Use of konjac glucomannan and derivative thereof, glucomannan caprylate, nanoparticles and coating obtained from glucomannan caprylate, preparation method for nanoparticles, and use of glucomannan caprylate, nanoparticles or coating

Abstract

A use of konjac glucomannan and a derivative thereof, glucomannan caprylate, nanoparticles and a coating obtained from the glucomannan caprylate, a preparation method for the nanoparticles, and a use of the glucomannan caprylate, the nanoparticles or the coating, relating to the technical field of medicine and material chemistry. The konjac glucomannan and the derivative thereof can be used as extracellular matrix fillers, and glucomannan caprylate obtained by modifying the konjac glucomannan by means of an esterification reaction, and nanoparticles or a glucomannan caprylate coating further obtained from the glucomannan caprylate can both be used for preparing drugs or materials for regulating functions of nucleus pulposus cells, and in particular, can be used for preparing drugs or materials for relieving or treating intervertebral disc degeneration. The glucomannan caprylate has the characteristics of being non-toxic, easily available and highly histocompatible, and can be prepared into different dosage forms according to requirements. The present disclosure provides a novel idea to improve the microenvironment of intervertebral disc degeneration, and also provides a novel use of a biomaterial based on a polysaccharide derivative.

Description

Application of konjac glucomannan and its derivatives, glucomannan caprylate and its nanoparticles, coating and its preparation method and application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202311098026X filed with the State Intellectual Property Office of China on August 29, 2023, entitled “Application of konjac glucomannan and its derivatives, glucomannan caprylate and its nanoparticles, coating and its preparation method and application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The present disclosure relates to the technical fields of medicine and material chemistry, and in particular to applications of konjac glucomannan and its derivatives, glucomannan caprylate and its nanoparticles, a coating and a preparation method and application thereof. Background Art

[0004] Lumbar disc degeneration is one of the most important causes of low back pain. Epidemiological surveys show that approximately 80% of adults have experienced more than one episode of low back pain. As a musculoskeletal disease closely related to age and physical activity, with the rapid aging of the population and the increasing popularity of exercise, more and more individuals will face disc degeneration and the associated symptoms of nerve entrapment. Disc degeneration is typically characterized by progressive degeneration of disc tissue, particularly the nucleus pulposus. Currently, medication and physical therapy can only improve symptoms in some patients but cannot effectively intervene in the progression of tissue degeneration, leaving most patients with surgery as the only definitive treatment. However, local mechanical instability caused by postoperative tissue loss, persistent degeneration of the remaining degenerated nucleus pulposus tissue, and postoperative microenvironmental disturbances are significant factors that exacerbate tissue degeneration, leading to reherniation and subsequent surgery. Effectively regulating the postoperative degenerative microenvironment to prevent recurrence and promote nucleus pulposus repair is currently a mainstream strategy for accelerated recovery.

[0005] Pathological studies have found that when the intervertebral disc degenerates, the extracellular matrix (ECM) of the nucleus pulposus tissue will accelerate degradation, the number of nucleus pulposus cells will decrease, and the phenotype will begin to be lost. Therefore, in clinical practice, typical changes such as weakening of the nucleus pulposus MR T2 signal and reduced intervertebral space height can be observed. Basic research further reveals that as degeneration progresses, matrix decomposition-related enzymes (such as MMP2, HYAL2, etc.) in the nucleus pulposus tissue gradually become highly expressed, while the content of ECM components such as collagen and hyaluronic acid will gradually decrease. In view of the changing characteristics of ECM, researchers have proposed the use of injectable and filling materials to fill the defects after intervertebral disc surgery in order to maintain height, improve the degenerative microenvironment, and improve treatment efficiency. However, there are currently no reports of effective related materials.

[0006] In view of this, the present disclosure is proposed.

[0007] Summary of the Invention

[0008] The present disclosure provides an application of konjac glucomannan and its derivatives as extracellular matrix fillers.

[0009] Optionally, the derivative of konjac glucomannan is a modified konjac glucomannan.

[0010] Optionally, the modification is carried out by modifying the polysaccharide molecules to introduce additional functional groups; optionally, the functional groups are acetyl groups or octanoyl groups.

[0011] Optionally, the derivative of konjac glucomannan is glucomannan caprylate.

[0012] Optionally, the substitution rate of glucomannan caprylate is 1.0-2.5; optionally, the substitution rate of glucomannan caprylate is 1.5-2.5; optionally, the substitution rate of glucomannan caprylate is 1.6-1.8.

[0013] Optionally, the konjac glucomannan and its derivatives are in the form of nanoparticles or coatings.

[0014] The present disclosure provides glucomannan caprylate, which is obtained by modifying konjac glucomannan through an esterification reaction.

[0015] Optionally, the substitution rate of glucomannan octanoate is 1.0-2.5; Optionally, the substitution rate of glucomannan octanoate is 1.5-2.5; Optionally, the substitution rate of glucomannan octanoate is 1.6-1.8

[0016] The present disclosure provides a method for synthesizing glucomannan caprylate according to the aforementioned embodiment, comprising the following steps: modifying konjac glucomannan with an octanoyl group.

[0017] Alternatively, konjac glucomannan is reacted with trifluoroacetic anhydride and octanoic acid, the reaction solution obtained by the reaction is subjected to solid-liquid separation, the liquid phase is collected for a first alcohol precipitation, a first precipitate obtained by the first alcohol precipitation is dissolved in chloroform to remove products insoluble in chloroform, and then the liquid phase dissolved in chloroform is subjected to a second alcohol precipitation, and the second precipitate obtained is collected.

[0018] Optionally, konjac glucomannan is added to a first reaction solution obtained by reacting trifluoroacetic anhydride and octanoic acid, and the reaction is continued to obtain a second reaction solution; the second reaction solution is subjected to solid-liquid separation, the liquid phase is collected and subjected to a first alcohol precipitation, a first precipitate obtained by the first alcohol precipitation is dissolved in chloroform to remove products insoluble in chloroform, and then the liquid phase dissolved in chloroform is subjected to a second alcohol precipitation, and the obtained second precipitate is collected.

