Application of blocking exogenous mitochondria into cells in promoting skin wound repair

Abstract

The present application belongs to the field of medicine and health, and relates to the application of blocking exogenous mitochondria into cells in promoting skin wound repair. Specifically, the present application provides a composition comprising mitochondria and the application thereof in repairing skin damage. In addition, the present application also provides a combination comprising mitochondria and a mitochondria entry blocker and the use thereof in repairing skin damage.

Description

Technical Field

[0001] This invention belongs to the field of medicine and health, specifically relating to the application of blocking exogenous mitochondrial entry into cells in promoting skin wound repair. Background Technology

[0002] As the largest organ in the human body, the skin is the first line of defense against external physical, chemical, and biological damage. However, because it is directly exposed to the external environment, the skin is extremely susceptible to various injuries, including acute trauma (such as surgical incisions, burns, mechanical abrasions, and lacerations) and chronic, difficult-to-heal wounds (such as diabetic foot ulcers, venous ulcers, and pressure injuries).

[0003] Skin injuries (including trauma, burns / scalds, and chronic, difficult-to-heal wounds) are prevalent in clinical practice. Oxidative stress caused by excessive reactive oxygen species (ROS) is considered a key factor hindering the repair process and exacerbating inflammatory responses and cell damage. Globally, the number of patients with skin injuries is enormous, causing immense physical and psychological suffering and a decline in quality of life, while also constituting a heavy public health and economic burden. Therefore, developing effective and safe drugs for the treatment of skin injuries remains a research hotspot and urgent need in the medical and pharmaceutical fields.

[0004] However, skin healing is a highly coordinated, complex, and orderly biological process, and existing treatments remain insufficient in addressing the growing prevalence of chronic and refractory skin lesions. The market urgently needs a new drug that can overcome the limitations of current therapies.

[0005] Ideal novel therapeutic agents should possess the following characteristics: They should be able to precisely regulate key pathophysiological processes in the healing process, such as effectively inhibiting excessive inflammation, efficiently activating fibroblasts and keratinocytes, and promoting collagen deposition and epithelial regeneration. Simultaneously, they should have the ability to promote angiogenesis and improve local blood perfusion to ensure a continuous supply of oxygen and nutrients during wound repair, supporting cell metabolism and tissue reconstruction. Furthermore, they should possess good biocompatibility and stability, and be able to adapt to the complex microenvironment of the wound.

[0006] Therefore, developing a skin injury treatment drug that is both highly effective and safe not only has significant scientific theoretical value, but also holds enormous clinical application prospects and market potential, bringing new hope to hundreds of millions of skin injury patients worldwide. Summary of the Invention

[0007] This invention provides a method and its application for enhancing extracellular antioxidant activity and promoting skin wound repair by blocking exogenous mitochondrial entry into cells.

[0008] In a first aspect of the invention, the use of mitochondria is provided for preparing a composition or formulation for repairing skin damage.

[0009] In another preferred embodiment, the composition is a pharmaceutical composition.

[0010] In another preferred embodiment, the composition or formulation is a topical composition or formulation.

[0011] In another preferred embodiment, the mitochondria are derived from autologous or exogenous cells.

[0012] In another preferred embodiment, the mitochondria are derived from immortalized cell lines, mesenchymal stem cells, or platelets.

[0013] In another preferred embodiment, the mitochondria are mitochondria derived from one or more of the following mesenchymal stem cells (MSCs): bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, umbilical cord or umbilical cord mesenchymal stem cells, placental or amnion-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, skin-derived mesenchymal stem cells, synovial-derived mesenchymal stem cells, and mesenchymal stem cells obtained by induced pluripotent stem cell differentiation.

[0014] In another preferred embodiment, the skin injury includes one or more injuries selected from the group consisting of: mechanical injury, thermal injury, chemical injury, radiation injury, infectious injury, inflammatory or immune injury.

[0015] In another preferred embodiment, the damage refers to thermal damage.

[0016] In another preferred embodiment, the thermal injury includes burns, scalds, or blast injuries.

[0017] In another preferred embodiment, the skin injury includes acute skin trauma or chronic, non-healing skin lesions.

[0018] In another preferred embodiment, the skin injury includes extensive skin damage.

[0019] In another preferred embodiment, the composition or formulation is one that facilitates mitochondrial diffusion and release.

[0020] In another preferred embodiment, the composition or formulation comprises: liquid, spray, gel, nanoparticle formulation, microsphere formulation, liposome formulation, hydrogel formulation, patch, film, dressing formulation, lyophilized powder formulation, injection, or topical ointment.

[0021] In another preferred embodiment, the concentration of the active ingredient in the gelling agent is ≤15%, more preferably ≤12%, and most preferably ≤10%.

[0022] In another preferred embodiment, the mitochondria are free mitochondria.

[0023] In another preferred embodiment, the mitochondria are isolated mitochondria.

[0024] In another preferred embodiment, the concentration of mitochondria in the composition or formulation is ≥0.6 μg / μL, more preferably ≥0.8 μg / μL, and most preferably ≥1.0 μg / μL.

[0025] In another preferred embodiment, it is also used for one or more sub-uses selected from the group consisting of:

[0026] (Z1) Improves blood perfusion;

[0027] (Z2) enhances tissue metabolism;

[0028] (Z3) alleviates oxidative stress;

[0029] (Z4) improves collagen deposition;

[0030] (Z5) reduces inflammatory infiltration;

[0031] (Z6) reduces fibroblast apoptosis;

[0032] (Z7) reduces oxidative damage to fibroblasts.

[0033] In another preferred embodiment, the improvement in blood perfusion means that, after the application of the composition or preparation, the blood flow rate A1 flowing through a unit volume of tissue per unit time is greater than or equal to the blood flow rate A0 flowing through a unit volume of tissue per unit time before the application of the composition or preparation, with A1 / A0 ≥ 2, preferably ≥ 3, and most preferably ≥ 4.

[0034] In another preferred embodiment, the enhancement of tissue metabolism means that, after the application of the composition or preparation, the ATP level B1 in the peri-wound tissue is B1 / B0 ≥ 1.5, preferably ≥ 2, and most preferably ≥ 3, compared with the ATP level B0 in the peri-wound tissue before the application of the composition or preparation.

[0035] In another preferred embodiment, the relief of oxidative stress means that, after application of the composition or formulation, the MDA level C1 in the peri-wound tissue is ≤2 / 3, preferably ≤1 / 2, and most preferably ≤1 / 3, compared with the MDA level C0 in the peri-wound tissue before application of the composition or formulation.

[0036] In another preferred embodiment, the improvement in collagen deposition means that, after application of the composition or formulation, the Col1a1 level D1 is ≥1.5 compared to the Col1a1 level D0 before application of the composition or formulation, preferably ≥2, and most preferably ≥3.

[0037] In another preferred embodiment, the improvement in collagen deposition means that, after applying the composition or preparation, the collagen fiber deposition G1 in the wound is ≥1.5 compared to the collagen fiber deposition G0 in the wound before applying the composition or preparation, preferably ≥2, and most preferably ≥2.5.

[0038] In another preferred embodiment, the inflammatory infiltration includes: the degree of inflammatory cell infiltration, the degree of lymphocyte infiltration, or a combination thereof.

[0039] In another preferred embodiment, the degree of inflammatory cell infiltration includes the degree of inflammatory cell infiltration in the wound tissue.

[0040] In another preferred embodiment, the degree of lymphocyte infiltration includes the degree of lymphocyte infiltration in the center of the wound.

[0041] In another preferred embodiment, the inflammatory infiltration is characterized by one or more of the following indicators: lymphocyte density, average number of cells per high power field (HPF), area of ​​inflammatory cells, or a combination thereof.

[0042] In another preferred embodiment, the reduction of inflammatory infiltration means that, after application of the composition or formulation, the expression level M1 of pro-inflammatory cytokines is M1 / M0 ≤ 0.8, preferably ≤ 0.7, and most preferably ≤ 0.5, compared with the expression level M0 of pro-inflammatory cytokines before application of the composition or formulation.

[0043] In another preferred embodiment, the pro-inflammatory cytokines comprise: IL-12p70, MCP-1, IFN-γ, IL-6, or combinations thereof.

