CN115531291B - A drug-carrying lysine rhein self-assembled hydrogel and its preparation method and application - Google Patents
A drug-carrying lysine rhein self-assembled hydrogel and its preparation method and application Download PDFInfo
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- CN115531291B CN115531291B CN202211069958.7A CN202211069958A CN115531291B CN 115531291 B CN115531291 B CN 115531291B CN 202211069958 A CN202211069958 A CN 202211069958A CN 115531291 B CN115531291 B CN 115531291B
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- 239000000017 hydrogel Substances 0.000 title claims abstract description 137
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 title claims abstract description 100
- 239000004472 Lysine Substances 0.000 title claims abstract description 73
- 238000002360 preparation method Methods 0.000 title abstract description 21
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- 125000003118 aryl group Chemical group 0.000 claims abstract description 12
- 239000002994 raw material Substances 0.000 claims abstract description 6
- 239000002904 solvent Substances 0.000 claims abstract description 4
- 125000001288 lysyl group Chemical group 0.000 claims description 102
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 85
- 238000001132 ultrasonic dispersion Methods 0.000 claims description 34
- BPEGJWRSRHCHSN-UHFFFAOYSA-N Temozolomide Chemical compound O=C1N(C)N=NC2=C(C(N)=O)N=CN21 BPEGJWRSRHCHSN-UHFFFAOYSA-N 0.000 claims description 31
- 229960004964 temozolomide Drugs 0.000 claims description 30
- QELUYTUMUWHWMC-UHFFFAOYSA-N edaravone Chemical compound O=C1CC(C)=NN1C1=CC=CC=C1 QELUYTUMUWHWMC-UHFFFAOYSA-N 0.000 claims description 14
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
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- RZVHIXYEVGDQDX-UHFFFAOYSA-N 9,10-anthraquinone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3C(=O)C2=C1 RZVHIXYEVGDQDX-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
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- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 description 2
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- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 1
- DLGOEMSEDOSKAD-UHFFFAOYSA-N Carmustine Chemical compound ClCCNC(=O)N(N=O)CCCl DLGOEMSEDOSKAD-UHFFFAOYSA-N 0.000 description 1
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- 208000025698 brain inflammatory disease Diseases 0.000 description 1
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- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
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- 229910052739 hydrogen Inorganic materials 0.000 description 1
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- 210000002865 immune cell Anatomy 0.000 description 1
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- 239000008176 lyophilized powder Substances 0.000 description 1
- 229960003646 lysine Drugs 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
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- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
- A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
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- A—HUMAN NECESSITIES
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/415—1,2-Diazoles
- A61K31/4152—1,2-Diazoles having oxo groups directly attached to the heterocyclic ring, e.g. antipyrine, phenylbutazone, sulfinpyrazone
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- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/4164—1,3-Diazoles
- A61K31/4188—1,3-Diazoles condensed with other heterocyclic ring systems, e.g. biotin, sorbinil
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- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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Abstract
本发明公开了一种可载药的赖氨大黄酸自组装水凝胶，未载药赖氨大黄酸自组装水凝胶是以赖氨大黄酸为原料，将赖氨大黄酸溶解于磷酸盐缓冲溶液中，超声分散得到赖氨大黄酸自组装水凝胶。本发明还公开了一种载药赖氨大黄酸自组装水凝胶，载药赖氨大黄酸自组装水凝胶是以赖氨大黄酸、带有芳香环的药物为原料，以磷酸盐缓冲溶液为溶剂，将带有芳香环的药物配制成含药溶液，将含药溶液与赖氨大黄酸混合，涡旋使赖氨大黄酸充分溶解，得到包含药物和赖氨大黄酸的溶液，再经超声分散得到载药赖氨大黄酸自组装水凝胶。与大黄酸相比，赖氨大黄酸在广泛的pH范围内均可形成均匀、稳定的可载药的赖氨大黄酸自组装水凝胶，制备方法简单且无需有机溶剂。
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 ultrasonically disperses to obtain the rhein lysine self-assembled hydrogel. 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 ultrasonically disperses to obtain the drug-carrying rhein lysine self-assembled hydrogel. 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
The invention belongs to the field of traditional Chinese medicines, and relates to a drug-loaded self-assembled lysyl rhein hydrogel, a preparation method and application thereof, and a drug-loaded self-assembled lysyl rhein hydrogel, a preparation method and application thereof.
Background
Gliomas are a common primary intracranial tumor with high invasiveness, and even if chemotherapy, radiotherapy and other treatments are performed after surgical excision, the survival time is only 14 months. When the surgery is finished, if the therapeutic drug bypasses the blood brain barrier and is directly delivered to the local part of the excision cavity, the curative effect can be improved, the toxic and side effects of the drug can be reduced, and the survival chance of a patient with brain glioma can be improved. Currently, wafer-based topical implants such as carmustine wafers have been put into clinical trials. However, the permanent shape and rigidity of the wafer is detrimental to drug diffusion and penetration into the tumor depth. Therefore, the development of new drug local delivery vehicles is of paramount importance.
Much evidence suggests that brain glioma progression is affected by complex interactions between tumor cells and immune cells within the tumor microenvironment. The immune system response to tumors promotes the formation of neuroinflammatory tumor microenvironment, and neuroinflammation is one of the key factors responsible for glioma survival and progression. Surgical removal of tumors can also lead to the occurrence of brain inflammation.
