Genetically engineered oriented electrostatic spinning fiber as well as preparation method and application thereof

Abstract

The invention provides a genetically engineered oriented electrostatic spinning fiber as well as a preparation method and application thereof, and belongs to the technical field of biomedical materials. Gene TSPAN9 is packaged in liposome through a micro-fluidic chip technology, then oriented electrostatic spinning fibers of a shell-core structure are prepared through an oriented micro-sol electrostatic spinning technology, and the liposome containing the TSPAN9 is protected on a core layer by hyaluronic acid; and the polylactic acid fibers are arranged in an oriented manner to form a shell layer. An in-vitro experiment shows that the oriented fiber closely simulates a fascia oriented arrangement structure, and the cell migration rate is remarkably improved through contact guidance; by slowly releasing lipidosome loaded with genes into cells and maintaining the mitochondrial steady state, mitochondrial respiration is effectively recovered, the active oxygen level is reduced, and the mitochondrial membrane potential function is maintained. In-vivo experiments show that the genetically engineered fiber can effectively inhibit inflammatory response and promote fascia tissue regeneration by promoting mitochondrial exocytosis to discharge damaged mitochondria.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to a genetically engineered oriented electrospun fiber and its preparation method and application. Background Art

[0002] Mitochondria play a key role in a variety of cellular processes, including generating adenosine triphosphate (ATP) for energy supply, regulating catabolic and anabolic processes, and maintaining cellular calcium and redox balance. At the same time, they are also the coordination and regulation center of the signal cascade of cell survival and death pathways. Although mitochondria are crucial for cell survival, they are extremely vulnerable to damage or destruction. Damaged mitochondria not only impair cell function, induce mitochondrial-related inflammation, but even threaten human life.

[0003] Mitochondria have high dynamicity, and a variety of mitochondrial quality control mechanisms maintain the balance of their functions. Damaged mitochondria can self-repair by fusing and dividing with healthy mitochondria, or be cleared through mitophagy. However, when the damage is severe or persistent, the self-repair mechanism of mitochondria is insufficient to restore their own functions, and external intervention means are needed to achieve this.

[0004] Currently, the intervention measures for protecting damaged mitochondria mainly include mitochondrial protectants such as coenzyme Q10, mitochondrial permeability transition pore inhibitors such as Sirtuin-1, and iron chelators such as deferoxamine, etc. However, these drugs have limitations such as instability, low bioavailability, and inaccurate drug distribution. For example, α-lipoic acid is a common mitochondrial protectant. Due to liver degradation, low solubility, and gastric instability, its half-life is short and its bioavailability is only 30%, which limits its clinical application. In addition, these intervention measures can only partially protect or maintain the structure and function of damaged mitochondria and cannot fundamentally solve the problem. Therefore, promoting the clearance of damaged mitochondria from cells may be another effective treatment strategy.

[0005] Damaged mitochondria can be expelled through migrasome-mediated mitocytosis to maintain the stability of the cellular mitochondrial pool and protect cells from mitochondrial membrane potential (MMP) fluctuations and respiratory dysfunction. Migrasomes are extracellular vesicles with a diameter of 0.5 to 3 μm that mediate the release of cellular contents and are involved in various biological processes, including intercellular communication, maintaining intracellular homeostasis, embryonic development, tissue repair, and immune responses. The adhesion of damaged mitochondria to intracellular dynein decreases, and they accumulate in domains rich in tetraspanin (TSPAN) on the periphery, and then form migrasomes to be expelled from the cell. TSPAN members play a key role in regulating migrasome formation, especially TSPAN9. Studies have shown that overexpression of TSPAN9 induces migrasome formation and promotes the clearance of damaged mitochondria. Therefore, introducing the TSPAN9 gene into cells may help expel damaged mitochondria and regulate cellular mitochondrial quality.

[0006] Tissue injury is a complex process closely related to mitochondria. After tissue injury, an inflammatory response is rapidly initiated. Inflammation is a complex biological response that is crucial for tissue repair after injury. Over-activated inflammation can impede tissue regeneration. It is currently believed that tissue injury, as a mitochondrial stressor, leads to mitochondrial oxidative stress and subsequent inflammation-related damage. Damaged mitochondria are activators of inflammation, which can stimulate the expression of inflammatory genes, activate the nuclear factor κB (NF-κB) signaling pathway, and trigger the NLRP3 inflammasome. Damage-associated molecular patterns (DAMPs) from mitochondria, including mitochondrial DNA (mtDNA) and mitochondrial RNA (mtRNA), can activate the cGAS-STING1 signaling pathway, leading to the synthesis of cytokines such as tumor necrosis factor (TNF) and interleukin-6 (IL-6). In addition, mitochondria also play a role in controlling the development, activation, differentiation, and survival of various immune cell types, including T lymphocytes. Due to the imbalance of the mitochondrial quality control mechanism in damaged tissues, damaged mitochondria accumulate in damaged tissues, continuously exacerbating inflammation-related damage. In this case, they produce excessive reactive oxygen species (ROS) and other inflammatory stimulatory factors, further aggravating tissue injury and hindering regeneration.

[0007] The rapid development of nanotechnology has provided new biomaterial science strategies for the treatment of diseases targeting mitochondria. Recently, researchers designed a new self-assembled nanodrug, pGBEMA22-b-pSSPPT9, whose main active ingredient is etoposide. This nanodrug actively interferes with mitochondria and promotes mitochondrial exocytosis. Mitochondrial exocytotic vesicles containing etoposide are then re-internalized by cells, delivering etoposide to neighboring cells, promoting deep drug penetration inside the tumor, and ultimately damaging tumor cells. However, in the field of tissue regeneration, current research focuses on the use of liposomes, microspheres, and hydrogels to deliver mitochondrial protective drugs to improve mitochondrial function and promote tissue regeneration. However, in the case of severe mitochondrial damage, these biomaterials are unable to expel damaged mitochondria and can only partially restore the function of mildly damaged mitochondria.

[0008] In addition, these biomaterial forms lack the necessary mechanical strength for deep tissue injuries (such as fascia injuries). In recent years, electrospinning technology has developed rapidly and has shown superior application potential in fascia regeneration compared to other nanotechnologies. This technology can prepare nanofibrous scaffolds with high specific surface area, high drug loading and simulated extracellular matrix (ECM) structure. These scaffolds have good mechanical properties and can mimic natural fascia structure. In addition, electrospinning is customizable, allowing optimization through surface modification, internal drug loading and surface morphology changes. However, this fiber membrane cannot achieve mitochondrial quality control.

[0009] It can be seen that most of the current materials and drugs are mainly aimed at reducing mitochondrial oxidative stress to achieve the recovery of mitochondrial function. This method has problems such as instability, low bioavailability, and inaccurate drug distribution. These intervention measures can only partially protect or maintain the structure and function of damaged mitochondria, and cannot fundamentally solve the problem for severely damaged mitochondria. Its efficacy in repairing damaged fascia is still insufficient.

