High-water-solubility fullerol and application thereof in treating intervertebral disc degeneration

Abstract

The invention belongs to the field of biomedical engineering, and particularly relates to high-water-solubility fullerol and application of the high-water-solubility fullerol in treatment of intervertebral disc degeneration. The chemical formula of the high-water-solubility fullerol is as shown in formula 1. Compared with the prior art, the preparation method at least has the following beneficial effects: (1) a novel fullerol is synthesized, and a branched chain of the fullerol contains a plurality of hydroxyl groups, so that the water solubility of the fullerol is enhanced, and cytotoxicity caused by aggregation due to poor water solubility is avoided; (2) the use mode and the use dosage of the fullerol in a rat IDD model are defined, and a foundation is laid for subsequent clinical transformation of the fullerol. And (3) the action target of the fullerol is determined to be Rap1, and downstream Nrf2 protein can be activated, so that the result proves that the fullerol is a potential Rap1 and Nrf2 agonist, which is not determined in the past.

Description

Technical Field

[0001] The invention belongs to the field of biomedical engineering, and specifically relates to a highly water-soluble fullerol and an application thereof in treating intervertebral disc degeneration. Background Art

[0002] Intervertebral disc degeneration (IDD) is the leading cause of low back pain and disability worldwide, placing a heavy burden on the socioeconomic system. Currently, clinical treatments for IDD mainly include drug therapy and surgical intervention, which can relieve symptoms but cannot prevent further degeneration of the disc. In the pathogenesis of IDD, nucleus pulposus degeneration is a key factor, so delaying the degeneration of nucleus pulposus cells has become a potential effective treatment strategy. During the development of IDD, nucleus pulposus cells are often in an oxidative stress state caused by ROS accumulation, causing cell senescence or death. Similarly, iron ion accumulation in the degenerative microenvironment can also promote ROS generation, leading to nucleus pulposus cell ferroptosis, a programmed cell death process characterized by iron overload and lipid peroxidation. Therefore, biomaterials designed to scavenge excess ROS and inhibit ferroptosis can theoretically improve the functional state of cells and thus contribute to the repair of IDD. However, the further application of these materials is limited by the uncertainty of long-term safety, dosage, and administration route.

[0003] Fullerene is a highly symmetrical icosahedral molecule composed of 60 carbon atoms. It is known as a "free radical sponge" for its excellent free radical scavenging ability and has a significant antioxidant effect. After fullerene enters the cell membrane, the π bonds on its surface react with free radicals, thereby enhancing its ability to scavenge ROS. However, fullerene has poor water solubility and is prone to aggregation, which increases its biological toxicity and limits its potential for application in antioxidant therapy. In order to solve this problem, a polyhydroxyl functionalization method is usually used to prepare fullerene derivatives, namely fullerols. This modification not only improves its water solubility and biocompatibility, but also does not weaken its ability to scavenge free radicals.

[0004] As a derivative of fullerene, fullerol has shown great potential in research and application in the field of biomedicine. It improves its water solubility and biocompatibility by introducing hydroxyl groups on fullerene molecules, thereby reducing cytotoxicity. The preparation method of fullerol has undergone many years of development, from the initial alkaline synthesis to the current more gentle and efficient Bingel reaction, which has mild reaction conditions, pure products and relatively high yields, laying the foundation for the further application of fullerol.

[0005] The research on the biomedical application of fullerol has made remarkable progress. It has shown high application value in many aspects such as tumor treatment, neuroprotection, anti-cell oxidation, drug carrier, and immunomodulation. Especially in the field of tumor treatment, fullerol can not only be used as a carrier to load traditional anticancer drugs to improve the biocompatibility of drugs and reduce cytotoxicity, but can also be used directly as an anticancer drug to inhibit angiogenesis and the invasive ability of cancer cells. In addition, fullerol has also been studied for novel applications such as myocardial infarction treatment, anti-radiation and vaccine preparation.

[0006] Although the research on fullerol has made significant progress, the current understanding of its structural modification, free radical scavenging and therapeutic effects in the biomedical field is still limited, which shows that there is still a lot of room for development in the research and application of fullerol. Especially in the field of intervertebral disc, no relevant fullerol has been used to treat IDD and the mechanism has been elucidated.