[0019] Optionally, the reaction of trifluoroacetic anhydride and octanoic acid is carried out at 50-55° C. for 15-25 min.

[0020] Optionally, the reaction of konjac glucomannan with the first reaction solution is carried out at 50-55° C. for 3-4 hours.

[0021] Optionally, the usage ratio of konjac glucomannan, trifluoroacetic anhydride and octanoic acid is 500 mg: (8-12) mL: (12-14) mL.

[0022] The present disclosure provides a nanoparticle obtained by nano-processing the glucomannan caprylate according to the aforementioned embodiment.

[0023] The present disclosure provides a method for preparing nanoparticles according to the aforementioned embodiment, comprising the following steps: subjecting glucomannan caprylate to nano-processing.

[0024] Optionally, glucomannan caprylate is subjected to ultrasonic nano-processing in the presence of polyvinyl alcohol, and the organic solvent is removed and the product is dried.

[0025] The present disclosure provides a glucomannan caprylate coating, which is obtained by coating the glucomannan caprylate according to the aforementioned embodiment.

[0026] The present disclosure provides a use of the glucomannan caprylate according to the aforementioned embodiment, or the nanoparticles according to the aforementioned embodiment, or the glucomannan caprylate coating according to the aforementioned embodiment in preparing a drug or material for regulating the function of nucleus pulposus cells.

[0027] Optionally, glucomannan caprylate or nanoparticles or glucomannan caprylate coating is used to prepare drugs or materials for regulating the adhesion and physiological functions of nucleus pulposus cells.

[0028] Alternatively, glucomannan caprylate or nanoparticles or glucomannan caprylate coating is used to prepare drugs or materials that protect the phenotype of nucleus pulposus cells by intervening in the cell-matrix and cell-cell interactions.

[0029] Optionally, glucomannan octanoate or nanoparticles or glucomannan octanoate coating is used to prepare a drug or material that highly expresses a nucleus pulposus phenotype. Optionally, the nucleus pulposus phenotype includes Collagen 2.

[0030] Optionally, glucomannan caprylate or nanoparticles or glucomannan caprylate coating is used to prepare a drug or material enriched in milk fat globule epidermal growth factor 8 secreted by nucleus pulposus cells; wherein the nucleus pulposus phenotype includes Collagen 2.

[0031] In an alternative embodiment, the material is a repair material.

[0032] The present disclosure provides a use of the glucomannan caprylate according to the aforementioned embodiment, or the nanoparticles according to the aforementioned embodiment, or the glucomannan caprylate coating according to the aforementioned embodiment in preparing a drug or material for alleviating or treating intervertebral disc degeneration.

[0033] Optionally, glucomannan caprylate or nanoparticles or glucomannan caprylate coating is used to prepare a drug for delaying water loss in nucleus pulposus tissue, or for preparing a drug for alleviating reduction in intervertebral disc height.

[0034] Optionally, the material is a repair material.

[0035] The present disclosure provides a drug, wherein the active ingredient of the drug includes the glucomannan caprylate of the aforementioned embodiment, or includes the glucomannan caprylate nanoparticles of the aforementioned embodiment, or the glucomannan caprylate coating of the aforementioned embodiment.

[0036] The present disclosure provides a restorative material, wherein the active component of the restorative material includes the glucomannan octanoate of the aforementioned embodiment, or includes the nanoparticles of the aforementioned embodiment, or the glucomannan octanoate coating of the aforementioned embodiment.

[0037] The present disclosure provides a method for regulating nucleus pulposus cell function, comprising administering to a subject in need thereof or applying to a subject in need thereof a therapeutically effective amount of glucomannan caprylate according to the aforementioned embodiment, or the nanoparticles according to the aforementioned embodiment, or the glucomannan caprylate coating according to the aforementioned embodiment.

[0038] Optionally, the administering or applying is performed in the form of a drug or material, respectively.

[0039] Optionally, the regulation of nucleus pulposus cell function is selected from one or more of the following:

[0040] Regulates the adhesion and physiological functions of nucleus pulposus cells;

[0041] Protecting the phenotype of nucleus pulposus cells by intervening in cell-matrix and cell-cell interactions;

[0042] Highly expressing nucleus pulposus phenotype; optionally, the nucleus pulposus phenotype includes Collagen2;

[0043] Enriched with milk fat globule epidermal growth factor 8 secreted by nucleus pulposus cells; wherein the nucleus pulposus phenotype includes Collagen 2.

[0044] Optionally, the material is a repair material.

[0045] The present disclosure provides a method for alleviating or treating intervertebral disc degeneration, comprising administering to a subject in need thereof or applying to a subject in need thereof a therapeutically effective amount of glucomannan caprylate according to the aforementioned embodiment, or the nanoparticles according to the aforementioned embodiment, or the glucomannan caprylate coating according to the aforementioned embodiment.

[0046] Optionally, the administering or applying is performed in the form of a drug or material, respectively.

[0047] Optionally, the relief or treatment of intervertebral disc degeneration is selected from delaying the loss of water content in the nucleus pulposus tissue and / or reducing the decrease in intervertebral disc height.

[0048] Optionally, the material is a repair material.

[0049] The present disclosure provides the glucomannan caprylate according to the aforementioned embodiment, the nanoparticle according to the aforementioned embodiment, or the glucomannan caprylate coating according to the aforementioned embodiment for use in regulating nucleus pulposus cell function.

[0050] Optionally, the administering or applying is performed in the form of a drug or material, respectively.

[0051] Optionally, the regulation of nucleus pulposus cell function is selected from one or more of the following:

[0052] Regulates the adhesion and physiological functions of nucleus pulposus cells;

[0053] Protecting the phenotype of nucleus pulposus cells by intervening in cell-matrix and cell-cell interactions;

[0054] Highly expressing nucleus pulposus phenotype; optionally, the nucleus pulposus phenotype includes Collagen2;

[0055] Enriched with milk fat globule epidermal growth factor 8 secreted by nucleus pulposus cells; wherein the nucleus pulposus phenotype includes Collagen 2.

