Application of functionalized cerium oxide nanoparticles in treatment of intervertebral disc degeneration

Abstract

The invention discloses an application scheme of functionalized cerium oxide nanoparticles in treatment of intervertebral disc degeneration. The cerium oxide nano-particles are subjected to surface modification through a high polymer material containing carboxyl or amino, so that the drug carrier performance of the cerium oxide nano-particles can be improved, and the cerium oxide nano-particles have good biocompatibility and water solubility; and carrying out an amidation reaction through a carboxyl or amino-containing high polymer material to obtain the functionalized cerium oxide nano-particles with stable covalent bonds, and applying the functionalized cerium oxide nano-particles to preparation of drugs for treating intervertebral disc degeneration caused by oxidative stress. According to the scheme, effective loading and continuous release of the medicine can be realized, the stability is good, the medicine carrying capacity is high, a technical guarantee is provided for accurate delivery of the treatment medicine, the degenerated intervertebral disc can be fundamentally repaired, and no side effect exists.

Description

Technical Field

[0001] The invention relates to the technical field of biological treatment, in particular to the application of functionalized cerium oxide nanoparticles in treating intervertebral disc degeneration. Background Art

[0002] Intervertebral disc degeneration is one of the main causes of spinal diseases, especially lumbar disc herniation and chronic low back pain. As people age or the waist is overloaded, the intervertebral disc gradually undergoes degenerative degeneration, which is manifested as dehydration of the nucleus pulposus, destruction of the annulus fibrosus structure and disordered chondrocyte metabolism, which eventually leads to decreased intervertebral disc function, compression of nerve roots or spinal cord, and clinical symptoms such as pain, numbness, muscle weakness and motor dysfunction. The pathogenesis of intervertebral disc degeneration is complex and involves multiple biological processes, including cell apoptosis, matrix degradation, inflammatory response and oxidative stress. At present, the treatment of intervertebral disc degeneration is mainly conservative treatment. Surgical treatment is only used when symptoms are severe or conservative treatment is ineffective. Conservative treatment cannot repair intervertebral disc tissue. Therefore, finding a treatment method that can promote intervertebral disc repair and regeneration has become a major challenge in the field of orthopedics.

[0003] Studies have found that oxidative stress is closely related to disc degeneration. With external stimulation, the nucleus pulposus cells and annulus fibrosus cells of the intervertebral disc are easily exposed to excessive reactive oxygen species (ROS). Reactive oxygen species can damage cell membranes, DNA, proteins and lipids, leading to cell dysfunction, apoptosis or necrosis. This damage accelerates the degeneration of the intervertebral disc. Oxidative stress activates the inflammatory response pathway by upregulating the expression of proinflammatory factors (such as TNF-α, IL-1β, etc.), exacerbating the inflammatory environment in disc degeneration; inflammatory factors not only directly damage disc cells, but also further damage the extracellular matrix, leading to structural damage to the disc. Further studies have shown that oxidative stress can activate cell apoptosis through different signaling pathways, leading to the loss of disc cells through multiple channels; moreover, oxidative stress may also delay the efficiency of cell autophagy, which will have adverse effects on maintaining the normal function of disc cells and removing damaged substances.

[0004] At present, nanomedicine, as an emerging technology, has shown great potential in drug delivery, tissue engineering and cell therapy due to its unique physical and chemical properties at the nanoscale. Among them, cerium oxide nanoparticles can regulate the redox state in the body, scavenge free radicals, relieve oxidative stress, reduce inflammation and promote cell survival due to their significant antioxidant activity, good biocompatibility and strong cell protection. Therefore, cerium oxide nanoparticles have broad application prospects in the treatment of intervertebral disc degeneration, especially in slowing down cell aging, inhibiting inflammatory response and protecting intervertebral disc cells.

[0005] After in-depth research and multiple experiments, the inventor team of this case proposed an application plan for functionalized cerium oxide nanoparticles in the treatment of intervertebral disc degeneration, and provided detailed drug-carrying material basis and related treatment strategies to improve biocompatibility, adjustable antioxidant properties and therapeutic effects on potential intervertebral disc degeneration. Summary of the invention

[0006] The purpose of the present invention is to provide an application scheme of functionalized cerium oxide nanoparticles in the treatment of intervertebral disc degeneration, so as to make such application research involved in the prior art more specific, effective and feasible, thereby better overcoming the problem that intervertebral disc degeneration leads to damage to intervertebral disc cells, destruction of extracellular matrix, and ultimately affects the structure of the intervertebral disc.

