Use of adipose tissue apoptotic vesicles, formulations and methods of preparation thereof

Abstract

The application provides application of fat tissue apoptosis vesicles, preparation and a preparation method thereof, relates to the field of biomedical basic research, and can culture fat tissue and induce apoptosis in the embodiment of the application, and then the apoptotic extracellular vesicles (ApoEVs) of the fat tissue are obtained through gradient centrifugal separation, the particle size of the fat tissue apoptosis vesicles is nanoscale in the embodiment of the application, and various clinical applications are facilitated, the fat tissue apoptosis vesicles in the embodiment of the application are derived from fat tissue, the source is extensive, the acquisition method is simple, and practical popularization and application are facilitated. The ApoEVs provided in the embodiment of the application can be directly applied to promote skin wound healing, reduce scar formation, and avoid problems such as post-transplantation necrosis, immune rejection, ethical issues and tumor risks that may be caused by cell therapy, and can be widely applied to clinical treatment of skin wounds.

Description

Technical Field

[0001] This invention relates to the field of basic biomedical research, and in particular to the application, formulation, and preparation method of apoptotic vesicles in adipose tissue. Background Technology

[0002] Skin, the largest organ in the human body, plays a crucial role in the interaction between the external environment and the body in this invention embodiment; therefore, it is inevitably subject to various forms of injury. Due to the importance of dermal adipocytes in skin development and remodeling, the transplantation of adipose tissue and its derivatives is considered one of the most advanced treatment methods for skin diseases. Related studies have used adipose tissue, nanofat, stromal adipose cells (SVF), adipose stem cells, and other cellular components to treat skin trauma and scars, demonstrating good therapeutic effects. However, cell therapy is limited by factors such as post-transplant necrosis, immune rejection, ethical concerns, and tumor risks.

[0003] Therefore, there is an urgent need for a new strategy for treating skin trauma and scars. Summary of the Invention

[0004] To address the problems existing in the above-mentioned background art, the present invention provides an application, formulation, and preparation method of adipose tissue apoptotic vesicles.

[0005] In a first aspect, the present invention provides the application of adipose tissue apoptotic vesicles in the preparation of a formulation that promotes skin wound healing. The adipose tissue apoptotic vesicles are derived from adipose tissue and are obtained by gradient centrifugation after culturing and inducing apoptosis in the adipose tissue. The adipose tissue apoptotic vesicles are spherical, have a phospholipid bilayer structure, and have a particle size of 50-500 nm. The adipose tissue apoptotic vesicles express apoptosis-related specific proteins. The adipose tissue apoptotic vesicles are also used to promote skin wound healing and reduce scar formation.

[0006] Optionally, the adipose tissue apoptotic vesicles are used to enhance the proliferation and migration of fibroblasts and endothelial cells, and promote the adipogenic differentiation of fibroblasts; the adipose tissue apoptotic vesicles reduce the expression of fibroblast-related genes Col 1, Col 3 and α-SMA, and increase the expression of adipogenic genes PPARγ and C / EBPα.

[0007] Secondly, the present invention provides an adipose tissue apoptosis vesicle formulation, comprising: adipose tissue apoptosis vesicles and a sustained-release material. The adipose tissue apoptosis vesicles are derived from adipose tissue, obtained by gradient centrifugation after culturing and inducing apoptosis in adipose tissue. The adipose tissue apoptosis vesicles are spherical, have a typical phospholipid bilayer structure, and have a particle size of 50–500 nm. The adipose tissue apoptosis vesicles express apoptosis-related specific proteins. The adipose tissue apoptosis vesicles are also used to promote skin wound healing and reduce scar formation. The sustained-release material includes one or more of hyaluronic acid, collagen, and chitosan.

[0008] Optionally, the adipose tissue apoptotic vesicles are used to enhance the proliferation and migration of fibroblasts and endothelial cells, and promote the adipogenic differentiation of fibroblasts; the adipose tissue apoptotic vesicles reduce the expression of fibroblast-related genes Col 1, Col 3 and α-SMA, and increase the expression of adipogenic genes PPARγ and C / EBPα;

[0009] The sustained-release material is used as a scaffold material to encapsulate the apoptotic vesicles in the adipose tissue.

[0010] Thirdly, the present invention provides a method for preparing an adipose tissue apoptosis vesicle formulation, the method comprising:

[0011] Collect apoptotic vesicles from adipose tissue, which are derived from adipose tissue and obtained by gradient centrifugation after culturing and inducing apoptosis in adipose tissue.

[0012] The adipose tissue apoptotic vesicles were mixed with a sustained-release material;

[0013] The adipose tissue apoptotic vesicles are spherical and have a phospholipid bilayer structure. The particle size of the adipose tissue apoptotic vesicles is 50-500 nm. The adipose tissue apoptotic vesicles express apoptosis-related specific proteins. The adipose tissue apoptotic vesicles are also used to promote skin wound healing and reduce scar formation.

