Mitochondria-phospholipid gel as well as preparation method and application thereof

Abstract

The invention provides mitochondria-phospholipid gel as well as a preparation method and application thereof, and belongs to the technical field of low-temperature medicine. The mitochondria-phospholipid gel is prepared from the following raw materials: mitochondria, high-molecular sodium hyaluronate, trehalose, egg yolk lecithin, ergosterol and deionized water. The phospholipid gel prepared from high-molecular sodium hyaluronate, trehalose, egg yolk lecithin and ergosterol has a protective effect on mitochondria and can maintain the activity and morphological structure of the mitochondria, so that when the mitochondria-phospholipid gel acts on damaged nerves, nerve regeneration and repair can be promoted.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cryomedicine and relates to a mitochondria-phospholipid gel, a preparation method and application. BACKGROUND

[0002] Nerve injury refers to dysfunction of the nervous system caused by brain trauma, cerebrovascular disease or infection, etc., and is mainly manifested as abnormal sensation, movement or autonomic nerve, etc., which is a kind of disease that can cause devastating consequences. Nerve injury not only deprives people of basic motor and sensory functions, causing paralysis, numbness, chronic pain, but also can seriously affect autonomic function, leading to problems such as incontinence and respiratory dysfunction. These functional disorders not only destroy the physical health of patients, making them lose the ability to live independently, but also bring heavy psychological burden and social isolation, seriously affecting the quality of life.

[0003] For the treatment of nerve injury, although neurosurgery can attempt to repair obvious structural damage, the medical community still has no solution for nerve cell regeneration and functional reconstruction. Drug treatment and physical rehabilitation can only alleviate some symptoms or achieve limited functional compensation, and it is difficult to achieve fundamental cure. In addition, the process of nerve regeneration is extremely slow and full of obstacles, and the formation of scar tissue and the lack of neurotrophic support often make the regeneration process fail. These bottlenecks force a large number of patients to live with the disease for a long time, bearing huge economic and spiritual pressure, and highlight the urgency of developing efficient nerve repair and regeneration strategies.

[0004] In recent years, studies have shown that mitochondria play a core protective role in nerve injury, and its mechanism far exceeds the traditional energy supply, covering multiple aspects such as maintaining calcium homeostasis, regulating reactive oxygen species signals, and mediating apoptosis pathways. For example, after axon injury, mitochondria can quickly recruit to the injury site to provide energy to support the massive biosynthesis required for axon regeneration and buffer calcium ion overload to maintain neuronal excitability. More importantly, mitochondria actively regulate epigenetic reprogramming and expression of pro-regenerative genes by releasing specific signals and metabolites, creating a favorable intracellular environment for nerve repair. Based on these findings, the strategy for treating nerve injury is shifting from "passive support" to "active regulation", such as using mitochondrial transplantation, antioxidants targeting mitochondria, and enhancing mitochondrial biogenesis, aiming to directly enhance the self-repairing ability of neurons, providing new hope for the treatment of neurodegenerative diseases and traumatic injuries. SUMMARY

[0005] Based on the above purpose, the application provides a mitochondria-phospholipid gel, a preparation method and application.

[0006] To achieve the above purpose, the application adopts the following technical solutions: In a first aspect, the application provides a mitochondria-phospholipid gel, which is prepared from mitochondria and a phospholipid gel, wherein the phospholipid gel is prepared from the following ingredients at a final concentration: 0.3-0.8% of high-molecular-weight sodium hyaluronate, 2-5% of trehalose, 0.5-2% of egg yolk lecithin, 0.05-0.2% of ergosterol, and deionized water.

[0007] In a second aspect, the application provides a method for preparing a mitochondria-phospholipid gel, which comprises the following steps: mixing high-molecular-weight sodium hyaluronate with deionized water, and standing at 4°C overnight to obtain a gel; adding trehalose, egg yolk lecithin, and ergosterol to the gel to obtain a phospholipid gel; adding mitochondria to the phospholipid gel to obtain a mitochondria-phospholipid gel.

