An engineered modification method for enhancing the immunomodulatory capacity of lt-evs with egcg

Abstract

The application relates to the technical field of tissue engineering, and discloses an engineering modification method for enhancing the immune regulation capacity of LT-EVs by using EGCG, S1, liver tissue-derived extracellular vesicles (LT-EVs) are extracted from liver tissue by adopting an enzymolysis method combined with a differential centrifugation method; S2, the LT-EVs are incubated with DSPE-PEG-PBA, the DSPE-PEG-PBA is inserted into the membrane of the LT-EVs and presents a phenylboronic acid group on the surface, and phenylboronic acid-modified LT-EVs are obtained; S3, the phenylboronic acid-modified LT-EVs are incubated with EGCG, a pH-responsive borate ester bond is formed between the phenylboronic acid and the catechol structure, the coupling of the EGCG and the LT-EVs is realized, and liver tissue-derived extracellular vesicles (EGCG-LT-EVs) modified by the EGCG chemical coupling are obtained; the application realizes the anti-infection effect by relying on the natural active molecule EGCG, and does not need to depend on high-concentration antibiotics.

Description

Technical Field

[0001] This invention relates to the field of tissue engineering technology, and more specifically to an engineered modification method that utilizes EGCG to enhance the immunomodulatory capacity of LT-EVs. Background Technology

[0002] In clinical practice, jawbone defects are often accompanied by bacterial infections such as Staphylococcus aureus (SA), resulting in infectious bone defects. Conventional bone repair materials are prone to failure in infected environments, and may even exacerbate the infection. While commonly used antibiotics can kill bacteria, high concentrations of antibiotics can inhibit osteoblast activity, which is detrimental to bone tissue healing. Extracellular vesicles (EVs) are non-immunogenic and biocompatible, and can serve as natural delivery carriers. Liver-derived extracellular vesicles (LT-EVs) have good osteogenic activity, but their direct antibacterial and anti-inflammatory immunomodulatory abilities are weak, making it difficult to exert stable therapeutic effects in an infected microenvironment. Existing extracellular vesicle modification methods, such as electroporation and extrusion, easily damage the vesicle membrane structure and reduce bioactivity. Antibiotic-released bone cement has drawbacks such as high heat generation, non-degradability, the need for secondary surgery for removal, and poor osteogenic induction. Therefore, developing a mild and efficient engineered modification method for LT-EVs that combines anti-infection, immunomodulatory, and osteogenic functions is of significant clinical value in solving the problem of healing infectious jawbone defects. Summary of the Invention

[0003] The purpose of this invention is to solve the above problems by designing an engineered modification method that utilizes EGCG to enhance the immunomodulatory capacity of LT-EVs.

[0004] This invention provides an engineered modification method for enhancing the immunomodulatory capacity of LT-EVs using EGCG, the method comprising the following steps: S1. Liver tissue-derived extracellular vesicles (LT-EVs) were extracted from liver tissue using an enzymatic hydrolysis method combined with differential centrifugation. S2. Incubate LT-EVs with DSPE-PEG-PBA to allow DSPE-PEG-PBA to insert into the LT-EVs film and present phenylboronic acid groups on the surface, thus obtaining phenylboronic acid modified LT-EVs. S3. The phenylboronic acid-modified LT-EVs were incubated with EGCG. The EGCG and LT-EVs were coupled by forming a pH-responsive borate ester bond between phenylboronic acid and catechol, thus obtaining EGCG-LT-EVs derived from liver tissue after chemical coupling modification with EGCG.

[0005] Optionally, in the first implementation of the present invention, in step S2, LT-EVs are diluted with pre-cooled PBS to a protein concentration of 500 μg / mL, and the mass ratio of DSPE-PEG-PBA to LT-EVs protein is 1:30 to 1:50.

[0006] Optionally, in the second implementation of the present invention, the incubation conditions in step S2 are 4°C, in the dark, and rotating incubation for 60 minutes.

[0007] Optionally, in a third implementation of the present invention, the final concentration of EGCG in the reaction system in step S3 is 10–50 μM.

[0008] Optionally, in the fourth implementation of the present invention, the incubation conditions in step S3 are 4°C, in the dark, and rotating incubation for 4 hours.

[0009] Optionally, in the fifth implementation of the present invention, the reaction systems for steps S2 and S3 are both PBS buffer solution with pH 7.4 or 1% Tris balanced salt solution.

[0010] Optionally, in the sixth implementation of the present invention, after the coupling in step S3 is completed, the reaction system is centrifuged to remove the uncoupled free EGCG in the supernatant, and the precipitate is resuspended and washed with pre-cooled PBS.