[0014] The sustained-release material includes one or more of hyaluronic acid, collagen, and chitosan.

[0015] Optionally, the collection of apoptotic vesicles from adipose tissue includes:

[0016] Obtaining adipose tissue;

[0017] α-MEM medium and astrococcus were added to the adipose tissue to obtain a mixture. The mixture was then transferred to a suspension culture bottle and placed in a 37°C, 5% CO2 incubator for suspension culture to induce apoptosis in the adipose tissue.

[0018] The cultured mixture was filtered to remove tissue fragments and obtain the culture supernatant.

[0019] The culture supernatant was centrifuged to remove cell debris and large vesicles, and the vesicle supernatant was collected.

[0020] The supernatant of the vesicles was centrifuged to collect apoptotic vesicles from adipose tissue.

[0021] Optionally, the culture supernatant is centrifuged to remove cell debris and large-diameter vesicles, and the vesicle supernatant is collected, comprising:

[0022] The culture supernatant was centrifuged at a force of 700g-900g for 8-12 minutes to remove cell debris and collect the cell supernatant.

[0023] The cell supernatant was centrifuged at a force of 1900g-2100g for 13-17 minutes to remove large-diameter vesicles and collect the vesicle supernatant.

[0024] Optionally, the vesicle supernatant is centrifuged to collect apoptotic vesicles from adipose tissue, including:

[0025] The vesicle supernatant was centrifuged at a force of 15000g-17000g for 28-32 minutes. The supernatant was removed, and the precipitated adipose tissue apoptotic vesicles were collected.

[0026] Optionally, the volume / mass ratio of the α-MEM culture medium is 9 to 11, and the concentration of the astrosporin is 200-300 nmol.

[0027] Optionally, the rotation speed of the suspension culture is 150-250 rpm / min.

[0028] Compared with the prior art, the present invention has the following advantages:

[0029] In this embodiment of the invention, adipose tissue can be cultured in suspension and apoptosis induced, and then apoptotic extracellular vesicles (ApoEVs) of adipose tissue can be obtained by gradient centrifugation. In this embodiment of the invention, the particle size of the apoptotic vesicles of adipose tissue is in the nanometer range, which is beneficial for various clinical applications. The apoptotic vesicles of adipose tissue in this embodiment of the invention are derived from adipose tissue, which is widely available and easy to obtain, thus facilitating practical application.

[0030] The ApoEVs provided in this invention can be directly applied to promote skin wound healing, reduce scarring, and avoid problems such as cell transplantation necrosis, immune rejection, ethical issues, and tumor risks that may occur with cell therapy. They can be widely used in the clinical management of skin trauma. Attached Figure Description

[0031] Figure 1 This is a schematic diagram showing the analysis results related to the preparation and characterization of apoptotic vesicles in adipose tissue provided in the embodiments of the present invention;

[0032] Figure 2 This is a schematic diagram illustrating the correlation analysis results of ApoEVs accelerating skin wound healing in the rat skin defect model experiment provided in this embodiment of the invention.

[0033] Figure 3 This is a schematic diagram of the relevant analysis results of ApoEVs-induced high-quality granulation tissue formation in the rat skin defect model experiment provided in the embodiments of the present invention;

[0034] Figure 4 This is a schematic diagram illustrating the correlation analysis results of ApoEVs reducing scar formation in a rat skin defect model experiment provided in this embodiment of the invention;

[0035] Figure 5 This is a schematic diagram illustrating the correlation analysis results of ApoEVs regulating fibroblast behavior provided in the embodiments of the present invention;

[0036] Figure 6 This is a schematic diagram illustrating the correlation analysis results of ApoEVs regulating the behavior of endothelial cells provided in the embodiments of the present invention. Detailed Implementation

[0037] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any formulation that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0038] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents and other instruments whose manufacturers are not specified are all commercially available reagents and preparations.

[0039] In this embodiment of the invention, the inventors obtained ApoEVs-AT from whole adipose tissue and characterized the obtained ApoEVs-AT. Then, ApoEVs-AT was used to explore the treatment of full-thickness skin wounds to investigate its ability to promote wound healing and reduce scar formation. Furthermore, the inventors also explored the effects of ApoEVs-AT on the behavior of fibroblasts and endothelial cells. Based on all the exploration results, this embodiment of the invention proposes a novel strategy for treating skin trauma and scars: utilizing apoptotic vesicles in adipose tissue to promote wound healing and improve scarring.

[0040] Specifically, this invention proposes the application of adipose tissue apoptotic vesicles in the preparation of agents that promote skin wound healing. The adipose tissue apoptotic vesicles are derived from adipose tissue, obtained by culturing and inducing apoptosis, followed by gradient centrifugation. The adipose tissue apoptotic vesicles are spherical, possess a phospholipid bilayer structure, and have a particle size of 50–500 nm. These vesicles express apoptosis-related specific proteins. Furthermore, the adipose tissue apoptotic vesicles are used to promote skin wound healing and reduce scar formation.