[0008] In a third aspect, the application provides a use of the mitochondria-phospholipid gel, i.e., the mitochondria-phospholipid gel is used for preparing a medicament for promoting nerve regeneration, repair, and functional reconstruction.

[0009] The application has the following advantages: (1) Trehalose forms hydrogen bonds with the phospholipid head group and membrane proteins, "disguises" as water molecules, maintains the double-layer structure of the mitochondrial biomembrane and the natural three-dimensional conformation of proteins, and prevents the disintegration and denaturation of proteins.

[0010] (2) Egg yolk lecithin wraps mitochondria, reduces the direct contact of mitochondria with the external environment, and avoids protein aggregation and membrane fusion caused by hydrophobic interactions.

[0011] (3) The rigid steroidal ring structure in ergosterol can be embedded in the phospholipid bilayer, filling the gap between the phospholipid fatty acid chains, increasing the mechanical strength of the mitochondrial membrane, reducing the fluidity of the mitochondrial membrane, and making the mitochondrial membrane less likely to rupture and permeate.

[0012] (4) The mitochondria-phospholipid gel can promote nerve regeneration, repair, and functional reconstruction. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 a comparison chart of behavior observation of rats in each group; Figure 2 a comparison chart of nerve sections of rats in each group. DETAILED DESCRIPTION

[0014] The application provides a mitochondrion-phospholipid gel, and raw materials for preparing the phospholipid gel include mitochondria and phospholipid, wherein the raw materials for preparing the phospholipid gel include, according to a final concentration, 0.3-0.8% of high-molecular-weight sodium hyaluronate, 2-5% of trehalose, 0.5-2% of egg yolk lecithin, 0.05-0.2% of ergosterol and deionized water.

[0015] Trehalose is a non-reducing disaccharide, and the hydration shell of biological membranes and proteins is crucial to the stability of trehalose in a normal hydration state. When in a water-deficient environment such as freezing, drying or gel solidification, the hydroxyl groups in trehalose can perfectly replace water molecules to form hydrogen bonds with the head groups of phospholipids and membrane proteins, thus “disguising” as water molecules, so as to maintain the bilayer structure of biological membranes and the natural three-dimensional conformation of proteins, and prevent them from collapsing and denaturing.

[0016] Egg yolk lecithin can form a lipophilic environment simulating the natural outer membrane of mitochondria, which can “accommodate” and “wrap” mitochondria, reduce the direct contact of mitochondria with the external environment, and avoid protein aggregation and membrane fusion caused by hydrophobic interaction.

[0017] Ergosterol is a sterol in fungi, and has a rigid steroidal ring structure. The rigid steroidal ring structure in ergosterol can be embedded in the phospholipid bilayer to fill the gap between the phospholipid fatty acid chains, so as to increase the mechanical strength of the mitochondrial membrane, reduce the fluidity of the mitochondrial membrane, make the mitochondrial membrane more difficult to break and permeate, and thus better withstand physical stress in the extraction, processing and delivery processes.

[0018] The phospholipid gel prepared by the high-molecular-weight sodium hyaluronate, trehalose, egg yolk lecithin and ergosterol in the application has a protective effect on mitochondria, can maintain the activity and morphological structure of mitochondria, and thus when the mitochondrion-phospholipid gel is applied to damaged nerves, can promote nerve regeneration and repair.

[0019] In the application, the concentration of mitochondria in the mitochondrion-phospholipid gel is 0.05-0.2 mg / mL. More preferably, the concentration of mitochondria in the mitochondrion-phospholipid gel is 0.1 mg / mL.

[0020] In the application, the raw materials for preparing the phospholipid gel include, according to a final concentration, 0.5% of high-molecular-weight sodium hyaluronate, 3% of trehalose, 1% of egg yolk lecithin, 0.1% of ergosterol and deionized water.

