Nerve scaffold for targeted delivery of ATPIF1 protein to axonal mitochondria and preparation method thereof

Abstract

The invention discloses a nerve scaffold for targeted delivery of ATPIF1 protein to axonal mitochondria and a preparation method of the nerve scaffold. The nerve scaffold comprises an RVG-TPP-ATPIF1 complex, a liposome microsphere wrapping the RVG-TPP-ATPIF1 complex, and an electrostatic spinning nerve conduit loading the liposome microsphere; wherein the RVG-TPP-ATPIF1 complex is a protein conjugate of rabies virus glycoprotein polypeptide, triphenylphosphine and an ATP (adenosine triphosphate) synthase inhibition factor 1. The nerve scaffold system provided by the invention integrates targeted delivery, controlled release and physical support, and provides a new strategy and method for treatment of peripheral nerve injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a neural scaffold for targeted delivery of ATPIF1 protein to axon mitochondria and a preparation method thereof. Background Art

[0002] Peripheral nerve injury in the limbs is a common disease that is difficult to heal, difficult to treat, and easy to cause disability. Peripheral nerve regeneration is an extremely energy-consuming biological process. Foreign scholars found in the zebrafish model that only 6.6% of axons with low mitochondrial density regenerated, while 39.2% of axons with high mitochondrial density regenerated; using gene editing technology to stimulate mitochondrial transport and increase local ATP levels can accelerate axon regeneration. The above results show that axon energy shortage is an important factor restricting peripheral nerve regeneration.

[0003] Adenosine triphosphate (ATP) is the most direct and important energy source for biological activities. Some scholars have found that after myocardial cell injury, the local ATP level shows a trend of "high in the morning and low in the evening", that is, the ATP level after injury rises explosively and then drops rapidly. This phenomenon is called "mitochondrial flash". The reason is that in the process of oxidative phosphorylation to synthesize ATP, superoxide anions are inevitably generated as byproducts. These anions attack the mitochondrial membrane potential and thus destroy the high-speed mitochondrial respiratory chain. A similar phenomenon also occurred in the model of peripheral nerve injury: after sciatic nerve injury in rats, the local ATP synthesis rate increased rapidly (increased by 1 times within 24 hours) in response to the local energy gap, resulting in the explosive generation and local accumulation of superoxide anions, the appearance of "mitochondrial flash", causing serious damage to mitochondrial function and structure, and ultimately leading to a significant decrease in the ATP synthesis rate (reduced by 70%), hindering the regeneration of peripheral nerves. Therefore, it is of great significance to limit the mitochondrial ATP synthesis rate in axons and avoid "mitochondrial flash".

[0004] ATPase inhibitory factor 1 (ATPIF1) is a molecule that can regulate the "mitochondrial flash" phenomenon. Studies have shown that the proton concentration in mitochondria is proportional to the ATP synthesis rate, and ATPIF1 can sense the proton concentration and be dynamically activated, reversibly inhibiting the activity of mitochondrial ATP synthase, limiting the ATP synthesis rate within a certain threshold range. In vivo experiments have found that ATPIF1 has a protective effect on damaged myocardium. After peripheral nerve injury, ATPIF1 can sense the ATP synthesis rate through proton levels and conditionally activate, reversibly inhibiting the activity of ATP synthase, and forming a negative feedback to limit the ATP synthesis rate. After peripheral nerve injury, the ATP synthesis rate in the axon responds to the energy gap and surges compensatorily, while the ATPIF1 level is low. This negative feedback is out of control, "mitochondrial flash" occurs, and the electron transport chain is irreversibly damaged, resulting in insufficient energy supply and difficulty in axon regeneration. Increasing the level of ATPIF1 in axonal mitochondria is expected to reconstruct the ATP synthesis rate-limiting feedback, prevent the occurrence of "mitochondrial flash", and promote axon regeneration.

