VPA-Exo drug-loaded exosome-based in-vitro evaluation model for promoting skin wound healing as well as construction method and application of VPA-Exo drug-loaded exosome-based in-vitro evaluation model

Abstract

The invention belongs to the field of medicines, and particularly relates to an in-vitro evaluation model for promoting skin wound healing based on a VPA-Exo drug-loaded exosome as well as a construction method and application of the in-vitro evaluation model. The construction method of the in-vitro evaluation model for promoting skin wound healing comprises the following steps: S1, after passage of resuscitated HSF cells and HUVEC cells, taking HSF cells or HUVEC cell suspension in a logarithmic phase, inoculating the HSF cells or HUVEC cell suspension on a culture plate, and culturing on a real-time label-free cell analyzer for 20-28 hours; and S2, then adding VPA-EXO with the concentration of 12.5 mu g / mL-400 mu g / mL to act on the HSF cells or the HUVEC cells.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to an in vitro evaluation model for promoting skin wound healing based on VPA-Exo drug-loaded exosomes, its construction method, and its application. Background Technology

[0002] Skin wound healing is a complex process involving the coordinated action of many different tissues and cell lineages. It requires close coordination of cell migration, proliferation, matrix deposition and remodeling, as well as inflammation and angiogenesis. Small skin wounds heal within days, while larger injuries, such as those caused by trauma, acute illness, or major surgery, may take weeks to heal. Interference in the wound healing process can lead to delayed healing, resulting in chronic wounds that can persist for three months and may never fully heal. The wound repair process is divided into the coagulation phase, inflammation phase, repair phase, and maturation phase. Traditional wound care methods typically involve using gauze or pressure dressings, simply isolating the wound from the outside world to prevent infection. These methods do not effectively promote wound healing and rely solely on the body's natural healing processes. Patients with diabetes, vascular disease, aging, and hemoglobinopathies are particularly prone to abnormal wound healing. Improper care can lead to wound recurrence, and in severe cases, even amputation or death. This not only seriously affects patients' quality of life and mental and physical health but also increases treatment costs. Therefore, there is an urgent need for alternative, effective, and safe methods to promote wound healing.

[0003] In recent years, cell therapy has entered the therapeutic field as a potential treatment. Mesenchymal stem cells (MSCs) are pluripotent mesenchymal cells that can differentiate into mesenchymal tissue lineages such as osteoblasts, chondrocytes, myocytes, and adipocytes, but cannot differentiate into hematopoietic stem cells. MSCs are present in most tissues, primarily in bone marrow and adipose tissue; however, their diversity and lineage are not fully understood. MSCs have been used in disease models to control several steps in wound healing and regeneration processes. Overall, the active role of MSCs in this process appears to be paracrine function.

[0004] Exosomes are a component of paracrine signaling and a major contributor to stem cell efficacy. As nanoscale membrane-bound vesicles, ranging in size from 30-150 nm, exosomes possess advantages such as high stability, low immunogenicity, good biocompatibility, and long circulation time, making them natural drug carriers. Exosomes are also believed to have various activities, such as remodeling the extracellular matrix and transmitting signals and molecules to other cells. Their use avoids many of the drawbacks of stem cells. Mesenchymal stem cells can produce / secrete various therapeutic cytokines / growth factors, miRNAs, and mRNAs, and exosomes also carry certain therapeutic molecules. This means that stem cell exosomes can not only serve as drug carriers but also exert therapeutic effects. With the continuous development of exosome research, the unique biological activities and medicinal value of exosomes are constantly being explored. Combined with small molecule drugs, the development potential of drug-loaded exosomes in the medical field is increasingly evident, and their application prospects are very broad.

