A drug-carrying lysine rhein self-assembled hydrogel and its preparation method and application

Abstract

The invention discloses a drug-carrying rhein lysine self-assembled hydrogel. The drug-free rhein lysine self-assembled hydrogel uses rhein lysine as a raw material, dissolves rhein lysine in a phosphate buffer solution, and obtains the rhein lysine self-assembled hydrogel by ultrasonic dispersion. The invention also discloses a drug-carrying rhein lysine self-assembled hydrogel. The drug-carrying rhein lysine self-assembled hydrogel uses rhein lysine and a drug with an aromatic ring as raw materials, uses a phosphate buffer solution as a solvent, prepares the drug with an aromatic ring into a drug-containing solution, mixes the drug-containing solution with rhein lysine, vortexes to fully dissolve rhein lysine, obtains a solution containing the drug and rhein lysine, and then obtains the drug-carrying rhein lysine self-assembled hydrogel by ultrasonic dispersion. Compared with rhein, rhein lysine can form a uniform and stable drug-carrying rhein lysine self-assembled hydrogel in a wide pH range, and the preparation method is simple and does not require an organic solvent.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine, and relates to a drug-carrying rhein lysine self-assembled hydrogel, a preparation method and application thereof, and a drug-carrying rhein lysine self-assembled hydrogel, a preparation method and application thereof. Background Art

[0002] Gliomas are common, highly invasive primary intracranial tumors. Even with chemotherapy and radiotherapy after surgical resection, the survival period is only 14 months. At the end of the surgical operation, if the therapeutic drugs are delivered directly to the local resection cavity bypassing the blood-brain barrier, the efficacy can be improved, the toxic side effects of the drugs can be reduced, and the survival chances of patients with gliomas can be improved. Currently, local implants using chips as drug carriers, such as carmustine chips, have entered clinical trials. However, the permanent shape and rigidity of the chip are not conducive to the diffusion of drugs and their penetration deep into the tumor. Therefore, it is crucial to develop new local drug delivery carriers.

[0003] A large amount of evidence shows that the progression of brain glioma is affected by the complex interaction between tumor cells and immune cells in the tumor microenvironment. The immune system's response to tumors promotes the formation of a neuroinflammatory tumor microenvironment, and neuroinflammation is one of the key factors causing the survival and progression of gliomas. Surgical removal of tumors can also lead to brain inflammation.

[0004] As a polymer network system, hydrogel has the characteristics of injectability and adjustable mechanical strength, and is considered to be an ideal drug carrier for local drug delivery. In recent years, a variety of supramolecular hydrogel delivery systems carrying drugs have been used in the post-tumor resection cavity. However, the gel matrices currently available on the market generally have poor biocompatibility and biodegradability, and low drug loading, which poses a great obstacle to clinical application. Therefore, the drug self-assembly gel delivery system that does not require any drug carrier, is injectable and has stimulus responsiveness has attracted widespread attention.

[0005] Rhein, an anthraquinone derivative isolated from the traditional Chinese medicine rhubarb, is used to treat neurodegenerative diseases and nerve damage caused by brain trauma because of its anti-neuroinflammatory effect. Studies have shown that rhein can also inhibit the growth of glioma cells and has the potential to fight glioma. The presence of anthraquinone rings and carboxyl groups in the planar structure of rhein gives it the potential to self-assemble into hydrogels. However, the extremely poor water solubility of rhein makes its gelation conditions very strict. It can only be dissolved in the pH range of 8.0 to 9.4 and then dispersed by ultrasonic dispersion to form a gel, which greatly limits its application in the local resection cavity after brain glioma resection. In addition, combined drug use can improve the therapeutic effect of drugs on tumors and is widely used in the treatment of tumors, but the strict gelation conditions of rhein greatly limit its drug loading possibilities.

[0006] Therefore, it is very important to find a rhein derivative that improves the water solubility of rhein and retains its gel-forming potential without affecting its pharmacological activity, and prepare it into a drug-loaded self-assembled hydrogel for application in the local resection cavity after brain glioma resection. Summary of the invention

[0007] The purpose of the present invention is to solve the problem that the self-assembly gelation conditions of rhein are strict due to its poor solubility, and to provide a drug-carrying rhein lysine self-assembled hydrogel, which can form a uniform and stable hydrogel in the pH range of 6 to 10 without the participation of an organic solvent. The hydrogel has anti-tumor and anti-neuroinflammatory activities and has drug-carrying properties, and can load drugs with different pharmacological activities.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] The invention discloses a drug-carrying rhein lysine self-assembled hydrogel (RHL-hydrogel). The drug-free rhein lysine self-assembled hydrogel uses rhein lysine as a raw material. The rhein lysine is dissolved in a phosphate buffer solution and ultrasonically dispersed to obtain the rhein lysine self-assembled hydrogel.

[0010] Rheinlysinate (RHL) is a salt compound formed by the combination of the carboxyl group of rhein and the amino group of lysine. The rheinlysinate is prepared by the following method: mixing rhein and water, adding lysine in batches, and when the lysine is added, the rhein in the system should be completely dissolved; stirring and reacting at a temperature of 32°C for 24 hours, after the reaction is completed, acetone is dripped into the reaction solution, and after the dripping is completed, crystallization is carried out at -20°C, the crystals are collected, washed with anhydrous ethanol, and vacuum dried to obtain rheinlysinate.