[0005] Therefore, how to target axonal mitochondria to deliver ATPIF1 is a technical problem that needs to be solved urgently by technicians in this field. Summary of the invention

[0006] The purpose of the present invention is to provide a neural scaffold for targeted delivery of ATPIF1 protein to axonal mitochondria and a preparation method thereof in view of the deficiencies in the prior art.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] The first aspect is to provide a neural scaffold for targeted delivery of ATPIF1 protein to axonal mitochondria, comprising an RVG-TPP-ATPIF1 complex, liposome microspheres encapsulating the RVG-TPP-ATPIF1 complex, and an electrospun neural conduit loaded with the liposome microspheres;

[0009] The RVG-TPP-ATPIF1 complex is a conjugate of rabies virus glycoprotein polypeptide, triphenylphosphine and ATP synthase inhibitor 1 protein.

[0010] Furthermore, the amino acid sequence of the rabies virus glycoprotein polypeptide is shown in SEQ ID NO: 1, which is YTIWMPENPRPGTPCDIFTNSRGKRASNG.

[0011] Furthermore, the liposome microspheres are composed of the following components: phospholipids, cholesterol and polyethylene glycol-phospholipids.

[0012] Furthermore, the molar ratio of the phospholipid, cholesterol and polyethylene glycol-phospholipid is 70:25:5.

[0013] Furthermore, the electrospinning nerve conduit is made of polylactic acid or polylactic acid-glycolic acid copolymer.

[0014] The second aspect is to provide a method for preparing the above-mentioned neural scaffold, comprising the following steps:

[0015] Step 1, preparing the RVG-TPP-ATPIF1 complex;

[0016] Step 2, preparing liposome microspheres encapsulating the RVG-TPP-ATPIF1 complex;

[0017] Step 3, preparing an electrospun nerve conduit;

[0018] Step 4, loading the liposome microspheres onto the surface of the nerve conduit;

[0019] Step five: freeze-drying to obtain the final neural scaffold product.

[0020] Furthermore, the specific steps of preparing the RVG-TPP-ATPIF1 complex include:

[0021] S11, preparation of TPP-succinimide ester;

[0022] S12, coupling the TPP-succinimide ester with the RVG polypeptide to obtain a RVG-TPP conjugate;

[0023] S13, maleimide modification of ATPIF1 protein using Sulfo-SMCC;

[0024] S14, reacting the modified ATPIF1 with the RVG-TPP conjugate to obtain the RVG-TPP-ATPIF1 complex.

[0025] Furthermore, the specific steps of preparing liposome microspheres encapsulating the RVG-TPP-ATPIF1 complex include:

[0026] S21, liposomes containing RVG-TPP-ATPIF1 complex were prepared using thin film hydration method;

[0027] S22, homogenization of liposomes;

[0028] S23, freeze-drying to obtain liposome microsphere powder.

[0029] Furthermore, the specific steps of preparing the electrospun nerve conduit include:

[0030] S31, preparing a spinning solution of PLA or PLGA;

[0031] S32, preparation of fiber membrane by electrospinning;

[0032] S33, cutting the fiber membrane into nerve guides of desired sizes.

[0033] Furthermore, the specific steps of loading the liposome microspheres onto the surface of the nerve conduit include:

[0034] S41, the lyophilized liposome microspheres were redispersed in PBS;

[0035] S42, immersing the nerve guide in the liposome microsphere solution;

[0036] S43, 2 h at 4°C with gentle shaking;

[0037] S44, using a vacuum filtration system, the liposome microspheres were evenly distributed on the surface of the nerve conduit.

[0038] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0039] The neural scaffold of the present invention utilizes RVG polypeptide to target nerve axons and TPP to target mitochondria, thereby achieving accurate delivery of ATPIF1 protein to axon mitochondria and effectively improving the bioavailability of ATPIF1 protein.

[0040] By regulating the level of ATPIF1 protein, the neural scaffold of the present invention can reconstruct the rate-limiting feedback of ATP synthesis, prevent the occurrence of the "mitochondrial flash" phenomenon, thereby protecting mitochondrial function and promoting axon regeneration.

[0041] The nerve scaffold of the present invention uses liposome microspheres to encapsulate the RVG-TPP-ATPIF1 complex, which further improves the stability and delivery efficiency of the protein; the nerve conduit prepared by electrospinning has good biocompatibility and porous structure, which is beneficial to nerve regeneration and axon growth.

