Use of platelet exosomes in treating diabetic wound healing

Abstract

This application relates to the fields of biomedicine and wound repair technology, and particularly to the application of platelet exosomes in the treatment of diabetic wound healing. This application discloses the use of platelet exosomes in the preparation of products having one or more of the following functions: 1) prevention and / or treatment of diabetic foot; 2) promotion of wound healing and / or tissue repair; 3) shortening wound healing time; 4) promoting granulation tissue growth; 5) enhancing wound angiogenesis; 6) alleviating inflammatory responses; 7) inhibiting the expression of pro-inflammatory factors; 8) promoting the expression of anti-inflammatory factors; 9) reducing MMP-9 expression; and 10) increasing MMP-2 expression. It is particularly suitable for the treatment of diabetic foot ulcers, delayed wound healing, and related complications, and also covers the use of this product in the preparation of diabetic foot treatment drugs and medical dressings.

Description

Technical Field

[0001] This application relates to the fields of biomedicine and wound repair technology, and in particular to the application of platelet exosomes in the treatment of diabetic wound healing. Background Technology

[0002] Diabetic foot is one of the most serious chronic complications of diabetes. Its core pathological features are peripheral neuropathy, vascular disease, and immune dysfunction caused by diabetes, leading to impaired wound healing, susceptibility to infection, and tissue necrosis in the foot. In severe cases, it can cause osteomyelitis, sepsis, and even require amputation to save the patient's life. Statistics show that the incidence of diabetic foot among diabetic patients worldwide is as high as 15%-25%, and its high disability and recurrence rates place a heavy burden on patients' families and the healthcare system.

[0003] The healing process of diabetic foot ulcers is constrained by multiple factors: the hyperglycemic microenvironment triggers continuous oxidative stress, leading to an imbalance in the inflammatory response and prolonging its duration; vascular stenosis or occlusion causes insufficient blood supply to the wound, hindering the delivery of nutrients and oxygen; reduced fibroblast proliferation activity and insufficient collagen synthesis result in slow granulation tissue growth; peripheral neuropathy leads to sensory loss, making the wound susceptible to external force damage and difficult to detect early. Current clinical treatments include glycemic control, anti-infection, debridement, vascular reconstruction, and negative pressure wound therapy. However, for chronic and refractory diabetic foot ulcers, existing treatments have limited effectiveness, exhibiting low healing rates and high recurrence rates. There is an urgent need to develop highly efficient and targeted biological therapeutic products.

[0004] Exosomes are membranous vesicles secreted by cells, carrying a variety of bioactive molecules such as proteins, nucleic acids, and lipids. They can regulate target cell function by mediating intercellular communication, showing great potential in the field of tissue repair. Platelets, as a natural reservoir of bioactive factors, secrete exosomes (platelet-derived exosomes, PDEs) that are rich in various repair-promoting factors such as platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF), as well as anti-inflammatory cytokines and signal-regulating molecules. They play a key role in hemostasis, inflammation regulation, angiogenesis, fibroblast proliferation, and collagen synthesis.

[0005] Existing research has confirmed that platelet exosomes have a significant healing-promoting effect on ordinary wounds. However, given the complex pathological microenvironment of diabetic foot, whether platelet exosomes can overcome the repair barriers caused by hyperglycemia and vascular neuropathy to effectively promote wound healing, their mechanisms of action (such as synergistic effects on nerve repair, angiogenesis, and inflammation regulation), optimal application methods, and dosages remain unclear. Related products and application patents are still lacking. Therefore, developing diabetic foot treatment products centered on platelet exosomes and clarifying their therapeutic effects and application methods is of significant practical importance and clinical value for filling clinical treatment gaps and improving the cure rate of diabetic foot.

[0006] C57BL / 6 mice are a commonly used strain for constructing diabetic animal models. Insulin-dependent diabetes mellitus can be stably established through methods such as streptozotocin (STZ) induction. The wound healing pathology of C57BL / 6 mice is highly similar to that of human diabetic wounds, making them an ideal model for evaluating the efficacy of wound repair drugs. Current clinical treatments for diabetic wounds include blood glucose control, debridement, anti-infection measures, and negative pressure wound therapy, but highly effective and targeted biological agents for promoting wound healing are still lacking.

[0007] Therefore, developing a product with platelet exosomes as the core active ingredient and clarifying its therapeutic effect on wound healing in C57BL / 6 diabetic mice would fill the application gap of platelet exosomes in the field of diabetic wound repair, which has important clinical value and market prospects. Summary of the Invention

[0008] To address the aforementioned technical problems, this application provides the use of platelet exosomes in the preparation of products having one or more of the following functions: 1) Prevention and / or treatment of diabetic foot; 2) Promotes wound healing and / or tissue repair; 3) Shorten wound healing time; 4) Promotes the growth of granulation tissue in the wound; 5) Enhances wound angiogenesis; 6) Relieves inflammatory response; 7) Inhibits the expression of pro-inflammatory factors; 8) Promotes the expression of anti-inflammatory factors; 9) Reduce MMP-9 expression; 10) Increase MMP-2 expression.

