Platelet-rich plasma exosome gel as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, and discloses a platelet-rich plasma exosome gel preparation method, which comprises: carrying out exosome separation on a PRP sample by using a high-speed centrifuge; stuffing the exosome precipitate into the shell-sphere structure to prepare core-shell structure particles so as to coat the exosome and form a thermal insulation layer; mixing thrombin and calcium gluconate according to a mass ratio of 10: 1, and adding the mixture into PRP to prepare a gel matrix; adding a photoinitiator, the magnetic coloring particles and the core-shell structure particles into the gel matrix, and uniformly mixing to prepare exosome gel; the invention further discloses application of the composition in repairing diabetic foot wounds. According to the invention, the irreversible influence of temporary environment change on the system can be weakened, meanwhile, the double mechanisms of healing are disclosed, during application, secondary arrangement is carried out by using a magnetic field, the release efficiency of PRP-Exo is improved under the later photo-thermal cooperation effect, and accurate formulation of a long-term treatment strategy is facilitated.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to a platelet-rich plasma exosome gel, its preparation method and application, and more particularly to a magnetic-controlled photothermal-responsive platelet-rich plasma exosome gel based on a core-shell structure, its preparation method and its application in the repair of diabetic foot wounds. Background Art

[0002] Diabetic foot ulcers (DFUs) are a serious complication that poses a major threat to the limbs of diabetic patients and may lead to systemic damage or even death. For patients with diabetic foot ulcers, due to the immune microenvironment disorder caused by hyperglycemia (such as persistent inflammation and angiogenesis disorder), existing treatment means (such as conventional PRP gels) have problems such as uncontrollable growth factor release, inconsistent preparation standards, susceptibility to drugs, poor patient compliance, large individual differences in efficacy, and unclear action mechanisms.

[0003] In particular, due to the multiplicity of the pathogenesis of DFUs, the latest research shows that the obstacle of macrophage transformation from the M1 to the M2 phenotype is the key factor leading to the formation of chronic refractory ulcers in DFUs. In the medical field, platelet-rich plasma-derived exosomes (PRP-Exos) have great therapeutic potential in promoting the healing of DFUs. However, in the existing technology, the application of PRP gels still has certain limitations, which are specifically manifested as follows:

[0004] I. Uncontrollable release of active ingredients and insufficient stability:

[0005] Existing PRP gels (such as CN102573943A) are activated by calcium chloride or type I collagen. Although they can form a gel structure, the release rate of growth factors cannot be precisely regulated, resulting in unpredictable efficacy. In addition, in the existing technology, nanofiber dressings (such as CN117503984A) use copper sulfide to compound with PRP to enhance antibacterial properties, but their core-shell structure relying on electrospinning can only delay platelet release and cannot solve the problem of exosome (Exos) inactivation at room temperature.

[0006] II. Technical limitations in the application of exosomes:

[0007] Although exosomes have significant potential in tissue repair, existing technologies are mostly limited to stem cell-derived exosomes (such as CN106727700A), which are costly to prepare and difficult to standardize. More critically, current exosome gels (such as CN117503984A) do not form a synergistic effect with PRP and lack a specific regulatory mechanism for the immune microenvironment of diabetic wounds (such as macrophage polarization M1→M2), resulting in low angiogenesis efficiency.

[0008] III. Lack of controlled release technology:

[0009] In the prior art, the release of PRP or exosome gel relies on passive diffusion or simple temperature response (such as gelatin melting), and it is impossible to achieve precise delivery to the lesion site. For example, CN102573943A activates PRP gel through type I collagen, but its release cannot be regulated again after curing. In addition, the existing solutions do not integrate magnetic field or photothermal response design, making it difficult to achieve on-demand release and adapt to long-term treatment strategies. SUMMARY OF THE INVENTION

[0010] The purpose of the present invention is to provide a platelet-rich plasma exosome gel, its preparation method and application, so as to solve the problems that the active ingredient release of PRP gel is uncontrollable and the immune microenvironment cannot be regulated in the treatment of diabetic wounds, and at the same time provide a technical solution of a PRP-Exos composition with low cost and standardized production and its stability application.

