Preparation and application of methotrexate-loaded silk fibroin gel drug release system

Abstract

The invention relates to preparation and application of a methotrexate-loaded silk fibroin gel drug release system, in particular to a methotrexate-containing gel preparation which contains methotrexate, silk fibroin and BDDE in a weight ratio of (1-2): (30-50): (6-10), the invention further provides a preparation method of the gel preparation, and the preparation method comprises the following steps: preparing the methotrexate-loaded silk fibroin gel drug release system by using the methotrexate-loaded silk fibroin gel drug release system, and preparing the methotrexate-loaded silk fibroin gel drug release system by using the methotrexate-loaded silk fibroin gel drug release system. The preparation method comprises the following steps: dissolving lithium bromide in water, adding silk fibroin and methotrexate, adding BDDE, and standing to form gel; the method specifically comprises the following steps: dissolving lithium bromide in water, adding silk fibroin and methotrexate, stirring at 50-70 DEG C for 0.5-2 hours, adding BDDE, and standing at 50-70 DEG C for 2-4 hours to form gel.

Description

Technical Field

[0001] The invention belongs to the field of medicine and biology, and relates to the preparation and application of a silk fibroin gel drug release system loaded with methotrexate. Background Art

[0002] Methotrexate is an immunosuppressant that mainly inhibits dihydrofolate reductase to prevent the reduction of dihydrofolate into physiologically active tetrahydrofolate, thereby blocking the biosynthesis of purine nucleotides and pyrimidine nucleotides and inhibiting the production of DNA, thereby exerting immune regulation and anti-inflammatory effects.

[0003] Methotrexate is the first drug used successfully to treat juvenile idiopathic arthritis. However, its application is limited by its gastrointestinal reactions, bone marrow suppression, liver damage and other adverse reactions. In addition, its short duration of action is also an obvious drawback of methotrexate.

[0004] Methotrexate currently sold on the market is available in injection, tablet and other dosage forms. It is administered systemically. Long-term treatment of the disease will cause varying degrees of damage to organs throughout the body and requires weekly administration.

[0005] As a natural protein, silk fibroin has good biocompatibility, biodegradability and a variety of crystallization and solidification methods. It is easy to maintain a high degree of bioaffinity and a special porous network structure, and can be used as a drug sustained-release carrier. The Chinese patent application number: CN201911300215.4 discloses a method for preparing a doxorubicin-loaded methylcellulose / silk fibroin composite hydrogel, which controls the sol-gel transition and drug release of the silk fibroin hydrogel by adding methylcellulose. The obtained DOX-loaded MC / SF composite hydrogel has good sustained-release and controlled-release properties and has good drug release performance. The structure of doxorubicin is as follows:

[0006]

[0007] Since methotrexate is different from doxorubicin in physical and chemical properties, satisfactory technical results cannot be achieved using the same method.

[0008] The structure of methotrexate is as follows:

[0009]

[0010] Its specific physical and chemical properties, such as its solubility is easily affected by pH value, and the hydrogel structure prepared by physical methods is relatively loose, making it difficult to achieve a good sustained-release effect.

[0011] Therefore, the present invention aims at the characteristics of methotrexate and also uses silk fibroin to prepare a gel preparation containing methotrexate, achieving unexpected technical effects. Summary of the invention

[0012] In clinical practice, methotrexate tablets have problems such as large individual differences, systemic adverse reactions and short duration of action. In order to solve the above problems, the present invention uses silk fibroin gel to encapsulate methotrexate, which can improve bioavailability while reducing the frequency of administration and continuously exerting drug efficacy. Local administration can reduce systemic adverse reactions.

[0013] The object of the present invention is to provide a silk fibroin gel preparation containing methotrexate, wherein BDDE (butylene glycol glycidyl ether) is used as a fusing agent.

[0014] To this end, an object of the present invention is to provide a gel preparation, which contains methotrexate, silk fibroin and BDDE, the weight ratio of the three is 1-2:30-50:6-10, preferably the weight ratio of the three is 1-2:30:6. The most preferred weight ratio of the three is 1:30:6.

