An epsilon-polylysine-based kartogenin cartilage delivery material, and methods and kits for making the same

Abstract

The application discloses a kind of Kartogenin cartilage delivery materials based on epsilon-polylysine and preparation method thereof, the cartilage delivery material is prepared by the covalent bond connection of Kartogenin molecule and epsilon-polylysine.The cartilage delivery material of the application can prolong the residence time of KGN in joint cavity, increase the solubility of KGN, and the material has cartilage permeability and cell membrane permeability, penetrates into cartilage tissue and stem cells and chondrocytes in cartilage tissue in the form of whole material, and degrades under the action of intracellular enzyme, releases KGN, improves the bioavailability of KGN;And have excellent tissue and cell compatibility, will not cause toxic side effects.

Description

Technical Field

[0001] This invention relates to the field of biomaterials, and more particularly to a ε-polylysine-based Kartogenin cartilage delivery material, its preparation method, and a kit. Background Technology

[0002] Osteoarthritis is a disease characterized by degenerative changes in articular cartilage tissue, with joint pain and limited mobility as its main clinical manifestations. With the increasing aging of my country's population, the number of osteoarthritis patients is rising sharply. Statistics show that the incidence of arthritis in people over 40 years of age in my country exceeds 40%. Currently, interventions for arthritis mainly aim to relieve pain and restore function, but there are no clinically effective drugs to improve the pathological manifestations of arthritis. Kartogenin (KGN), a small-molecule inhibitor of the TGF-β and Smad4 / 5 pathways discovered in recent years, can effectively promote the differentiation of mesenchymal stem cells into chondrocytes, inhibit chondrocyte inflammation, and protect chondrocytes in an inflammatory state, making it a highly promising intervention agent for the pathological manifestations of arthritis.

[0003] Currently, in relevant in vivo studies, KGN is administered via intra-articular injection. For the treatment of arthritis, intra-articular injection is superior to systemic administration: firstly, cartilage tissue lacks blood vessels, preventing the drug from reaching that area via blood circulation; secondly, intra-articular injection reduces interference with non-target tissues. However, for small molecule intervention agents like KGN, their half-life in the joint cavity is only 1-4 hours, making it difficult to maintain effective concentrations over a long period; furthermore, KGN molecules themselves have poor water solubility. These problems significantly reduce the bioavailability of intra-articularly injected KGN, severely limiting the clinical application of KGN in arthritis intervention.

[0004] Currently, researchers have proposed using KGN combined with controlled-release materials to address the aforementioned problems. Milan Kang et al. (Biomaterials, 35, 9984-9994) grafted KGN molecules onto natural polymeric chitosan to prepare intra-articular injectable nano- and micro-sized KGN sustained-release microparticles, improving the water solubility of KGN and achieving in vitro sustained-release of KGN for up to 24 weeks and intra-articular residence in rat knee joints for 24 days; their animal experiments showed that this drug delivery method had a better intervention effect on arthritis compared to simple intra-articular KGN injection. Patent CN108079371A discloses a three-dimensional scaffold material for sustained-release KGN, first loading KGN into nano-silica, and then loading it onto a macroscopic three-dimensional scaffold to achieve long-term sustained release of KGN. Patent CN104587531A uses KGN-loaded PLGA nanospheres to prepare a photocrosslinked hyaluronic acid hydrogel, which can be in situ filled at cartilage injury sites and release KGN to promote cartilage repair.

[0005] The above-mentioned technical solutions all provide some solutions for the long-term sustained release of KGN in the joint cavity. However, literature research shows that the mechanism of action of KGN molecules occurs within stem cells and chondrocytes. Therefore, more effectively delivering KGN molecules to stem cells and chondrocytes distributed in cartilage tissue to improve bioavailability is key to its efficient effect. Although existing technologies prolong the residence time of KGN in the joint cavity, KGN is still released into the joint cavity in the form of a small molecule reagent before penetrating into the cartilage tissue to exert its effect. Therefore, existing technologies do not fundamentally change the problems of KGN's water solubility and rapid clearance in the joint cavity, making it difficult to truly improve the bioavailability of KGN in the intervention of arthritis. Summary of the Invention