[0011] To solve the above technical problems, the present invention specifically provides the following technical solutions:

[0012] The present invention provides a preparation method of a platelet-rich plasma exosome gel, comprising the following steps:

[0013] S100. Separating exosomes from the PRP sample by using a high-speed centrifuge to obtain exosome precipitate;

[0014] S200. Inserting the exosome precipitate into a shell sphere structure with a particle size of 0.01 - 0.2 mm to prepare core-shell structure particles. The shell sphere structure can coat the exosomes and form a thermal insulation layer, and the shell sphere structure melts and breaks at a temperature of 30 - 35 °C;

[0015] S300. Mixing thrombin and calcium gluconate in a mass ratio of 10:1 and adding them to PRP to prepare a gel matrix;

[0016] S400. Cooling the gel matrix to 0 - 15 °C at a cooling rate of 5 °C / min, adding a photoinitiator, magnetic coloring particles and the core-shell structure particles to the cooled gel matrix, and mixing evenly to prepare an exosome gel derived from PRP.

[0017] As a preferred embodiment of the present invention, in step S200, the following steps are further included:

[0018] Filling sterile PBS into the core-shell structure particles so that the exosomes are suspended in the shell sphere structure.

[0019] As a preferred embodiment of the present invention, the shell sphere structure is composed of gelatin material.

[0020] As a preferred embodiment of the present invention, in step S400, the preparation method of the magnetic coloring particles is as follows:

[0021] Spray a biocompatible dye on the magnetic nanoparticles to obtain the magnetic coloring particles.

[0022] As a preferred embodiment of the present invention, the particle size of the coloring particles is 500 - 1000 nanometers.

[0023] In addition, the present invention also provides a platelet-rich plasma exosome gel prepared by the above-mentioned preparation method of the platelet-rich plasma exosome gel. Under an external magnetic field environment, the magnetic coloring particles and the core-shell structure particles in the exosome gel can be rearranged to form an upper and lower double-layer structure. When the exosome gel with the double-layer structure is cured, the core-shell structure particles located at the bottom layer are melted, so that the thermal insulation layer is damaged to release exosomes.

[0024] Furthermore, the present invention also provides an application of the above-mentioned platelet-rich plasma exosome gel. When repairing Wagner grade II-IV diabetic foot wounds, the exosomes inhibit the polarization of macrophages at the diabetic wound site to the M1 phenotype and promote the transformation of macrophages to the M2 phenotype to promote angiogenesis in diabetic wounds.

[0025] The present invention has the following beneficial effects compared with the prior art:

[0026] In the present invention, by inserting PRP-Exo into the shell sphere structure, a microenvironment for heat preservation and insulation of PRP-Exo is formed within the shell sphere structure, maintaining the microenvironment in which it is located within a certain limit and weakening the irreversible influence of temporary environmental changes on it. The present invention for the first time reveals that PRP-Exos accelerate healing through the dual mechanisms of regulating macrophage polarization (M1→M2) and promoting angiogenesis. In specific applications, secondary alignment is carried out using the magnetic field effect, and the release efficiency of PRP-Exo is improved under the combined action of photothermal in the later stage;

[0027] The present invention optimizes the technical bottlenecks of easy inactivation and uncontrollable release of exosomes through "core-shell structure thermal insulation + magnetic field synergy", strengthens its anticipation for diabetic wound healing, and facilitates the accurate formulation of long-term treatment strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only exemplary, and for those of ordinary skill in the art, without creative efforts, other implementation drawings can be obtained according to the provided drawings.

[0029] Figure 1 This is a schematic flow chart of the preparation method of platelet-rich plasma exosome gel provided by the present invention.