[0009] The beneficial effects of this application include, but are not limited to, the following: the platelet exosomes prepared in this application have the following functions for diabetic individuals: shortening wound healing time; promoting the growth of granulation tissue in the wound; enhancing wound angiogenesis; improving the proliferation of fibroblasts and collagen synthesis in the wound; regulating wound inflammatory response; and reducing the risk of wound infection. Attached Figure Description

[0010] This application will be further described by way of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not limiting, wherein: Figure 1 The figure shows the particle size distribution of platelet exosomes as determined by this invention.

[0011] Figure 2 The image shown is a transmission electron microscope image of platelet exosomes of the present invention.

[0012] Figure 3 The results show the characteristic protein assays for artificial platelet exosomes.

[0013] Figure 4 Image of lyophilized exosomes derived from artificial platelets.

[0014] Figure 5 A flowchart for constructing a diabetic mouse model using high-fat diet combined with STZ and for creating a back wound model.

[0015] Figure 6 Images of wounds in diabetic mice.

[0016] Figure 7 The curve and bar chart show the wound healing rate of diabetic mice.

[0017] Figure 8 A bar chart showing the expression levels of inflammatory factors in newly formed mouse skin tissue.

[0018] Figure 9 A bar chart showing the expression levels of genes related to skin tissue remodeling in mice. Detailed Implementation

[0019] To more clearly illustrate the technical solutions of the embodiments in this specification, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are merely some examples or embodiments of this specification. For those skilled in the art, these drawings can be applied to other similar scenarios without creative effort. Unless obvious from the context or otherwise specified, the same reference numerals in the drawings represent the same structures or operations.

[0020] As indicated in this specification and claims, unless the context clearly indicates otherwise, the words "a," "an," "an," and / or "the" do not specifically refer to the singular and may also include the plural. Generally speaking, the terms "comprising" and "including" only indicate the inclusion of expressly identified steps and elements, which do not constitute an exclusive list, and the method or apparatus may also include other steps or elements.

[0021] Flowcharts are used in this specification to illustrate the operations performed by the system according to embodiments of this specification. It should be understood that the preceding or following operations are not necessarily performed in exact order. Instead, the steps can be processed in reverse order or simultaneously. Furthermore, other operations can be added to these processes, or one or more steps can be removed from them.

[0022] This application provides the use of platelet exosomes in the preparation of products having one or more of the following functions: 1) Prevention and / or treatment of diabetic foot; 2) Promotes wound healing and / or tissue repair; 3) Shorten wound healing time; 4) Promotes the growth of granulation tissue in the wound; 5) Enhances wound angiogenesis; 6) Relieves inflammatory response; 7) Inhibits the expression of pro-inflammatory factors; 8) Promotes the expression of anti-inflammatory factors; 9) Reduce MMP-9 expression; 10) Increase MMP-2 expression.

[0023] The term "prevention and / or treatment" (and its grammatical variations) refers to an attempt to alter the natural course of disease in an individual being treated, and can be a clinical intervention implemented for prevention or during a clinicopathological process. The desired effects of treatment include, but are not limited to, prevention... Preventing the occurrence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, and slowing down the progression of the disease. The rate of disease progression, improvement or mitigation of disease status, and elimination or improvement of prognosis.

[0024] In some embodiments, the product can promote wound healing and / or tissue repair in diabetic individuals.

[0025] In some embodiments, the product can shorten wound healing time for diabetic individuals.

[0026] In some embodiments, the product can promote the growth of granulation tissue in wounds of diabetic individuals.

[0027] In some embodiments, the product can enhance wound angiogenesis in diabetic individuals.

[0028] In some embodiments, the product can alleviate diabetes-related inflammatory responses.

[0029] In some embodiments, the product can inhibit the expression of pro-inflammatory factors in diabetic individuals.

[0030] In some embodiments, the product can promote the expression of anti-inflammatory factors in diabetic individuals.

[0031] In some embodiments, the product can reduce the expression of MMP-9 in diabetic individuals.

[0032] In some embodiments, the product can enhance the expression of MMP-2 in individuals with diabetes.

[0033] In some embodiments, the pro-inflammatory factors may include TNF-α and IL-6.

[0034] In some embodiments, the anti-inflammatory factor may be IL-10.

[0035] In some embodiments, the main peak particle size of the platelet exosomes can be 120.9 nm; In some embodiments, the platelet exosomes may contain exosome marker proteins CD63 and CD9; In some embodiments, the platelet exosomes do not contain Calnexin protein; In some embodiments, the platelet exosomes may be derived from artificially regenerated platelets and / or natural platelets.

[0036] In some embodiments, the artificially regenerated platelets may be produced by megakaryocytes.

[0037] In some embodiments, the artificially regenerated platelets can be generated by megakaryocytes through a directed differentiation system.

[0038] In some embodiments, the megakaryocytes may be genetically engineered megakaryocytes.

[0039] In some embodiments, the efficient differentiation of megakaryocytes into artificial platelets requires a specifically optimized culture medium system to ensure differentiation efficiency, obtain artificial platelets with the required function and purity.

[0040] In some embodiments, the genetic engineering modification may be any one or more of the following: knockout of epigenetic factors SETD2, B2M (β2 microglobulin) or CIITA, overexpression of ABCB1, overexpression of uPAR or von Willebrand factor (vWF), or knockout of EGLN1 (prolyl hydroxylase).