[0010] Therefore, how to provide a drug delivery carrier with mitochondrial exocytosis function in order to promote the removal of damaged mitochondria from cells, thereby better facilitating the repair and regeneration of severely damaged mitochondria and other fascia tissues, has become a technical problem that needs to be solved urgently. Summary of the invention

[0011] The present invention is to solve the above technical problems, thereby providing a genetically engineered oriented electrospun fiber and its preparation method and application. The technical purpose of the present invention is to solve the problem that the existing mitochondrial drugs are mainly used to reduce the oxidative stress of mitochondria, and can only repair slightly damaged mitochondria, but cannot be used for fundamental treatment of severely damaged mitochondria; and the current drugs have poor efficacy in repairing damaged fascia tissue, and cannot achieve efficient repair effects on severely damaged fascia tissue.

[0012] To achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

[0013] The present invention first provides a method for preparing genetically engineered oriented electrospun fibers, comprising the following steps:

[0014] (1) Prepare a liposome solution using lecithin, cholesterol, and octadecylamine as lipid raw materials, then mix the liposome solution with the gene, and obtain a liposome solution loaded with the gene through a microfluidic chip;

[0015] (2) Dissolve the liposome solution obtained in step (1) in a hyaluronic acid solution, then mix it with a polylactic acid solution to form an electrospinning solution, and prepare the genetically engineered oriented electrospun fibers through electrospinning.

[0016] Furthermore, in step (1), the weight ratio of the lecithin, cholesterol, and octadecylamine is ().

[0017] Furthermore, the gene in step (1) is TSPAN9.

[0018] Furthermore, the concentration of the gene in step (1) is (78.1:16.7:2).

[0019] Furthermore, in step (1), the ratio of the flow rate of the central channel to the flow rate of the side channel of the microfluidic chip is 1:10.

[0020] Furthermore, in step (2), the concentration of the hyaluronic acid is 1% (w / v), the concentration of the polylactic acid solution is 10% (w / v), and the liposome contains 120 μg of the gene.

[0021] Furthermore, the process parameters of the electrospinning in step (2) are: the distance between the collector and the needle is 15 cm, the applied voltage is 15 kV, the flow rate is 5 mL / h, and the rotation speed of the collector is 4000 rpm.

[0022] The second object of the present invention is to provide the genetically engineered oriented electrospun fibers prepared by the above method.

[0023] The third object of the present invention is to provide the application of the genetically engineered oriented electrospun fibers as described above in the preparation of drugs for treating mitochondrial damage repair.

[0024] The fourth object of the present invention is to provide the application of the genetically engineered oriented electrospun fibers as described above in the preparation of drugs for fascia repair and regeneration.

[0025] The present invention uses emulsion electrospinning technology to prepare a core-shell structure gene-electrospun membrane for fascia regeneration. The core layer contains hyaluronic acid (HA) and TSPAN9-plasmid liposomes, which can be stably transduced into the target tissue. The outer shell layer is composed of orderly arranged poly(lactic acid) (PLA) fibers, which can promote the orderly arrangement and migration of cells. In vitro experiments verified the ability of this novel fiber membrane to mediate cell adhesion, proliferation and migration. In vivo experiments confirmed its excellent ability to maintain mitochondrial homeostasis, and the ability of this genetically engineered oriented fiber membrane to inhibit early inflammation at the injury site through mitochondrial exocytosis, and evaluated its potential to promote tissue regeneration according to late tissue remodeling. Therefore, the gene-transduced electrospun membrane provided by the present invention can promote the exocytosis of damaged mitochondria, thereby alleviating inflammation after implantation and promoting tissue regeneration. It shows great promise in clinical applications and provides a new treatment strategy for fascia regeneration.

[0026] Many difficulties were faced during the preparation of the materials of the present invention. First of all, in order to more fully carry the gene liposomes, the inventors extensively explored the amount of gene carried by the liposomes. After a large number of experiments, the appropriate amount was finally determined. Secondly, the inventors also explored the amount of hyaluronic acid (HA) carried by electrospinning under the system of the present invention. It was found that if the amount of HA was excessive, rough characteristics such as droplets would appear on the surface of the electrospun membrane. Thirdly, the inventors also explored the effects of different voltages and rotation speeds on the formed electrospun membrane. It was found that if the voltage and rotation speed were too low, the orientation of the formed fiber membrane would be very poor. The method of the present invention successfully constructed a genetically engineered electrospun fiber with good orientation, which can be well used for the repair of fascia tissue.

[0027] Another difficulty in the construction of the fiber material of the present invention is that the effects of the present invention cannot be achieved by ordinary liposome construction methods. Existing ordinary liposome preparation methods such as the thin film dispersion method, most of the electrospinning carried materials load liposomes on the surface of the scaffold by surface adsorption, which results in poor drug stability, low drug utilization rate and poor curative effect. The electrospinning material of the present invention is prepared by the method of micro-sol electrospinning, which can load liposomes inside the scaffold, thereby greatly improving the drug stability. For severely damaged mitochondria, the efficient repair and regeneration of damaged fascia tissue can be well achieved through the mitochondrial exocytosis function.

[0028] The beneficial effects of the present invention are as follows:

[0029] The abnormal accumulation of damaged mitochondria severely hinders the tissue repair process, and traditional treatment methods represented by drugs are difficult to excrete damaged mitochondria through the mechanism of mitochondrial exocytosis. The present invention combines microfluidic chips and micro-sol electrospinning technology to develop genetically engineered aligned electrospun fibers, and for the first time promotes mitochondrial exocytosis to repair damaged fascia by upregulating tetraspanin 9 (TSPAN9) in the periphery. First, the key gene TSPAN9 for mitochondrial exocytosis is encapsulated in liposomes by microfluidic chip technology. Subsequently, aligned electrospun fibers with a core-shell structure are prepared by aligned micro-sol electrospinning technology, in which liposomes containing TSPAN9 are protected in the core layer by hyaluronic acid (HA); while the polylactic acid (PLA) fibers are aligned to form the outer shell layer. In vitro experiments show that: the aligned fibers closely mimic the structure of fascia alignment, and significantly improve the cell migration rate through contact guidance; by slowly releasing the liposomes loaded with genes into the cells, by maintaining mitochondrial homeostasis, effectively restore mitochondrial respiration, reduce the level of reactive oxygen species, and maintain the function of mitochondrial membrane potential. In vivo experiments show that: the genetically engineered fibers can excrete damaged mitochondria by promoting mitochondrial exocytosis, effectively inhibit the inflammatory response and promote the regeneration of fascia tissue. Description of the Drawings

[0030] Figure 1 Liposomes containing plasmids can protect cells from oxidative stress and maintain mitochondrial quality; A) Using 3% agarose gel electrophoresis to characterize liposomes containing plasmid DNA; the 1st and 8th lanes are markers, and the 2nd to 6th lanes represent different concentrations of plasmid contents in the liposome system, with concentrations of 10, 5, 3, 2, 1, 0 μg / μL respectively; B) Particle size distribution and Zeta potential of liposomes loaded with different proportions of plasmid DNA; C) TEM image of DNA@liposomes; D) Flow cytometry results after treating C2C12 cells with liposomes containing different concentrations of plasmid content; E-F) Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) results after treating C2C12 cells with liposomes containing different concentrations of DNA; G) Parameter analysis based on OCR results, including basal respiration, maximum respiratory capacity and spare respiratory capacity (n = 3, *p < 0.05, **p < 0.01, ****p < 0.0001). Data are expressed as mean ± standard deviation (mean ± SD), and one-way ANOVA is used to determine statistical differences.