[0011] The mass ratio of the rhein to water is 1:250; the volume ratio of the water to acetone is 1:4.

[0012] The molar ratio of rhein to lysine is 1:2-1:3. Whether rhein lysine can form gel is closely related to the molar ratio of rhein to lysine. According to Example 1, the inventor only adjusted the molar ratio of rhein to lysine (1:2, 1:3, 1:4) to obtain rhein lysine salts with different ratios of lysine to rhein, and investigated the gelling ability under pH 7.4: 6 mg of rhein lysine was weighed in a screw bottle, 1 mL of pH 7.4 phosphate buffer solution was added, vortexed to dissolve, and a rhein lysine solution with a concentration of 6 mg / mL was obtained, which was ultrasonically dispersed for 10 minutes under an ultrasonic power of 100 W and an ice bath. The test results show that compared with rhein, the solubility of rhein lysine prepared at each molar ratio is significantly increased, but only the rhein lysine salt prepared at a feed ratio of 1:2-1:3 can form gel after ultrasonic dispersion. The specific results are as follows: the lysine rhein salt prepared according to the feed molar ratio of rhein to lysine of 1:2 is easily prepared into a uniform, stable, and invertible hydrogel after ultrasonic dispersion; the lysine rhein salt prepared according to the feed molar ratio of rhein to lysine of 1:3 can form a gel after ultrasonic dispersion, but the gel formation is more difficult than that of the lysine rhein salt prepared at a feed molar ratio of 1:2. Specifically, the lysine rhein salt prepared at two different feed ratios is prepared into a solution with a concentration of 6 mg / mL using a phosphate buffer solution of pH 7.4, The time required for ultrasonic dispersion and gelation of lysine rhein synthesized at a feed ratio of 1:3 is longer than that at a feed ratio of 1:2, and after ultrasonic dispersion, it needs to be left to stand for a period of time or stored in a refrigerator for a period of time before it can be stably inverted, that is, the stability after gelation is not as good as that at a feed ratio of 1:2 (lysine rhein prepared at a feed ratio of 1:2 can form a uniform, stable and invertible gel after ultrasonic dispersion, and the time required for ultrasonic dispersion is shorter; lysine rhein prepared according to a molar ratio of rhein to lysine of 1:4 cannot form a gel after ultrasonic dispersion.

[0013] Preferably, the molar ratio of rhein to lysine is 1:2.

[0014] The higher the concentration of rhein lysine, the better the stability of the hydrogel after ultrasonic dispersion. As the concentration of rhein lysine increases, the amount of drug that can be loaded also increases. The concentration of rhein lysine is 4 to 14 mg / mL, preferably 4 to 12 mg / mL; the pH of the phosphate buffer is 6 to 10.

[0015] The power of the ultrasonic dispersion is 75 to 150 W, preferably 100 W, and the time of the ultrasonic dispersion is 5 to 30 minutes.

[0016] The ultrasonic dispersion is carried out in an ice bath.

[0017] Another object of the present invention is to provide a method for preparing the drug-carrying rhein lysine self-assembled hydrogel, comprising: dissolving rhein lysine in a phosphate buffer solution, and ultrasonically dispersing to obtain the rhein lysine self-assembled hydrogel. The preparation method is simple in process and does not require an organic solvent.

[0018] Another object of the present invention is to provide the use of the drug-carrying lysine rhein self-assembled hydrogel in the preparation of drugs for treating brain gliomas or microglia-mediated neurodegenerative diseases, and in the preparation of drugs for treating nerve damage caused by brain trauma.

[0019] As a further preferred embodiment of the present invention, another object of the present invention is a drug-loaded rhein lysine self-assembled hydrogel. The drug-loaded rhein lysine self-assembled hydrogel is prepared from rhein lysine and a drug with an aromatic ring as raw materials, and a phosphate buffer solution as a solvent. The drug with an aromatic ring is prepared into a drug-containing solution, the drug-containing solution is mixed with rhein lysine, and vortexed to fully dissolve the rhein lysine to obtain a solution containing the drug and rhein lysine, and then ultrasonically dispersed to obtain the drug-loaded rhein lysine self-assembled hydrogel.

[0020] The drug with an aromatic ring is 5-fluorouracil, temozolomide or edaravone.

[0021] The pH of the phosphate buffer is 6-10.

[0022] The preparation method of the drug-containing solution is as follows: weigh the drug with an aromatic ring, add a phosphate buffer solution, vortex for 30 seconds, and perform ultrasound to fully dissolve the drug to obtain the drug-containing solution.

[0023] In the solution containing the drug and rhein lysine, the concentration of rhein lysine is 4-14 mg / mL, and the concentration of the drug with an aromatic ring is 0.1-2.5 mg / mL.

[0024] The power of the ultrasonic dispersion is 100-200W, and the time of the ultrasonic dispersion is 10-40min.