[0006] To address the problems of the prior art, this invention provides a ε-polylysine-based Kartogenin cartilage delivery material, its preparation method, and a kit. This delivery material can increase the retention time of KGN in the joint cavity, improve its solubility, and deliver KGN to cartilage tissue cells.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The first aspect of this invention is to provide a Kartogenin cartilage delivery material based on ε-polylysine, having a structure as shown in formula (I):

[0009]

[0010] Where R is:

[0011]

[0012] R1 is:

[0013]

[0014] Where p and q are the set of positive integers and 0, and 0≤p≤50, 0≤q≤20;

[0015] R2 is: -H or

[0016]

[0017] Furthermore, in the above formula (I), n is a positive integer, and 5 ≤ n ≤ 200.

[0018] Furthermore, in the above formula (I), a, b, and c are sets of positive integers and 0, and a ≥ 0, and at least one of b and c is not 0.

[0019] Furthermore, in equation (I), a+b+c=n.

[0020] A second aspect of the present invention is to provide a method for preparing the Kartogenin cartilage delivery material, comprising the following steps:

[0021] S1: Kartogenin and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine hydrochloride were mixed and dissolved in N,N-dimethylformamide (DMF), stirred at room temperature, and then the DMF was evaporated. The mixture was then separated by column chromatography to obtain a compound with the structure of formula (II).

[0022]

[0023]

[0024] Kartogenin, the PEG polymer shown in formula (III), dicyclohexylcarbodiimide and 4-dimethylaminopyridine were dissolved in an organic solvent and stirred at 25℃-60℃ for 20-30 hours; the resulting reaction solution was reprecipitated and filtered to obtain a compound with the structure of formula (IV).

[0025]

[0026] S2: Dissolve ε-polylysine hydrochloride fully in the mixed solvent and adjust the pH to 8-9 to obtain an ε-polylysine hydrochloride solution;

[0027] S3: The compound with structure (II) and / or the compound with structure (IV) obtained in S1 is dissolved in a solvent and then slowly added dropwise to an ε-polylysine hydrochloride solution. The reaction is stirred, deionized water is added to the reaction solution, and then the solution is dialyzed and freeze-dried to obtain Kartogenin cartilage delivery material.

[0028] Further, in S1, the molar ratio of Kartogenin and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine hydrochloride is 1:1-1.5.

[0029] More preferably, in S1, the molar ratio of Kartogenin and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine hydrochloride is 1:1.2.

[0030] Further, in S1, the stirring time at room temperature is 1-3 hours; the column chromatography separation conditions are: 200-300 mesh silica gel column, and the eluent is a 1:2 volume ratio of dichloromethane / petroleum ether mixed solvent.

[0031] Furthermore, in S2, the molar ratio of Kartogenin, the PEG polymer shown in formula (III), dicyclohexylcarbodiimide and 4-dimethylaminopyridine is 1:1.2:2:1.5.

[0032] Further, in S2, the reprecipitation specifically involves adding the obtained reaction solution dropwise into 10-20 times its volume of diethyl ether.

[0033] Further, in S3, the mixed solvent is N,N-dimethylformamide / water or dimethyl sulfoxide / water; the volume of the deionized water is 2-5 times the volume of the reaction solution.

[0034] More preferably, the volume ratio of N,N-dimethylformamide or dimethyl sulfoxide to water is 1:1-5, preferably 1:3.

[0035] Furthermore, in S3, the stirring reaction temperature is 25-60℃, preferably 25℃; the stirring reaction time is 2-10 hours, preferably 5 hours.

[0036] Furthermore, in S3, the dialysis is performed using a dialysis bag with a molecular weight cutoff of 1000 Da.

[0037] Further, in S3, the molar ratio of the compound of formula (II) to the amino group in the ε-polylysine molecule is 1:0.1-5; the molar ratio of the compound of formula (IV) to the amino group in the ε-polylysine molecule is 1:0.1-5.

[0038] The third invention is to provide a kit in which a lyophilized powder of a compound of formula (I) is used as component A and a biocompatible solvent is used as component B.