[0041] In some embodiments, the genetically engineered megakaryocytes may be derived from induced pluripotent stem cells.

[0042] In some embodiments, the platelet exosomes are prepared through the following steps: 1) Inducing pluripotent stem cells to differentiate into hematopoietic progenitor stem cells; 2) Inducing hematopoietic progenitor stem cells to differentiate into megakaryocytes; 3) Culture megakaryocytes and collect platelet exosomes.

[0043] In some embodiments, artificial platelets can be produced by culturing megakaryocytes.

[0044] In some embodiments, the supernatant from which platelets are produced is collected for further purification of exosomes.

[0045] In some embodiments, the differentiation induction step in step 1) specifically includes: a. Induced pluripotent stem cells were cultured in Essentia18 medium containing VEGF-A165, bFGF, Y-27632, Chir-99021 and Activin A for 1 to 3 days, preferably for 2 days, to obtain the first cells; b. The first cells are cultured in IMDM medium containing ITS, glutamax, monothioglycerol, ascorbic acid, KO-SRM, VEGF-A165, bFGF, BMP4, Y-27632 and Activin A for 3 to 5 days, preferably 4 days, to obtain the second cells; c. The second cells are cultured in IMDM medium containing ITS, glutamax, monothioglycerol, ascorbic acid, KO-SRM, VEGF-A165, 10 ng / ml bFGF, 50 ng / ml SCF, thrombopoietin, erythropoietin, Y-27632, IL-3 and IL-6 for 7 to 9 days, preferably 8 days, to obtain hematopoietic progenitor stem cells.

[0046] In some embodiments, the differentiation induction step in step 2) can specifically be: d. Hematopoietic progenitor stem cells were cultured in IMDM medium containing ITS, glutamax, monothioglycerol, ascorbic acid, KO-SRM, VEGF-A165, bFGF, thrombopoietin, IL-3, IL-6, IL-21, Tazemetostat, Eltrombopag, and iBET151 for 15–20 days, and suspended megakaryocytes were collected. e. The suspended megakaryocytes are cultured in a low-adsorption culture vessel using IMDM medium containing ITS, glutamax, monothioglycerol, ascorbic acid, KO-SRM, thrombopoietin, SCF, IL-21 and Y-27632 with shaking for 3 to 10 days, and passaged every 2 to 3 days to obtain suspended megakaryocytes adapted for shaker culture.

[0047] In some embodiments, the suspension megakaryocytes suitable for shaker culture are statically cultured for 1 day in IMDM medium containing ITS, glutamax, thioglycerol, ascorbic acid, heparin, human plasma, thrombopoietin, and Y-27632, and then statically cultured for 1 day in IMDM medium containing 1x ITS, 1x glutamax, 0.45mM thioglycerol, 50g / ml ascorbic acid, 10U heparin, 5% human plasma, 50ng / ml TPO, 0.5ng / ml collagen, 5nM Fingolimod HCl, and 5uM Y-27632, followed by suspension culture, and platelet exosomes are collected by centrifugation.

[0048] This invention provides a product characterized in that the product contains an effective amount of platelet exosomes, and the product has the function of promoting wound healing in C57BL / 6 diabetic mice, specifically including any one or more of the following effects: shortening wound healing time; promoting the growth of granulation tissue in the wound; enhancing angiogenesis in the wound; improving the proliferation of fibroblasts and collagen synthesis in the wound; regulating the inflammatory response in the wound; and reducing the risk of wound infection.

[0049] The platelet exosomes are produced after the megakaryocytes differentiate from IPSCs and are activated; the separation involves collecting the supernatant from the platelet preparation process, centrifuging it and collecting the precipitate, which is the platelet exosome.

[0050] The activated megakaryocytes were treated with thrombopoietin, the ROCK inhibitor Y-27632, and plasma.

[0051] The product contains ≥1×10^8 platelet exosomes / mL; the main peak particle size is 120~125nm, and the average particle size is 135nm~140nm.

[0052] The product may be a topical preparation, an injection, or a dressing. Administration method: Five-week-old male C57BL / 6 mice were acclimatized to a normal diet for one week; 20 mice were fed a high-fat diet with 60% fat for energy for 6 weeks, and their weight and the presence of polydipsia and polyuria were monitored; 5 mice were kept on a normal diet. After 6 weeks of feeding, STZ solution was injected intraperitoneally into mice that had been fasted for 18 hours at a dose of 40 mg / kg for 4 consecutive days to establish a diabetic mouse model. Blood glucose levels were measured using a glucometer 10 days after STZ induction; a blood glucose level above 16.7 mmol / L was considered a successful and stable diabetic mouse model. The diabetic mice were randomly divided into 4 groups according to their blood glucose levels, with 5 mice fed a normal diet serving as the blank control group. After grouping, the mice were anesthetized with sodium pentobarbital, and a circular wound (D0) was created on the back of the mouse using a 1 cm skin punch. The wound healing was monitored, and the back wound was photographed on D6, D10, and D14, and the wound area was calculated. The wound area was measured using ImageJ software, and the healing rate was calculated: Healing rate (%) = (Initial wound area - (Remaining wound area) / Initial wound area × 100%; ; At the end of the experiment, mice were euthanized and new skin from the wound was harvested for subsequent detection of relevant mRNA levels in the skin.