[0044] In another preferred embodiment, the reduction in fibroblast apoptosis means that, after administration of the composition or formulation, Vimentin... + / TUNEL + The proportion of double-positive cells E1 and Vimentin before application of the composition or formulation + / TUNEL + Compared to E0, the proportion of double-positive cells is E1 / E0 ≤ 3 / 4, preferably ≤ 2 / 3, and optimally ≤ 1 / 2.

[0045] In another preferred embodiment, it is also used to reduce the level of intramitochondrial ROS (mtROS).

[0046] In another preferred embodiment, the reduction in mtROS level means that after application of the composition or formulation, the mtROS level F1 is ≤3 / 4, more preferably ≤2 / 3, and most preferably ≤1 / 2, compared with the reduction in mtROS level F0 before application of the composition or formulation.

[0047] In another preferred embodiment, the reduction of fibroblast oxidative DNA damage means that, after application of the composition or formulation, Vimentin... + / 8-OHG + The proportion of double-positive cells H1 and Vimentin before application of the composition or formulation + / 8-OHG + Compared to the proportion of double-positive cells H0, H1 / H0 ≤ 1 / 2, preferably ≤ 1 / 3, and optimally ≤ 1 / 4.

[0048] In a second aspect of the invention, a composition or formulation is provided, said composition or formulation comprising the following substances:

[0049] (i) mitochondria; and

[0050] (ii) Pharmaceutically acceptable carriers.

[0051] In another preferred embodiment, the composition or formulation comprises: liquid, spray, gel, nanoparticle formulation, microsphere formulation, liposome formulation, hydrogel formulation, patch, film, dressing formulation, lyophilized powder formulation, injection, or topical ointment.

[0052] In another preferred embodiment, the pharmaceutically acceptable carrier is a substance that facilitates mitochondrial diffusion and release.

[0053] In another preferred embodiment, the carrier is a solution.

[0054] In another preferred embodiment, the solution is a physiological buffer for mitochondria.

[0055] In another preferred embodiment, the solution comprises: a PBS solution.

[0056] In another preferred embodiment, the concentration of mitochondria in the composition or formulation is ≥0.5 μg / μL; more preferably ≥0.6 μg / μL, even more preferably ≥0.8 μg / μL, and most preferably ≥1.0 μg / μL.

[0057] In another preferred embodiment, the carrier is a gel.

[0058] In another preferred embodiment, the gel is a thermosensitive gel.

[0059] In another preferred embodiment, the concentration of the active ingredient in the gel is ≤15%, more preferably ≤12%, and most preferably ≤10%.

[0060] In another preferred embodiment, the mitochondria are free mitochondria.

[0061] In another preferred embodiment, the composition or formulation does not contain any organelles other than mitochondria.

[0062] In another preferred embodiment, the mitochondria are the only active ingredient.

[0063] In a third aspect of the invention, a pharmaceutical composition is provided, comprising:

[0064] mitochondria; and

[0065] Mitochondrial entry blockers.

[0066] In another preferred embodiment, after the administration of the mitochondrial entry blocker, the proportion of mitochondria entering the cell is reduced by ≥60%, more preferably by ≥70%, and most preferably by ≥80%.

[0067] In another preferred embodiment, the mitochondrial entry blocker is Dynasore and its derivatives.

[0068] In another preferred embodiment, the mitochondrial entry blocker comprises: Dynasore, Dyngo-4a, or a combination thereof.

[0069] In a fourth aspect of the invention, the use of the pharmaceutical combination described in the third aspect of the invention is provided for preparing a medicine box for repairing skin damage.

[0070] In a fourth aspect of the invention, the use of a mitochondrial entry blocker is provided for preparing a formulation or material for enhancing the ability of mitochondria to repair skin damage.

[0071] In another preferred embodiment, the mitochondrial entry blocker comprises a physical barrier material.

[0072] In another preferred embodiment, the mitochondrial entry blocker comprises Dynasore and its derivatives.

[0073] In another preferred embodiment, it is also used for one or more sub-uses selected from the group consisting of:

[0074] (Z1') inhibits mitochondrial entry into cells;

[0075] (Z2') promotes increased cell survival rate;

[0076] (Z3') promotes the reduction of mitochondrial ROS levels.

[0077] In another preferred embodiment, the inhibition of mitochondrial entry into cells means that, after the application of the preparation and mitochondria, the proportion J1 of mitochondria entering cells is ≤20%, preferably ≤15%, and most preferably ≤10%, compared with the proportion J0 of mitochondria entering cells when mitochondria are applied alone.

[0078] In another preferred embodiment, the improvement in cell survival rate means that after applying the preparation and mitochondria, the cell survival rate K1 is K1 / K0 ≥ 1.1, preferably ≥ 1.3, and most preferably ≥ 1.5, compared with the cell survival rate K0 when mitochondria are applied alone.

[0079] In another preferred embodiment, the promotion of reducing mitochondrial ROS levels means that after applying the formulation and mitochondria, the mitochondrial ROS level L1 is L1 / L0 ≤ 0.95, preferably ≤ 0.9, and most preferably ≤ 0.8, compared with the mitochondrial ROS level L0 when mitochondria are applied alone.

[0080] In another preferred embodiment, the cell refers to a fibroblast.

[0081] In another preferred embodiment, the derivative comprises Dynasore, Dyngo-4a, or a combination thereof.

[0082] In another preferred embodiment, the derivative comprises a compound with an overall skeleton of "N-acylhydrazine-hydrazone condensed from 3-hydroxy-naphthalene-2-carboxylic acid hydrazine and polyhydroxy aromatic aldehyde".

[0083] In another preferred embodiment, the derivative comprises a pharmaceutically acceptable salt, solvate (including hydrate), prodrug, isotope label, stereo / geometric / tautomer (including E / Z-hydrazone isomers), polymorph, cocrystal, or inclusion compound of a compound whose overall skeleton is “N-acylhydrazine-hydrazone condensed with a polyhydroxy aromatic aldehyde”, or any combination thereof.

[0084] In another preferred embodiment, the stereo / geometric / tautomer includes: E / Z-hydrazone isomers.

[0085] In another preferred embodiment, the compound whose overall skeleton is “N-acylhydrazine-hydrazone condensed from 3-hydroxy-naphthalene-2-carboxylic acid hydrazine and polyhydroxy aromatic aldehyde” includes Dynasore, Dyngo-4a, or a combination thereof.

[0086] In a fifth aspect of the invention, a medicine box is provided, the medicine box comprising:

[0087] (I) Mitochondria;

[0088] (II) Mitochondrial entry blockers.

[0089] In another preferred embodiment, the mitochondrial entry blocker comprises: a physical barrier material or a chemical reagent.

[0090] In another preferred embodiment, the physical barrier material allows small molecules to pass through.

[0091] In another preferred embodiment, the physical barrier material prevents mitochondria from entering the cell.

[0092] In another preferred embodiment, the pore size of the physical barrier material is 100–800 nm, more preferably 200–600 nm, even more preferably 300–500 nm, and most preferably 400 nm.

[0093] In another preferred embodiment, the mitochondria and the mitochondrial entry blocker are packaged separately.

[0094] In another preferred embodiment, mitochondria and mitochondrial entry blockers are packaged together.

[0095] In another preferred embodiment, the medicine box also includes an instruction manual.

[0096] In another preferred embodiment, the instructions guide the use of the medicine box for repairing skin damage.

[0097] In another preferred embodiment, the specification states that a mitochondrial entry blocker is first administered to the test subject, followed by the administration of a pharmaceutically effective amount of mitochondria to the test subject, thereby repairing the test subject's skin damage.

[0098] In another preferred embodiment, the application includes: smearing, dripping, spraying / spraying, rinsing / irrigating, soaking / wet dressing, applying / bandaging, applying gel / ointment / emulsion, sprinkling foam or powder, reconstituted lyophilized powder for external use; and percutaneous enhancement methods used when necessary, including but not limited to microneedling / needle roller transdermal, iontophoresis, electroosmosis, ultrasound delivery; and local injection administration, including intradermal, subcutaneous, or perilesional infiltration injection.

[0099] In another preferred embodiment, the effective amount of the mitochondria is 1–800 μg / cm³. 2 The preferred concentration is 2–500 μg / cm³. 2 More preferably 5–300 μg / cm 2 The optimal concentration is 10–150 μg / cm³. 2 The optimal value is 15–60 μg / cm³. 2 .

[0100] In another preferred embodiment, the chemical reagent comprises Dynasore, Dyngo-4a, or a combination thereof.