The hydrogel is used as a polymer network system, has the characteristics of injectability, adjustable mechanical strength and the like, and is considered as an ideal drug carrier for local administration. In recent years, a variety of drug-carrying supramolecular hydrogel delivery systems have been used in post-tumor resection cavities. However, the gel matrix currently marketed is generally poor in biocompatibility and biodegradability, and low in drug loading, which brings great obstacle to clinical application. Therefore, drug self-assembled gel delivery systems that are injectable and stimulus responsive without any drug carrier are of great interest.
Rhein is an anthraquinone derivative separated from rhubarb, and can play the role of resisting neuroinflammation, so that it is used in treating neurodegenerative diseases and cerebral trauma. Studies have shown that rhein can also inhibit the growth of glioma cells, and has the potential of resisting glioma. The presence of anthraquinone ring and carboxylate on rhein plane structure makes rhein possess the potential of self-assembling to form hydrogel. However, the extremely poor water solubility of rhein makes the gelling condition very strict, and the rhein can be dissolved and dispersed in the pH range of 8.0-9.4 to form gel, which greatly limits the application of rhein to the local excision cavity after the glioma excision. In addition, the combined medicine can improve the treatment effect of the medicine on the tumor, and is widely applied to the treatment of the tumor, but the possibility of medicine loading is greatly limited by the adhesive tape forming piece which is Huang Suanyan in size.
Therefore, it is important to find a rhein derivative with improved rhein water solubility, which retains its gelling potential without affecting its pharmacological activity, and to apply the self-assembled hydrogel prepared as drug-carrying agent to the resection cavity part after the glioma resection.
Disclosure of Invention
The invention aims to solve the problem that rhein is severely assembled into a rubber strip part due to poor solubility, and provides a drug-carrying self-assembled lysyl rhein hydrogel which can form uniform and stable hydrogel within the pH range of 6-10 under the condition of no participation of an organic solvent, and the hydrogel has anti-tumor and anti-neuroinflammation activities, has drug-carrying performance and can carry drugs with different pharmacological activities.
In order to solve the technical problems, the invention adopts the following technical scheme:
a self-assembled hydrogel (RHL-hydrogel) of medicine-carried lysine is prepared from the material of lysine through dissolving it in phosphate buffer solution, and ultrasonic dispersing.
Lys rhein (RHEINLYSINATE, RHL) is a salt compound formed by combining the carboxyl group of rhein with the amino group of lysine. The lys rhein is prepared by mixing rhein and water, adding lysine in batches, stirring at 32 deg.c for 24 hr to react, dripping acetone into the reaction liquid, crystallizing at-20 deg.c, collecting crystal, washing with absolute alcohol, and vacuum drying.
The mass ratio of the rhein to the water is 1:250, and the volume ratio of the water to the acetone is 1:4.
The feeding mole ratio of rhein to lysine is 1:2-1:3. Whether the rhein can form gel is related to the feeding mole ratio of rhein to lysine. According to example 1, the inventors only adjusted the molar ratio of rhein to lysine (1:2, 1:3, 1:4) to prepare lysine and Huang Suanbi cases of different rhein salts, examined the gelling ability under the condition of pH7.4, weighed 6mg of rhein in a screw bottle, added 1mL of phosphate buffer solution of pH7.4, dissolved by vortex to obtain rhein solution with concentration of 6mg/mL, and dispersed by ultrasound for 10 minutes under the condition of ultrasonic power of 100W and ice bath. Test results show that compared with rhein, the solubility of the rhein prepared by each molar ratio is obviously increased, but the rhein prepared by the material adding ratio of 1:2-1:3 can be gelled only after ultrasonic dispersion. The preparation method comprises the steps of preparing uniform and stable and invertible hydrogel after the rhein prepared according to the feeding molar ratio of 1:2 of rhein and lysine is subjected to ultrasonic dispersion, and performing stable inversion after the rhein prepared according to the feeding molar ratio of 1:3 of rhein and lysine is subjected to ultrasonic dispersion, wherein the stability after the glue is not as high as that of the rhein prepared by feeding according to the feeding ratio of 1:2, particularly, the rhein prepared by adopting a phosphate buffer solution with the pH of 7.4 is difficult to prepare the rhein prepared at two different feeding ratios into a solution with the concentration of 6mg/mL, the time required for performing ultrasonic dispersion on the rhein synthesized according to the feeding ratio of 1:3 is longer than that required by feeding ratio of 1:2, and the rhein prepared according to the feeding ratio of 1:3 needs to be stood for a period of time or stored in a refrigerator after ultrasonic dispersion is subjected to stable inversion, namely the stability after the glue is not as high as that of the rhein 1:2 (the rhein prepared according to the feeding ratio of 1:2, the rhein can be formed into stable and invertible gel after ultrasonic dispersion is subjected to ultrasonic dispersion, the rhein accordance with the required time of the feeding ratio of 1:2).
Preferably, the feeding mole ratio of rhein to lysine is 1:2.
The higher the concentration of the lysine rhein, the better the stability of the hydrogel after ultrasonic dispersion into gel, and the increase of the concentration of the lysine rhein increases the amount of the drug loaded by the hydrogel, wherein the concentration of the lysine rhein is 4-14 mg/mL, preferably 4-12 mg/mL, and the pH of the phosphate buffer solution is 6-10.