[0031] Figure 2Construction and characterization of electrospun fibers; A) After adding the DNA@HA solution and stirring at high speed, the transparent PLA solution was transformed into an emulsion-like DNA@HA solution; B) Water contact angles of PLA electrospun membranes with oriented core-shell structures, membranes with different DNA@HA contents; C) Atomic force microscopy (AFM) images of oriented fibers; D) Scanning electron microscopy (SEM) images and their color mapping images of R-PLA, A-PLA, A-PLA-HA, and DNA@A-PLA-HA fiber membranes, and the angular frequency distribution of orientation of the four fiber membranes based on the SEM images; E) Transmission electron microscopy (TEM) results of the core-shell structures of R-PLA, A-PLA, A-PLA-HA, and DNA@A-PLA-HA fiber membranes; F) Tensile tests and Young's moduli of electrospun membranes (n = 3); H) Quantitative analysis of the fiber diameters of electrospun membranes based on TEM images (n = 3). (*P<0.05; **P<0.01; ***P<0.001).

[0032] Figure 3 The DNA@A-PLA-HA membrane has good biocompatibility and the ability to promote cell migration; A) Live / dead cell staining was performed on cells cultured on the membrane; The cells were stained with CalceinAM (showing green fluorescence) and PI (showing red fluorescence) to distinguish live cells and dead cells; B) Cytoskeleton staining of cells on the fiber membrane; C) Schematic diagram of the cell migration experiment on the electrospun membrane; D) Observation of the cell migration experiment on the electrospun membrane using an optical microscope; E) Representative confocal microscope images of the cell migration experiment on the electrospun membrane; F) CCK-8 detection results of cells co-cultured with R-PLA, A-PLA, and DNA@A-PLA-HA (ns: no significant difference); G) Quantitative analysis of the wound healing rate in the cell migration experiment (**p<0.01, ***p<0.001, ****p<0.0001).

[0033] Figure 4The DNA@A-PLA-HA membrane can protect cells from oxidative stress and maintain mitochondrial mass; A) Schematic diagram of cell staining cultured on the fibrous membrane to show mitochondrial cell division; TMRM (red) indicates mitochondrial membrane potential; MitoTracker (green) indicates the location of mitochondria; DAPI (blue) marks the location of the nucleus; WGA (cyan) marks the location of the cell membrane; B) Expression levels of TSPAN9 mRNA in cells cultured on different fibrous membranes (n = 3, ns: not significant, ***p < 0.001, ****p < 0.0001); C) Representative fluorescence images of ROS in L929 cells cultured on different membranes after induction with 100 μM H2O2; D) Semi-quantitative analysis of ROS staining results to evaluate the antioxidant stress ability of different membranes (n = 3, ns: not significant, *p < 0.05, **p < 0.01); Data are presented as mean ± standard deviation (mean ± SD), and one-way ANOVA is used to determine statistical differences; E) Representative fluorescence images of mitochondrial membrane depolarization analyzed by TMRM staining in L929 cells cultured on different membranes after being interfered with H2O2 (100 μMol); F) Semi-quantitative analysis of TMRM staining results to evaluate the ability of different membranes to maintain mitochondrial mass (n = 3, ns: not significant, *p < 0.05); G) Representative transmission electron microscopy (TEM) images of intracellular mitochondria on different fibrous membranes. Data are presented as mean ± standard deviation (mean ± SD), and one-way ANOVA is used to determine statistical differences.

[0034] Figure 5 The DNA@A-PLA-HA membrane promotes in vivo regeneration; A) Flow chart of the in vivo experimental process for inducing an abdominal wall hernia model in female SD rats; B) Staining results of the abdominal wall defect area in rats treated by HE staining, using R-PLA and DNA@A-PLA-HA fibrous membranes respectively, untreated (sham operation) and non-modeled as controls (control group); C) Quantitative results of exogenous macrophage counts; (n = 3, ***p < 0.001, ****p < 0.0001); D) Masson staining results of the four groups; E) Quantitative results of relative collagen density; (n = 3, **p < 0.01, ***p < 0.001, ****p < 0.0001); Data are presented as mean ± standard deviation (mean ± SD), and one-way ANOVA is used to determine statistical differences.

[0035] Figure 6The DNA@A-PLA-HA membrane promotes neovascularization and extracellular matrix remodeling in vivo. A) Representative immunofluorescence (IF) images of α-smooth muscle actin (α-SMA). B) Semi-quantitative statistical analysis of α-SMA immunofluorescence staining (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). C) Representative immunofluorescence (IF) images of platelet endothelial cell adhesion molecule-1 (CD31). D) Semi-quantitative statistical analysis of CD31 immunofluorescence staining (n = 3, *p < 0.05, **p < 0.01). E) Representative immunohistochemistry (IHC) images of type I collagen (COL1). F) Statistical analysis of COL1 immunohistochemical scores (n = 3, **p < 0.01, ***p < 0.001, ****p < 0.0001). G) Representative immunohistochemistry (IHC) images of type III collagen (COL3). H) Statistical analysis of COL3 immunohistochemical scores (n = 3, ns: no significant difference, *p < 0.05, **p < 0.01). Data are presented as mean ± standard deviation (mean ± SD), and one-way ANOVA was used to determine statistical differences.

[0036] Figure 7 The DNA@A-PLA-HA membrane inhibits inflammation in vivo; A) Immunofluorescence staining of ROS (red) and DAPI (blue) on day 7; B) Semi-quantitative analysis of the optical density of ROS on day 7; (n = 3, ns = no significant difference, **p < 0.01, ***p < 0.001, ****p < 0.0001); C) Images of immunofluorescence staining of ROS (red) and DAPI (blue) on day 28 after surgery; D) Semi-quantitative analysis of the optical density of ROS on day 28 after surgery; (n = 3, *p < 0.05, **p < 0.01, ****p < 0.0001); E) Representative immunohistochemistry (IHC) images of interleukin-6 (IL-6); F) Statistical analysis of IL-6 immunohistochemical scores (n = 3, *p < 0.05, **p < 0.01); G) Representative immunohistochemistry (IHC) images of tumor necrosis factor-α (TNF-α); H) Statistical analysis of TNF-α immunohistochemical scores (n = 3, **p < 0.01). Data are presented as mean ± standard deviation (mean ± SD), and one-way ANOVA was used to determine statistical differences.

[0037] Figure 8 Photo of the aligned fiber electrospun membrane.

[0038] Figure 9Parameter analysis based on ECAR results, including glycolytic capacity and glycolytic reserve (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001); data are presented as mean ± standard deviation (SD), and one-way ANOVA was used for statistical difference analysis.