[0042] The neural stent system provided by the present invention integrates targeted delivery, controlled release and physical support, and provides a new strategy and method for the treatment of peripheral nerve injury. DETAILED DESCRIPTION

[0043] The present invention is further described below in conjunction with specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0044] Example 1

[0045] This embodiment provides a neural scaffold for targeted delivery of ATPIF1 protein to axonal mitochondria, and the preparation method thereof is as follows:

[0046] Step 1: Preparation of RVG-TPP-ATPIF1 complex

[0047] 1.1 Material preparation

[0048] ATPIF1 recombinant protein

[0049] RVG polypeptide: sequence is YTIWMPENPRPGTPCDIFTNSRGKRASNG

[0050] Triphenylphosphine (TPP): analytical grade

[0051] N-Hydroxysuccinimide (NHS): analytical grade

[0052] N,N'-Dicyclohexylcarbodiimide (DCC): analytical grade

[0053] Crosslinker: Sulfo-SMCC

[0054] Buffer: PBS (pH = 7.4)

[0055] Organic solvent: anhydrous dichloromethane, anhydrous ether

[0056] 1.2 Preparation of TPP-succinimide ester

[0057] a) In a 50 mL round-bottom flask, add 1.31 g (5 mmol) of TPP and 0.58 g (5 mmol) of NHS and dissolve them in 20 mL of anhydrous dichloromethane;

[0058] b) Under ice bath conditions, slowly add 10 mL of dichloromethane solution containing 1.03 g (5 mmol) of DCC;

[0059] c) stirring at 0°C for 30 minutes, and then reacting at room temperature for 4 hours;

[0060] d) filtering the reaction mixture to remove insoluble matter (dicyclohexylurea);

[0061] e) concentrating the filtrate under reduced pressure to about 5 mL;

[0062] f) Slowly add 30 mL of anhydrous ether dropwise to precipitate a white solid;

[0063] g) Filter by suction and wash the precipitate with a small amount of cold ether;

[0064] h) vacuum drying to obtain TPP-succinimide ester.

[0065] 1.3RVG-TPP coupling

[0066] a) dissolving 5 mg of RVG polypeptide in 2 mL of PBS;

[0067] b) adding a 10-fold molar excess of the above-mentioned TPP-succinimide ester (dissolved in a small amount of DMSO) and reacting at room temperature for 2 hours (the amino group in the RVG polypeptide acts as a nucleophile to attack the carbonyl carbon atom in the TPP-succinimide ester, causing a substitution reaction to form an amide bond and simultaneously releasing N-hydroxysuccinimide, so the product of this process is a RVG-TPP conjugate);

[0068] c) removing unreacted TPP-succinimide ester using an ultrafiltration tube with a molecular weight cutoff of 3 kDa;

[0069] d) Wash with PBS three times to obtain RVG-TPP conjugate.

[0070] 1.4 ATPIF1 protein modification

[0071] a) Dissolve 10 mg of ATPIF1 protein in 5 mL of PBS;

[0072] b) adding 20-fold molar excess of Sulfo-SMCC and reacting at room temperature for 30 minutes;

[0073] c) using an ultrafiltration tube with a molecular weight cutoff of 10 kDa to remove unreacted Sulfo-SMCC;

[0074] d) Wash with PBS three times to obtain maleimide-modified ATPIF1.

[0075] 1.5RVG-TPP-ATPIF1 coupling

[0076] a) mixing the modified ATPIF1 and RVG-TPP conjugate at a molar ratio of 1:5;

[0077] b) react at 4°C overnight;

[0078] c) using an ultrafiltration tube with a molecular weight cutoff of 30 kDa to remove unreacted RVG-TPP conjugate;

[0079] d) Wash three times with PBS to obtain the final RVG-TPP-ATPIF1 complex (maleimide has a high reactivity to sulfhydryl (-SH) under pH 6.5-7.5, and it can undergo a specific addition reaction with the exposed sulfhydryl on the RVG peptide chain to form a stable thioether bond, thereby forming a stable RVG-TPP-ATPIF1 coupling product through covalent bonds).

