureus.

[0027] More preferably, the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA).

[0028] The product exerts its therapeutic effect through one or more of the following mechanisms: a. The released EGCG can directly inhibit or kill pathogens and destroy their biofilm structure.

[0029] b. EGCG and PS work synergistically to regulate the polarization of macrophages at the site of infection from the pro-inflammatory M1 phenotype to the anti-inflammatory and repair-promoting M2 phenotype, thereby alleviating excessive inflammatory response and creating a favorable microenvironment for tissue repair.

[0030] The significant advantages of this invention compared to the prior art are as follows: (1) This invention successfully obtained PS-EGCG liposomes with uniform particle size distribution (approximately 20-60 nm), stable zeta potential (approximately -42.8 mV), high encapsulation efficiency (>82%), and good dispersibility by limiting the molar ratio of phosphatidylserine, phosphatidylcholine, and cholesterol to (1~2):2:1 and using a thin-film hydration method. This effectively solves the problems of instability and easy metabolism of EGCG itself, providing a stable carrier for efficient drug delivery.

[0031] (2) In this invention, phosphatidylserine (PS) modification enables liposomes to be specifically recognized and efficiently internalized by macrophages, and upregulates the expression of phagocytosis-related receptors. The loaded EGCG has significant inhibitory and killing effects on pathogens such as MRSA at a safe concentration (≤50 μM). More importantly, EGCG successfully delivered into cells can effectively drive inflammatory macrophages in the infection lesion to convert to the anti-inflammatory repair M2 type, thereby achieving active regulation of the immune microenvironment.

[0032] (3) The present invention further provides a pH-responsive delivery system and bone repair material comprising the above-mentioned liposomes. By encapsulating liposomes in chitosan hydrogel, the system can rapidly release drugs in response to the slightly acidic environment of the infection site, achieving early intensive treatment; after infection control, it switches to a gradual release to maintain a long-lasting effect. This provides a comprehensive solution for treating infectious bone defects caused by Staphylococcus aureus (such as MRSA), which combines local antibacterial, immunomodulatory, and bone repair support functions. Attached Figure Description

[0033] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0034] Figure 1The images shown are transmission electron microscope (TEM) images of the liposomes prepared in Examples 1 to 3, showing their lipid bilayer vesicle structure and particle size distribution.

[0035] Figure 2 The results show the drug encapsulation efficiency analysis of the liposomes prepared in Examples 1 to 3.

[0036] Figure 3 The results of zeta potential analysis are for the PS-EGCG liposomes prepared in Example 2.

[0037] Figure 4 The Tyndall effect diagram is shown in the dynamic light scattering experiment of the PS-EGCG liposomes prepared in Example 2 in PBS.

[0038] Figure 5 The effects of different concentrations of PS-EGCG liposomes on the viability of bone marrow mesenchymal stem cells (BMSCs) and RAW264.7 macrophages were investigated.

[0039] Figure 6 Fluorescence microscopy images of live / dead bacteria stained after co-incubation of PS-EGCG liposomes with different concentrations.

[0040] Figure 7 Scanning electron microscopy (SEM) images of methicillin-resistant Staphylococcus aureus and Escherichia coli after treatment with PS-EGCG liposomes.

[0041] Figure 8 The curves show the inhibition of proliferation of methicillin-resistant Staphylococcus aureus and Escherichia coli by different concentrations of PS-EGCG liposomes.

[0042] Figure 9 Confocal laser scanning microscopy images of RAW264.7 macrophages taking up DiI fluorescently labeled PS-EGCG liposomes at different time points.

[0043] Figure 10 To Figure 9 Quantitative analysis results of fluorescence intensity in macrophages.

[0044] Figure 11 The results of RT-qPCR detection show the effects of different treatment groups on the expression of phagocytosis-related receptor genes (Mertk, Trem2, Tim4, Stab2) in RAW264.7 macrophages.

[0045] Figure 12 Immunofluorescence staining images showing the effects of different treatment groups on the expression of RAW264.7 macrophage phenotypic markers CD86 and CD206.

[0046] Figure 13 To Figure 12 Quantitative analysis results of the fluorescence intensity of CD86 and CD206.