[0039] Figure 10 It is a schematic diagram of a gene electrospun nanofiber membrane, which promotes the exocytosis of damaged mitochondria through gene transduction, thereby promoting fascia regeneration. Detailed implementation manners

[0040] In order to make the objectives, technical solutions and advantages of the present invention more clear and understandable, the following describes the present invention in detail with reference to the embodiments. It should be noted that the following embodiments are only used to explain and illustrate the present invention, and are not used to limit the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above inventive content still fall within the protection scope of the present invention.

[0041] Example 1

[0042] I. Experimental methods and steps

[0043] 1. Preparation of liposomes

[0044] First, 78.1 mg of lecithin, 16.7 mg of cholesterol and 2 mg of octadecylamine were dissolved in 3 mL of absolute ethanol to prepare a liposome solution. Then, 1 mL syringes were used to aspirate the liposome solution and the plasmid solution respectively. In order to determine the optimal content of the plasmid in this system, plasmid solutions with different concentration gradients were used in the experiment. The microfluidic chip was set up by connecting the liposome solution to the central microchannel and the plasmid solution to the side microchannel. The syringes were respectively installed on two microinjection pumps, and the flow rate of the central channel was adjusted to 1 mL / h, and the flow rate of the side channel was adjusted to 10 mL / h. The liposomes during the process were collected through the outlet of the microfluidic chip. In order to obtain uniformly dispersed liposomes, the collected liposomes were treated by ultrasonic cell disruption at 10% power, with an interval of 1 second running and 1 second stopping, and the total treatment time was 5 minutes. Finally, the obtained product was filtered through a 0.22 μm filter membrane to obtain a uniform liposome solution.

[0045] 2. Characterization of liposomes

[0046] The binding ability of liposomes and plasmid DNA was evaluated by agarose gel electrophoresis (Beyotime Biotechnology, China). Samples were electrophoresed on a 3% (w / v) agarose gel at a voltage of 70 V for 30 minutes. The electrophoresis results were analyzed by capturing the red-stained bands in the gel using a UVIPro Gel Documentation System (Tanon 2500, China) imaging system. The particle size and Zeta potential of plasmid-loaded liposomes were measured by dynamic light scattering on a Malvern Zetasizer Nano ZS90 (Malvern, UK) instrument at a measurement temperature of 25 °C.

[0047] 3. Preparation of R-PLA and R-PLA fiber membranes

[0048] A 10% (w / v) polylactic acid (PLA) solution was prepared by dissolving 1 g of PLA in a mixture of 8 g of dichloromethane (DCM) and 2 g of N,N-dimethylformamide (DMF) and stirring overnight at room temperature. Subsequently, the electrospinning solution was loaded into a 10 mL syringe and equipped with a stainless-steel needle. The distance between the needle and the collector was set at 15 cm, the applied voltage was 15 kV, and the flow rate was 5 mL / h. The rotation speed of the collector for randomly electrospun PLA membranes was set at 400 rpm, while for aligned electrospun PLA membranes it was set at a higher 4000 rpm. After electrospinning, the prepared membranes were volatilized of residual organic solvents in a ventilated place and stored at 4 °C.

[0049] 4. Preparation of A-PLA-HA and DNA@A-PLA-HA fiber membranes

[0050] First, 0.020 g of Span-80 was dissolved in 8 g of DCM. The prepared liposomes were dispersed in a 1% hyaluronic acid (HA) solution to prepare a 1% DNA@HA solution. Different volumes of the 1% HA solution or 1% DNA@HA solution were added dropwise to DCM using a micro syringe and sonicated to obtain a uniformly dispersed solution. Then, 1 g of PLA was dissolved in this DCM mixed solution and stirred until completely dissolved under magnetic stirring. Then 2 g of DMF was added and stirred evenly to obtain the electrospinning solution. The prepared solution was loaded into a 10 mL syringe and equipped with a stainless-steel needle. The electrospinning process parameters were as follows: the distance between the collector and the needle was 15 cm, the applied voltage was 15 kV, the flow rate was 5 mL / h, and the rotation speed of the collector was 4000 rpm. The prepared membranes were dried in a ventilated place to volatilize the organic solvents and stored at 4 °C.

[0051] 5. Characterization of fiber membranes

[0052] The hydrophilicity of electrospun membranes with different HA contents was evaluated by measuring the static water contact angle (WCA, ZT-705SB). The physical morphology of the fiber membrane surface after preparation was observed by scanning electron microscopy (SEM, Hitachi S4800, Japan), and the samples were treated with gold coating. The SEM images were analyzed using the Orientation J plugin of Image J software. The surface morphology of the fiber membrane was observed by atomic force microscopy (AFM, Bruker Dimension ICON). Single electrospun fibers were collected on copper grids, and their core-shell structure and fiber diameter were observed by transmission electron microscopy (TEM, Talos L120CG2). The mechanical properties were tested using a universal material testing machine (equipped with a 500 g model 31 sensor, Honeywell) at a tensile rate of 5 mm / min. Before testing, the fiber membranes were cut into dumbbell-shaped samples, and each test was performed three times. The stress-strain curves of the fiber membranes were plotted using Origin software.

[0053] 6. Cell culture

[0054] In vitro experiments were performed using the mouse fibroblast cell line L929 and the mouse skeletal muscle cell line C2C12. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin (PS, Gibco, USA). Cells were cultured in an incubator at 37 °C with 5% CO2.

[0055] 7. Flow cytometry and Seahorse metabolic analysis

[0056] An oxidative stress model of C2C12 cells was established by adding 100 μM H2O2. Then, liposomes containing the gene were added to make the gene concentration in the medium reach 0.5 μg / mL, 1 μg / mL, and 2 μg / mL. After 48 hours of treatment, the ROS level was stained with DCFH-DA (Beyotime, China), and the fluorescence intensity was measured by flow cytometry (BD FACSVerse, USA). In addition, the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the Seahorse XF Cell Mito Stress Test Kit and Seahorse XF-24 Flux Analyzer (Agilent Technologies). Briefly, oligomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), and rotenone were added sequentially to detect OCR, while glucose, oligomycin, and 2-deoxyglucose (2-DG) were added sequentially to measure ECAR. The OCR and ECAR values were calculated after normalization. Each group contained n = 3, and the results were expressed as mean ± standard deviation (mean ± SD).

[0057] 8. Live / Dead Staining of Cells on the Fiber Membrane

[0058] After sterilizing each group of electrospun fiber membranes, place them at the bottom of a 6-well plate and press an appropriate sterilized steel ring (Tako, China) on top to fix the fiber membranes. Inoculate L929 fibroblasts onto the fiber membranes. When the cells grow to nearly 90%, perform staining according to the operating steps of the Live / Dead staining kit. In addition, after culturing C2C12 cells on the fiber membranes, use phalloidin (Bestbio, China) and DAPI for cytoskeleton staining. The stained cells are observed under a confocal microscope and representative images are taken. To evaluate cell proliferation and cytotoxicity, inoculate L929 cells (1×104 cells / well) into a 48-well plate and fix the electrospun fiber membrane at the bottom of the well. The blank well serves as the control group. Set three replicate wells for each group. On the 1st, 3rd, and 5th days after inoculation, remove the culture medium, wash the wells with 1xPBS, and then add the CCK-8 working solution for a 1-hour incubation. Then, collect 100 μL of the supernatant from each well, transfer it to a 96-well plate, and measure the absorbance using a microplate reader.