[0005] Current wound care methods primarily focus on infection prevention and control. This is achieved through the use of materials like silver ion antibacterial agents to reduce the risk of infection, and by using pressure-reducing pads to alleviate pressure on the wound and surrounding tissues. Physical isolation methods maintain the wound's local closure, preventing contamination from secretions and excretions. Depending on the wound's size, depth, color, and amount of exudate, occlusive dressings are selected to provide a low-oxygen, moist healing environment, such as foam dressings, hydrocolloid dressings, and hydrogel dressings. These methods can treat skin wounds but do not accelerate wound closure. Human epidermal growth factor (HGF) can directly act on the skin and mucous membranes, promoting cell growth and accelerating epidermal damage repair. It can effectively treat burns, scalds, and other skin wounds or mucous membrane ulcers caused by various reasons. However, its use and dosage must be strictly controlled; otherwise, it can easily trigger allergic reactions, causing local redness, swelling, pain, itching, and in severe cases, tissue hyperplasia. Some drugs have limitations in application. Large molecule drugs are poorly absorbed in the human body, easily causing cytotoxicity and failing to promote wound healing, thus hindering their clinical trials. Mesenchymal stem cell-derived exosomes, however, contain abundant bioactive substances and offer several advantages. They possess natural targeting capabilities, offer various drug delivery methods, and are easy to cryopreserve, transport, quantify, and transplant. Mesenchymal stem cell exosomes can act as drug carriers while also exerting therapeutic effects. Exosome-based drug delivery systems can overcome biological barriers, resulting in excellent therapeutic outcomes. As drug carriers, exosomes can increase drug solubility, stability, and bioavailability, improve drug delivery efficiency, and promote wound healing. However, exosome drug delivery methods still need optimization; current methods have low drug loading capacities and cannot achieve large-scale production. Summary of the Invention

[0006] To address the needs of existing technologies, this invention encapsulates VPA into exosomes to form drug-loaded exosomes VPA-EXO, thereby solving the problem of poor wound healing effects when using VPA alone. It also addresses the issues of slow onset of action and difficulty in controlling drug dosage associated with other wound care methods.

[0007] The inventive point of this invention is as follows: After passage of resuscitated HSF cells and HUVEC cells, HSF cells and HUVEC cells in the logarithmic growth phase are seeded into E-plate16 culture plates and cultured for 20-28 hours on a real-time label-free cell analyzer. Then, different concentrations of VPA-EXO are added to the HSF cells and HUVEC cells. The evaluation model is constructed using drug-loaded exosomes with a concentration of 25-200 μg / mL, preferably 100 μg / mL protein, that affects the cell proliferation of HSF cells and HUVEC cells and whose overall survival rate changes relatively stably. The maximum applicable range is between 12.5 μg / mL and 400 μg / mL; the preferred range is 25-200 μg / mL; and the optimal concentration is 100 μg / mL.

[0008] Therefore, the present invention provides a method for constructing an in vitro evaluation model to promote skin wound healing, which includes the following steps:

[0009] S1 After passage of resuscitated HSF cells and HUVEC cells, HSF cells or HUVEC cell suspensions in the logarithmic growth phase were seeded into culture plates and cultured for 20-28 hours on a real-time label-free cell analyzer.

[0010] S2 is obtained by adding VPA-EXO at a concentration of 12.5 μg / mL to 400 μg / mL to HSF cells or HUVEC cells.

[0011] Preferably, the mass concentration of the added VPA-EXO is 25-200 μg / mL, and more preferably 100 μg / mL.

[0012] Specifically, HSF cells or HUVEC cells are cultured to a cell mass of 85%-90%.

[0013] In a specific implementation, the resuscitation method for HSF cells and HUVECs in S1 is as follows: when the cell mass is 85%-90% in DMEM high glucose medium containing a mixture of 8-12 vol.% fetal bovine serum and 0.8-1.3 vol.% penicillin and streptomycin, they can be passaged and subjected to subsequent experiments.

[0014] More preferably, during inoculation in S1, the cell suspension is diluted to 2-15 × 10⁻⁶. 4 Cells / mL, preferably 2-15×10⁻⁶ 4 per mL.

[0015] In a specific embodiment, after adding VPA-EXO to S2, the mixture is placed in an incubator and cultured for 12-48 hours, preferably 24 hours.

[0016] This invention provides an in vitro evaluation model for promoting skin wound healing obtained by the method described.

[0017] The present invention further provides a kit for detecting whether a sample to be tested has the ability to promote wound healing, comprising the in vitro evaluation model for promoting skin wound healing as described in claim 7, and optionally further comprising other reagents, such as probiotics, traditional Chinese medicine and active ingredients.