[0047] Figure 14 The results of RT-qPCR detection show the effects of different treatment groups on the expression of polarization-related cytokine genes in RAW264.7 macrophages. Detailed Implementation

[0048] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0049] The term "embodiment" as used herein means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in multiple embodiments of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a mutually exclusive, independent, or alternative embodiment. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0050] Example 1: A phosphatidylserine-modified EGCG liposome was prepared according to the following steps: Phosphatidylserine (PS), phosphatidylcholine (PC), and cholesterol (Chol) were weighed in a molar ratio of 1:2:1 and dissolved in chloroform. The resulting solution was dried under vacuum using a rotary evaporator for 2 hours to completely remove the organic solvent, forming a uniform lipid film. The lipid film was placed in a 1M EGCG solution and hydrated in a 37°C water bath at 200 rpm for 1 hour to obtain a crude liposome suspension. To improve dispersibility, the liposome suspension was dispersed using an ultrasonic homogenizer at 20 kW and 20 kHz for 10 minutes. Finally, the crude liposomes were extruded through a 100 nm polycarbonate membrane to obtain uniformly sized EGCG liposome particles.

[0051] Example 2: A phosphatidylserine-modified EGCG liposome was prepared according to the following steps: Phosphatidylserine (PS), phosphatidylcholine (PC), and cholesterol (Chol) were weighed in a molar ratio of 2:2:1 and dissolved in chloroform. The resulting solution was dried under vacuum using a rotary evaporator for 2 hours to completely remove the organic solvent, forming a uniform lipid film. The lipid film was placed in a 1M EGCG solution and hydrated in a 37°C water bath at 200 rpm for 1 hour to obtain a crude liposome suspension. To improve dispersibility, the liposome suspension was dispersed using an ultrasonic homogenizer at 20 kW and 20 kHz for 10 minutes. Finally, the crude liposomes were extruded through a 100 nm polycarbonate membrane to obtain uniformly sized EGCG liposome particles.

[0052] Example 3: A phosphatidylserine-modified EGCG liposome was prepared according to the following steps: Phosphatidylserine (PS), phosphatidylcholine (PC), and cholesterol (Chol) were weighed in a molar ratio of 3:2:1 and dissolved in chloroform. The resulting solution was dried under vacuum using a rotary evaporator for 2 hours to completely remove the organic solvent, forming a uniform lipid film. The lipid film was placed in a 1M EGCG solution and hydrated in a 37°C water bath at 200 rpm for 1 hour to obtain a crude liposome suspension. To improve dispersibility, the liposome suspension was dispersed using an ultrasonic homogenizer at 20 kW and 20 kHz for 10 minutes. Finally, the crude liposomes were extruded through a 100 nm polycarbonate membrane to obtain uniformly sized EGCG liposome particles.

[0053] The liposomes obtained in Examples 1-3 were characterized. (1) Zeta potential: detected by a nanoparticle size potentiometer ZetasizerNanoZSP.

[0054] (2) Encapsulation efficiency: Liposomes were separated from free drug by centrifugation at 10,000 rpm, and the supernatant was collected. Subsequently, the liposome precipitate was resuspended in a suitable solvent, completely dissolved in ethanol, and the concentration of free epigallocatechin gallate (EGCG) in the solution was determined by UV-Vis spectroscopy to calculate the encapsulation efficiency. (3) Particle size and morphology: Observed using a transmission electron microscope.

[0055] Please see Figure 1Transmission electron microscopy images showed that the liposomes prepared in Example 1 (PS:PC:Chol=1:2:1) and Example 2 (PS:PC:Chol=2:2:1) both exhibited clear and complete lipid bilayer vesicle structures, with particle size distributions ranging from 25-50 nm and 20-40 nm, respectively. This indicates that both formulations can successfully prepare nanoscale liposomes, and the liposomes prepared in Example 2 have smaller particle sizes, more concentrated distribution, and better uniformity.

[0056] Please see Figure 2 Further analysis of drug encapsulation efficiency showed that the drug loading efficiencies of Example 1 and Example 2 were 83.2±0.83% and 82.6±0.71%, respectively, both exhibiting high drug loading capacity with no significant difference. Given that Example 2 has a more uniform particle size distribution and the expected enhanced PS surface modification effect, the formulation of Example 2 was selected for subsequent verification experiments.