The power of ultrasonic dispersion is 75-150W, preferably 100W, and the time of ultrasonic dispersion is 5-30 min.
The ultrasonic dispersion is carried out under ice bath conditions.
The invention also provides a preparation method of the drug-loaded self-assembled lysyl rhein hydrogel, which comprises the steps of dissolving lysyl rhein in phosphate buffer solution, and performing ultrasonic dispersion to obtain the self-assembled lysyl rhein hydrogel. The preparation method has simple process and no need of organic solvent.
The invention also aims to provide the application of the self-assembled hydrogel of the drug-loaded lysyl rhein in preparing drugs for treating brain glioma or microglial cell mediated neurodegenerative diseases and preparing drugs for treating nerve injury caused by brain trauma.
As a further preferable scheme of the invention, the invention further aims at providing the drug-loaded rhein self-assembled hydrogel, wherein the drug-loaded rhein self-assembled hydrogel takes rhein and drugs with aromatic rings as raw materials, phosphate buffer solution is used as a solvent, the drugs with the aromatic rings are prepared into drug-containing solution, the drug-containing solution is mixed with the rhein, the rhein is fully dissolved by vortex, the solution containing the drugs and the rhein is obtained, and the drug-loaded rhein self-assembled hydrogel is obtained by ultrasonic dispersion.
The medicine with the aromatic ring is 5-fluorouracil, temozolomide or edaravone and the like.
The pH value of the phosphate buffer solution is 6-10.
The preparation method of the medicine-containing solution comprises the steps of weighing the medicine with aromatic rings, adding phosphate buffer solution, swirling for 30s, and fully dissolving the medicine by ultrasonic wave to obtain the medicine-containing solution.
In the solution containing the drug and the lysine rhein, the concentration of the lysine rhein is 4-14 mg/mL, and the concentration of the drug with an aromatic ring is 0.1-2.5 mg/mL.
The power of ultrasonic dispersion is 100-200W, and the time of ultrasonic dispersion is 10-40 min.
The ultrasonic dispersion is carried out under ice bath conditions.
The invention also aims to provide the application of the drug-loaded rhein self-assembled hydrogel in preparing drugs for treating brain glioma or protecting nerve injury.
Specifically, in the self-assembled lysyl rhein hydrogel loaded with temozolomide, the temozolomide and lysyl rhein can play a synergistic effect, have remarkable effect and obviously inhibit proliferation of tumor cells, and are expected to become an ideal brain glioma postoperative cavity local implant. The invention also provides application of the temozolomide-loaded lysyl rhein self-assembled hydrogel in preparation of a medicine for treating brain glioma.
The invention has the beneficial effects that:
Compared with rhein, the rhein has better water solubility, can form uniform and stable self-assembled hydrogel capable of carrying medicine in a wide pH range, and has simple preparation method and no need of organic solvent. The gel is formed to greatly delay the release of the lysine rhein, and retain the pharmacological activities of the lysine rhein such as anti-tumor and anti-neuroinflammation, and the like, thereby having the potential of local application of the cavity after glioma operation. The method has the advantages that the method is simple and easy to implement under the gelling condition, and the wide gelling range enables the self-assembled hydrogel of the lysyl rhein to be loaded with medicines with different pharmacological activities, such as antitumor medicines of 5-fluorouracil, temozolomide, neuroprotectants of edaravone and the like, and the lysyl rhein can play a synergistic effect with the loaded medicines, so that a new thought is provided for the drug delivery after glioma operation and other local parts.
Drawings
FIG. 1 is a digital photograph of a self-assembled hydrogel of lysyl rhein solution and lysyl rhein prepared in example 2.
FIG. 2 is a fluorescence emission spectrum of the self-assembled hydrogel of lysyl rhein and lysyl rhein prepared in example 2.
FIG. 3 is an infrared spectrum of the self-assembled hydrogel lyophilized powder of lysyl rhein powder and lysyl rhein in example 2.
FIG. 4 is a digital photograph of the self-assembled hydrogel of lysyl rhein prepared under different pH conditions of example 3.
FIG. 5 shows a transmission electron microscope (A) and a scanning electron microscope (B) of the self-assembled lysyl rhein hydrogel prepared in example 4.
FIG. 6 shows the in vitro drug release behavior of the self-assembled hydrogel of lys rhein, wherein A is the in vitro drug release behavior of the self-assembled hydrogel of lys rhein prepared from lys rhein solution with a concentration of 6mg/mL, and B is the in vitro drug release behavior of the self-assembled hydrogel of lys rhein prepared from lys rhein solution with a concentration of 12 mg/mL.
FIG. 7 shows the proliferation inhibition effect of the self-assembled hydrogel of lysyl rhein and lysyl rhein on U87 cells, wherein A is the proliferation inhibition effect of 24h administration on U87 cells, and B is the proliferation inhibition effect of 72h administration on U87 cells.
FIG. 8 is the inhibition of secretion of tumor necrosis factor alpha (TNF-alpha) by LPS activated BV2 cells by lysyl rhein solution and lysyl rhein self-assembled hydrogel.
FIG. 9 is the inhibition of interleukin 6 (IL-6) secretion by LPS activated BV2 cells by lysyl rhein solution and lysyl rhein self-assembled hydrogels.
