A composition containing mesenchymal stem cell-derived extracellular vesicles, and methods of making and uses thereof

Abstract

The present application relates to a kind of extracellular vesicle compositions and its preparation method and use, specifically relates to a kind of extracellular vesicle compositions in the use of preparation for preventing and / or treating hyperalgesia and / or promoting wound healing, the active ingredients of the extracellular vesicle composition include mesenchymal stem cell-derived extracellular vesicle and lidocaine.The present application can achieve the double goal of "promoting wound healing" and "preventing hyperalgesia caused by scald, hyperalgesia", fundamentally improve the long-term prognosis of infant scald patient.

Description

Technical Field

[0001] This invention relates to the field of extracellular vesicle technology, and in particular to a composition containing extracellular vesicles derived from mesenchymal stem cells, its preparation method, and its uses. Background Technology

[0002] Infants and young children have delicate skin, and their nervous systems are in a critical stage of rapid development and plasticity. Severe burns not only cause local tissue damage, but the intense noxious stimulation they produce can also easily lead to pathological remodeling of the spinal cord and higher central nervous systems, known as "central sensitization." This can result in behavioral disorders such as long-term hyperalgesia, chronic pain hypersensitivity, and abnormal pain in children. This long-term neurological dysfunction caused by early injury has become a prominent challenge in the field of pediatric pain management.

[0003] Currently, the core of burn treatment focuses on wound debridement, infection control, and promoting healing, with pain management largely limited to acute analgesia. However, simple acute analgesia (such as systemic use of opioids or local anesthetics) is often insufficient to effectively block the formation of central sensitization, and long-term use can easily lead to numerous side effects. Mesenchymal stem cell-derived extracellular vesicles (MSC-Exo) have excellent anti-inflammatory effects, but research on their intervention in post-burn neuropathic pain, particularly regarding their long-term effects on the developing nervous system, remains lacking.

[0004] Therefore, there is an urgent need in this field for a new strategy that can simultaneously intervene in peripheral tissue repair and central nervous system sensitization in the early stages of burns, thereby fundamentally improving the long-term prognosis of infant patients. Summary of the Invention

[0005] In view of the above-mentioned technical problems in the prior art, the purpose of this invention is to provide a new scheme for the combined application of extracellular vesicles derived from mesenchymal stem cells and lidocaine, so as to achieve the dual goals of "promoting wound healing" and "preventing hyperalgesia and hyperalgesia caused by burns", thereby fundamentally improving the long-term prognosis of infants and young children with burns.

[0006] The present invention provides the use of an extracellular vesicle composition in the preparation of a medicament for the prevention and / or treatment of hyperalgesia and / or wound healing, wherein the active ingredients of the extracellular vesicle composition include extracellular vesicles derived from mesenchymal stem cells and lidocaine.

[0007] Preferably, the active ingredient of the extracellular vesicle composition consists of extracellular vesicles derived from mesenchymal stem cells and lidocaine.

[0008] Preferably, the mesenchymal stem cells are selected from one or more of human bone marrow mesenchymal stem cells, human adipose mesenchymal stem cells, and human umbilical cord blood mesenchymal stem cells.

[0009] Preferably, in the extracellular vesicle composition, the concentration of the extracellular vesicles derived from mesenchymal stem cells is 1 × 10⁻⁶. 8 ~1×10 12 Particles / mL; And / or, in the extracellular vesicle composition, the lidocaine content is 10wt% to 40wt%.

[0010] Preferably, in the extracellular vesicle composition, the concentration of the extracellular vesicles derived from mesenchymal stem cells is 1 × 10⁻⁶. 9 ~1×10 11 Particles / mL; And / or, in the extracellular vesicle composition, the lidocaine content is 20wt% to 30wt%.

[0011] Preferably, the method for preparing the extracellular vesicles derived from mesenchymal stem cells includes: culturing mesenchymal stem cells, collecting the culture supernatant, and further extracting extracellular vesicles to obtain the extracellular vesicles derived from mesenchymal stem cells.

[0012] Preferably, the extraction method is selected from one or more of density gradient centrifugation, ultracentrifugation, tangential flow filtration, PEG precipitation, and fractional filtration.

[0013] Preferably, the specific steps of the tangential flow filtration include: (a) clarifying the culture supernatant using a tangential flow filtration system and collecting the permeate; (b) concentrating and purifying the filtrate obtained in step (a) using a hollow fiber column to obtain the extracellular vesicles derived from the mesenchymal stem cells. And / or, the specific steps of the ultracentrifugation method include: centrifuging the culture supernatant at 200~400×g for 10~20 minutes and collecting the supernatant; centrifuging at 2000~3000×g for 10~20 minutes and collecting the supernatant; centrifuging at 10000~15000×g for 30~40 minutes and collecting the supernatant; centrifuging at 100000~150000×g for 70~120 minutes and collecting the precipitate to obtain the extracellular vesicles derived from the mesenchymal stem cells.