[0056] Optionally, the material is a repair material.

[0057] The present disclosure provides the glucomannan caprylate according to the aforementioned embodiment, or the nanoparticles according to the aforementioned embodiment, or the glucomannan caprylate coating according to the aforementioned embodiment, for use in alleviating or treating intervertebral disc degeneration.

[0058] Optionally, the administering or applying is performed in the form of a drug or material, respectively.

[0059] Optionally, the relief or treatment of intervertebral disc degeneration is selected from delaying the loss of water content in the nucleus pulposus tissue and / or reducing the decrease in intervertebral disc height.

[0060] Optionally, the material is a repair material. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present disclosure and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without creative work.

[0062] FIG1 is a mass spectrogram of the enzymatic hydrolysis product of hyaluronic acid in Example 1;

[0063] FIG2 is a mass spectrogram of the enzymatic hydrolysis product of KGM in Example 1;

[0064] FIG3 is a comparison diagram of infrared spectra of KGM and GMOC in Example 2;

[0065] FIG4 is a comparison of the nuclear magnetic resonance images of KGM and GMOC in Example 2;

[0066] FIG5 is a diagram showing the cell morphology changes after co-culture of GMOC and nucleus pulposus cells in Example 3;

[0067] FIG6 is a diagram showing the results of GMOC regulation of cell phenotypes in Example 3;

[0068] FIG7 is a diagram showing the enrichment of Mfge8 protein by GMOC in Example 3;

[0069] FIG8 is a diagram showing the results of nuclear magnetic resonance imaging in Example 4;

[0070] FIG9 is a diagram showing the X-ray imaging results in Example 4. DETAILED DESCRIPTION

[0071] To make the purpose, technical solutions, and advantages of the embodiments of the present disclosure more clear, the technical solutions in the embodiments of the present disclosure are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer were followed. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0072] Unless otherwise specified, the terms "konjac glucomannan" and "konjac polysaccharide" are used interchangeably in the present disclosure. Both refer to a water-soluble high molecular weight polysaccharide, which is a polymer of glucose and mannan linked by β-1,4-glycosidic bonds.

[0073] Unless otherwise specified, the "nanoparticles" or "nanoparticles of glucomannan octanoate" disclosed herein can be used interchangeably, and both refer to substances obtained by nano-processing glucomannan octanoate by methods known in the art. The manner and conditions of the nano-processing are not limited to those listed in the present invention, and similar processing can also be performed with reference to relevant prior art.

[0074] Unless otherwise specified, the substitution rate disclosed herein refers to the number of hydroxyl groups at positions 2, 3, and 6 of the sugar ring replaced by corresponding groups during the esterification reaction of konjac glucomannan. It is understood that when the hydroxyl groups at positions 2, 3, and 6 of the sugar ring are completely replaced by corresponding groups, the substitution rate is 3.

[0075] The substitution rate can be calculated by using hydrogen nuclear magnetic resonance spectroscopy, and the specific formula is: (integral peak area of ​​the ester methyl proton peak in the reaction product / 3) / (integral peak area of ​​the proton peak on the sugar ring / 7).

[0076] The following describes in detail the applications of konjac glucomannan and its derivatives, glucomannan octanoate (GMOC) and its nanoparticles, coatings, and preparation methods and applications thereof provided by the present invention.

[0077] The present invention provides applications of konjac glucomannan and its derivatives, glucomannan caprylate and its nanoparticles, a coating and a preparation method and application thereof.

[0078] The present disclosure provides a use of konjac glucomannan (KGM) and its derivatives as extracellular matrix fillers.

[0079] The rich and varied molecular configurations endow polysaccharides with enormous potential in tissue repair. Konjac glucomannan, a water-soluble high-molecular-weight polysaccharide, is a polymer of glucose and mannan linked by β-1,4-glycosidic bonds found in konjac tubers. Furthermore, specific biological effects can be achieved by modifying the polysaccharide molecules with additional functional groups. For example, multiple acetyl groups can be added to the polysaccharide through esterification to modulate cellular function.

[0080] At the same time, konjac glucomannan has excellent anti-enzymatic degradation properties and can stay stably in the local area for a long time to replenish the ECM lost due to degeneration. Ultimately, it achieves the therapeutic goal of delaying intervertebral disc degeneration by improving the microenvironment of intervertebral disc degeneration tissue.

[0081] The present invention prepares a filling material by selecting konjac polysaccharide (KGM) and its derivatives composed of non-nucleus pulposus ECM, which can effectively resist degradation by common matrix-degrading enzymes in situ and achieve long-term retention of the filling material in the defect site.

[0082] In an optional embodiment, the derivative of konjac glucomannan is a modified konjac glucomannan.

[0083] In an optional embodiment, the modification is to introduce additional functional groups into the polysaccharide molecules.

[0084] The functional group may be an acetyl group or an octanoyl group.

[0085] In an optional embodiment, the derivative of konjac glucomannan is glucomannan caprylate.

[0086] In an optional embodiment, the substitution rate of the glucomannan caprylate is 1.0-2.5. For example, the substitution rate of the glucomannan caprylate is 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 or 2.4, or any range therein, such as 1.5-2.5, optionally 1.6-1.8. In a specific embodiment, the substitution rate of glucomannan caprylate is about 1.7, for example, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79 or 1.80, or any range therebetween.

[0087] It is believed, without being bound by theory, that the substitution rate will have a certain impact on subsequent biological effects. If the substitution rate is too low, the polarity of the product will change. During the purification process of dissolving in chloroform and then precipitating with alcohol, the low-substitution product will be eliminated in the chloroform solution as an insoluble form. At the same time, too low a degree of substitution also poses certain challenges to the preparation process during subsequent coating and nanoparticle preparation. It is known in the art that the substitution rate can be controlled by fixing the reaction time and reactant equivalents.

[0088] In an alternative embodiment, the konjac glucomannan and its derivatives are in the form of nanoparticles or coatings.