[0025] The ultrasonic dispersion is carried out in an ice bath.

[0026] Another object of the present invention is to provide the use of the drug-loaded lysine rhein self-assembled hydrogel in the preparation of drugs for treating brain gliomas or protecting nerve damage.

[0027] Specifically, in the temozolomide-loaded rhein lysine self-assembled hydrogel, temozolomide and rhein lysine can play a synergistic role, with significant effects, significantly inhibiting the proliferation of tumor cells, and is expected to become an ideal local implant for postoperative glioma cavities. The present invention also provides the use of the temozolomide-loaded rhein lysine self-assembled hydrogel in the preparation of drugs for treating glioma.

[0028] The beneficial effects of the present invention are:

[0029] Compared with rhein, rhein lysine has better water solubility and can form uniform and stable drug-loaded rhein lysine self-assembled hydrogels in a wide pH range. The preparation method is simple and does not require organic solvents. The formation of the gel greatly delays the release of rhein lysine and retains its own anti-tumor and anti-neuroinflammatory pharmacological activities, which has the potential for local application in the cavity after glioma surgery. The simple and easy gelation conditions and wide gelation range allow rhein lysine self-assembled hydrogels to load drugs with different pharmacological activities, such as anti-tumor drugs 5-fluorouracil, temozolomide, and neuroprotectant edaravone. Rhein lysine can play a synergistic role with the loaded drugs, providing new ideas for drug delivery after glioma surgery and other local areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a digital photo of the lysine rhein solution and the lysine rhein self-assembled hydrogel prepared in Example 2.

[0031] Figure 2 It is a fluorescence emission spectrum diagram of the lysine rhein solution and the lysine rhein self-assembled hydrogel prepared in Example 2.

[0032] Figure 3 It is the infrared spectra of the lysine rhein powder and the lysine rhein self-assembled hydrogel freeze-dried powder in Example 2.

[0033] Figure 4 These are digital photos of the lysine-rhein self-assembled hydrogels prepared under different pH conditions in Example 3.

[0034] Figure 5 The transmission electron microscope image (A) and the scanning electron microscope image (B) of the lysine-rhein self-assembled hydrogel prepared in Example 4 are shown.

[0035] Figure 6 It is the in vitro drug release behavior of rhein lysine self-assembled hydrogel; wherein, A is the in vitro drug release behavior of rhein lysine self-assembled hydrogel prepared from rhein lysine solution with a concentration of 6 mg / mL, and B is the in vitro drug release behavior of rhein lysine self-assembled hydrogel prepared from rhein lysine solution with a concentration of 12 mg / mL.

[0036] Figure 7 The results are as follows: The results are as follows: The inhibitory effect of rhein lysine solution and rhein lysine self-assembled hydrogel on the proliferation of U87 cells; wherein A is the inhibitory effect of rhein lysine on the proliferation of U87 cells after 24 hours of administration, and B is the inhibitory effect of rhein lysine on the proliferation of U87 cells after 72 hours of administration.

[0037] Figure 8The inhibitory effect of lysine rhein solution and lysine rhein self-assembled hydrogel on the secretion of tumor necrosis factor α (TNF-α) by BV2 cells activated by LPS.

[0038] Fig. 9 The inhibitory effect of rhein lysine solution and rhein lysine self-assembled hydrogel on the secretion of interleukin 6 (IL-6) by BV2 cells activated by LPS.

[0039] Fig.10 These are digital photographs of the self-assembled hydrogels of lysine-rhein loaded with 5-fluorouracil, temozolomide and edaravone at room temperature for 0 days and 30 days.

[0040] Fig.11 These are transmission electron micrographs of self-assembled hydrogels of lysine-rhein loaded with 5-fluorouracil, temozolomide, and edaravone, respectively.

[0041] Fig.12 The inhibitory effects of unloaded rhein lysine self-assembled hydrogel and temozolomide-loaded rhein lysine self-assembled hydrogel on the proliferation of U87 cells DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention is further described below in conjunction with specific embodiments and drawings. It should be understood that these embodiments are used to illustrate the present invention and are not limited to the scope of the present invention.

[0043] The reagents used in the present invention are: rhein, lysine, temozolomide, 5-fluorouracil (Shanghai Aladdin Biochemical Technology Co., Ltd.), and edaravone (Pharmaceutical Chemistry Laboratory of Nanjing Medical University).