[0014] Preferably, the subject of the drug is an infant, a young child, or an adult; And / or, the pain sensation includes one or more of the following: pain following nerve injury, pain following tissue injury, or neuropathic pain; wherein, the pain following tissue injury includes: pain following burns or scalds; And / or, the hyperalgesia includes one or more of the following: mechanosensitive hyperalgesia, thermal hyperalgesia, and cold hyperalgesia.

[0015] Preferably, the drug further includes a pharmaceutically acceptable carrier or excipient; And / or, the extracellular vesicle composition in the drug comprises 1 wt% to 50 wt% by mass. And / or, the drug is formulated into a pharmaceutically permissible dosage form, wherein the dosage form includes injections, creams, ointments, patches, sprays, or gels.

[0016] The effects of the invention: This invention targets specific pathological processes and applicable populations, and for the first time focuses a combined intervention on the key scientific issue of early blocking of central sensitization after burns in infants and young children. By using a burn model in P7-14 day old juvenile mice to simulate the pathological state of infants and young children, the research conclusions have greater guiding value in pediatric clinical practice. Based on previous studies, extracellular vesicles derived from mesenchymal stem cells have shown poor efficacy in inhibiting excessive activation of spinal cord microglia and neurons. This invention presents a novel synergistic mechanism: at the peripheral level, MSC-Exo can regulate inflammatory factors to inhibit excessive activation of the inflammatory response, creating a favorable microenvironment for wound repair and the effectiveness of lidocaine. Simultaneously, its active ingredients can promote benign nerve fiber repair. At the central level, lidocaine can directly block the transmission of noxious signals to the spinal cord in the early stages, reducing the intensity of stimuli that induce central sensitization from the source. Meanwhile, MSC-Exo can transmit anti-inflammatory and neurotrophic signals to the dorsal horn of the spinal cord through humoral circulation or neuro-immune regulatory pathways. It can downregulate the abnormally high expression of pain-related molecules in dorsal horn neurons and inhibit the expression of neuroactive transcription factors such as c-Fos in immune-related microglia. Furthermore, this invention inhibits the formation of central sensitization through a dual-pathway, multi-target approach. It clarifies the critical early intervention time window, emphasizing the initiation of combined intervention within 24-72 hours of the acute phase after burns. This stage is a critical window for blocking the formation of nociceptive memories and preventing irreversible neural remodeling. Validated in a juvenile mouse burn model, compared to the model group and the lidocaine hydrogel group, the experimental dressing containing a combination of extracellular vesicles derived from mesenchymal stem cells and lidocaine significantly promoted wound healing, improved skin lesion severity, and reduced inflammatory factor levels. Simultaneously, it effectively prevented the occurrence of pain hypersensitivity in the long term after injury, significantly increased the mechanical pain threshold, reduced the body's abnormal sensitivity to cold stimuli, and decreased spontaneous pain behavior. In addition, this invention is adaptable to various dosage forms such as hydrogels, creams, and sprays, possessing broad application prospects. Attached Figure Description

[0017] Figure 1 This is an electron micrograph (TEM) of extracellular vesicles derived from mesenchymal stem cells prepared in Example 1. Figure 2 The particle size distribution of extracellular vesicles derived from mesenchymal stem cells prepared in Example 1 is shown. Figure 3 Electrophoresis diagrams showing the expression analysis of CD9, CD81, TSG101 and Calnexin in extracellular vesicles (MSC-Exo) and MSC cell lysate (MSC-Cel) obtained in Example 1. Figure 4 The effect of extracellular vesicles derived from mesenchymal stem cells prepared in Example 1 on the level of the inflammatory factor IL-6; where ****P<0.0001 indicates that the difference is statistically significant and extremely statistically significant compared with the negative control group; Figure 5 To detect the mechanical pain behavior in Example 3, specifically the mechanical pain threshold test in mice after drug administration; *P<0.05 and **P<0.01 indicate significant or highly significant differences compared with the normal group; #P<0.05 and ##P<0.01 indicate significant and highly significant differences compared with the model group. Figure 6 To detect the thermal pain behavior in Example 4, specifically the thermal pain threshold of mice after drug administration; *P<0.05 and **P<0.01 indicate significant or highly significant differences compared with the normal group; #P<0.05 and ##P<0.01 indicate significant and highly significant differences compared with the model group. Figure 7 To detect spontaneous pain behavior in Example 5, the spontaneous pain threshold of mice after drug administration was measured; *P<0.05 and **P<0.01 indicate significant or highly significant differences compared with the normal group; #P<0.05 and ##P<0.01 indicate significant and highly significant differences compared with the model group. Figure 8 To detect wound healing in the skin burn model mice in Case 6 (day 7 after intervention); Figure 9 To verify the mechanism of prevention and / or treatment of pain hypersensitivity in Case 7, the expression of ion-calcium binding adaptor molecule 1 (IBA-1) in mouse spinal cord and the number of positive cells for neuronal activation marker (c-Fos) were specifically measured; *P<0.05 and **P<0.01 indicate statistically significant and highly statistically significant differences compared with the normal group, respectively. # P<0.05 and ## P<0.01 indicates that the difference compared with the model group is statistically significant and extremely statistically significant, respectively. Detailed Implementation

[0018] To make the technical solution and beneficial effects of the present invention more apparent and understandable, a detailed description is provided below by listing specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application pertains.