[0030] Figure 2 This is a chart showing different blood components in PRP and whole blood samples provided by the present invention;

[0031] Figure 3 This is a schematic diagram of the mechanism of action of PRP-Exo regulating macrophage polarization to promote wound healing provided by the present invention;

[0032] Figure 4 This is a schematic diagram showing that platelet-rich plasma-derived exosomes (PRP-Exos) accelerate the healing of diabetic mouse ulcers by promoting tissue regeneration and reducing inflammation provided by the present invention;

[0033] Figure 5 This is a schematic diagram showing that PRP-Exos accelerate the healing of ulcer tissues in diabetic mice by promoting the polarization of macrophages to the M2 type. Detailed implementation manners

[0034] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0035] As Figure 1 shown, the present invention provides a preparation method of platelet-rich plasma exosome gel, including the following steps:

[0036] (1) Preparation of auxiliary materials: Iron oxide nanoparticles are synthesized by the co-precipitation method, and a flesh-colored dye is sprayed on the iron oxide nanoparticles to obtain magnetic colored particles, and the particle size of the magnetic colored particles is 500-1000 nanometers;

[0037] (2) Preparation of platelet-rich plasma exosomes (PRP-Exo): The PRP sample is separated by a high-speed centrifuge to obtain exosome precipitate;

[0038] (3) Establishment of microenvironment: The exosome precipitate is divided into several parts, and the exosomes are stuffed into a plurality of shell sphere structures composed of gelatin materials in a specific amount to obtain core-shell structure particles. The shell sphere structure can coat the exosomes and form a thermal insulation layer. The particle size of the core-shell structure particles is 0.01-0.2 millimeters, and the melting temperature of the shell sphere structure is 30-35 °C;

[0039] (4) Preparation of gel matrix: Thrombin and calcium gluconate are mixed in a mass ratio of 10:1 and added to PRP to prepare the gel matrix;

[0040] (5) Preparation of the drug: The gel matrix is cooled to 0 - 15°C at a cooling rate of 5°C / min, and the photoinitiator, magnetic coloring particles, and the core-shell structure particles are added to the gel matrix and mixed evenly to prepare the exosome gel.

[0041] In step (3), several shell sphere structures need to be prepared in advance. This shell sphere structure has a certain thickness and a vacancy is provided in the center. PRP-Exo can be injected into the shell sphere structure through injection without breaking the shell to ensure the integrity of the shell sphere structure itself. Such core-shell structure particles are theoretically a microenvironment isolated from the external environment. The shell sphere structure in the core-shell structure particles can protect the PRP-Exo inside to a certain extent, isolate the entry of heat, and reduce the inactivation rate of PRP-Exo.

[0042] Especially, this core-shell structure can melt rapidly after the temperature rises. After the outer layer of the core-shell structure particles is damaged, the internal PRP-Exo can be slowly released, thus realizing the slow release effect of PRP-Exo on the patient's skin surface. It not only extends the storage period of PRP-Exo, but also can protect its drug efficacy from being affected by the coating.

[0043] The shell sphere structure is composed of gelatin material. The optional preparation steps of the gelatin material are as follows:

[0044] (1) Preparation of gelatin solution:

[0045] Weigh 5 - 10 parts by weight of gelatin and place it in a beaker, then add 80 - 100 parts by weight of distilled water. First, let it swell at room temperature for 30 min, and then stir it in a 50°C constant temperature water bath until the gelatin is completely dissolved. After filtering to remove impurities, a gelatin solution with a concentration range of 10% - 20% w / v is obtained;

[0046] (2) Preparation of shell sphere structure:

[0047] The gelatin solution and glutaraldehyde are added to a container, stirred and stored at a low temperature of 0 - 10°C for 2 - 5 hours, and then stirred in a 30 - 70°C constant temperature water bath for 4 hours. The uniformly mixed liquid is poured into a shell sphere mold and allowed to flow naturally, and the mixed liquid is dried at room temperature to obtain a shell sphere structure of gelatin material crosslinked with glutaraldehyde solution.

[0048] The dosage of glutaraldehyde is 0.01% - 0.06% of the total weight of the gelatin solution, and the shell sphere structure of gelatin material can melt at 30 - 35°C.

[0049] During specific use, the storage condition of this gel is 0°C - 80°C, and it needs to be thawed in advance before use. In the exosome gel without the protection of the "microenvironment", after the gel is taken out, the gel at the edge of the container will absorb heat in advance, which will cause the exosomes to be inactivated quickly. However, the exosomes of the present invention are placed in a shell-sphere structure, and the shell-sphere structure can keep the exosomes warm to a certain extent, reduce the entry of heat, and thus delay the decline rate of the activity of the exosome gel.

