Arginine polyesteramide polymers, methods of making and using the same

Abstract

The application discloses an arginine polyester amide polymer, a preparation method and application thereof, and belongs to the technical field of medicines.The arginine polyester amide polymer combines the advantages of polyesters and polyamides, has good biocompatibility and biodegradability, and has positive charges.The arginine polyester amide polymer can penetrate the cartilage endplate into the intervertebral disc tissue without damaging the integrity of the intervertebral disc, and plays a role in treating intervertebral disc degeneration by virtue of the positive charge characteristics of the polymer and the negative charge property of the cartilage endplate.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and in particular, relates to an arginine polyester amide polymer, a preparation method and application thereof. BACKGROUND

[0002] Intervertebral disc (IVD) is the center of load activity of the basic functional unit of the spine, which has three main components: nucleus pulposus (NP), annulus fibrosus (AF) and cartilage endplate (such as Figure 1 ). The nucleus pulposus is the most central tissue in the structure of the IVD, mainly composed of type II collagen, and contains a considerable amount of elastin and proteoglycans. The annulus fibrosus is in the periphery of the NP tissue, and its main function is to provide a tough fibrous wrapping and stabilize the NP tissue. When the AF is intact, the highly hydrated NP tissue can maintain its hydrostatic pressure to maintain the functional integrity of the IVD, which also illustrates the interdependence of the various IVD tissues. The cartilage endplate is composed of hyaline cartilage, located between the AF and the NP, and between the upper and lower vertebral bodies (VB) around the IVD. The function of the endplate is to act as a transition structure between the NP and the vertebral body, increasing the mechanical stability. In addition to being a load-bearing structure of the intervertebral disc, the cartilage endplate also manages the exchange of nutrients between the intervertebral disc tissue and the outside world. The vascular structure in the IVD is mostly located in the cartilage endplate, and nutrients enter the intervertebral disc through the cartilage endplate. Proteoglycans and collagen are important structural components of the extracellular matrix of the cartilage endplate, and have an important influence on the physiological function of the cartilage endplate. Studies have shown that the proteoglycans carried by the cartilage endplate have a negative charge, which controls the distribution of charged solutes and the osmotic pressure in the matrix. Therefore, the biochemical properties of the cartilage endplate are crucial to maintaining the integrity of the intervertebral disc, especially the regulation of solute transport by proteoglycans. Due to the high concentration of proteoglycans, the intervertebral disc is mainly negatively charged, and small solutes such as glucose and oxygen can freely diffuse into the intervertebral disc matrix. Negatively charged molecules such as sulfate and chloride ions can relatively easily pass through the endplate, but it is difficult to enter the nucleus pulposus. Sodium and calcium ions can be exchanged freely with the atomic nucleus.

[0003] Intervertebral disc degeneration is an independent risk factor for low back pain and is closely related to spinal musculoskeletal diseases. At present, traditional various conservative treatment and surgical treatment are difficult to fundamentally treat and reverse the already occurred intervertebral disc degeneration. Tissue engineering based on bioactive materials aims to better target the pathogenesis of intervertebral disc degeneration, maintain the intervertebral disc phenotype, restore the function of the extracellular matrix, and thus provide functional mechanical support.

[0004] However, due to the complexity of the intervertebral disc tissue structure, the application of the bioactive material in the early mild intervertebral disc degeneration is mainly by direct puncture of the annulus fibrosus into the intervertebral disc injection, which will destroy the integrity of the intervertebral disc structure and cause more serious intervertebral disc degeneration.

[0005] Therefore, how to make the biomaterials enter the intervertebral disc tissue to treat intervertebral disc degeneration without damaging the integrity of the intervertebral disc is a key problem to be solved at present. SUMMARY

[0006] In order to solve the above technical problems, the present application provides an arginine-based poly(ester amide) (Arg-PEA) which combines the advantages of polyesters and polyamides, has good biocompatibility and biodegradability, and has a positive charge. The polymer can penetrate the cartilage endplate into the intervertebral disc tissue without damaging the integrity of the intervertebral disc, and play a role in treating intervertebral disc degeneration. The scheme adopted by the present application is as follows:

[0007] The first aspect of the present application provides a preparation method of arginine-based poly(ester amide), which utilizes monomer Nx and arginine monomer Arg-y-S to condense and polymerize by amidation reaction under the catalysis of triethylamine, and the reaction formula is as follows:

[0008]

[0009] In the formula, x = 2-10, y = 2-8.

[0010] In the present application, different values of x and y can obtain different arginine-based poly(ester amide) polymers, for example, when x = 8 and y = 6, 8A6S is prepared; for another example, when x = 6 and y = 6, 6A6S is prepared; for another example, when x = 2 and y = 6, 2A6S is prepared.

[0011] In some embodiments of the present application, the monomer Nx is prepared by the following steps:

[0012] Triethylamine and p-nitrophenol are added to acetone, and the reaction is kept at 0℃ by ice bath; Nx is prepared by adding the following solution dropwise to the above cooled solution, stirring at 0℃ for 2h, and overnight at room temperature; the generated product is precipitated in distilled water, completely washed, recrystallized in 4:1 (v:v) ethyl acetate / DMF, and vacuum dried to constant weight, and the reaction formula is as follows:

[0013]

[0014] ​In some embodiments of the present application, the arginine monomer Arg-y-S is prepared by the following steps:

[0015] L-arginine and are directly mixed, followed by the addition of toluene and p-toluenesulfonic acid monohydrate; the solid-liquid reaction mixture is heated to 120-130°C, then stirred and refluxed for 16-24 hours, and cooled to room temperature; after toluene is precipitated, it is poured, the product is dissolved in isopropanol at 75°C under stirring, then precipitated at 4°C to obtain arginine monomer Arg-y-S, the reaction formula is as follows:

[0016]

[0017] The second aspect of the present application provides an arginine polyester amide polymer prepared by the preparation method of any one of the first aspect of the present application.

[0018] The third aspect of the present application provides use of the arginine polyester amide polymer of the second aspect of the present application in the preparation of a medicament for treating intervertebral disc degeneration-related diseases.