[0007] The technical solution of the present invention is: the application of functionalized cerium oxide nanoparticles in the treatment of intervertebral disc degeneration, which is characterized in that: the intervertebral disc degeneration is caused by oxidative stress, the functionalized cerium oxide nanoparticles are synthesized based on amide reaction, and the preparation process of the functionalized cerium oxide nanoparticles combined with loaded drugs is as follows:

[0008] (i) preparing cerium oxide nanoparticles by a solvothermal method or a coprecipitation method, and fully dissolving the cerium oxide nanoparticles in an organic solvent to obtain an aqueous solution of cerium oxide nanoparticles;

[0009] (ii) adding an appropriate amount of loaded drug A to the prepared aqueous solution of cerium oxide nanoparticles under the action of ultrasound at a frequency of 20 to 40 kHz, and fully mixing for 0.5 to 1.5 hours, and then adding ultrapure water to redissolve the cerium oxide nanoparticles after fully evaporating the organic solvent in the mixture by an electric rotary heating device, and removing the residual loaded drug A by centrifugal separation to obtain an aqueous solution of cerium oxide nanoparticles coated with loaded drug A, and dropping an appropriate amount of loaded drug B into the aqueous solution of cerium oxide nanoparticles coated with loaded drug A, and shaking overnight at room temperature to obtain an intermediate product;

[0010] (iii) removing the unbound loaded drug B in the obtained intermediate product by centrifugal separation to obtain the final functionalized cerium oxide nanoparticles for treating intervertebral disc degeneration caused by oxidative stress.

[0011] In the above technical solution, the loaded drug B can also be added first and then the loaded drug A is added. The specific process is as follows:

[0012] (i) preparing cerium oxide nanoparticles by a solvothermal method or a coprecipitation method, and fully dissolving the cerium oxide nanoparticles in an organic solvent to obtain an aqueous solution of cerium oxide nanoparticles.

[0013] (ii) adding an appropriate amount of loaded drug B to the prepared aqueous solution of cerium oxide nanoparticles under the action of ultrasound at a frequency of 20 to 40 kHz, and fully mixing for 0.75 to 1.25 hours. After fully evaporating the organic solvent in the mixture by an electric rotary heating device, adding ultrapure water to re-dissolve the cerium oxide nanoparticles, removing the residual loaded drug B by centrifugal separation, and obtaining an aqueous solution of cerium oxide nanoparticles coated with loaded drug B. Dropping an appropriate amount of loaded drug A into the aqueous solution of cerium oxide nanoparticles coated with loaded drug B, shaking overnight at room temperature of 22 to 24°C, and obtaining an intermediate product.

[0014] (iii) removing the unbound loaded drug B in the obtained intermediate product by centrifugal separation to obtain the final functionalized cerium oxide nanoparticles for treating intervertebral disc degeneration caused by oxidative stress.

[0015] Furthermore, in the use of the functionalized cerium oxide nanoparticles in treating intervertebral disc degeneration, the organic solvent is dichloromethane.

[0016] Furthermore, the above-mentioned functionalized cerium oxide nanoparticles are used in the treatment of intervertebral disc degeneration, wherein: the loaded drug A is a carboxyl-containing polymer material, and the carboxyl-containing polymer material includes but is not limited to polyethylene glycol-carboxyl and palmitic acid.

[0017] Furthermore, the above-mentioned functionalized cerium oxide nanoparticles are used to treat intervertebral disc degeneration, wherein: the loaded drug B is an amino-containing polymer material, and the amino-containing polymer material includes but is not limited to polyethylene glycol-amino, deferoxamine mesylate, and dopamine.

[0018] Furthermore, in the application of the functionalized cerium oxide nanoparticles in the treatment of intervertebral disc degeneration, the molar mass of the loaded drug A and the loaded drug B is 1:1.

[0019] Furthermore, in the application of the functionalized cerium oxide nanoparticles in the treatment of intervertebral disc degeneration, the temperature range of the shaking table overnight is 21-25° C., and the rotation speed of the shaking table is 100-300 rpm.

[0020] By adopting the technical scheme of the present invention, first, the surface of cerium oxide nanoparticles is modified by a polymer material containing carboxyl or amino groups, which can improve its drug carrier performance and have good biocompatibility and water solubility; then, the carboxyl or amino-containing polymer material is chemically reacted to obtain functionalized cerium oxide nanoparticles, which have stable covalent bonds and can not only achieve effective loading and sustained release of drugs, but also improve stability and drug loading capacity, providing more possibilities for the precise delivery of therapeutic drugs.