FIG. 10 is a photograph of digital photographs of lysyl rhein self-assembled hydrogels loaded with 5-fluorouracil, temozolomide, and edaravone, respectively, left at room temperature for 0 days and 30 days.
FIG. 11 is a transmission electron microscope image of a self-assembled hydrogel of lysyl rhein loaded with 5 fluorouracil, temozolomide and edaravone, respectively.
FIG. 12 shows the proliferation inhibition effect of non-drug-loaded self-assembled lysyl rhein hydrogel and temozolomide-loaded self-assembled lysyl rhein hydrogel on U87 cells
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be further described with reference to specific embodiments and drawings. It should be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
The invention adopts the reagents of rhein, lysine, temozolomide, 5-fluorouracil (Shanghai Ala Biotechnology Co., ltd.) and edaravone (Nanjing medical university pharmaceutical laboratory).
Example 1
Preparation of lysyl rhein
Adding 100mg rhein into 250mL eggplant-shaped bottle, adding 25mL double distilled water into the eggplant-shaped bottle, placing on a magnetic stirrer for stirring for 30min, weighing 103mg lysine, slowly adding into the eggplant-shaped bottle in batches, observing the dissolution condition of rhein while adding, controlling the adding time to be 140 min, completely dissolving rhein in the reaction solution after adding lysine, presenting a reddish brown transparent solution, stirring the reaction solution at 32 ℃ for 24 h, slowly dripping acetone into the reaction solution, controlling the dripping speed to be about 6 drops/min, observing the change of the reaction solution while dripping 100mL acetone, stopping adding, placing the eggplant-shaped bottle in a refrigerator at-20 ℃ for overnight, observing the precipitation of purple crystals on the inner wall of the eggplant-shaped bottle the next day, precipitating part of purple crystals on the bottom of the eggplant-shaped bottle, filtering the reaction solution, vacuum drying filter cakes and the eggplant-shaped bottle, scraping the wall crystals and the filter cakes of the reaction bottle, washing 3 times with absolute ethyl alcohol, centrifuging to remove washing liquid, and drying the product in a vacuum drying box to obtain the rhein which is the salt compound formed by combining the carboxyl of rhein and the lysine with the amino group of the lysine.
Example 2
Preparation of self-assembled hydrogel of lysyl rhein
(1) 3.63G of disodium hydrogen phosphate, 0.24g of monopotassium phosphate, 0.2g of potassium chloride and 8.0g of sodium chloride are weighed, 1L of double distilled water is added, and the pH is regulated to 7.4 by using hydrochloric acid and sodium hydroxide solution, so as to prepare a phosphate buffer solution.
(2) Weighing 6mg of lysine rhein in a screw bottle, adding 1mL of phosphate buffer solution with pH of 7.4, performing vortex dissolution to obtain a rhein solution with the concentration of 6mg/mL, and performing ultrasonic dispersion for 10 minutes under the conditions of ultrasonic power of 100W and ice bath to prepare the rhein self-assembled hydrogel.
The appearance of the lys rhein solution and the lys rhein self-assembled hydrogel was recorded with a digital camera, as shown in fig. 1, the lys rhein self-assembled hydrogel was reddish brown, uniform and stable, and reversible.
And absorbing a proper amount of the lys rhein solution and the self-assembled hydrogel in the embodiment, scanning the emitted light intensity of all samples at 450-700 nm by taking 434nm as an excitation wavelength, and respectively drawing fluorescence emission spectra of the lys rhein solution and the lys rhein self-assembled hydrogel. As shown in FIG. 2, when the lysine rhein solution in the free state is converted into the hydrogel state by ultrasonic dispersion, its fluorescence intensity is greatly reduced, which is mainly due to pi-pi interaction of anthraquinone ring on the lysine rhein, which suppresses conformational rotation of the lysine rhein and quenches fluorescence. This result demonstrates that pi-pi accumulation plays an important role in the formation of self-assembled hydrogels of lysyl rhein.
The self-assembled hydrogel of lys rhein prepared in this example was freeze-dried to obtain xerogel, and equal mass xerogel and lys rhein powder were weighed and pressed with potassium bromide to obtain tablet samples, and fourier infrared spectrograms of the lys rhein self-assembled hydrogel and lys rhein powder were detected. As shown in FIG. 3, the telescopic vibration peaks of the lysine rhein powder at 2919cm -1 and 2850cm -1 and the carboxyl ion strong peak at 1623cm -1 are shifted after the hydrogel is formed, the telescopic vibration peaks of the lysine rhein powder are shifted to 2942cm -1 and 2869cm -1, and the carboxyl ion strong peak is shifted to 1629cm -1. This result demonstrates that hydrogen bonding plays an important role in the formation of self-assembled hydrogels of lysyl rhein.
Example 3
Influence of pH on the gelling ability of lysine rhein
Weighing 6mg of lysyl rhein in a screw bottle, adding 1mL of phosphate buffer solution with pH of 4,5, 6, 7.4, 8, 9, 10 and 11 into the bottle in parallel with 8 parts, performing vortex dissolution to obtain lysyl rhein solution with concentration of 6mg/mL and different pH, performing ultrasonic dispersion for 10 minutes under the conditions of ultrasonic power of 100W and ice bath, and observing the influence of the pH on the gelling capacity of the lysyl rhein.