[0011] Optionally, in the seventh implementation of the present invention, the centrifugation conditions are 4°C, 16800g for 30 minutes, and the washing is performed 1 to 2 times.

[0012] Optionally, in an eighth implementation of the present invention, an EGCG-modified extracellular vesicle derived from liver tissue is prepared by any of the methods described above, and the vesicle surface is coupled with EGCG via borate ester bonds.

[0013] Optionally, in the ninth implementation of the present invention, the EGCG-modified liver tissue-derived extracellular vesicles are used in the preparation of drugs for repairing infectious bone defects.

[0014] In the technical solution provided by this invention, EGCG is stably modified onto the surface of extracellular vesicles (LT-EVs) derived from liver tissue through mild chemical coupling mediated by DSPE-PEG-PBA, achieving a synergistic effect of "anti-infection-immune regulation-osteoproliferation," overcoming the limitations of single-function methods. This invention organically combines the natural antibacterial and anti-inflammatory activities of EGCG with the inherent osteoproliferative activity of LT-EVs. In the microenvironment of infectious bone defects, EGCG can directly inhibit the proliferation of Staphylococcus aureus and disrupt the bacterial biofilm structure, while simultaneously regulating macrophage polarization towards the M2 anti-inflammatory phenotype, reducing the levels of pro-inflammatory factors such as IL-6 and TNF-α, and improving local inflammation. Microenvironment; LT-EVs continuously exert osteogenic induction effects, upregulating the expression of osteogenic-related genes and proteins such as RUNX2 and ALP, achieving simultaneous progress in infection control and bone regeneration and repair, solving the clinical pain point of traditional materials' difficulty in simultaneously achieving anti-infection and osteogenic effects; This invention adopts a combination of lipid insertion and borate ester covalent coupling, and the entire process is carried out under low temperature, neutral pH, and no severe mechanical external force conditions. It does not use strong chemical cross-linking agents, nor does it employ physical methods that easily damage the vesicle membrane such as electroporation and extrusion, which can effectively avoid LT-EVs membrane structure damage, content leakage, and loss of bioactivity. The modified vesicles have high yield, stable structure, and uniform particle size, and can be used in vivo and in vitro. Safety is significantly improved; the borate ester bond between EGCG and LT-EVs, formed by phenylboronic acid-catechol, is firmly bound under normal physiological pH, making it less prone to dissociation and non-specific shedding; it can rapidly cleave in the acidic microenvironment of the infection site, achieving targeted and controllable release of EGCG, increasing local effective drug concentration, prolonging the duration of action, and reducing problems such as easy metabolism of free EGCG, short in vivo retention time, and poor targeting; the DSPE-PEG-PBA used combines membrane anchoring, spatial stability, and specific binding capabilities. The DSPE fragment can firmly insert into the vesicle lipid membrane, and the PEG chain segment can reduce vesicle aggregation, reduce immune clearance, and prolong the duration of action. The internal circulation time allows for precise coupling of the PBA group, resulting in a highly biocompatible system with no significant cytotoxicity. The reaction conditions used in this invention are all achievable in conventional biological laboratories, including low-temperature, light-protected, and rotary incubation. Reaction parameters are well-defined, quantifiable, and easily reproducible, requiring no complex or sophisticated equipment. LT-EVs are derived from liver tissue, offering high yield, wide availability, and controllable costs. This invention relies on the natural active molecule EGCG to achieve anti-infective effects, eliminating the need for high-concentration antibiotics and avoiding side effects such as antibiotic inhibition of osteoblasts, disruption of bone healing, induction of bacterial resistance, and damage to surrounding normal tissues. This provides a safer, greener, and longer-lasting treatment pathway for infectious bone defects. Attached Figure Description

[0015] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention.

[0016] Figure 1 A flowchart of an engineered modification method for enhancing the immune regulation capacity of LT-EVs using EGCG, provided in an embodiment of the present invention. Detailed Implementation

[0017] The terms “first,” “second,” “third,” “fourth,” etc. (if present) in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a particular order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms “comprising” or “having,” and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, apparatus, product, or device that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or devices.