[0101] In a sixth aspect of the invention, a method for repairing skin damage is provided, the method comprising: administering to a test subject an effective amount of the composition or preparation of the second aspect of the invention, the pharmaceutical combination of the third aspect of the invention, or the medicament of the fifth aspect of the invention.

[0102] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0103] Figure 1 This showed abnormal tissue metabolism and decreased fibroblast activity in clinically extensive burn wounds. Among them, Figure 1 A- Figure 1 D shows the impaired energy and antioxidant function of refractory wound tissue compared to normal skin tissue. Figure 1 A); Reduced regenerative factors and increased inflammation ( Figure 1 B); Histological abnormalities ( Figure 1 C); Decreased fibroblasts ( Figure 1 D).

[0104] Figure 2 This study demonstrates the identification of MSC-derived mitochondria (MSC-mt) and their evaluation of their role in skin wound repair. Among other things, Figure 2 The NTA results of A showed that the MSC-mt particles were relatively concentrated, with an average diameter of 438.5 ± 199.3 nm. Figure 2 The TEM results of B show a complete bilayer membrane and a clear cristae structure, proving that the ultrastructure of MSC-mt is intact. Figure 2 The TMRE flow cytometry results of C showed that, compared with the decoupling control (CCCP), MSC-mt exhibited a significant rightward shift / increase in TMRE fluorescence intensity, indicating that the mitochondrial membrane potential (ΔΨm) was preserved and the activity was good. Figure 2 The immunoblot of D showed clear enrichment of the OXPHOS complex (I–V) and the mitochondrial outer membrane marker TOM20 in the isolated MSC-mt, supporting the mitochondrial identity and functional composition of MSC-mt. Figure 2 The mouse full-thickness skin wound model of E showed that after local surface application of MSC-mt, the mtL group showed a trend of accelerated healing compared with the control, and the mtH group had a significantly faster healing rate than the control group; Schematic diagram of wound area ( Figure 2 F) and quantitative statistics Figure 2 G) is consistent with the trend described above.

[0105] Figure 3 This study demonstrated that MSC-MT improved blood perfusion and enhanced metabolism in a mouse skin wound model. Among these effects, Figure 3 A shows that MSC-MT improves blood perfusion; Figure 3 B showed that MSC-mt increased ATP in perioperative tissues; Figure 3 C showed that MSC-mt alleviated oxidative stress; Figure 3 D、 Figure 3E showed that the infiltration of inflammatory cells in the tissue below the wound in the mtH group was significantly reduced compared with the ctrl group; Figure 3 F showed that the levels of inflammatory factors IL-12p70, MCP1, IFN-γ, and IL-6 were all significantly reduced in the mtH group.

[0106] Figure 4 The study demonstrated that MSC-MT improved collagen deposition and protected fibroblasts in a mouse skin wound model. Figure 4 A and Figure 4 B shows that the matrix remodeling in the mtH group was more ordered than that in the ctrl group; Figure 4 C and Figure 4 D showed that collagen synthesis dominated by dermal fibroblasts was effectively promoted in the mtH group; Figure 4 E and Figure 4 F shows that mtH treatment effectively inhibits fibroblast apoptosis in the wound environment; Figure 4 G and Figure 4 H showed that mtH alleviated oxidative damage in fibroblasts.

[0107] Figure 5 This study demonstrated that MSC-mt protects fibroblasts from H2O2-induced oxidative damage and functional decline in vitro. Among other things, Figure 5 A shows the dose-effect screening of MSC-mt; Figure 5 B showed that MSC-mt enhanced metabolic support: Figure 5 C and Figure 5 D showed that MSC-mt had a significant anti-apoptotic effect; Figure 5 E showed that MSC-mt enhanced antioxidant capacity, manifested by a decrease in intramitochondrial ROS (mitoSOX); Figure 5 F shows that MSC-mt maintains mitochondrial membrane potential; Figure 5 G showed that MSC-mt delayed cell senescence; Figure 5 H, Figure 5 I showed the proliferative potential of fibroblasts; Figure 5 J shows that the mt(R) function is impaired.

[0108] Figure 6 The study showed a negative correlation between MSC-mt uptake in fibroblasts and cytoprotective effects. Specifically, Figure 6 A showed that Dynasore (Dyn) specifically inhibits mitochondrial uptake; Figure 6 B showed that Dyn treatment actually increased cell survival; Figure 6 C shows a negative correlation between mitochondrial uptake and cell viability: Figure 6 D shows mt transfer - Higher cell survival rate; Figure 6E shows the flow cytometry results of H2O2-stimulated apoptosis in primary fibroblasts; Figure 6 F showed that Dyn significantly inhibited mitochondrial translocation in primary cells; Figure 6 G shows mt transfer - The survival rate of the subpopulation was consistently higher than that of the mt transfer. + Subgroup; Figure 6 H and Figure 6 I shows the consistency validation in PX-12 and Rotenone-induced oxidative stress models; Figure 6 J showed that Dyn enhances the antioxidant effect of MSC-mt.

[0109] Figure 7 The effects of different dynein inhibitors on MSC-mt entry and cytoprotective effects were demonstrated. Figure 7 A and Figure 7 B showed that mt significantly reduced H2O2-induced cell damage; Dynasore and Dyngo-4a further enhanced the protective effect.

[0110] Figure 8 This demonstrated the cytoprotective effect of pharmacological or physical blockade of mitochondrial entry into cells, thereby enhancing MSC-mtocytes. Among these, Figure 8 A shows a schematic diagram of the experimental design; Figure 8 B and Figure 8 C showed that H2O2 exposure significantly reduced cell viability, with partial recovery in the MSC-mt treatment group; in the Dynasore and Transwell groups (especially the (H2O2+mt) group), cell viability was further improved, suggesting that blocking cell entry actually enhanced the protective effect; Figure 8 D showed through flow cytometry results that the Dynasore and Transwell models effectively inhibited mitochondrial entry into cells; Figure 8 E shows mt transfer - Cell survival rate was significantly higher than that of mt transfer. + cell; Figure 8 F, as shown by MitoSOX staining, revealed that mtROS levels were further reduced after cell entry was blocked; Figure 8 G showed that MSC-mt can directly scavenge extracellular H2O2.

[0111] Figure 9 The results showed that in PWD8, both the mtH and mtH+Dyn groups significantly promoted wound healing, and mtH and Dyn synergistically promoted wound healing.

[0112] Figure 10 The study showed that the cell protection function of MSC-mt cells was impaired after hydrogel immobilization. Figure 10A shows the gel temperature sensitivity results; Figure 10 B shows the scanning electron microscope results; Figure 10 C shows the effect of different gel concentrations on mt release; Figure 10 D、 Figure 10 E and Figure 10 F shows gross photographs of the wound at different time points in a mouse full-thickness skin wound model, a schematic diagram of the wound size, and statistics, illustrating that gel concentration affects the wound healing speed. Figure 10 G and Figure 10 H shows the results and statistics of wound blood perfusion in each group on the 7th postoperative day.

[0113] Figure 11 The study showed that gel restriction weakens extracellular ROS scavenging and cell protection. Figure 11 A showed that 50% gel-fixed MSCs-mts significantly reduced H2O2 scavenging ability, with supernatant residual H2O2 levels comparable to those in the Transwell isolation group; Figure 11 B showed a significant decrease in intracellular MSC-mt fluorescence signal in the 50% gel+mt group; Figure 11 C shows the intracellular ROS (DCFH-DA) detection results for each group. Detailed Implementation

[0114] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time that mitochondria promote skin wound repair. However, the more exogenous mitochondria fibroblasts take up, the higher their mortality rate becomes. In other words, the ability of mitochondria to repair skin damage is negatively correlated with their mitochondrial entry rate; conversely, mitochondrial entry inhibitors promote mitochondrial repair of skin damage. When exogenous mitochondria are used in combination with agents that inhibit exogenous mitochondrial entry, they exhibit a synergistic effect, significantly increasing the skin wound repair effect. Based on this, the present invention was completed.