[0041] Specifically, in this embodiment of the invention, the adipose tissue apoptotic vesicles are used to enhance the proliferation and migration of fibroblasts and endothelial cells, and promote the adipogenic differentiation of fibroblasts; the adipose tissue apoptotic vesicles reduce the expression of fibroblast-related genes Col 1, Col 3 and α-SMA, and increase the expression of adipogenic genes PPARγ and C / EBPα.

[0042] In this embodiment of the invention, an adipose tissue apoptosis vesicle formulation is also proposed. The adipose tissue apoptosis vesicle formulation comprises: adipose tissue apoptosis vesicles and a sustained-release material. The adipose tissue apoptosis vesicles are derived from adipose tissue and are obtained by gradient centrifugation after culturing and inducing apoptosis in adipose tissue. The adipose tissue apoptosis vesicles are spherical and have a typical phospholipid bilayer structure. The particle size of the adipose tissue apoptosis vesicles is 50-500 nm. The adipose tissue apoptosis vesicles express apoptosis-related specific proteins. The adipose tissue apoptosis vesicles are also used to promote skin wound healing and reduce scar formation. The sustained-release material includes one or more of hyaluronic acid, collagen, and chitosan.

[0043] Specifically, the adipose tissue apoptosis vesicles enhance the proliferation and migration of fibroblasts and endothelial cells, and promote adipogenic differentiation of fibroblasts; the adipose tissue apoptosis vesicles reduce the expression of fibroblast-related genes Col 1, Col 3 and α-SMA, and increase the expression of adipogenic genes PPARγ and C / EBPα;

[0044] The sustained-release material is used as a scaffold material to encapsulate the apoptotic vesicles in the adipose tissue.

[0045] In this embodiment of the invention, a method for preparing an adipose tissue apoptosis vesicle formulation is also proposed, the method comprising:

[0046] S101, collects apoptotic vesicles from adipose tissue.

[0047] Specifically, in this embodiment of the invention, the adipose tissue apoptotic vesicles are derived from adipose tissue, obtained by culturing and inducing apoptosis in adipose tissue followed by gradient centrifugation. The adipose tissue apoptotic vesicles are spherical, possess a phospholipid bilayer structure, and have a particle size of 50–500 nm. These adipose tissue apoptotic vesicles express apoptosis-related specific proteins. Furthermore, the adipose tissue apoptotic vesicles also promote skin wound healing and reduce scar formation.

[0048] Specifically, step S101 includes the following sub-steps:

[0049] S1011, Obtain adipose tissue.

[0050] Specifically, in this embodiment of the invention, the adipose tissue can be adipose tissue from various genera such as rat, pig, and human.

[0051] Specifically, in this embodiment of the invention, the adipose tissue can be cut into pieces of 0.8-1.2 mm. 3 The tissue block is of a certain size, preferably 1 mm. 3 .

[0052] S1012, α-MEM medium and astrosporin are added to the adipose tissue to obtain a mixture. The mixture is then transferred to a suspension culture bottle and placed in a 37°C, 5% CO2 incubator for suspension culture to induce apoptosis in the adipose tissue.

[0053] Specifically, in this embodiment of the invention, the volume / mass of the α-MEM culture medium is 9 to 11, preferably 10.

[0054] Specifically, in this embodiment of the invention, the concentration of asteroidin is 200-300 nmol, preferably 250 nmol.

[0055] Specifically, in this embodiment of the invention, the rotation speed of the suspension culture is 150-250 rpm / min. Preferably, the rotation speed of the suspension culture is 200 rpm / min.

[0056] Specifically, in this embodiment of the invention, the suspension culture time is 46-50 hours, preferably 48 hours.

[0057] S1013, filter the cultured mixture to remove tissue blocks and obtain culture supernatant.

[0058] S1014, the culture supernatant is centrifuged to remove cell debris and large-diameter vesicles, and the vesicle supernatant is collected.

[0059] Specifically, in this embodiment of the invention, the culture supernatant is centrifuged using gradient centrifugation, which may specifically include two centrifugations:

[0060] First centrifugation: The culture supernatant is centrifuged at a force of 700g-900g for 8-12 minutes to remove cell debris and collect the cell supernatant. Preferably, the centrifugation force is 800g and the centrifugation time is 10 minutes.

[0061] Second centrifugation: The cell supernatant is centrifuged at a force of 1900g-2100g for 13-17 minutes to remove large-diameter vesicles and collect the vesicle supernatant. Preferably, the centrifugation force is 2000g and the centrifugation time is 15 minutes.

[0062] S1015, the vesicle supernatant is centrifuged to collect adipose tissue apoptotic vesicles.