[0018] This invention also provides the application of the aforementioned in vitro evaluation model for promoting skin wound healing in detecting whether a test sample has the ability to promote wound healing;

[0019] The sample to be tested is a compound, a pharmaceutical composition, or a mixture.

[0020] The specific application method is as follows: the sample to be tested is applied to the in vitro evaluation model for promoting skin wound healing as described in claim 7, and the effect of promoting wound healing is determined by detecting cell proliferation and / or migration.

[0021] More specifically, the aforementioned effect refers to the following steps: after the cells of the in vitro evaluation model for promoting skin wound healing adhere and grow, the sample to be tested is diluted to a safe concentration and added to the cell culture medium. More specifically, the cells of the in vitro evaluation model for promoting skin wound healing are cultured for 24 hours, the sample solution is diluted to a safe concentration and added to the cell culture medium, and the cell growth trend is observed within 48 hours using the in vitro evaluation model for promoting skin wound healing. The positive and progressive effects of this invention are: it can regulate cell proliferation and migration ability, promoting wound healing. When VPA-EXO at a concentration of 100 μg / mL was applied to HSF cells and HUVEC cells, it was observed that 100 μg / mL VPA-EXO promoted the proliferation and migration of both HSF cells and HUVEC cells, thus promoting wound healing. Drug-loaded exosomes VPA-EXO can be taken up by target cells. The factors carried by the exosomes themselves and VPA can exert therapeutic effects simultaneously, promoting the proliferation and migration of HSF cells and HUVEC cells. HSF cells and HUVEC cells play a crucial role in the four stages of wound healing. Promoting the proliferation and migration of HSF cells and HUVEC cells indicates that VPA-EXO can promote wound contraction, promote the formation of granulation tissue, accelerate tissue reconstruction, and promote angiogenesis, thereby achieving the effect of promoting wound healing. Attached Figure Description

[0022] Figure 1 To investigate the cytotoxic effects of different concentrations of VPA-EXO on human skin fibroblasts and human vascular endothelial cells.

[0023] Figure 2 Optical images and quantitative results of cell scratch assay (A) HSF cell scratch assay (B) Quantitative results of HSF cell scratch assay (C) HUVEC cell scratch assay (D) Quantitative results of HUVEC cell scratch assay

[0024] Figure 3 Optical images and quantitative results of Transwell assay for cell migration (A) Transwell assay of HSF cells (B) Transwell assay of HUVEC cells (C) Quantitative results of Transwell assay of HSF cells (D) Quantitative results of Transwell assay of HUVEC cells. Detailed Implementation

[0025] The present invention will be further described below through specific embodiments in order to better understand the present invention, but this does not constitute a limitation on the present invention.

[0026] Example 1:

[0027] Materials preparation:

[0028] Human skin fibroblasts (HSF cells)

[0029] Human umbilical vein endothelial cells (HUVECs)

[0030] The preparation method of VPA-EXO is described in Chinese patent application No. 202411253654.5, entitled "Preparation Method and Application of Valproic Acid Complex Loaded on Exosomes Derived from Mesenchymal Stem Cells." The specific steps are as follows: Exosomes MSC-EXO and VPA are mixed at a mass ratio of 1:1. The mixture of exosomes and VPA is then treated with an ultrasonic cell disruptor for drug loading. The ultrasonic parameters are set as follows: 20% amplitude, 30s on / off, for a total of 6 cycles, with a 2-minute cooling time between each cycle. After ultrasonic treatment, the mixture is incubated at 37°C for 60 minutes to restore the exosome membrane. The free drug is separated by centrifugation at 5000×g for 10 minutes using an ultrafiltration tube to obtain the drug-loaded exosomes.

[0031] The steps for VPA-EXO to act on HSF cells and HUVEC cells are as follows:

[0032] 1. Resuscitate HSF and HUVEC cells in DMEM high-glucose medium containing 8–12 vol.% fetal bovine serum and 0.8–1.3 vol.% penicillin-streptomycin mixture. Culture until the cell mass reaches 85%–90% before passage and subsequent experiments.