[0115] Specifically, this invention aims to propose a novel skin injury treatment strategy based on "blocking cell entry and enhancing extracellular antioxidant capacity." It focuses on the key mechanism of exogenous mitochondria (preferably mesenchymal stem cell-derived mitochondria, MSC-mt) directly scavenging ROS extracellularly. This is achieved by inhibiting the uptake of mitochondria by recipient cells through pharmacological or physical means, thereby enhancing their antioxidant and tissue-protective effects and promoting skin wound healing. Simultaneously, this invention proposes a novel application for dynein inhibitors (such as Dynasore and its analogues) to enhance the extracellular antioxidant function of exogenous mitochondria, applicable to various oxidative stress-related pathological conditions.

[0116] the term

[0117] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0118] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0119] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0120] As used in this article, "large-area" injury refers to the clinically defined large-area injury. In a specific implementation, large-area injury refers to large-area burns. In a specific implementation, large-area burns are defined as burns covering more than 10% of the total surface area. In a specific implementation, large-area burns are determined using the "nine-point method".

[0121] As used herein, "free mitochondria" refers to mitochondria that are freely dispersed in the system. In specific embodiments, the mitochondria are not contained within a carrier, which comprises vesicles, exosomes, liposomes, and synthetic nanoparticles.

[0122] As used in this article, the term "peri-wound tissue" refers to the area of ​​damaged healthy tissue extending a certain distance outward from the wound edge, typically including the skin and subcutaneous tissue within approximately 1–5 mm lateral to the wound edge. This area plays a crucial role in wound healing, including cell source, matrix remodeling, and angiogenesis, and is therefore often used as a key area for assessing wound repair status.

[0123] As used herein, the term “repairing skin damage” includes preventing wound deterioration, slowing the rate of wound deterioration, improving wound condition, increasing the levels of substances that promote wound healing, decreasing the levels of substances that inhibit wound healing, or combinations thereof.

[0124] As used herein, substances that facilitate mitochondrial diffusion and release do not contain mitochondrial nanocarriers. In specific embodiments, substances that facilitate mitochondrial diffusion and release do not contain common nanocarriers or transfer carriers, such as vesicles, exosomes, or carriers that load or encapsulate mitochondria.

[0125] mitochondria

[0126] Mitochondria, as the core of cellular energy and redox metabolism, have been explored in recent years for cell protection and tissue repair.

[0127] Previous studies have shown that supplementing with exogenous mitochondria (MT) can alleviate oxidative stress damage and promote tissue repair. Current technology generally holds that exogenous mitochondria must be endocytosed or transported into the cell by a recipient cell to exert their effects through mechanisms such as metabolic compensation.

[0128] The method of the present invention

[0129] Through systematic in vitro and in vivo experiments, the inventors discovered that exogenous mitochondria can directly scavenge ROS and produce significant cell protection effects outside the cell without entering the recipient cell; and blocking mitochondrial entry into the cell actually enhances its extracellular antioxidant and tissue protection effects.

[0130] Specifically, dynamin inhibitors (such as Dynasore, MedChemExpress, Cat#HY-15304; Dyngo-4a, MedChemExpress, Cat#HY-13863) can pharmacologically inhibit mitochondrial entry into cells, or physical methods such as Transwell can isolate mitochondria from direct contact with cells, thereby enhancing the antioxidant and cell-protective effects of exogenous mitochondria in various oxidative stress models.

[0131] Conversely, immobilizing mitochondria in high-concentration hydrogels (e.g., 50% by volume) limits their effective diffusion in tissue / culture systems, weakens their ability to scavenge extracellular ROS, and thus reduces their protective effect. However, low-concentration hydrogels are feasible.

[0132] These findings overturn the traditional understanding that "entry into cells is a prerequisite for exerting its effects" and provide a new intervention pathway for skin damage repair.

[0133] The main advantages of this invention include:

[0134] (a) This invention provides the application of free mitochondria in repairing skin damage, and provides compositions or preparations containing free mitochondria and their application in repairing skin damage. These compositions or preparations exhibit good biocompatibility and stability, can adapt to the complex microenvironment of wounds, and demonstrate high safety and significant therapeutic effects.

[0135] (b) This invention provides the use of mitochondria and mitochondrial entry blockers in the repair of skin damage. Dynamite inhibitors (such as Dynasore, Dyngo-4a) or physical barriers (such as 400nm Transwell) can block mitochondrial entry, allowing mitochondria to maintain their diffusivity and antioxidant capacity in the extracellular environment, thereby enhancing ROS clearance, reducing apoptosis, and improving tissue repair.

[0136] (c) This invention provides a method for effectively improving skin damage by regulating key pathophysiological processes in the healing process, such as effectively improving blood perfusion; enhancing tissue metabolism; alleviating oxidative stress; improving collagen deposition; reducing inflammation; reducing fibroblast apoptosis; and reducing oxidative DNA damage in fibroblasts.

[0137] (d) This invention is the first to discover that mitochondria have a clear optimal dose window (bell response curve), and high doses may reduce efficacy, possibly due to excessive uptake, local crowding effect or receptor cell stress leading to decreased efficacy.

[0138] (e) Through systematic in vitro and in vivo experiments, this invention unexpectedly discovered that for exogenous mitochondria to fully exert their ability to scavenge extracellular ROS, they must be able to maintain open contact and free spatial diffusion in the wound microenvironment. Regardless of the delivery method, as long as the structural design significantly restricts the diffusion of mitochondria or their accessibility to the environment, their antioxidant function will be significantly weakened or even completely lost. Specifically: (1) Carriers that restrict the diffusion of mitochondria will weaken their function. When the density, viscosity, or spatial structure of the carrier is too dense, such as high-concentration (≥20–50%) hydrogels, occlusive dressings, dense matrices, etc., it will block the contact between mitochondria and external H2O2 / ROS. The results are: decreased ROS scavenging ability; continuous accumulation of intracellular ROS; increased apoptosis rate; and significantly weakened tissue repair. Experiments of this invention have shown that the protective effect of mitochondria fixed by high-density carriers is equivalent to that of completely isolated (such as Transwell separation), suggesting that restricted diffusion will lead to the basic loss of mitochondrial function. (ii) Carriers that allow free diffusion of mitochondria can enhance the therapeutic effect. Conversely, low-concentration, low-density carriers with open structures (such as 10% hydrogels, sprays, and liquid formulations) can maintain the diffusion capacity of mitochondria, enabling them to: more effectively contact and remove extracellular ROS; protect tissues more broadly; significantly improve blood perfusion, inflammatory response, and collagen deposition; and accelerate wound repair. (iii) This invention proposes a universal biophysical design principle applicable to all mitochondrial drug delivery systems. This principle is not limited to a certain type of carrier but is applicable to, but includes, but is not limited to: hydrogels (thermosensitive, natural, polymeric, etc.); dressings, sprays, patches, films; injectable scaffolds, biodegradable matrices; microspheres, nanoparticles, encapsulation materials; bioinks, biomembranes, and bioscaffolds. As long as the carrier allows mitochondria to maintain sufficient diffusion and accessibility in the wound environment, its antioxidant and tissue repair effects can be maintained or enhanced; conversely, if the carrier hinders mitochondrial movement or contact with ROS, it should be avoided. (iv) Therefore, this invention proposes a carrier design principle that is clearly defined for the first time in the field of mitochondrial therapy: "Free diffusion is the fundamental condition for exogenous mitochondria to exert their extracellular antioxidant and tissue repair capabilities."

[0139] This discovery not only applies to the treatment of skin lesions, but also provides important methodological guidance and theoretical foundation for the future development of mitochondrial-based drugs.

[0140] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0141] Example 1: Abnormal tissue metabolism and decreased fibroblast activity in clinical large-area burn wounds.

[0142] A comparative study was conducted using clinically derived human skin tissue samples. Normal control tissue was obtained from excess skin tissue removed during surgery in children with polydactyly; wound tissue was obtained from the wound edges of children with extensive burns or blast injuries during debridement surgery.

[0143] Tissue samples were lysed using RIPA lysis buffer, and total protein concentration was determined using the BCA method. Subsequently, flow cytometry combined with a cytokine assay kit (CBA, BioLegend) was used to quantitatively detect the levels of multiple cytokines, including tumor necrosis factor-α (TNF-α), epidermal growth factor (EGF), fibroblast growth factor (FGF), and matrix metalloproteinase inhibitor 1 (TIMP1), and the results were normalized for protein content. Simultaneously, the antioxidant indicators superoxide dismutase (SOD) activity and ATP levels were measured. Histological features were observed after hematoxylin-eosin (H&E) staining of tissue sections, and immunofluorescence staining was used to detect the fibroblast marker Vimentin to evaluate fibroblast activity.