[0007] Therefore, the present invention prepares a highly water-soluble fullerol, and uses it to remove ROS in the nucleus pulposus, promote the recovery of nucleus pulposus cell function, and then treat IDD. Summary of the invention

[0008] The present invention first provides a highly water-soluble fullerol, the chemical formula of which is shown in Formula 1:

[0009] Formula 1.

[0010] The present invention also provides a method for preparing the above-mentioned highly water-soluble fullerol, the method comprising the following steps:

[0011] (1) Weigh fullerene and CBr 4 In a round-bottom flask, add ODCB, sonicate to dissolve it completely, and treat with nitrogen;

[0012] (2) DBU was then diluted in ODCB and slowly added dropwise to the round-bottom flask; the reaction was carried out in the dark; after the reaction was completed, ODCB was removed by rotary evaporation, and toluene / dichloromethane was added to dissolve the product; the obtained dissolved product was used for thin layer chromatography to separate the target product 1; the target product 1 was mixed with MeOH / H 2 O mixed, then Na 2 CO 3 Dissolve in water and add to the reaction system;

[0013] (3) Add 50 ml of H 2 O and 10 ml of MeOH were used for dilution, and then 1 g of cation exchange resin was added to react overnight, and the solution was filtered to obtain the final product, which was the highly water-soluble fullerenol shown in Formula 1.

[0014] In certain embodiments, the nitrogen treatment is to introduce nitrogen at 0° C. for 30 minutes.

[0015] In certain embodiments, the light-protected reaction time is 6 hours.

[0016] In certain embodiments, the target product 1 is mixed with MeOH / H2O in a ratio of 4:1.

[0017] In certain embodiments, the Na 2 CO 3 The weight is 200~250 mg.

[0018] Finally, the present invention provides an application of the highly water-soluble fullerol described above, wherein the application is one or more of a) to d);

[0019] a) Used to remove ROS in the nucleus pulposus;

[0020] b) Preparation of a medicament for treating degenerative disc disease;

[0021] c) Used to promote the structural and functional repair of the nucleus pulposus or degenerative intervertebral disc;

[0022] d) Anti-ferroptosis;

[0023] e) Enhance the antioxidant capacity of cells;

[0024] f) Activate the downstream Nrf2 protein;

[0025] g) Preparation of agonists of Rap1 and Nrf2;

[0026] h) Protect mitochondria;

[0027] The use is for non-therapeutic purposes.

[0028] Compared with the prior art, the present invention has at least the following beneficial effects:

[0029] (1) A new type of fullerene alcohol was synthesized, which contains multiple hydroxyl groups on its side chains, thereby enhancing its water solubility and avoiding aggregation and cytotoxicity caused by poor water solubility.

[0030] (2) The use and dosage of fullerol in the rat IDD model were clarified, laying the foundation for its subsequent clinical transformation.

[0031] (3) We determined that the target of fullerol is Rap1, and that it can activate the downstream Nrf2 protein. Therefore, our results demonstrate that fullerol is a potential agonist of Rap1 and Nrf2, which has not been identified before. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a diagram showing the synthesis steps of fullerene alcohol;

[0033] Figure 2 H NMR spectrum of fullerene alcohol;

[0034] Figure 3 Fourier transform infrared spectrum of fullerene alcohol;

[0035] Figure 4 The ability of fullerol to scavenge DPPH free radicals;

[0036] Figure 5 Fullerol scavenges H 2 O 2 Capability map;

[0037] Figure 6 The ability of fullerol to chelate ferrous ions; the left picture is the Zeta potential, and the right picture is the X-ray photoelectron spectroscopy, both of which prove that fullerol can chelate ferrous ions.

[0038] Figure 7 The left picture shows the activation of Rap1 protein by fullerol; the right picture shows the results of protein imprinting.

[0039] Figure 8 Fullerol-activated Rap1 and Keap1 protein binding diagram;

[0040] Fig. 9 Therapeutic effect 1——Fulerol can inhibit TBHP-induced ferroptosis of nucleus pulposus cells.