[0021] The application also provides a preparation method of the mitochondrion-phospholipid gel, which comprises the following steps: S01: uniformly mixing high-molecular-weight sodium hyaluronate with deionized water, standing at 4°C overnight to form a gel, and then adding trehalose, egg yolk lecithin and ergosterol into the gel to uniformly mix, so as to obtain a phospholipid gel.

[0022] S02: culturing mesenchymal stem cells to the 3rd generation, collecting mitochondria of the mesenchymal stem cells by using the method of ultracentrifugation. The collected mitochondria are added to the phospholipid gel, and after adjusting the concentration of the mitochondria to 0.05-0.2 mg / mL, the mitochondria-phospholipid gel is obtained.

[0023] In addition, the application further provides an application of the mitochondria-phospholipid gel, namely, the mitochondria-phospholipid gel is used for preparing a drug for promoting nerve regeneration repair and function reconstruction.

[0024] The technical solutions of the application are further explained and described below through specific embodiments.

[0025] Embodiment 1 The embodiment of the application provides a mitochondria-phospholipid gel, and raw materials for preparing the mitochondria-phospholipid gel include mitochondria and a phospholipid gel. The concentration of the mitochondria in the mitochondria-phospholipid gel is 0.1 mg / mL. The raw materials for preparing the phospholipid gel include, according to the final concentration, 0.5% of high-molecular-weight sodium hyaluronate, 3% of trehalose, 1% of egg yolk lecithin, 0.1% of ergosterol and deionized water.

[0026] The embodiment of the application further provides a preparation method of the mitochondria-phospholipid gel, and the method comprises the following steps. S101: uniformly mixing high-molecular-weight sodium hyaluronate and deionized water, and after standing at 4 ℃ overnight, a gel is formed. Trehalose, egg yolk lecithin and ergosterol are added to the gel and uniformly mixed to obtain a phospholipid gel.

[0027] S102: culturing mesenchymal stem cells to the 3rd generation, collecting mitochondria of the mesenchymal stem cells by using the method of ultracentrifugation. The collected mitochondria are added to the phospholipid gel, and after adjusting the concentration of the mitochondria to 0.1 mg / mL, the mitochondria-phospholipid gel is obtained.

[0028] Embodiment 2 The embodiment of the application provides a mitochondria-phospholipid gel, and raw materials for preparing the mitochondria-phospholipid gel include mitochondria and a phospholipid gel. The concentration of the mitochondria in the mitochondria-phospholipid gel is 0.05 mg / mL. The raw materials for preparing the phospholipid gel include, according to the final concentration, 0.3% of high-molecular-weight sodium hyaluronate, 5% of trehalose, 1% of egg yolk lecithin, 0.05% of ergosterol and deionized water.

[0029] The preparation method of the mitochondria-phospholipid gel provided by the embodiment of the application is the same as that in the embodiment 1.

[0030] Embodiment 3 The embodiment of the present application provides a kind of mitochondria-phospholipid gel, its preparation raw material includes mitochondria and phospholipid gel, wherein the concentration of mitochondria in mitochondria-phospholipid gel is 0.15mg / mL, and the preparation raw material of phospholipid gel includes the following according to final concentration: high molecular weight sodium hyaluronate 0.5%, trehalose 4%, egg yolk lecithin 0.5%, ergosterol 0.15% and deionized water.

[0031] The embodiment of the present application provides a kind of mitochondria-phospholipid gel, its preparation raw material includes mitochondria and phospholipid gel, wherein the concentration of mitochondria in mitochondria-phospholipid gel is 0.15mg / mL, and the preparation raw material of phospholipid gel includes the following according to final concentration: high molecular weight sodium hyaluronate 0.5%, trehalose 4%, egg yolk lecithin 0.5%, ergosterol 0.15% and deionized water.

[0032] Example 4 The embodiment of the present application provides a kind of mitochondria-phospholipid gel, its preparation raw material includes mitochondria and phospholipid gel, wherein the concentration of mitochondria in mitochondria-phospholipid gel is 0.15mg / mL, and the preparation raw material of phospholipid gel includes the following according to final concentration: high molecular weight sodium hyaluronate 0.5%, trehalose 4%, egg yolk lecithin 0.5%, ergosterol 0.15% and deionized water.