[0053] Application frequency: once every two days, 200 g / animal each time, 100 g / animal for the positive drug group, and continue to be applied until the wound is completely healed.

[0054] Experimental conclusion: This experiment confirms that platelet exosomes can significantly promote wound healing in C57BL / 6 diabetic mice. The mechanism of action may be related to enhancing the growth of granulation tissue, promoting angiogenesis, increasing collagen synthesis, and regulating inflammatory response. Both topical application and subcutaneous injection can significantly promote wound healing in diabetic mice.

[0055] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent companies. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0056] Example 1: Preparation of platelets derived from human induced pluripotent stem cells (iPSCs) (1) Differentiate iPSCs into hematopoietic progenitor cells (iHPCs) S1. Digest iPSCs into a single-cell suspension, count them, and adjust the cell concentration to 1×10⁶. 5 ~ 2×10 5 Cells / mL were evenly seeded in culture dishes pre-packaged with recombinant human laminin-521 (rhLaminin-521, Thermofisher). The culture medium consisted of Essential8 (Thermofisher) supplemented with 0-60 ng / mL VEGF, 0-50 ng / mL bFGF, and 0-100 ng / mL LSCCF. The culture time was 2-3 days.

[0057] S2. Replace the culture medium with HDM, with the following formulation: HDM medium: Iscove modified Dulbecco (IMDM) with 1x ITS, 1x glutamax, 0-1 mM monothioglycerol, 50-100 g / mL ascorbicacid, and 2% KO-SRM. Then, add 0-60 ng / mL VEGF-A165, 0-20 ng / mL bFGF, 0-50 ng / mL BMP4, 0-10 μM Y-27632, and 0-50 ng / mL Activin A, and continue culturing for 3-5 days.

[0058] S3. Replace the culture medium with HDM medium and add 0~60 ng / mL VEGF-A165, 0~20 ng / mL bFGF, 0~100 ng / mL stem cell factor (SCF), 0~100 ng / mL thrombopoietin (TPO), 0~4 U / mL erythropoietin (EPO), 0~10 μMY-27632, 0~20 ng / mL IL-3, and 0~20 ng / mL IL-6, and continue culturing for 8 days.

[0059] S4. Collect the suspension-grown hematopoietic progenitor stem cells (iHPCs), collect the culture supernatant containing iHPCs, and centrifuge at 1000 rpm for 5 minutes.

[0060] (2) Differentiate hematopoietic progenitor stem cells (iHPCs) into megakaryocytes (iMKs). S1. Collect the hematopoietic progenitor stem cells obtained in (1) of this embodiment and seed them in a culture dish pre-prepared with 0.1% gelatin. The culture medium is HDM medium supplemented with 0-50 ng / mL VEGF-A165, 0-10 ng / mL bFGF, 0-100 ng / mL thrombopoietin (TPO), 0-50 ng / mL IL-3, 0-50 ng / mL IL-6, 0-100 ng / mL IL-21, 0-10 nM Tazemetostat, 0-400 nM Eltrombopag, and 200 nM iBET151. Continue culturing for 15-20 days. Then collect the suspended megakaryocytes.

[0061] S2. The obtained megakaryocytes were transferred to low-absorption material culture flasks / shake flasks, and the concentration was adjusted to 2×10⁻⁶. 5 Cells / mL culture medium. Replace with HDM and add 0~100 ng / mL thrombopoietin (TPO), 0~100 ng / mL stem cell factor (SCF), 0~100 ng / mL IL-21 and 0~10 μMY-27632, and place on a horizontal shaking table for amplification, adjusting the rotation speed to 120~150 rpm.

[0062] (3) Production of artificial platelets S1. The megakaryocytes obtained in Example 1(2) were suspended in PM medium and 0-100 ng / mL thrombopoietin (TPO), 0-10 μM Y-27632, 0-1 ng / mL collagen, and 0-10 nM Fingolimod HCl were added and the cells were incubated statically for 1 day. Then, the cells were incubated on a horizontal shaking table for another day at a speed of 120-150 rpm.

[0063] S2. Transfer the megakaryocytes obtained in the above steps to a bioreactor and perform platelet maturation in a 20 L system. Continue culturing for 5-6 days in a culture environment of 37℃ and 5% CO2, with a maximum horizontal liquid flow velocity of 0-60 cm / s in the reactor.

[0064] In this embodiment, the in vitro maturation and shedding process of platelets, in a parallel control between a static environment and a reactor environment, showed that the proportion of platelets produced by a single megakaryocyte under reactor conditions could reach more than 1:100, and the overall size was closer to that of platelets from peripheral blood.

[0065] Example 2: Preparation of platelet exosomes derived from human induced pluripotent stem cells (iPSCs) (1) Collection of exosomes Collect the supernatant from the platelet production process in Example 1 (3) and perform gradient centrifugation. First centrifugation: 0-400×g at 4℃ for 5-10 min to remove large cells; Second centrifugation: 0-5000×g at 4℃ for 5-10 min to remove small cells; Third centrifugation: 0-15000×g at 4℃ for 10-30 min, collect the supernatant to remove large vesicles; Fourth ultracentrifugation: 0-150000×g at 4℃ for 30-90 min, concentrate, collect the precipitate, which is the purified exosome. Aliquot with D-PBS and store at -80℃.

