Highly water-soluble fullerenols and their use in the treatment of intervertebral disc degeneration

Abstract

The application belongs to the field of biomedical engineering, and particularly relates to a high water-soluble fullerol and application thereof in treatment of intervertebral disc degeneration. The chemical formula of the high water-soluble fullerol is shown as formula 1. Compared with the prior art, the application has at least the following beneficial effects: (1) a novel fullerol is synthesized, which contains multiple hydroxyl groups on the branched chain, thereby enhancing the water solubility and avoiding cell toxicity caused by poor water solubility and aggregation; (2) the use mode and use dose of the fullerol in the IDD model of rats are determined, thereby laying a foundation for subsequent clinical conversion; and (3) the action target of the fullerol is determined as Rap1, and the downstream Nrf2 protein can be activated, and therefore, the results prove that the fullerol is a potential agonist of Rap1 and Nrf2, which has not been determined in the past.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering, specifically relating to a highly water-soluble fullerol and its application in the treatment of intervertebral disc degeneration. Background Technology

[0002] Intervertebral disc degeneration (IDD) is a leading cause of lower back pain and disability worldwide, imposing a heavy socioeconomic burden. Currently, clinical treatments for IDD primarily include drug therapy and surgery. While these methods can alleviate symptoms, they cannot prevent further disc degeneration. Degeneration of the nucleus pulposus is a key factor in the pathogenesis of IDD; therefore, delaying nucleus pulposus cell degeneration has become a potentially effective therapeutic strategy. During IDD development, nucleus pulposus cells are often under oxidative stress due to ROS accumulation, leading to cell senescence or death. Similarly, iron ion accumulation in the degenerative microenvironment also promotes ROS generation, leading to ferroptosis in nucleus pulposus cells—a programmed cell death process characterized by iron overload and lipid peroxidation. Therefore, biomaterials designed to scavenge excess ROS and inhibit ferroptosis could theoretically improve cellular function, thereby contributing to IDD repair. However, the further application of these materials is limited due to uncertainties regarding long-term safety, dosage, and route of administration.

[0003] Fullerenes are highly symmetrical icosahedral molecules composed of 60 carbon atoms, renowned as "free radical sponges" due to their exceptional free radical scavenging capabilities, exhibiting significant antioxidant effects. After entering the cell membrane, the π bonds on the fullerene surface interact with free radicals, thereby enhancing its ability to scavenge reactive oxygen species (ROS). However, fullerenes have poor water solubility and are prone to aggregation, which increases their biotoxicity and limits their potential application in antioxidant therapy. To address this issue, polyhydroxyl functionalization is commonly used to prepare fullerene derivatives, namely fullerols. This modification not only improves their water solubility and biocompatibility but also does not diminish their free radical scavenging ability.

[0004] Fullerols, as derivatives of fullerenes, have shown great potential in biomedical research and applications. They are synthesized by introducing hydroxyl groups onto the fullerene molecule to improve its water solubility and biocompatibility, thereby reducing cytotoxicity. The preparation methods of fullerols have undergone many years of development, from the initial alkaline synthesis to the more moderate and efficient Bingel reaction. This reaction offers mild conditions, pure products, and relatively high yields, laying the foundation for further applications of fullerols.

[0005] Significant progress has been made in the research of fullerol's biomedical applications. It has demonstrated high application value in multiple areas, including tumor therapy, neuroprotection, anti-oxidation, drug delivery, and immunomodulation. Particularly in tumor therapy, fullerol can not only serve as a carrier for loading traditional anticancer drugs, improving drug biocompatibility and reducing cytotoxicity, but it can also be used directly as an anticancer drug, exerting its effects by inhibiting angiogenesis and the invasiveness of cancer cells. Furthermore, fullerol is also being investigated for novel applications such as myocardial infarction treatment, radiation protection, and vaccine development.

[0006] Despite significant progress in fullerol research, our understanding of its structural modification, free radical scavenging, and therapeutic effects in the biomedical field remains limited, indicating substantial room for further development and application. Particularly in the intervertebral disc field, there is currently no evidence of fullerol being used to treat intervertebral disc disease (IDD) and the underlying mechanisms have been elucidated.