[0063] Specifically, in this embodiment of the invention, the vesicle supernatant is centrifuged at a force of 15000g-17000g for 28-32 minutes, the supernatant is removed, and the precipitated adipose tissue apoptotic vesicles are collected. Preferably, the centrifugation force is 16000g and the centrifugation time is 30 minutes.

[0064] S102, the adipose tissue apoptotic vesicles are mixed with the sustained-release material.

[0065] Specifically, in this embodiment of the invention, the sustained-release material includes one or more of hyaluronic acid, collagen, and chitosan.

[0066] In this embodiment of the invention, the sustained-release material is used as a scaffold material to encapsulate the apoptotic vesicles of the adipose tissue.

[0067] To enable those skilled in the art to better understand the present invention, the following specific embodiments illustrate the application, formulation, and preparation method of adipose tissue apoptosis vesicles provided by the present invention.

[0068] Example 1: Preparation and characterization of apoptotic vesicles in adipose tissue

[0069] In this embodiment of the invention, inguinal adipose tissue was isolated from 4-week-old SD rats, cut into small pieces, and transferred to suspension culture flasks. Serum-free α-modified Eagle medium (α-MEM, HyClone) containing 250 nM astrococcal spores (STS, Beyptime) was added to the suspension culture flasks. The mixture was incubated at 37°C under 5% CO2-95% atmospheric conditions at 200 rpm for 2 days. Tissue blocks were removed by filtration through gauze, and the supernatant was collected. Tissue fragments and larger extracellular vesicles were removed by centrifugation at 800 g for 10 minutes at 4°C and at 2000 g for 15 minutes at 4°C. The supernatant was further collected and centrifuged at 16000 g for 30 minutes at 4°C to obtain ApoEVs-AT, as shown in the figure. Figure 1 As shown in section A, it illustrates a schematic diagram of the preparation process of adipose tissue apoptotic vesicles used in an embodiment of the present invention.

[0070] In this embodiment of the invention, apoptosis in adipose tissue was assessed using H&E and TUNEL staining. Compared to native adipose tissue, apoptotic adipose tissue has a loose and irregular structure (e.g., Figure 1 Part B shows representative images of native and apoptotic adipose tissue with H&E staining. TUNEL-stained images show apoptosis occurring in many cells of the apoptotic adipose tissue, whereas native adipose tissue lacks TUNEL-labeled nuclei (e.g., Figure 1 Section C shows representative images of native and apoptotic adipose tissue stained with TUNEL, where green represents TUNEL-stained apoptotic cells and blue represents DAPI-stained cell nuclei.

[0071] To further evaluate the characteristics of ApoEVs-AT, morphology, particle size distribution, and specific marker proteins were analyzed in this embodiment of the invention. First, by transmission electron microscopy (TEM), ApoEVs-AT exhibited a typical double-membrane spherical structure (e.g., ...). Figure 1 As shown in section D, which presents a representative image of ApoEVs-AT under a transmission electron microscope. Nanoparticle tracking analysis indicates that most ApoEVs-AT range from 25 to 400 nm. The average size is 155.4 nm (e.g., ...). Figure 1 As shown in section E, which illustrates the particle size distribution of ApoEVs-AT as measured by nanoparticle tracking analysis. Immunofluorescence images then revealed that ApoEVs-AT was positive for the apoptosis-specific marker phosphatidylserine (PtdSer), as shown by Annexin V staining (e.g., ...). Figure 1Section F shows a representative image of ApoEVs-AT stained with Annexin V, where the white dashed box indicates the magnified area in the right-hand magnification panel, and green indicates Annexin V-stained ApoEVs-AT. Furthermore, western blot analysis showed that ApoEVs-AT express apoptosis-specific marker proteins such as cleaved caspase-3, caveolin-1 (Cav-1), RhoA, and histone H3 (e.g., ...). Figure 1 As shown in section G, which illustrates Western blot analysis of apoptosis-specific marker proteins, cleaved caspase-3, caveolin-1 (Cav-1), RhoA, and histone H3. These results demonstrate that ApoEVs can be successfully prepared from adipose tissue after 2 days of culture with STS, and that ApoEVs-AT possess apoptotic vesicle characteristics.