[0019] The present invention provides the use of an extracellular vesicle composition in the preparation of a medicament for the prevention and / or treatment of hyperalgesia and / or wound healing, wherein the active ingredients of the extracellular vesicle composition include extracellular vesicles derived from mesenchymal stem cells and lidocaine.

[0020] In some embodiments, the active ingredient of the extracellular vesicle composition consists of extracellular vesicles derived from mesenchymal stem cells and lidocaine.

[0021] In some embodiments, the mesenchymal stem cells are selected from one or more of human bone marrow mesenchymal stem cells, human adipose mesenchymal stem cells, and human umbilical cord blood mesenchymal stem cells.

[0022] In some embodiments, the concentration of the mesenchymal stem cell-derived extracellular vesicles in the extracellular vesicle composition is 1 × 10⁻⁶. 8 ~1×10 12 Particles / mL, for example, 1×10 8 Particles / mL, 1×10 9 Particles / mL, 5×10 9 Particles / mL, 1×10 10 Particles / mL, 1×10 11 Particles / mL, 1×10 12 Particles / mL, etc.

[0023] In some embodiments, the lidocaine content in the extracellular vesicle composition is 10wt% to 40wt%, for example, 10wt%, 15wt%, 20wt%, 25wt%, 30wt%, 35wt%, 40wt%, etc.

[0024] In some embodiments, the concentration of the mesenchymal stem cell-derived extracellular vesicles in the extracellular vesicle composition is 1 × 10⁻⁶. 9 ~1×10 11 Particles / mL.

[0025] In some embodiments, the lidocaine content in the extracellular vesicle composition is 20 wt% to 30 wt%.

[0026] In some embodiments, the lidocaine content in the extracellular vesicle composition is 25 wt%.

[0027] In some embodiments, the method for preparing the extracellular vesicles derived from mesenchymal stem cells includes: culturing mesenchymal stem cells, collecting the culture supernatant, and further extracting extracellular vesicles to obtain the extracellular vesicles derived from mesenchymal stem cells.

[0028] In some embodiments, the extraction method is selected from one or more of density gradient centrifugation, ultracentrifugation, tangential flow filtration, PEG precipitation, and fractional filtration.

[0029] In some embodiments, the specific steps of the tangential flow filtration include: (a) clarifying the culture supernatant using a tangential flow filtration system and collecting the permeate; and (b) concentrating and purifying the filtrate obtained in step (a) using a hollow fiber column to obtain the extracellular vesicles derived from the mesenchymal stem cells.

[0030] In some embodiments, the ultracentrifugation method specifically includes the following steps: centrifuging the culture supernatant at 200-400×g for 10-20 minutes and collecting the supernatant; centrifuging again at 2000-3000×g for 10-20 minutes and collecting the supernatant; centrifuging again at 10000-15000×g for 30-40 minutes and collecting the supernatant; and centrifuging again at 100000-150000×g for 70-120 minutes and collecting the precipitate to obtain the extracellular vesicles derived from mesenchymal stem cells.

[0031] In some embodiments, the subject of the drug is an infant, a young child, or an adult.

[0032] In some embodiments, the subject of the drug is an infant or young child.

[0033] In some embodiments, the pain sensation includes one or more of the following: pain following nerve injury, pain following tissue injury, or neuropathic pain; wherein, pain following tissue injury includes: pain following burns or scalds.

[0034] In some embodiments, the hyperalgesia includes one or more of mechanical hyperalgesia, thermal hyperalgesia, and cold hyperalgesia.

[0035] In some embodiments, the wound includes one or more of the following: lacerations, abrasions, tears, crush injuries, puncture wounds, burns, scalds, frostbite, electric shock injuries, radiation injuries, skin and mucous membrane erosions, infected wounds, lower extremity venous ulcers, arterial ulcers, diabetic foot ulcers, pressure ulcers, tumorous wounds, bedsores, and surgical wounds.

[0036] In some embodiments, the drug may also include a pharmaceutically acceptable carrier or excipient.

[0037] In some embodiments, the pharmaceutically acceptable carrier or excipient is selected from one or more of thickeners, emulsifiers, complexing agents, pH adjusters, humectants, plasticizers, and neutralizers.

[0038] In some embodiments, the thickener is selected from one or more of carbomer, xanthan gum, guar gum, locust bean gum, hydroxyethyl cellulose, sodium carboxymethyl cellulose, acrylate cross-linked polymer-2 sodium, acrylate / C10-30 alkanol acrylate cross-linked polymer, and polyacrylate cross-linked polymer-6.

[0039] In some embodiments, the thickener is selected from carbomer.

[0040] In some embodiments, the neutralizing agent is selected from one or more of triethanolamine, sodium hydroxide, and potassium hydroxide.

[0041] In some embodiments, the neutralizing agent is selected from triethanolamine.