[0015] Another object of the present invention is to provide a method for preparing a gel formulation of methotrexate, wherein the method comprises the following steps:

[0016] Take lithium bromide, dissolve it in water, add silk fibroin and methotrexate, add BDDE, and let it stand to form a gel.

[0017] Preferably, the preparation method of the present invention comprises the following steps:

[0018] Take lithium bromide, dissolve it in water, add silk fibroin and methotrexate, stir at 50-70°C for 0.5-2h, add BDDE, and let stand at 50-70°C for 2-4h to form a gel.

[0019] Most preferably, the preparation method of the present invention comprises the following steps:

[0020] Take lithium bromide, dissolve it in water, add silk fibroin and methotrexate, stir at 60°C for 1 hour, add BDDE, and let stand at 60°C for 3 hours to form a gel.

[0021] The most preferred preparation method of the present invention is, wherein the ratio of silk fibroin to BDDE is 5:1, the concentration of lithium bromide is 9.3M, and 5 mg of methotrexate is added to every 1 ml of lithium bromide solution.

[0022] In the gel preparation of the present invention, the silk fibroin can be prepared according to the prior art or purchased on the market. The present invention uses the soluble freeze-dried silk fibroin of Suzhou Silkmate Company, with a molecular weight of 50,000-100,000 Daltons.

[0023] Among them, the role of lithium bromide is to fully stretch the silk protein molecular chain so that it can fully react with BDDE.

[0024] Among them, the role of BDDE is to induce structural changes in silk fibroin and form a gel.

[0025] The gel preparation of the present invention may be further added with necessary drug carriers as required to achieve molding, so that it can be used for injection and is convenient for packaging, storage, sales and the like.

[0026] Compared with the prior art, the gel preparation and preparation method of the present invention have the following beneficial effects:

[0027] The obtained drug-loaded gel has suitable strength, can be used for injection, has good sustained-release effect and can be regulated as needed, and can be used for local treatment of juvenile idiopathic arthritis, rheumatoid arthritis and other diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 , Degradation diagram of silk fibroin gel with different concentrations of chemical cross-linking

[0029] Figure 2 , Porosity diagram of different concentrations of chemically cross-linked silk fibroin gel

[0030] Figure 3 , Swelling curves of chemically cross-linked silk fibroin at different concentrations

[0031] Figure 4 , Viscosity curves of chemically cross-linked silk fibroin at different concentrations

[0032] Figure 5 , 15% concentration of silk fibroin drug loading and blank gel viscosity curve

[0033] Figure 6 , Modulus curve of chemically cross-linked silk fibroin at different concentrations

[0034] Figure 7 , 15% concentration of silk fibroin drug loading and blank gel modulus curve

[0035] Figure 8 , Scanning electron microscopy images of different concentrations of chemically cross-linked silk protein gel

[0036] Fig. 9 , in vitro release diagram of different concentrations of chemically cross-linked silk protein gel

[0037] Fig.10 , Release rate of chemically cross-linked silk fibroin at different concentrations

[0038] Fig.11 , Cell uptake diagram of different drug groups DETAILED DESCRIPTION

[0039] The present invention is further illustrated by the following examples.

[0040] The product and preparation method of the present invention are further described below in conjunction with specific examples, but the present invention is not limited to the following examples. The methods are conventional methods unless otherwise specified. The raw materials can be obtained from public commercial channels unless otherwise specified.

[0041] Example 1. Gel preparation containing methotrexate

[0042] (1) Preparation of gel

[0043] In 1 ml of lithium bromide solution, add 150 mg or 200 mg or 250 mg of silk fibroin and 5 mg of methotrexate, stir at 60°C for 1 hour, then add 30 μl / 40 μl / 50 μl BDDE and incubate at 60°C for 3 hours to obtain gel.