[0050] The exosome gel disclosed in the present invention can reduce the influence of temperature rise on exosomes during drug transfer and storage, so as to delay the service life of the gel. Even if the drug has been opened, the core-shell structure can also protect the internal active substances to a certain extent and reduce the influence of bacteria on exosomes.

[0051] Since the shell-sphere structure is composed of a gelatin structure, the injection point of the shell-sphere structure can be locally heated and resealed by melting, without affecting the sealing performance of the whole shell-sphere structure.

[0052] Further, during injection, PRP-Exo can be dissolved in sterile PBS with a concentration of 0.1M and a pH of 7.4 in advance. The exosomes can be suspended in the sterile PBS, and then this mixed liquid is filled into the shell-sphere structure. An ultrasonic instrument is used to perform ultrasonic vibration on the shell-sphere structure during the loading process.

[0053] The core-shell structure particles are stored by quick freezing, and the storage temperature of the quick-frozen core-shell structure particles is lower than 0°C. After the whole gel is "frozen", the sterile PBS can further protect PRP-Exo after freezing, that is, a double-layer heat preservation structure is formed, and the heat preservation effect is further enhanced.

[0054] During the production, storage, transportation and use of the currently applied exosome gel, in order to maintain the activity of the product, the whole chain process needs to be stored and transported in an environment of 0°C to -80°C. When the exosome gel is taken out of the low-temperature environment, the activity of its product is extremely easy to be affected, thus affecting the stability of the product performance. Moreover, in the storage, transportation and even use scenarios of multiple links, the product will inevitably be temporarily taken out of the low-temperature environment, and there is a large problem of activity reduction, resulting in the imbalance of the product performance stability. When the product is affected by the environment, even if it is stored again, its product performance will be greatly affected, that is, the influence of the environment on the product performance is irreversible.

[0055] PRP-Exos can be cryopreserved and the preparation process is standardized. Utilizing the industrialization advantages, its stability can be better than that of traditional PRP gels. In the present invention, by stuffing PRP-Exo into the shell sphere structure, a microenvironment for heat preservation and insulation of PRP-Exo is formed inside the shell sphere structure. The multi-layer shell structure can, to a certain extent, keep the exosomes warm. Even when the gel leaves the storage environment, the microenvironment can play a role in heat insulation within a certain period of time, thereby delaying the decline rate of the active substances in the exosome gel. At the same time, the active substances are separately sealed to, to a certain extent, protect the internal active substances and delay the service life of the gel.

[0056] On the exosomes in the platelet-rich plasma exosome gel, there are multi-layer protection structures. Such structures can help with the heat preservation of PRP-Exo, delay the thawing time during transfer storage, and thus protect its activity. However, this will cause the drug to require a long thawing time during use. If some of the core-shell structures cannot be opened, it will instead affect the release process of PRP-Exo.

[0057] In order to eliminate the problem of difficult release caused by overprotection of PRP-Exo, the gel of the present invention contains a photoinitiator, such as UV glue. After the gel is extruded, the gel is irradiated with an ultraviolet lamp. The photoinitiator will undergo a chemical reaction in seconds under ultraviolet irradiation, releasing heat. The rapid release of heat can quickly melt the core-shell structure, making the core-shell structure become liquid, and PRP-Exo is quickly released, solving the problem of low release efficiency of PRP-Exo.

[0058] In the present invention, through magnetic particles and photoinitiator, after applying an external magnetic field, the flow of the gel is driven, so that the core-shell structure particles are arranged at a position away from the magnetic field, that is, attached to the skin at the application site, reducing the movement path of PRP-Exo after release, helping PRP-Exo to quickly aggregate on the surface of the attachment site, and at the same time releasing heat during lamp curing, making the core-shell structure become liquid, solving the problem of low release efficiency of PRP-Exo.