[0041] Fig.10 Therapeutic effect 2: Fullerol can maintain the normal morphology of mitochondria.

[0042] Fig.11 Treatment effect 3: Fullerol can delay the progression of IDD in rats Fig.11 A: Flowchart for evaluating the therapeutic effect of fullerenol in the acupuncture-induced rat coccygeal IDD model. Fig.11 B: X-ray and MRI examination results of the rat's coccygeal vertebrae. Fig.11 C: Diagram of the intervertebral height index (DHI) in the treatment group. Fig.11 E: MRI examination knot map. Fig.11 D, F: Staining by hematoxylin and eosin (HE) and safranin fast green (SO / FG). DETAILED DESCRIPTION

[0043] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0044] List of abbreviations, English and definitions of key terms:

[0045] 1. IDD: Intervertebral Disc Degeneration

[0046] 2. ROS: Reactive oxygen species

[0047] 3. ODCB: o-dichlorobenzene

[0048] 4. DBU: 1,8-diazabicycloundec-7-ene

[0049] 5. MeOH: methanol

[0050] Example 1 Synthesis of fullerene alcohol

[0051] The synthesis of fullerene alcohol is shown in the synthesis steps of Figure 1. Weigh fullerene and CBr 4 In a round-bottom flask, add ODCB and sonicate to dissolve it completely, and then pass nitrogen at 0°C for 30 minutes. Then dilute DBU in 1 ml of ODCB and slowly add it dropwise to the round-bottom flask. After the addition is complete, react in the dark for 6 hours. After the reaction is complete, remove ODCB by rotary evaporation, and add toluene / dichloromethane to dissolve the product. The obtained dissolved product is used for thin layer chromatography to separate the target product. The product is mixed with MeOH / H 2 O (4:1), and then 200-250 mg Na 2 CO 3 Dissolve in water and add to the reaction system. Add 50 ml of H 2 O and 10 ml of MeOH, and then 1 g of cation exchange resin was added to react overnight, and the solution was filtered to obtain the final product.

[0052] The fullerene alcohol was identified and the results were as follows Figure 2-Figure 6 shown.

[0053] Example 2 Fullerol in the treatment of IDD in vivo

[0054] Eight-week-old Sprague Dawley rats were selected, and a 21G needle was used to percutaneously puncture into the nucleus pulposus under X-ray guidance, rotated 360 degrees, and then held for 30 seconds to construct an acupuncture-induced rat coccygeal IDD model. The rat coccygeal C5 / 6 was used as a self-control, while C7 / 8 and C8 / 9 were surgically treated segments. For the fullerol treatment group, 10 μL of a 25 mg / mL fullerol solution was injected into C6 / 7 and C8 / 9 using a 31G microsyringe 1 week and 2 weeks after surgery, respectively. The rats were euthanized 4 weeks after surgery to obtain intervertebral disc samples for treatment effect evaluation.