[0018] For ease of understanding, the specific process of the embodiments of the present invention is described below. Please refer to [link / reference]. Figure 1 The flowchart of the engineered modification method for enhancing the immune regulation ability of LT-EVs using EGCG provided in this embodiment of the invention includes the following steps: S1. Liver tissue-derived extracellular vesicles (LT-EVs) were extracted from liver tissue using an enzymatic hydrolysis method combined with differential centrifugation. S2. Incubate LT-EVs with DSPE-PEG-PBA to allow DSPE-PEG-PBA to insert into the LT-EVs film and present phenylboronic acid groups on the surface, thus obtaining phenylboronic acid modified LT-EVs. S3. The phenylboronic acid-modified LT-EVs were incubated with EGCG. The EGCG and LT-EVs were coupled by forming a pH-responsive borate ester bond between phenylboronic acid and catechol, thus obtaining EGCG-LT-EVs derived from liver tissue after chemical coupling modification with EGCG.

[0019] In this embodiment, liver tissue-derived extracellular vesicles (LT-EVs) were extracted from liver tissue using an enzymatic digestion method combined with differential centrifugation: Fresh mammalian liver tissue was taken and repeatedly rinsed with pre-cooled sterile phosphate-buffered saline (PBS) to remove residual blood and connective tissue from the surface. The liver tissue was then cut into 1mm pieces. 3The following small tissue blocks were added to a digestion solution containing collagenase IV and digested at 37°C with constant temperature shaking to fully dissociate the interstitial tissue and release extracellular vesicles. After digestion, undigested tissue residue was removed by filtration through a cell sieve to obtain the tissue digestion supernatant. The supernatant was then centrifuged sequentially at different speeds. First, centrifugation at low temperature and low speed removed intact cells, cell debris, and large tissue impurities. The supernatant was then collected by further centrifugation at low temperature and high speed to collect the precipitate. The precipitate was resuspended and washed with pre-cooled sterile PBS and purified by centrifugation again to obtain liver tissue-derived extracellular vesicles (LT-EVs) with high purity and intact structure. The protein content of LT-EVs was quantified using a BCA protein concentration assay kit for later use.

[0020] In this embodiment, LT-EVs were incubated with DSPE-PEG-PBA to allow DSPE-PEG-PBA to insert into the LT-EVs membrane and present phenylboronic acid groups on its surface, thus obtaining phenylboronic acid-modified LT-EVs. First, the LT-EVs obtained in step S1 were diluted with pre-cooled sterile phosphate-buffered saline (PBS) or 1% Tris balanced salt solution to a protein concentration of 500 μg / mL and placed in sterile centrifuge tubes for later use. Then, DSPE-PEG-PBA powder was accurately weighed at a mass ratio of 1:30 to 1:50 between DSPE-PEG-PBA and LT-EVs protein, completely dissolved in a small amount of anhydrous ethanol or sterile PBS, and added dropwise to the LT-EVs membrane. Add the solution to the s dilution buffer while gently vortexing to avoid vesicle aggregation caused by excessively high local concentrations. Place the mixture in a constant temperature environment of 4°C and incubate slowly for 60 minutes using a rotary mixer at a speed of 30-50 r / min under light-protected conditions. This allows the hydrophobic DSPE fragment in the DSPE-PEG-PBA molecule to fully insert into the lipid bilayer membrane of LT-EVs through hydrophobic interactions, while the hydrophilic PEG chain extends outward and the terminal phenylboronic acid (PBA) active group is exposed on the vesicle membrane surface, completing the membrane modification of the phenylboronic acid group. After incubation, a suspension of LT-EVs with surface-modified phenylboronic acid groups is obtained, i.e., phenylboronic acid-modified LT-EVs, which can be directly used for the next step of EGCG coupling reaction.

[0021] In this embodiment, phenylboronic acid-modified LT-EVs were incubated with EGCG in a pH 7.4 buffer system. The coupling of EGCG and LT-EVs was achieved through the formation of pH-responsive borate ester bonds between phenylboronic acid and catechol, resulting in EGCG-LT-EVs derived from liver tissue after chemical coupling modification with EGCG. The phenylboronic acid-modified LT-EVs suspension obtained in step S2 was placed in a sterile phosphate-buffered saline (PBS) or 1% Tris balanced salt solution system at pH 7.4, maintaining a stable temperature and uniform ionic strength. Sterilely filtered EGCG stock solution was added to the suspension to achieve a final EGCG concentration of 10–50 μM. The mixture was gently pipetted to ensure thorough dispersion of EGCG. The mixture was placed in a low-temperature environment of 4℃, protected from light, and continuously incubated by rotation for 4 hours. This allowed the exposed phenylboronic acid groups on the surface of LT-EVs to specifically recognize and bind to the catechol groups in the EGCG molecular structure, forming a stable borate ester covalent bond with pH-responsive characteristics. This achieved a mild and efficient site-specific coupling of EGCG on the surface of LT-EVs. After the reaction, the mixture was centrifuged at 4℃ and 16800g for 30 minutes. The uncoupled free EGCG in the supernatant was discarded. The resulting precipitate was resuspended in pre-cooled sterile PBS and washed 1–2 times. Finally, the purified precipitate was resuspended in an appropriate amount of pre-cooled PBS to obtain structurally intact and stably coupled EGCG-LT-EVs, which were stored at low temperature and protected from light for later use.