[0019] In some embodiments of the present application, the arginine polyester amide polymer treats intervertebral disc degeneration-related diseases by at least one of the following functions:

[0020] (1) reducing the degeneration of nucleus pulposus cells;

[0021] (2) restoring the physiological function of degenerated nucleus pulposus cells;

[0022] (3) promoting the proliferation of nucleus pulposus cells;

[0023] (4) promoting the regeneration of nucleus pulposus tissue;

[0024] (5) promoting the repair of nucleus pulposus tissue;

[0025] (6) reducing the expression levels of MMP13 and ADAMTS5 genes and proteins in nucleus pulposus cells;

[0026] (7) increasing the expression levels of ACAN, Collagen II and SOX9 genes and proteins in nucleus pulposus cells.

[0027] Further, the arginine polyester amide polymer functions by inhibiting the activation of the IL-17 signaling pathway.

[0028] Still further, the inhibition of the activation of the IL-17 signaling pathway refers to reducing the expression levels of IL-17R, TRAF6, Act1 and RELA genes and proteins.

[0029] In the present application, the medicament is injected into the upper and lower vertebrae, and can penetrate the cartilage bone plate into the intervertebral disc tissue.

[0030] The fourth aspect of the present application provides a drug for treating intervertebral disc degeneration related diseases, comprising the arginine polyester amide polymer of the second aspect of the present application.

[0031] Advantages of the present application

[0032] Compared with the prior art, the present application has the following advantages:

[0033] The arginine polyester amide polymer of the present application combines the advantages of polyesters and polyamides, has good biocompatibility and biodegradability, and has a positive charge. By taking advantage of the positive charge characteristic of the arginine polyester amide polymer and the negative charge property of the cartilage endplate, the polymer can penetrate the cartilage endplate into the intervertebral disc tissue without damaging the integrity of the intervertebral disc, and play a role in treating intervertebral disc degeneration.

[0034] The arginine polyester amide polymer of the present application can promote the proliferation of nucleus pulposus cells and the regeneration of nucleus pulposus tissue by resisting the IL-17 signaling pathway, save intervertebral disc degeneration, and provide a new idea for the design and development of bioactive materials, and also provide a new method and approach for tissue engineering in the treatment of early mild intervertebral disc degeneration. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 An intervertebral disc anatomical diagram is shown.

[0036] Figure 2 A preparation method of monomer I, monomer II (arginine monomer) and arginine polyester amide is shown.

[0037] Figure 3 A preparation method of arginine polyester amide polymer is shown. 1 H-NMR and FT-IR results for Arg-6-S and 8A6S.

[0038] Figure 4 A method for applying arginine polyester amide polymer is shown.

[0039] Figure 5 Penetration detection of arginine polyester amide polymers with different structures and mass ratios is shown, scale: 500 μm.

[0040] Figure 6 Comparison of cy7 radiation efficiency of 8A6S in intervertebral discs at different time points is shown.

[0041] Figure 7 Effect of 8A6S with different masses on the activity of rat nucleus pulposus cells is shown.

[0042] Figure 8The effect of 8A6S of different quality on the apoptosis of rat nucleus pulposus cells is shown.

[0043] Figure 9 The toxicity of 8A6S of different quality on rat nucleus pulposus cells is shown, scale: 100 pm.

[0044] Figure 10 The expression level of degeneration-related genes and proteins of nucleus pulposus cells after 8A6S treatment is shown.

[0045] Figure 11 The expression level of degeneration-related genes of nucleus pulposus cells after 8A6S treatment is shown, scale: 100 pm, 50 pm (enlarged).

[0046] Figure 12 The schematic diagram of rat intervertebral disc compression degeneration model is shown, A: sham operation group, B: operation group.

[0047] Figure 13 The rat in vivo experiment process is shown.

[0048] Figure 14 The results of rat intervertebral disc imaging at different time points and statistical results are shown.

[0049] Figure 15 The rat intervertebral disc pathological tissue sections at different time points are shown, scale: 400 pm.

[0050] Figure 16 The expression of degeneration-related proteins in rat intervertebral discs at different time points is shown, scale: 500 pm.

[0051] Figure 17 The results of RNA sequencing analysis are shown.

[0052] Figure 18 The results of qPCR detection of IL-17 signaling pathway-related gene expression levels are shown.

[0053] Figure 19 The expression level of IL-17 signaling pathway-related proteins is detected by immunofluorescence, scale: 100 pm.

[0054] Figure 20 The results of qPCR and Western Blot detection of IL-17 signaling pathway-related gene and protein expression levels are shown. DETAILED DESCRIPTION

[0055] Unless otherwise indicated, all parts and percentages expressed herein are based upon weight and all tests and measurements are conducted at the date of filing of this application. To the extent that any patent, patent application, or publication is cited in this application, the same is hereby incorporated by reference in its entirety into this application, and the equivalent thereof is intended to be expressly incorporated by reference into this application. If any definition contained in this application is contrary to or otherwise inconsistent with any definition of the same term in a patent, patent application, or publication that is cited herein, the definition contained in this application prevails over the definition in the cited patent, patent application, or publication.

[0056] Numerical ranges expressed in the present application in terms of "from about" or "from between" are approximate ranges and include endpoints, unless otherwise indicated. Numerical ranges include all values from and including the lower and to and including the upper range values. In this context, a numerical range includes all values and / or subsets of values that fall within the indicated ranges. In the numerical ranges implementing "from between", the individual numerical values are included in the range. For example, a range from between 1 and 5 includes 1, 2, 3, 4, and 5, and also includes ranges such as 2 to 4, 2 to 5, 4 to 5, and 3 to 5. Similarly, a range from about 1 to about 5 includes all values from 1 to 5, and also includes ranges such as 2 to 4, 2 to 5, 4 to 5, and 3 to 5.