[0033] 2. Remove cells with a fusion rate of 85%-90% from the incubator and conduct the experiment in a clean bench. Remove the liquid in the culture flask, wash with PBS 2-3 times, discard the liquid in the flask, and add 2mL of trypsin for digestion.

[0034] 3. Observe the cell morphology under a microscope. When the cells are spherical and in a suspended and mobile state, add an equal amount of complete culture medium to stop the digestion.

[0035] 4. Transfer the cell suspension to a centrifuge tube and centrifuge at 1000 rpm for 4 minutes. Aspirate the supernatant, add an appropriate amount of complete culture medium and mix well. Add an appropriate amount of complete culture medium / endothelial cell culture medium and cell suspension to the cell culture flask (HSF cells are cultured in complete culture medium, while HUVEC cells are cultured in endothelial cell culture medium. The two cell culture media are different, but both involve adding their respective culture medium and cell suspension to the culture flask), and place it in an incubator (37℃, 5% CO2) for incubation.

[0036] 5. Add 50 μL of complete culture medium to each well of the E-Plate 16 plate, place the E-Plate 16 plate in the RTCA DP system, set the relevant experimental information and check the baseline. Wells with a baseline value less than 0.01 can be used for subsequent experiments.

[0037] 6. Count the HSF and HUVEC cells obtained in step 4, and dilute the cell suspension to 8 × 10⁻⁶. 4 Cells / mL: Add 100 μL of cell suspension to each well of an E-Plate 16 plate to achieve a cell count of 8 × 10⁶ cells per well. 3 indivual.

[0038] 7. Place the E-Plate 16 plate back into the cell culture incubator and let it stand for 30 minutes. Once the cells have stably contacted the bottom electrode of the well, start the RTCA DP system and culture for 20–28 hours. Add different concentrations of VPA-EXO, and set up a negative control group. Each group has 3 replicates. Continue culturing in an incubator (37℃, 5% CO2) for 24 hours, and observe the cell growth trend within 48 hours. The concentration of added VPA-EXO ranges from 12.5 μg / mL to 400 μg / mL.

[0039] Cell viability and VPA-EXO concentration showed a dose-dependent relationship. As the VPA-EXO protein concentration increased, the VPA content in the drug-loaded exosomes also increased, affecting cell viability. A VPA-EXO protein concentration of 12.5 μg / mL had no significant effect on cell growth. Within the concentration range of 25-200 μg / mL, the drug promoted cell proliferation, with the highest cell viability observed at a concentration of 100 μg / mL. At a concentration of 200 μg / mL, cell viability was lower than at 100 μg / mL, indicating that the increased VPA concentration at this level affected cell status and inhibited cell viability. At a concentration of 400 μg / mL, the cell growth curve was lower than the negative control, indicating that high VPA concentrations affected cell viability, leading to a certain number of cell deaths. Within the ideal effect range, a drug-loaded exosome protein concentration of 100 μg / mL was selected for subsequent experiments.

[0040] Example 2

[0041] Cell scratch assay: Place the 2-well scratch assay insert into a 6-well plate, one insert per well. After digesting and centrifuging HSF and HUVEC cells, add complete culture medium to prepare a cell suspension. Dilute the suspension to 5 × 10⁻⁶. 5Cells / mL: Add 70 μL of cell suspension to each well of the wound healing insert and incubate at 37°C, 5% CO2 for 24 h. Remove the wound healing insert using sterile forceps. Wash cells three times with PBS to remove free cells, add 1 mL of complete culture medium, then add 1 mL of different drugs (exosome EXO, VPA, drug-loaded exosome VPA-EXO) for treatment, and a blank control. Incubate in an incubator, and take pictures at the same location at 0, 4, 8, 12, and 24 hours. Figure 2 The results shown indicate that, as confirmed by optical microscopy, VPA-EXO promoted the proliferation of vascular endothelial cells and fibroblasts at 12 and 24 hours in the scratch assay.