[0072] Example 2: Rat skin defect model experiment

[0073] In this embodiment of the invention, to evaluate the role of ApoEVs-AT in the healing of full-thickness skin wounds, two full-thickness skin wounds (2 cm in diameter) were created on the back of each SD rat. In the ApoEVs-AT group, 100 μl of PBS containing 200 μg of ApoEVs-AT was subcutaneously injected around the wound every 4 days. In the blank control group, 100 μl of PBS was used as a control (e.g., ...). Figure 2 Part A shows a schematic diagram of the experimental procedure for the rat skin defect model experiment used in this embodiment of the invention. Digital photographs were taken on days 0, 4, 8, 12, and 16 to measure the healing rate. A schematic diagram was created to more clearly indicate the speed of wound healing (e.g., Figure 2 Part B shows representative digital photographs of the wound area on days 0, 4, 8, 12, and 16. The dashed circles represent the initial wound area on day 0, and the colored areas represent the area of ​​unhealed wounds at different time points. In this embodiment, the wound area (%) is defined as: unhealed wound area on day X / initial wound area on day 0 × 100%. Statistical analysis showed that after two injections (day 0 and day 4), ApoEVs-AT demonstrated an ability to accelerate wound healing. This was based on the result that adding ApoEVs-AT reduced the average wound area on day 8 from 48.27% (blank group) to 34.03% (ApoEVs-AT group). Figure 2As shown in Section C, which illustrates the statistical analysis of wound area in mice in the control group and the ApoEVs-AT group. Similarly, after three injections (days 0, 4, and 8), the addition of ApoEVs-AT reduced the mean wound area on day 12 from 36.87% (control group) to 20.57% (ApoEVs-AT group) (as shown in Section C). Figure 2 (As shown in Section C). Furthermore, after four injections (days 0, 4, 8, and 12), all full-thickness skin wounds in the ApoEVs-AT group healed by day 16, compared to a mean wound area of ​​25.87% in the control group on day 16, which was significantly different from the wound area in the ApoEVs-AT group (e.g., ...). Figure 2 (As shown in section C). These results indicate that ApoEVs-AT demonstrates the ability to accelerate the healing of full-thickness skin wounds.

[0074] In this embodiment of the invention, the quality of granulation tissue in the unhealed wound area was also evaluated, which significantly affects the quality of subsequent skin healing. Wounds were collected on day 8 and sections were prepared for subsequent staining. Through H&E staining of longitudinal sections, this embodiment of the invention found that ApoEVs-AT accelerated the rate of re-epithelialization, based on the fact that the area without epithelialization in the ApoEVs-AT group was narrower than that in the control group (e.g., ...). Figure 3 Part A shows a representative image of a longitudinal section of the wound stained with H&E on day 8. The black inverted triangles indicate the edges of the wound area / re-epithelialization, and the solid black boxes represent magnified areas. In the magnified area on the right, the black dashed lines indicate the areas of regenerated epidermis, and the black dashed boxes represent areas of... Figure 3 (Magnified H&E stained area in part B), consistent with the results of the digital photograph.

[0075] In this embodiment of the invention, it was also found that the unhealed wound area was mainly connected by granulation tissue, which was covered by a scab (e.g. Figure 3 Part B shows representative images of granulation tissue on day 8 using H&E and Massen staining. Dashed lines indicate the edges of the granulation tissue, solid lines with double arrows represent the thickness of the granulation tissue, and solid black boxes indicate the areas within the granulation tissue. Figure 3 (Magnified Masson staining region in part D). Through H&E and Masson staining, embodiments of the present invention revealed that in the ApoEVs-AT group, granulation tissue was thicker and more diverse in composition than in the control group (e.g., ...). Figure 3 (As shown in Part B). Statistical analysis showed that the average thickness of granulation tissue in the ApoEV-AT group was 1342.26 μm. However, the average thickness in the control group was 826.32 μm, a statistically significant difference (e.g., ...). Figure 3Section C shows the statistical results of granulation tissue thickness. Masson staining revealed that fibers were the main component of granulation tissue in both the ApoEVs-AT group and the control group. However, the ApoEVs-AT group showed more blood vessels, which was further confirmed by immunofluorescence staining of CD31 (a universal marker for vascular endothelial cells) (e.g.). Figure 3 Part D shows a representative magnified image of the area in Part B of the Massen staining and immunofluorescence staining, where yellow arrows point to blood vessels, green indicates CD31-stained blood vessels, red indicates adipocytes, and blue indicates DAPI-stained nuclei. Furthermore, the number of blood vessels per field of view in the ApoEVs-AT group was statistically different from that in the control group (e.g., ...). Figure 3 As shown in section E, which presents statistical results on the number of blood vessels.

[0076] Because adipocytes are an extremely important cell type in skin development and regeneration, adipocytes were present in every wound in the ApoEVs-AT group, while no adipocytes were found in the control group (e.g., ...). Figure 3 As shown in section D, yellow stars indicate areas of adipocytes, green represents CD31-stained blood vessels, red represents adipocytes, and blue represents DAPI-stained cell nuclei. Therefore, these results confirm that ApoEVs-AT can promote the formation of high-quality granulation tissue during skin wound healing, which may contribute to the quality of later-healing skin.