[0057] The terms "comprising", "including", "containing", and variations thereof, do not exclude the presence of other components, steps or processes, and are used synonymously with the term "including". For the avoidance of doubt, unless expressly stated otherwise, all compositions comprising a term "comprising", "including", or "having" of the present application can comprise any additional additive, adjuvant, or compound. In contrast, the term "consisting essentially of excludes from the scope of any succeeding recitation a component, step, or process not specifically recited. The term "consisting of excludes any component, step, or process not specifically recited. The term "or", unless otherwise indicated, is used in the conjunctive sense, i.e. the term "or" is used in the inclusive sense.

[0058] In order to make the technical problems solved by the present application, the technical solutions and the beneficial effects clearer, the present application will be further explained in detail below with reference to the embodiments.

[0059] Embodiments

[0060] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present application, and are not intended to limit the scope of what the inventors regard as their application. One skilled in the art will recognize that the examples set forth herein represent techniques that can be used in carrying out the present application, and that changes can be made in the art without departing from the spirit and scope of the present application.

[0061] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, and the meaning ascribed to in the materials incorporated herein by reference.

[0062] Those of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the application described herein. Such equivalents are intended to be encompassed by the following claims.

[0063] The experimental methods in the following examples are routine unless otherwise stated. The instruments used in the following examples are routine laboratory instruments unless otherwise stated; the test materials used in the following examples are purchased from routine biochemical reagent stores unless otherwise stated.

[0064] Example 1 Preparation of arginine polyester amide polymers

[0065] (1) Synthesis of monomer I (N8)

[0066] The synthesis route of monomer N8 is described as follows:

[0067] Triethylamine (0.32 mol) and p-nitrophenol (0.31 mol) were added to 200 mL of acetone and the reaction was kept at 0 °C using an ice bath. Sebacoyl chloride was diluted in 100 mL of cold acetone and then added dropwise to the above cooled solution, stirred at 0 °C for 2 h and at room temperature overnight. The resulting p-nitrophenyl sebacate was precipitated in distilled water, washed thoroughly, recrystallized 3 times in ethyl acetate / DMF (4:1, v:v) and dried to constant weight under vacuum to yield monomer N8 with a yield of 95%, which was stored under seal.

[0068] (2) Synthesis of monomer II (Arg-6-S)

[0069] The synthesis route of monomer Arg-6-S is described as follows:

[0070] L-arginine (0.04 mol) and 1,6-hexanediol (0.02 mol) were directly mixed, followed by the addition of toluene (400 mL) and p-toluenesulfonic acid monohydrate (0.082 mol). The solid-liquid reaction mixture was heated to 130°C and then stirred and refluxed for 24 hours. The reaction mixture (a viscous solid) was then cooled to room temperature. After the toluene precipitated and poured out, the product was dissolved in isopropanol at 75°C with stirring and then precipitated at 4°C. This process was repeated three times to obtain monomer Arg-6-S with a yield of 75%. The product was then sealed and stored. The structure of the purified monomer Arg-6-S was confirmed by NMR and IR spectroscopy.

[0071] (3) Synthesis of L-arginine-based polyester amide Arg-PEA (8A6S)

[0072] L-arginine-based polyesteramide 8A6S is synthesized by polycondensation of monomers N8 and Arg-6-S catalyzed by triethylamine. The specific synthesis method is as follows: Dissolve N8 (2 mmol) and Arg-6-S (2 mmol) in anhydrous DMSO (5 mL). Heat at 120°C, thoroughly mix by vortexing and stirring. Once fully dissolved, triethylamine (3 mL) is added dropwise. Stirring is stopped, and the mixture is maintained at 80°C overnight. The mixed solution is added dropwise to pre-cooled ethyl acetate for precipitation. After thorough stirring, the initial product is obtained, which is washed three times with methanol and dried under vacuum at 60°C to constant weight. This yields purified 8A6S with a yield of 25%, which is then sealed and stored. The structure of the purified 8A6S is confirmed by nuclear magnetic resonance (NMR) and infrared spectroscopy.

[0073] The reaction equations for each step are as follows Figure 2 shown.

[0074] The NMR and IR results of Arg-6-S and 8A6S are as follows Figure 3 As shown, the results show that arginine polyester amide Arg-PEA was successfully prepared by amidation condensation polymerization using monomer N8 and arginine monomer Arg-6-S under the catalysis of triethylamine.

[0075] Example 2: Cartilage endplate penetration test of arginine polyesteramide 8A6S

[0076] 1.8 Application of A6S

[0077] (1) GFP fluorescent protein was mixed with 2A2S and 8A6S at different ratios (1:100 and 1:200), and GFP was mixed with PBS as a blank control. Each mixture was vortexed and mixed thoroughly.

[0078] (2) After ether anesthesia of rats, operation was performed, and Kirschner wire was used to punch the upper and lower vertebral bodies of Co8 / 9 segment of the rat tail vertebrae, and a 50 μL mixed solution was injected through the hole by using a syringe, and finally bone wax was used to close the hole to prevent the leakage of the polymer Figure 4

[0079] (3) After 7 days, the rats were sacrificed by the method of spinal dislocation, and the corresponding vertebral body-disc-vertebral body segment was taken, and fixed with 4% PFA overnight in the dark;

[0080] (4) Decalcification with EDTA decalcification solution in the dark for 14 days.

[0081] 2. Decalcified frozen sections

[0082] (1) The corresponding tissue block after decalcification was transferred to a frozen section embedding box, embedded with OCT, and placed in a-80℃ ultra-low temperature refrigerator for 2h to make the OCT fully solidify;

[0083] (2) The fixed OCT frozen tissue block was cut into a section with a thickness of 10 μm by using a frozen section machine, and was attached to a glass slide;

[0084] (3) The tissue section should be stored in a-20℃ low temperature refrigerator.