[0044] Example 1

[0045] Preparation of Lysine-Rhein

[0046] 100 mg of rhein was added to a 250 mL eggplant-shaped bottle, 25 mL of double distilled water was added to the eggplant-shaped bottle, and the bottle was placed on a magnetic stirrer and stirred for 30 minutes; 103 mg of lysine was weighed and slowly added to the eggplant-shaped bottle in batches, and the dissolution of rhein was observed while adding, and the addition time was controlled to 140 minutes. After the addition of lysine, the rhein in the reaction solution should be completely dissolved to present a reddish brown transparent solution; the reaction solution was stirred at 32°C for 24 hours, and acetone was slowly added to the reaction solution at a rate of about 6 drops / min The reaction mixture was added dropwise for 10 minutes while observing the changes in the reaction liquid. After adding 100 mL of acetone, the addition was stopped. The eggplant-shaped bottle was placed in a -20°C refrigerator overnight. The next day, purple crystals were observed to be precipitated on the inner wall of the eggplant-shaped bottle, and some of the purple crystals sank to the bottom of the eggplant-shaped bottle. The reaction liquid was filtered, the filter cake and the eggplant-shaped bottle were vacuum dried, the crystals on the wall of the reaction bottle and the filter cake were scraped off, and washed with anhydrous ethanol for 3 times, and the washing liquid was removed by centrifugation. The product was dried in a vacuum drying oven overnight to obtain lysine rhein, which is a salt compound formed by the combination of the carboxyl group of rhein and the amino group of lysine.

[0047] Example 2

[0048] Preparation of Lysine-Rhein Acid Self-Assembled Hydrogel

[0049] (1) Weigh 3.63 g of disodium hydrogen phosphate, 0.24 g of potassium dihydrogen phosphate, 0.2 g of potassium chloride, and 8.0 g of sodium chloride, add 1 L of double distilled water, and adjust the pH to 7.4 with hydrochloric acid and sodium hydroxide solution to prepare a phosphate buffer solution.

[0050] (2) Weigh 6 mg of rhein lysine into a screw-cap bottle, add 1 mL of pH 7.4 phosphate buffer solution, vortex to dissolve, and obtain a 6 mg / mL rhein lysine solution. Ultrasonic dispersion is performed at an ultrasonic power of 100 W in an ice bath for 10 minutes to prepare a rhein lysine self-assembled hydrogel.

[0051] The appearance of the lysine-rhein solution and the lysine-rhein self-assembled hydrogel was recorded with a digital camera. Figure 1 As shown, the lysine-rhein self-assembled hydrogel is reddish brown, uniform, stable, and invertible.

[0052] Appropriate amounts of the rhein lysine solution and the self-assembled hydrogel of this example were taken, and the emission light intensity of all samples at 450-700 nm was scanned with an excitation wavelength of 434 nm to plot the fluorescence emission spectra of the rhein lysine solution and the self-assembled hydrogel of rhein lysine, respectively. Figure 2 As shown in the figure, when the free state of rhein lysine solution is transformed into a hydrogel state by ultrasonic dispersion, its fluorescence intensity decreases significantly, which is mainly attributed to the π-π interaction of the anthraquinone ring on rhein lysine, which inhibits the conformational rotation of rhein lysine and quenches the fluorescence. This result proves that π-π stacking plays an important role in the formation of rhein lysine self-assembled hydrogel.

[0053] The rhein lysine self-assembled hydrogel prepared in this example was freeze-dried to obtain a xerogel, and equal masses of the xerogel and rhein lysine powder were weighed, and the xerogel and rhein lysine powder were pressed into tablets with potassium bromide to obtain sheet samples, and the Fourier infrared spectra of the rhein lysine self-assembled hydrogel and the rhein lysine powder were detected. Figure 3 As shown, lysine rhein powder is at 2919 cm -1 and 2850cm -1 The stretching vibration peaks of the ammonium salt at 1623 cm -1 The strong peaks of carboxyl ions at 2942 cm-1 and 2950 cm-2 shifted after the formation of hydrogels, and the stretching vibration peak of ammonium salt shifted to 2942 cm-1. -1 and 2869cm -1 The strong peak of carboxyl ion shifted to 1629 cm -1 This result proves that hydrogen bonding plays an important role in the formation of lysine-rhein self-assembled hydrogel.

[0054] Example 3

[0055] Effect of pH on the gel-forming ability of rhein-lysine

[0056] Weigh 6 mg of rhein lysine into a screw-cap bottle, add 1 mL of phosphate buffer solution with a pH of 4, 5, 6, 7.4, 8, 9, 10, and 11 to each of 8 parallel portions, vortex to dissolve, and obtain 6 mg / mL rhein lysine solutions with different pH values. Ultrasonic dispersion was performed at an ultrasonic power of 100 W in an ice bath for 10 min to observe the effect of pH on the gelling ability of rhein lysine.

[0057] like Figure 4 As shown, a 6 mg / mL lysine rhein solution can self-assemble to form a uniform, stable, and invertible hydrogel within a pH range of 6 to 10. As the pH gradually increases, the sample gradually changes from yellow to dark reddish brown. Excessive acidity (pH < 6) and excessive alkalinity (pH > 10) are not conducive to the formation of hydrogels. When the pH of the solvent is less than 6, the structure of lysine rhein will be destroyed, and the yellow rhein raw material will precipitate. This result proves that lysine rhein can form a gel in a wider pH range.

[0058] Example 4 Microstructure Investigation of Lysine-Rhein Acid Self-Assembled Hydrogel

[0059] Preparation of 4 mg / mL rhein lysine solution and self-assembled hydrogel: weigh 4 mg of rhein lysine into a screw-cap bottle, add 1 mL of pH 7.4 phosphate buffer solution, vortex to dissolve to obtain a 4 mg / mL rhein lysine solution, and ultrasonically disperse for 10 minutes under an ultrasonic power of 100 W and an ice bath to prepare a rhein lysine self-assembled hydrogel.