[0077] To assess the quality of subsequent healing, in this embodiment of the invention, healed skin from the wound site was collected on day 40 and sections were prepared for subsequent staining. Based on digital radiographs, scar area (%) was defined as: scar area on day 40 / initial wound area on day 0 × 100%. Statistical analysis showed that, through ApoEVs-AT injection, the average scar area decreased from 55.93% (blank group) to 22.43% (ApoEVs-AT group). Figure 4 As shown in Section A, it presents representative digital photographs of the scars from both experimental groups on day 40 and the statistical analysis results of the scar area (with black dashed circles indicating the edges of the scars). H&E-stained images also show that the scars in the ApoEVs-AT group were narrower than those in the control group (e.g., ...). Figure 4 Part B shows a representative image of a longitudinal section of the scar stained with H&E on day 40, where the black dashed box indicates the area... Figure 4(Enlarged area in section C). In the ApoEVs-AT group, there were numerous hair follicles in the non-scarred areas, indicating similarity to natural skin, while the scarred areas had fewer hair follicles. The control group had largely no hair follicles or other appendages, being occupied by fibrous tissue. However, both the ApoEVs-AT and control groups had intact epidermal structures (e.g., Figure 4 Part C shows a representative image of the magnified area shown in Part B, based on H&E staining, Masson staining, and immunofluorescence staining. The dashed lines indicate the edges of the epidermis and dermis, where Ep. represents the epidermis, De. represents the dermis, yellow represents α-SMA, green represents Col 1, and red represents Col 3. Statistical analysis showed that ApoEVs-AT treatment significantly increased the number of hair follicles per field of view (e.g., ...). Figure 4 As shown in section D, which illustrates the statistical analysis results of hair follicle count. Masson staining indicates that fibers are the main component of the scar area. This embodiment of the invention further performed immunofluorescence staining analysis on these fibers to evaluate different components. Here, this embodiment of the invention found that ApoEVs-AT can reduce the expression of α-SMA (a myofibroblast marker, highly expressed in scars) (e.g., Figure 4 As shown in section C of Figure 4). The relative fluorescence density (α-SMA / DAPI) in each field of view of the ApoEVs-AT group was significantly reduced (as shown in section E of Figure 4, which illustrates the statistical analysis of the relative fluorescence density of α-SMA / DAPI). Furthermore, in this embodiment of the invention, collagen 1 (Col 1) and collagen 3 (Col 3) were labeled by immunofluorescence staining, and it was found that the positive signal of Col 3 was greater in the ApoEVs-AT group than in the blank group (as shown in section C of Figure 4). In addition, ApoEVs-AT treatment increased the relative fluorescence density of Col 3 / Col 1 in each field of view from 0.10 (blank group) to 0.26 (ApoEVs-AT group) (as shown in Figure 4). Figure 4 As shown in section F, which presents the statistical analysis results of the relative fluorescence density of Col 3 / Col 1, an increased Col 3 / Col 1 ratio has been reported to be closely associated with scar reduction and increased skin softness. These results indicate that ApoEVs-AT demonstrates the ability to promote high-quality, full-thickness skin wound healing while reducing scar formation and hair follicle regeneration.

[0078] To explore the cellular mechanism by which ApoEVs-AT promotes high-quality skin wound healing, this invention selected two important cell types in skin regeneration (fibroblasts and endothelial cells) for in vitro experiments. After culturing fibroblasts with Dio-labeled ApoEVs-AT for 8 hours, ApoEVs-AT (e.g., ...) could be effectively detected within the fibroblasts. Figure 5Section A shows the analysis of fibroblast uptake of ApoEVs-AT, where red represents fibroblasts stained with phalloidin, green represents Dio-labeled ApoEVs-AT, and blue represents DAPI-stained cell nuclei. CCK8 assays indicated that 50 μg / ml ApoEVs-AT significantly increased fibroblast proliferation compared to the control group (e.g., ...). Figure 5 Part B shows the analysis of fibroblast proliferation results measured by the CCK8 assay. Both scratch and transwell assays indicated that ApoEVs-AT significantly promoted fibroblast migration (e.g., Figure 5 Parts C, D, E, and F are shown in the figure. Part C shows representative images of the migration region at 0 and 16 hours in the scratch assay, with the yellow line indicating the edge of the migrating cells. Part D shows the statistical analysis results of the migration region. Part E shows representative images of the migrating cells at 12 hours in the transwell assay. Part F shows the statistical analysis results of the migrating cells. Furthermore, after fibroblasts were treated with ApoEVs-AT for 20 days, the expression of fibroblast-related genes (Col1, Col3, and α-SMA) and adipogenesis-related genes (PPARγ and C / EBPα) was assessed by qRT-PCR. The results showed that ApoEVs-AT reduced the expression of fibroblast-related genes (Col1, Col3, and α-SMA) and increased the expression of adipogenesis-related genes (PPARγ and C / EBPα). Figure 5 As shown in section G, it illustrates the results of a relative expression analysis of fibrosis-related genes (Col 1, Col 3, and α-SMA) and adipose-related genes (PPARγ and C / EBPα) detected by qRT-PCR, which may contribute to reducing scar formation. Furthermore, the results also indicate that ApoEVs-AT treatment increased the Col 3 / Col 1 ratio (e.g., ...). Figure 5 The middle section H shows the analytical results of the Col 3 / Col 1 ratio, which is consistent with the in vivo experimental results (e.g., Figure 4 (As shown in section F).