[0085] 3. Immunofluorescence of tissue sections

[0086] (1) The tissue section stored in the-20℃ low temperature refrigerator was taken out and thawed at room temperature for 30 min;

[0087] (2) The rewarmed section was placed in a 60℃ oven for 15 min;

[0088] (3) 4% PFA was added to completely cover the tissue, and after fixation for 10 min, PBS was used for rinsing 3 times, each for 5 min;

[0089] (4) The surface liquid was gently wiped off, and the area where the tissue was located was circled with a water-repellent pen;

[0090] (5) 0.05% Triton X-100 was added to completely cover the tissue, and the tissue was permeabilized, and after permeabilization for 10 min, PBS was used for rinsing 3 times, each for 5 min;

[0091] (6) Antigen repair solution was added to completely cover the tissue, and after incubation at room temperature for 5 min, PBS was used for rinsing 3 times, each for 5 min;

[0092] (7) Goat serum was added to completely cover the tissue, and incubation was performed at room temperature for 1h;

[0093] ​(8) Absorb goat serum, drop DAPI-containing anti-fluorescence quenching sealing agent to completely cover the tissue, cover glass sealing, avoid bubble generation, after sealing, place in a humid box and avoid light;

[0094] (9) Observe the staining results on the laser confocal microscope LSM880, and take pictures of the tissue at 10x magnification.

[0095] The immunofluorescence results are shown in Figure 5 As shown, GFP and 8A6S at a mass ratio of 1:200, GFP significantly aggregated in the intervertebral disc, and since GFP was combined with 2A2S or 8A6S by electrostatic adsorption, it was shown that 8A6S could penetrate the cartilage endplate into the intervertebral disc, in comparison, 2A2S had relatively weak penetration.

[0096] 4. In vivo imaging system analysis

[0097] In order to clarify the penetration size and action time of 8A6S in rats in vivo, the inventors further observed the retention time and penetration effect of 8A6S in the rat tail vertebrae at different time points by using a small animal in vivo imaging instrument. Specifically:

[0098] (1) After cy7-NHS dye and BSA protein were purified, they were combined with 8A6S by electrostatic adsorption at a mass ratio of 1:200 to obtain a Cy7-NHS-BSA+8A6S mixture, and a Cy7-NHS-BSA+PBS mixture was prepared as a control;

[0099] (2) After the rats were anesthetized, the upper and lower vertebrae of the Co8 / 9 segment of the rat tail were punctured with a k-wire, and 50 μL of Cy7-NHS-BSA+PBS / 8A6S mixture was injected, and the bone wax was sealed;

[0100] (3) The small animal in vivo imaging instrument was used to take pictures of the Cy7 fluorescence of the corresponding segment of the rat tail vertebrae at the corresponding time points.

[0101] (4) The picture parameters were adjusted by using the imaging system program, and statistical analysis was performed, and statistical calculation was performed using Graphpad Prism9 version, and single factor analysis of variance was used when comparing whether there were statistical (significant) differences between groups in multiple groups, in this embodiment, P value> 0.05 was considered meaningless (ns), P value≤0.05 was (*), P value≤0.01 was (**) and P value≤0.001 was (***).

[0102] The results are shown in Figure 6As shown, compared with the control group (cy7-NHS-BAS+PBS) injected with cy7 dye only, the 8A6S group (Cy7-NHS-BSA+8A6S) showed no obvious change within 24 hours, a tendency of peripheral diffusion at the third day, and part of the 8A6S diffused into the intervertebral disc at the seventh day. The cy7 fluorescence intensity in the intervertebral disc was the strongest at the fourteenth day, gradually weakened after 7 days, and the fluorescence dissipated at the twenty-eighth day. The average radiation efficiency of the cy7 fluorescence in the intervertebral disc was statistically analyzed. There was no statistically significant difference in the average radiation efficiency before 3 days. Starting from the seventh day, the average radiation efficiency of the 8A6S group was significantly higher than that of the control group.

[0103] The above results show that 8A6S has strong penetration and can penetrate the rat caudal vertebral body and intervertebral disc cartilage endplate and finally enter the intervertebral disc, which also shows that it can be used for in vivo treatment without damaging the intervertebral disc.

[0104] Example 3 Application of arginine polyester amide polymer in promoting proliferation of rat nucleus pulposus cells and reducing cell degeneration

[0105] 1. Obtaining and culturing of rat primary nucleus pulposus cells

[0106] (1) Healthy adult male Sprague Dawley (SD) rats (8 weeks old, weighing 200-250 g) were selected, anesthetized with ether, sacrificed by spinal dislocation, and the tail was cut off with a bone rongeur. After the skin was peeled off, it was soaked in 0.1% PVP-I disinfectant for 10 minutes, and then transferred into a biosafety cabinet covered with sterile gauze;

[0107] (2) Wear surgical gloves and strictly follow the sterile principle. All operating instruments such as ophthalmic scissors, straight forceps, curved forceps, and hemostats need to be sterilized and dried in advance before use;

[0108] (3) The tail soaked in 0.1% PVP-I disinfectant was removed and soaked in PBS to remove excess disinfectant. A scalpel blade was inserted into the annulus fibrosus, and the jelly-like nucleus pulposus tissue was observed to pop out of the intervertebral disc. The tissue was transferred into a 6mm culture dish containing PBS. Each tail could obtain about 8-10 segments of nucleus pulposus tissue;

[0109] (4) The PBS containing the nucleus pulposus tissue was transferred into a 15ml centrifuge tube with a barreled tube;

[0110] (5) Centrifugation was performed at room temperature at 1000 rpm for 5 min, the supernatant was removed, and the operation was repeated twice;

[0111] (6) The total volume of the tissue was estimated, and an equal volume of sterile 0.2% collagenase type II solution was added;

[0112] (7) Transfer into a constant temperature shaking box for digestion, set the program at 37℃, 180 rpm, 30 min;

[0113] (8) Take out the centrifuge tube, observe the degree of tissue digestion, and appropriately extend the digestion time if there are too many tissue clumps;

[0114] (9) Add an equal volume of DMEM / F12 complete medium to terminate digestion;

[0115] (10) Further remove undigested tissue residues through a 70 μm filter screen;

[0116] (11) Centrifuge at room temperature, 1300 rpm, 5 min, remove the supernatant;

[0117] (12) Resuspend the tissue / cell precipitate in 5 mL of DMEM / F12 complete medium, count the single cells using a cell counting plate, and transfer into a T25 culture flask at 2.0 x 10 5 cells / mL;

[0118] (13) Place the culture flask in a 37℃, 5% CO2 constant temperature incubator for culture;

[0119] (14) After 3-5 days, observe under a microscope for cell adhesion and growth, and then change the medium every 2-3 days;

[0120] (15) When the cells grow to 80-90% confluence, add 0.25% EDTA-containing trypsin to digest the cells, and subculture into a T75 culture flask at 1-3. Use the 4th to 7th generation cells for experiments.