[0022] In this embodiment, EGCG, short for epigallocatechingallate, is a catechin polyphenol compound naturally found in green tea leaves. It is also the most abundant and bioactive monomeric component in green tea. Its core molecular structure contains three aromatic rings: A, B, and C. The unique catechol structure (3',4'-dihydroxyphenyl) of ring B is its key functional site. In this invention, EGCG is not simply an auxiliary additive, but rather a core functional modification tool: on the one hand, its catechol group can specifically bind to the phenylboronic acid (PBA) group to form a dynamically reversible borate bond, achieving precise coupling with LT-EVs; on the other hand, EGCG itself possesses broad-spectrum antibacterial, anti-inflammatory, antioxidant, and osteogenic activities. It can directly inhibit the proliferation of Staphylococcus aureus, disrupt bacterial biofilms, and regulate the immune microenvironment, thereby endowing engineered vesicles with a triple synergistic function of "anti-infection + immune regulation + osteogenic," significantly improving the treatment efficiency of infectious bone defects.

[0023] In this embodiment, LT-EVs, short for Liver Tissue-derived Extracellular Vesicles, are nanoscale membranous vesicles secreted and released by liver parenchymal or interstitial cells under physiological or pathological conditions. They typically range in diameter from 30 to 200 nm and possess a typical lipid bilayer membrane structure, encapsulating abundant bioactive substances such as proteins, nucleic acids (miRNA, mRNA), lipids, and metabolites. As the core substrate material and natural delivery carrier of this invention, LT-EVs offer advantages over extracellular vesicles from other sources, including wide availability, low immunogenicity, good biocompatibility, and strong inherent osteogenic activity. This invention utilizes enzymatic hydrolysis combined with differential centrifugation to extract and purify LT-EVs, obtaining high-purity, structurally intact LT-EVs. This provides a stable carrier framework for subsequent engineering modifications, ensuring the safety and efficiency of the loaded active substance (EGCG) during in vivo delivery.

[0024] In this embodiment, DSPE-PEG-PBA, short for distearylphosphatidylethanolamine-polyethylene glycol-phenylboronic acid, is a well-defined amphiphilic triblock functional lipid derivative composed of three functional segments linked by covalent bonds. The DSPE (distearylphosphatidylethanolamine) segment is a hydrophobic fatty acid chain that can efficiently insert into the lipid bilayer membrane of LT-EVs through hydrophobic interactions, forming a strong "membrane anchoring" effect to ensure the modified molecule does not detach. The PEG (polyethylene glycol) segment is a hydrophilic long chain with good water solubility and steric hindrance, effectively preventing LT-EVs aggregation and adhesion, prolonging the half-life of vesicles in vivo, reducing the probability of clearance by the mononuclear phagocytic system, and improving system stability. The PBA (phenylboronic acid) segment is the terminal active functional group, which is key to achieving EGCG targeted coupling. This lipid molecule has a mild overall design and excellent biocompatibility, serving as the core connecting bridge for constructing the "EGCG-LT-EVs" coupling system and achieving functional modification in this invention.

[0025] In this embodiment, PBA stands for phenylboronic acid, which is the terminal active functional group of the DSPE-PEG-PBA lipid molecule and the core reactive group of the chemical coupling mechanism of this invention. The phenylboronic acid group possesses unique Lewis acidity, allowing it to specifically recognize the catechol group (cis-dihydroxy) of the B ring in the EGCG molecule under neutral or weakly alkaline pH conditions (such as the pH 7.4 system described in this invention), forming a borate ester bond (BOC) through dehydration condensation. This binding method combines the stability of covalent bonds with dynamic and reversible pH response characteristics: under normal physiological neutral conditions, the borate ester bond remains stable, ensuring that EGCG is firmly attached to the vesicle surface; while in the acidic microenvironment unique to the infected lesion area, the borate ester bond breaks, thereby achieving targeted and controllable release of EGCG. The introduction of the PBA group solves the problems of weak or non-specific binding in traditional modification methods, providing a structural basis for precision treatment.