[0089] The present disclosure also provides glucomannan caprylate, which is obtained by modifying konjac glucomannan through an esterification reaction.

[0090] In an optional embodiment, the substitution rate of the glucomannan caprylate is 1.0-2.5. For example, the substitution rate of the glucomannan caprylate is 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 or 2.4, or any range therein, such as 1.5-2.5, optionally 1.6-1.8. In a specific embodiment, the substitution rate of glucomannan caprylate is about 1.7, for example, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79 or 1.80, or any range therebetween.

[0091] Accordingly, the synthesis method of the glucomannan caprylate may include the following steps: modifying konjac glucomannan with an octanoyl group.

[0092] In some embodiments, konjac glucomannan is reacted with trifluoroacetic anhydride (TFAA) and octanoic acid, the reaction solution obtained by the reaction is subjected to solid-liquid separation, the liquid phase is collected for a first alcohol precipitation, the first precipitate obtained by the first alcohol precipitation is dissolved in chloroform to remove products insoluble in chloroform, and then the liquid phase dissolved in chloroform is subjected to a second alcohol precipitation, and the second precipitate obtained is collected. The second precipitate can be used as glucomannan octanoate.

[0093] The reaction equations involved in the above process are shown below:

[0094] The trifluoroacetic anhydride is primarily used as a catalyst in the synthesis reaction, efficiently producing the target product. The use of octanoic acid ensures the simplicity, stability, and feasibility of the reaction while also avoiding the formation of byproducts.

[0095] It should be noted that the product obtained by the first alcohol precipitation is glucomannan caprylate containing more impurities, and a relatively pure product can be obtained by repurifying the product.

[0096] In some embodiments, konjac glucomannan can be added to a first reaction solution obtained by reacting trifluoroacetic anhydride and octanoic acid, and the reaction is continued to obtain a second reaction solution; the second reaction solution is separated into solid and liquid, the liquid phase is collected for a first alcohol precipitation, the first precipitate obtained by the first alcohol precipitation is dissolved in chloroform to remove products insoluble in chloroform, and then the liquid phase dissolved in chloroform is subjected to a second alcohol precipitation, and the obtained second precipitate is collected.

[0097] For reference, the reaction of trifluoroacetic anhydride and octanoic acid can be carried out at 50-55°C, such as 50°C, 51°C, 52°C, 53°C, 54°C or 55°C, for 15-25 minutes, such as 15 minutes, 18 minutes, 20 minutes, 22 minutes or 25 minutes. For example, the reaction temperature can be in the range of 51-54°C, 52-53°C, 51-53°C or any value in the defined range. For example, the reaction time can be 18-22 minutes, 20-22 minutes, 15-22 minutes or any value in the defined range. In some embodiments, the reaction of trifluoroacetic anhydride and octanoic acid is carried out at 50°C for 20 minutes.

[0098] The reaction of konjac glucomannan with the first reaction solution can be carried out at 50-55°C (such as 50°C, 51°C, 52°C, 53°C, 54°C or 55°C, etc.) for 3-4h (such as 3h, 3.2h, 3.5h, 3.8h or 4h, etc.). For example, the reaction temperature can be in the range of 51-54°C, 52-53°C, 51-53°C or any value in the defined range. For example, the reaction time can be 3-3.8h, 3.2-3.5h, 3.2-3.7h or any value in the defined range. In some embodiments, the reaction of konjac glucomannan with the first reaction solution is carried out at 50°C for 3.5h.

[0099] The usage ratio of konjac glucomannan, trifluoroacetic anhydride and caprylic acid can be 500 mg:(8-12) mL:(12-14) mL, such as 500 mg:8 mL:12 mL, 500 mg:8 mL:13 mL, 500 mg:8 mL:14 mL, 500 mg:10 mL:12 mL, 500 mg:10 mL:13 mL, 500 mg:10 mL:14 mL, 500 mg:12 mL:12 mL, 500 mg:12 mL:13 mL, 500 mg:12 mL:14 mL, etc. For example, the usage ratio of konjac glucomannan, trifluoroacetic anhydride and octanoic acid can be 500 mg: (10-12) mL: (13-14) mL or 500 mg: (9-11) mL: (12-14) mL, etc., or any value within the defined range. In some embodiments, the usage ratio of konjac glucomannan, trifluoroacetic anhydride and octanoic acid is 500 mg: 10 mL: 13 mL.

[0100] The alcohol used in each of the above alcohol precipitation processes can be ethanol, for example but not limited thereto, and its amount can be 3-5 times the volume of the solution to be treated.

[0101] Furthermore, the collected second precipitate can be vacuum dried.

[0102] It should be noted that, in other embodiments, the above reaction conditions can be appropriately adjusted as needed. In addition, the second precipitate can be further dissolved in chloroform, and the liquid phase dissolved in chloroform can be subjected to a third alcohol precipitation. In other words, the number of alcohol precipitations can be adjusted as needed.

[0103] Based on the above-mentioned glucomannan caprylate, the present disclosure further provides a nanoparticle, which is obtained by nano-processing the above-mentioned glucomannan caprylate.

[0104] For reference, the method for preparing the nanoparticles may include the following steps: subjecting glucomannan caprylate to nano-processing.

[0105] In some embodiments, glucomannan caprylate may be subjected to ultrasonic nano-processing in the presence of polyvinyl alcohol (PVA), and the nanoparticles may be collected, and then the organic solvent may be removed and dried.

[0106] In some embodiments, glucomannan caprylate can be first dissolved in chloroform to obtain a glucomannan caprylate solution. The glucomannan caprylate solution is then added dropwise to a 4-6% polyvinyl alcohol solution at a volume ratio of 1:8-12. Ultrasonication is then performed at 15-20 Hz for 2-4 minutes. Excess PVA is added and stirred overnight. The organic solvent is removed by rotary evaporation, and finally, the nanoparticles are obtained by freeze-drying.

[0107] The above-mentioned PVA can utilize its surfactant properties to promote the nanoparticles generated by ultrasound to be more evenly dispersed in the solution, maintain stability, and avoid the fusion or rupture of the nanoparticles.