[0021] Compared with the prior art, the technical solution of the present invention is used to prepare a drug for treating intervertebral disc degeneration caused by oxidative stress by using functionalized cerium oxide nanoparticles, which can treat intervertebral disc degeneration and fundamentally repair the degenerated intervertebral disc without side effects, thereby relieving intervertebral disc-derived pain. At the same time, the technical solution of the present invention can also be combined with other treatment methods, such as immunomodulators, biological agents, etc., to achieve a better recovery effect through synergistic treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a synthetic electron microscope image of the functionalized cerium oxide nanoparticles of the present invention;

[0023] Figure 2 is the ultraviolet absorption spectrum of the functionalized cerium oxide nanoparticles of the present invention;

[0024] Figure 3 This is a trend comparison imaging diagram of the intervertebral disc structure of rats after X-ray treatment of control group 1 and control group 2 of the present invention;

[0025] Figure 4 The trend comparison imaging diagram of the intervertebral disc structure of rats after nuclear magnetic resonance (MRI) treatment of control group 1 and control group 2 of the present invention;

[0026] Figure 5 This is a trend comparison imaging diagram of rat intervertebral disc tissue after hematoxylin-eosin staining of control group 1 and control group 2 of the present invention;

[0027] Figure 6 This is a trend comparison imaging diagram of rat intervertebral disc tissue after the control group 1 and the control group 2 of the present invention were stained with safranin fast green;

[0028] Figure 7 This is a trend comparison imaging diagram of type II collagen staining in intervertebral disc tissues of control group 1 and control group 2 detected by immunohistochemistry technology in the present invention;

[0029] Figure 8 This is a trend comparison imaging diagram of matrix metalloproteinase 13 staining in the intervertebral disc tissue of control group 1 and control group 2 detected by immunohistochemistry technology in the present invention. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with specific examples, but it should not be understood that the protection scope of the above subject matter of the present invention is limited to the following examples. Among them, the raw materials used are commercially available or homemade using methods known in the industry.

[0031] First, cerium oxide nanoparticles are prepared by a solvothermal method or a coprecipitation method, and then the prepared cerium oxide nanoparticles are fully dissolved in dichloromethane. Under the intermittent action of ultrasound with a frequency of 30 kHz (i.e., ultrasound is generated alternately with working for three seconds and stopping for one second, and intermittent operation can effectively save energy, extend the service life of ultrasonic equipment, and can also improve the uniformity of material mixing, increase the particle dispersion and accelerate chemical reactions), an excess of polyethylene glycol-carboxyl (PEG-COOH) is added, and ultrasonic mixing is performed for 1 hour. The organic solvent is evaporated by rotary heating, and ultrapure water is added to dissolve the oxygen again. CeO2 nanoparticles were prepared and the residual polyethylene glycol-carboxyl (PEG-COOH) was removed by centrifugation to obtain cerium oxide nanoparticles coated with polyethylene glycol-carboxyl (PEG-COOH); under the intermittent action of ultrasound at a frequency of 40 kHz, deferoxamine mesylate (DFOM) was dropped into the aqueous solution of cerium oxide nanoparticles coated with polyethylene glycol-carboxyl (PEG-COOH), and the mixture was shaken at 25°C overnight with a shaker speed of 300 rpm. Finally, the unbound loaded drug was removed by centrifugation to obtain functionalized cerium oxide nanoparticles (DFOM@CeO2).

[0032] like Figure 1 As shown, the successful synthesis of functionalized cerium oxide nanoparticles was confirmed by electron microscopy. Figure 2 As shown, the successful synthesis of the functionalized cerium oxide nanoparticles and the normal law of the corresponding amide reaction were confirmed by UV absorption spectroscopy experiments.

[0033] Control group 1

[0034] Comparative Example 1-A

[0035] A rat coccygeal acupuncture model of intervertebral disc degeneration was established without coccygeal puncture or drug intervention and maintained for 4 weeks.

[0036] Comparative Example 2-A

[0037] A rat coccygeal acupuncture model of intervertebral disc degeneration was established by coccygeal puncture without drug intervention for 4 weeks.

[0038] Comparative Example 3-A

[0039] A rat coccygeal acupuncture disc degeneration model was established. After coccygeal puncture, 2 μl DFOM was injected into the rat coccygeal disc for treatment; the injection was performed once a week for 4 weeks.

[0040] Comparative Example 4-A

[0041] A rat coccygeal acupuncture model of intervertebral disc degeneration was established. After coccygeal puncture, 2 μl CeO2 was injected into the rat coccygeal disc for treatment; the injection was given once a week for 4 weeks.