[0033] The embodiment of the present application provides a kind of mitochondria-phospholipid gel, its preparation raw material includes mitochondria and phospholipid gel, wherein the concentration of mitochondria in mitochondria-phospholipid gel is 0.15mg / mL, and the preparation raw material of phospholipid gel includes the following according to final concentration: high molecular weight sodium hyaluronate 0.5%, trehalose 4%, egg yolk lecithin 0.5%, ergosterol 0.15% and deionized water.

[0034] To verify the mitochondria-phospholipid gel provided by the embodiment of the present application has the effect of promoting nerve regeneration repair and function reconstruction, the mitochondria-phospholipid gel in example 1 is used in the sciatic nerve injury mice in the embodiment of the present application to observe its effect.

[0035] Specific experimental process is as follows: Female SD rats are divided into normal group, injury group and treatment group, 6 rats in each group. After anesthesia, the rats in injury group and treatment group are operated to expose right sciatic nerve, and the nerve is squeezed for 10 seconds with hemostatic forceps, the continuity of nerve is reserved, and the wound is sutured after the operation is finished, to cause sciatic nerve injury. After 4 days of sciatic nerve injury, each rat in treatment group is injected with 0.5mL mitochondria-phospholipid gel in example 1, and each rat in injury group is injected with 0.5mL normal saline. After injection, the hind limb movement and wound infection of each group of rats are observed, and the following Figure 1 is obtained. In addition, after 14 days, each group of rats is killed, and the repair of sciatic nerve is observed, and the following Figure 2 is obtained.

[0036] As shown in the following Figure 1 , the wound of rats in injury group appears edema, and the body is emaciated and moves slowly; while the wound edema of rats in treatment group is improved, and the body shape and movement ability are consistent with rats in normal group.

[0037] As shown in the following Figure 2It can be seen that the sciatic nerve structure of the rats in the damage group is disordered, the epineurium is slightly thickened, the inter-fascicular space is significantly widened, and mild edema is accompanied; and the structure disorder and edema of the rats in the treatment group are improved.

[0038] The above shows that the mitochondrial-phospholipid gel group in Example 1 has a significant repairing effect on the sciatic nerve damage of rats, and can be used for preparing a drug for promoting nerve regeneration repair and functional reconstruction.

[0039] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Those skilled in the art can make various modifications and changes to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A mitochondrial-phospholipid gel, characterized in that, The raw materials for preparation include mitochondria and phospholipid gel. The raw materials for preparing the phospholipid gel, according to the final concentration, include: 0.3-0.8% sodium hyaluronate, 2-5% trehalose, 0.5-2% egg yolk lecithin, 0.05-0.2% ergosterol, and deionized water.

2. The mitochondrial-phospholipid gel according to claim 1, characterized in that, The raw materials for preparing the phospholipid gel, according to the final concentration, include: 0.5% sodium hyaluronate, 3% trehalose, 1% egg yolk lecithin, 0.1% ergosterol, and deionized water.

3. The mitochondrial-phospholipid gel according to claim 1, characterized in that, The concentration of mitochondria in the mitochondrial-phospholipid gel is 0.05-0.2 mg / mL.

4. The mitochondrial-phospholipid gel according to claim 1, characterized in that, The concentration of mitochondria in the mitochondrial-phospholipid gel was 0.1 mg / mL.

5. The method for preparing the mitochondrial-phospholipid gel according to any one of claims 1-4, characterized in that, include: Sodium hyaluronate with high molecular weight was mixed with deionized water and allowed to stand overnight at 4°C. Trehalose, egg yolk lecithin and ergosterol were then added and mixed to obtain phospholipid gel. Mitochondria were added to the phospholipid gel to obtain a mitochondrial-phospholipid gel.

6. The mitochondrial-phospholipid gel according to any one of claims 1-4 is used to prepare a drug that promotes nerve regeneration, repair and functional reconstruction.

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