Temperature-sensitive hydrogel loaded with nitric oxide nanoparticles and fat extract and application of temperature-sensitive hydrogel in diabetes wound treatment

Abstract

The invention provides temperature-sensitive hydrogel loaded with nitric oxide nanoparticles and fat extract and application of the temperature-sensitive hydrogel in diabetes wound treatment, and belongs to the technical field of biomedicine. The preparation method comprises the following steps: mixing egg yolk lecithin and dichloromethane, carrying out rotary evaporation until a lipid membrane is formed, and adding a phosphate buffer solution to obtain a liposome suspension; adding perfluorobromooctane into the liposome suspension, carrying out emulsification treatment to obtain a liposome wrapped with perfluorobromooctane, and loading NO gas in a closed space to obtain a liposome loaded with NO; and adding F-127 powder, dissolving the F-127 powder, and adding the fat extract FE to obtain the temperature-sensitive hydrogel loaded with the nitric oxide nanoparticles and the fat extract. The gel is a flowable liquid at normal temperature, can form a stable gel structure at the body temperature of 37 DEG C, and synergistically promotes the repair processes of angiogenesis, fibroblast migration, collagen deposition and the like; the closure of the diabetic wound surface is accelerated.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a thermosensitive hydrogel loaded with nitric oxide nanoparticles and fat extracts and its application in the treatment of diabetic wounds. Background Technology

[0002] Diabetic wounds are a common complication of diabetes, primarily manifesting as chronic, difficult-to-heal wounds such as diabetic foot ulcers (DFU). Diabetic wounds are characterized by long healing periods, a high risk of secondary infection, and in severe cases, even the risk of amputation, significantly impacting patients' quality of life and imposing a substantial economic burden on society and the healthcare system. Normal wound repair involves stages such as hemostasis, inflammation, proliferation, and tissue remodeling, requiring the coordinated action of multiple cell types and bioactive factors. However, in the diabetic state, factors such as hyperglycemia, chronic inflammation, and oxidative stress lead to numerous problems, including decreased angiogenesis, insufficient growth factor expression, abnormal macrophage function, and impaired extracellular matrix remodeling, resulting in prolonged wound healing failure.

[0003] In recent years, fat extract (FE), as a bio-based therapeutic agent rich in various endogenous growth factors, has shown promising prospects in the fields of tissue repair and regenerative medicine. It contains multiple pro-angiogenic and tissue regeneration factors such as VEGF, bFGF, IGF-1, and TGF-β, exhibiting effects such as promoting angiogenesis, anti-apoptosis, and immunomodulation. Furthermore, FE is widely available and has high biocompatibility. However, the protein-based active factors in FE are rapidly metabolized in vivo and have a short half-life, making it difficult to maintain a stable release over a long period.

[0004] Nitric oxide (NO), as an important gaseous signaling molecule, plays a crucial physiological role in vasodilation, pro-inflammatory and immune regulation, and angiogenesis. Especially in the early stages of diabetic wounds, it helps regulate the inflammatory response and initiate angiogenesis. However, in diabetic patients, NO production and bioavailability are generally reduced, limiting its endogenous therapeutic potential. Furthermore, NO has an extremely short half-life and strong concentration dependence; low concentrations of NO are beneficial for repair, while high concentrations can significantly enhance toxicity. Therefore, exogenous NO delivery systems face the dual challenges of stability and dosage control.

[0005] Although FE and NO each have their own advantages in the treatment of diabetic wounds, a highly efficient treatment system capable of safely, stably, and synergistically delivering both is currently lacking. Existing FE or NO delivery systems often suffer from insufficient carrier biocompatibility, difficulty in controlling drug release rates, or weak targeting capabilities, failing to meet the practical needs of local treatment for chronic wounds. Therefore, there is an urgent need to develop a novel combined delivery platform that can stably release NO while protecting the activity of growth factors in FE, synergistically leveraging its effects in promoting angiogenesis, regulating inflammatory responses, and promoting tissue repair, thereby improving the treatment efficiency and safety of diabetic wounds. Summary of the Invention

[0006] The purpose of this invention is to provide a synergistic delivery system that, while maintaining the active release of growth factors, achieves controlled release of nitric oxide, thereby significantly enhancing angiogenesis, cell migration and tissue repair capabilities, and accelerating the healing of diabetic wounds.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for preparing a thermosensitive hydrogel loaded with nitric oxide nanoparticles and a fat extract, comprising the following steps:

[0009] (1) Mix egg yolk lecithin and dichloromethane, rotary evaporate until a lipid membrane is formed, and add phosphate buffer to obtain a liposome suspension;

[0010] (2) Add perfluorobromooctane to the liposome suspension and emulsify to obtain liposomes Lips-PFOB encapsulated with perfluorobromooctane. Load NO gas in a closed space to obtain NO-loaded liposomes Lips-PFOB(NO).