[0042] In some embodiments, the humectant is selected from one or more of the following: glycerin, 1,3-propanediol, butylene glycol, 1,2-pentanediol, 1,2-hexanediol, dipropylene glycol, panthenol, sorbitol, erythritol, sodium hyaluronate, trehalose, allantoin, betaine, β-glucan, ethylhexylglycerin, glyceryl polyether-26, glyceryl glucoside, saccharide isomers, panthioethylamine, aloe polysaccharide, tremella polysaccharide, and dendrobium polysaccharide.

[0043] In some embodiments, the humectant is selected from glycerin.

[0044] In some embodiments, the extracellular vesicle composition in the drug comprises 1 wt% to 50 wt% by mass.

[0045] In some embodiments, the volume percentage of the extracellular vesicle composition in the drug is 1% (v / v) to 50% (v / v).

[0046] In some embodiments, the drug is formulated into a pharmaceutically permissible dosage form, wherein the dosage form includes an injection, cream, ointment, patch, spray, or gel.

[0047] In some embodiments, the extracellular vesicles derived from mesenchymal stem cells have a particle size of 30-150 nm.

[0048] In some implementations, the hyperalgesia includes short-term hyperalgesia and / or long-term hyperalgesia.

[0049] In some implementations, the hyperalgesia is short-term hyperalgesia.

[0050] In some implementations, the hyperalgesia is long-term hyperalgesia.

[0051] In some implementations, the short-term hyperalgesia includes hyperalgesia within 48 hours of injury.

[0052] In some implementations, the short-term hyperalgesia includes hyperalgesia within 24 hours of injury.

[0053] In some implementations, the long-term hyperalgesia includes hyperalgesia that occurs more than 30 days after the injury.

[0054] In some embodiments, the long-term hyperalgesia includes hyperalgesia that occurs 30 to 90 days after injury.

[0055] In some implementations, the pain hypersensitivity includes short-term pain hypersensitivity and / or long-term pain hypersensitivity.

[0056] In some implementations, the pain hypersensitivity is short-term pain hypersensitivity.

[0057] In some implementations, the pain hypersensitivity is long-term pain hypersensitivity.

[0058] In some implementations, the short-term hyperalgesia includes hyperalgesia that occurs 7 to 30 days after injury.

[0059] In some implementations, the long-term hyperalgesia includes hyperalgesia that occurs more than 90 days after the injury.

[0060] In some embodiments, the long-term hyperalgesia includes hyperalgesia that occurs 90 to 180 days after injury.

[0061] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0062] The present invention will be further described below through specific embodiments. Unless otherwise specified, "%" represents a mass percentage. The materials and reagents used in the following embodiments are all commonly used materials or reagents in the art, and can be obtained commercially or synthesized by known methods. Experimental methods in the following embodiments without specified conditions are generally performed according to conventional experimental conditions or the conditions recommended by the manufacturer of the relevant reagent (kit).

[0063] Example 1. Preparation of extracellular vesicles (MSC-Exo) derived from mesenchymal stem cells 1.1 Cultivation Human umbilical cord mesenchymal stem cells from passages P2 to P5 were cultured using serum-free and animal-free culture media with clearly defined components. When the cell confluence reached 80% to 90%, the cell culture supernatant was collected for later use.

[0064] 1.2 Extraction 1.2.1 Extraction using tangential flow filtration technology: The first step, clarification: The collected supernatant was subjected to tangential flow filtration (TFF) using a membrane with a pore size of 5 μm. Cell debris and impurity particles larger than the membrane pore size were retained in the retentate, while extracellular vesicles (exosomes) were present in the permeate, thus achieving preliminary separation. The second step is concentration and liquid exchange: the permeate is concentrated and purified by tangential flow ultrafiltration (TFF) equipment to retain components with a particle size of less than 200 nm, thereby achieving the initial capture and enrichment of vesicle particles. Finally, the concentrate was filtered through a 0.22 μm filter membrane for sterilization to obtain sterile extracellular vesicle stock solution derived from mesenchymal stem cells. The obtained extracellular vesicle stock solution was stored in a -80°C freezer or freeze-dried with the addition of a freeze-drying protectant to prepare lyophilized powder for long-term preservation.

[0065] 1.2.2 Differential centrifugation extraction: The collected cell culture supernatant was centrifuged at 4°C and 300×g for 10 minutes to remove live cells, dead cells, and large cell debris. The supernatant was carefully aspirated, and the intact cells that had settled at the bottom of the tube were discarded, leaving only the supernatant. The supernatant was then centrifuged again at 4°C and 2000×g for 10 minutes to remove apoptotic bodies and larger vesicles. The supernatant was carefully aspirated, and the intact cells that had settled at the bottom of the tube were discarded, leaving only the supernatant. The supernatant was then centrifuged again at 4°C and 10000×g. Centrifuge for 30 minutes under the specified conditions, carefully aspirate the supernatant, discard the intact cells precipitated at the bottom of the tube, and retain the supernatant; finally, centrifuge for 90 minutes at a temperature of 4℃ and a relative centrifugal force of 100000×g, carefully aspirate the supernatant, retain the precipitate, resuspend the precipitate in sterile PBS solution, and filter it through a 0.22μm pore size filter membrane to obtain sterile extracellular vesicle stock solution derived from mesenchymal stem cells; store the obtained extracellular vesicle stock solution in a -80℃ freezer, or freeze-dry with the addition of a lyophilization protectant to prepare lyophilized powder for long-term preservation.