As shown in FIG. 4, the solution of the lysine and the rheum officinale with the concentration of 6mg/mL can be self-assembled to form uniform, stable and invertible hydrogel within the pH range of 6-10. As the pH gradually increased, the sample gradually changed from yellow to dark reddish brown. Peracids (pH < 6), overbases (pH > 10) all do not favor hydrogel formation. When the pH of the solvent is less than 6, the structure of the lysyl rhein is destroyed, and yellow rhein raw materials are separated out. This result demonstrates that lysyl rhein can gel over a broad pH range.
EXAMPLE 4 microstructure investigation of self-assembled hydrogels of lysyl rhein
The preparation method comprises the steps of weighing 4mg of lysyl rhein in a screw bottle, adding 1mL of phosphate buffer solution with pH of 7.4, performing vortex dissolution to obtain lysyl rhein solution with concentration of 4mg/mL, and performing ultrasonic dispersion for 10 minutes under the conditions of ultrasonic power of 100W and ice bath to obtain the lysyl rhein self-assembled hydrogel.
And (3) a transmission electron microscope, namely sucking a proper amount of self-assembled lysyl rhein hydrogel on a glass slide, clamping a copper mesh by forceps, slightly fishing back and forth in a sample, placing the copper mesh on filter paper with the front face upwards, naturally airing at a shade place, and observing the microstructure of the hydrogel by using the transmission electron microscope (figure 5A), wherein the lysyl rhein self-assembled hydrogel presents a network-shaped structure formed by coherent nanofibers.
And (3) a scanning electron microscope, namely sucking a proper amount of lysyl rhein self-assembled hydrogel sample into a penicillin bottle, placing the penicillin bottle in a-80 ℃ to be pre-frozen to be solid, and freeze-drying the hydrogel sample for 24 hours by a freeze dryer to obtain hydrogel powder. After a proper amount of hydrogel powder is subjected to metal spraying treatment, the microstructure of the hydrogel powder is observed under a scanning electron microscope (figure 5B), and the self-assembled lysyl rhein hydrogel presents a porous three-dimensional space structure.
Example 5
In vitro drug release behavior of self-assembled hydrogel of lysyl rhein
The preparation method comprises the steps of weighing 6mg of lysyl rhein in a screw bottle, adding 1mL of phosphate buffer solution with pH of 7.4, performing vortex dissolution to obtain lysyl rhein solution with concentration of 6mg/mL, and performing ultrasonic dispersion for 10 minutes under the conditions of ultrasonic power of 100W and ice bath to obtain the lysyl rhein self-assembled hydrogel.
12Mg/mL of lysyl rhein solution and self-assembled hydrogel are prepared by weighing 12mg of lysyl rhein in a screw bottle, adding 1mL of phosphate buffer solution with pH of 7.4, performing vortex dissolution to obtain 12mg/mL of lysyl rhein solution, and performing ultrasonic dispersion for 15 minutes under the conditions of ultrasonic power of 100W and ice bath to prepare the lysyl rhein self-assembled hydrogel.
PBS with pH of 7.4 is used as a release medium, and the in-vitro drug release behavior of the self-assembled hydrogel of the lysyl rhein is inspected by simulating the physiological environment in the body. And respectively taking a proper amount of lys rhein solution with the concentration of 6mg/mL and 12mg/mL and the prepared lys rhein self-assembled hydrogel with the respective concentrations, placing the hydrogel at the bottom of a 15mL centrifuge tube, adding 10mL of release medium preheated to 37 ℃, placing each centrifuge tube in a shaking table at 37 ℃, vibrating at constant temperature at the frequency of 100rpm, sucking 150 mu L of release medium at preset time points of 0.25, 0.5, 1, 2,4, 6, 9, 12, 24, 36, 48, 72 hours and the like, and supplementing the same amount of preheated fresh release medium. The release medium taken out at different time points is diluted to proper concentration by PBS, the ultraviolet absorbance is detected at 434nm by a full-wavelength enzyme-labeled instrument, the accumulated release percentage is calculated according to the standard curve of the rhein, and the in-vitro drug release behavior is examined. Triplicate runs were performed.
As shown in FIG. 6, the hydrogel (FIG. 6A) prepared from the rhein solution at a concentration of 6mg/mL can release 80% of rhein after 4 days, while the rhein solution (at a concentration of 6 mg/mL) releases the same amount only for 36 hours, and the hydrogel (FIG. 6B) prepared from the rhein solution at a concentration of 12mg/mL, which slowly releases 80% of rhein, takes 20 days, which is much longer than 7 days required for the rhein solution (at a concentration of 12 mg/mL) to release the same drug. The results show that the in vitro release of the lys rhein is obviously delayed after the lys rhein solution self-assembles to form hydrogel.
Example 6
1. Proliferation inhibition effect of lysyl rhein self-assembled hydrogel on U87 cells
Preparing a liquid medicine:
The preparation of the self-assembled hydrogel medium containing lys rhein comprises diluting a self-assembled hydrogel (prepared in reference to example 2) containing lys rhein with DMEM medium to give a self-assembled hydrogel medium containing lys rhein with concentration of Cheng Laian rhein (calculated as lys rhein) of 20. Mu.M (8.6. Mu.g/mL), 40. Mu.M (17.2. Mu.g/mL), 80. Mu.M (34.4. Mu.g/mL) and 160. Mu.M (68.8. Mu.g/mL), respectively.