[0066] (2) Identification of exosomes Particle size and concentration were determined by nanoparticle tracking analysis (NTA); CD9, CD81, and Alix exosome markers were validated by Western blotting; and cup-shaped morphology was observed by transmission electron microscopy to ensure exosome quality.

[0067] A portion of exosomes was subjected to nanoparticle tracking analysis (NTA) to detect the hydrodynamic particle size distribution and transmission electron microscopy results. Figure 1 and Figure 2 The results showed that the main peak particle size was 120.9 nm, and the EV concentration was 3.60 E11 particles / mL. Example 3: Western blot detection of expression in artificial platelet exosomes (1) Sample Preparation: Prepare a lysis buffer with the following components: 50 mM Tris (pH 7.4), 40 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 50 mM NaF, 10 mM sodium pyrophosphate, and 10 mM sodium glycerophosphate. Add an appropriate amount of a mixture of protease inhibitors and phosphatase inhibitors. Add this buffer to the adipogenic induced adipose-derived stem cell exosomes (ADSC-Exos) prepared in Example 1, lyse on ice for 30 minutes, then transfer to a 1.5 ml EP tube, centrifuge at 10,000 rpm for 10 minutes, and collect the supernatant. Use the BCA protein quantification kit described above, strictly following the instructions, to determine the protein concentration of ADSC-Exos. Mix an appropriate amount of ADSC-Exos sample with 5× loading buffer, heat at 100°C for 10 minutes to denature the protein, and store at -80°C for later use.

[0068] (2) Electrophoresis: Prepare SDS-PAGE gels and load them into the electrophoresis tank. Add electrophoresis buffer to both the inner and outer tanks. Use a micropipette to add protein markers and samples to the wells, with a sample loading volume of 30 μg per well. Start electrophoresis at a constant voltage of 80 V. After the bromophenol blue leading edge enters the separating gel, adjust the voltage to 120 V until the protein marker bands are clearly separated, then stop electrophoresis.

[0069] (3) Transfer: Cut the PVDF membrane to 6.6×8.5cm size, activate it in methanol for 5 minutes, and then place it in transfer buffer for later use. Remove the gel after electrophoresis and rinse with pure water to remove residual electrophoresis solution. Assemble the transfer system in the following order: cathode clamp → sponge → filter paper → gel → PVDF membrane → filter paper → sponge → anode clamp, ensuring that the gel is close to the negative electrode and the PVDF membrane is close to the positive electrode. Place the transfer device in a foam ice box, add transfer buffer, and transfer at a constant voltage of 100V for 1.5 hours.

[0070] (4) Blocking: After the transfer, the PVDF membrane was blocked by immersing it in 5% skim milk. The 5% skim milk was prepared with 1×TBST, which was prepared by dissolving 500 μl of Tween 20 in 500 ml of 1×TBS. The PVDF membrane was placed in the blocking solution and incubated on a shaker at room temperature for 1 hour.

[0071] (5) Primary antibody incubation: Discard the blocking solution and wash the PVDF membrane with 1×TBST for 5 minutes each time, for a total of 5 washes. Dilute the primary antibody according to the following ratios: TSG101 (Abcam ab125011, 1:1000 dilution), CD9 (Abcam ab92726, 1:1000 dilution), CD81 (Abcam ab109201, 1:1000 dilution). Incubate the PVDF membrane with the diluted primary antibody overnight at 4°C.

[0072] (6) Secondary antibody incubation: After recovering the primary antibody, wash the membrane 5 times with 1×TBST (5 minutes each time), dilute the secondary antibody at a ratio of 1:5000, and incubate on a shaker at room temperature for 1 hour.

[0073] (7) Chemiluminescence and development: Exposure and development are performed using a chemiluminescence system. Key data such as sample sequence and antibody information are recorded in detail, and experimental results are properly preserved.

[0074] Table 1 Western blot results are as follows Figure 3 The results showed that CD9 and CD63 proteins were expressed, while the negative Calnexin protein was not expressed, which met the core criteria for exosome identification, indicating that the exosome extraction was of high purity, free from intracellular protein contamination, and of qualified quality.

[0075] Example 4: Preparation of lyophilized platelet exosome powder derived from human induced pluripotent stem cells (iPSCs) (1) Addition of exosome concentration and freeze-drying protectant S1. Exosomes were further concentrated to a concentration ≥1×10¹ using a 100kDa ultrafiltration membrane. 0To improve freeze-drying efficiency and product stability, particles / ml are controlled. A protective agent is added; the basic formulation consists of 5%-10% trehalose and 1%-2% mannitol to maintain membrane integrity. The protective agent is thoroughly mixed with the exosome suspension and incubated at 4°C for 30 minutes to ensure uniform coating by the protective agent.