[0011] (3) Add F-127 powder to the Lips-PFOB(NO), dissolve it, and then add the fat extract FE to obtain the thermosensitive hydrogel Gel@FE-LP(NO) loaded with nitric oxide nanoparticles and fat extract.

[0012] Preferably, in step (1), the mass-to-volume ratio of egg yolk lecithin to dichloromethane is 4-6 mg: 1 mL.

[0013] Preferably, the conditions for rotary evaporation in step (1) are: a rotation speed of 100-500 rpm and a water bath heating temperature of 45-55°C;

[0014] The concentration of the liposome suspension is 8–12 mg / mL.

[0015] Preferably, in step (2), the volume ratio of perfluorooctane added to the liposome suspension is 1 mL: 15-25 μL.

[0016] Preferably, the emulsification conditions in step (2) are: power 100-200W, working mode 2 seconds on / 3 seconds off, and cumulative time 5-10 minutes.

[0017] Preferably, in step (2), the concentration of NO gas in the enclosed space is 150-250 ppm and the NO loading is 1-20 nM.

[0018] Preferably, the mass percentage of F-127 in step (3) is 15-20 wt%.

[0019] The final concentration of FE is 50–200 μg / mL.

[0020] Preferably, the preparation method of the fat extract FE in step (3) is as follows: collect fresh fat aspirate, wash it, and cut it into pieces of about 1-2 cm. 3 The fat tissue block was added to PBS and homogenized until the fat tissue was a fine and uniform paste. The first centrifugation was performed to separate the layers. The middle layer of fat tissue was taken and emulsified to obtain emulsified fat. The emulsified fat was freeze-thawed and then centrifuged to separate the layers a second time. The middle pink aqueous phase was collected, filtered to remove bacteria, and freeze-dried to obtain the fat extract FE.

[0021] The conditions for the two centrifugation separations were: 3-5℃, centrifugation at 1000-1500×g for 3-5 min;

[0022] The amount of PBS added is 1 / 3 to 1 / 2 of the total weight of the adipose tissue block;

[0023] The homogenization conditions are: 4500-5500 rpm, homogenization for 1-3 min;

[0024] The freeze-thaw method involves rapidly freezing the emulsified fat at -85 to -75°C for 30 to 40 minutes, and then immediately transferring it to thaw at 35 to 40°C.

[0025] The present invention provides a thermosensitive hydrogel containing nitric oxide-loaded nanoparticles and a fat extract prepared by the above preparation method.

[0026] The present invention also provides the application of the thermosensitive hydrogel containing nitric oxide-loaded nanoparticles and fat extracts in the preparation of drugs or dressings for diabetic wound repair.

[0027] The thermosensitive gel @FE-LP(NO) provided by this invention is a flowable liquid at room temperature and can form a stable gel structure at body temperature of 37°C, ensuring in-situ gelation, adhesion and moist environment of the wound area, and is suitable for local treatment of irregular skin wounds.

[0028] This invention provides a synergistic treatment system for refractory diabetic wounds, specifically a thermosensitive hydrogel complex that co-delivers fat extract (FE) and nitric oxide (NO). The thermosensitive hydrogel complex, Gel@FE-LP(NO), continuously releases growth factors after administration, prolonging the in vivo half-life of FE; it also controls the release of NO, avoiding excessive concentrations that could induce cytotoxicity, and synergistically promotes angiogenesis, fibroblast migration, collagen deposition, and other repair processes, accelerating wound closure. This invention addresses key issues in existing diabetic wound treatments, such as insufficient angiogenesis, low growth factor activity, poor NO stability, and difficulty in controlling local release, thereby achieving rapid, safe, and efficient diabetic wound repair. Attached Figure Description

[0029] Figure 1 GO analysis results for the main protein components of FE;

[0030] Figure 2 To evaluate the cell proliferation-promoting capacity of FE;