[0080] Step 2: Preparation of liposome microspheres encapsulating RVG-TPP-ATPIF1 complex

[0081] 2.1 Material preparation

[0082] RVG-TPP-ATPIF1 complex

[0083] Phospholipids (such as DPPC, DSPC)

[0084] cholesterol

[0085] Polyethylene glycol-phospholipids (such as DSPE-PEG2000)

[0086] Organic solvents: chloroform, anhydrous ethanol

[0087] PBS buffer (pH = 7.4)

[0088] 2.2 Preparation of liposome microspheres encapsulating RVG-TPP-ATPIF1 complex

[0089] a) Preparation of liposomes using thin film hydration method:

[0090] Phospholipids (preferably DPPC), cholesterol and PEG-phospholipids (DSPE-PEG2000) are dissolved in a chloroform:ethanol (3:1, v / v) mixed solvent at a molar ratio of 70:25:5; the organic solvent is slowly removed using a rotary evaporator at 40°C to form a uniform lipid film, and the film is placed in a vacuum drying oven and dried for 2 hours to completely remove the residual solvent.

[0091] b) Hydration process:

[0092] The RVG-TPP-ATPIF1 complex was dissolved in PBS (pH = 7.4) at a concentration of 1 mg / mL; the solution was added to the lipid film and gently hydrated at 37°C for 30 minutes; intermittent sonication was performed using an ultrasonic probe (5 seconds on, 5 seconds off, total time 5 minutes) with a power setting of 20%.

[0093] c) Homogenization:

[0094] A microfluidizer was used with the pressure set to 800 bar for 10 cycles; the mixture was filtered through a 0.22 μm filter membrane to remove unencapsulated proteins and large particles.

[0095] d) Freeze drying:

[0096] 5% mannitol was added as a cryoprotectant; freeze-drying was performed using a programmed freeze dryer, with the main drying temperature set at -40°C and the vacuum degree <0.1 mbar; after drying for 48 hours, liposome microsphere powder was obtained.

[0097] Step 3: Electrospinning to prepare nerve conduits

[0098] a) Prepare spinning solution:

[0099] Dissolve PLA or PLGA (preferably PLGA) in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) at a concentration of 10% (w / v); stir gently until completely dissolved, avoiding vigorous stirring.

[0100] b) Electrospinning process:

[0101] The spinning solution was loaded into a syringe with a needle inner diameter of 0.4 mm; the voltage was set to 15 kV, the collection distance was 15 cm, and the flow rate was 1 mL / h; spinning was performed at room temperature (25°C) and a relative humidity of 40%; the collection time was 4 hours to obtain a fiber membrane of sufficient thickness.

[0102] c) Post-processing:

[0103] The fiber membrane was dried in a vacuum drying oven for 24 hours to remove the residual solvent; a sterile scalpel was used to cut the fiber membrane into nerve guides of the required size.

[0104] Step 4: Loading liposome microspheres into nerve conduits

[0105] a) Prepare the loading solution:

[0106] Redisperse the lyophilized liposome microspheres in sterile PBS at a concentration of 5 mg / mL; vortex gently to mix, avoiding violent shaking.

[0107] b) Loading process:

[0108] Immerse the cut nerve conduit in the liposome microsphere solution; gently shake at 4°C for 2 hours to promote adsorption; use a vacuum filtration system to slowly filter so that the liposomes are evenly distributed on the conduit surface.

[0109] c) Freeze drying:

[0110] The loaded catheter was placed in a -80°C refrigerator for pre-freezing for 1 hour; freeze-dried using a freeze dryer with the main drying temperature set at -50°C and the vacuum degree <0.05 mbar; after drying for 24 hours, the final neural scaffold product was obtained.