[0026] In this embodiment, PBS stands for Phosphate Buffered Saline, the most widely used isotonic buffer system in biomedical experiments. Its main components include sodium dihydrogen phosphate (NaH₂PO₄), disodium hydrogen phosphate (Na₂HPO₄), and sodium chloride (NaCl). The phosphate acid-base balance system precisely maintains a stable pH (typically 7.2-7.4), while sodium chloride provides isotonic ionic strength, mimicking the physiological environment in vivo. In this invention, PBS is used throughout the entire preparation and processing flow: for washing, resuspending, and diluting LT-EVs after extraction, maintaining the integrity and bioactivity of the vesicle membrane structure; as the main buffer medium for the EGCG coupling reaction, providing the optimal pH environment for the binding of PBA and catechol; and also for the purification and washing steps after the reaction to thoroughly remove unbound free EGCG and impurities, ensuring the purity of the final product.

[0027] In this embodiment, Tris, short for Tris(hydroxymethyl)aminomethane, is a commonly used biological buffer. Its aqueous solution is weakly alkaline and has good buffering capacity and biocompatibility. In this invention, Tris is mainly used to prepare a 1% Tris balanced salt solution as an auxiliary buffer medium for the coupling reaction of EGCG and LT-EVs. Compared with PBS, the Tris buffer system provides a milder weakly alkaline environment (pH 7.4-8.0), which is more advantageous for the moderate auto-oxidative polymerization of EGCG and its interaction with membrane proteins under certain experimental conditions (such as the one-step coupling described in Example 5). Its function is to stabilize the pH of the reaction system, avoid damage to the LT-EVs membrane structure caused by local pH fluctuations, and provide a stable and friendly chemical environment for the binding of EGCG and vesicle carriers, ensuring that the reaction proceeds efficiently and gently, and preserving the natural activity of the vesicles to the greatest extent.

[0028] In this embodiment, SA stands for Staphylococcus aureus, a Gram-positive coccus and one of the most common pathogens in clinical orthopedics, oral and maxillofacial surgery, and the treatment of infected bone defects. This bacterium is highly invasive and drug-resistant, easily forming a dense bacterial biofilm on the bone defect wound surface, thus evading the host immune system's clearance and the bactericidal effect of antibiotics, leading to persistent and chronic infections, and even serious complications such as osteomyelitis and sepsis. The therapeutic target of this invention is clearly SA infection. It utilizes the natural anti-Staphylococcus aureus activity of EGCG to disrupt its biofilm structure, combined with the bone-promoting function of LT-EVs, to achieve precise clearance of the infection focus and simultaneous repair of bone tissue. Therefore, SA is the key pathogenic microorganism defining the application scenario and technical value of this invention.

[0029] Example 1: Extraction and purification of extracellular vesicles (LT-EVs) derived from liver tissue Fresh liver tissue from SPF-grade mice was taken and placed in a sterile laminar flow hood. It was rinsed three times repeatedly with pre-cooled sterile phosphate-buffered saline (PBS) at 4°C to thoroughly remove residual blood, connective tissue, and impurities from the tissue surface. The washed liver tissue was then cut into small pieces approximately 1 mm in size. 3Small tissue fragments were transferred to centrifuge tubes, and a digestion solution containing 0.1% collagenase IV was added. The tubes were then placed in a 37°C constant temperature shaker at 120 rpm for 30 min to fully dissociate the interstitial tissue structure and release intracellular and extracellular vesicle structures. Immediately after digestion, an equal volume of complete culture medium was added to terminate the digestion reaction. The mixture was then filtered through a 70 μm sterile cell sieve to remove undigested tissue fragments, large tissue debris, and intact cells, and the supernatant was collected. The supernatant was then subjected to differential centrifugation. First, centrifugation was performed at 4°C and 300 g for 10 min to discard the precipitate and remove cellular impurities. The supernatant was then further centrifuged at 4°C and 20 rpm. Centrifuge at 00g for 20 min to remove cell debris and apoptotic bodies; take the supernatant and centrifuge again at 4℃ and 10000g for 30 min to remove large vesicles and impurities; finally, take the supernatant and ultracentrifuge at 4℃ and 120000g for 70 min, discard the supernatant and collect the bottom precipitate; resuspend the precipitate in 4℃ pre-cooled sterile PBS, and wash once more by ultracentrifugation to obtain high-purity liver tissue-derived extracellular vesicles (LT-EVs); use the BCA protein concentration assay kit to quantify the protein in LT-EVs, adjust the protein concentration to 500 μg / mL, and store at 4℃ protected from light for later use.