[0044] Embodiment 2,

[0045] The gel preparation of Example 1 was used as the test object and the corresponding tests were performed:

[0046] 1. In vitro protease degradation:

[0047] Take 15%, 20%, and 25% concentrations of silk fibroin drug-loaded gel and freeze-dry them. After freeze-drying, take 50 mg of each and place them in a centrifuge tube. Place the centrifuge tube in a shaker at 37°C at 60 rpm. Take out the remaining gel at different time points, rinse with PBS, absorb excess water and weigh it. The degradation rate of chemically cross-linked silk fibroin gel at different concentrations is as follows: Figure 1 shown.

[0048] There was no significant difference in the degradation rate of 15% and 20% silk fibroin gels, and the degradation rate of 25% silk fibroin gel was slightly slower, which may be due to the tighter structure of 25% silk fibroin, smaller contact area with the enzymatic solution, and slower degradation rate.

[0049] 2. Porosity and swelling of gel:

[0050] Take 15%, 20%, and 25% concentration silk fibroin drug-loaded gel and freeze-dry it. Immerse the freeze-dried drug-loaded gel in a hexane solution with a volume of V1 for 10 minutes. The volume of the hexane solution after complete immersion is V2. Take out the sample and record the remaining hexane solution volume as V3. The porosity is calculated by the following equation: (P) = (V1-V3) / (V2-V3). The porosity of chemically cross-linked silk fibroin gels with different concentrations is shown in Figure 2 shown.

[0051] With the increase of silk fibroin concentration, the porosity tends to decrease, especially the porosity of 25% silk fibroin drug-loaded gel is significantly reduced, which reflects the compactness of its structure.

[0052] Take 15%, 20%, and 25% concentrations of silk fibroin drug-loaded gel and freeze-dry it. Weigh the freeze-dried drug-loaded gel and record it as W0. Immerse the dried hydrogel in PBS solution. After immersing in PBS solution for 24 hours, take out the swollen gel after absorbing water, wipe off the excess water and weigh it, record it as Wt. The swelling rate is calculated according to the following formula: SR = (wt-w0) / w0. The swelling rate of chemically cross-linked silk fibroin gel with different concentrations is as follows: Figure 3 As shown, with the increase of silk fibroin concentration, the swelling property tends to decrease, especially the 25% silk fibroin drug-loaded gel has obviously poor swelling property, which reflects the compactness of its structure.

[0053] 3 Rheology

[0054] 3.1 Shear rate-viscosity curve

[0055] Take appropriate amount of freshly prepared gel and drug-loaded gel, use rheometer, scan at 0.1~100s-1, see Figure 4 , 5 shown.

[0056] The viscosity of silk fibroin gels of different concentrations is significantly different, and with the increase of concentration, the viscosity gradually increases, reflecting the increase of viscosity, but they all have the characteristics of shear thinning, which proves their injectability. The viscosity increases slightly after drug loading, but no significant change is observed. The drug loading concentration can be adjusted according to clinical drug needs.

[0057] The methotrexate-loaded silk fibroin gel reached a lower viscosity at a silk fibroin concentration of 15% and had better injectability, which met the requirements for injectable drug-loaded gels in this study and was the preferred gel in this experiment.

[0058] 3.2 Frequency change-modulus curve

[0059] Take appropriate amount of freshly prepared gel and drug-loaded gel, use rheometer, shear rate is 1%, observe in 1~0.01Hz, see Figure 6 , 7 shown.

[0060] The elastic modulus (G') measured under different conditions was higher than the viscous modulus (G") and did not intersect, which reflected that the prepared samples had gel properties and verified that the drug release system was a gel.

[0061] The viscous modulus of silk fibroin gels with different concentrations are somewhat different, while the elastic modulus is quite different. With the increase of concentration, the elastic modulus gradually increases, reflecting the improvement of its viscoelasticity and the correspondingly greater mechanical strength. For gels without drug loading, both the viscous modulus and elastic modulus are reduced, reflecting the effect of adding other non-gel materials on the properties of gels.