[0121] 2. Cryopreservation of rat primary nucleus pulposus cells

[0122] When the cells in the T75 culture flask are in the logarithmic growth phase and grow to 80-90% confluence, some are cryopreserved for preservation, and the specific steps are as follows:

[0123] (1) Remove the old medium and add 2 mL of PBS for washing once;

[0124] (2) Add 1 mL of 0.25% EDTA-containing trypsin, place in a 37℃ cell incubator for digestion for 1 min, and after observing under a light microscope that most cells have detached from the wall, add 2 mL of DMEM / F12 complete medium to terminate digestion;

[0125] (3) Transfer the cell suspension to a centrifuge tube, mix well by blowing, take 10 μL of the cell suspension, and count the single cells using a cell counting plate;

[0126] (4) Centrifuge (4℃, 1000 rpm, 5 min) to remove the supernatant;

[0127] (5) 1.0 x 10 6 The cell freezing solution was added to the cell suspension, and the cell suspension was resuspended by adding an appropriate amount of cell freezing solution, and then transferred to a cell freezing tube and labeled;

[0128] (6) The freezing tube was placed in a programmed cooling box and placed in a -80°C ultra-low temperature refrigerator overnight;

[0129] (7) The next day, the freezing tube was transferred to a liquid nitrogen bottle for freezing.

[0130] 3. Recovery of rat primary nucleus pulposus cells

[0131] (1) The freezing tube was removed from the liquid nitrogen and placed in a 37°C constant temperature water bath to quickly thaw, and then transferred to a 15 mL centrifuge tube;

[0132] (2) 3 times the volume of preheated DMEM / F12 medium was added to the centrifuge tube as a buffer;

[0133] (3) Centrifugation (room temperature, 1000 rpm, 5 min) to remove the supernatant;

[0134] (4) The cell pellet was resuspended by adding preheated DMEM / F12 complete medium;

[0135] (5) The cells were inoculated in a suitable culture bottle according to the number of cells;

[0136] (6) The culture bottle was placed in a 37°C, 5% CO2 cell incubator for culture.

[0137] 4. Treatment of rat nucleus pulposus cells

[0138] (1) The 3rd to 6th generation of rat nucleus pulposus cells (RNPC) were passaged, and the cell suspension was inoculated in a well plate;

[0139] (2) After the cells adhered, different amounts of 8A6S were added and incubated for 24 h;

[0140] (3) After the cells adhered, 175 μM 3% hydrogen peroxide solution prepared with complete culture medium was added, and after incubation for 24 h, the original culture medium was removed, washed once with PBS, and 200 μg / mL 8A6S solution prepared with complete culture medium was added and incubated for 24 h.

[0141] 5. Detection of the biocompatibility of 8A6S

[0142] To verify the effect of 8A6S on normal RNPC, the inventors added different amounts of 8A6S into primary RNPC, and used CCK-8 solution, Annexin V / PI and Calcein AM / PI to detect the changes of RNPC cell activity, apoptosis and toxicity, respectively.

[0143] 5.1 CCK-8 cell activity detection

[0144] (1) After the RNPC in the 96-well plate was treated, the original culture medium was removed, and PBS was washed once, 10 μL of CCK-8 solution was added to each well to avoid air bubbles;

[0145] (2) Set up a blank control: add the corresponding amount of complete culture medium and CCK-8 solution to the wells without cells;

[0146] (3) Incubate in the cell incubator for 1-4 hours, and measure the absorbance at 450 nm every 1 hour using a microplate reader, and select the time point with appropriate absorbance range for further experiment;

[0147] (4) Statistics the data and calculates the cell survival rate = (experimental group OD value-blank group OD value) / (control group OD value-blank group OD value).

[0148] The results of CCK-8 cell activity experiment are shown in Figure 7 It can be seen that 8A6S can promote the proliferation of RNPC, and the promoting effect is most obvious when 300 μg / mL of 8A6S is added.

[0149] 5.2 Annexin V / PI cell apoptosis detection

[0150] (1) After the RNPC in the 12-well plate was treated, the original culture medium was removed, and PBS was washed once, and then an appropriate amount of trypsin cell digestion solution was added to digest the cells, and attention should be paid to avoid excessive digestion of the cells. Gently blowing can make the cells fall off;

[0151] (2) The suspended cells after digestion were transferred to a 1.5 mL EP tube, centrifuged (room temperature, 1000 rpm, 5 min), and the supernatant was discarded, and PBS was washed three times;

[0152] (3) Add 195 μL of Annexin V-FITC binding solution to resuspend the cells; add 5 μL of Annexin V-FITC and 10 μL of propidium iodide staining solution, mix gently, and incubate at room temperature for 10 min in the dark;

[0153] (4) Immediately detect by flow cytometry. Annexin V-FITC is green fluorescence, and propidium iodide (PI) is red fluorescence.

[0154] Annexin V / PI cell apoptosis experiment results are shown in Figure 8 As can be seen, 8A6S has no obvious effect on the apoptosis of RNPC.

[0155] 5.3 Calcein AM / PI cell viability detection

[0156] (1) After treatment, RNPC in the 24-well plate was removed from the original culture medium, and PBS was washed once;

[0157] (2) 500 μL of Calcein AM detection buffer, 0.5 μL of Calcein AM and PI were added to each well, and incubated at 37°C in the dark for 30 min;

[0158] (3) After incubation, inverted fluorescence microscope DMI8 was used to take pictures and record (Calcein AM is green fluorescence, Ex / Em = 494 / 517 nm; PI is red fluorescence, Ex / Em = 535 / 617 nm).