[0030] Example 2: Phenylboronic acid (PBA) group modification of LT-EVs The LT-EVs suspension prepared in Example 1 was diluted with sterile PBS pre-cooled at 4°C to a protein concentration of 500 μg / mL. Following a DSPE-PEG-PBA to LT-EVs protein ratio of 1:40, an appropriate amount of DSPE-PEG-PBA reagent was dissolved in a small amount of sterile PBS and slowly added dropwise to the LT-EVs suspension while gently vortexing to avoid excessive local concentrations that could cause vesicle aggregation. The uniformly mixed suspension was placed in a constant temperature environment at 4°C and incubated slowly for 60 min at 40 r / min using a rotary mixer under complete darkness. This allowed the hydrophobic DSPE fragment in the DSPE-PEG-PBA molecule to stably insert into the lipid bilayer membrane structure of LT-EVs through hydrophobic interactions, with the PEG long chain extending outwards and the terminal phenylboronic acid (PBA) active group fully exposed on the outer surface of the vesicle membrane. After incubation, LT-EVs with uniformly modified phenylboronic acid groups (PBA-LT-EVs) were obtained, which could be directly used for the next EGCG coupling reaction without further purification.

[0031] Example 3: Mild Coupling and Purification of EGCG and PBA-LT-EVs The PBA-LT-EVs suspension obtained in Example 2 was placed in a sterile PBS buffer system at pH 7.4 to maintain stable ionic strength and pH. EGCG stock solution filtered through a 0.22 μm sterile membrane was slowly added to the suspension to bring the final EGCG concentration in the reaction system to 30 μM. The mixture was gently blown to ensure uniform distribution of EGCG molecules. The reaction mixture was then incubated at 4°C under complete darkness for 4 hours to allow the PBA groups on the vesicle surface to specifically bind to the catechol structure in the EGCG molecules, forming a stable pH. Responsive borate ester bonds were used to complete the site-specific covalent coupling of EGCG on the surface of LT-EVs. After the coupling reaction was completed, the mixture was centrifuged at 16800g for 30 min at 4℃, and the supernatant was carefully discarded to remove uncoupled free EGCG. The bottom precipitate was collected, resuspended in sterile PBS pre-cooled at 4℃, and centrifuged and washed twice to completely remove residual free impurities. Finally, the purified product was resuspended in an appropriate amount of pre-cooled PBS to obtain EGCG-modified liver tissue-derived extracellular vesicles (EGCG-LT-EVs), which were stored at 4℃ in the dark for later use.

[0032] Example 4: Functional validation of EGCG-LT-EVs in an infected jawbone defect model SPF-grade mice were selected to construct a mouse model of jawbone defects caused by Staphylococcus aureus (SA) infection: 3 mm diameter circular critical bone defects were created in the bilateral mandibles of mice, and 1 × 10⁻⁶ mcg of urea solution was injected into the defect area. 6A model of infectious bone defect was established using CFU Staphylococcus aureus suspension. Mice with successfully induced bone defects were randomly divided into four groups: model group, LT-EVs group, EGCG group, and EGCG-LT-EVs group (n=6 per group). The EGCG-LT-EVs group received an injection of the EGCG-LT-EVs suspension prepared in Example 3 into the defect area. The LT-EVs group received an equal volume of unmodified LT-EVs, the EGCG group received an equal volume of free EGCG, and the model group received an equal volume of sterile PBS. Administration was repeated every 3 days for 21 consecutive days. Samples were collected on postoperative days 7, 14, and 21 for relevant indicator detection: the number of Staphylococcus aureus colonies in the defect area was detected using the plate count method, and the bacterial count was determined using an ELISA kit. The levels of IL-6 and TNF-α inflammatory factors were measured, macrophage polarization was observed using immunofluorescence staining, the expression levels of osteogenic genes RUNX2 and ALP were detected using qRT-PCR, and the bone defect repair effect was observed using Micro-CT and histological staining. The results showed that compared with the model group, LT-EVs group, and EGCG group, the number of bacteria in the defect area was significantly reduced, the level of inflammatory factors was significantly decreased, the proportion of M2 macrophages was increased, the expression of RUNX2 and ALP was significantly upregulated, and the bone defect area had more new bone and more complete trabecular structure, indicating that EGCG-LT-EVs can achieve synergistic effects of anti-infection, anti-inflammatory immune regulation, and bone regeneration and repair.