[0039] Furthermore, the biocompatible solvent is one or a mixture of several of physiological saline, physiological buffer, or cell culture medium.

[0040] A fourth aspect of the present invention is to provide the application of the kit in arthritis intervention and cartilage damage repair.

[0041] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0042] This invention relates to a ε-polylysine-based Kartogenin cartilage delivery material that can prolong the residence time of KGN in the joint cavity and increase the solubility of KGN. At the same time, the material has cartilage permeability and cell membrane permeability, allowing it to penetrate into cartilage tissue and stem cells and chondrocytes within the cartilage tissue in the form of a whole material. Under the action of intracellular enzymes, it degrades and releases KGN, thereby improving the bioavailability of KGN.

[0043] The Kartogenin cartilage delivery material of the present invention has excellent tissue and cell compatibility and does not cause toxic side effects.

[0044] The preparation method of this invention is simple, the ε-polylysine used is low cost, and the Kartogenin cartilage delivery material prepared is very easy to use, and can be directly injected into the joint cavity. Attached Figure Description

[0045] Figure 1 These are microscope images of the cartilage permeability test of the KGN cartilage delivery material of this invention;

[0046] Figure 2 This is the kinetic curve of in vitro release of KGN from the KGN cartilage delivery material of the present invention;

[0047] Figure 3 This is a safranin-O stained microscope image of the KGN cartilage delivery material of this invention used to intervene in arthritis in mice. Detailed Implementation

[0048] The present invention will now be described in detail and specifically through specific embodiments to enable a better understanding of the invention. However, the following embodiments do not limit the scope of the invention.

[0049] Example 1

[0050] This embodiment provides a Kartogenin cartilage delivery material based on ε-polylysine, having a structure as shown in formula (I):

[0051]

[0052] Where R is:

[0053]

[0054] R1 is:

[0055]

[0056] Where p and q are the set of positive integers and 0, and 0≤p≤50, 0≤q≤20;

[0057] R2 is: -H or

[0058]

[0059]

[0060] In a preferred embodiment, in equation (I), n is a positive integer, and 5 ≤ n ≤ 200.

[0061] In a preferred embodiment, in equation (I), a, b, and c are sets of positive integers and 0, and a ≥ 0, and at least one of b and c is not 0.

[0062] In a preferred embodiment, in equation (I), a+b+c=n.

[0063] Example 2

[0064] This embodiment provides a method for preparing a ε-polylysine-based Kartogenin cartilage delivery material with the structure of formula (I) when c=0 in Embodiment 1 above:

[0065] Accurately weigh 0.317 g (1 mmol) of KGN and 0.26 g (1.2 mmol) of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM), and fully dissolve them in 15 mL of DMF. Stir at 25 °C for 2 hours. Then, evaporate the organic solvent DMF at 5 Pa and 60 °C. Dissolve the resulting solid powder in 2 mL of dichloromethane and perform column chromatography separation. The separation conditions are: 200-300 mesh silica gel column, and the eluent is a 1:2 volume ratio of dichloromethane / petroleum ether mixed solvent to obtain the yellow powder active intermediate with the structure of formula (II).

[0066]

[0067] Accurately weigh 1 g (the molar amount of amino group is approximately 6.1 mmol) of ε-polylysine hydrochloride (degree of polymerization 25-30), dissolve it completely in 50 mL of a water / DMF mixed solvent with a volume ratio of 1.5:1, and adjust the pH of the solution to 8.5 using 2 M NaOH aqueous solution to obtain an ε-polylysine hydrochloride solution.

[0068] Accurately weigh 0.144 g (0.45 mmol) of the yellow powder active intermediate with the structure of formula (II) above, dissolve it in 10 mL of DMF, and add it dropwise to the above ε-polylysine hydrochloride solution. Stir the reaction at 25 °C for 5 hours. Then, add 150 mL of water to the reaction solution, use a dialysis bag with a molecular weight cutoff of 1000 Da, and dialyze with pure water for 48 hours, changing the dialysate every 6 hours. Finally, freeze-dry the dialyzed liquid to obtain 1.1 g of Kartogenin cartilage delivery material with the structure of formula (I) (c=0).