Preparation of a Rheum acid solution containing solution of lysine culture medium A Rheum acid solution (prepared in reference example 2) with concentration of 6mg/mL was taken as mother liquor, and DMEM culture medium was added to dilute Cheng Laian Rheum acid solution containing solution of lysine culture medium with concentration of 20. Mu.M (8.6. Mu.g/mL), 40. Mu.M (17.2. Mu.g/mL), 80. Mu.M (34.4. Mu.g/mL) and 160. Mu.M (68.8. Mu.g/mL), respectively.
Administration:
Taking a U87 cell strain with good growth condition in logarithmic growth phase, re-suspending the cell strain by using a DMEM culture medium, uniformly inoculating the cell strain on a 96-well plate at the density of 5 multiplied by 10 3 per well, performing conventional culture for 24 hours in a cell culture box, discarding the culture medium, respectively adding a non-drug-containing culture medium, a self-assembled hydrogel culture medium containing lysyl rhein with different concentrations and a solution culture medium containing lysyl rhein, taking the inoculated cell hole as a negative control group by adding the same volume of non-drug-containing culture, taking the non-inoculated cell hole as a blank control group by adding the same volume of non-drug-containing culture, respectively culturing for 24 hours and 72 hours, adding 10 mu L of MTT solution prepared by PBS (phosphate buffer solution) into each hole, continuously culturing for 4 hours in an incubator, carefully discarding the upper culture medium, adding 150 mu L of dimethyl sulfoxide into each hole, completely dissolving purple crystals at the bottom of the plate by shaking for 10 minutes in a dark place, detecting the absorbance value of each hole at 570nm on an enzyme marker, and calculating the survival rate of the cell according to formula (1), thereby checking the inhibition of the self-assembled hydrogel of the cell rhein U87.
As shown in fig. 7A, after 24h of administration, the activity of U87 cells is significantly reduced by both 20-160 μm of the lysyl rhein solution and the lysyl rhein self-assembled hydrogel (**** P <0.0001, compared with the negative control group), and the inhibition effect of lysyl rhein solution and hydrogel on the proliferation of U87 cells is equivalent. As shown in fig. 7B, the viability of U87 cells continued to decrease as the administration time was prolonged to 72 hours, further demonstrating the proliferation inhibitory effect of lysyl rhein on U87 cells. As can be seen from fig. 7, the lysyl rhein solution exhibited a stronger proliferation inhibition effect on U87 cells compared to hydrogels after 72 hours of administration (**** P <0.0001 compared to negative control; NS: no statistical significance of differences, ###P<0.001,#### P <0.0001 compared between groups), due to the prolonged drug onset time of the sustained release of hydrogels.
2. Inhibition of LPS-activated BV2 cells secreting tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) by lysyl rhein self-assembled hydrogels
Preparing a liquid medicine:
The preparation of the self-assembled hydrogel medium containing the lysyl rhein comprises the steps of taking the lysyl rhein self-assembled hydrogel (prepared in reference example 2) as a mother solution, adding the DMEM medium to dilute the drug-containing medium 1 with the concentration of Cheng Laian rhein self-assembled hydrogel (calculated by lysyl rhein) of 20 mu M (8.6 mu g/mL).
Preparation of a Rheum acid solution containing solution, wherein a Rheum acid solution (prepared in reference to example 2) with a concentration of 6mg/mL is taken as a mother solution, and DMEM medium is added to dilute Cheng Laian Rheum acid solution with a concentration of 20 μm (8.6. Mu.g/mL) of medicated medium 2.
BV2 cell lines in logarithmic growth phase and good growth condition are taken, uniformly inoculated on a 24-well plate at the density of 8X 10 4 cells/well after cell counting, the culture medium is discarded after conventional culture for 24 hours in a cell culture box, the non-drug-containing culture medium is respectively added, BV2 cells are pretreated by drug-containing culture medium 1 and 2 for 1 hour, LPS solution is added to the final concentration of 100ng/mL for incubation for 24 hours, BV2 cells which are pretreated by the same volume of non-drug-containing culture medium and are not stimulated by LPS are taken as a control group, BV2 cells which are pretreated by the same volume of non-drug-containing culture medium and stimulated by LPS are taken as a vehicle group, IL-6 and TNF-alpha content in the BV2 cell culture medium are respectively detected according to ELISA kit detection instructions of IL-6 and TNF-alpha, cells on the well plate are lysed by using 0.1% of triton, after centrifugation for 20 minutes at 3000 revolutions, supernatant is collected, the protein content of each well is measured according to BCA kit detection instructions, and the ratio of IL-6 content detected and IL-alpha content of IL-6 and the IL-alpha content of the protein content in the well is measured.
As shown in fig. 8 and 9, the self-assembled hydrogel of lysyl rhein exhibited a superior ability to inhibit TNF- α and IL-6 secretion by BV2 cells compared to lysyl rhein (NS: no statistical significance of differences, ***P<0.001,**** P <0.0001 compared to control group; #P<0.05,##P<0.01,###P<0.001,#### P <0.0001 compared between groups). This result verifies the anti-neuroinflammatory activity of lysyl rhein, and the formation of hydrogel can exert better efficacy.