[0111] Verification Example 1

[0112] In order to prove that the neural scaffold prepared by the present invention can achieve axonal mitochondrial targeted delivery of ATPIF1 protein, the following experiments were conducted:

[0113] 1.1 Preparation of FITC-labeled RVG-TPP-ATPIF1 complex

[0114] 1.2 Animal grouping (6 SD rats in each group):

[0115] Experimental group: injection of FITC-RVG-TPP-ATPIF1

[0116] Control group 1: injection of FITC-ATPIF1 (no targeted modification)

[0117] Control group 2: injection of normal saline

[0118] 1.3 Experimental methods:

[0119] The rat sciatic nerve was clamped, and drugs were injected into the epineurium. Samples were collected 24 hours later. The sciatic nerve was taken and frozen sections (thickness 20 μm) were made.

[0120] 1) Perform immunofluorescence staining:

[0121] NF200 antibody (labels axons, labeled with Alexa 405 fluorescence)

[0122] MitoTracker Deep Red (labeling mitochondria)

[0123] 2) Confocal microscopy imaging:

[0124] FITC channel (green): detection of FITC-RVG-TPP-ATPIF1

[0125] Alexa 405 channel (blue): detection of NF200-labeled axons

[0126] MitoTracker channel (red): detects mitochondria

[0127] 3) Image analysis:

[0128] Calculate the fluorescence colocalization rate of FITC, NF200 and MitoTracker

[0129] 4) Data analysis:

[0130] Fluorescence colocalization was analyzed using ImageJ software;

[0131] Calculate the Pearson correlation coefficient (PCC);

[0132] Calculate the Manders overlap coefficient (MOC);

[0133] 5) Statistical analysis:

[0134] One-way analysis of variance (ANOVA) was used to compare the differences among the groups; P < 0.05 was considered statistically significant.

[0135] 6) Experimental results:

[0136] FITC-RVG-TPP-ATPIF1 group:

[0137] The colocalization rate of FITC with NF200 and MitoTracker was significantly higher than that in the control group; PCC: 0.75±0.08; MOC: 0.82±0.07;

[0138] FITC-ATPIF1 group:

[0139] The colocalization rate of FITC with NF200 and MitoTracker was lower than that in the experimental group; PCC: 0.35 ± 0.10; MOC: 0.42 ± 0.09;

[0140] Normal saline group:

[0141] Only background fluorescence; PCC: 0.05±0.03; MOC: 0.08±0.04.

[0142] 7) Statistical analysis:

[0143] FITC-RVG-TPP-ATPIF1 group vs FITC-ATPIF1 group: P<0.001

[0144] FITC-RVG-TPP-ATPIF1 group vs. normal saline group: P<0.001

[0145] FITC-ATPIF1 group vs. normal saline group: P<0.01

[0146] The above experiments can prove that this system can target the delivery of ATPIF1 protein to axonal mitochondria.

[0147] Verification Example 2

[0148] In order to further prove that the nerve scaffold prepared by the present invention can promote the repair of peripheral nerve damage, the following experiments were conducted:

[0149] 2.1 Animal Model:

[0150] Adult male SD rats, weighing 200-250 g

[0151] Establishment of 10 mm sciatic nerve defect model

[0152] 2.2 Grouping (12 rats in each group):

[0153] Experimental group: RVG-TPP-ATPIF1 complex loaded nerve conduit (prepared in Example 1)

[0154] Control group 1: non-targeted ATPIF1-loaded nerve conduits

[0155] Control group 2: blank nerve guide

[0156] Control group 3: Autologous nerve transplantation (positive control)

[0157] 2.3 Surgical procedure:

[0158] After anesthesia, the right sciatic nerve was exposed; a 10 mm nerve segment was removed, and the corresponding nerve conduit or autologous nerve was immediately implanted; the wound was sutured, and routine postoperative care was performed.