[0059] As a cleavage-type initiator, the photoinitiator can absorb the ultraviolet quanta emitted by a strong ultraviolet lamp, thereby initiating polymerization crosslinking and grafting reactions, and forming a solid film within a fraction of a second for liquids, such as 1173, 184, 907, 369, 1490, 1700, etc.

[0060] The concentration of the photoinitiator in the gel is 0.1% - 1.0% w / w. If the entire gel becomes solid, it will also affect the release of PRP-Exo.

[0061] Preferably, the concentration of the photoinitiator in the gel is 0.2% - 0.8% w / w.

[0062] To solve this problem, the gel of the present invention contains a quantitative amount of magnetic coloring particles, and the preparation method of the magnetic coloring particles is as follows:

[0063] Iron oxide nanoparticles are synthesized by the co-precipitation method, and the surface of the iron oxide nanoparticles is modified with chitosan to obtain composite particles;

[0064] Natural pigments β-carotene, beetroot red, safflower yellow, and titanium white are combined in a specific ratio to form a biocompatible dye for flesh color;

[0065] Adjust the parameters of the spraying machine, and spray the biocompatible dye on the surface of the composite particles at a pressure of 0.5 - 1.0 MPa and a temperature of 25 - 30 °C. The biocompatible dye is covalently coupled to the surface of the composite particles to form magnetic coloring particles.

[0066] The biocompatible dye is prepared by mixing FDA-approved edible pigments and titanium white, and has good biological safety, enabling it to directly contact the human body.

[0067] After coloring, the diameter of the magnetic particles is between 500 - 1000 nanometers, which enables them to be uniformly dispersed in the exosome gel and form a specific arrangement structure when subjected to magnetic force.

[0068] After the gel is extruded, spread the gel flat on the affected area. After spreading, apply an external magnetic field to the gel with a magnetic sheet. The magnetic coloring particles are rearranged under the influence of the external magnetic field and move to the surface of the entire gel to form a coloring layer. After the coloring layer is formed, irradiate the gel with an ultraviolet lamp to protect the fluidity at the bottom of the gel.

[0069] The functions of the coloring layer are as follows:

[0070] (1) During the formation process, it can drive the flow of the gel, causing the core-shell structure particles to be arranged at a position far from the magnetic field, that is, fitting on the skin at the application site, reducing the movement path after the release of PRP-Exo, and helping PRP-Exo to quickly aggregate on the surface of the fitting site;

[0071] (2) The coloring layer is closely arranged and can block ultraviolet rays from entering the bottom, preventing the entire gel material from being cured to a certain extent;

[0072] (3) The coloring layer is flesh-colored and can be used as a covering to cover the affected area of the patient. If the affected area of the patient is on the exposed skin (such as the face), it can be used as a cosmetic to a certain extent.

[0073] During the lamp irradiation process, the lamp irradiation time is 2 - 3 s to prevent the gel from being overly cured.

[0074] In the present invention, the platelet concentration in the PRP sample prepared by a blood cell separator is significantly higher than that in the whole blood sample, and its concentration is about 3.9 times that of the platelet concentration in the whole blood sample. The results are shown in Figure 2 as follows. Figure 2 Table of different blood components in PRP and whole blood samples.

[0075] In Figure 2 , A. Platelet count in whole blood and PRP; B. Red blood cell count in whole blood and PRP; C. White blood cell count in whole blood and PRP. n = 28. ****, P < 0.0001.

[0076] The present invention further provides a platelet-rich plasma exosome gel, and the usage method of this gel is as follows:

[0077] Take out the PRP, prepare the PRP-Exo gel after thawing, and apply the gel to the affected area;

[0078] Place the magnetic sheet above the applied area until the coloring layer is formed;

[0079] Remove the magnetic sheet, irradiate with an ultraviolet lamp for 2 - 3 seconds, the gel quickly releases heat and releases exosomes. The exosomes promote the healing of the affected area, and at the same time, the surface of the coloring layer solidifies to cover the wound. The following provides an example to illustrate the treatment mechanism of the platelet-rich plasma exosome gel on diabetic foot wounds. The active ingredient in the platelet-rich plasma exosome gel is mainly PRP-Exo. The PRP-Exo regulation mechanism is shown in Figure 3 as follows.