[0059] 9. Scratch Assay on the Fiber Membrane

[0060] After sterilizing the electrospun membrane, place it at the bottom of a 6-well plate and add a sterilized steel ring (Tako, China) on top. Then inoculate L929 fibroblasts onto the membrane. When the cell confluence is approximately 90%, gently remove the steel ring, wash away the detached cells and debris, and rinse with 1x PBS. Subsequently, add fresh serum-containing medium and place the culture plate in the cell culture incubator. Observe the cell migration at different time points using an optical microscope and a confocal microscope. Under the confocal microscope, use Phalloidin (red) to label the cytoskeletal protein and DAPI (blue) to label the cell nucleus for observation.

[0061] 10. Staining of Mitochondrial Exocytosis in Cells on the Fiber Membrane

[0062] After sterilizing each group of electrospun fiber membranes, place them at the bottom of the well plate and press an appropriate sterilized steel ring (Tako, China) on top to fix the fiber membranes. Then inoculate a small amount of L929 fibroblasts. After culturing for 48 hours, perform immunofluorescence staining. Prepare working solutions of corresponding concentrations using dyes such as TMRM, MitoTracker, WGA, and DAPI for staining. Finally, wash with 1x PBS multiple times to remove the excess dyes. Observe the staining results using a confocal microscope.

[0063] 11. qPCR Experiment of Cells on the Fiber Membrane

[0064] Place the fiber membrane at the bottom of a 6-cm dish, seed L929 cells at an appropriate concentration on it, and after culturing for 48 hours, the cells reach about 90% confluence at the bottom of the well plate. Collect the cells cultured on the fiber membranes of each group. Digest and lyse the cells using TRIzol reagent to extract total RNA. Determine the RNA concentration through the Nanodrop system, and then reverse transcribe the RNA into cDNA. Subsequently, perform real-time quantitative PCR to detect the transcriptional levels of related genes. The specific genes and primer sequences are shown in Table 1.

[0065] Table 1 Primer sequences for RT-PCR amplification

[0066]

[0067] 12. Observation of mitochondrial-related indicators on the fiber membrane

[0068] Cut the sterilized random fiber membranes, oriented fiber membranes, and fiber membranes with DNA content into small pieces, place them at the bottom of a 6-well plate, and flatten them with a customized iron ring (Tako, China). Inoculate fibroblasts and treat them with 100 μM H2O2 to establish an oxidative stress model. To detect the ROS level, all cells are stained using a ROS kit (Bestbio, China), and after washing three times, they are observed using a confocal microscope. To measure the mitochondrial membrane potential, the cells are incubated with a TMRM membrane potential kit (Bestbio, China) for 15 minutes and observed under the same microscope. In addition, after establishing the H2O2-induced oxidative stress model, the cells cultured on the fiber membrane for 48 hours are collected, digested with trypsin, and fixed with glutaraldehyde fixative. Subsequently, the morphology of intracellular mitochondria is observed under a transmission electron microscope (TEM).

[0069] 13. In vivo animal model study

[0070] In this study, a rat ventral hernia model was established for in vivo experiments. The animal experiments were approved by the Animal Research Committee of Shanghai Jiao Tong University School of Medicine (A-2020036). Forty-eight female Sprague-Dawley rats, with an average body weight of 200–250 g, were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). The rats were randomly divided into four groups (n = 12 per group), and the animals were sacrificed at 7 days and 28 days after surgery. The rats were anesthetized by intraperitoneal injection of 3 mg / mL sodium pentobarbital (Sigma, America) at a dose of 1 mL / 100 g, and then a full-thickness ventral hernia model was established. The specific operation was as follows: The abdominal hair of the rats was shaved, and a longitudinal incision was made near the linea alba. In the exposed surgical area, a 4 cm × 1 cm abdominal muscle defect was prepared by removing the superficial muscle layer and fascia on one side of the abdominal wall, while retaining the transverse fascia and peritoneum. Subsequently, the sterile R-PLA and DNA@A-PLA-HA fiber membranes were covered on the defect and fixed with 4 / 0 Monocryl suture. The sham operation group only washed the wound with normal saline, while the control group did not receive any surgical treatment. Finally, the skin wound was sutured, and the rats were returned to a pathogen-free breeding environment.

[0071] 14. Histological analysis

[0072] At 7 days and 28 days after surgery, the animals were sacrificed, and the implants and the abdominal wall tissues within 1 cm around them were removed. A part of the samples was fixed with 4% paraformaldehyde (PFA), embedded in paraffin, and cut into 5-μm-thick sections. The tissue morphology and repair of the defect site were observed by hematoxylin-eosin (H&E) staining. In addition, Masson's trichrome staining was performed to evaluate collagen deposition. Observation was carried out using an optical microscope (Nikon, Japan). Immunofluorescence (IF) staining was used to evaluate angiogenesis. In addition, after the paraffin sections were dewaxed, immunohistochemical (IHC) analysis was further performed. The results of IHC staining were evaluated by the immunoreactive score method, which comprehensively considered the staining intensity and the proportion of positive cells. A brown-yellow signal in the cytoplasm was determined as a positive reaction. The IHC score was obtained by multiplying the staining intensity score by the positive cell proportion score, and its value range was 0-12. The scoring criteria were as follows: The staining intensity was divided into 0 (negative), 1 (weak), 2 (medium), 3 (strong); the proportion of positive cells was divided into 0 (<5%), 1 (5%-25%), 2 (26%-50%), 3 (51%-75%), 4 (>75%). All observations were carried out under an optical microscope (Nikon, Japan).

[0073] 15. Detection of ROS in tissues

[0074] To detect the ROS level in tissues, a part of the samples were quickly frozen after being taken out and cut into 10-μm-thick sections. Staining was performed according to the DHE-ROS kit (BestBio, China), and then observed with a fluorescence microscope (Nikon, Japan).

[0075] 16. Statistical analysis

[0076] All experiments were performed with at least three replicates. All data were expressed as mean ± standard deviation (SD). Student's t-test was used for comparison between two groups, and one-way analysis of variance (ANOVA) was used for comparison among three groups or more. All data were processed using GraphPad Prism 9.4.1 for Mac. A P value less than 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, no significant difference). All images were analyzed using Image J for Mac.