[0060] Transmission electron microscopy: Pipette an appropriate amount of lysine-rhein self-assembled hydrogel onto a glass slide, use tweezers to pick up a copper mesh and gently scoop it back and forth in the sample a few times, place the copper mesh face up on filter paper, and air dry it in a cool place. Then use a transmission electron microscope to observe the microstructure of the hydrogel ( Figure 5 A), Lysine-rhein self-assembled hydrogel exhibits a network-like structure composed of coherent nanofibers.

[0061] Scanning electron microscope: Pipette an appropriate amount of lysine rhein self-assembled hydrogel sample into a vial, pre-freeze at -80℃ until solid, and freeze-dry in a freeze dryer for 24 hours to obtain hydrogel powder. Spray gold on an appropriate amount of hydrogel powder and observe its microstructure under a scanning electron microscope ( Figure 5 B), Lysine-rhein self-assembled hydrogel presents a porous three-dimensional spatial structure.

[0062] Example 5

[0063] In vitro drug release behavior of lysine-rhein self-assembled hydrogel

[0064] Preparation of 6 mg / mL rhein lysine solution and self-assembled hydrogel: weigh 6 mg of rhein lysine into a screw-cap bottle, add 1 mL of pH 7.4 phosphate buffer solution, vortex to dissolve to obtain a 6 mg / mL rhein lysine solution, and ultrasonically disperse for 10 minutes under an ultrasonic power of 100 W and an ice bath to prepare a rhein lysine self-assembled hydrogel.

[0065] Preparation of 12 mg / mL rhein lysine solution and self-assembled hydrogel: weigh 12 mg of rhein lysine into a screw-cap bottle, add 1 mL of pH 7.4 phosphate buffer solution, vortex to dissolve to obtain a 12 mg / mL rhein lysine solution, and ultrasonically disperse for 15 minutes under an ultrasonic power of 100 W and an ice bath to prepare a rhein lysine self-assembled hydrogel.

[0066] PBS with pH 7.4 was used as the release medium to simulate the physiological environment in vivo to investigate the in vitro drug release behavior of lysine rhein self-assembled hydrogel. Appropriate amounts of lysine rhein solutions with concentrations of 6 mg / mL and 12 mg / mL and lysine rhein self-assembled hydrogels prepared at their respective concentrations were taken and placed at the bottom of a 15 mL centrifuge tube. 10 mL of release medium preheated to 37 ° C was added, and each centrifuge tube was placed in a shaker at 37 ° C and oscillated at a constant temperature of 100 rpm. At preset time points such as 0.25, 0.5, 1, 2, 4, 6, 9, 12, 24, 36, 48, and 72 hours, 150 μL of release medium was aspirated, and an equal amount of preheated fresh release medium was added at the same time. The release medium taken out at different time points was diluted with PBS to an appropriate concentration, and its ultraviolet absorbance was detected at 434 nm using a full-wavelength microplate reader. Its cumulative release percentage was calculated based on the lysine rhein standard curve to investigate its in vitro drug release behavior. Three replicates were operated in parallel.

[0067] like Figure 6 As shown, the hydrogel prepared from the lysine rhein solution with a concentration of 6 mg / mL ( Figure 6 A) can release 80% of lysine rhein after 4 days, while lysine rhein solution (concentration of 6 mg / mL) releases the same amount in only 36 hours; the hydrogel prepared from lysine rhein solution with a concentration of 12 mg / mL ( Figure 6 B), it takes 20 days to slowly release 80% of rhein lysine, which is much longer than the 7 days required for rhein lysine solution (concentration of 12 mg / mL) to release the same drug. The above results show that after the rhein lysine solution self-assembles to form a hydrogel, the in vitro release of rhein lysine is significantly delayed.

[0068] Example 6

[0069] 1. Inhibitory effect of lysine-rhein self-assembled hydrogel on the proliferation of U87 cells

[0070] Preparation of liquid medicine:

[0071] Preparation of culture medium containing rhein lysine self-assembled hydrogel: Using rhein lysine self-assembled hydrogel (prepared with reference to Example 2) as the mother liquor, DMEM culture medium was added to dilute it into culture medium containing rhein lysine self-assembled hydrogel (calculated as rhein lysine) with concentrations of 20 μM (8.6 μg / mL), 40 μM (17.2 μg / mL), 80 μM (34.4 μg / mL), and 160 μM (68.8 μg / mL), respectively.

[0072] Preparation of culture medium containing rhein lysine solution: Take a 6 mg / mL rhein lysine solution (prepared with reference to Example 2) as a mother solution, add DMEM culture medium to dilute it into culture medium containing rhein lysine solution with rhein lysine concentrations of 20 μM (8.6 μg / mL), 40 μM (17.2 μg / mL), 80 μM (34.4 μg / mL), and 160 μM (68.8 μg / mL), respectively.