[0159] Calcein AM / PI cell toxicity experiment results are shown in Figure 9 As can be seen, 8A6S has no obvious effect on the cell death of RNPC below 200 μg.

[0160] The above results show that 8A6S has good biocompatibility, can promote the proliferation of RNPC, and has no obvious effect on the cell death of RNPC below 200 μg, so 200 μg of 8A6S is selected for subsequent experiments.

[0161] 6.8A6S rescue effect on degeneration of rat nucleus pulposus cells

[0162] The gene and protein expression levels of Aggrecan (ACAN), Collagen II and SOX9 decrease with the increase of degeneration of nucleus pulposus, while the gene and protein expression levels of matrix metalloproteinase-13 (MMP13) and aggrecanase-5 (ADAMTS5) are inversely related to the degeneration of nucleus pulposus.

[0163] In order to determine whether 8A6S can restore the physiological function of degenerated rat nucleus pulposus cells, the inventors detected the cell morphology, gene level or protein level of Aggrecan, Collagen II, SOX9, MMP13 and ADAMTS5 of rat nucleus pulposus cells after hydrogen peroxide stimulation and 8A6S treatment by real-time fluorescence quantitative PCR, Western blotting and immunofluorescence, respectively.

[0164] (1) Real-time fluorescence quantitative PCR

[0165] RNA of RNPC was extracted using the TRIzol method, and cDNA was synthesized on a T100 ThermA cycler using the PrimeScript RT Reagent Kit (Takara).

[0166] Using PowerUp TM SYBR TM Real-time fluorescence quantitative PCR was performed using Green Master Mix and CFX96 Real-Time System, and repeated three times in three independent experiments. -ΔΔCt Method determination.

[0167] The primer and probe sequences of each gene are shown in Table 1:

[0168] Table 1 Primer and probe sequence information

[0169]

[0170] The qRT-PCR reaction system was as follows: 5 μL of 2× PowerUp SYBR Green Master Mix; 0.4 μL of upstream primer; 0.4 μL of downstream primer; 0.8 μL of diluted cDNA template, and ddH2O to 10 μL. The reaction conditions are shown in Table 2:

[0171] Table 2 qRT-PCR reaction procedure

[0172]

[0173] The CT value of each sample was recorded, and GAPDH was used as the internal reference for mRNA and lncRNA expression. -ΔΔCt The relative expression levels were calculated by the method, and the experiment was repeated 3 times.

[0174] (2) Immunoblotting

[0175] Total cell protein was obtained using the RIPA lysis method, and protein concentration was determined using the BCA assay. Protein sample and 5× loading buffer were added at a ratio of 1:4, and the mixture was boiled in a 100°C metal bath for 10 minutes to denature the protein. The protein was then placed on ice or stored in a -20°C freezer. Western blotting was then performed using specific antibodies to detect the expression levels of ACAN (Proteintech, 13880-1-AP), SOX9, COL2a1 (Thermo, PA1-26206), MMP13 (absin, abs154975), and ADAMTS5 (absin, abs124645). GAPDH was used as an internal control.

[0176] (3) Cell immunofluorescence

[0177] (1) After removing the original culture medium of the treated RNPCs on the cell climbing sheet in the 24-well plate, wash with PBS for 3 times, add 250 μl 4% PFA to fix at room temperature for 10 min, and then wash with PBS for 3 times, each for 5 min;

[0178] (2) Perform permeabilization treatment on the cells by using PBS containing 0.05% Triton X-100, and after permeabilization for 10 min, wash with PBS for 3 times, each for 5 min;

[0179] (3) Add goat serum to completely cover the cells, and incubate at room temperature for 1 h;

[0180] (4) Absorb the goat serum, and add the diluted primary antibody solution according to the proportion in the instruction, and incubate in the refrigerator at 4°C for 12-16 h;

[0181] (5) Absorb the primary antibody solution, and wash with PBS for 3 times, each for 5 min;

[0182] (6) Drop the secondary antibody solution diluted at 1:500, and incubate at room temperature for 1 h;

[0183] (7) Absorb the secondary antibody solution, and wash with PBS for 3 times, each for 5 min;

[0184] (8) Take out the cell climbing sheet in each well, and place it upside down on the glass slide to which the anti-fluorescence quenching sealing agent containing DAPI is added, so as to avoid the generation of air bubbles, and then place it in a wet box in the dark;

[0185] (9) Observe the staining results on the laser confocal microscope LSM880, and take photos of the cells at 10x / 20x magnification.

[0186] The qPCR and Western Blot results are shown in Figure 10 Compared with the normal rat nucleus pulposus cells, the gene and protein expression levels of Aggrecan and Collagen II of the nucleus pulposus cells after hydrogen peroxide stimulation decreased obviously, while the expression levels of MMP13 and ADAMTS5 increased. After the addition of 8A6S, the gene and protein expression levels of Aggrecan and Collagen II increased, while the expression levels of MMP13 and ADAMTS5 decreased obviously.

[0187] The immunofluorescence results are shown in Figure 11As shown, the morphology of rat nucleus pulposus cells changed significantly after hydrogen peroxide stimulation, the cell volume decreased, and the cell number decreased. Second, the fluorescence semi-quantitative result analysis showed that the expression levels of Aggrecan and Collagen II decreased significantly. After the addition of 8A6S, the cell morphology gradually returned to normal levels, the cell number increased, and the expression levels of Aggrecan and Collagen II returned to normal levels.

[0188] In summary, 8A6S can treat the degeneration of rat nucleus pulposus cells by reducing the degeneration of rat nucleus pulposus cells and restoring the physiological function of the cells.