[0031] Figure 3 Characterization of the gel-forming properties of Gel@FE-LP(NO);

[0032] Figure 4 To promote HUVEC migration results using Gel@FE-LP(NO);

[0033] Figure 5 This describes the healing of skin wounds in diabetic rats. Detailed Implementation

[0034] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1: Method for extracting FE

[0036] This embodiment provides a method for extracting fat extract (FE) from human fat aspirate, the specific steps of which are as follows:

[0037] (1) Raw material preparation: Collect fresh fat aspirates obtained from clinical liposuction surgery and immediately rinse the fat aspirates multiple times with sterile phosphate buffered saline (PBS) until all impurities such as red blood cells (RBC) and fascia mixed in are completely removed.

[0038] (2) Homogenization of fat: After cleaning the adipose tissue, cut it into small pieces of about 1-2 cm3. Place the fat pieces in a food-grade stainless steel mixer, add a small amount of PBS (about 1 / 3-1 / 2 of the tissue weight), set the mixer to low speed to gradually loosen the tissue and avoid splashing. Gradually increase the speed to 5000 rpm and homogenize for 3 minutes until the adipose tissue is a fine and uniform paste.

[0039] (3) Centrifugation and layering (first centrifugation and layering): The homogenized adipose tissue was centrifuged at 1200×g for 3 minutes at 4℃ to completely remove the upper oil and lower aqueous phase, while retaining the middle layer of adipose tissue.

[0040] (4) Emulsification treatment: Take the middle layer of adipose tissue mentioned above, place it between two 10mL syringes, connect the two syringes with Luer-Lok connectors, and push the mixture back and forth 70 times to fully emulsify the adipose tissue and obtain emulsified fat.

[0041] (5) Freeze-thaw destruction of tissue structure: After the emulsified fat is placed in an ultra-low temperature freezer at -80℃ for 30 minutes, it is immediately transferred to a water bath or constant temperature box at 37℃ to thaw, completing a complete freeze-thaw cycle to further destroy the fat cell membrane structure.

[0042] (6) Fat separation (second centrifugation layering): The frozen and thawed sample was centrifuged at 1200×g for 5 minutes at 4℃. The middle pink aqueous phase layer (i.e., fat extract rich in cytokines and bioactive proteins) was aspirated. This part is the required FE solution.

[0043] (7) Post-processing and storage: The obtained FE solution was sterilized by passing it through a 0.22μm sterile filter and the protein concentration was detected by BCA protein quantification method to be 5mg / mL. Then it was freeze-dried with a freeze-drying vacuum degree of less than 50Pa, a freeze-drying temperature of -40℃ and a freeze-drying time of 48h until the sample was completely dry and there was no obvious residual liquid. Finally, the dried FE powder was stored at -20℃ for later use.

[0044] The extraction method described in this embodiment can effectively remove lipid impurities and cell debris, obtaining a uniform, cell-free fat extract rich in bioactive proteins, which is suitable for subsequent applications in biomaterial composites, tissue repair, and other fields.

[0045] The main protein components of the obtained FE were subjected to proteomic sequencing, and the proteomic results were analyzed using bioinformatics. The GO analysis results are as follows: Figure 1As shown, the keywords indicating significant enrichment of the main protein components in FE include Fibroblast migration, collagen biosynthetic process, and wound healing. These components are primarily related to biological processes involving tissue repair, extracellular matrix remodeling, and dynamic cell behavior. Their core functions are concentrated in the activation, proliferation, and migration of fibroblasts, as well as the synthesis and organization of collagen. This suggests that the proteins in FE obtained in this invention have the potential to promote wound healing and angiogenesis.

[0046] The effects of different concentrations of FE (0–800 μg / ml) on the proliferation of human umbilical vein endothelial cells (HUVECs), mouse embryonic fibroblasts (NIH 3T3), and mouse fibroblasts (L929) were evaluated using the CCK-8 assay.

[0047] The method was as follows: Different cell types were digested with complete culture medium, counted, and the density was adjusted to 8 × 10³ cells / well. Cells were then evenly seeded into 96-well plates and cultured at 37°C in a 5% CO₂ incubator for 24 h to allow cell adhesion. Different concentrations of FE were added, and the cells were cultured for another 24 h. Subsequently, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated at 37°C for 30 min. The absorbance (OD value) of each well was measured using a microplate reader at 450 nm, and the relative cell viability (%) was calculated.