[0108] It should be noted that the methods and conditions for nano-processing are not limited to those listed above, and similar processing can also be performed with reference to relevant existing technologies.

[0109] Based on the glucomannan caprylate proposed above, the present disclosure further provides a glucomannan caprylate coating, which is obtained by coating the glucomannan caprylate.

[0110] It should be noted that conventional methods can be used for the coating substrate and coating method, and no further limitations are imposed herein.

[0111] In addition, the above-mentioned glucomannan caprylate can also be prepared into different dosage forms according to needs, such as capsules, soft capsules, powders, granules, tablets, pills, honey pastes, oral liquids, alcoholic drinks or injections.

[0112] Furthermore, the present disclosure also provides the use of the above-mentioned glucomannan caprylate or glucomannan caprylate nanoparticles or glucomannan caprylate coating in the preparation of drugs or materials for regulating the function of nucleus pulposus cells.

[0113] In some embodiments, the aforementioned glucomannan octanoate or nanoparticles or glucomannan octanoate coating can be used to prepare drugs or materials for regulating the adhesion and physiological functions of nucleus pulposus cells.

[0114] In some embodiments, the glucomannan octanoate or nanoparticles or glucomannan octanoate coating can be used to prepare drugs or materials that protect the phenotype of nucleus pulposus cells by intervening in the cell-matrix and cell-cell interactions.

[0115] In some embodiments, the aforementioned glucomannan octanoate or nanoparticles or glucomannan octanoate coating can be used to prepare drugs or materials that highly express nucleus pulposus phenotype.

[0116] In some embodiments, the above-mentioned glucomannan octanoate or nanoparticles or glucomannan octanoate coating can be used to prepare drugs or materials enriched in milk fat globule epidermal growth factor 8 (Mfge8 protein) secreted by nucleus pulposus cells; wherein the nucleus pulposus phenotype can exemplarily but not limitatively include Collagen2, etc.

[0117] In addition, the present disclosure also provides the use of the above-mentioned glucomannan caprylate or glucomannan caprylate nanoparticles or glucomannan caprylate coating in the preparation of drugs or materials for alleviating or treating intervertebral disc degeneration.

[0118] In some embodiments, the aforementioned glucomannan octanoate, nanoparticles, or glucomannan octanoate coating can be used to prepare a drug for delaying water loss in nucleus pulposus tissue. In other embodiments, the aforementioned glucomannan octanoate, nanoparticles, or glucomannan octanoate coating can be used to prepare a drug for reducing loss of intervertebral disc height.

[0119] Accordingly, the present disclosure also provides a drug, the active ingredient of which includes the above-mentioned glucomannan caprylate or the above-mentioned nanoparticles or the above-mentioned glucomannan caprylate coating.

[0120] Similarly, the present disclosure also provides a restorative material, the active ingredient of which includes the aforementioned glucomannan octanoate or the aforementioned nanoparticles or the aforementioned glucomannan octanoate coating.

[0121] Accordingly, the present disclosure provides a method for regulating nucleus pulposus cell function, comprising administering to a subject in need thereof or applying to a subject in need thereof a therapeutically effective amount of glucomannan caprylate according to the aforementioned embodiments, or the nanoparticles according to the aforementioned embodiments, or the glucomannan caprylate coating according to the aforementioned embodiments.

[0122] In some embodiments, the administering or applying is in the form of a drug or material, respectively.

[0123] In some embodiments, the regulation of nucleus pulposus cell function is selected from one or more of the following:

[0124] Regulates the adhesion and physiological functions of nucleus pulposus cells;

[0125] Protecting the phenotype of nucleus pulposus cells by intervening in cell-matrix and cell-cell interactions;

[0126] Highly expressing nucleus pulposus phenotype; optionally, the nucleus pulposus phenotype includes Collagen2;

[0127] Enriched with milk fat globule epidermal growth factor 8 secreted by nucleus pulposus cells; wherein the nucleus pulposus phenotype includes Collagen 2.

[0128] In some embodiments, the material is a repair material.

[0129] Accordingly, the present disclosure provides a method for alleviating or treating intervertebral disc degeneration, comprising administering to a subject in need thereof or applying to a subject in need thereof a therapeutically effective amount of glucomannan caprylate according to the aforementioned embodiments, or the nanoparticles according to the aforementioned embodiments, or the glucomannan caprylate coating according to the aforementioned embodiments.

[0130] In some embodiments, the administering or applying is in the form of a drug or material, respectively.

[0131] In some embodiments, the alleviating or treating intervertebral disc degeneration is selected from the group consisting of delaying the loss of nucleus pulposus tissue moisture and / or reducing the decrease in intervertebral disc height.

[0132] In some embodiments, the material is a repair material.

[0133] Accordingly, the present disclosure provides glucomannan caprylate according to the aforementioned embodiment, or the nanoparticles according to the aforementioned embodiment, or the glucomannan caprylate coating according to the aforementioned embodiment, for use in regulating nucleus pulposus cell function.

[0134] In some embodiments, the administering or applying is performed in the form of a drug or material, respectively;

[0135] In some embodiments, the regulation of nucleus pulposus cell function is selected from one or more of the following:

[0136] Regulates the adhesion and physiological functions of nucleus pulposus cells;

[0137] Protecting the phenotype of nucleus pulposus cells by intervening in cell-matrix and cell-cell interactions;

[0138] Highly expressing nucleus pulposus phenotype; optionally, the nucleus pulposus phenotype includes Collagen2;

[0139] Enriched with milk fat globule epidermal growth factor 8 secreted by nucleus pulposus cells; wherein the nucleus pulposus phenotype includes Collagen 2.

[0140] In some embodiments, the material is a repair material.

[0141] Accordingly, the present disclosure provides glucomannan caprylate according to the aforementioned embodiment, or the nanoparticles according to the aforementioned embodiment, or the glucomannan caprylate coating according to the aforementioned embodiment, for use in alleviating or treating intervertebral disc degeneration.