[0189] Example 4 Arginine polyester amide polymer treats rat intervertebral disc degeneration by promoting the regeneration of nucleus pulposus tissue

[0190] 1. Construction of a rat intervertebral disc compression degeneration model

[0191] (1) The rats were anesthetized by intraperitoneal injection of 10% chloral hydrate;

[0192] (2) The positions of the 8th and 10th vertebral disc between the tail vertebrae of the rats were observed and judged, and were marked. The positions of the three blood vessels in the tail of the rats were also observed and judged, and were marked;

[0193] (3) A Kirschner needle was used to vertically punch holes in the upper and lower vertebrae of the intervertebral disc, and care was taken to avoid the blood vessels;

[0194] (4) An Ilizarov-type device with a spring was used for fixation, as shown in Figure 12 ;

[0195] (5) An axial force was applied from the distal end to generate a compressive stress of 1.3 MPa, which is close to the instantaneous intervertebral disc load force generated by lifting a medium weight in a human lumbar vertebra to induce intervertebral disc degeneration;

[0196] (6) The Ilizarov-type device was removed after 7 days, and MRI was used to observe the intervertebral disc degeneration.

[0197] 2. In vivo animal experiment

[0198] The flow chart of the in vivo animal experiment is shown in Figure 13 .

[0199] (1) The rats were randomly divided into three groups: a normal control group, a sham operation control group, and a surgery group. The rats in the normal group were not treated specially. The rats in the sham operation group were fixed with the device as described above, but were not subjected to pressure. The rats in the surgery group were subjected to pressure on the intervertebral disc of the tail vertebrae by the method described above;

[0200] (2) 7 days later, MRI was used to detect the three groups of rats respectively. After the statistical data, 3 rats were randomly taken from the operation group, and the rats were treated according to the method of Example 2. After the intervertebral disc tissue was decalcified, frozen sections and paraffin sections were prepared;

[0201] (3) The remaining rats in the operation group were injected with 50 μL of 8A6S solution on the cartilage endplate of the upper and lower vertebrae according to the method of Example 2, and the bone wax was sealed. Another three rats in the operation group were injected with PBS as the operation control group. 7 days later, the experiment continued according to the method of step (2);

[0202] (4) Similarly, 7 days later, steps (2) and (3) were repeated;

[0203] (5) On the 28th day, after the MRI experiment was completed, all the animals were sacrificed, and the materials were taken according to step (2) for subsequent experiments.

[0204] 3. Image data acquisition

[0205] (1) The MRI image data of all animals was obtained from a 3.0T MRI system SIGNA PIONEER. Magic sequence was directly processed on the scanning console to automatically generate T2WI, T2 mapping and other sequences. The scanning sequence and main parameters are shown in Table 3:

[0206] Table 3 Magic scanning sequence and parameters

[0207]

[0208]

[0209] According to Figure 13 The time point, the inventors obtained the T2WI and T2 mapping sequence image results of the tail vertebrae of the rats at each time point, and simultaneously performed statistical analysis on the intervertebral disc nucleus pulposus region in the T2 mapping sequence (such as Figure 14 ).

[0210] The MRI T2WI results showed that after the operation and compression, the height of the rat intervertebral disc decreased significantly, and the water content of the nucleus pulposus decreased. After 8A6S treatment, the physiological state of the intervertebral disc gradually recovered over time, the height gradually increased, and the nucleus pulposus gradually recovered. Injection of PBS had no significant effect on the degenerated intervertebral disc, proving that the degenerated intervertebral disc could not recover naturally.

[0211] The inventors further statistically analyzed the T2 mapping values. In order to better reflect the degree of intervertebral disc degeneration of each rat, the inventors respectively delineated the ROI of the intervertebral disc after treatment and the ROI of the normal intervertebral disc on the same caudal vertebra, and statistically analyzed the ratio of the two. It was found that the T2 mapping values were significantly reduced after the operation. After injection of 8A6S, the T2 mapping values gradually increased over time and returned to normal levels.

[0212] 3. Staining of tissue frozen sections

[0213] In order to better reflect the pathophysiological changes of each tissue structure of the intervertebral disc, the inventors took the intervertebral disc of the treated rat, and observed and studied the distribution and changes of each tissue of the intervertebral disc by hematoxylin-eosin staining, Safranin O-fast green staining and Alcian blue staining.

[0214] The results are shown in Table 2. Figure 15 Compared with the normal group and the sham operation group, the nucleus pulposus of the rat intervertebral disc after compression was less or even disappeared, but the integrity of the annulus fibrosus was not destroyed. After injection of 8A6S, the annulus fibrosus gradually opened, the nucleus pulposus cartilage tissue gradually began to regenerate, and the normal tissue structure of the intervertebral disc was restored. However, injection of PBS alone could not restore the degenerated intervertebral disc tissue.

[0215] 5. Immunofluorescence detection

[0216] In order to further prove the recovery of the physiological function of the rat intervertebral disc under the action of 8A6S, the inventors observed the expression of Aggrecan, Collagen II (COL2A1), MMP13 and ADAMTS5 related to intervertebral disc degeneration by immunofluorescence method.

[0217] The results are shown in Table 3. Figure 16 Compared with the normal group and the sham operation group, the average fluorescence intensity of Aggrecan and Collagen II in the nucleus pulposus tissue of the rat intervertebral disc was significantly decreased after compression operation, and on the contrary, the expression levels of MMP13 and ADAMTS5 were significantly enhanced. After injection of 8A6S, the expression levels of Aggrecan and Collagen II gradually increased over time, and the average fluorescence intensity of MMP13 and ADAMTS5 was on the contrary.

[0218] In summary, the results of the in vivo animal experiment of the rat showed that after 8A6S penetrated the endplate cartilage into the intervertebral disc, it promoted the regeneration and repair of the nucleus pulposus tissue, and gradually restored the physiological function and tissue structure of the intervertebral disc.

[0219] Example 5 Arginine polyester amide polymer regulates the proliferation of nucleus pulposus cells and tissues by affecting the IL-17 signaling pathway

[0220] 1.8 The mechanism of A6S in treating nucleus pulposus cell degeneration

[0221] To explore the possible mechanism of 8A6S in treating nucleus pulposus cell degeneration, rat nucleus pulposus cells were stimulated with hydrogen peroxide, and then one group was added with PBS and the other group was added with 8A6S, and each group was repeated three times.