[0073] Dosage:

[0074] Take the U87 cell line in the logarithmic growth phase and in good growth condition, count the cells and resuspend them in DMEM medium to 5×10 3 The cells were uniformly inoculated on a 96-well plate at a density of 100 μL / well. After conventional culture in a cell culture incubator for 24 hours, the culture medium was discarded and medium without drug, medium containing rhein lysine self-assembled hydrogel at different concentrations, and medium containing rhein lysine solution were added respectively. The wells inoculated with the same volume of medium without drug were used as negative control groups, and the wells without drug culture were used as blank control groups. After culture for 24 and 72 hours, 10 μL of 5 mg / mL MTT solution prepared with PBS was added to each well. The cells were cultured in an incubator for 4 hours. The upper culture medium was carefully discarded and 150 μL of dimethyl sulfoxide was added to each well. The plates were shaken in the dark for 10 minutes to completely dissolve the purple crystals at the bottom of the plate. The absorbance of each well was measured at 570 nm on an ELISA reader. The cell survival rate was calculated according to formula (1), thereby investigating the inhibitory effect of rhein lysine self-assembled hydrogel on the proliferation of U87 cells.

[0075]

[0076] like Figure 7 As shown in A, 24 hours after administration, 20-160 μM lysine rhein solution and lysine rhein self-assembled hydrogel significantly reduced the viability of U87 cells ( **** P<0.0001, compared with the negative control group), and the inhibitory effects of rhein lysine solution and hydrogel on the proliferation of U87 cells were comparable. Figure 7 B, As the administration time was extended to 72 hours, the viability of U87 cells continued to decline, further proving the inhibitory effect of rhein on the proliferation of U87 cells. Figure 7 It can be seen that after 72 hours of administration, the lysine rhein solution showed a stronger inhibitory effect on the proliferation of U87 cells compared with the hydrogel ( **** P<0.0001, compared with the negative control group; NS: no statistically significant difference, ### P < 0.001, #### P < 0.0001, between-group comparison), which was attributed to the sustained release of the hydrogel prolonging the onset of drug action.

[0077] II. Inhibitory effect of lysine-rhein self-assembled hydrogel on the secretion of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) by BV2 cells activated by LPS

[0078] Preparation of liquid medicine:

[0079] Preparation of medium containing rhein lysine self-assembled hydrogel: Using rhein lysine self-assembled hydrogel (prepared with reference to Example 2) as the mother liquor, add DMEM medium to dilute it into a drug-containing medium 1 with a rhein lysine self-assembled hydrogel concentration (calculated as rhein lysine) of 20 μM (8.6 μg / mL).

[0080] Preparation of medium containing rhein lysine solution: Take a 6 mg / mL rhein lysine solution (prepared with reference to Example 2) as a mother solution, add DMEM medium to dilute it into a drug-containing medium 2 with a rhein lysine concentration of 20 μM (8.6 μg / mL).

[0081] Take the BV2 cell line in the logarithmic growth phase and in good growth condition, count the cells and calculate the number of cells to 8×10 4 The cells were uniformly inoculated at a density of 100 / well on a 24-well plate. After conventional culture in a cell culture incubator for 24 hours, the culture medium was discarded, and non-drug-containing culture medium and drug-containing culture medium 1 and 2 were added to pretreat BV2 cells for 1 hour, and then LPS solution was added to a final LPS concentration of 100 ng / mL and incubated for 24 hours. BV2 cells pretreated with only the same volume of non-drug-containing culture medium and not stimulated with LPS were used as the control group, and BV2 cells pretreated with only the same volume of non-drug-containing culture medium and stimulated with LPS were used as the vehicle group. The IL-6 and TNF-α contents in the BV2 cell culture medium were detected according to the ELISA kit detection instructions for IL-6 and TNF-α, the cells on the well plate were lysed with 0.1% Triton, and the supernatant was collected after centrifugation at 3000 rpm for 20 minutes. The protein content of each well was determined according to the BCA kit detection instructions, and the level of IL-6 and TNF-α produced in the well was measured by the ratio of the detected IL-6 and TNF-α content to the protein content of the well.

[0082] like Figure 8 , Fig. 9 As shown in the figure, compared with rhein lysine, rhein lysine self-assembled hydrogel showed a better ability to inhibit the secretion of TNF-α and IL-6 by BV2 cells (NS: no statistically significant difference, *** P < 0.001, **** P<0.0001, compared with the control group; # P<0.05, ## P<0.01, ###P < 0.001, #### P<0.0001, inter-group comparison). This result verifies the anti-neuroinflammatory activity of rhein lysine, and the formation of hydrogel can better exert the drug effect.

[0083] Example 7

[0084] Preparation of self-assembled hydrogels of lysine-rhein loaded with 5-fluorouracil, temozolomide and edaravone

[0085] Weigh 2 mg of 5-fluorouracil into an EP tube, add 1 mL of pH 7.4 phosphate buffer, vortex for 30 seconds, and sonicate to fully dissolve the drug to obtain a drug-containing phosphate buffer solution; weigh 8 mg of rhein lysine into an EP tube, add the drug-containing phosphate buffer solution, vortex to fully dissolve the rhein lysine, and ultrasonically disperse for 20 minutes at an ultrasonic power of 125 W and in an ice bath to obtain a lysine rhein self-assembled hydrogel loaded with 5-fluorouracil.