[0077] II. Results and discussion

[0078] 1. Liposomes of TSPAN9 gene can play a role in promoting mitochondrial exocytosis in vitro

[0079] First, we prepared liposomes containing TSPAN9 plasmid using microfluidic technology. To determine the optimal plasmid loading, we prepared liposomes with different plasmid concentrations (0, 1, 2, 3, 5, 10 μg / μL). The results of agarose gel electrophoresis experiments showed that when the plasmid concentration was between 1 and 2 μg / μL, the encapsulation effect of the plasmid was the best ( Figure 1 in A). Subsequently, we prepared liposomes with plasmid concentrations of 1.2, 1.4, and 1.8 μg / μL, and used dynamic light scattering (DLS) technology to evaluate their particle size and zeta potential ( Figure 1 in B). We found that with the increase of plasmid concentration, the particle size of liposomes increased and the zeta potential decreased, which might be due to the influence of the negative charge of the plasmid. Considering that liposomes between 100 nm and 200 nm are more easily taken up by cells, and the higher the zeta potential, the better the stability of liposomes, we selected a plasmid concentration of 1.2 μg / μL for subsequent material preparation. At this time, the TEM image of the liposomes containing the optimal amount of TSPAN9 plasmid is as shown in Figure 1 C. At this time, the average particle size of the liposomes was 188.73 ± 6.99 nm, the zeta potential was 47.50 ± 0.95 mV, and the polydispersity index (PDI) was 0.158 ± 0.003. These results indicate that the prepared liposomes have an ideal particle size and uniform distribution, and are easy to be internalized by cells.

[0080] To investigate whether TSPAN9 liposomes can promote the excretion of damaged mitochondria, we induced oxidative stress in cells in vitro using H2O2 and evaluated the effects of these liposomes on ROS levels. In the experiment, the control group received no treatment, the blank group was only treated with H2O2, and the other groups were treated with different concentrations of TSPAN9 liposomes. ROS levels were measured using the 2',7'-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe and flow cytometry (see Figure 1 D in the figure). The results showed that as the concentration of liposomes increased, the ROS level decreased, indicating that the liposomes were successfully transfected into cells, could resist oxidative stress, and played a role in scavenging ROS.

[0081] Similarly, since mitochondrial respiratory function is closely related to mitochondrial function, we stimulated cells with H2O2 and treated the cells with TSPAN9 liposomes, and evaluated the oxygen consumption rate (OCR) of the cells through the Seahorse experiment (see Figure 1 E in the figure). H2O2 significantly inhibited mitochondrial respiration, while treatment with TSPAN9 liposomes could alleviate this effect and improve basal respiration, maximum respiration, and spare respiratory capacity (see Figure 1 G in the figure). At the same time, we measured the extracellular acidification rate (ECAR) to evaluate the glycolytic state. The results showed that treatment with TSPAN9 liposomes could also enhance glycolytic ability and reserve, indicating that cell metabolism was improved (see Figure 1 F in the figure; Figure 9 ). These results indicate that TSPAN9 liposomes can maintain mitochondrial function and enhance cell metabolic activity.

[0082] 2. Genetically engineered oriented fibers that promote mitochondrial exocytosis

[0083] Next, we added the prepared TSPAN9 gene liposomes to a 1% HA solution and mixed them with the PLA solution at high speed at room temperature to form an emulsion for electrospinning (see Figure 2 A in the figure). It was observed that when the volume of the 1% HA solution exceeded 200 μL, the surface of the electrospun membrane became rough and showed dot-like protrusions, which might be due to the increased viscosity of the HA solution. Subsequently, to investigate whether the HA content could be incorporated into the electrospun membrane without compromising its hydrophilicity, membranes containing 0, 50, 100, 150, and 200 μL of HA with different volumes were prepared respectively. During the preparation of electrospun fibers, the electrospinning voltage and the collector speed are key parameters affecting the fiber morphology and the overall membrane quality. The electrospinning voltage determines the stability and stretching degree of the polymer jet, while the collection speed affects the arrangement of the fibers and the uniformity of the membrane. After systematic optimization, the electrospinning voltage was set at 15 kV to maintain a stable jet, and the rotational speed of the collector was adjusted to 4000 rpm to produce a fiber membrane with neatly arranged fibers (macroscopic view seeFigure 9 )。

[0084] To evaluate the potential effects of surface wettability and HA integration on the hydrophilicity of electrospun membranes, water contact angle (WCA) measurements were conducted. The WCAs of PLA electrospun membranes with different HA contents all exhibited good hydrophilicity and showed no significant differences ( Figure 2 B). This indicates that the change in HA content did not significantly alter the surface wettability, suggesting that HA was likely uniformly encapsulated in the PLA matrix rather than exposed on the membrane surface. To enhance plasmid encapsulation, a system containing 200 μL of 1% HA was selected for subsequent experiments, and the resulting electrospun membranes showed superior hydrophilicity with an average WCA of 120.63 ± 0.35°.

[0085] To study the surface morphology of the obtained materials, we used scanning electron microscopy (SEM) to image the fiber membranes of different groups, and representative images are shown in Figure 2 D. Using Image J software, we performed pseudo-color processing and statistical analysis of the fibers based on the fiber orientation angle. In the simulation diagram, fibers with the same orientation angle are represented by the same color. If the colors remain consistent after simulation, it indicates that the angular orientation of the fibers is uniform, highlighting effective alignment. Compared with the randomly oriented PLA electrospun membrane (R-PLA group), the oriented membranes (regardless of whether they contain HA or DNA@liposome, i.e., A-PLA, A-PLA-HA, and DNA@A-PLA-HA groups) all showed good alignment characteristics. Subsequently, we used ImageJ software to analyze and quantify the fiber diameter distribution. The statistical results of the fiber diameters of each group are shown in Figure 2 G, with diameters of 0.667 ± 0.01, 0.604 ± 0.03, 0.614 ± 0.01, and 0.642 ± 0.01 μm, respectively. The diameters of the fibers with consistent orientation were slightly smaller than those of the R-PLA group, which may be due to the higher rotational speed of the collector during the electrospinning process, stretching the fibers and reducing their diameters. In addition, atomic force microscopy (AFM) observation of the DNA@A-PLA-HA group confirmed that the fiber surface was smooth and the fibers were neatly arranged, indicating the successful preparation of the oriented fiber membrane ( Figure 2 C).

[0086] Transmission electron microscopy (TEM) was used to further observe the internal morphology of the fibers (see Figure 2 E). The TEM images showed that DNA@A-PLA-HA presented an obvious core-shell bilayer structure, with PLA on the outer layer and HA on the inner layer, confirming the successful formation of electrospun fibers with a core-shell structure.

[0087] Next, the mechanical properties of the electrospun membranes were systematically evaluated (see Figure 2In (F), the R-PLA group showed better mechanical properties than the other groups, which might be attributed to its larger fiber diameter. Although the performance of the A-PLA group was slightly better than that of the A-PLA-HA and DNA@A-PLA-HA groups, probably due to the addition of HA, there was no statistically significant difference in the Young's modulus among the groups, which were 31.54 ± 8.16, 24.01 ± 2.37, 25.32 ± 1.09, and 25.36 ± 1.07 MPa, respectively. We believe that these values indicate that the membranes can provide sufficient mechanical support for cell adhesion and tissue growth, making all the groups suitable for biomedical applications.