[0033] Example 5: One-step coupling preparation of EGCG-LT-EVs based on 1% Tris equilibrium salt solution Weigh Tris dry powder and components such as sodium chloride, potassium chloride, and sodium dihydrogen phosphate, dissolve them in ultrapure water, stir thoroughly to dissolve, adjust the pH to weakly alkaline, sterilize under high temperature and high pressure, and prepare a 1% Tris balanced salt solution as the reaction medium for subsequent mild coupling reactions; take the high-purity LT-EVs suspension obtained in Example 1, determine the protein concentration using a BCA protein quantification kit, and dilute the LT-EVs to a protein concentration of 100 μg / mL with the above-mentioned 1% Tris balanced salt solution; add the sterile filtered EGCG stock solution dropwise to the LT-EVs dilution, mix thoroughly by pipetting, and ensure uniform distribution of all components in the system; place the mixed reaction solution in a low-temperature environment of 4°C, wrap it in aluminum foil to protect it from light throughout the process, and incubate it at low speed for 1-2 hours. The mild, weakly alkaline environment provided by the 1% Tris balanced salt solution promotes moderate auto-oxidative polymerization of EGCG and forms stable hydrogen bonds and hydrophobic interactions with proteins on the surface of LT-EVs, achieving non-covalent and damage-free attachment of EGCG to the LT-EVs surface. After incubation, the reaction mixture is transferred to an ultracentrifuge tube and ultracentrifuged at 4°C and 120,000g for 70 min. Unbound free EGCG and small molecule impurities in the supernatant are discarded, and the bottom precipitate is collected. The precipitate is resuspended in pre-cooled sterile PBS and washed 1-2 times by ultracentrifugation to remove residual impurities and Tris solution components, finally obtaining EGCG-LT-EVs prepared in one step based on 1% Tris balanced salt solution, which is stored at 4°C in the dark for later use.

[0034] Comparative Example 1: Unmodified LT-EVs for the repair of infected jawbone defects Liver tissue-derived extracellular vesicles (LT-EVs) were extracted and purified using the enzymatic hydrolysis combined with differential centrifugation method as described in Example 1. No DSPE-PEG-PBA modification or EGCG coupling treatment was performed; the LT-EVs were simply resuspended in pre-cooled sterile PBS and the protein concentration was adjusted to 500 μg / mL. A mouse jawbone defect model induced by Staphylococcus aureus infection was constructed as described in Example 4. Mice with successfully modeled LT-EVs were randomly divided into two groups. The comparative group received a local injection of an equal volume of unmodified LT-EVs suspension at the bone defect site. The dosage, frequency of administration, observation period, rearing environment, and sampling time were consistent with those in Example 4. Tissue samples were collected from the defect area on days 7, 14, and 21 post-surgery. The same detection methods were used for colony counting, inflammatory factor detection, macrophage polarization analysis, osteogenic gene detection, and Micro-CT and histological evaluation. The results showed that unmodified LT-EVs had no direct antibacterial ability, could not destroy Staphylococcus aureus biofilm, had no significant inhibitory effect on pro-inflammatory factors such as IL-6 and TNF-α, and could not induce macrophages to polarize to the M2 anti-inflammatory phenotype. In the infection microenvironment, the upregulation of osteogenic related genes RUNX2 and ALP was limited, the amount of new bone tissue was small, the trabecular bone structure was sparse, the infection symptoms were not significantly relieved, and the bone defect repair effect was far lower than that of the EGCG-LT-EVs group prepared in Example 3.

[0035] Comparative Example 2: Free EGCG for the repair of infected jawbone defects Free EGCG solution was prepared according to the EGCG concentration used in Example 3, filtered through a 0.22 μm sterile filter membrane for sterilization, and was not coupled with LT-EVs or used with any carrier. A mouse jawbone defect model infected with Staphylococcus aureus was constructed according to Example 4. The above-mentioned free EGCG solution was injected locally into the defect area. The administration volume, administration frequency, treatment cycle and detection indicators were completely consistent with Example 4. Postoperative dynamic observation showed that free EGCG was metabolized rapidly in vivo, had a short local retention time, and weak targeted enrichment ability. It could not maintain an effective concentration in the microenvironment of infected bone defects for a long time. It only showed a transient antibacterial and anti-inflammatory effect in the early stage of administration, and the effect disappeared rapidly over time. The detection results showed that free EGCG could not continuously regulate macrophage polarization, had no stable promoting effect on the osteogenic process, and the infection in the bone defect area was prone to recurrence. The amount of new bone tissue formation was significantly lower than that in the EGCG-LT-EVs treatment group, and it could not achieve a synergistic effect of anti-infection and bone repair.