[0057] Please see Figure 3 Zeta potential analysis showed that the average surface potential of the PS-EGCG liposomes obtained in Example 2 was -42.82 ± 1.43 mV. This indicates that the PS-EGCG liposomes have a stronger negative charge on their surface, which helps to improve the colloidal stability of the nanoparticle system through electrostatic repulsion.

[0058] Please see Figure 4 Dynamic light scattering experiments performed on the liposomes of Example 2 in PBS revealed a significant Tyndall effect, indicating that they formed a highly dispersed and stable colloidal solution in an aqueous medium. This excellent colloidal stability and dispersibility promises to reduce the rapid clearance of liposomes due to non-specific aggregation in complex physiological environments, thereby improving the efficiency of delivery to target sites and bioavailability.

[0059] Example 4: Biosafety and antibacterial activity assay of PS-EGCG liposomes 1. Experimental Materials Cells: Mouse mononuclear macrophage line RAW264.7 and bone marrow mesenchymal stem cells (BMSCs) were purchased from Shanghai Beyotime Biotechnology Co., Ltd. Strains: Methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli strains were purchased from Beyotime Biotechnology Co., Ltd.

[0060] PS-EGCG liposomes: The preparation method is as described in Example 2, wherein the molar ratio of phosphatidylserine (PS), phosphatidylcholine (PC), and cholesterol (Chol) is 2:2:1.

[0061] 2. Experimental Methods 2.1 Cytotoxicity assay (CCK-8 assay) BMSCs and RAW264.7 cells in the logarithmic growth phase were seeded in 96-well plates at a density of 5 × 10³ cells per well, with a culture volume of 100 μL per well.

[0062] After the cells adhered overnight, the old culture medium was discarded. The experiment was set up with the following groups: Control group: Contains fresh complete culture medium and cells, without the addition of any liposomes.

[0063] Blank liposome group (PS): contains blank phosphatidylserine liposomes diluted with fresh complete culture medium (i.e., without EGCG, with the same lipid composition and concentration as the 50 μM PS-EGCG group).

[0064] PS-EGCG experimental group: containing PS-EGCG liposomes diluted with fresh complete culture medium (prepared in Example 2), with final concentration gradients of 0, 10, 20, 50, 75, and 100 μM.

[0065] Each group should have at least 5 duplicate wells, and incubate for another 24 hours.

[0066] After incubation, add 10 μL of CCK-8 reagent to each well and continue incubation for 2 hours. Use a microplate reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm and calculate cell viability.

[0067] 2.2 Evaluation of antibacterial activity Methicillin-resistant Staphylococcus aureus and Escherichia coli were revived in culture medium and cultured to the logarithmic growth phase, and the bacterial concentration was adjusted to approximately 1 × 10⁻⁶. 8 CFU / mL available for use.

[0068] The two bacteria were co-incubated with different concentrations (10 μM, 50 μM and 100 μM) of PS-EGCG liposomes for 2 hours.

[0069] The bacterial suspension was treated with a live / dead bacteria staining kit, and stained with SYTO-9 and propidium iodide (PI) for 15 minutes in the dark.

[0070] Take the stained bacterial solution onto a glass slide and immediately observe and photograph it using a fluorescence microscope or confocal microscope. Green fluorescence indicates live bacteria, and red fluorescence indicates dead bacteria.

[0071] 2.3 Scanning electron microscopy observation Different concentrations (10 μM, 50 μM and 100 μM) of PS-EGCG liposomes were co-incubated for 2 hours with methicillin-resistant Staphylococcus aureus and Escherichia coli. The bacteria were collected by centrifugation, fixed with glutaraldehyde overnight, dehydrated by ethanol gradient, dried and sputter-coated with gold, and the surface morphology of the bacteria was observed by scanning electron microscopy.

[0072] 2.4 Bacterial proliferation inhibition test: Log-phase methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli) bacterial suspensions were diluted with fresh complete culture medium and the following groups were set up: Control group: Contains fresh complete culture medium and cells, without the addition of any liposomes.

[0073] Blank liposome group (PS): contains blank phosphatidylserine liposomes diluted with fresh complete culture medium (i.e., without EGCG, with the same lipid composition and concentration as the 50 μM PS-EGCG group).