[0007] Therefore, this invention prepares a highly water-soluble fullerol, which is used to clear ROS in the nucleus pulposus, promote the recovery of nucleus pulposus cell function, and thus treat IDD. Summary of the Invention

[0008] This 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 CBr4 into a round-bottom flask, add ODCB and sonicate to dissolve them completely, then treat with nitrogen.

[0012] (2) Then DBU was diluted in ODCB and slowly added dropwise to a round-bottom flask; after the addition was complete, 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 dissolved product was used for thin-layer chromatography to separate target product 1; target product 1 was mixed with MeOH / H2O, and then Na2CO3 was dissolved in water and added to the reaction system.

[0013] (3) Add 50 mL of H2O and 10 mL of MeOH to the system after the reaction is completed for dilution, then add 1 g of cation exchange resin and react overnight. Filter the solution to obtain the final product, which is the highly water-soluble fullerol shown in Formula 1.

[0014] In some embodiments, the nitrogen treatment is performed by introducing nitrogen at 0°C for 30 minutes.

[0015] In some embodiments, the light-shielding reaction takes 6 hours.

[0016] In some embodiments, the ratio of the target product 1 to MeOH / H2O is 4:1.

[0017] In some embodiments, the weight of the Na2CO3 is 200-250 mg.

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

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

[0020] b) Prepare drugs for treating intervertebral disc degenerative diseases;

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

[0022] d) Used to combat ferrodegeneration;

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

[0024] f) Activate the downstream Nrf2 protein;

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

[0026] h) Protect mitochondria;

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

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

[0029] (1) A novel fullerol was synthesized, which has multiple hydroxyl groups on its side chain, thereby enhancing its water solubility and avoiding cytotoxicity caused by aggregation due to poor water solubility.

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

[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 in the past. Attached Figure Description

[0032] Figure 1. Schematic diagram of the synthesis steps of fullerol;

[0033] Figure 2 The 1H NMR spectrum of fullerol;

[0034] Figure 3 Fourier transform infrared spectrum of fullerol;

[0035] Figure 4 A graph illustrating the ability of fullerols to scavenge DPPH free radicals;

[0036] Figure 5 Graph of the ability of fullerol to scavenge H2O2;

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

[0038] Figure 7 Fullerol activation of Rap1 protein; left image shows gene set enrichment analysis; right image shows protein imprinting results.

[0039] Figure 8 Diagram showing the binding of fullerol-activated Rap1 to Keap1 protein;

[0040] Figure 9 Treatment effect 1 - Fullerol can inhibit TBHP-induced ferroptosis in nucleus pulposus cells.

[0041] Figure 10 Treatment effect 2 - Fullerol can maintain the normal morphology of mitochondria.

[0042] Figure 11 Treatment effect 3 – Fullerol can delay the progression of IDD in rats (Figure) Figure 11 A: Flowchart for evaluating the efficacy of fullerol treatment in a rat caudal vertebra IDD model induced by acupuncture (Puncture). Figure 11 B: X-ray and MRI results of rat tail vertebrae. Figure 11 C: Diagram of intervertebral height index (DHI) in the treatment group. Figure 11 E: MRI findings. Figure 11 D, F: Staining diagrams by hematoxylin and eosin (HE) and safranin-fixing green (SO / FG). Detailed Implementation

[0043] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0044] List of abbreviations, their English equivalents, and key term definitions:

[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 Fullerol

[0051] The synthesis of fullerol is shown in Figure 1. Fullerene and CBr4 were weighed into a round-bottom flask, ODCB was added, and the mixture was sonicated until completely dissolved. Nitrogen gas was then introduced at 0°C for 30 minutes. DBU was then diluted in 1 mL of ODCB and slowly added dropwise to the round-bottom flask. The reaction was carried out in the dark for 6 hours. After the reaction was complete, ODCB was removed by rotary evaporation, and toluene / dichloromethane was added to dissolve the product. The dissolved product was used for thin-layer chromatography to separate the target product. The product was mixed with MeOH / H2O (4:1), and 200–250 mg of Na2CO3 dissolved in water was added to the reaction system. After the reaction was complete, 50 mL of H2O and 10 mL of MeOH were added to the diluted solution, followed by the addition of 1 g of cation exchange resin and reaction overnight. The solution was filtered to obtain the final product.

[0052] The results of the identification of fullerol are as follows: Figures 2-6 As shown.