[0048] like Figure 2 As shown, different FE concentrations had varying effects on different cell types, with cell viability generally showing a trend of first increasing and then decreasing. FE concentrations of 200–400 μg / ml significantly promoted the proliferation of NIH 3T3, while a concentration of 400 μg / ml significantly promoted the proliferation of L929. FE concentrations of 50–200 μg / ml significantly promoted the proliferation of HUVECs. Overall, a concentration of 100 μg / ml significantly promoted the proliferation of all three bacteria.

[0049] Example 2: Preparation and characterization of Gel@FE-LP(NO)

[0050] This embodiment provides a method for preparing a thermosensitive hydrogel (Gel@FE-LP(NO)) loaded with nitric oxide nanoparticles and a fat extract, specifically including the following steps:

[0051] (1) Preparation of lipid membrane: Weigh 50mg of egg yolk lecithin and dissolve it in 10mL of dichloromethane. Place it in a round-bottom flask and connect it to a rotary evaporator. Perform rotary evaporation at 100rpm and 50℃ water bath heating to evaporate the organic solvent and finally form a white lipid membrane that is uniformly distributed on the flask wall.

[0052] (2) Hydrate liposomes: Add 5 mL of phosphate buffer (PBS) to the lipid membrane and shake thoroughly to hydrate the lipid membrane to obtain a liposome suspension with a concentration of 10 mg / mL;

[0053] (3) Emulsification preparation of PFOB liposomes: Take 1 mL of the liposome suspension, add 20 μL of perfluorooctyl bromide (PFOB), place it in an ice bath, and emulsify it using an ultrasonic homogenizer (power 100W, working mode 2 seconds on / 3 seconds off, cumulative time 5 minutes) to obtain liposomes encapsulated with perfluorooctyl bromide (Lips-PFOB).

[0054] (4) Forming NO carrier by gas filling: The Lips-PFOB is placed in a sealed container and NO gas is introduced to a concentration of 200 ppm to obtain NO-loaded liposomes Lips-PFOB(NO) with a NO loading of 5 nM.

[0055] (5) Construction of composite hydrogel system: F-127 powder was slowly added to the Lips-PFOB(NO), stored in the dark at 4°C and allowed to dissolve completely to obtain a 17.5% (w / w) F-127 solution. After mixing, the fat extract (FE) described in Example 1 was added to a concentration of 100 μg / mL and mixed quickly and evenly to obtain the thermosensitive hydrogel Gel@FE-LP(NO).

[0056] Observe the gels (e.g., Gel@FE-LP(NO)) prepared under different temperature conditions (37℃ and 25℃) and different amounts of F-127 (10, 15, 17.5, 20, 23.5, 30% (w / w)). Figure 3 (a) Gel groups were obtained by dissolving equal volumes of phosphate buffer (without FE and LP(NO)) in different amounts of F-127 at 4°C. In the Gel@FE-LP(NO) and F-127 mixtures obtained in this invention, at concentrations of 17.5% and 20% (w / w), the mixture was in solution at room temperature (25°C) and in gel form at body temperature (37°C), representing the optimal addition amounts. In contrast, the pure gel group at a concentration of 17.5% did not gel at body temperature (37°C), demonstrating that the addition of FE and LP(NO) may contribute to gel formation.

[0057] The surface morphology of the hydrogel was observed using SEM. The specific method was as follows: Gel@FE-LP(NO) and gel hydrogel were placed in a 6-well plate and gelled at 37℃. The gel was then pre-frozen at -80℃ and followed by lyophilization. The sample was fixed on a SEM sample holder, coated with a layer of gold, and the focus, brightness, and contrast were adjusted to observe the pore structure, pore size, and surface morphology of the gel. Figure 3 As shown in (b), both Gel@FE-LP(NO) and Gel hydrogel have porous structures, and nano-sized particles can be observed adhering to the surface in Gel@FE-LP(NO), proving that LP(NO) can be effectively loaded into the gel.

[0058] The temperature-sensitive rheology of the gel was tested using a viscometer. The sample was loaded into the viscometer's sample chamber, ensuring uniform contact and no air bubbles. The temperature range was set to 10-40℃. Figure 3 As shown in Figure c, the Gel@FE-LP(NO) and Gel hydrogel system described in Example 2 are in a solution state at low temperature (4°C), while their viscosity increases rapidly as the temperature rises. It can be seen that the gelation temperature of Gel@FE-LP(NO) is slightly higher than that of Gel, which facilitates local wound drug administration at body temperature. It can quickly form a gel at 37°C, realizing the continuous release of FE and local supply of NO, providing synergistic therapeutic support for the repair of diabetic wounds.