[0062] The methotrexate-loaded silk fibroin gel reached a lower viscoelasticity at a silk fibroin concentration of 3%, which met the requirements for injectable drug-loaded gels in this study and was the preferred gel in this experiment.

[0063] 4 Scanning electron microscopy

[0064] A small amount of sample was taken from freeze-dried gel and drug-loaded gel and photographed using a field emission scanning electron microscope at a voltage of 3 kV and a current of 10 μA. The gold injection current was set to 10 mA and the gold injection time was 90 s. Figure 8 shown.

[0065] Silk fibroin gel presents a honeycomb network structure without drug loading. The mesh becomes tighter with increasing concentration, and there is no obvious change in morphology after drug loading, which is consistent with the expected structure.

[0066] 5 In vitro release

[0067] The same volume of drug-loaded gel with different concentrations was taken and placed in EP tubes. The same enzyme-containing PBS was added to each EP tube as the release medium. Samples were taken at different time points and all the release medium was replaced. The drug concentration in the sample was determined by HPLC. The release rate of chemically cross-linked silk protein with different concentrations was as follows: Fig. 9 shown.

[0068] As the concentration of silk fibroin increased, the release rate showed a downward trend, especially the 25% silk fibroin gel release rate is particularly slow. On the 9th day, 15% and 20% silk fibroin gel concentrations almost all released, 25% concentration of silk fibroin gel only released about 50%. Fig.10 shown.

[0069] 6 Cellular uptake

[0070] DIR was loaded into silk fibroin gels of different concentrations. RAW264.7 cells were plated into confocal microplates and induced with lipopolysaccharide (LPS) for 24 hours after overnight attachment. The drugs were administered (PBS, blank gel, DIR, gel loaded with DIR). Fluorescence intensity was detected by flow cytometry on days 1, 3, and 7, respectively. Cell uptake in different groups was as follows: Fig.11 shown.

[0071] The cells in the PBS and blank gel groups had no fluorescence absorption on days 1, 3, and 7. The fluorescence absorbed by the cells in the DIR group showed a rapid growth trend, especially on the 7th day, with a more obvious fluorescence absorption. The DIR gel group always showed a lower fluorescence absorption, reflecting the sustained release of the gel.

[0072] The above description is only a preferred embodiment of the present invention. It should be noted that the embodiments of the present invention are not limited to the embodiments. Without departing from the principle of the present invention, several changes, modifications, substitutions, combinations, and simplifications can be made, all of which should be equivalent replacement methods, and these should also be regarded as the scope of protection of the present invention.

Claims

1. A gel preparation containing methotrexate, characterized in that: It contains methotrexate, silk fibroin and BDDE, and the weight ratio of the three is 1-2:30-50:6-10.

2. The gel preparation according to claim 1, characterized in that The weight ratio of methotrexate, silk fibroin and BDDE is 1-2:30:

6.

3. The gel preparation according to claim 1, characterized in that The weight ratio of methotrexate, silk fibroin and BDDE is 1:30:

6.

4. The method for preparing the gel preparation according to claim 1, characterized in that: The method comprises the following steps: taking lithium bromide, dissolving it in water, adding silk fibroin and methotrexate, adding BDDE, and standing to form a gel.

5. The preparation method according to claim 4, characterized in that: The method comprises the following steps: Take lithium bromide, dissolve it in water, add silk fibroin and methotrexate, stir at 50-70°C for 0.5-2h, add BDDE, and let stand at 50-70°C for 2-4h to form a gel.

6. The preparation method according to claim 4, characterized in that: The method comprises the following steps: Take lithium bromide, dissolve it in water, add silk fibroin and methotrexate, stir at 60°C for 1 hour, add BDDE, and let stand at 60°C for 3 hours to form a gel.

7. The method for preparing the gel preparation according to claim 4, characterized in that: The method comprises the following steps: the ratio of silk fibroin to BDDE is 5:1, and the concentration of lithium bromide is 9.3M.

8. The method for preparing the gel preparation according to claim 4, characterized in that: in, Add 5 mg of methotrexate to every 1 ml of lithium bromide solution.

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