[0076] S2. Pre-freezing: Gradient pre-freezing: -40℃ → -80℃, cooling rate 1℃ / min, hold for 2-3 hours to reduce ice crystal damage to the membrane. First drying (sublimation drying): Temperature -50℃ → -30℃, heating rate ≤1℃ / h to ensure ice crystal sublimation without collapse. Maintain vacuum at 5-10 Pa to provide sublimation motive force and prevent exosome oxidation. Time: 24-30 hours, adjusted according to sample volume, with real-time monitoring of sample temperature and vacuum. Second drying (desorption drying): Temperature rises to 20-25℃ to remove bound water. Time 4-6 hours to ensure residual moisture ≤2% and improve long-term stability. Sealed storage: After drying, pack the lyophilized powder into vials, 1 mg / vial, and store at 4℃ or room temperature. After reconstitution, exosome activity can be maintained for several months to several years (see artificial platelet exosomes). Figure 4 ).

[0077] Example 5: Diabetes in C57BL / 6 mice and the construction of its wound surface (1) C57BL / 6 mice were acclimatized to a standard diet for at least one week; (2) Twenty mice were fed a high-fat diet with 60% fat for energy for 4 to 7 weeks. The weight of the mice and whether they showed signs of polydipsia and polyuria were monitored. Five mice were fed a normal diet. (3) After the high-fat diet was completed, STZ solution was injected intraperitoneally into mice that had been fasting for 18 hours for 4 to 5 consecutive days at a dose of 0 to 60 mg / kg to establish a diabetic mouse model. (4) Use a blood glucose meter to detect the blood glucose level of mice 10-12 days after STZ induction. A blood glucose value higher than 16.7 mmol / L indicates that a stable diabetic mouse model has been successfully constructed. (5) Twenty diabetic mice were randomly divided into four groups based on their blood glucose levels (model control group, positive drug group, platelet exosome administration group, and platelet exosome subcutaneous administration group), and five mice fed with normal diet served as blank control group mice; (6) After grouping, the mice were anesthetized with sodium pentobarbital and a circular wound (denoted as D0) was created on the back of the mice using a 1 cm skin punch. (7) Results: The procedure for constructing a diabetic mouse model by high-fat diet combined with STZ and the back wound modeling procedure can be found in [link to documentation]. Figure 5 .

[0078] Example 6: Drug administration and wound healing in C57BL / 6 diabetic mice (1) Model control group (Diabitic): The wound was incubated with 0.2% trehalose dripped onto sterile gauze for 30-60 min / animal, every 1-2 days.

[0079] (2) Positive drug group (Diabetic+positive drug): Yifu-recombinant human growth factor (drug identification code: 8384265), 100 μg / mouse per day, 30-60 min / mouse, every 1-2 days. Use sterile cotton swabs to evenly apply the ointment to the wound site of the mouse each time.

[0080] (3) Artificial platelet exosome incubation group (Diabetic+PLT Exos): 75 μL of 0.2% trehalose was used to dissolve the lyophilized powder of platelet exosomes. After it was fully dissolved and clear, the wound was incubated with sterile gauze and the platelet exosome solution was added drop by drop. The incubation time was 30-60 min / animal, 200 μg / animal, every 1-2 days.

[0081] (4) Subcutaneous administration of artificial platelet exosomes (Diabetic+PLT Exos / SC): 75 μL of 0.2% trehalose was used to dissolve the lyophilized powder of platelet exosomes. After it was fully dissolved and clear, the platelet exosome solution was drawn with an insulin injection needle and injected at multiple points around the wound, 200 μg / animal, every 1-2 days.

[0082] (5) Monitor the wound healing of mice and take photos of the back wounds on D6, D10 and D14 respectively, and calculate the wound area; use ImageJ software to measure the wound area and calculate the healing rate: healing rate (%) = (initial wound area - remaining wound area) / initial wound area × 100%. At the end of the experiment, euthanize the mice and take the newly formed skin from the wound for subsequent detection of relevant mRNA levels in the skin.

[0083] Throughout the experiment, images of the mouse's dorsal wounds were taken at D0, D6, D10, and D14. Figure 6 ); and used ImageJ to calculate the wound healing rate in mice ( Figure 7 ).

[0084] During the wound inflammation phase (D6), the wound area in the Diabetic+PLT Exos group was significantly smaller than that in the other four groups. During the wound proliferation phase (D10), there was no visible difference in wound area between the Diabetic+PLT Exos group and the positive drug and blank control groups. During the wound proliferation phase (D14), the wound area in the Diabetic+PLT Exos group and the Diabetic+PLT Exos / SC group was significantly smaller than that in the Diabetic group. Furthermore, the images show that the Diabetic+PLT Exos group mice produced more hair around the wound, but further experimental verification is needed.

[0085] Example 7: Real-time PCR detection of mRNA expression in mouse skin tissue (1) Extraction of RNA from mouse skin tissue S1. Homogenize the tissue: Take fresh tissue, cut it into small pieces with sterilized scissors, add 500 μL Buffer RL, and homogenize with an electric homogenizer until there are no obvious tissue blocks. S2. Transfer the lysed sample to FastPure gDNA-Filter Columns 11, centrifuge at 12,000 rpm for 30 sec, discard FastPure gDNA-Filter Columns 11, and collect the filtrate; S3. Add 0.5 times the volume of anhydrous ethanol to the filtrate and mix thoroughly; S4. Transfer all the mixture from step 2 to FastPureRNAColumns11, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate; S5. Add 700 μL of Buffer RW1 to FastPure RNAColumns ll, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate; S6. Add 700 μL of Buffer RW2 to FastPure RNA Columns ll, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate; S7. Add 500 μL of Buffer RW2 to FastPure RNA Columns l, centrifuge at 12,000 rpm for 2 min, and carefully remove the adsorption column from the collection tube to avoid contact with the filtrate and contamination. S8. Carefully transfer the adsorbed RNA to a new RNase-free Coleclion Tube 1.5 ml centrifuge tube, add 50 μL of RNase-free ddH2O to the center of the adsorption column, incubate at room temperature for 1 min, centrifuge at 12,000 rpm for 1 min, and elute the RNA.