[0144] Experimental results are as follows Figure 1 As shown.

[0145] Figure 1 A showed that, compared with normal skin tissue, the energy and antioxidant functions of refractory wound tissue were impaired. Specifically, ATP levels were significantly reduced, indicating insufficient local tissue energy metabolism; and SOD activity was decreased, indicating weakened antioxidant defense.

[0146] Figure 1B showed that, compared with normal skin tissue, refractory wound tissue had reduced regenerative factors and enhanced inflammation. Specifically, compared with normal skin, TIMP1 levels were significantly decreased, suggesting insufficient fibroblast activity; key growth factors EGF and FGF were both significantly reduced, while the inflammatory factor TNF-α was significantly increased.

[0147] Figure 1 C showed histological abnormalities in the tissue of refractory wounds compared to normal skin tissue. Specifically, H&E staining results showed that the epidermal layer of normal skin was clear and intact, and appendages (hair follicles, sebaceous glands) were preserved, while the tissue of refractory wounds showed thinning or absence of the epidermis and lack of normal skin appendages.

[0148] Figure 1 D showed a reduction in fibroblasts in refractory wound tissue. Specifically, immunofluorescence results showed that the number of Vimentin-positive fibroblasts in refractory wound tissue was significantly lower than in normal skin.

[0149] Example 2: Identification of MSC-derived mitochondria and evaluation of their role in skin wound repair.

[0150] Mitochondrial isolation and identification: Mouse bone marrow-derived mesenchymal stem cells (MSCs) were cultured, and mitochondria (MSC-mts) were isolated using differential centrifugation. Nanoparticle analysis (NTA) was used to detect the particle size distribution of the isolated mitochondria; transmission electron microscopy (TEM) was used to observe the ultrastructure of the mitochondria; the membrane potential difference between normal mitochondria and CCCP-treated mitochondria was detected by flow cytometry using the membrane potential fluorescent probe TMRE; Western blot was used to detect proteins in MSCs and isolated mitochondria, including OXPHOS cocktail (mitochondrial respiratory chain complex) and mitochondrial outer membrane protein TOM20 (…). Figure 2 D).

[0151] To investigate the effect of MSC-mt on the repair of full-thickness skin wounds in mice. First, a large-area full-thickness skin defect model (15 mm in diameter) was established on the back of mice. The mice were randomly divided into a control group (ctrl), a low-dose MSC-mt group (mtL, 50 μg MSC-mt dispersed in 100 μL PBS), and a high-dose MSC-mt group (mtH, 100 μg MSC-mt dispersed in 100 μL PBS). After modeling, PBS (100 μL PBS) or different doses of MSC-mt were dripped onto the wound surface to cover the entire wound. Macroscopic images of the wound were taken at different time points, and a schematic diagram of wound area changes was plotted. The wound healing rate was statistically analyzed.

[0152] The results are as follows Figure 2 As shown.

[0153] NTA analysis showed that MSC-mt was mainly distributed around 500nm. Figure 2 A); TEM showed that the MSC-mt bilayer membrane was intact and the cristae structure was clear. Figure 2 B); TMRE flow cytometry analysis showed that normal MSC-mt had intact mitochondrial membrane potential, while the membrane potential decreased significantly after CCCP treatment. Figure 2 C); Western blot results confirmed that the isolated MSC-mt was rich in OXPHOS complex protein and mitochondrial marker TOM20 (C); Figure 2 D).

[0154] In addition, animal experiments included gross photographs of the wound at different time points ( Figure 2 E), Wound size diagram ( Figure 2 F) and statistics Figure 2 G) showed that MSC-MT treatment generally promoted skin wound closure. Specifically, the high-dose group (mtH) had a significantly higher wound healing rate than the control group, and its trend was superior to that of the low-dose group (mtL). Although the low-dose group (mtL) showed a better healing trend than the control group, the difference was not statistically significant. Notably, the data from the high-dose group (mtH) were more stable and had lower dispersion.

[0155] Example 3: In a mouse skin wound model, MSC-mt improved blood perfusion, enhanced metabolism, promoted collagen deposition, and protected fibroblasts.

[0156] Animal models and drug administration were performed using the same method as in Example 2, and blood perfusion, tissue metabolism and oxidative stress, collagen deposition and fibrosis, fibroblast apoptosis and oxidative damage performance were evaluated.

[0157] Blood perfusion assessment:

[0158] On postoperative day 7 (PWD7), laser speckle contrast imaging (LSCI) was used to detect blood perfusion in the wound area; the perfusion signal was displayed and quantitatively analyzed using pseudo-color thermograms.

[0159] The results are as follows Figure 3 As shown in Figure A, at PWD8 (LSCI was completed at PWD7 and the tissue was taken at PWD8 according to the preset procedure), the blood perfusion in the wound area of ​​the mtH group was significantly higher than that of the control group, indicating improved angiogenesis and perfusion status.

[0160] Tissue metabolism and oxidative stress detection:

[0161] On postoperative day 8 (PWD8), peri-wound tissue was harvested, and ATP levels (normalized to total protein) were measured using an ATP assay kit; lipid peroxidation product MDA (normalized to total protein) was measured using an MDA assay kit.

[0162] The results are as follows Figure 3 As shown in Figure B, in the peri-injury tissue of PWD8, the ATP level in the mtH group was significantly increased, indicating enhanced local metabolic activity; at the same time, the MDA level was significantly decreased, indicating that lipid peroxidation was inhibited and oxidative damage was reduced. Figure 3 C).

[0163] Tissue inflammation detection:

[0164] On postoperative day 8 (PWD8), peri-wound tissue was taken for H&E staining. The wound area was observed under low magnification, and the infiltration of inflammatory cells was observed under high magnification. The expression of inflammatory factors in the tissue was detected using the CBA kit (BD Biosciences, Cat#552364).

[0165] The results are as follows Figure 3 As shown in DE, the wound area in the mtH group was significantly smaller than that in the control group (ctrl) (indicated by green triangle arrows and lines); under high magnification, the infiltration of inflammatory cells in the tissue beneath the wound in the mtH group was significantly reduced compared to the ctrl group. Figure 3 D and Figure 3 E); inflammatory factors IL-12p70, MCP1, IFN-γ, and IL-6 were all significantly reduced in the mtH group. Figure 3 F).

[0166] Assessment of collagen deposition and fiber formation:

[0167] Masson staining was performed on the 8th day after surgery to compare collagen fiber deposition and arrangement between the control group and the mtH group; immunohistochemistry was used to detect Col1a1 expression and quantify its positive area.

[0168] Masson staining results are as follows: Figure 4 A and Figure 4 As shown in Figure B, the mtH group showed more abundant and more regularly arranged collagen fibers in the wound, which was significantly improved compared with the control group, indicating that the matrix remodeling was more orderly.

[0169] Immunohistochemical results as follows Figure 4 C and Figure 4 As shown in Figure D, consistent with Masson's results, Col1a1 expression was significantly increased in the mtH group (positive area increased), suggesting that collagen synthesis dominated by dermal fibroblasts was effectively promoted.

[0170] Assessment of fibroblast apoptosis and oxidative damage:

[0171] The apoptosis of fibroblasts was detected by dual immunofluorescence of Vimentin (a fibroblast marker) + TUNEL assay, and the Vimentin levels were statistically analyzed. + / TUNEL +The proportion of double-positive cells; co-staining with Vimentin and 8-hydroxyguanine (8-OHG) to assess the level of oxidative DNA damage in fibroblasts.

[0172] The results of Vimentin+TUNEL double staining are as follows: Figure 4 E and Figure 4 As shown in F, mtH group Vimentin + / TUNEL + The proportion of double-positive cells was significantly reduced, suggesting that MSC-mt treatment effectively inhibited fibroblast apoptosis in the wound environment.

[0173] The results of co-staining Vimentin with 8-OHG are as follows: Figure 4 G and Figure 4 As shown in Figure H, the 8-OHG signal in MSC-mt cells was significantly reduced, suggesting that MSC-mt alleviated oxidative damage in fibroblasts.

[0174] Example 4: MSC-mt protects fibroblasts from H2O2-induced oxidative damage and functional decline in vitro.

[0175] We selected skin fibroblasts L929 as the research subject and used a hydrogen peroxide (H2O2) induced oxidative stress model to simulate the wound microenvironment in order to construct a wound cell model.