[0080] It can be seen from Figure 3 that the differentially expressed microRNA in PRP-Exo can bind to the target protein, promote the polarization of macrophages from the M1 type to the M2 type, and then promote neovascularization and wound re-epithelialization, and participate in the healing of DFU. The present invention clarifies the role of PRP-Exo in regulating macrophage polarization in the repair of DFU wounds and reveals its specific molecular mechanism, providing a new theoretical and experimental basis for the biological treatment of DFU, and at the same time providing new ideas for the treatment of DFU.

[0081] Currently, the mechanism of exosome gel in diabetic wound healing is not clear, and it is unable to specifically regulate the immune microenvironment, resulting in insufficient or unpredictable efficacy in diabetic wound healing. The efficacy has a certain randomness, which affects the formulation of long-term treatment strategies.

[0082] In the following experiment, a digital camera was used to measure the ulcer area every 3 days, and the wound closure rate was calculated. ImageJ software was used to analyze the wound area measurement images.

[0083] The animal model of diabetic skin wound and its treatment are as follows:

[0084] Adult male Balb / c mice (6-8 weeks old, 26-28 g, grade: clean, license number: SCXK2023-005) were purchased from Nanjing Qinglongshan Laboratory Animal Center and housed in a specific pathogen-free (SPF) mouse colony.

[0085] Mice were housed in a controlled environment with a 12-hour light / dark cycle and maintained at 22 ± 2°C. Standard rodent chow and free access to drinking water were provided. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ, 50 mg / kg) for 5 consecutive days. Blood glucose levels were monitored after 4 weeks, and mice with blood glucose levels consistently above 16.8 mmol / L were considered to have a successful diabetic model. Serum glucose levels were measured using a Roche glucometer.

[0086] Subsequently, a full-thickness skin ulcer with a diameter of 6 mm was created on the back of the mice as a diabetic wound ulcer model.

[0087] (1) Preparation of PRP-Exos

[0088] ①PRP preparation: PRP was separated using a fully automatic blood cell separator (COM.TEC; FreseniusKabi, Germany);

[0089] ② The PRP sample was centrifuged at 3000 × g for 15 minutes at 4°C to remove cell debris. The supernatant was then centrifuged at 10,000 × g for 30 minutes at 4°C to remove large particles. Finally, the supernatant was ultracentrifuged at 100,000 × g for 70 minutes at 4°C to collect exosomes. The exosome pellet was resuspended in PBS and centrifuged again at 100,000 × g for 70 minutes to wash the exosomes.

[0090] (2) PRP-Exos identification

[0091] The isolated PRP exosomes were identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western Blot (WB). TEM showed that the exosomes were often cup-shaped or spherical, with a double-layer membrane structure, and were mostly round vesicles with a diameter of about 100 nm ( Figure 4 A). NTA detection showed that the average particle size of the nanoparticles was 105 nm ( Figure 4 B). WB results confirmed that PRP-Exos expressed highly efficient exosome markers, namely Alix, CD63, CD81 and Flot-1 ( Figure 4 C). These results collectively confirm the existence of PRP-Exos.

[0092] (3) In vivo efficacy of PRP-Exos

[0093] ① The significant efficacy of PRP-Exos in promoting epithelial regeneration and collagen remodeling

[0094] PRP exosomes were identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot (WB) methods. TEM showed that exosomes were often cup-shaped or spherical, with a double-membrane structure, mostly round vesicles, and a diameter of about 100 nm( Figure 4 A). NTA detection showed that the average particle size of nanoparticles was 105 nm( Figure 4 B). WB results confirmed that PRP-Exos expressed high levels of exosome markers, namely Alix, CD63, CD81, and Flot-1( Figure 4 C). These results together confirmed the existence of PRP-Exos.

[0095] A wound model simulating human full-thickness skin defects was established in a streptozotocin (STZ)-induced diabetic mouse model. The study included two treatment groups: the PRP gel group and the PRP-Exos group. Digital photographs of the wound healing process were recorded at days 0, 5, 7, 9, and 11 after treatment, and the wound area was quantified using ImageJ software.