[0086] S9. Reverse transcription to synthesize cDNA Establish a 10 µL reverse transcription system. Experimental RNA (up to 5 µg / reaction) × µL, Primer Oligo (dT) 15 (0.5 µg / reaction) 0.5 µL, Nuclease-Free Water X µL, Final volume 5.55 µL (2) After heat blocking at 70 °C for 5 min in the PCR instrument, immediately place on ice for 5 min, and then centrifuge for a short time. Prepare the Real-time reaction mixture, add 4.45 µL to each reaction system, and mix on ice.

[0087] S10. Mix all components thoroughly, centrifuge, and place in a PCR instrument. Anneal for 5 min at 25 °C; extend for 60 min at 42 °C; and thermally block for 15 min at 70 °C.

[0088] The obtained cDNA from S11 can be used for further analysis or frozen and stored at -80 °C.

[0089] (2) Real-time PCR detection of the expression of related genes S1. Construct a 10 µL Real-time PCR system using the obtained cDNA template: 2 µL Primers, 1 µL cDNA, 5 µL qPCR Master Mix, and 2 µL Nuclease-Free Water. S2. Reaction Procedure: Step 1: 95 °C, 2 min, 1 cycle. Step 2: 95 °C, 15 s; 60 °C, 20 s; 60 °C, 20 s; 44 cycles. Step 3: Dissociation curve analysis.

[0090] According to the experimental statistics ( Figure 8This indicates that, compared with the normal control group (Control), the mRNA expression of pro-inflammatory cytokines TNF-α and IL-6 was significantly increased in the diabetic group (Diabetic), suggesting a significant inflammatory response in the diabetic state. The positive control group (Diabetic + positive drug), the platelet-derived exosome group (Diabetic + PLTExos), and the platelet-derived exosome group (Diabetic + PLT Exos / SC) all showed some inhibitory effect on the pro-inflammatory factors TNF-α and IL-6. Simultaneously, the platelet-derived exosome group (Diabetic + PLT Exos / SC) also showed the strongest promotion of the expression of the anti-inflammatory cytokine IL-10.

[0091] In a comprehensive comparison of effects, platelet-derived exosomes, whether administered via dressing or multi-point subcutaneous injection around the wound, demonstrated excellent performance in alleviating diabetes-related inflammation, suggesting that artificial platelet-derived exosomes may be a more effective treatment strategy for this disease.

[0092] According to the experimental statistics ( Figure 9 The results showed that, compared with the normal control group, the expression levels of MMP-2 and MMP-9 mRNA were significantly increased in the diabetic group. This suggests that the diabetic state promotes the expression of matrix metalloproteinases, which may be related to pathological processes associated with tissue damage and inflammation.

[0093] The positive control group (Diabetic + positive drug), the artificial platelet-derived exosome group (Diabetic + PLTExos), and the artificial platelet-derived exosome group (Diabetic + PLT Exos / SC) all reduced MMP-9 expression to varying degrees, inhibiting the abnormal increase in MMP-9 induced by diabetes. For MMP-2, both the positive control group and the artificial platelet-derived exosome group significantly increased its mRNA expression level, with the artificial platelet-derived exosome group (Diabetic + PLT Exos) showing the most significant increase, and the platelet-derived exosome group (Diabetic + PLT Exos / SC) also exhibited a strong upregulation effect. Therefore, artificial platelet-derived exosomes have a function in promoting wound healing in the treatment of diabetes.

[0094] The basic concepts have been described above. Obviously, for those skilled in the art, the detailed disclosure above is merely illustrative and does not constitute a limitation of this specification. Although not explicitly stated herein, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are suggested in this specification and therefore remain within the spirit and scope of the exemplary embodiments described herein.

[0095] Furthermore, this specification uses specific terms to describe embodiments thereof. For example, "an embodiment," "one embodiment," and / or "some embodiments" refer to a particular feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "an embodiment," "one embodiment," or "an alternative embodiment" in different locations throughout this specification do not necessarily refer to the same embodiment. Moreover, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0096] In some embodiments, numbers describing the quantity of components and attributes are used. It should be understood that such numbers used in the description of embodiments are modified in some examples with the terms "approximately," "approximately," or "generally." Unless otherwise stated, "approximately," "approximately," or "generally" indicates that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may be changed depending on the characteristics required by individual embodiments. In some embodiments, numerical parameters should take into account specified significant digits and employ a general method of digit reservation. Although the numerical ranges and parameters used to confirm their breadth of range in some embodiments of this specification are approximate values, in specific embodiments, such values ​​are set as precisely as feasible.