[0074] PS-EGCG experimental group: containing PS-EGCG liposomes diluted with fresh complete culture medium (prepared in Example 2), with final concentration gradients of 0, 10, 20, 50, 75, and 100 μM.

[0075] The absorbance (OD value) of each well was measured at a wavelength of 600 nm using a microplate reader at 0h, 2h, 4h, 6h, 8h, 10h, 12h, and 14h of incubation, and the data were recorded.

[0076] 3. Experimental Results Please see Figure 5 When the concentration of PS-EGCG liposomes was as low as 50 μM, the viability of bone marrow mesenchymal stem cells (BMSCs) and RAW264.7 cells remained almost 100%; this indicates that within this concentration range, PS-EGCG liposomes did not exhibit significant toxicity to either of the two key cell types, demonstrating excellent biocompatibility. This result proves that the PS-EGCG liposomes prepared in this invention can be safely used for subsequent cell research at a set safe dose (≤50 μM), providing important safety evidence for its application as a biomaterial. However, concentrations exceeding 50 μM induce dose-dependent cytotoxicity, indicating that excessive liposome exposure may impair cell viability.

[0077] Please see Figure 6Live / dead staining revealed a large number of viable bacteria (green fluorescence) in the control group and 10 μM PS-EGCG liposomes, indicating limited bactericidal efficacy at low concentrations. In contrast, at 50 μM, the number of dead bacteria (red fluorescence) increased significantly, indicating a strong antibacterial effect at this concentration and above.

[0078] Please see Figure 7 Scanning electron microscopy revealed that in the control group, the cell surfaces of both MRSA and E. coli remained intact and smooth. However, after exposure to 50 μM PS-EGCG liposomes, the morphology of MRSA underwent significant changes, including cell membrane shrinkage, disruption, and partial dissolution, indicating severe damage. These phenomena are likely due to polyphenol-induced intracellular oxidative stress, which disrupts cell membrane integrity.

[0079] Please see Figure 8 The proliferation inhibition assay showed that neither the control group (Control) nor the blank liposome group (PS) inhibited bacterial growth, while the increase in PS-EGCG liposome concentration limited bacterial proliferation. Notably, the proliferation of methicillin-resistant Staphylococcus aureus (MRSA) was more significantly inhibited compared to Escherichia coli (E. coli).

[0080] In summary, these results demonstrate that the PS-EGCG liposomes provided by this invention have a stable and uniform structure and exhibit effective inhibition of methicillin-resistant Staphylococcus aureus (MRSA) within a safe concentration range, showing potential role as a regulator in the early infection environment.

[0081] Example 5: Detection of the regulatory effect of PS-EGCG liposomes on macrophages 1. Experimental Materials and Methods 1.1 Experimental Materials Same as in Example 4, all other materials are conventional commercially available products.

[0082] 1.2 Macrophage uptake of PS-EGCG experiment DiI fluorescently labeled PS-EGCG liposomes were prepared using the same method as in Example 2 above, except that DiI dye was added during the lipid film hydration stage. RAW264.7 macrophages were seeded into confocal culture dishes. After adhesion, 50 μM of DiI-labeled PS-EGCG liposomes were added and incubated for 10, 30, 60 and 120 min respectively.

[0083] Unuptaken liposomes were removed by washing with pre-cooled PBS, fixed with 4% paraformaldehyde, and the nuclei were stained with DAPI. Intracellular fluorescence signals were observed by confocal laser scanning microscopy, and the average fluorescence intensity was quantified using ImageJ software.

[0084] 1.3 Detection of phagocytosis-related receptor and cytokine gene expression RAW264.7 macrophages were set into the following groups: Control group: Contains only culture medium, without the addition of any liposomes.

[0085] Unmodified liposomes (Lip): containing blank liposomes diluted with culture medium (i.e., without PS).

[0086] PS-EGCG group (PS-Lip): contains PS-EGCG liposomes diluted with complete culture medium (prepared in Example 2, concentration controlled at 50 μM).

[0087] Total RNA was extracted from each group 24 hours after treatment, and reverse transcribed into cDNA. The expression of phagocytic receptor genes Mertk, Trem2, Tim4, and Stab2 was then detected.