[0036] Comparative Example 3: Electroporation modification of EGCG and LT-EVs The LT-EVs extracted in Example 1 were modified with EGCG using a conventional electroporation method instead of the gentle coupling method of DSPE-PEG-PBA of the present invention: LT-EVs and EGCG were mixed and added to an electroporation cup, and electroporation was performed using conventional electroporation parameters: voltage 200–400V, capacitance 100–300μF, and resistance 100–300Ω, in an attempt to load or bind EGCG onto the surface of LT-EVs; after electroporation, unbound free EGCG was removed under the same centrifugation and washing conditions as in Example 3, obtaining EGCG modified by electroporation. CG-LT-EVs; An infectious jawbone defect model was constructed according to Example 4, and drug administration and efficacy evaluation were performed. The results showed that the strong electric field and instantaneous high voltage generated during the electroporation process severely damaged the lipid bilayer structure of LT-EVs, causing vesicle rupture, leakage of contents, uneven particle size, and a significant reduction in biological activity. The method had low EGCG binding rate and poor binding stability, and the antibacterial, anti-inflammatory, and immunomodulatory effects of the obtained products were significantly reduced. It could not effectively exert its osteogenic function in the infected environment, and the repair effect was far lower than that of EGCG-LT-EVs prepared by the mild chemical coupling method described in this invention.

[0037] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. An engineered modification method for enhancing the immunomodulatory capacity of LT-EVs using EGCG, characterized in that, The method includes the following steps: S1. Liver tissue-derived extracellular vesicles (LT-EVs) were extracted from liver tissue using an enzymatic hydrolysis method combined with differential centrifugation. S2. Incubate LT-EVs with DSPE-PEG-PBA to allow DSPE-PEG-PBA to insert into the LT-EVs film and present phenylboronic acid groups on the surface, thus obtaining phenylboronic acid modified LT-EVs. S3. The phenylboronic acid-modified LT-EVs were incubated with EGCG. The EGCG and LT-EVs were coupled by forming a pH-responsive borate ester bond between phenylboronic acid and catechol, thus obtaining EGCG-LT-EVs derived from liver tissue after chemical coupling modification with EGCG.

2. The engineered modification method for enhancing the immunomodulatory capacity of LT-EVs using EGCG as described in claim 1, characterized in that, In step S2, LT-EVs are diluted with pre-cooled PBS to a protein concentration of 500 μg / mL, and the mass ratio of DSPE-PEG-PBA to LT-EVs protein is 1:30 to 1:

50.

3. The engineered modification method for enhancing the immunomodulatory capacity of LT-EVs using EGCG as described in claim 1, characterized in that, The incubation conditions in step S2 are 4°C, in the dark, and rotating incubation for 60 minutes.

4. The engineered modification method for enhancing the immunomodulatory capacity of LT-EVs using EGCG as described in claim 1, characterized in that, In step S3, the final concentration of EGCG in the reaction system is 10–50 μM.

5. The engineered modification method for enhancing the immunomodulatory capacity of LT-EVs using EGCG as described in claim 1, characterized in that, The incubation conditions in step S3 are 4℃, in the dark, and rotating incubation for 4 hours.

6. The engineered modification method for enhancing the immunomodulatory capacity of LT-EVs using EGCG as described in claim 1, characterized in that, The reaction systems for steps S2 and S3 are both PBS buffer at pH 7.4 or 1% Tris balanced salt solution.

7. The engineered modification method for enhancing the immunomodulatory capacity of LT-EVs using EGCG as described in claim 1, characterized in that, After the coupling in step S3 is completed, the reaction system is centrifuged to remove uncoupled free EGCG from the supernatant. The precipitate is then resuspended in pre-cooled PBS and washed.

8. The engineered modification method for enhancing the immunomodulatory capacity of LT-EVs using EGCG as described in claim 7, characterized in that, Centrifugation conditions: 4℃, 16800g for 30 minutes, and washing 1-2 times.

9. The engineered modification method for enhancing the immunomodulatory capacity of LT-EVs using EGCG as described in claim 1, characterized in that, An EGCG-modified extracellular vesicle derived from liver tissue is prepared by the method described in any one of claims 1-8, wherein EGCG is coupled to the surface of the vesicle via borate ester bonds.

10. The engineered modification method for enhancing the immunomodulatory capacity of LT-EVs using EGCG as described in claim 9, characterized in that, The application of the EGCG-modified extracellular vesicles derived from liver tissue in the preparation of drugs for repairing infectious bone defects.

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