[0066] Example 1. Identification of extracellular vesicles derived from mesenchymal stem cells. The typical cup-shaped morphology was observed by transmission electron microscopy, the particle size distribution was detected by nanoflow cytometry, and the marker proteins CD81, CD9, and TSG101 were detected by Western blotting to identify the extracted vesicles as extracellular vesicles.

[0067] 1.1 TEM The morphological characteristics of extracellular vesicles derived from mesenchymal stem cells obtained in Example 1 were observed using transmission electron microscopy (TEM). Depending on the sample conditions, the extracellular vesicles derived from mesenchymal stem cells obtained in Example 1 were diluted to an appropriate concentration. Approximately 15 μL of the solution was pipetted onto a copper grid and allowed to stand for 1 min. Excess sample was blotted dry from one side with filter paper, and approximately 15 μL of 2% uranium acetate staining solution was added, followed by staining at room temperature for 1 min. If significant adsorption was visible on the copper grid, pure water was added to the surface and quickly aspirated; this process was repeated several times. The morphology and structure of the extracellular vesicles derived from mesenchymal stem cells were observed and images were acquired using a 120 kV transmission electron microscope, and the data were saved.

[0068] The results are as follows Figure 1 As shown, extracellular vesicles derived from mesenchymal stem cells exhibit a cup-shaped structure under TEM.

[0069] 1.2 Particle size distribution The particle size distribution of extracellular vesicles derived from mesenchymal stem cells was detected using nanoflow cytometry. Depending on the sample conditions, the extracellular vesicles derived from mesenchymal stem cells obtained in Example 1 were diluted to an appropriate concentration, and the sample loading concentration was controlled to be approximately 1 × 10⁻⁶. 8 Particles / mL.

[0070] The results are as follows Figure 2 As shown, the particle size of extracellular vesicles derived from mesenchymal stem cells is mainly distributed in the range of 30~150nm, with an average particle size of 82.7nm.

[0071] 1.3 Biomarker Proteins Human umbilical cord mesenchymal stem cell lysis buffer was used as a control group, referred to as the MSC-Cel group. Extracellular vesicles derived from mesenchymal stem cells prepared in Example 1 were lysed using RIPA and protein quantified using the BCA method. After denaturation by boiling with loading buffer, 20-40 μg of protein and protein standards (protein markers) were loaded onto each well of the precast gel. Electrophoresis was performed at a constant voltage of 120-150 V for approximately 1 hour, until the bromophenol blue indicator was near the bottom of the gel. Using a rapid transfer buffer wet transfer method, the protein was transferred to a methanol-activated PVDF membrane and transferred at a constant current of 300 mA for 25-40 minutes (cooled in an ice bath). The membrane was blocked with 5% BSA at room temperature for 1 hour. Primary antibodies (such as CD9, CD81, TSG101, and Calnexin) were added and incubated overnight at 4°C. After washing with TBST, HRP-labeled secondary antibody was added and incubated at room temperature for 1 hour. Finally, ECL chemiluminescence was used for development.

[0072] The results are as follows Figure 3As shown, no obvious band was detected in the negative control marker (calnexin, ~90kDa), indicating that there was no significant cell or organelle debris contamination in the sample and the extracellular vesicles were of good purity. At the same time, three classic extracellular vesicle markers were successfully detected: transmembrane proteins CD9 (~25kDa) and CD81 (~25kDa) both showed clear single or tight bands, indicating that an extracellular vesicle subpopulation rich in tetraspanmembrane protein family was isolated; cytoplasmic protein TSG101 (~44kDa) also showed a clear positive signal, further confirming that the isolated extracellular vesicles were derived from mesenchymal stem cells.

[0073] Example 2. Effect of extracellular vesicles derived from mesenchymal stem cells on the level of the inflammatory cytokine IL-6. The experimental groups are as follows: Control group: The control group without an established inflammation model used complete culture medium; Negative control group: An inflammation model was established using complete culture medium containing 100 ng / mL LPS; Positive control group: Inflammation was induced by using complete culture medium containing 100 ng / mL LPS and 100 μg / mL dexamethasone was added for anti-inflammatory purposes; Experimental group (MSC-Exo group): using 1×10 9 The culture medium containing extracellular vesicles (MSC-Exo) derived from mesenchymal stem cells at a concentration of granules / mL and 100 ng / mL LPS was designated as the MSC-Exo group. RAW264.7 macrophages in good growth condition were harvested and cultured at a concentration of 1 × 10⁶ cells / well. 6 Cells were seeded at a density of 100 cells per well in 24-well plates and cultured at 37°C and 5% CO2 for 12 hours to allow the cells to adhere fully.

[0074] Subsequent interventions were performed: After discarding the original culture medium, the blank group was replaced with 500 μL of fresh complete culture medium; the negative control group was replaced with 500 μL of complete culture medium containing 100 ng / mL LPS; the positive control group was replaced with 500 μL of complete culture medium containing 100 ng / mL LPS and 100 μg / mL dexamethasone; and the experimental group (MSC-Exo group) was replaced with 500 μL of culture medium containing the same concentration of LPS and 1×10⁻⁶ LPS. 9 Complete medium containing particles / mL of MSC-Exo. Each group was incubated in an incubator for another 24 hours.