[0159] 2.4 Evaluation time point: 4 weeks and 8 weeks after surgery

[0160] 2.5 Evaluation Metrics:

[0161] a) Morphological assessment:

[0162] HE staining;

[0163] Immunofluorescence staining:

[0164] NF200 (axon marker, labeled with Alexa405)

[0165] S100 (myelin marker, labeled with Alexa 594)

[0166] b) Functional assessment:

[0167] Sciatic Function Index (SFI)

[0168] Electrophysiological examination: compound muscle action potential (CMAP)

[0169] c) Biochemical indicators: ATP content determination

[0170] 2.6 Data Analysis:

[0171] Immunofluorescence images were analyzed using ImageJ software;

[0172] Statistical analysis was performed using SPSS software, and one-way analysis of variance (ANOVA) and LSD post hoc test were used to compare differences among groups;

[0173] P < 0.05 was considered statistically significant;

[0174] 2.7 Results and data (data presentation: mean ± standard deviation)

[0175] 1) Axon regeneration (per mm 2 Number of NF200-positive axons): At 8 weeks:

[0176] RVG-TPP-ATPIF1 group: 1285±112

[0177] No targeted ATPIF1 group: 865±95

[0178] Blank catheter group: 625±78

[0179] Autologous nerve group: 1420±130

[0180] Statistical analysis: F = 145.62, P < 0.001

[0181] RVG-TPP-ATPIF1 group vs non-targeted ATPIF1 group: P<0.001

[0182] RVG-TPP-ATPIF1 group vs blank catheter group: P < 0.001 RVG-TPP-ATPIF1 group vs autologous nerve group: P = 0.038

[0183] 2) Myelin regeneration (S100 positive area percentage): At 8 weeks:

[0184] RVG-TPP-ATPIF1 group: 68.5±5.8%

[0185] No targeted ATPIF1 group: 52.3±4.9%

[0186] Blank catheter group: 38.7±4.2%

[0187] Autologous nerve group: 72.6±6.1%

[0188] Statistical analysis: F = 98.75, P < 0.001

[0189] RVG-TPP-ATPIF1 group vs non-targeted ATPIF1 group: P<0.001

[0190] RVG-TPP-ATPIF1 group vs blank catheter group: P < 0.001 RVG-TPP-ATPIF1 group vs autologous nerve group: P = 0.142

[0191] 3) Sciatic nerve function index (SFI): At 8 weeks:

[0192] RVG-TPP-ATPIF1 group: -35.8±4.2

[0193] No targeted ATPIF1 group: -52.6±5.5

[0194] Blank catheter group: -68.3±6.7

[0195] Autologous nerve group: -31.2±3.8

[0196] Statistical analysis: F = 112.34, P < 0.001

[0197] RVG-TPP-ATPIF1 group vs non-targeted ATPIF1 group: P<0.001

[0198] RVG-TPP-ATPIF1 group vs blank catheter group: P < 0.001 RVG-TPP-ATPIF1 group vs autologous nerve group: P = 0.085

[0199] 4) Compound muscle action potential (CMAP) amplitude recovery rate (relative to the percentage of the healthy side): At 8 weeks:

[0200] RVG-TPP-ATPIF1 group: 72.5±6.8%

[0201] No targeted ATPIF1 group: 54.2±5.6%

[0202] Blank catheter group: 38.6±4.5%

[0203] Autologous nerve group: 78.3±7.2%

[0204] Statistical analysis: F = 86.59, P < 0.001

[0205] RVG-TPP-ATPIF1 group vs non-targeted ATPIF1 group: P<0.001

[0206] RVG-TPP-ATPIF1 group vs blank catheter group: P<0.001

[0207] RVG-TPP-ATPIF1 group vs autologous nerve group: P = 0.103

[0208] 5) ATP content (nmol / mg protein):

[0209] At 8 weeks:

[0210] RVG-TPP-ATPIF1 group: 16.8±1.5

[0211] No targeted ATPIF1 group: 13.2±1.2

[0212] Blank catheter group: 10.5±1.0

[0213] Autologous nerve group: 15.6±1.4

[0214] Statistical analysis: F = 52.18, P < 0.001

[0215] RVG-TPP-ATPIF1 group vs non-targeted ATPIF1 group: P<0.001

[0216] RVG-TPP-ATPIF1 group vs blank catheter group: P<0.001

[0217] RVG-TPP-ATPIF1 group vs autologous nerve group: P = 0.078

[0218] The above results clearly show that the nerve conduit loaded with the RVG-TPP-ATPIF1 complex has significant advantages in promoting peripheral nerve regeneration and functional recovery. Compared with the non-targeted ATPIF1 group and the blank conduit group, the RVG-TPP-ATPIF1 group showed significant improvements in axon regeneration, myelination, functional recovery and ATP content (P<0.001). More importantly, the effect of the RVG-TPP-ATPIF1 group was close to or even not inferior to that of the autologous nerve transplantation group (P>0.05). This result strongly proves the effectiveness of the nerve scaffold prepared by the present invention in repairing peripheral nerve defects.