[0176] MSC-MT co-culture and dose screening:

[0177] Under H2O2 stimulation, different doses of MSC-derived mitochondria (MSC-mt) were added to the culture system and cultured for 24 hours. Intracellular ATP levels were then measured.

[0178] The results are as follows Figure 5 As shown in Figure A, ATP exhibits a bell-shaped response curve at 0.67 μg / cm². 2 Peak levels were reached at higher doses (1.33–2.66 μg / cm³), while higher doses (1.33–2.66 μg / cm³) showed higher peak levels. 2 This leads to a decrease in ATP, indicating the existence of an optimal window for action. Therefore, subsequent experiments all used 0.67 μg / cm³. 2 dose.

[0179] Antioxidant function assessment:

[0180] MSC-mt were cultured for 24 hours at different H2O2 concentrations (125–1000 μM) and ATP levels were measured to evaluate the metabolic support effect of MSC-mt.

[0181] The results are as follows Figure 5As shown in Figure B, the results indicate that within the range of 125–1000 μM H2O2, MSC-mt significantly increases cellular ATP content, demonstrating its ability to maintain energy metabolism under strong oxidative stress.

[0182] Apoptosis and cell death analysis:

[0183] After stimulation with H2O2, cells were collected at set time points and analyzed by flow cytometry using Annexin V / PI double staining to calculate the proportions of early apoptosis, late apoptosis, and cell death.

[0184] The results are as follows Figure 5 C and Figure 5 As shown in Figure D, MSC-mt significantly reduced the H2O2-induced apoptosis rate, while the protective effect of mt(R) pretreated with the mitochondrial respiratory chain complex I inhibitor Rotenone was significantly weakened.

[0185] Oxidative stress and membrane potential measurement:

[0186] The level of intracellular ROS (mtROS) in cells was detected using MitoSOX dye (DAPI-gated for live cells), and the mitochondrial membrane potential (ΔΨ) was detected using the TMRE kit. m ).

[0187] mtROS results are as follows Figure 5 As shown in E, MSC-mt significantly reduced oxidative stress-induced mtROS accumulation, while mt(R) failed to produce the same effect.

[0188] Mitochondrial membrane potential results are as follows Figure 5 As shown in F, MSC-mt maintains a high ΔΨm under oxidative stress, while mt(R) has a weak protective effect.

[0189] Assessment of cell senescence and proliferation:

[0190] β-galactosidase flow cytometry (Thermo Fisher Scientific, Cat#C10841) was used to evaluate stress-induced senescence; clonogenic assays were used to detect the proliferative potential of fibroblasts.

[0191] Cellular senescence assessment results such as Figure 5 G and H2O2 stimulation significantly increased β-galactosidase activity, which was significantly inhibited by MSC-mt, with the effect weakened in the mt(R) group. These results indicate that MSC-derived mitochondria delay oxidative stress-induced cellular senescence.

[0192] The proliferative potential of fibroblasts, such as Figure 5 H and Figure 5As shown in Figure I, H2O2 significantly inhibited the ability of fibroblasts to form colonies, while MSC-mt significantly restored this ability. However, this effect of MSC-mt was weakened after Rotenone pretreatment. The results indicate that MSC-mt promotes the recovery of cell proliferation.

[0193] Mitochondrial function verification:

[0194] To verify mitochondrial function dependence, mitochondria were treated with the mitochondrial respiratory chain complex I inhibitor Rotenone [denoted as mt(R)]. Western blot analysis of the expression levels of OXPHOS complex protein, PINK1, and TOM20 confirmed impaired mt(R) function.

[0195] Western blot results are as follows Figure 5 As shown in J, the results indicate that the expression of OXPHOS, PINK1, USP30, and TOM20 in mt(R) is downregulated, confirming their impaired function.

[0196] Example 5: MSC-mt uptake in fibroblasts is negatively correlated with cell protection effect.

[0197] To investigate the mechanism by which MSC-derived mitochondria (MSC-mt) enter recipient cells, several major pathways were considered, including macropinocytosis, clathrin-mediated endocytosis, and dynein-dependent endocytosis, and corresponding pharmacological inhibitors were selected.

[0198] EIPA and LY294002 (macrophosome inhibitors);

[0199] Chloroquine (CQ, a clathrin-mediated endocytosis inhibitor);

[0200] Dynasore (Dyn, a dynein-dependent endocytosis inhibitor);

[0201] MNS (Src / Syk kinase inhibitors, which interfere with membrane dynamics and vesicle transport).

[0202] After co-culturing L929 fibroblasts with MitoTracker Deep Red-labeled MSCs for 16 hours, the proportion of mitochondrial signaling in recipient cells was detected by flow cytometry to assess the effect of each drug on mitochondrial uptake.

[0203] The results are as follows Figure 6 As shown in Figure A, among various endocytosis inhibitors, only Dyn significantly reduced the uptake level of MSC-mt in L929 fibroblasts, suggesting that it mainly enters receptor cells through the dynein-dependent endocytosis pathway.

[0204] In addition, such as Figure 6As shown in B, although Dyn significantly reduced mitochondrial uptake, the Annexin V- / PI- cell ratio increased, and cell survival improved.

[0205] Apoptosis analysis and correlation calculation:

[0206] After co-culture, cell viability was assessed by Annexin V / PI double staining flow cytometry (Annexin V- / PI- population). The Pearson correlation coefficient between mitochondrial translocation rate and cell viability was calculated by comparing the different drug treatment groups.

[0207] The results are as follows Figure 6 As shown in Figure C, correlation analysis revealed that the higher the mitochondrial uptake rate, the lower the cell survival rate, suggesting that mitochondrial endocytosis may be associated with increased cellular stress or apoptosis.

[0208] MT Transfer + / - Subgroup analysis:

[0209] Based on flow cytometry gating, cells were divided into mitochondrial transfer-positive (MT transfer) cells. + MitoTracker + ) and negative (mt transfer) - MitoTracker - Subgroups were selected, and the differences in apoptosis rate and survival rate were compared.

[0210] The results are as follows Figure 6 As shown in D, in each experimental group, mt transfer - The proportion of surviving cells in the population was significantly higher than that in mt transfer. + The population further supports the association between endocytosis and cell damage.

[0211] Primary fibroblast validation experiment:

[0212] The above experiment was repeated in primary mouse skin fibroblasts. Co-culture times were set at 90 min and 180 min. The following treatment groups were established:

[0213] H2O2-induced oxidative damage model control group;

[0214] MSC-MT co-culture group;

[0215] Rotenone treatment of mitochondria (mt(R) group);

[0216] MSC-mt+Dyn co-treatment group;

[0217] Apoptosis rates in each group were detected by Annexin V / PI flow cytometry.

[0218] Results of primary fibroblasts as follows Figure 6 As shown in Figure E, MSC-mt significantly alleviated H2O2-induced apoptosis in primary fibroblasts, while the protective effect was weakened in the mt(R) group. Notably, Dyn treatment further enhanced the protective effect of MSC-mt, leading to a further increase in cell survival.

[0219] Streaming results as follows Figure 6 As shown in F, at both 90 min and 180 min time points, Dyn significantly reduced the uptake of MSC-mt by primary fibroblasts.

[0220] also, Figure 6 G shows that under different conditions, mt transfer - The survival rate of the subpopulation was consistently higher than that of mttransfer. + The subgroups validated the principle that "the more you ingest, the lower your survival rate."

[0221] Validation of different oxidative stress models:

[0222] Model extension validation was performed using two classic oxidative stress inducers:

[0223] PX-12 (a thioredoxin pathway inhibitor);

[0224] Rotenone (a mitochondrial complex I inhibitor).

[0225] The effects of different stress conditions on apoptosis in L929 cells were analyzed using mt, mt(R), and mt+Dyn treatments.

[0226] The results are as follows Figure 6 H and Figure 6 As shown in Figure I, in both PX-12 and Rotenone-induced oxidative stress models, mt significantly promoted cell survival; the protection of the mt(R) group was weakened, while the survival rate of the mt+Dyn group was further improved.

[0227] mtROS detection:

[0228] mtROS accumulation in L929 was detected using MitoSOX dye under Rotenone-induced oxidative stress.