[0075] After incubation, the cell supernatant from each well was collected, centrifuged to remove cell debris, and stored at -80℃. The concentration of interleukin-6 (IL-6) in the supernatant was measured using an ELISA kit, strictly following the instructions. The specific IL-6 concentration for each sample was calculated using a standard curve, and the inhibition rate of other groups was calculated using the negative control group as a baseline.

[0076] The results are as follows Figure 4 As shown, compared with the negative control group stimulated only by LPS, the MSC-Exo group, which was co-added with extracellular vesicles MSC-Exo and LPS, significantly inhibited the secretion of macrophage inflammatory factor IL-6, with an inhibition rate of 69.40%.

[0077] Example 2. Preparation of the composition The extracellular vesicles derived from mesenchymal stem cells prepared in Example 1 were compounded with commercially available lidocaine according to different particle numbers and contents to obtain a composition, as follows.

[0078]

[0079] Example 3. Preparation of composite hydrogel Weigh 0.5 g of carbomer and add it to 50 mL of purified water. Stir thoroughly until it swells and dissolves completely. Add 10 mL of glycerol and stir well. Use a pH meter to detect the pH value of the system. While stirring continuously, add 10 mL of triethanolamine (0.68 g) dropwise to adjust the pH to the suitable range for gel formation (usually 5.0–6.0). Then add 10 mL of the mixture of composition (Formula 1), mix gently, and add purified water to a final volume of 100 mL to obtain the initial gel. To remove air bubbles introduced during stirring, centrifuge the gel at 2000 × g for 5 minutes to obtain a homogeneous and transparent composite hydrogel containing extracellular vesicles derived from mesenchymal stem cells and lidocaine, as shown in Table 1.

[0080] Table 1 Composite Hydrogel Formulation

[0081] Comparative Example 1. Preparation of lidocaine hydrogel The only difference between this comparative example and Example 3 is that the composition (Formulation 1) is replaced with a 25wt% lidocaine solution, and 10mL is prepared into a lidocaine hydrogel according to the method of Example 3.

[0082] Example 4. Establishment, treatment, and detection of a scald model in juvenile mice. A scald model was established using juvenile Balb / c mice (P7-P14 days old). Twenty-four hours prior to modeling, hair was removed from the backs of the mice using a depilatory cream. During modeling, the mice were anesthetized, and a 2cm diameter copper rod was placed in boiling water at 100℃ for 30 minutes. After removal and drying, the rod was vertically pressed against the mouse's back for 5 seconds to complete the scald model. The scalded area was left exposed without covering. After modeling, the mice were returned to their cages for routine care.

[0083] Mice after scalding were randomly divided into a model group, a model + lidocaine hydrogel group, and a model + composite hydrogel group. All groups received appropriate treatment immediately after the burn, and the hydrogel was changed every 3 days for a total of 2 changes. Wound healing was observed and recorded regularly. At 28 days post-injury (long-term observation point), behavioral tests (including mechanical pain threshold, thermal pain threshold, and spontaneous pain threshold tests) were performed on mice in each group to observe wound healing and verify the mechanisms of prevention and / or treatment of hyperalgesia in the skin burn model mice.

[0084] Example 3. Behavioral testing for mechanical pain Before testing, prepare a Von Frey fiber stimulation frame and a set of progressively stronger fibers. Place the mouse in a transparent observation box to acclimate to the environment for about 10 minutes. Once the mouse is calm, use the Von Frey fibers to vertically stimulate the sole of the mouse's hind paw starting from the lowest intensity, for about 2-3 seconds. Observe for any pain response such as lifting, retracting, or licking the paw; if so, record the intensity of that fiber. If there is no response, replace with a fiber with a higher intensity and repeat the stimulation. Test each mouse and each paw at least 5 times, with an interval of at least 3 minutes between each test.

[0085] The number of times each group of mice exhibited a positive response to filaments of varying force was recorded. The mechanical pain threshold (expressed as the minimum force required to elicit a positive response, unit: g) was calculated, and the differences in pain thresholds between groups were compared. Experimental data are expressed as mean ± standard error, and one-way ANOVA was used for statistical comparisons between groups. *P<0.05 and **P<0.01 indicate statistically significant and highly significant differences compared to the normal group, respectively; #P<0.05 and ##P<0.01 indicate statistically significant and highly significant differences compared to the model group, respectively.

[0086] The results are as follows Figure 5 As shown, the mechanical withdrawal threshold of the model + composite hydrogel group was significantly higher than that of the model group and the model + lidocaine hydrogel group, indicating that the composition containing extracellular vesicles derived from mesenchymal stem cells and lidocaine can improve mechanical pain sensitivity caused by burns, and the two have a synergistic effect.