[0079] The cellular uptake of ApoEVs-AT by endothelial cells was assessed, and the same phenomena as those observed in fibroblasts were detected (e.g., Figure 6 Section A shows the analysis results of endothelial cell uptake of ApoEVs-AT, where red indicates endothelial cells stained with phalloidin, green indicates Dio-labeled ApoEVs-AT, and blue indicates DAPI-stained cell nuclei. ApoEVs-AT also promotes endothelial cell proliferation and migration (e.g., Figure 6Parts B, C, and D are shown in the figure. Part B shows the analysis of endothelial cell proliferation results measured by the CCK8 assay; Part C shows representative images of migrating cells at 12 hours in the transwell assay; and Part D shows the analysis results of the migrating cells. Furthermore, tube formation assays showed that ApoEVs-AT significantly induced angiogenesis in endothelial cells, based on the increased node number, mesh number, and total length after pretreatment of endothelial cells with 50 μg / ml ApoEVs-AT (e.g., ...). Figure 6 As shown in sections E and F, section E presents representative images of endothelial cell tubular structure formation, with the image in the upper left region representing the results of ImageJ software analysis. Section F shows the results of cell number analysis. In summary, ApoEVs-AT promotes high-quality healing of skin wounds, which may be related to the behavioral regulation of fibroblasts and endothelial cells.

[0080] In this embodiment of the invention, ApoEVs can be directly isolated from adipose tissue. Under real-world conditions, tissue-derived ApoEVs more closely resemble the internal microenvironment than single-cell-derived ApoEVs. In this embodiment of the invention, the ApoEVs-AT extracted from adipose tissue also exhibit high purity.

[0081] This invention creates a skin defect with a diameter of 2 cm, which can be considered a large defect in a wound healing model and is more likely to form a scar. This invention ensures treatment effectiveness by injecting ApoEVs-AT every 4 days. This invention uses tissue-derived ApoEVs to promote skin wound healing and further improves the quality of subsequent wound healing. Specifically, this invention also proposes a drug delivery method, such as encapsulating ApoEVs-AT in a sustained-release material, which not only reduces iatrogenic trauma and pain caused by local injection but also slowly releases ApoEVs-AT and improves the antibacterial and adhesive properties of the formulation.

[0082] In this embodiment of the invention, ApoEVs-AT extracted directly from adipose tissue exhibits a nanoscale size, making it suitable for various drug delivery methods. Therefore, ApoEVs-AT can also be combined with more efficient delivery methods to maximize its value in the treatment of skin wounds.

[0083] For the sake of simplicity, the method embodiments are described as a series of actions. However, those skilled in the art should understand that the present invention is not limited to the described order of actions, as some steps can be performed in other orders or simultaneously according to the present invention. Furthermore, those skilled in the art should also understand that the embodiments described in the specification are preferred embodiments, and the actions and components involved are not necessarily essential to the present invention.

[0084] The application, formulation, and preparation method of adipose tissue apoptosis vesicles provided by the present invention have been described in detail above. Specific examples have been used to illustrate the principle and implementation of the present invention. The description of the above embodiments is only for the purpose of helping to understand the method and core idea of ​​the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation and application scope based on the idea of ​​the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. The application of adipose tissue apoptotic vesicles in the preparation of formulations that promote skin wound healing and reduce scarring after skin wound healing, characterized in that, The adipose tissue apoptotic vesicles are directly derived from adipose tissue, obtained by gradient centrifugation after culturing and inducing apoptosis in adipose tissue. These apoptotic vesicles are spherical, possess a phospholipid bilayer structure, and have a particle size of 50-500 nm. They express apoptosis-related specific proteins. These apoptotic vesicles also promote skin wound healing and reduce scar formation; they enhance the proliferation and migration of fibroblasts and endothelial cells, and promote adipogenic differentiation of fibroblasts. The adipose tissue apoptosis-inducing mechanism is as follows: α-MEM medium and astrococcus were added to the adipose tissue to obtain a mixture. The mixture was then transferred to a suspension culture bottle and placed in a 37°C, 5% CO2 incubator for suspension culture to induce apoptosis in the adipose tissue. Wherein, the volume of the α-MEM culture medium / the mass of the adipose tissue = 9~11; The concentration of the asteroidin is 200-300 nmol; The rotation speed of the suspension culture is 150-250 rpm / min; The suspension culture time is 46-50 hours.