[0097] Finally, it should be understood that the embodiments described in this specification are merely illustrative of the principles of the embodiments described herein. Other variations may also fall within the scope of this specification. Therefore, alternative configurations of the embodiments described herein are intended to be illustrative rather than limiting, and should be considered consistent with the teachings of this specification. Accordingly, the embodiments described herein are not limited to those explicitly introduced and described herein.

Claims

1. Use of platelet exosomes in the preparation of products having one or more of the following functions: 1) Prevention and / or treatment of diabetic foot; 2) Promotes wound healing and / or tissue repair; 3) Shorten wound healing time; 4) Promotes the growth of granulation tissue in the wound; 5) Enhances wound angiogenesis; 6) Relieves inflammatory response; 7) Inhibits the expression of pro-inflammatory factors; 8) Promotes the expression of anti-inflammatory factors; 9) Reduce MMP-9 expression; 10) Increase MMP-2 expression.

2. The use as described in claim 1, characterized in that, The product promotes wound healing and / or tissue repair in diabetic individuals; And / or, the product shortens wound healing time in diabetic individuals; And / or, the product promotes the growth of granulation tissue in wounds of diabetic individuals; And / or, the product enhances wound angiogenesis in diabetic individuals; And / or, the product alleviates diabetes-related inflammatory responses; And / or, the product inhibits the expression of pro-inflammatory factors in diabetic individuals; And / or, the product promotes the expression of anti-inflammatory factors in diabetic individuals; And / or, the product reduces the expression of MMP-9 in diabetic individuals; And / or, the product enhances MMP-2 expression in diabetic individuals; And / or, the pro-inflammatory factors include TNF-α and IL-6; And / or, the anti-inflammatory factor is IL-10.

3. The use as described in claim 1, characterized in that, The main peak particle size of the platelet exosomes was 120.9 nm; And / or, the platelet exosomes contain exosome marker proteins CD63 and CD9; And / or, the platelet exosomes do not contain Calnexin protein; And / or, the platelet exosomes are derived from artificially regenerated platelets and / or natural platelets.

4. The use as described in claim 3, characterized in that, The artificially regenerated platelets are produced by megakaryocytes.

5. The use as described in claim 4, characterized in that, The megakaryocytes mentioned are genetically engineered megakaryocytes.

6. The use as described in claim 5, characterized in that, The genetically engineered megakaryocytes were derived from induced pluripotent stem cells.

7. The use as described in claim 1, characterized in that, The platelet exosomes are prepared through the following steps: 1) Inducing pluripotent stem cells to differentiate into hematopoietic progenitor stem cells; 2) Inducing hematopoietic progenitor stem cells to differentiate into megakaryocytes; 3) Culture megakaryocytes and collect platelet exosomes.

8. The use as described in claim 7, characterized in that, The induced differentiation step in step 1) specifically refers to: a. Induced pluripotent stem cells were cultured in Essentia18 medium containing VEGF-A165, bFGF, Y-27632, Chir-99021 and Activin A for 1 to 3 days, preferably for 2 days, to obtain the first cells; b. The first cells are cultured in IMDM medium containing ITS, glutamax, monothioglycerol, ascorbic acid, KO-SRM, VEGF-A165, bFGF, BMP4, Y-27632 and Activin A for 3 to 5 days, preferably 4 days, to obtain the second cells; c. The second cells are cultured in IMDM medium containing ITS, glutamax, monothioglycerol, ascorbic acid, KO-SRM, VEGF-A165, 10 ng / ml bFGF, 50 ng / ml SCF, thrombopoietin, erythropoietin, Y-27632, IL-3 and IL-6 for 7 to 9 days, preferably 8 days, to obtain hematopoietic progenitor stem cells.

9. The use as described in claim 7, characterized in that, The induced differentiation step in step 2) specifically refers to: d. Hematopoietic progenitor stem cells were cultured in IMDM medium containing ITS, glutamax, monothioglycerol, ascorbic acid, KO-SRM, VEGF-A165, bFGF, thrombopoietin, IL-3, IL-6, IL-21, Tazemetostat, Eltrombopag, and iBET151 for 15–20 days, and suspended megakaryocytes were collected. e. The suspended megakaryocytes are cultured in a low-adsorption culture vessel using IMDM medium containing ITS, glutamax, monothioglycerol, ascorbic acid, KO-SRM, thrombopoietin, SCF, IL-21 and Y-27632 with shaking for 3 to 10 days, and passaged every 2 to 3 days to obtain suspended megakaryocytes adapted for shaker culture.

10. The use as described in claim 9, characterized in that, The suspension megakaryocytes suitable for shaker culture were statically cultured for 1 day in IMDM medium containing ITS, glutamax, thioglycerol, ascorbic acid, heparin, human plasma, thrombopoietin, and Y-27632. Then, they were statically cultured for 1 day in IMDM medium containing 1x ITS, 1x glutamax, 0.45mM thioglycerol, 50g / ml ascorbic acid, 10U heparin, 5% human plasma, 50ng / ml TPO, 0.5ng / ml collagen, 5nM Fingolimod HCl, and 5uM Y-27632. After suspension culture, platelet exosomes were collected by centrifugation.

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