[0069] According to the calculation based on the feed ratio, all the intermediates were grafted onto the molecular backbone of ε-polylysine.

[0070] Example 3

[0071] This example provides a method for preparing a ε-polylysine-based Kartogenin cartilage delivery material with the structure of formula (I) when a=0.6, b=0.2, and c=0.2 in Example 1 above:

[0072] Kartogenin (0.32 g, 0.1 mmol), the PEG polymer of formula (III) (p = 5, q = 1, 0.36 g, 0.12 mmol), dicyclohexylcarbodiimide (0.25 g, 0.12 mmol) and 4-dimethylaminopyridine (0.15 g, 0.12 mmol) were dissolved in anhydrous dichloromethane and stirred at 25 °C for 25 hours. The resulting reaction solution was reprecipitated and filtered to obtain 0.5 g of the compound with the structure of formula (IV), with a yield of 80%.

[0073]

[0074] 1 g of polylysine hydrochloride (the molar amount of amino group is approximately 6.1 mmol) was accurately weighed and dissolved in 50 mL of water / N'N-dimethylformamide at a volume ratio of 1:1. The pH of the solution was adjusted to between 8 and 9 using 2 M NaOH. Subsequently, 0.56 g (1.22 mmol) of compound (II) and 1.4 g (2.44 mmol) of compound (IV) were added, and the mixture was stirred at room temperature for 30 hours. Then, 50 mL of deionized water was added to the reaction solution, and the mixture was dialyzed for 4 days using a dialysis bag with a molecular weight cutoff of 3500. The solution was then freeze-dried to obtain 1.88 g of KGN cartilage delivery material.

[0075] Verification Example

[0076] 1.1 Verification experiment showing that the ε-polylysine-based Kartogenin cartilage delivery material of this invention can significantly improve the solubility of KGN in water:

[0077] In the 1.1g Kartogenin cartilage delivery material prepared in Example 2, the mass of KGN molecular structure accounted for 0.1g.

[0078] Accurately weigh 0.11 g of the Kartogenin cartilage delivery material prepared in Example 2 and dissolve it in 5 mL of physiological saline. A dynamic light scattering experiment was performed on the solution, and no nanoparticles of any size were detected. This result indicates that the Kartogenin cartilage delivery material prepared in this invention completely dissolves 0.01 g of KGN in water, resulting in a KGN concentration of 2 mg / mL. Furthermore, using a UV-Vis spectrophotometer, the solubility of KGN molecules at the same temperature was measured to be 1.25 μg / mL. Therefore, the Kartogenin cartilage delivery material disclosed in this invention can significantly improve the solubility of KGN in water.

[0079] 1.2 Cartilage Permeability Test

[0080] The KGN cartilage delivery material prepared in Example 2 was labeled with the amino-reactive fluorescent dye FITC and dissolved in physiological saline to prepare a 10 μg / mL solution. Freshly extracted miniature pig joints were used to extract articular cartilage tissue slices with a diameter of 4 mm and a thickness of 1 mm using a bone drill. A custom-made polytetrafluoroethylene mold with several 4 mm diameter through-holes was used. The cartilage tissue slices were placed in the holes of the mold, with the prepared fluorescently labeled KGN cartilage delivery material added above the mold and physiological saline added below, ensuring the liquid was in contact with the lower part of the cartilage tissue slices. After 24 hours, the tissue samples were frozen sectioned and observed under a confocal microscope. Figure 1 As shown.

[0081] Figure 1 Experimental results showed that after 24 hours, the KGN cartilage delivery material efficiently penetrated into the cartilage tissue and was distributed throughout the entire cartilage tissue; simultaneously, fluorescent signals were observed in the cells of the cartilage tissue. This demonstrates that the KGN delivery material constructed in this invention can efficiently penetrate cartilage tissue and enter the cells within it.