2. A fat tissue apoptotic vesicle formulation for promoting skin wound healing and reducing scar formation after skin wound healing, characterized in that, The adipose tissue apoptosis vesicle formulation comprises: adipose tissue apoptosis vesicles and a sustained-release material. The adipose tissue apoptosis vesicles are derived from adipose tissue, obtained by culturing and inducing apoptosis in adipose tissue followed by gradient centrifugation. The adipose tissue apoptosis vesicles are spherical, possess a typical phospholipid bilayer structure, and have a particle size of 50-500 nm. The adipose tissue apoptosis vesicles express apoptosis-related specific proteins. The adipose tissue apoptosis vesicles are also used to promote skin wound healing and reduce scar formation. The sustained-release material includes one or more of hyaluronic acid, collagen, and chitosan. The adipose tissue apoptosis vesicles are used to enhance the proliferation and migration of fibroblasts and endothelial cells, and promote adipogenic differentiation of fibroblasts. The adipose tissue apoptosis-inducing mechanism is as follows: α-MEM medium and astrococcus were added to the adipose tissue to obtain a mixture. The mixture was then transferred to a suspension culture bottle and placed in a 37°C, 5% CO2 incubator for suspension culture to induce apoptosis in the adipose tissue. Wherein, the volume of the α-MEM culture medium / the mass of the adipose tissue = 9~11; The concentration of the asteroidin is 200-300 nmol; The rotation speed of the suspension culture is 150-250 rpm / min; The suspension culture time is 46-50 hours.

3. The adipose tissue apoptosis vesicle formulation according to claim 2, characterized in that, The adipose tissue apoptotic vesicles enhance the proliferation and migration of fibroblasts and endothelial cells, and promote adipogenic differentiation of fibroblasts; the adipose tissue apoptotic vesicles reduce the expression of fibroblast-related genes Col 1, Col 3 and α-SMA, and increase the expression of adipogenic genes PPARγ and C / EBPα. The sustained-release material is used as a scaffold material to encapsulate the apoptotic vesicles in the adipose tissue.

4. A method for preparing an adipose tissue apoptosis vesicle formulation for promoting skin wound healing and reducing scar formation after skin wound healing, characterized in that, The method includes: Collect apoptotic vesicles from adipose tissue, which are derived from adipose tissue and obtained by gradient centrifugation after culturing and inducing apoptosis in adipose tissue. The adipose tissue apoptotic vesicles were mixed with a sustained-release material; The adipose tissue apoptotic vesicles are spherical with a phospholipid bilayer structure and a particle size of 50-500 nm. They express apoptosis-related specific proteins. The adipose tissue apoptotic vesicles also promote skin wound healing and reduce scar formation. They enhance the proliferation and migration of fibroblasts and endothelial cells and promote adipogenic differentiation of fibroblasts. The sustained-release material includes one or more of hyaluronic acid, collagen, and chitosan; The adipose tissue apoptosis-inducing mechanism is as follows: α-MEM medium and astrococcus were added to the adipose tissue to obtain a mixture. The mixture was then transferred to a suspension culture bottle and placed in a 37°C, 5% CO2 incubator for suspension culture to induce apoptosis in the adipose tissue. Wherein, the volume of the α-MEM culture medium / the mass of the adipose tissue = 9~11; The concentration of the asteroidin is 200-300 nmol; The rotation speed of the suspension culture is 150-250 rpm / min; The suspension culture time is 46-50 hours.

5. The preparation method according to claim 4, characterized in that, The collection of apoptotic vesicles from adipose tissue includes: Obtaining adipose tissue; α-MEM medium and astrococcus were added to the adipose tissue to obtain a mixture. The mixture was then transferred to a suspension culture bottle and placed in a 37°C, 5% CO2 incubator for suspension culture to induce apoptosis in the adipose tissue. Wherein, the volume of the α-MEM culture medium / the mass of the adipose tissue = 9~11; The concentration of the asteroidin is 200-300 nmol; The rotation speed of the suspension culture is 150-250 rpm / min; The suspension culture time is 46-50 hours; The cultured mixture was filtered to remove tissue fragments and obtain the culture supernatant. The culture supernatant was centrifuged to remove cell debris and large vesicles, and the vesicle supernatant was collected. The supernatant of the vesicles was centrifuged to collect apoptotic vesicles from adipose tissue.

6. The preparation method according to claim 5, characterized in that, The culture supernatant was centrifuged to remove cell debris and large vesicles, and the vesicle supernatant was collected, comprising: The culture supernatant was centrifuged at a force of 700g-900g for 8-12 minutes to remove cell debris and collect the cell supernatant. The cell supernatant was centrifuged at a force of 1900g-2100g for 13-17 minutes to remove large-diameter vesicles and collect the vesicle supernatant.

7. The preparation method according to claim 5, characterized in that, The vesicle supernatant was centrifuged to collect apoptotic vesicles from adipose tissue, including: The vesicle supernatant was centrifuged at a force of 15000g-17000g for 28-32 minutes. The supernatant was removed, and the precipitated adipose tissue apoptotic vesicles were collected.

8. The preparation method according to claim 5, characterized in that, The volume / mass ratio of the α-MEM medium is 9~11, and the concentration of the astrocytocin is 200-300 nmol.

9. The preparation method according to claim 5, characterized in that, The rotation speed for suspension culture is 150-250 rpm / min.

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