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

[0054] Eight-week-old Sprague Dawley rats were used. A 21G needle was used to percutaneously puncture the nucleus pulposus under X-ray guidance, rotate 360 ​​degrees, and hold for 30 seconds to establish a needle-induced rat caudal vertebral intervertebral disc degeneration (IDD) model. C5 / 6 of the rat caudal vertebrae served as the control, while C7 / 8 and C8 / 9 were the 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 at 1 and 2 weeks post-surgery. The rats were euthanized 4 weeks post-surgery to obtain intervertebral disc samples for evaluating treatment efficacy.

[0055] Example 3: Molecular mechanism of action of fullerol

[0056] The activation effect of fullerol on Rap1 was confirmed by transcriptome sequencing, bioinformatics analysis, and protein immunoblotting. Figure 7 As shown, fullerol can activate the Rap1 protein; the left figure shows gene set enrichment analysis, indicating that the Rap1 signaling pathway is activated in cells treated with fullerol; the right figure shows protein imprinting results demonstrating that fullerol can activate Rap1, and the effect of fullerol can be counteracted by using the Rap1 inhibitor ESI-05. Figure 8As shown, fullerol-activated Rap1 can bind to the Keap1 protein, relieving Keap1's inhibition of Nrf2, thereby upregulating Nrf2 protein levels. That is, fullerol's target is Rap1. Further analysis revealed that Rap1 binds to the Keap1 protein, relieving Keap1's inhibition of Nrf2 protein, thereby activating intracellular Nrf2 signaling and exerting an anti-ferroptosis effect. Figure 9 As shown, fullerol can inhibit TBHP-induced ferroptosis in nucleus pulposus cells. During TBHP-induced ferroptosis, oxidized fluorescence is enhanced, while non-oxidized fluorescence is weakened; the fullerol treatment group (T+F) inhibited these changes. Figure 10 As shown, fullerol can maintain the normal morphology of mitochondria. Transmission electron microscopy reveals that oxidative stress caused by TBHP damages the normal structure of mitochondria, causing them to shrink and become high-density round. In contrast, the mitochondrial morphology of the fullerol treatment group (T+F) is similar to that of the normal group (Ctrl), with clearly visible mitochondrial cristae and a normal oval shape. Figure 11 As shown, fullerol can delay the progression of IDD in rats; Figure 11 A: Flowchart for evaluating the efficacy of fullerol treatment in a rat caudal vertebra IDD model induced by acupuncture (Puncture). Figure 11 B: X-ray and MRI results of rat tail vertebrae. Figure 11 C: Compared with the Puncture group, the fullerol treatment group significantly maintained intervertebral height, and the intervertebral height index (DHI) of this group was close to that of the normal control group (Ctrl). Figure 11 E: Based on MRI results, the Pfirrmann degeneration grade of the intervertebral discs in each group was determined. The Puncture group had the highest degeneration grade, while the fullerol treatment group showed a significant decrease. Figure 11 D, F: Histological and pathological changes in each group were observed using hematoxylin and eosin (HE) and safranin-fast green (SO / FG) staining. The Puncture group showed significant nucleus pulposus fibrosis, with the boundary between the nucleus pulposus and the annulus fibrosus disappearing; its histopathological score was significantly higher than that of the Ctrl group and the fullerol treatment group. Conversely, the fullerol treatment group showed normal-morphological and viable cells in the nucleus pulposus, rich in proteoglycans, with a clear boundary between the nucleus pulposus and the annulus fibrosus; its histopathological score was close to that of the Ctrl group. It also enhanced the antioxidant capacity of cells.

[0057] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of highly water-soluble fullerols as shown in Formula 1, characterized in that, The application is one or more of a) to b): a) Prepare drugs for treating intervertebral disc degeneration. b) Prepare agonists of Rap1 and Nrf2, the application of which is for non-therapeutic purposes; Formula 1.

2. The application according to claim 1, characterized in that, The highly water-soluble fullerol is used to remove reactive oxygen species (ROS) from the nucleus pulposus.

3. The application according to claim 1, characterized in that, The highly water-soluble fullerol is used to promote the structural and functional repair of nucleus pulposus cells or degenerated intervertebral discs.

4. The application according to claim 1, characterized in that, The highly water-soluble fullerol is used to enhance the antioxidant capacity of nucleus pulposus cells.

5. The application according to claim 1, characterized in that, The highly water-soluble fullerol is used to protect the mitochondria of nucleus pulposus cells.

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