Example 7
Preparation of self-assembled hydrogel of lysyl rhein loaded with 5-fluorouracil, temozolomide and edaravone
Weighing 2mg of 5-fluorouracil in an EP tube, adding 1mL of phosphate buffer solution with pH of 7.4, vortexing for 30s, and fully dissolving the medicine by ultrasound to obtain a medicine-containing phosphate buffer solution, weighing 8mg of lysine rhein in the EP tube, adding the medicine-containing phosphate buffer solution, vortexing to fully dissolve the lysine rhein, and performing ultrasonic dispersion for 20 minutes under the conditions of ultrasonic power of 125W and ice bath to obtain the self-assembled lysine rhein hydrogel loaded with 5-fluorouracil.
The method comprises the steps of weighing 2mg of temozolomide in an EP tube, adding 1mL of phosphate buffer with pH of 7.4, swirling for 30s, fully dissolving the drug by ultrasound to obtain a drug-containing phosphate buffer solution, weighing 8mg of lysine rhein in the EP tube, adding the drug-containing phosphate buffer solution, swirling to fully dissolve the lysine rhein, and performing ultrasonic dispersion for 20 minutes under the conditions of ultrasonic power of 125W and ice bath to obtain the self-assembled lysine rhein hydrogel loaded with temozolomide.
And (3) weighing 1.33mg of edaravone in an EP tube, adding 1mL of phosphate buffer with pH of 7.4, vortexing for 30s, and sufficiently dissolving the medicine by ultrasound to obtain a medicine-containing phosphate buffer solution, weighing 8mg of lysine rhein in the EP tube, adding the medicine-containing phosphate buffer solution, vortexing to sufficiently dissolve the lysine rhein, and performing ultrasonic dispersion for 30 minutes under the conditions of ultrasonic power of 125W and ice bath to obtain the edaravone-loaded self-assembled hydrogel of the lysine rhein.
The appearance of drug-loaded rhein self-assembled hydrogel was recorded with a digital camera immediately after 30 days of drug loading with room temperature. As shown in figure 10, the self-assembled hydrogel of the lysyl rhein has drug carrying capacity and can carry small molecular drugs such as 5-fluorouracil, temozolomide, edaravone and the like. And the drug-loaded gel is still in a gel state after being placed for 30 days at room temperature and can be stably inverted, which proves that the drug-loaded gel has better stability.
Example 8
Transmission electron microscope image of drug-loaded lysyl rhein self-assembled hydrogel
Weighing 0.2mg of 5-fluorouracil in an EP tube, adding 1mL of phosphate buffer solution with pH of 7.4, vortexing for 30s, and fully dissolving the medicine by ultrasound to obtain a medicine-containing phosphate buffer solution, weighing 4mg of lysine rhein in the EP tube, adding the medicine-containing phosphate buffer solution, vortexing to fully dissolve the lysine rhein, and performing ultrasonic dispersion for 20 minutes under the conditions of ultrasonic power of 125W and ice bath to obtain the self-assembled lysine rhein hydrogel loaded with 5-fluorouracil.
The method comprises the steps of weighing 0.2mg of temozolomide in an EP tube, adding 1mL of phosphate buffer solution with pH of 7.4, vortexing for 30s, and fully dissolving the drug by ultrasound to obtain a drug-containing phosphate buffer solution, weighing 4mg of lysine rhein in the EP tube, adding the drug-containing phosphate buffer solution, vortexing to fully dissolve the lysine rhein, and carrying out ultrasonic dispersion for 20 minutes under the condition of ultrasonic power of 125W/ice bath to obtain the self-assembled hydrogel of the lysine rhein loaded with temozolomide.
And (3) weighing 0.1mg of edaravone in an EP tube, adding 1mL of phosphate buffer solution with pH of 7.4, vortexing for 30s, and fully dissolving the medicine by ultrasound to obtain a medicine-containing phosphate buffer solution, weighing 4mg of lysine rhein in the EP tube, adding the medicine-containing phosphate buffer solution, vortexing to fully dissolve the lysine rhein, and performing ultrasonic dispersion for 30 minutes under the conditions of ultrasonic power of 125W and ice bath to obtain the edaravone-loaded self-assembled hydrogel of the lysine rhein.
And respectively sucking a proper amount of self-assembled lysyl rhein hydrogel loaded with different medicines on a glass slide, clamping a copper mesh by forceps, slightly fishing back and forth in a sample, placing the copper mesh on filter paper with the right side upwards, naturally air-drying in a shade, and observing the microstructure of the hydrogel by using a transmission electron microscope. As shown in fig. 11, temozolomide-loaded self-assembled lysyl rhein hydrogel, and edaravone-loaded self-assembled lysyl rhein hydrogel all exhibited a coherent nanofiber network structure similar to that of non-drug-loaded lysyl rhein self-assembled hydrogel (example 4). The method shows that the microstructure of the self-assembled hydrogel of the lysyl rhein is not affected by adding a proper amount of small molecular drugs with aromatic rings such as 5-fluorouracil, temozolomide, edaravone and the like.
Example 9
According to the method for preparing temozolomide-loaded self-assembled lysyl rhein hydrogel in reference to example 7, the amount of lysyl rhein is adjusted from 8mg to 6mg, so that temozolomide-loaded self-assembled lysyl rhein hydrogel is prepared.