[0142] In some embodiments, the administering or applying is in the form of a drug or material, respectively.

[0143] In some embodiments, the alleviating or treating intervertebral disc degeneration is selected from the group consisting of delaying the loss of nucleus pulposus tissue moisture and / or reducing the decrease in intervertebral disc height.

[0144] In some embodiments, the material is a repair material.

[0145] It should be noted that, in existing technologies, supplementing the intervertebral disc with exogenous collagen or hyaluronic acid can increase tissue water content and improve the tissue microenvironment in the short term. However, the abundant expression of matrix-degrading enzymes will further degrade the supplemented exogenous ECM, potentially leading to repair failure.

[0146] In the cell-cell and cell-ECM interactions, Mfge8 is secreted by cells and expressed in the cell membrane and ECM as a "bridge protein", participating in mediating cell recognition and adhesion behavior, thereby further regulating physiological functions. The present invention uses an esterification reaction to add multiple acetyl groups to the polysaccharide, and the modified product exhibits an extremely high Mfge8 protein enrichment ability. By preparing polysaccharide coatings and nanoparticles, they are brought into contact with nucleus pulposus cells in vitro and in vivo, respectively, and the changes in nucleus pulposus cell function are mediated by enriching Mfge8 protein. At the same time, the polysaccharide material has excellent resistance to enzyme degradation properties and can remain stably in the local area for a long time to supplement the ECM lost due to degeneration. Ultimately, by improving the microenvironment of the degenerative disc tissue, the therapeutic goal of delaying intervertebral disc degeneration is achieved.

[0147] The konjac glucomannan and its derivatives provided by the present disclosure can be used as extracellular matrix fillers. The glucomannan octanoate obtained by modifying the konjac glucomannan octanoate through an esterification reaction, as well as the nanoparticles or glucomannan octanoate coating further obtained from the glucomannan octanoate, can be used to prepare drugs or materials that regulate the function of nucleus pulposus cells, especially drugs or materials that can be used to alleviate or treat intervertebral disc degeneration. The above-mentioned glucomannan octanoate has the characteristics of being non-toxic, easy to obtain, and having high tissue compatibility, and can be prepared into different dosage forms according to needs. The present disclosure provides a new idea for improving the microenvironment of intervertebral disc degeneration, and also provides a new use of biomaterials based on polysaccharide derivatives.

[0148] The features and performance of the present disclosure are further described in detail below with reference to the embodiments.

[0149] Example 1

[0150] Verify the anti-enzymatic performance of KGM

[0151] 50 mg of KGM and hyaluronic acid (molecular weight 180K) were added to 10 mL of buffer solution (PBS saline solution, pH = 5.3) and stirred to dissolve. 2 mg of hyaluronidase was then added to each sample. The mixture was allowed to react at 37 ° C for 6 h, then heated to 90 ° C to inactivate the enzyme for 5 min, ending the reaction. After cooling, the sample was filtered through a 0.22 μm membrane to remove insoluble particles. Mass spectrometry was used to detect the presence of enzymatically hydrolyzed oligosaccharide products in the reaction solution to verify the substrate's resistance to enzymatic hydrolysis.

[0152] The mass spectrometry results are shown in Figures 1 and 2. The hyaluronic acid in Figure 1, used as the control group, showed a large number of oligosaccharide peaks after enzymatic hydrolysis, indicating that it was hydrolyzed. However, the KGM in Figure 2 showed no significant oligosaccharide peaks, indicating that it has good resistance to enzymatic hydrolysis.

[0153] Example 2

[0154] Synthesis of GMOC by esterification reaction

[0155] Weigh 13.2 mL of octanoic acid and slowly add it dropwise to 10 mL of TFAA. React in an oil bath at 50°C for 20 minutes to obtain the first reaction solution. Add 500 mg of KGM to the first reaction solution and react for 3.5 hours to obtain the second reaction solution. Centrifuge the second reaction solution, take the supernatant and add it dropwise to four times the volume of ethanol solution for the first alcohol precipitation. Centrifuge and collect the first precipitate, which is further dissolved in chloroform to remove the insoluble product. Then, the liquid phase dissolved in chloroform is subjected to a second alcohol precipitation. Collect the second precipitate to obtain the modified reaction product GMOC, which is vacuum dried for later use.

[0156] 1 mg of the obtained GMOC was characterized by infrared spectroscopy and nuclear magnetic resonance, and it was observed that the modification was successful, as shown in Figures 3 and 4. It was calculated that by controlling the reaction time, a GMOC product with a substitution rate of approximately 1.77 could be stably obtained.

[0157] Example 3

[0158] Verify the role of GMOC in regulating nucleus pulposus cell function

[0159] The GMOC prepared in Example 2 was coated on a slide and co-cultured with nucleus pulposus cells. Immunofluorescence staining was used to observe changes in nucleus pulposus cell phenotype and cell morphology, as shown in Figure 5. This figure shows that nucleus pulposus cells exhibited significant colony-like growth on the GMOC and highly expressed nucleus pulposus phenotypes, including Collagen 2.

[0160] Further proteomic analysis suggested that the GMOC coating could significantly adsorb the Mfge8 protein secreted by nucleus pulposus cells, which was confirmed in subsequent verification experiments (Figure 7).

[0161] Proteomic analysis involves using 4D-label-free technology to detect the types of nucleus pulposus cell membrane proteins specifically adsorbed by GMOCs. The steps involved incubating nucleus pulposus cell membranes with GMOCs for 24 hours, then washing three times with PBS. Proteins adsorbed on the GMOC surface were then digested with trypsin into peptides, which were then desalted and analyzed using a flow cytometer.

[0162] The experiment corresponding to Figure 7 is as follows: Nucleus pulposus cells were seeded onto the surfaces of glass and GMOC materials and cultured for 24 hours. Adherent cells were removed using EDTA. The samples were washed three times with PBS. The glass and GMOC materials were incubated with the primary antibody against mfGE8 at 4°C overnight and washed three times with PBS. An HRP-conjugated secondary antibody was added, incubated at room temperature for 1 hour, and washed three times with PBS. ECL chemiluminescent solution was added and the samples were exposed to light to observe the adsorption of mfGE8 protein on the surfaces.