[0042] Example 1-A

[0043] A rat coccygeal acupuncture model of intervertebral disc degeneration was established. After coccygeal puncture, 2 μl DFOM@CeO2 was injected into the rat coccygeal disc for treatment; the injection was performed once a week for 4 weeks.

[0044] Control group 2

[0045] Comparative Example 1-B

[0046] A rat coccygeal acupuncture model of intervertebral disc degeneration was established without coccygeal puncture or drug intervention and maintained for 8 weeks.

[0047] Comparative Example 2-B

[0048] A rat coccygeal acupuncture model of intervertebral disc degeneration was established by coccygeal puncture without drug intervention for 8 weeks.

[0049] Comparative Example 3-B

[0050] A rat coccygeal acupuncture disc degeneration model was established. After coccygeal puncture, 2 μl DFOM was injected into the rat coccygeal disc for treatment; the injection was performed once a week for 8 weeks.

[0051] Comparative Example 4-B

[0052] A rat coccygeal acupuncture model of intervertebral disc degeneration was established. After coccygeal puncture, 2 μl CeO2 was injected into the rat coccygeal disc for treatment; the injection was given once a week for 8 weeks.

[0053] Example 1-B

[0054] A rat coccygeal acupuncture model of intervertebral disc degeneration was established. After coccygeal puncture, 2 μl DFOM@CeO2 was injected into the rat coccygeal disc for treatment; the injection was performed once a week for 8 weeks.

[0055] like Figure 3 and Figure 4 As shown, imaging was used to evaluate the application effect. The intervertebral disc structure of rats in control group 1 and control group 2 was imaged by X-ray and MRI (magnetic resonance imaging) to evaluate the morphological changes, degeneration degree and disc height changes of the intervertebral disc. In comparative example 1B and comparative example 2B, the X-ray showed a decrease in disc height, and the MRI image showed a decrease in signal intensity, indicating that the disc was dehydrated; as time went on, the signal intensity of control group 1 and control group 2 decreased to varying degrees; but compared with comparative example 3-A, comparative example 4-A, and example 1-A, and comparative example 3-B, comparative example 4-B, and example 1-B, the disc height of each injection group recovered to varying degrees; the MRI results showed that example 1-A and example 1-B showed the strongest signal intensity, showing the best in vivo therapeutic effect.

[0056] like Figure 5and Figure 6 As shown, the application effect was evaluated by histology, and the intervertebral disc tissues of rats in control group 1 and control group 2 were respectively stained with hematoxylin-eosin and safranin fast green to observe the cell morphology, matrix degradation, and changes in the number of cells in each intervertebral disc tissue. Among them, safranin fast green staining at two time points showed that the nucleus pulposus and annulus fibrosus tissue boundaries of Comparative Example 1-A and Comparative Example 1-B were obvious, the nucleus pulposus and cartilage endplates were mainly stained with safranin, and the lamellar annulus fibrosus was mainly stained with fast green. In contrast, the boundaries between the nucleus pulposus and the cartilage endplates of Comparative Example 1B and Comparative Example 2B were unclear, the safranin staining area was significantly reduced, most of it was replaced by a lighter green, and the intervertebral disc height was significantly reduced; the boundaries between the nucleus pulposus and the cartilage endplates of Example 1-A and Example 1-B were clear, and the safranin staining area was significantly larger than the corresponding Comparative Example 3-A, Comparative Example 4-A and Comparative Example 3-B, Comparative Example 4-B, and Example 1-A and Example 1-B showed that the recovery effect of its intervertebral disc tissue in vivo treatment was the best.

[0057] like Figure 7 and Figure 8 As shown, the application effect was evaluated histologically, and the intervertebral disc tissue of rats was taken and immunohistochemistry was used to detect type II collagen and matrix metalloproteinase 13 in the intervertebral disc tissue. Type II collagen and matrix metalloproteinase 13 are markers of intervertebral disc matrix metabolism. Type II collagen positively promotes matrix synthesis, while matrix metalloproteinase 13 promotes matrix degradation. Compared with Comparative Example 1-A and Comparative Example 1-B, the expression of type II collagen in the remaining Comparative Example 2-A, Comparative Example 2-B, Comparative Example 3-A, Comparative Example 3-B, Comparative Example 4-A, Comparative Example 4-B and Example 1-A and Example 1-B all decreased, but the expression of type II collagen in Example 1-A and Example 1-B was the highest; on the contrary, the expression of matrix metalloproteinase 13 in Comparative Example 2-A, Comparative Example 2-B, Comparative Example 3-A, Comparative Example 3-B, Comparative Example 4-A, Comparative Example 4-B and Example 1-A and Example 1-B all increased, but the expression level of matrix metalloproteinase 13 in Example 1-A and Example 1-B was the lowest. It can be seen that Example 1-A and Example 1-B show the best recovery effect of their intervertebral disc tissue in vivo treatment.