[0055] Example 3 Molecular mechanism of action of fullerol

[0056] Transcriptome sequencing, bioinformatics analysis, and protein immunoblotting techniques confirmed the activation of Rap1 by fullerol. Figure 7 As shown in the figure, fullerol can activate Rap1 protein; the left figure is a gene set enrichment analysis, showing that the Rap1 signaling pathway is activated in cells treated with fullerol; the right figure is a protein imprint result proving that fullerol can activate Rap1, and the use of Rap1 inhibitor ESI-05 can offset the effect of fullerol. Figure 8 As shown in the figure, Rap1 activated by fullerol can bind to Keap1 protein, relieve Keap1's inhibitory effect on Nrf2, and thus upregulate Nrf2 protein levels. That is, the target of fullerol is Rap1. Further analysis found that Rap1 will bind to Keap1 protein, relieve Keap1's inhibitory effect on Nrf2 protein, and then activate Nrf2 signaling in cells, exerting an anti-ferroptosis effect, such as Fig. 9 As shown in the figure, fullerol can inhibit TBHP-induced ferroptosis of nucleus pulposus cells. When TBHP induces ferroptosis, the fluorescence of oxidized cells increases, while the fluorescence of non-oxidized cells decreases. The fullerol treatment group (T+F) inhibited the above changes. Fig.10 As shown in the figure, fullerol can maintain the normal morphology of mitochondria. Transmission electron microscopy shows that the oxidative stress caused by TBHP damages the normal structure of mitochondria, causing them to shrink and become high-density round. The mitochondrial morphology of the fullerol treatment group (T+F) is similar to that of the normal group (Ctrl), with clear mitochondrial cristae and a normal oval shape. Fig.11 As shown, fullerol can delay the progression of IDD in rats; Fig.11 A: Flowchart for evaluating the therapeutic effect of fullerenol in the acupuncture-induced rat coccygeal IDD model. Fig.11 B: X-ray and MRI examination results of the rat's coccygeal vertebrae. Fig.11 C: Compared with the Puncture group, the fullerol-treated group significantly maintained intervertebral height, and the intervertebral height index (DHI) of this group was close to that of the normal control group (Ctrl). Fig.11 E: Based on the MRI results, the Pfirrmann degeneration grade of each group's intervertebral disc was determined. The degeneration grade was the highest in the Puncture group, while it was significantly lower in the fullerol treatment group. Fig.11 D, F: The tissue structure and pathological changes of each group were observed by hematoxylin eosin (HE) and safranin fast green (SO / FG) staining. The nucleus pulposus in the Puncture group was obviously fibrotic, and the boundary between the nucleus pulposus and the annulus fibrosus disappeared. Its histopathological score was significantly higher than that of the Ctrl group and the fullerol treatment group. In contrast, the nucleus pulposus in the fullerol treatment group had normal morphology and viable cells, rich in proteoglycans, and a clear boundary between the nucleus pulposus and the annulus fibrosus. Its histopathological score was close to that of the Ctrl group. At the same time, it also enhanced the antioxidant capacity of cells.

[0057] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A highly water-soluble fullerol, characterized in that: The chemical formula of the highly water-soluble fullerol is shown in Formula 1, Formula 1.

2. A method for preparing the highly water-soluble fullerol according to claim 1, characterized in that: The method comprises the following steps: (1) Weigh fullerene and CBr4 in a round-bottom flask, add ODCB, perform ultrasonication to completely dissolve it, and treat with nitrogen; (2) DBU is then diluted in ODCB and slowly added dropwise to a round-bottom flask; after the addition is complete, the reaction is carried out in the dark; after the reaction is complete, ODCB is removed by rotary evaporation, and toluene / dichloromethane is added to dissolve the product; the obtained dissolved product is used for thin layer chromatography to separate the target product 1; the target product 1 is mixed with MeOH / H2O, and then Na2CO3 is dissolved in water and added to the reaction system; (3) 50 ml of H2O and 10 ml of MeOH were added to the reaction system for dilution, and then 1 g of cation exchange resin was added to react overnight. The solution was filtered to obtain the final product, which is the highly water-soluble fullerol shown in Formula 1.

3. The preparation method according to claim 2, characterized in that: The nitrogen treatment is to introduce nitrogen at 0°C for 30 minutes.

4. The preparation method according to claim 2, characterized in that: The light-proof reaction time is 6 hours.

5. The preparation method according to claim 2, characterized in that: The target product 1 is mixed with MeOH / H2O in a ratio of 4:

1.

6. The preparation method according to claim 2, characterized in that: The weight of the Na2CO3 is 200-250 mg.

7. A use of the highly water-soluble fullerol according to claim 1, characterized in that: The application is one or more of a) to d); a) Used to remove ROS in the nucleus pulposus; b) Preparation of a medicament for treating degenerative disc disease; c) Used to promote the structural and functional repair of the nucleus pulposus or degenerative intervertebral disc; d) Anti-ferroptosis; e) Enhance the antioxidant capacity of cells; f) Activate the downstream Nrf2 protein; g) Preparation of agonists of Rap1 and Nrf2; h) Protect mitochondria; The use is for non-therapeutic purposes.

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