[0163] In Figures 6 and 7, Control represents a conventionally used cell culture plate; Glass represents a cell culture plate, i.e., a glass slide; and GMOC represents the glucomannan coating provided by the present disclosure, which is coated on the surface of the cell culture plate.

[0164] Example 4

[0165] Animal model validates the therapeutic effect of GMOC in alleviating intervertebral disc degeneration

[0166] Eight-week-old, 250g SD female rats (obtained from the Animal Center of the Faculty of Health Sciences, University of Macau) were selected and anesthetized. A 21G needle was used to puncture the intervertebral space halfway to establish an intervertebral disc degeneration model (IDD). A microsyringe was used to inject 10μL of 5% (w / v) GMOC PBS solution in situ into the defect site as the treatment group, wherein the GMOC was prepared by Example 2. The defect segment injected with an equal amount of PBS was used as the control group. 3.0t nuclear magnetic resonance and X-ray were used to detect the water content of the nucleus pulposus tissue and the height of the intervertebral space after treatment for 3 days and 7 days, respectively, to evaluate the therapeutic effect of GMOC. The results are shown in Figures 8 and 9.

[0167] Among them, control is the healthy control group, that is, the control group without any treatment; IDD group is the degeneration group, that is, only puncture is performed without any treatment; PBS group is injected with PBS solution after puncture; GMOC group is injected with GMOC solution after puncture.

[0168] As shown in Figures 8 and 9, GMOC treatment can significantly delay the water loss of nucleus pulposus tissue and reduce the degree of reduction in intervertebral disc height compared with the IDD group.

[0169] Figure 8 shows the assessment of nucleus pulposus water content using MR T2 images. By calculating the grayscale value of the nucleus pulposus, changes in nucleus pulposus water content can be reflected. Compared to the PBS and IDD groups, GMOC maintained tissue water content seven days after treatment, as reflected by minimal signal loss.

[0170] Figure 9 shows that the height between adjacent intervertebral spaces (DHI) can be measured through X-RAY detection to reflect the degree of intervertebral disc tissue degeneration. For the PBS and IDD groups, a certain intervertebral space height can still be maintained on the seventh day after GMOC treatment, indicating that there is sufficient nucleus pulposus tissue as support.

[0171] In summary, the sugar-like biomaterial provided by the present disclosure is obtained by modifying konjac polysaccharide (KGM) through an esterification reaction to obtain glucomannan octanoate (GMOC), which can regulate the adhesion and physiological function of nucleus pulposus cells by enriching milk fat globule epidermal growth factor 8 (Mfge8), and further protect the phenotype of nucleus pulposus cells by intervening in cell-matrix and cell-cell interactions, thereby improving the microenvironment of intervertebral disc degeneration. This sugar derivative material (GMOC) has the characteristics of being non-toxic, easy to obtain, and having high tissue compatibility, and can be prepared into different dosage forms according to needs. On this basis, the present disclosure provides a new idea for improving the microenvironment of intervertebral disc degeneration, as well as a new use of biomaterials based on the above-mentioned polysaccharide derivatives.

[0172] The above are merely preferred embodiments of the present disclosure and are not intended to limit the present disclosure. Those skilled in the art will readily appreciate that various modifications and variations of the present disclosure are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present disclosure shall be included within the scope of protection of the present disclosure. Industrial Applicability

[0173] The present disclosure provides an application of konjac glucomannan and its derivatives. Konjac glucomannan and its derivatives can be used as extracellular matrix fillers. Glucomannan octanoate obtained by modifying the konjac glucomannan through an esterification reaction, as well as nanoparticles or glucomannan octanoate coatings further obtained from the konjac glucomannan octanoate, can be used to prepare drugs or materials that regulate the function of nucleus pulposus cells, particularly drugs or materials that alleviate or treat intervertebral disc degeneration. The konjac glucomannan octanoate is non-toxic, readily available, and highly tissue compatible. It can be prepared into different dosage forms as needed, and has excellent industrial applicability.

Claims

1. Application of konjac glucomannan and its derivatives as extracellular matrix fillers.

2. The use according to claim 1, characterized in that: The derivative of konjac glucomannan is a modified konjac glucomannan; Optionally, the modification is to introduce additional functional groups by modifying the polysaccharide molecules therein; Optionally, the functional group is acetyl or octanoyl; Optionally, the derivative of konjac glucomannan is glucomannan caprylate; Optionally, the substitution rate of glucomannan caprylate is 1.0-2.5; Optionally, the substitution rate of glucomannan caprylate is 1.5-2.5; Optionally, the substitution rate of glucomannan caprylate is 1.6-1.8; Optionally, the konjac glucomannan and its derivatives are in the form of nanoparticles or coatings.

3. A glucomannan caprylate, characterized in that The glucomannan caprylate is obtained by modifying konjac glucomannan through an esterification reaction; Optionally, the substitution rate of glucomannan caprylate is 1.0-2.5; optionally, the substitution rate of glucomannan caprylate is 1.5-2.5; optionally, the substitution rate of glucomannan caprylate is 1.6-1.

8.

4. A method for synthesizing glucomannan caprylate as claimed in claim 3, characterized in that: The following steps are involved: The konjac glucomannan is modified with an octanoyl group; Optionally, konjac glucomannan is reacted with trifluoroacetic anhydride and octanoic acid, the reaction solution obtained by the reaction is subjected to solid-liquid separation, the liquid phase is collected for a first alcohol precipitation, a first precipitate obtained by the first alcohol precipitation is dissolved in chloroform to remove products insoluble in chloroform, and then the liquid phase dissolved in chloroform is subjected to a second alcohol precipitation, and the obtained second precipitate is collected; Optionally, konjac glucomannan is added to a first reaction solution obtained by reacting trifluoroacetic anhydride with octanoic acid, and the reaction is continued to obtain a second reaction solution; the second reaction solution is separated into solid and liquid, the liquid phase is collected for a first alcohol precipitation, a first precipitate obtained by the first alcohol precipitation is dissolved in chloroform to remove products insoluble in chloroform, and then the liquid phase dissolved in chloroform is subjected to a second alcohol precipitation, and the obtained second precipitate is collected; Optionally, the reaction of the trifluoroacetic anhydride and the octanoic acid is carried out at 50-55° C. for 15-25 min; Optionally, the reaction of the konjac glucomannan with the first reaction solution is carried out at 50-55° C. for 3-4 hours; Optionally, the usage ratio of the konjac glucomannan, the trifluoroacetic anhydride and the caprylic acid is 500 mg:(8-12) mL:(12-14) mL.