[0088] 1.4 Polarization-related gene detection To evaluate the regulatory effect of PS-EGCG on the inflammatory phenotype of macrophages, the following experiments were conducted: RAW264.7 macrophages were divided into four groups for culture and intervention: Control group: No LPS stimulation was used; cultured only in complete culture medium.

[0089] Model group (LPS): The M1 model was established by stimulating the medium with 10 ng / mL LPS for 24 h.

[0090] Unencapsulated liposome group (LPS+PS): After stimulation with LPS, the culture medium was replaced with blank phosphatidylserine liposomes (i.e., without EGCG, with a lipid concentration similar to the experimental group).

[0091] PS-EGCG experimental group (LPS+PS-EGCG): After LPS stimulation, the culture medium was replaced with PS-EGCG liposomes (prepared in Example 2, with the concentration controlled at 50 μM).

[0092] After all the above interventions were continued for 24 hours, the expression of M1 markers (TNF-α, IL-6) and M2 markers (IL-4, IL-10) was detected by RT-qPCR.

[0093] 1.5 Immunofluorescence detection of macrophage phenotypic markers After the cells were fixed, permeabilized, and blocked following the grouping intervention in Example 5, Section 1.4, anti-CD86 primary antibody and anti-CD206 primary antibody were added and incubated overnight at 4°C. Then, fluorescent secondary antibody was added and incubated at room temperature in the dark for 1 hour. After DAPI staining of the nuclei, the cells were observed under a confocal microscope, and the fluorescence intensity was quantified using ImageJ.

[0094] 2. Experimental Results Please see Figure 9 Confocal imaging showed that in the PS-EGCG uptake experiment by macrophages, the fluorescence intensity within the macrophages gradually increased with prolonged co-culture time. Please see [link to related documentation]. Figure 10 Quantitative analysis showed that the fluorescence intensity reached a stable state during the 120-minute co-incubation period, indicating that macrophages achieved maximum liposome uptake in a relatively short time.

[0095] Please see Figure 11 RT-qPCR results showed that, compared with the blank control group (Control) and the unmodified liposome group (Lip), the expression of Mertk, Trem2, Tim4 and Stab2 genes in the PS-EGCG group (PS-Lip) was significantly upregulated, verifying the effectiveness of PS-EGCG in promoting the effective recognition and phagocytosis of macrophages.

[0096] Please see Figure 12 Immunofluorescence showed that CD86 signal was significantly enhanced in the model group (LPS), while CD86 expression was significantly decreased and CD206 expression was significantly upregulated in the PS-EGCG group (LPS+PS-EGCG). Please refer to [link to relevant documentation]. Figure 13 Quantitative analysis further validated this trend, indicating that PS-EGCG can reverse LPS-induced M1 polarization and promote macrophage conversion to the M2 phenotype.

[0097] Please see Figure 14 RT-qPCR confirmed that TNF-α and IL-6 gene expression was downregulated and IL-4 and IL-10 gene expression was upregulated in the PS-EGCG group (LPS+PS-EGCG). This result further confirms at the gene transcription level that PS-EGCG not only inhibits the pro-inflammatory response of macrophages but also actively activates their anti-inflammatory and tissue repair programs, clarifying its role in driving the conversion of macrophage functional phenotypes from M1 to M2 at the molecular level.

[0098] The above results indicate that the PS-EGCG liposomes provided by this invention can be effectively taken up by macrophages, enhance their phagocytic function by upregulating phagocytosis-related receptors, and reprogram LPS-induced M1 pro-inflammatory macrophages into M2 anti-inflammatory / repair phenotypes, demonstrating their potential as an immune regulator.

[0099] Example 6: A composite bone repair scaffold for the treatment of infected bone defects and its preparation method. Combining the above-mentioned PS-EGCG liposomes with pH-responsive carriers and bone repair scaffolds includes: Pretreatment of bone repair scaffolds: The material of the bone repair scaffold is not particularly limited in this embodiment. In some embodiments, it can be bioceramics such as hydroxyapatite and β-tricalcium phosphate. In other embodiments, it may also be a biodegradable polymer such as polylactic acid, polycaprolactone, or a composite material thereof; Clean and dry the bone repair scaffold before use to ensure good adhesion of the subsequent coating.

[0100] Construction of pH-responsive chitosan hydrogel coating: Chitosan was dissolved in acetic acid solution to prepare a chitosan solution with a concentration of 0.5%-2.0wt%.