[0088] 3. In vitro cytological study of genetically engineered electrospun membranes promoting mitochondrial exocytosis

[0089] To investigate whether the fiber membranes we prepared had good biocompatibility, we conducted cell culture on the sterilized fiber membranes and performed live-dead staining on them. Representative images under a confocal microscope are shown in Figure 3 Figure A. Among them, the green fluorescence is Calcein Acetoxymethyl Ester (CalceinAM), indicating live cells; the red fluorescence is Propidium Iodide (PI), indicating dead cells. As can be seen from the figure, the prepared electrospun fiber membranes all had good biocompatibility. In addition, the adhesion and proliferation of cells on the fiber membranes were evaluated by cytoskeleton staining and Cell Counting Kit-8 (CCK-8) assay. The results of cytoskeleton staining after cell culture showed that the cells could adhere well to the fiber membranes of each group ( Figure 3 Figure B). The surface morphology of the fiber membranes provided different physical signals for cell adhesion and morphology. The cells were arranged orderly on the oriented fiber membranes, while they were randomly distributed on the random fiber membranes. As shown in Figure 3 Figure F, the CCK-8 assay results further indicated that the cells showed good adhesion and strong proliferation ability on the fiber membranes, suggesting that all the fiber membranes had good biocompatibility.

[0090] To investigate whether our materials had good biocompatibility and promoted cell migration on their surfaces, we designed a cell migration experiment on the electrospun fiber membranes, as shown in Figure 3 Figure C. The sterilized electrospun fiber membranes were placed in cell culture dishes and fixed with sterilized steel rings, and then cell culture was started. After the cells completely covered the culture dishes, the steel rings were removed, leaving blank imprints. After culturing for another 48 hours, the cell cytoskeletons were stained with phalloidin and observed under an optical microscope and a confocal microscope ( Figure 3 Figures D-E), and the statistical data are shown in Figure 3G. The image shows that cells grew well on the membranes of all groups, indicating that these fibers have good biocompatibility. Since the steel ring imprints were circular, the cell growth showed a certain degree of curvature. The cells in the A-PLA group and the DNA@A-PLA-HA group showed faster migration and directional growth trends, which may be due to the surface topology of the aligned fibers providing a direction for cell growth. In addition, the enhanced migration ability in the DNA@A-PLA-HA group suggests that TSPAN9 transfection may increase cell activity and migration ability.

[0091] 4. Genetically engineered electrospun membranes promote mitochondrial exocytosis and maintain mitochondrial function in vitro

[0092] To evaluate whether electrospun fiber membranes can promote the clearance of damaged mitochondria by promoting mitochondrial exocytosis, we cultured cells on the sterilized fiber membranes and performed immunohistochemical staining. The confocal images are shown in Figure 4 A as follows. Among them, the red fluorescence is TMRM, which is a cell membrane-permeable dye that accumulates in active mitochondria, thus reflecting the mitochondrial membrane potential (MMP). When mitochondria are damaged, the MMP decreases, resulting in a decrease in the fluorescence intensity of TMRM. The green fluorescence is MitoTractor, which can show the location of mitochondria in cells. The blue dye is 4',6-Diamidino-2-phenylindole (DAPI) to reflect the condition of the cell nucleus. The cyan color is Wheat Germ Agglutinin (WGA), which is often used to show the location and structure of the cell membrane. As can be seen from the figure, on the three fiber membranes, the cells can spread and adhere well. The cells on A-PLA and DNA@A-PLA-HA show a more oriented shape. And obvious mitochondrial exocytosis can be seen in the cells on DNA@A-PLA-HA. Subsequently, we tested the mRNA expression level of TSPAN9 in cells cultured on different fiber membranes for 48 h by QPCR to evaluate whether the fiber membranes can carry genes into cells. The primer sequences used in the experiment are shown in Table 1, and the experimental results are shown in Figure 4 B as follows. As can be seen from the figure, after culturing for 48 h, the mRNA expression level of TSPAN9 in the cells on DNA@A-PLA-HA increased significantly, increasing by about 7 times compared with other groups.

[0093] Next, to further evaluate the ability of electrospun fiber membranes to maintain mitochondrial function by promoting the clearance of damaged mitochondria, we cultured cells on the prepared electrospun fiber membranes of each group and induced oxidative stress with H2O2. Subsequently, the cells were stained with reactive oxygen species (ROS) (see Figure 4 C, E) and tetramethylrhodamine methyl ester (TMRM) (see Figure 4In Figures D and F). It can be seen from the images that an oxidative stress model was successfully established in all cells. Among the three groups, there was no significant difference in the ROS fluorescence intensity between the first two groups (R-PLA and A-PLA), while the ROS fluorescence intensity in the DNA@A-PLA-HA group was significantly reduced. Similarly, there was no obvious difference in the TMRM fluorescence intensity between the R-PLA and A-PLA groups, while the TMRM fluorescence intensity in the DNA@A-PLA-HA group was significantly increased. In addition, transmission electron microscopy (TEM) imaging technology was used to evaluate the mitochondrial morphology in cells cultured on different fibrous membranes ( Figure 4 G). The results showed that the mitochondria in the DNA@A-PLA-HA group maintained the most complete and normal ultrastructure. In contrast, the mitochondria in the R-PLA group and the A-PLA group showed varying degrees of swelling and structural damage, with the R-PLA group being the most severely affected. These results indicate that the DNA@A-PLA-HA fibrous membrane significantly reduces the ROS level and MMP loss, thereby protecting the cultured cells from oxidative stress-induced damage and maintaining mitochondrial mass homeostasis.

[0094] 5. Histological Evaluation of Genetically Engineered Electrospun Membranes for Promoting Abdominal Wall Hernia Repair in Vivo

[0095] To evaluate the biological function of the electrospun membrane in vivo, we established a rat abdominal wall hernia model, and the experimental flow chart is shown in Figure 5 Figure A. Normal rats served as the control group, and the untreated modeled rats were designated as the sham operation group. The R-PLA group and the DNA@A-PLA-HA group were implanted with the corresponding materials to close the abdominal wall defect. Four weeks after implantation, the tissues at the implantation site were taken and stained with hematoxylin and eosin (H&E) to evaluate the inflammatory and regenerative status (see Figure 5 Figure B). Compared with the control group, the H&E staining results showed differences in tissue regeneration and inflammation levels in the implantation groups. In the R-PLA group and the DNA@A-PLA-HA group, macrophage phagocytosis and inflammatory cell aggregation could be observed around the implanted materials, which is an inevitable phenomenon in foreign body implantation. The statistical results of foreign body giant cells in each group are shown in Figure 5 Figure C. Although no materials were implanted in the sham operation group, injury-induced inflammation also recruited some foreign body giant cells. In addition, compared with the R-PLA group, the DNA@A-PLA-HA group had significantly fewer foreign body giant cells and less inflammatory encapsulation, indicating a milder inflammatory response in this group.

[0096] Collagen deposition is the main form of fascia regeneration. We further performed Masson staining to track the distribution of collagen (see Figure 5In Figure D-E). Compared with the implantation group, the collagen deposition in the sham operation group was significantly reduced, indicating that although the material might cause an inflammatory response, it also promoted better and faster regeneration of the defect. In addition, the DNA@A-PLA-HA group showed a more ordered collagen distribution, mainly on the surface of the material, which was beneficial for forming a denser tissue at the defect site after degradation. These results indicated that the DNA@A-PLA-HA group had a stronger tissue regeneration ability.