The preparation of the liquid medicine to be tested, namely taking the self-assembled hydrogel of the lysyl rhein prepared in the example 2, the temozolomide, the self-assembled hydrogel of the lysyl rhein loaded with the temozolomide prepared in the example, and adopting a DMEM culture medium to prepare a diluted solution of the lysyl rhein with the concentration of 40 mu M (calculated by lysyl rhein) and a diluted solution of the temozolomide with the concentration of 30 mu M, wherein the concentration of the lysyl rhein is 40 mu M and the concentration of the temozolomide is 30 mu M.
Proliferation inhibition effect of temozolomide-loaded lysyl rhein self-assembled hydrogel on U87 cells
Taking a U87 cell strain with good growth condition in logarithmic growth phase, re-suspending the cell strain by using a DMEM culture medium, uniformly inoculating the cell strain on a 96-well plate with the density of 5 multiplied by 10 3 cells/well, after conventional culture for 24 hours in a cell culture box, discarding the culture medium, respectively adding a self-assembled hydrogel dilution of lysyl rhein with the concentration of 40 mu M and a self-assembled hydrogel dilution of temozolomide with the concentration of 30 mu M, adding 150 mu L of dimethyl sulfoxide to each well, shaking the self-assembled hydrogel dilution of temozolomide loaded with temozolomide with the concentration of 40 mu M, taking the inoculated cell hole as a negative control group by adding the same-volume culture and taking the non-inoculated cell hole as a blank control group by adding the same-volume culture, respectively culturing for 24, 48, 72 and 120 hours, adding 10 mu L of MTT solution prepared by PBS (5 mg/mL) into each well, after continuous culture for 4 hours in the culture box, carefully adding 150 mu L of dimethyl sulfoxide into each well, completely shaking the crystal marker with the bottom of the plate for 10 minutes under dark shaking, completely detecting the crystal marker with the concentration of temozolomide, and detecting the absorbance value at each well at the position of 1nm, and detecting the enzyme, and checking the survival rate of the cell strain, and detecting the enzyme strain, and the enzyme strain.
As shown in fig. 12, with the extension of the administration time, the synergy between temozolomide and lysyl rhein in the aqueous carrier gel (TMZ-loaded hydregel) is better than that of the non-drug-loaded lysyl rhein self-assembled hydrogel, and the proliferation inhibition effect of the aqueous carrier gel on the proliferation of U87 cells is obviously better than that of the non-drug-loaded lysyl rhein self-assembled hydrogel (* P <0.05 compared with that of the drug-loaded lysyl rhein self-assembled hydrogel corresponding to the administration time) when the administration time is prolonged to 120 hours.
Claims (7)
1. The drug-loaded rhein self-assembled hydrogel is characterized in that the drug-loaded rhein self-assembled hydrogel takes rhein and drugs with aromatic rings as raw materials, phosphate buffer solution is taken as a solvent, the drugs with the aromatic rings are prepared into drug-containing solution, the drug-containing solution and the rhein are mixed, vortex is carried out to enable the rhein to be fully dissolved, so as to obtain a solution containing the drugs and the rhein, and then ultrasonic dispersion is carried out to obtain the drug-loaded rhein self-assembled hydrogel;
the lys rhein is a salt compound formed by combining the carboxyl of rhein and the amino of lysine, and the feeding mole ratio of rhein to lysine is 1:2-1:3;
the medicine with the aromatic ring is 5-fluorouracil, temozolomide or edaravone;
the pH value of the phosphate buffer solution is 6-10;
In the solution containing the drug and the lysyl rhein, the concentration of the lysyl rhein is 4-14 mg/mL, and the concentration of the drug with an aromatic ring is 0.1-2.5 mg/mL.
2. The drug-loaded rhein self-assembled hydrogel of claim 1, wherein the power of ultrasonic dispersion is 100-200W and the time of ultrasonic dispersion is 10-40 min.
3. The use of the drug-loaded rhein self-assembled hydrogel of claim 1 for preparing a drug for treating brain glioma or protecting nerve injury.
4. A drug-loaded self-assembled lysyl rhein hydrogel is characterized in that the drug-loaded self-assembled lysyl rhein hydrogel takes lysyl rhein as a raw material, and is obtained by dissolving lysyl rhein a phosphate buffer solution and performing ultrasonic dispersion;
the lys rhein is a salt compound formed by combining the carboxyl of rhein and the amino of lysine, and the feeding mole ratio of rhein to lysine is 1:2-1:3;
The concentration of the lysine and the rhein is 4-14 mg/mL, and the pH value of the phosphate buffer solution is 6-7.4.
5. The drug-loaded self-assembled hydrogel of lysine and rhein according to claim 4, wherein the concentration of the lysine and rhein is 4-12 mg/mL.
6. The drug-loaded self-assembled lysyl rhein hydrogel of claim 4, wherein the power of ultrasonic dispersion is 75-150W and the time of ultrasonic dispersion is 5-30 min.
7. The use of the drug-loadable lysyl rhein self-assembled hydrogel of claim 4 for preparing a drug for treating brain glioma or microglial cell-mediated neurodegenerative diseases and for preparing a drug for treating nerve injury caused by brain trauma.
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