[0229] MitoSOX test results are as follows Figure 6 As shown in J, mt can significantly reduce Rotenone-induced mtROS accumulation, while mt+Dyn treatment further enhances the antioxidant effect, that is, Dyn enhances the antioxidant effect of MSC-mt.

[0230] Example 6: Effects of different dynein inhibitors on MSC-mt entry and cell protection.

[0231] L929 cells were selected and routinely cultured in low-glucose DMEM containing 5% fetal bovine serum.

[0232] MSC-derived mitochondria (MSC-mt) were extracted using a commercially available mitochondrial isolation kit, at the experimental dose (0.67 μg / cm³). 2 It was added to L929 cells and co-cultured with the cells for 24 hours.

[0233] To verify the mechanism of action of different dynein inhibitors, Dynasore and Dyngo-4a (working concentration 10 uM) were added to MSC-mt, respectively.

[0234] Therefore, the experiment was divided into six groups:

[0235] ① Normal control group (ctrl);

[0236] ②H2O2 damage group (H2O2);

[0237] ③H2O2+Dynasore group (H2O2+Dyn);

[0238] ④H2O2+Dyngo-4a group (H2O2+Dyngo-4a);

[0239] ⑤ H2O2+mt group (H2O2+mt);

[0240] ⑥H2O2+mt+Dynasore group (H2O2+mt+Dyn);

[0241] ⑦H2O2+mt+Dyngo-4a group (H2O2+mt+Dyngo-4a);

[0242] After co-culturing for 24 hours, cells from each group were collected and analyzed by flow cytometry using the Annexin V–FITC / PI double staining kit. - / PI - Annexin V is needed to ensure cell survival. + / PI - For early apoptotic cells, Annexin V + / PI + These are late-stage apoptotic cells, Annexin V- / PI + Necrotic cells.

[0243] Figure 7 A and Figure 7B showed that mt could significantly reduce H2O2-induced cell death; Dynasore and Dyngo-4a have certain protective effects on their own, and their combination with mt can further synergistically enhance the cell protection effect.

[0244] Example 7: Pharmacological or physical blocking of mitochondrial entry enhances the cytoprotective effect of MSC-mt.

[0245] L929 cells were co-cultured with MSC-mt after being stimulated with H2O2. Figure 8 As shown in Figure A, the experiment consisted of five groups:

[0246] ①H2O2 group;

[0247] ②H2O2+mt group (direct co-culture);

[0248] ③ H2O2+mt+Dyn group (pharmacologically blocked cell entry);

[0249] ④H2O2+mt physical blocking group (H2O2+(mt) group, mitochondria are placed in the upper chamber of Transwell, and cells and H2O2 are in the lower chamber);

[0250] ⑤ (H2O2+mt) group (H2O2 and MSC-mt were placed together in the upper chamber of Transwell, and the cells were in the lower chamber).

[0251] The Transwell membrane has a pore size of 400 nm, allowing H₂O₂ (molecular weight approximately 34 Da) to diffuse freely, while most mitochondria have a diameter >400 nm. Figure 2 A), therefore, this design can effectively prevent mitochondria from entering the cell.

[0252] Cell penetration ratio and fluorescence intensity:

[0253] After co-culturing for 24 hours, Annexin V / PI double staining was used to detect cell apoptosis and distinguish live cells (Annexin V). - / PI - ), early apoptosis (Annexin V) + / PI - ), late apoptosis (Annexin V) + / PI + ), Necrotic cells (Annexin V- / PI) + The proportion of mitochondria entering cells was determined. Mitotracker Deep Red was used to label mitochondria, and the proportion and fluorescence intensity of mitochondria in each group of cells were detected.

[0254] Figure 8 B and Figure 8C showed that H2O2 exposure significantly reduced cell viability, with partial recovery in the MSC-mt treatment group. In the Dynasore and Transwell groups (especially the (H2O2+mt) group), cell viability was further improved, suggesting that blocking cell entry actually enhanced the protective effect.

[0255] Figure 8 The flow cytometry results showed that the Dynasore and Transwell models effectively inhibited mitochondrial entry into cells. Specifically, Dynasore significantly reduced intracellular MitoTracker signal, while Transwell showed almost no detection signal in both groups, proving that entry into cells was completely blocked.

[0256] Figure 8 E shows mt transfer - Cell survival rate was significantly higher than that of mt transfer. + Cells, among which, in all groups, cells that did not take up mitochondria (mt transfer) - All showed higher activity and survival rates.

[0257] mtROS and H2O2 detection:

[0258] The level of mtROS in cells was detected using MitoSOX Red. To analyze the extracellular antioxidant capacity of MSC-mt cells, the H2O2 concentration was measured in the culture supernatant at 60 min.

[0259] Figure 8 F indicates that mtROS levels were further reduced after blocking cell entry. Specifically, MitoSOX staining results showed that mt significantly reduced H2O2-induced mtROS accumulation, with more significant ROS reduction in the Dynasore and Transwell groups, especially the (H2O2+mt) group.

[0260] Figure 8 G showed that MSC-mt can directly scavenge extracellular H2O2. Specifically, the H2O2 content in the culture supernatant was measured, and at 60 min, the H2O2 concentration in all MSC-mt-added groups decreased significantly, with the (H2O2+mt) group showing the best effect.

[0261] In vivo experiments:

[0262] A full-thickness skin wound model was established in mice (wound diameter approximately 15 mm). Immediately after surgery, 100 μL of PBS (ctrl group), MSC-mt (mtH group, 100 μg MSC-mt dispersed in 100 μL PBS), mtH+Dyn (10 mg / kg), or Dyn (10 mg / kg) solution were applied to the wound surface. Wound area was photographed and measured on postoperative days 2, 4, 6, and 8, and the healing rate was calculated.

[0263] Figure 9 The results showed that in PWD8, both the mtH and mtH+Dyn groups significantly promoted wound healing, with the mtH+Dyn group showing a significantly better healing rate than the mtH group; the healing rates of the Dyn group and the ctrl group were comparable, meaning that mtH and Dyn synergistically promoted wound healing.

[0264] Example 8: Cell protection function was impaired after MSC-mt was fixed by hydrogel.

[0265] Hydrogel preparation and mitochondrial immobilization and release detection:

[0266] The selected material is a clinically usable thermosensitive hydrogel (Intelligel, Horizon International (Beijing) Medical Devices Co., Ltd., Registration Certificate No. 20202140315), approved by the National Medical Products Administration. Its main components are hydroxybutyl chitosan, glycerol, and purified water. It is liquid at 4°C and rapidly solidifies into a gel at 37°C. Samples embedded with a mixture of 50% gel and MSC-mt were fixed, dehydrated, embedded, and ultra-thinly sectioned using scanning electron microscopy (SEM) to verify the integration of MSC-mt with the gel.

[0267] To evaluate the effect of different concentrations of hydrogel on MSC-mt release and diffusion, MSCs were first incubated with MitoTrackerGreen (CST, Cat#9074) at 37°C for 30 min to specifically label mitochondria, followed by thorough washing with PBS to remove free dye. Labeled MSCs were obtained through mitochondrial separation and resuspended in PBS. The MSCs were then thoroughly mixed with thermosensitive hydrogels at final concentrations of 10%, 20%, and 50% and incubated at 37°C. At different time points, 200 μL of the supernatant was collected and transferred to black opaque 96-well plates. Fluorescence intensity was measured at 485 / 528 nm using a microplate reader to evaluate the amount of mitochondria released from gels of different concentrations.

[0268] Figure 10 A shows the temperature sensitivity of the gel. The gel is a thermosensitive hydrogel, which is solid at 37 degrees Celsius and liquid at 4 degrees Celsius.

[0269] Figure 10 Scanning electron microscopy results of B showed that the MSC-mt suspended in the gel had an intact morphology.

[0270] Figure 10 C shows the effect of different gel concentrations on MT release. The results indicate that 20% and 50% gel concentrations significantly inhibited MT diffusion, while 10% gel did not significantly affect release, suggesting that there is a diffusion threshold at gel concentration.

[0271] In vivo wound model and management:

[0272] A mouse full-thickness skin wound model (wound diameter approximately 15 mm) was established. Immediately after surgery, the following four treatment solutions were applied to the wound surface: simple gel group (gel group, 100 μL gel), free MSC-mt (mtH group, 100 μg MSC-mt dispersed in 100 μL PBS), 10% gel + mtH group (10 μL hydrogel was mixed with 90 μL PBS to form a 10% (v / v) hydrogel solution, and mtH was resuspended in the mixture before application to the wound), and 50% gel + mtH group (50 μL hydrogel was mixed with 50 μL PBS to form a 50% (v / v) hydrogel solution, and mtH was resuspended in the mixture before application to the wound).