[0222] The inventors extracted and sequenced the cell RNA of three groups of rats. Figure 17 ), and found that compared with the control group, the downregulated differentially expressed genes were significantly associated with the IL-17 signaling pathway. 4.2.2 Functional verification of the IL-17 signaling pathway

[0223] To verify the role of the IL-17 signaling pathway in the treatment of 8A6S, we detected the expression of the receptor IL-17RC, signal transduction factors Act1 and TRAF6, and downstream factor RELA in the IL-17 signaling pathway by real-time quantitative fluorescence PCR and immunofluorescence.

[0224] 2. Functional Verification of IL-17 Signaling Pathway

[0225] To verify the role of the IL-17 signaling pathway in the treatment of 8A6S, the inventors detected the expression of the receptor IL-17RC, signal transduction factors Act1 and TRAF6, and downstream acting factor RELA in the IL-17 signaling pathway by real-time quantitative fluorescence PCR and immunofluorescence.

[0226] qPCR results are as follows Figure 18 The results showed that compared with normal RNPC, hydrogen peroxide stimulation upregulated the expression of IL-17R, TRAF6, Act1, and RELA genes, activating the IL-17 signaling pathway. 8A6S treatment reduced the expression of IL-17R, TRAF6, Act1, and RELA genes, inhibiting the activation of the IL-17 signaling pathway.

[0227] Immunofluorescence images such as Figure 19 As shown in the figure, the mean fluorescence intensity of TRAF6, Act1, and IL-17R decreased in RNPCs stimulated by hydrogen peroxide. After treatment with 8A6S, the expression levels of TRAF6, Act1, and IL-17R increased, demonstrating the inhibitory effect of 8A6S on the IL-17 signaling pathway.

[0228] The above experimental results confirm that the IL-17 signaling pathway plays a key role in the treatment of nucleus pulposus cells, and 8A6S may exert a therapeutic effect on RNPC by inhibiting the IL-17 signaling pathway.

[0229] 3.8A6S treats RNPC by affecting IL-17 signaling pathway

[0230] To confirm the key role of IL-17 signaling pathway in 8A6S treatment of degenerative nucleus pulposus cells, the inventors added IL-17A recombinant protein to the RNPC after hydrogen peroxide stimulation to activate the IL-17 signaling pathway, and used qPCR and Western Blot detection to observe the changes in the treatment effect of 8A6S.

[0231] As shown in FIG. 8A, the results show that, compared with normal rat nucleus pulposus cells, the gene and protein expression levels of MMP13 and ADAMTS5 in the hydrogen peroxide-stimulated nucleus pulposus cells were significantly up-regulated, while the gene expression levels of ACAN and SOX9 were down-regulated. After the addition of 8A6S alone, the gene and protein expression levels of MMP13 and ADAMTS5 were reduced, while the gene expression levels of ACAN and SOX9 were significantly increased. However, after the addition of IL-17A and 8A6S at the same time, the expression levels of MMP13, ADAMTS5, ACAN and SOX9 in the nucleus pulposus cells did not change significantly compared with the degenerative nucleus pulposus cells. Figure 20

[0232] The above results show that 8A6S promotes cell proliferation by inhibiting the IL-17 signaling pathway to treat degenerative RNPC.

[0233] All the documents mentioned in the present application are incorporated by reference in the present application as if each document was individually incorporated by reference. In addition, it should be understood that various changes and modifications to the present application can be made by those skilled in the art upon the reading and understanding of the present teaching as contained herein. Such modifications are intended to fall within the scope of the appended claims.​

Claims

1. Use of an arginine polyesteramide polymer in the preparation of a drug for treating intervertebral disc degeneration-related diseases. The arginine polyesteramide polymer is prepared by condensation polymerization of a monomer Nx and an arginine monomer Arg-yS under the catalysis of triethylamine through an amidation reaction, as shown in the following reaction formula: in, x=2, 4, 8, y=2-10.

2. The use according to claim 1, characterized in that The monomer Nx is prepared by the following steps: Add triethylamine and p-nitrophenol to acetone and keep the reaction at 0℃ using ice bath. Dilute in cold acetone, then add dropwise to the above cooled solution, stir at 0°C for 2 hours, and allow to stand overnight at room temperature; the resulting product is precipitated in distilled water, washed thoroughly, recrystallized in 4:1 (v:v) ethyl acetate / DMF, and vacuum dried to constant weight to obtain monomer Nx. The reaction formula is as follows:

3. The use according to claim 1, characterized in that The arginine monomer Arg-yS is prepared by the following steps: L-arginine and Directly mix, then add toluene and p-toluenesulfonic acid monohydrate; heat the solid-liquid reaction mixture to 120-130°C, then stir and reflux for 16-24 hours, and cool to room temperature; pour out the toluene after precipitation, and dissolve the product in isopropanol at 75°C under stirring, and then precipitate at 4°C to obtain arginine monomer Arg-yS. The reaction formula is as follows:

4. The use according to claim 1, characterized in that The arginine polyesteramide polymer treats intervertebral disc degeneration-related diseases through at least one of the following functions: (1) Alleviate the degenerative reaction of nucleus pulposus cells; (2) Restore the physiological functions of degenerated nucleus pulposus cells; (3) Promote the proliferation of nucleus pulposus cells; (4) Promote the regeneration of nucleus pulposus tissue; (5) Promote the repair of nucleus pulposus tissue; (6) reduce the gene and protein expression levels of MMP13 and ADAMTS5 in nucleus pulposus cells; (7) Increase the expression levels of ACAN, Collagen II and SOX9 genes and proteins in nucleus pulposus cells.

5. The use according to claim 4, characterized in that The arginine polyesteramide polymer activates the function by inhibiting the IL-17 signaling pathway.

6. The use according to claim 5, characterized in that The inhibition of the activation of the IL-17 signaling pathway refers to reducing the expression levels of IL-17R, TRAF6, Act1 and RELA genes and proteins.

7. The use according to any one of claims 1 to 6, characterized in that: The drug is injected through the upper and lower vertebrae and can penetrate the cartilage plate into the intervertebral disc tissue.

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