[0101] The pretreated bone repair scaffold was immersed in the above-mentioned chitosan solution, and the chitosan adsorbed on the scaffold was cross-linked and cured using a cross-linking agent to form a stable hydrogel coating.

[0102] Loading PS-EGCG liposomes: The cross-linked and cleaned bone repair scaffold was immersed in the PS-EGCG liposome suspension prepared in Examples 1 and 2. After standing at a suitable temperature, the PS-EGCG liposomes were effectively captured by the hydrogel network through diffusion and electrostatic interaction, ultimately yielding a composite bone repair scaffold.

[0103] Since the composite bone repair scaffold is loaded with the PS-EGCG liposomes described in Example 1 or 2, the composite scaffold in this embodiment naturally inherits and integrates all the technical effects of the liposomes that have been proven above, so these technical effects will not be described in detail.

[0104] Most importantly, the bone repair scaffold is not a simple drug carrier; its design simulates an ideal process for repairing infected bone defects. Bone repair scaffolds provide immediate physical filling and support.

[0105] When the implantation site becomes acidic due to bacterial infection and inflammatory response, the chitosan hydrogel coating responds and swells rapidly, releasing PS-EGCG liposomes to form an effective therapeutic concentration, achieving early clearance of pathogens and timely regulation of excessive inflammation.

[0106] As the infection is controlled and the pH of the microenvironment approaches neutral, the chitosan hydrogel network remains relatively contracted, allowing for the slow release of PS-EGCG liposomes, which are close to normal physiological conditions. This property ensures that the therapeutic agent can take effect rapidly at the site of infection where it is most needed, while reducing systemic exposure, achieving site-specific enhanced drug delivery and long-lasting efficacy.

[0107] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A phosphatidylserine-modified EGCG liposome, characterized in that, The liposomes are loaded with epigallocatechin gallate. The lipid bilayer of the liposome comprises phosphatidylserine, phosphatidylcholine and cholesterol, wherein the molar ratio of phosphatidylserine, phosphatidylcholine and cholesterol is controlled at (1~2):2:

1.

2. The liposomes according to claim 1, characterized in that, The molar ratio of phosphatidylserine, phosphatidylcholine and cholesterol is 2:2:

1.

3. The liposomes according to claim 1, characterized in that, The liposomes have an average hydrated particle size of 20-60 nm and a zeta potential of -30 mV to -50 mV.

4. A method for preparing phosphatidylserine-modified EGCG liposomes as described in any one of claims 1-3, characterized in that, Includes the following steps: Phosphatidylserine, phosphatidylcholine and cholesterol are dissolved in an organic solvent, and the solvent is removed to form a lipid film. The lipid membrane was hydrated with an aqueous solution containing EGCG to obtain a crude liposome suspension; the hydrated crude liposome suspension was then homogenized to obtain the liposomes.

5. The preparation method according to claim 4, characterized in that, The hydration is carried out under oscillation conditions of 37-45℃ and 100-300 rpm for 0.5-2 hours; the homogenization process includes sonicating the crude liposome suspension and then squeezing it through a polycarbonate membrane with a pore size of 50-100 nm.

6. A drug delivery system, characterized in that, Comprising: a) EGCG liposomes modified with phosphatidylserine as described in any one of claims 1-3; b) pH-responsive chitosan hydrogel; The liposomes are encapsulated in the chitosan hydrogel, forming a composite structure that can release the liposomes under a slightly acidic environment.

7. A bone repair material, characterized in that, It includes a bone repair scaffold and a drug delivery system as described in claim 6, wherein the drug delivery system is disposed on the bone repair scaffold as a functional coating or filler.

8. The bone repair material according to claim 7, characterized in that, The bone repair scaffold is made of any one of bioinert metals, bioceramics, or polymers.

9. The use of the phosphatidylserine-modified EGCG liposomes as described in any one of claims 1-3, the drug delivery system as described in claim 6, or the bone repair material as described in any one of claims 7-8 in the preparation of a product for treating bone infections caused by Staphylococcus aureus.

10. The application according to claim 9, characterized in that, The product is used to inhibit or kill Staphylococcus aureus at the site of infection and / or promote the polarization of macrophages at the site of infection toward the M2 anti-inflammatory phenotype.

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