[0059] Example 3: Cell migration assay to evaluate the proliferative effect of Gel@FE-LP(NO)

[0060] To evaluate the effect of the Gel@FE-LP(NO) complex system constructed in this invention on the migration ability of human umbilical vein endothelial cells (HUVECs), an in vitro scratch assay was used for verification. The specific experimental steps are as follows:

[0061] (1) Cell seeding: HUVECs were seeded at a rate of 3 × 10⁶ cells per well. 4 Cells were seeded at a density of 10% in 24-well plates, and DMEM medium containing 10% fetal bovine serum (FBS) was added. The cells were cultured at 37°C and 5% CO2 until the cell confluence reached more than 90%.

[0062] (2) Scratching procedure: Draw two horizontal marking lines on the bottom of the well plate using a marker and ruler as positioning references; after 24 hours of incubation, use a 200μL pipette tip to draw a vertical straight scratch perpendicular to the marking lines, making it intersect the marking lines. Select 3 fixed fields of view for subsequent image comparison in each well plate;

[0063] (3) Washing treatment: Gently wash the well plate 3 times with PBS to completely remove detached cells and discard residual culture medium;

[0064] (4) Experimental Grouping and Treatment: The following treatment groups were established: Ctrl control group (no treatment); Gel group (F-127 hydrogel); LP(NO) group (NO-loaded liposomes); Free FE group (free fat extract); Gel@FE-LP(NO) group (the thermosensitive hydrogel composite system of this invention). In all treatment groups, the concentration of fat extract (FE) was uniformly 100 μg / mL, and the concentration of NO was uniformly 5 nM. Serum-free culture medium was used, with 400 μL added to each well. Each group had 3 replicate wells.

[0065] (5) Imaging and Analysis: Immediately after the scratching was completed, images of the selected area were taken using an inverted microscope and recorded at time point 0h. Subsequently, the cells were incubated at 37°C and 5% CO2 for 24 hours. Cell migration was then recorded again by photographing the same area. Figure 4 (a);

[0066] (6) Results Processing: The area of ​​the scratched region was measured using ImageJ image analysis software, and the cell migration rate was calculated according to the following formula. The results are as follows: Figure 4 As shown in (b):

[0067] Migration rate (%) = (0h scratch area - 24h scratch area) / 0h scratch area × 100% Figure 4 It is evident that the Gel@FE-LP(NO) group exhibited a more significant scratch closure trend compared to other groups, suggesting that the composite material described in this invention has good application potential in promoting endothelial cell migration and accelerating wound repair.

[0068] Example 4: Full-thickness skin wound model in diabetic rats and evaluation of the therapeutic effect of Gel@FE-LP(NO)

[0069] To evaluate the efficacy of Gel@FE-LP(NO) described in this invention in the treatment of diabetic wounds, a full-thickness skin injury model of Wistar diabetic rats was constructed, and intervention experiments were conducted in different treatment groups. The specific methods are as follows:

[0070] Animal modeling: Adult male Wistar rats were selected and a diabetic model was established by intraperitoneal injection of 1% streptozotocin (STZ) solution (55 mg / kg body weight, single intraperitoneal injection). A fasting blood glucose level ≥16.7 mmol / L for more than 3 days was considered a successful model.

[0071] Trauma model establishment: After hair removal and routine skin disinfection on the back of rats, a full-thickness skin defect was prepared in the middle of the back using a disposable sterile skin biopsy punch (MEDLINE) with a diameter of 7 mm. The wound depth was about 2 mm, penetrating the epidermis and dermis.

[0072] Experimental grouping: Rats were randomly divided into the following 7 groups:

[0073] Ctrl group: Healthy rats, no modeling or treatment;

[0074] DM group: a diabetes model group that did not receive hydrogel treatment;

[0075] Gel group: Diabetic model + 50μL blank F127 hydrogel;

[0076] Free FE group: Diabetic model + 50 μL free lipid extract (FE, concentration 100 μg / ml);

[0077] Gel@FE group: Diabetic model + 50 μL FE loaded on F127 hydrogel (unloaded NO, FE concentration of 100 μg / ml);

[0078] Gel@LP(NO) group: Diabetic model + 50μL NO liposome-loaded hydrogel (without FE added);

[0079] Gel@FE-LP(NO) group: Diabetes model + 50 μL Gel@FE-LP(NO) prepared in Example 2.