[0086] 2 mg of temozolomide was weighed into an EP tube, 1 mL of pH 7.4 phosphate buffer was added, vortexed for 30 s, and ultrasonicated to fully dissolve the drug to obtain a drug-containing phosphate buffer solution; 8 mg of rhein lysine was weighed into an EP tube, the drug-containing phosphate buffer solution was added, vortexed to fully dissolve the rhein lysine, and ultrasonically dispersed for 20 minutes under an ultrasonic power of 125 W and an ice bath to obtain a temozolomide-loaded rhein lysine self-assembled hydrogel.

[0087] 1.33 mg of edaravone was weighed into an EP tube, 1 mL of pH 7.4 phosphate buffer was added, the mixture was vortexed for 30 s, and the drug was fully dissolved by ultrasound to obtain a drug-containing phosphate buffer solution; 8 mg of rhein lysine was weighed into an EP tube, the drug-containing phosphate buffer solution was added, and the rhein lysine was fully dissolved. The rhein lysine self-assembled hydrogel loaded with edaravone was obtained by ultrasonic dispersion at an ultrasonic power of 125 W and in an ice bath for 30 minutes.

[0088] A digital camera was used to record the appearance of the drug-loaded lysine-rhein self-assembled hydrogel just prepared and after being placed at room temperature for 30 days. Fig.10 As shown, it shows that the self-assembled hydrogel of lysine rhein has drug-carrying capacity and can load small molecule drugs such as 5-fluorouracil, temozolomide and edaravone. Moreover, the drug-carrying hydrogel is still in a gel state after being placed at room temperature for 30 days and can be stably inverted, which verifies that the drug-carrying gel has good stability.

[0089] Example 8

[0090] Transmission electron microscopy image of drug-loaded lysine rhein self-assembled hydrogel

[0091] 0.2 mg of 5-fluorouracil was weighed into an EP tube, 1 mL of a phosphate buffer solution of pH 7.4 was added, the mixture was vortexed for 30 s, and the drug was fully dissolved by ultrasound to obtain a drug-containing phosphate buffer solution; 4 mg of rhein lysine was weighed into an EP tube, the drug-containing phosphate buffer solution was added, and the mixture was vortexed to fully dissolve the rhein lysine. The rhein lysine self-assembled hydrogel loaded with 5-fluorouracil was obtained by ultrasonic dispersion at an ultrasonic power of 125 W and in an ice bath for 20 minutes.

[0092] 0.2 mg of temozolomide was weighed into an EP tube, 1 mL of a phosphate buffer solution of pH 7.4 was added, vortexed for 30 s, and ultrasonically dissolved the drug to obtain a drug-containing phosphate buffer solution; 4 mg of rhein lysinate was weighed into an EP tube, the drug-containing phosphate buffer solution was added, vortexed to fully dissolve the rhein lysinate, and ultrasonically dispersed for 20 minutes under an ultrasonic power of 125 W\ice bath conditions to obtain a temozolomide-loaded rhein lysinate self-assembled hydrogel.

[0093] Weigh 0.1 mg of edaravone into an EP tube, add 1 mL of a phosphate buffer solution of pH 7.4, vortex for 30 seconds, and ultrasonically dissolve the drug fully to obtain a drug-containing phosphate buffer solution; weigh 4 mg of rhein lysinate into an EP tube, add the drug-containing phosphate buffer solution, vortex to dissolve the rhein lysinate fully, and ultrasonically disperse for 30 minutes under an ultrasonic power of 125 W and an ice bath to obtain a rhein lysinate self-assembled hydrogel loaded with edaravone.

[0094] Appropriate amounts of lysine-rhein self-assembled hydrogels loaded with different drugs were respectively drawn onto a glass slide, a copper mesh was picked up with tweezers and gently scooped back and forth in the sample a few times, and the copper mesh was placed face up on filter paper, and the hydrogel microstructure was observed using a transmission electron microscope after natural air drying in a cool place. Fig.11 As shown, the temozolomide-loaded rhein lysine self-assembled hydrogel, the temozolomide-loaded rhein lysine self-assembled hydrogel, and the edaravone-loaded rhein lysine self-assembled hydrogel all present a coherent nanofiber network structure similar to the unloaded rhein lysine self-assembled hydrogel (Example 4), indicating that the addition of an appropriate amount of small molecule drugs with aromatic rings such as 5-fluorouracil, temozolomide and edaravone does not affect the microstructure of the rhein lysine self-assembled hydrogel.

[0095] Example 9

[0096] Referring to the method for preparing the temozolomide-loaded rhein lysine self-assembled hydrogel in Example 7, only the amount of rhein lysine was adjusted from 8 mg to 6 mg to prepare the temozolomide-loaded rhein lysine self-assembled hydrogel.