[0087] Test Example 4. Behavioral Testing for Thermal Pain Before testing, clean the instrument plate and place a single mouse in the transparent observation cage of the testing instrument to acclimatize to the environment for about 10 minutes. When conducting the hot plate test, set and stabilize the temperature of the metal plate at 55°C, then gently place the mouse on the plate and immediately start timing to record the latency (in seconds) from placement to the first occurrence of a noxious response such as licking the hind paw, rapidly raising the paw, or jumping. After the test, immediately remove the mouse and clean the plate.

[0088] The response latency of mice in each group during the hot plate test was recorded in detail. Experimental data are expressed as mean ± standard error, and one-way ANOVA was used for statistical comparisons between groups. *P<0.05 and **P<0.01 indicate statistically significant and highly statistically significant differences compared to the normal group, respectively. # P<0.05 and ## P<0.01 indicates that the difference compared with the model group is statistically significant and extremely statistically significant, respectively.

[0089] The results are as follows Figure 6 As shown, the thermal pain threshold of mice in the model + composite hydrogel group was significantly higher than that in the model group and the model + lidocaine hydrogel group, indicating that the composition containing extracellular vesicles derived from mesenchymal stem cells and lidocaine can improve thermal pain sensitivity caused by burns, and the two have a synergistic effect.

[0090] Example 5. Spontaneous pain behavioral test During the test, the operator used a long cotton swab tip to gently brush the fur on the back, limbs, and healthy paws of the mouse with even and moderate pressure, taking special care to avoid the cotton swab directly touching the affected paw on the model side.

[0091] Closely observe and record the behavior of mice during and shortly after brushing stimulation, and score them according to the following criteria: 0 points indicates normal behavior, agility and alertness; 1 point indicates avoidance of stimulation or protection of the affected limb; 2 points indicates at least one of the following behaviors: fur standing on end, retraction of the affected paw, or occasional painful vocalizations; 3 points indicates at least two of the following behaviors: fur standing on end, retraction of paws, or occasional painful vocalizations; 4 points indicates at least one strong reaction: attacking the cotton swab, frequent or continuous hissing, circling, or licking and biting the affected limb.

[0092] Each mouse was tested three times, with at least 10 minutes between each test. The average score was taken as the final score for spontaneous pain behavior in that mouse, and the behavioral score data for each group of mice were recorded completely. Experimental data are expressed as mean ± standard error, and one-way ANOVA was used for statistical comparisons between groups. *P<0.05 and **P<0.01 indicate statistically significant and highly statistically significant differences compared to the normal group, respectively. # P<0.05 and ## P<0.01 indicates that the difference compared with the model group is statistically significant and extremely statistically significant, respectively.

[0093] The results are as follows Figure 7 As shown, the spontaneous pain behavior score of mice in the model + composite hydrogel group was significantly lower than that in the model group and the model + lidocaine hydrogel group, indicating that the composition containing extracellular vesicles derived from mesenchymal stem cells and lidocaine can significantly improve spontaneous pain behavior after burns, and the two have a synergistic effect.

[0094] Example 6. Observation of wound healing in a mouse model of skin burns. For mice with scald models, lidocaine hydrogel prepared in Comparative Example 1 and composite hydrogel prepared in Example 3 were applied topically to the mouse wounds. On the 7th day after intervention, the wound healing status of each group of mice was macroscopically observed and evaluated.

[0095] The results are as follows Figure 8 As shown, the skin tissue in the model + composite hydrogel group exhibited a significant regeneration trend, and its healing rate and quality were statistically significantly different from those in the model + lidocaine hydrogel group. This indicates that the early intervention strategy of the combination containing extracellular vesicles derived from mesenchymal stem cells and lidocaine has a significant advantage in promoting wound repair.

[0096] Example 7. Verification of mechanisms for the prevention and / or treatment of hyperalgesia. After perfusion fixation of normal mice and scalded model mice, the lumbar enlargement segment (L4–L6) of the spinal cord was harvested and soaked in JYBL-II decalcification solution for 24 h until the tissue softened. The tissue was then dehydrated using a gradient of ethanol (70%, 80%, 90%, 95%, and twice with 100% ethanol, 40 min each), cleared three times with xylene (1 h each), and impregnated with paraffin three times (1 h each), followed by routine embedding to prepare paraffin blocks. 4 μm continuous coronal sections were cut using a microtome, spread in a 45°C water bath, attached to glass slides, baked at 65°C for 1 h, and then oven-dried for 2 h. The sections were then routinely dewaxed to water using xylene and a gradient of ethanol (100% to 50%). Antigen retrieval was performed using a high-pressure retrieval method: after high-pressure steam injection, the time was 2 min, followed by natural cooling to room temperature. After rinsing with PBS, the sections were incubated with blocking serum in a humidified chamber at 37°C for 60 min. Rabbit anti-Iba-1 (dilution 1:500) and mouse anti-c-Fos (dilution 1:1500) were mixed as primary antibodies and incubated overnight at 4°C in a humidified chamber. After thorough washing with PBS, a mixture of corresponding species-specific fluorescent secondary antibodies was added and incubated at 37°C in the dark for 1 hour. After washing with PBS, nuclei were counterstained with DAPI in the dark for 10 minutes, and the slides were mounted with anti-fluorescence quenching mounting medium. Images of the superficial dorsal horn of the spinal cord were acquired using a fluorescence microscope or confocal microscope under the same parameters. The mean fluorescence intensity of ion-calcium binding adaptor molecule 1 (IBA-1) positive regions was measured using ImageJ software, and the nuclei of cells positive for neuronal activation markers (c-Fos) were counted. Experimental data are expressed as mean ± standard error, and one-way ANOVA combined with Tukey's post-hoc test was used for comparisons among multiple groups. IBA-1 expression in the spinal cord of normal mice remained at a physiologically low level; compared with the normal group, IBA-1 expression in the model group mice was significantly increased, suggesting that the burn model successfully induced a large number of microglia in the spinal cord, and significantly enhanced central nervous system inflammation.