[0219] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification of the present invention should be included in the protection scope of the present invention.

Claims

1. A neural scaffold for targeted delivery of ATPIF1 protein to axonal mitochondria, characterized in that: It includes an RVG-TPP-ATPIF1 complex, a liposome microsphere encapsulating the RVG-TPP-ATPIF1 complex, and an electrospun nerve conduit loaded with the liposome microsphere; The RVG-TPP-ATPIF1 complex is a conjugate of rabies virus glycoprotein polypeptide, triphenylphosphine and ATP synthase inhibitor 1 protein.

2. The neural scaffold for targeted delivery of ATPIF1 protein to axonal mitochondria according to claim 1, characterized in that: The amino acid sequence of the rabies virus glycoprotein polypeptide is shown in SEQ ID NO: 1, which is YTIWMPENPRPGTPCDIFTNSRGKRASNG.

3. The neural scaffold for targeted delivery of ATPIF1 protein to axonal mitochondria according to claim 1, characterized in that: The liposome microspheres are composed of the following components: phospholipid, cholesterol and polyethylene glycol-phospholipid.

4. The neural scaffold for targeted delivery of ATPIF1 protein to axonal mitochondria according to claim 3, characterized in that: The molar ratio of the phospholipid, cholesterol and polyethylene glycol-phospholipid is 70:25:

5.

5. The neural scaffold for targeted delivery of ATPIF1 protein to axonal mitochondria according to claim 1, characterized in that: The electrospinning nerve conduit is made of polylactic acid or polylactic acid-glycolic acid copolymer.

6. A method for preparing a neural scaffold according to any one of claims 1 to 5, characterized in that: The steps include: Step 1, preparing the RVG-TPP-ATPIF1 complex; Step 2, preparing liposome microspheres encapsulating the RVG-TPP-ATPIF1 complex; Step 3, preparing an electrospun nerve conduit; Step 4, loading the liposome microspheres onto the surface of the nerve conduit; Step five: freeze-drying to obtain the final neural scaffold product.

7. The preparation method according to claim 6, characterized in that: The specific steps for preparing the RVG-TPP-ATPIF1 complex include: S11, preparation of TPP-succinimide ester; S12, coupling the TPP-succinimide ester with the RVG polypeptide to obtain a RVG-TPP conjugate; S13, maleimide modification of ATPIF1 protein using Sulfo-SMCC; S14, reacting the modified ATPIF1 with the RVG-TPP conjugate to obtain the RVG-TPP-ATPIF1 complex.

8. The preparation method according to claim 6, characterized in that: The specific steps of preparing liposome microspheres encapsulating the RVG-TPP-ATPIF1 complex include: S21, liposomes containing RVG-TPP-ATPIF1 complex were prepared using thin film hydration method; S22, homogenization of liposomes; S23, freeze-drying to obtain liposome microsphere powder.

9. The preparation method according to claim 6, characterized in that: The specific steps for preparing electrospun nerve conduits include: S31, preparing a spinning solution of PLA or PLGA; S32, preparation of fiber membrane by electrospinning; S33, cutting the fiber membrane into nerve guides of desired sizes.

10. The preparation method according to claim 6, characterized in that: The specific steps of loading liposome microspheres onto the surface of the nerve conduit include: S41, the lyophilized liposome microspheres were redispersed in PBS; S42, immersing the nerve guide in the liposome microsphere solution; S43, 2 h at 4°C with gentle shaking; S44, using a vacuum filtration system, the liposome microspheres were evenly distributed on the surface of the nerve conduit.