[0082] 1.3 Joint cavity retention

[0083] The KGN cartilage delivery material prepared in Example 2 was labeled with the amino-reactive fluorescent dye Alex Fluor 680-NHS and dissolved in physiological saline to prepare a 10 μg / mL solution. 10 μL of this solution was injected into the right knee joint cavity of C57 mice using a microsyringe, while 10 μL of the same fluorescent intensity Alex Fluor 680 dye was injected into the left knee joint cavity. The fluorescence signal within the mouse joint cavity was analyzed using an in vivo imaging system at different time points. Experimental results showed that fluorescence signals were still observable in the joint cavity of the material group after 9 days, while almost no fluorescence signal was observed in the joint cavity of the dye-only group after 2 days.

[0084] Release of 1.4KGN

[0085] The KGN cartilage delivery material prepared in Example 2 was dissolved in physiological buffer solutions with pH = 7.4 containing and without 10U protease to prepare solutions of 1 mg / mL. 500 μL of each solution was then placed in a dialysis tube with a molecular weight cutoff of 1000 Da, and the tube was placed in 5 mL of PBS with pH = 7.4 for dialysis. The dialysate was extracted at fixed time points, and the KGN concentration in the dialysate was detected using a UV-Vis spectrophotometer. The results are as follows: Figure 2 As shown.

[0086] Experimental results show that KGN is barely detectable in the dialysate in the absence of protease, indicating that the KGN cartilage delivery material of the present invention has good hydrolytic stability; however, KGN molecules were detected in the dialysate in the presence of protease, indicating that the KGN cartilage delivery material of the present invention can be degraded and release KGN under the action of protease.

[0087] Application examples

[0088] 2.1 Preparation of Kartogenin Cartilage Delivery Material Kit

[0089] Prepare aqueous solutions of KGN cartilage delivery material prepared in Examples 2 and 3 at a concentration of 1.1 mg / mL; filter the solutions under sterile conditions using a 220 nm pore size filter membrane; take 2 mL of the sterilized aqueous solution and put it into a 5 mL vial, freeze-dry it under sterile conditions, and then seal the vial.

[0090] Take another vial, fill it with 2 mL of physiological saline, seal the vial directly, and sterilize it at 120℃ and 205.8 kPa for 15 minutes.

[0091] The two samples mentioned above constitute a kit, which is used as follows: Use a sterile syringe to draw out all the physiological saline and add it to the KGN cartilage delivery material lyophilized powder. Dissolve it at room temperature, and then use a sterile syringe to draw out the dissolved liquid for injection.

[0092] Evaluation of the effect of 2.2KGN cartilage delivery material on arthritis intervention in C57 mice

[0093] Fifteen female C57 mice weighing 25g ± 5g were used, and the cruciate ligaments of their left and right knee joints were severed. The mice were housed in an SPF-grade animal laboratory for one week to establish a mouse model of arthritis. The mice were randomly divided into three groups. Group 1 received an intra-articular injection of 10 μL of the KGN cartilage delivery material solution from the kit provided in the application example for both left and right knee joints. Group 2 received an intra-articular injection of 10 μL of physiological saline containing 1 μg of KGN solid for both left and right knee joints. Group 3 received an injection of 10 μL of physiological saline. On day 14, each group received a second injection at the same dosage. Twenty-eight days after the first injection, the animals were euthanized, and samples from the left and right knee joints of each mouse were extracted. These samples were then fixed in 4% formaldehyde solution, decalcified with EDTA, and dehydrated with ethanol. Finally, frozen sections were prepared, and the cartilage tissue from each group was histologically evaluated to assess the pathological manifestations of arthritis in different intervention groups. The results are as follows: Figure 3 As shown.

[0094] Experimental results showed that Group 1, treated with KGN cartilage delivery material, exhibited the mildest arthritis pathology. Cartilage tissue sections in this group showed intact cartilage matrix, extensive distribution, and deep staining, closely resembling normal mouse knee cartilage tissue. Group 2 showed better pathological manifestations than the control group (Group 3). This indicates that the KGN cartilage delivery material of this invention is significantly more effective than KGN intra-articular injection alone in treating arthritis pathology, confirming the high efficiency of the KGN cartilage delivery material of this invention.