[0097] The results are as follows Figure 9As shown, IBA-1 expression in the model + lidocaine hydrogel group was not significantly different from that in the model group, but was still significantly higher than that in the normal group, indicating that lidocaine dressing alone could not inhibit microglia activation. Compared with the model group, IBA-1 expression in the model + composite hydrogel group was significantly reduced, and the expression level was close to that in the normal group, indicating that extracellular vesicles derived from mesenchymal stem cells can significantly inhibit the excessive activation of spinal cord microglia and alleviate central nervous system inflammation.

[0098] The results are as follows Figure 9 As shown, c-Fos expression in the spinal cord of normal mice remained at a physiologically low level. Compared with the normal group, c-Fos expression in the model group was significantly increased, indicating that pain-transmitting neurons in the dorsal horn of the spinal cord were overactivated after burn modeling, resulting in hyperactive pain signal transmission. The c-Fos expression in the model + lidocaine hydrogel group was significantly higher than that in the normal group, indicating that lidocaine hydrogel alone could not inhibit neuronal overactivation. Compared with the model group, c-Fos expression in the model + composite hydrogel group was significantly decreased, with an expression level close to that of the normal group, indicating that extracellular vesicles derived from mesenchymal stem cells combined with lidocaine can significantly inhibit the overactivation of spinal cord neurons and block hyperactive pain signal transmission.

[0099] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.

Claims

1. The use of an extracellular vesicle composition in the preparation of a medicament for the prevention and / or treatment of hyperalgesia and / or hyperalgesia, characterized in that, The active ingredients of the extracellular vesicle composition include extracellular vesicles derived from mesenchymal stem cells and lidocaine; The mesenchymal stem cells are selected from human umbilical cord blood mesenchymal stem cells; the pain sensation is post-scald pain and / or post-burn pain.

2. The use according to claim 1, characterized in that, The active ingredient of the extracellular vesicle composition consists of extracellular vesicles derived from mesenchymal stem cells and lidocaine.

3. The use according to claim 1, characterized in that, In the extracellular vesicle composition, the concentration of the extracellular vesicles derived from mesenchymal stem cells is 1 × 10⁻⁶. 8 ~1×10 12 Particles / mL; And / or, in the extracellular vesicle composition, the lidocaine content is 10wt% to 40wt%.

4. The use according to claim 3, characterized in that, In the extracellular vesicle composition, the concentration of the extracellular vesicles derived from mesenchymal stem cells is 1 × 10⁻⁶. 9 ~1×10 11 Particles / mL; And / or, in the extracellular vesicle composition, the lidocaine content is 20wt% to 30wt%.

5. The use according to claim 1, characterized in that, The method for preparing extracellular vesicles derived from mesenchymal stem cells includes: culturing mesenchymal stem cells, collecting the culture supernatant, and further extracting extracellular vesicles to obtain the extracellular vesicles derived from mesenchymal stem cells.

6. The use according to claim 5, characterized in that, The extraction method is selected from one or more of density gradient centrifugation, ultracentrifugation, tangential flow filtration, PEG precipitation, and fractional filtration.

7. The use according to claim 6, characterized in that, The specific steps of the tangential flow filtration include: (a) clarifying the culture supernatant using a tangential flow filtration system and collecting the permeate; (b) concentrating and purifying the filtrate obtained in step (a) using a hollow fiber column to obtain the extracellular vesicles derived from the mesenchymal stem cells. And / or, the specific steps of the ultracentrifugation method include: centrifuging the culture supernatant at 200~400×g for 10~20 minutes and collecting the supernatant; centrifuging at 2000~3000×g for 10~20 minutes and collecting the supernatant; centrifuging at 10000~15000×g for 30~40 minutes and collecting the supernatant; centrifuging at 100000~150000×g for 70~120 minutes and collecting the precipitate to obtain the extracellular vesicles derived from the mesenchymal stem cells.

8. The use according to claim 1, characterized in that, The subjects of the drug are infants, young children, or adults; And / or, the hyperalgesia includes one or more of the following: mechanosensitive hyperalgesia, thermal hyperalgesia, and cold hyperalgesia.

9. The use according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable carriers or excipients; And / or, the extracellular vesicle composition in the drug comprises 1 wt% to 50 wt% by mass. And / or, the drug is formulated into a pharmaceutically permissible dosage form, wherein the dosage form includes injections, creams, ointments, patches, sprays, or gels.

Images