[0042] Example 3

[0043] Transwell migration: HSF cells and HUVEC cells in logarithmic growth phase were harvested, digested, centrifuged, and then added to serum-free culture medium to prepare a cell suspension. The cell density was adjusted to 5 × 10⁶ cells / year. 5 Cells / mL were added to the lower chamber of a 24-well plate with different drug solutions (exosome EXO, VPA, drug-loaded exosome VPA-EXO, and a blank control). 200 μL of cell suspension was added to the upper chamber, and the total volume of liquid in the lower chamber was 600 μL. The plates were then incubated for 20-24 hours. The chambers were removed, the culture medium in the upper chamber was aspirated, and the cells in the upper chamber were gently wiped with a cotton swab. A new 24-well plate was prepared and 600 μL of 4% tissue cell fixative was added for fixation for 20-30 min. The fixative was discarded, and the plates were stained with 0.1% crystal violet for 5-10 min. The plates were washed three times with PBS to remove unbound crystal violet. After appropriate air drying, five fields of view were selected under a high-power microscope to observe and count the cells. Results are as follows: Figure 3 As shown, optical microscopy confirmed that VPA-EXO promoted cell migration in the Transwell experiment, further demonstrating that VPA-EXO enhances cell proliferation and migration capabilities.

[0044] In summary, Examples 1, 2, and 3 all demonstrate that VPA-EXO can promote the proliferation of HSF cells and HUVEC cells, thereby promoting wound healing. First, cell growth curves reflect the increased cell activity and number of both cell types after VPA-EXO treatment, indicating that VPA-EXO can promote the proliferation of both cell types. Second, scratch assays demonstrate that VPA-EXO can promote the proliferation and migration (lateral migration) of both cell types. Third, Transwell assays demonstrate that VPA-EXO can promote the proliferation and migration (vertical migration) of both cell types. These three examples all demonstrate that VPA-EXO can promote cell proliferation and migration, thereby accelerating wound healing.

Claims

1. A method for constructing an in vitro evaluation model for promoting skin wound healing, characterized by, It comprises the following steps: S1 After the HSF cells and HUVEC cells are recovered, the HSF cells or HUVEC cells in the logarithmic growth phase are inoculated in a culture plate and cultured for 20-28h in a real-time label-free cell analyzer; S2 Then, VPA-EXO is added to the HSF cells or HUVEC cells at a concentration of 12.5 μg / mL-400 μg / mL.

2. The construction method of claim 1, wherein, The mass concentration of VPA-EXO added is 25-200 μg / mL, preferably 100 μg / mL.

3. The method of claim 1, wherein, The HSF cells or HUVEC cells are cultured to a cell amount of 85%-90%.

4. The method of claim 1, wherein, The recovery method of HSF cells and HUVEC in S1 is as follows: when the cells are cultured in DMEM high-sugar medium containing 8-12 Vol.% fetal bovine serum and 0.8-1.3 Vol.% penicillin-streptomycin mixture to a cell amount of 85%-90%, the cells can be passaged and used for subsequent experiments.

5. The method of claim 1, wherein, In S1 the inoculation is performed by diluting the cell suspension to 2-15 x 10 4 cells / mL, preferably 2-15 x 10 4 cells / mL.

6. The method of claim 1, wherein, After the VPA-EXO is added in S2, the cells are further cultured in an incubator for 12-48h, preferably 24h.

7. The skin wound healing promotion in vitro evaluation model obtained by the method of any one of claims 1-6.

8. A kit for detecting whether a sample to be tested has a wound healing promoting ability, characterized by, The skin wound healing promotion in vitro evaluation model of claim 7, optionally further comprising other agents, such as probiotics, traditional Chinese medicines or active ingredients thereof.

9. Use of the skin wound healing promotion in vitro evaluation model of claim 7 in detecting whether a test sample has the ability to promote wound healing. The test sample is a compound, a pharmaceutical composition or a mixture.

10. Use according to claim 9, wherein The specific application method is as follows: the test sample is added to the skin wound healing promotion in vitro evaluation model of claim 7, and the ability of the test sample to promote wound healing is determined by detecting cell proliferation and / or migration. More specifically, after the skin wound healing promotion in vitro evaluation model cells are adherent, the test sample is diluted to a safe concentration and added to the cell culture medium, and more specifically, the skin wound healing promotion in vitro evaluation model cells are cultured for 24h, the test sample solution is diluted to a safe concentration and added to the cell culture medium, and the skin wound healing promotion in vitro evaluation model is used to observe the cell growth trend within 48h.

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