[0273] Figure 10 D、 Figure 10 E and Figure 10 F shows gross photographs of the wound, wound area, and healing rate on days 0, 2, 4, 6, and 8. In a mouse full-thickness skin wound model, different gel concentrations significantly affected the wound-healing promoting effect of MSC-MT. The results showed that the 10% gel + mtH group had the fastest wound closure speed, with the highest healing rate on postoperative day 8 (PWD 8), which was significantly higher than that of the free mtH group. In contrast, the healing rates of the gel group and the 50% gel + mtH group were similar, both significantly lower than those of the mtH group and the 10% gel + mtH group.

[0274] Figure 10 G and Figure 10 H shows representative images and statistical results of laser speckle blood flow imaging on postoperative day 7 to assess local wound perfusion. On postoperative day 7, wound perfusion was similar in the 10% gel + mtH group and the mtH group, while blood flow was significantly reduced in the 50% gel + mtH group. Figure 10 G, 10H).

[0275] In vitro ROS scavenging and cell protection experiments:

[0276] To further investigate the effects of thermosensitive hydrogel immobilization on the extracellular antioxidant function and cell protection of MSC-mt cells, the following design was performed:

[0277] ① Ctrl group (control group without any stimulation);

[0278] ②H2O2 group (oxidative stress only);

[0279] ③H2O2+mt group (free MSC-mt cells directly co-cultured with cells);

[0280] ④H2O2+(mt) group (MSC-mt is placed in the upper chamber of Transwell, and H2O2 and cells are placed in the lower chamber, allowing ROS molecules to diffuse but preventing mitochondrial entry into cells).

[0281] ⑤ H2O2+(50% gel+mt) group (MSC-mt was resuspended in a 50% (v / v) hydrogel-PBS mixture and co-cultured with cells);

[0282] L929 cells were subjected to oxidative stress induced by 400 μM H2O2. Before co-culturing with cells, MSC-mt were incubated with MitoTracker Deep Red (Thermo Fisher, Cat#A66440) at 37°C for 30 min for mitochondrial-specific fluorescent labeling. The MSCs were then thoroughly washed with PBS to remove free dye before extraction to obtain fluorescently labeled MSC-mt. The labeled MSC-mt were added to the cell culture system according to the above-described groupings and co-cultured with cells. The culture supernatant was collected at 60 min, and the H2O2 concentration in the supernatant was detected using a commercial H2O2 detection kit (Beyotime, Cat#S0038) to quantitatively evaluate the extracellular ROS scavenging ability of MSC-mt under different conditions. Simultaneously, MitoTracker Deep Red signal was detected by flow cytometry to assess mitochondrial ingress in each group. The total ROS level in each group was detected using the DCFH-DA fluorescent probe.

[0283] The results are as follows Figure 11 A- Figure 11 As shown in C. In in vitro experiments, 50% gel-fixed MSC-mt significantly reduced H2O2 scavenging ability, and its supernatant residual H2O2 level was comparable to that of the Transwell isolation group. Figure 11 A). Flow cytometry analysis showed that the intracellular MSC-MT fluorescence signal was significantly reduced in the 50% gel+mt group ( Figure 11 B). DCFH-DA assays showed that free MSC-mt significantly reduced H2O2-induced intracellular ROS accumulation, while 50% gel+mt weakened the antioxidant effect of MSC-mt. Figure 11 C). In other words, gel restriction weakens the clearance of extracellular ROS and cell protection.

[0284] discuss

[0285] In this invention, under oxidative stress, exogenous mitochondria exhibit decreased cytoprotective capacity after entering recipient cells, while maintaining them in an extracellular state is more conducive to ROS clearance. This phenomenon may be related to the following mechanisms: when mitochondria are in the extracellular environment, their exposed respiratory chain complexes and antioxidant enzymes can directly and continuously contact surrounding ROS, acting like "free-diffusion antioxidant sponges," rapidly absorbing and neutralizing excess H2O2 and related free radicals from the environment; however, once mitochondria are endocytosed into cells, their accessible ROS sources are limited to the microenvironment surrounding endocytic vesicles or the cytoplasm, preventing access to a large amount of external ROS, resulting in a decrease in overall net clearance efficiency. Furthermore, the endocytosis process itself may activate the lysosomal pathway or induce mild stress, causing additional damage to recipient cells. Therefore, according to the conclusions of this invention, maintaining exogenous mitochondria in the wound fluid microenvironment, rather than allowing them to enter cells, is key to enhancing their antioxidant capacity and wound repair effects.

[0286] The full-thickness skin defect model used in this invention (approximately 15 mm in diameter) significantly exceeds the typical experimental wound area, representing a highly complex and extensive tissue defect characterized by significant inflammation, oxidative stress, tissue necrosis, and blood supply disruption. Its physiological features are highly similar to large-area wounds following clinical burns / scalds. Therefore, the accelerated healing effect achieved by this invention has high clinical relevance and better reflects the feasibility and advantages of exogenous mitochondrial therapy strategies in the context of severe skin injury.

[0287] This invention reveals that the concentration of hydrogel directly affects the distribution and function of mitochondria in wounds. At high gel concentrations (e.g., 20%–50%), mitochondria are confined within the gel and cannot fully diffuse into the wound margin tissue, significantly reducing their contact with external ROS and thus diminishing their antioxidant activity. At low gel concentrations (e.g., 10%), the gel provides a biocompatible adhesion environment while maintaining necessary fluidity, allowing mitochondria to be gradually released and diffused in the wound's fluid environment, still effectively scavenging extracellular ROS. Therefore, moderate fluidity and diffusivity (rather than fixing mitochondria) are key design principles for maintaining the efficacy of topical mitochondrial formulations.

[0288] The experimental results of this invention further demonstrate that intact and bioactive mitochondria have irreplaceable advantages in the skin damage repair process, while the application of mitochondrial components or functionally impaired mitochondrial materials cannot achieve the same effect. Intact mitochondria are a dynamic extracellular antioxidant system, relying on their complete electron transport chain, membrane potential (ΔΨm), and peroxidase system to continuously scavenge reactive oxygen species (ROS). In this invention, when mitochondria were pretreated with Rotenone, the expression of the mitochondrial outer membrane protein Tom20 and the OXPHOS complex was reduced, and their abilities to inhibit apoptosis, reduce intracellular mtROS, increase mitochondrial membrane potential, inhibit cell senescence, and promote cell proliferation were significantly weakened. This phenomenon indicates that damaged or inactivated mitochondria do not possess the functional antioxidant characteristics of intact mitochondria.

[0289] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The uses of mitochondria, characterized in that, Used to prepare a composition or formulation for repairing skin damage.

2. The use as described in claim 1, characterized in that, It is also used for one or more sub-uses selected from the following group: (Z1) Improves blood perfusion; (Z2) enhances tissue metabolism; (Z3) alleviates oxidative stress; (Z4) improves collagen deposition; (Z5) reduces inflammatory infiltration; (Z6) reduces fibroblast apoptosis; (Z7) reduces oxidative damage to fibroblasts.

3. A composition or formulation, characterized in that, The composition or preparation comprises the following substances: (i) mitochondria; and (ii) Pharmaceutically acceptable carriers.

4. A drug combination, characterized in that, Include: mitochondria; and Mitochondrial entry blockers.

5. The pharmaceutical combination as described in claim 4, characterized in that, The mitochondrial entry blockers include: Dynasore, Dyngo-4a, or combinations thereof.

6. The use of the pharmaceutical combination according to claim 4, characterized in that, This is used to prepare a medicine box for repairing skin damage.

7. The use of mitochondrial entry blockers, characterized in that, This is used to prepare a formulation or material for enhancing the ability of mitochondria to repair skin damage.

8. A medicine box, characterized in that, The medicine box contains: (I) Mitochondria; (II) Mitochondrial entry blockers.

9. The medicine box as described in claim 8, characterized in that, The mitochondrial entry blocker includes: physical barrier materials or chemical reagents.

10. The medicine box as described in claim 8, characterized in that, The chemical reagents include: Dynasore, Dyngo-4a, or combinations thereof.

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