[0097] Preparation of the test solution: Take the lysine rhein self-assembled hydrogel prepared in Example 2, temozolomide, and the lysine rhein self-assembled hydrogel loaded with temozolomide prepared in this example, and use DMEM culture medium to prepare a lysine rhein self-assembled hydrogel dilution with a concentration (in terms of lysine rhein) of 40 μM, a temozolomide solution with a concentration of 30 μM, and a lysine rhein self-assembled hydrogel dilution loaded with temozolomide with a lysine concentration of 40 μM and a temozolomide concentration of 30 μM.

[0098] Inhibitory effect of temozolomide-loaded rhein lysine self-assembled hydrogel on the proliferation of U87 cells

[0099] Take the U87 cell line in the logarithmic growth phase and in good growth condition, count the cells and resuspend them in DMEM medium at 5×10 3 The cells were uniformly inoculated on a 96-well plate at a density of 1000 cells / well. After conventional culture in a cell culture incubator for 24 hours, the culture medium was discarded, and a 40 μM lysine-rhein self-assembled hydrogel dilution, a 30 μM temozolomide solution, and a 40 μM lysine-rhein self-assembled hydrogel dilution loaded with 30 μM temozolomide were added, respectively. The wells inoculated with cells added with the same volume of culture medium were used as negative control groups, and the wells without cells added with the same volume of culture medium were used as blank controls. For the control group, 10 μL of 5 mg / mL MTT solution prepared with PBS was added to each well after culturing for 24, 48, 72, and 120 hours, respectively. After further culturing in the incubator for 4 hours, the upper culture medium was carefully discarded, 150 μL of dimethyl sulfoxide was added to each well, and the plate was shaken in the dark for 10 minutes to completely dissolve the purple crystals at the bottom of the plate. The absorbance of each well was measured at 570 nm on an ELISA reader, and the cell survival rate was calculated according to formula (1), thereby investigating the inhibitory effect of lysine-rhein self-assembled hydrogel on the proliferation of U87 cells.

[0100] like Fig.12 As shown in the figure, as the administration time increases, compared with the temozolomide solution and the un-drug-loaded lysine rhein self-assembled hydrogel, the temozolomide and lysine rhein in the drug-loaded hydrogel (TMZ-loaded hydregel) exert a synergistic effect and can better inhibit the proliferation of U87 cells. When the administration time is extended to 120 hours, the drug-loaded hydrogel has a significantly better inhibitory effect on the proliferation of U87 cells than the un-drug-loaded lysine rhein self-assembled hydrogel ( * P < 0.05, compared with the temozolomide-loaded rhein self-assembled hydrogel at the corresponding administration time).

Claims

1. A drug-loaded lysine rhein self-assembled hydrogel, characterized in that: The drug-loaded rhein lysine self-assembled hydrogel is prepared from rhein lysine and a drug with an aromatic ring as raw materials, and a phosphate buffer solution as a solvent. The drug with an aromatic ring is prepared into a drug-containing solution, and the drug-containing solution is mixed with rhein lysine, and vortexed to fully dissolve the rhein lysine to obtain a solution containing the drug and rhein lysine, and then ultrasonically dispersed to obtain the drug-loaded rhein lysine self-assembled hydrogel; Lysine rhein is a salt compound formed by the combination of the carboxyl group of rhein and the amino group of lysine; the molar ratio of rhein to lysine is 1:2 to 1:3; The drug with an aromatic ring is 5-fluorouracil, temozolomide or edaravone; The pH of the phosphate buffer is 6 to 10; In the solution containing the drug and rhein, the concentration of rhein is 4 to 14 mg / mL, and the concentration of the drug having an aromatic ring is 0.1 to 2.5 mg / mL.

2. The drug-loaded lysine rhein self-assembled hydrogel according to claim 1, characterized in that: The power of the ultrasonic dispersion is 100-200 W, and the time of the ultrasonic dispersion is 10-40 min.

3. Use of the drug-loaded lysine rhein self-assembled hydrogel according to claim 1 in preparing drugs for treating brain glioma or protecting nerve damage.

4. A drug-carrying lysine-rhein self-assembled hydrogel, characterized in that: The drug-carrying rhein lysine self-assembled hydrogel is made of rhein lysine as a raw material, and rhein lysine is dissolved in a phosphate buffer solution and ultrasonically dispersed to obtain the rhein lysine self-assembled hydrogel; Lysine rhein is a salt compound formed by the combination of the carboxyl group of rhein and the amino group of lysine; the molar ratio of rhein to lysine is 1:2 to 1:3; The concentration of the rhein lysine is 4-14 mg / mL; the pH of the phosphate buffer is 6-7.

4.

5. The drug-carrying lysine-rhein self-assembled hydrogel according to claim 4, characterized in that: The concentration of the lysine rhein is 4-12 mg / mL.

6. The drug-carrying lysine-rhein self-assembled hydrogel according to claim 4, characterized in that: The power of the ultrasonic dispersion is 75 to 150 W, and the time of the ultrasonic dispersion is 5 to 30 minutes.

7. Use of the drug-carrying lysine rhein self-assembled hydrogel according to claim 4 in the preparation of drugs for treating brain glioma or microglia-mediated neurodegenerative diseases, and in the preparation of drugs for treating nerve damage caused by brain trauma.

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