[0058] In summary, the use of functionalized cerium oxide nanoparticles to prepare drugs for treating intervertebral disc degeneration caused by oxidative stress can effectively treat intervertebral disc degeneration with significant therapeutic effects.

[0059] The technical scheme, working process and implementation effect of the present invention are described in detail above. It should be noted that what is described is only a typical example of the present invention. In addition, the present invention may also have many other specific implementation methods. Any technical scheme formed by equivalent replacement or equivalent transformation falls within the scope of protection required by the present invention.

Claims

1. Application of functionalized cerium oxide nanoparticles in the treatment of intervertebral disc degeneration, characterized by: The intervertebral disc degeneration is intervertebral disc degeneration caused by oxidative stress. The functionalized cerium oxide nanoparticles are synthesized based on amide reaction. The preparation process of the functionalized cerium oxide nanoparticles combined with loaded drugs is as follows: Step S1: preparing cerium oxide nanoparticles by a solvothermal method or a coprecipitation method, and fully dissolving the cerium oxide nanoparticles in an organic solvent to obtain a cerium oxide nanoparticle aqueous solution; Step S2: under the action of ultrasound at a frequency of 20 to 40 kHz, add an appropriate amount of loaded drug A to the aqueous solution of cerium oxide nanoparticles prepared in step S1, fully mix for 0.5 to 1.5 hours, fully evaporate the organic solvent in the mixture by an electric rotary heating device, then add ultrapure water to re-dissolve the cerium oxide nanoparticles, remove the residual loaded drug A by centrifugal separation, and obtain an aqueous solution of cerium oxide nanoparticles coated with loaded drug A, drop an appropriate amount of loaded drug B into the aqueous solution of cerium oxide nanoparticles coated with loaded drug A, and shake overnight at room temperature to obtain an intermediate product; Step S3: removing the unbound loaded drug in the intermediate product obtained in step S2 by centrifugal separation to obtain the final functionalized cerium oxide nanoparticles for treating intervertebral disc degeneration caused by oxidative stress.

2. The use of the functionalized cerium oxide nanoparticles in the treatment of intervertebral disc degeneration according to claim 1, characterized in that: The operation sequence of step S2 also includes: adding an appropriate amount of loaded drug B to the aqueous solution of cerium oxide nanoparticles prepared in step S1 under the action of ultrasound with a frequency of 20 to 40 kHz, fully mixing for 0.75 to 1.25 hours, fully evaporating the organic solvent in the mixture through an electric rotary heating device, then adding ultrapure water to re-dissolve the cerium oxide nanoparticles, removing the residual loaded drug B through centrifugal separation, obtaining an aqueous solution of cerium oxide nanoparticles coated with loaded drug B, dripping an appropriate amount of loaded drug A into the aqueous solution of cerium oxide nanoparticles coated with loaded drug B, shaking overnight at room temperature of 22 to 24° C., and obtaining an intermediate product.

3. The use of the functionalized cerium oxide nanoparticles according to claim 1 in treating intervertebral disc degeneration, characterized in that: In step S1, the organic solvent is dichloromethane.

4. The use of the functionalized cerium oxide nanoparticles in treating intervertebral disc degeneration according to claim 1, characterized in that: In step S2, the loaded drug A is a polymer material containing a carboxyl group.

5. The use of the functionalized cerium oxide nanoparticles in treating intervertebral disc degeneration according to claim 4, characterized in that: The carboxyl-containing polymer material includes but is not limited to polyethylene glycol-carboxyl and palmitic acid.

6. The use of the functionalized cerium oxide nanoparticles in treating intervertebral disc degeneration according to claim 1, characterized in that: In step S2, the loaded drug B is a polymer material containing amino groups.

7. The use of the functionalized cerium oxide nanoparticles in treating intervertebral disc degeneration according to claim 6, characterized in that: The amino-containing polymer material includes, but is not limited to, polyethylene glycol-amino, deferoxamine mesylate, and dopamine.

8. The use of the functionalized cerium oxide nanoparticles in treating intervertebral disc degeneration according to claim 1, characterized in that: The molar mass of the loaded drug A and the loaded drug B is 1:

1.

9. The use of the functionalized cerium oxide nanoparticles in treating intervertebral disc degeneration according to claim 1, characterized in that: The temperature range of the shaking table overnight is 21-25° C., and the rotation speed of the shaking table is 100-300 rpm.

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