[0095] The specific embodiments of the present invention have been described in detail above, but they are merely examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A Kartogenin cartilage delivery material based on ε-polylysine, characterized in that, It has a structure as shown in equation (I): ； Formula (I); Where n is a positive integer, and 25≤n≤30; a+b+c=n; Where a and b are positive integers, and a≥0, b≠0, and c=0; Where R is: ； Where R2 is: -H or ； The preparation method of the Kartogenin cartilage delivery material is as follows: Accurately weigh 0.317 g, 1 mmol KGN, and 0.26 g, 1.2 mmol 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM), and dissolve them thoroughly in 15 mL of DMF. Stir at 25 °C for 2 hours. Then, evaporate the organic solvent DMF under 5 Pa and 60 °C. Dissolve the resulting solid powder in 2 mL of dichloromethane and perform column chromatography separation. The separation conditions are: 200-300 mesh silica gel column, and the eluent is a 1:2 volume ratio of dichloromethane / petroleum ether mixed solvent to obtain the yellow powder active intermediate with formula (II). ； Formula (II); Accurately weigh 1g of ε-polylysine hydrochloride with a molar amount of 6.1mmol of amino group and a degree of polymerization of 25-30, and fully dissolve it in 50mL of water / DMF mixed solvent with a volume ratio of 1.5:

1. Adjust the pH of the solution to 8.5 using 2M NaOH aqueous solution to obtain ε-polylysine hydrochloride solution. Accurately weigh 0.144 g and 0.45 mmol of the yellow powder active intermediate with the above-mentioned formula (II) structure, dissolve it in 10 mL of DMF, and add it dropwise to the above-mentioned ε-polylysine hydrochloride solution. Stir the reaction at 25 °C for 5 hours. Then, add 150 mL of water to the reaction solution, use a dialysis bag with a molecular weight cutoff of 1000 Da, and dialyze with pure water for 48 hours, changing the dialysate every 6 hours. Finally, freeze-dry the dialyzed liquid to obtain 1.1 g of Kartogenin cartilage delivery material with the structure of formula (I) and c=0.

2. A method for preparing the Kartogenin cartilage delivery material as described in claim 1, characterized in that, Includes the following steps: Accurately weigh 0.317 g, 1 mmol KGN, and 0.26 g, 1.2 mmol 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM), and dissolve them thoroughly in 15 mL of DMF. Stir at 25 °C for 2 hours. Then, evaporate the organic solvent DMF under 5 Pa and 60 °C. Dissolve the resulting solid powder in 2 mL of dichloromethane and perform column chromatography separation. The separation conditions are: 200-300 mesh silica gel column, and the eluent is a 1:2 volume ratio of dichloromethane / petroleum ether mixed solvent to obtain the yellow powder active intermediate with formula (II). ； Formula (II); Accurately weigh 1g of ε-polylysine hydrochloride with a molar amount of 6.1mmol of amino group and a degree of polymerization of 25-30, and fully dissolve it in 50mL of water / DMF mixed solvent with a volume ratio of 1.5:

1. Adjust the pH of the solution to 8.5 using 2M NaOH aqueous solution to obtain ε-polylysine hydrochloride solution. Accurately weigh 0.144 g and 0.45 mmol of the yellow powder active intermediate with the above-mentioned formula (II) structure, dissolve it in 10 mL of DMF, and add it dropwise to the above-mentioned ε-polylysine hydrochloride solution. Stir the reaction at 25 °C for 5 hours. Then, add 150 mL of water to the reaction solution, use a dialysis bag with a molecular weight cutoff of 1000 Da, and dialyze with pure water for 48 hours, changing the dialysate every 6 hours. Finally, freeze-dry the dialyzed liquid to obtain 1.1 g of Kartogenin cartilage delivery material with the structure of formula (I) and c=0.

3. A kit comprising the Kartogenin cartilage delivery material as described in claim 1, characterized in that, The kit consists of lyophilized powder of the compound shown in formula (I) as component A and biocompatible solvent as component B.

4. The reagent kit according to claim 3, characterized in that, The biocompatible solvent is one or a mixture of several of physiological saline, physiological buffer, or cell culture medium.

5. The use of the kit according to any one of claims 3-4 in the preparation of a medicament for treating arthritis.

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