[0096] Compared with the control group, wound healing in the PRP-Exos group was significantly accelerated( Figure 4 D). By day 11, the wounds in the PRP-Exos group were almost completely re-epithelialized, showing a significant wound-healing effect of this treatment( Figure 4 E, F).

[0097] For histological evaluation, wound samples were stained with hematoxylin and eosin (H&E) at day 7 after treatment to evaluate the formation of new epithelium. Compared with the control group, epithelial regeneration in the PRP-Exos group was significantly enhanced( Figure 4 G). In addition, collagen deposition and organization were evaluated by Masson's trichrome staining at day 14. The PRP-Exos group showed more extensive collagen deposition, characterized by thick and wavy collagen fibers and a well-organized extracellular matrix, superior to the control PRP gel group( Figure 4 H).

[0098] The above description of the significant efficacy of PRP-Exos in promoting epithelial regeneration and collagen remodeling indicates its therapeutic potential in diabetic wound healing.

[0099] ② PRP-Exos accelerates the healing of diabetic foot ulcer wounds by promoting angiogenesis and M2 macrophage polarization

[0100] Immunofluorescence (IF) staining was used to illustrate the effect of PRP-Exos on angiogenesis on day 14. CD31 is a marker specifically expressed on the surface of endothelial cells, while α-smooth muscle actin (α-SMA) is localized in vascular smooth muscle cells. Both are reliable indicators of vascular network formation. In the IF analysis, the formation of new blood vessels was evaluated by quantifying the number of CD31-positive and α-SMA-positive blood vessels within each wound. Quantitative analysis showed that the PRP-Exos group exhibited significantly higher angiogenic activity compared to the PRP gel group, highlighting its superior effect in vascular regeneration ( Figure 5 A). The regulatory mechanism of PRP-Exos on the healing of ulcer wounds in diabetic mice was further elucidated by performing IF analysis on the collected ulcer tissues on day 11. The results showed that the number of cells co-positive for F4 / 80 (a macrophage marker) and Arg-1 (an M2 macrophage marker) was significantly increased in the PRP-Exos group compared to the PRP gel group. The results indicate that PRP-Exos effectively promotes the polarization of macrophages towards the anti-inflammatory M2 phenotype, thus significantly accelerating the healing process of ulcer wounds in diabetic mice ( Figure 5 B).

[0101] ③ PRP-Exos inhibits the transformation of M0 macrophages into the pro-inflammatory M1 phenotype

[0102] Starting from the isolation of bone marrow-derived macrophages, a comprehensive in vitro culture system was established ( Figure 5 C) to further clarify the mechanism by which PRP-Exos regulates macrophage polarization. Through systematic experimental analysis, it was found that PRP-Exos regulates macrophage polarization by inhibiting the transformation of M0 macrophages into the pro-inflammatory M1 phenotype and promoting their differentiation into the anti-inflammatory M2 phenotype, as Figure 5 shown in D.

[0103] The present invention for the first time reveals that PRP-Exos accelerates healing through the dual mechanisms of regulating macrophage polarization (M1→M2) and promoting angiogenesis. The present invention for the first time proposes that PRP-Exos accelerates the healing of diabetic foot ulcer wounds by inhibiting the transformation of M0 macrophages into the pro-inflammatory M1 phenotype, promoting angiogenesis and promoting macrophage polarization into M2 macrophages, shows a more significant effect in promoting diabetic wound healing than simply using PRP, promotes angiogenesis and blood vessel formation in diabetic wounds, clarifies the mechanism of action, and provides a novel and effective treatment strategy for DFUs.

[0104] The present invention proposes that PRP exosome gel exhibits a dual regulatory effect of regulating macrophage polarization and promoting blood vessel regeneration during the wound healing process. In the early stage, PRP exosome gel reduces inflammation by inhibiting M1 macrophages; in the later stage, it promotes the polarization of macrophages into the M2 phenotype, thereby accelerating tissue repair and creating a favorable microenvironment for regeneration. PRP exosomes (PRP-Exos) show a more significant effect in promoting diabetic wound healing than simply using PRP, and it successfully promotes angiogenesis and blood vessel formation in diabetic wounds, thus accelerating the healing process. It can be seen that PRP-Exos is a novel and effective treatment strategy for DFUs, providing solid theoretical support for future clinical translation.