[0097] Accurately identifying collagen types is crucial for fascia tissue remodeling because different types of collagen play different roles in the target tissue, and type I collagen (Col1) and type III collagen (Col3) are the key factors determining the strength and elasticity of fascia tissue. However, Masson's trichrome staining can only visualize collagen deposition and cannot distinguish specific collagen types. Therefore, we further carried out immunohistochemistry (IHC) staining to identify the collagen types involved in the extracellular matrix (ECM) remodeling process in each group. Representative images and statistical results are shown in Figure 6 Figure E-6H. The results showed that the control group exhibited ordered and high levels of Col1 and Col3, while the sham operation group showed sparse and disordered collagen deposition. Although the R-PLA group showed obvious Col1 deposition, the collagen mainly accumulated around the material, which might lead to scar formation during subsequent tissue healing. Although there was collagen, it might not provide sufficient mechanical strength. In contrast, the DNA@A-PLA-HA group induced the most significant collagen fiber deposition, including Col1 and Col3, exceeding the sham operation group and the R-PLA group in terms of structural organization and quantity. This significant enhancement indicated that the regenerated tissue would have greater strength and elasticity. In addition, the organization of collagen deposition was closely related to the inflammatory response. This might be attributed to the ability of the DNA@A-PLA-HA group to promote cell proliferation and regulate the related inflammatory response, thus effectively enhancing collagen deposition.

[0098] To further analyze the tissue remodeling process, we performed an immunofluorescence (IF) staining experiment on the 28th day after surgery. Angiogenesis is crucial for the newly formed tissue because it provides necessary nutrients and oxygen to ensure the success of tissue repair and regeneration. We performed IF staining for α-smooth muscle actin (α-SMA, a marker of smooth muscle cells) and platelet endothelial cell adhesion molecule-1 (CD31, a marker of newly formed endothelial cells) to evaluate and quantify angiogenesis in the fibrous membranes of each group during tissue remodeling. The results are shown in Figure 6As shown in A-6D. The control group in the steady state showed low vascular density. In contrast, the DNA@A-PLA-HA group showed higher neovascular density and stronger fluorescence signal compared with the sham operation group and the R-PLA group. These findings suggest that the DNA@A-PLA-HA group may have promoted cell proliferation, effectively regulated the early inflammatory response, and established a microenvironment that can both inhibit inflammation and facilitate angiogenesis and tissue regeneration, further confirming its advantageous role in promoting tissue repair and regeneration.

[0099] 6. Evaluation of the Promotion of Mitochondrial Exocytosis by Genetically Engineered Electrospun Membranes in Vivo

[0100] Reactive oxygen species (ROS) are a sensitive indicator of mitochondrial function. To investigate whether the DNA@A-PLA-HA membrane can promote the excretion of damaged mitochondria and enhance the clearance of ROS in vivo, we performed ROS staining at 1 week and 4 weeks after implantation. One week after implantation, a relatively clear material contour was visible in the implantation group (see Figure 7 A in the figure), which indicated that inflammatory cells had been recruited around the implanted material and the inflammatory response had been initiated. Semi-quantitative analysis (see Figure 7 B in the figure) showed that there was an obvious inflammatory response in all groups, and the DNA@A-PLA-HA group showed a milder inflammatory response. This may be attributed to the excretion of damaged mitochondria mediated by the TSPAN9 plasmid, thus reducing the production of ROS. By the 4th week, the ROS levels in all groups had decreased (see Figure 7 C-D in the figure). The contour of the material in the implantation group was more blurred than before, indicating that the material had been partially degraded and the inflammation had subsided as the regeneration stage began. Consistent with the previous results, the DNA@A-PLA-HA group showed the lowest ROS level, indicating that the mitochondria were effectively excreted and the inflammation was alleviated at the beginning of the regeneration stage.

[0101] In addition, to further evaluate the inflammatory response at the implantation site, immunohistochemistry (IHC) was used to detect the expression levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) at 4 weeks after surgery ( Figure 7 E-H). The results showed that the inflammatory response was most significant in the R-PLA group, followed by the sham operation group, while the DNA@A-PLA-HA group had the lowest inflammation level. These results further highlight the differential immune responses induced by different materials after implantation.

[0102] Overall, the implant material provides a scaffold for tissue adhesion, thus accelerating the tissue regeneration process. However, implantation inevitably triggers an inflammatory response, which may lead to scar formation and delayed healing. Therefore, the development of biomaterials capable of effectively regulating the local immune response remains a key focus of current research. The IHC results of this study further confirmed the excellent ability of DNA@A-PLA-HA to inhibit the local immune response by effectively reducing the levels of inflammatory factors IL-6 and TNF-α. This regulation helps create a microenvironment more conducive to tissue regeneration, which is of great significance for improving the quality and efficiency of tissue repair.

[0103] III. Conclusion

[0104] The present invention proposes a gene-electrospun fiber membrane that adopts an "inside-out" strategy to effectively promote the excretion of damaged mitochondria, thereby maintaining the stability of the mitochondrial pool. The material exhibits excellent biocompatibility, and its ability to maintain mitochondrial function, relieve inflammation, and promote tissue regeneration has been confirmed in both in vitro and in vivo experiments ( Figure 10 ). The biomaterials designed for mitochondrial exocytosis shown in the present invention have great potential in clinical applications. Such biomaterials have significant potential for clinical translation and provide a novel material-based treatment strategy for diseases related to fascia regeneration, such as abdominal wall hernia and pelvic organ prolapse.

Claims

1. A method for preparing genetically engineered oriented electrospun fibers, characterized in that, It includes the following steps: (1) Prepare a liposome solution using lecithin, cholesterol, and octadecylamine as lipid raw materials. Mix the liposome solution with the gene through a microfluidic chip to obtain a liposome solution loaded with the gene. (2) Disperse the liposomes obtained in step (1) in a hyaluronic acid solution, and then mix it with a polylactic acid solution to form an electrospinning solution. Prepare the genetically engineered oriented electrospun fiber through electrospinning.

2. The method according to claim 1, wherein In step (1), the weight ratio of lecithin, cholesterol, and octadecylamine is (78.1:16.7:2).

3. The method according to claim 1, characterized in that The gene in step (1) is TSPAN9.

4. The method according to claim 1 or 3, characterized in that, The concentration of the gene in step (1) is (1.2 μg / μL).

5. The method according to claim 1, wherein In step (1), the flow rate ratio of the central channel to the side channel of the microfluidic chip is 1:

10.

6. The method according to claim 1, wherein In step (2), the concentration of hyaluronic acid is 1% (w / v), the concentration of the polylactic acid solution is 10% (w / v), and the liposomes contain 120 μg of the gene.

7. The method according to claim 1, wherein The process parameters of electrospinning in step (2) are: the distance between the collector and the needle is 15 cm, the applied voltage is 15 kV, the flow rate is 5 mL / h, and the rotation speed of the collector is 4000 rpm.

8. The genetically engineered oriented electrospun fiber prepared by the method according to any one of claims 1-7.

9. The application of the genetically engineered oriented electrospun fiber according to claim 8 in the preparation of a drug for treating mitochondrial injury repair.

10. The application of the genetically engineered oriented electrospun fiber according to claim 8 in the preparation of a drug for fascia repair and regeneration.

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