5. A nanoparticle, characterized in that: The nanoparticles are obtained by subjecting the glucomannan caprylate described in claim 3 to nano-processing.

6. A method for preparing nanoparticles according to claim 5, characterized in that: The following steps are involved: The glucomannan caprylate is subjected to nano-processing; Optionally, the glucomannan caprylate is subjected to ultrasonic nano-processing in the presence of polyvinyl alcohol, and the organic solvent is removed and the product is dried.

7. A glucomannan caprylate coating, characterized in that: The glucomannan caprylate is obtained by coating the glucomannan caprylate according to claim 3.

8. Use of the glucomannan caprylate according to claim 3, the nanoparticles according to claim 5, or the glucomannan caprylate coating according to claim 7 in the preparation of a drug or material for regulating the function of nucleus pulposus cells; Optionally, the glucomannan caprylate or the nanoparticle or the glucomannan caprylate coating is used to prepare a method for regulating nucleus pulposus cells. Adhesion and physiological function of drugs or materials; Optionally, the glucomannan caprylate or the nanoparticle or the glucomannan caprylate coating is used to prepare a drug or material for protecting the phenotype of nucleus pulposus cells by intervening in the interactions between cells-matrix and cells-cells; Optionally, the glucomannan caprylate or the nanoparticle or the glucomannan caprylate coating is used to prepare a drug or material that highly expresses a nucleus pulposus phenotype; optionally, the nucleus pulposus phenotype includes Collagen2; Optionally, the glucomannan caprylate or the nanoparticles or the glucomannan caprylate coating is used to prepare a drug or material enriched in milk fat globule epidermal growth factor 8 secreted by nucleus pulposus cells; wherein, The nucleus pulposus phenotype includes Collagen2; Optionally, the material is a repair material.

9. Use of the glucomannan caprylate according to claim 3, the nanoparticles according to claim 5, or the glucomannan caprylate coating according to claim 7 in preparing a drug or material for alleviating or treating intervertebral disc degeneration; Optionally, the glucomannan caprylate or the nanoparticles or the glucomannan caprylate coating is used to prepare a drug for delaying water loss in nucleus pulposus tissue, or for preparing a drug for reducing the decrease in intervertebral disc height; Optionally, the material is a repair material.

10. A drug, characterized in that The active ingredient of the drug includes the glucomannan caprylate according to claim 3 or the nanoparticles according to claim 5 or the glucomannan caprylate coating according to claim 7.

11. A repair material, characterized in that: The active ingredient of the repair material includes the glucomannan octanoate according to claim 3 or the nanoparticles according to claim 5 or the glucomannan octanoate coating according to claim 7.

12. A method for regulating nucleus pulposus cell function, the method comprising administering to a subject in need thereof or applying to a subject in need thereof a therapeutically effective amount of the glucomannan caprylate of claim 3 or the nanoparticles of claim 5 or the glucomannan caprylate coating of claim 7; Optionally, the administering or applying is performed in the form of a drug or material, respectively; Optionally, the regulation of nucleus pulposus cell function is selected from one or more of the following: Regulates the adhesion and physiological functions of nucleus pulposus cells; Protecting the phenotype of nucleus pulposus cells by intervening in cell-matrix and cell-cell interactions; Highly expressing nucleus pulposus phenotype; optionally, the nucleus pulposus phenotype includes Collagen2; Enriched with milk fat globule epidermal growth factor 8 secreted by nucleus pulposus cells; wherein the nucleus pulposus phenotype includes Collagen2; Optionally, the material is a repair material.

13. A method for alleviating or treating intervertebral disc degeneration, the method comprising administering to a subject in need thereof or applying to a subject in need thereof a therapeutically effective amount of the glucomannan caprylate according to claim 3 or the nanoparticles according to claim 5 or the glucomannan caprylate coating according to claim 7; Optionally, the administering or applying is performed in the form of a drug or material, respectively; Optionally, the relief or treatment of intervertebral disc degeneration is selected from delaying the loss of water content in nucleus pulposus tissue and / or reducing the decrease in intervertebral disc height; Optionally, the material is a repair material.

14. A glucomannan caprylate as claimed in claim 3, or a nanoparticle as claimed in claim 5, or a glucomannan caprylate coating as claimed in claim 7, for use in regulating the function of nucleus pulposus cells; Optionally, the administering or applying is performed in the form of a drug or material, respectively; Optionally, the regulation of nucleus pulposus cell function is selected from one or more of the following: Regulates the adhesion and physiological functions of nucleus pulposus cells; Protecting the phenotype of nucleus pulposus cells by intervening in cell-matrix and cell-cell interactions; Highly expressing nucleus pulposus phenotype; optionally, the nucleus pulposus phenotype includes Collagen2; Enriched with milk fat globule epidermal growth factor 8 secreted by nucleus pulposus cells; wherein the nucleus pulposus phenotype includes Collagen2; Optionally, the material is a repair material.

15. The glucomannan octanoate according to claim 3, the nanoparticles according to claim 5, or the glucomannan octanoate coating according to claim 7, for alleviating or treating intervertebral disc degeneration; Optionally, the administering or applying is performed in the form of a drug or material, respectively; Optionally, the relief or treatment of intervertebral disc degeneration is selected from delaying the loss of water content in nucleus pulposus tissue and / or reducing the decrease in intervertebral disc height; Optionally, the material is a repair material.

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