[0105] Through the preparation method of platelet-rich plasma exosome gel in this embodiment and its application in repairing diabetic foot wounds, multiple sealing protections for the active substance exosomes can be achieved, making them not easily affected by temperature rise during storage or temporary handling, slowing down the rate of activity decline. At the same time, through operations such as magnetic attraction and curing during the use of the gel, the exosomes can be quickly released to the joint between the gel and the skin, improving the release efficiency. Through the synergistic effect of the "thermal insulation layer + magnetic control orientation" dual technology, the activity of exosomes is effectively maintained and their precise release at the lesion site is ensured.

[0106] The above embodiments are only exemplary embodiments of the present application and are not used to limit the present application. The protection scope of the present application is defined by the claims. Those skilled in the art can make various modifications or equivalent replacements within the essence and protection scope of the present application, and such modifications or equivalent replacements should also be regarded as falling within the protection scope of the present application.

Claims

1. A preparation method of platelet-rich plasma exosome gel, characterized in that, The method includes the following steps: S100: Separating exosomes from the PRP sample using a high-speed centrifuge to obtain exosome precipitate; S200: Stuffing the exosome precipitate into a gelatin shell sphere structure with a particle size of 0.01 - 0.2 mm to prepare core-shell structure particles. The shell sphere structure can coat the exosomes and form a thermal insulation layer, and the melting temperature of the shell sphere structure is 30 - 35 °C; S300: Mixing thrombin and calcium gluconate in a mass ratio of 10:1 and adding them to PRP to prepare a gel matrix; S400: Cooling the gel matrix to 0 - 15 °C, adding a photoinitiator, magnetic coloring particles and the core-shell structure particles to the cooled gel matrix, and mixing evenly to prepare an exosome gel derived from PRP.

2. The preparation method of a platelet-rich plasma exosome gel according to claim 1, characterized in that, In step S200, the following steps are further included: Filling 0.1M sterile PBS with a pH of 7.4 into the core-shell structure particles so that the exosomes are suspended in the shell sphere structure.

3. The preparation method of a platelet-rich plasma exosome gel according to claim 1, wherein the shell sphere structure is composed of a gelatin material.

4. The preparation method of a platelet-rich plasma exosome gel according to claim 1, wherein, In step S400, the preparation method of the magnetic coloring particles is as follows: Synthesizing iron oxide nanoparticles by the co-precipitation method, and performing surface modification on the iron oxide nanoparticles with chitosan to obtain composite particles; Spraying a biocompatible dye on the composite particles to prepare the magnetic coloring particles.

5. The preparation method of a platelet-rich plasma exosome gel according to claim 1, wherein the particle size of the coloring particles is 500 - 1000 nanometers.

6. The preparation method of a platelet-rich plasma exosome gel according to claim 1, wherein The photoinitiator is selected from Irgacure 1173, 184, 907, 369, 1490, 1700; The concentration of the photoinitiator in the exosome gel is 0.2% - 0.8% w / w.

7. A platelet-rich plasma exosome gel, characterized in that, Prepared by the preparation method of the platelet-rich plasma exosome gel according to any one of claims 1 - 6, in an external magnetic field environment, the magnetic coloring particles and the core-shell structure particles in the exosome gel can be rearranged to form an upper and lower double-layer structure. When curing the exosome gel with the double-layer structure, the core-shell structure particles at the bottom layer are melted, so that the thermal insulation layer is destroyed to release exosomes.

8. Use of the platelet-rich plasma exosome gel according to claim 7, characterized in that, When repairing Wagner grade II - IV diabetic foot wounds, the exosomes inhibit the polarization of macrophages at the diabetic wound site to the M1 phenotype and promote the transformation of macrophages to the M2 phenotype, so as to promote angiogenesis and vasculogenesis in diabetic wounds.

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