[0080] Administration: Immediately after surgery, apply the corresponding preparation for each group to the wound. The Healthy and DM groups require no treatment. For the other groups, apply 50 μL of the same amount of hydrogel each day during dressing changes. The treatment cycle continues until the wound is basically closed.

[0081] Wound healing monitoring: Wound images were taken using a high-resolution digital camera on days 0, 3, 7, 10, and 14. (See attached image.) Figure 5 (a) Recording the healing process. After the images were standardized, the changes in wound area were calculated using ImageJ image analysis software. See [link to image analysis]. Figure 5 (b) Evaluate the effect of different treatments on wound healing rate.

[0082] like Figure 5 As shown, the Gel@FE-LP(NO) group exhibited the most significant wound healing acceleration effect, with a significantly higher wound shrinkage rate than other groups, indicating that the NO and FE synergistic delivery system constructed in this invention has significant therapeutic potential in the repair of diabetic refractory wounds.

[0083] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a thermosensitive hydrogel loaded with nitric oxide nanoparticles and a fat extract, characterized in that, Includes the following steps: (1) Mix egg yolk lecithin and dichloromethane, rotary evaporate until a lipid membrane is formed, and add phosphate buffer to obtain a liposome suspension; (2) Add perfluorobromooctane to the liposome suspension and emulsify to obtain liposomes Lips-PFOB encapsulated with perfluorobromooctane. Load NO gas in a closed space to obtain NO-loaded liposomes Lips-PFOB(NO). (3) Add F-127 powder to the Lips-PFOB(NO), dissolve it, and then add the fat extract FE to obtain the thermosensitive hydrogel Gel@FE-LP(NO) loaded with nitric oxide nanoparticles and fat extract.

2. The preparation method according to claim 1, characterized in that, In step (1), the mass-to-volume ratio of egg yolk lecithin to dichloromethane is 4-6 mg: 1 mL.

3. The preparation method according to claim 2, characterized in that, The conditions for rotary evaporation in step (1) are: a rotation speed of 100-500 rpm and a water bath heating temperature of 45-55°C; The concentration of the liposome suspension is 8–12 mg / mL.

4. The preparation method according to claim 3, characterized in that, In step (2), the volume ratio of perfluorooctane added to the liposome suspension is 1 mL: 15-25 μL.

5. The preparation method according to claim 4, characterized in that, The emulsification conditions in step (2) are: power 100-200W, working mode 2 seconds on / 3 seconds off, and cumulative time 5-10 minutes.

6. The preparation method according to claim 5, characterized in that, In step (2), the concentration of NO gas in the enclosed space is 150-250 ppm and the NO loading is 1-20 nM.

7. The preparation method according to claim 6, characterized in that, In step (3), the mass percentage of F-127 is 15-20 wt%. The final concentration of FE is 50–200 μg / mL.

8. The preparation method according to claim 7, characterized in that, The preparation method of the fat extract FE in step (3) is as follows: collect fresh fat aspirate, wash it, cut it into fat tissue blocks of about 1-2 cm3, add PBS, homogenize it until the fat tissue is a fine and uniform paste, perform the first centrifugation and layering, take the middle layer of fat tissue and emulsify it to obtain emulsified fat, freeze-thaw the emulsified fat and perform the second centrifugation and layering, collect the middle pink aqueous phase layer, filter and sterilize it, freeze dry it to obtain the fat extract FE. The conditions for the two centrifugation separations were: 3-5℃, centrifugation at 1000-1500×g for 3-5 min; The amount of PBS added is 1 / 3 to 1 / 2 of the total weight of the adipose tissue block; The homogenization conditions are: 4500-5500 rpm, homogenization for 1-3 min; The freeze-thaw method involves rapidly freezing the emulsified fat at -85 to -75°C for 30 to 40 minutes, and then immediately transferring it to thaw at 35 to 40°C.

9. The thermosensitive hydrogel containing nitric oxide-loaded nanoparticles and a fat extract prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the thermosensitive hydrogel of claim 9, which is loaded with nitric oxide nanoparticles and a fat extract, in the preparation of a medicament or dressing for the repair of diabetic wounds.

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