Baicalin-sophocarpidine self-assembled injectable hydrogel and preparation method thereof

Abstract

The invention discloses baicalin-sophocarpidine self-assembled injectable hydrogel and a preparation method thereof, and belongs to the field of medicinal chemistry. The self-assembled injectable hydrogel consists of baicalin and matrine in a molar ratio of (2.5-6): 1, and is prepared by the following steps: (1) dissolving the baicalin and the matrine in a solvent to obtain a reaction solution, then adding alkali liquor until the baicalin is completely dissolved, finally heating and stirring, and cooling after the reaction is finished; and (2) adjusting the pH value of the product obtained in the step (1) to 3.5-5.5 by using a strong acid solution, and gelatinizing to obtain the product. The injectable hydrogel is formed by combining natural small molecule compounds in a self-assembly mode, the advantages of specificity, low toxicity and good biocompatibility of the injectable hydrogel can be exerted, the slow release effect of bioactive components is achieved, the injectable hydrogel is self-assembled into nano-particles in the release process, the drug solubility can be improved, and the bioavailability of the injectable hydrogel is improved. The treatment effect of the medicine is also enhanced.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and particularly relates to a baicalin - matrine self - assembled injectable hydrogel and a preparation method thereof. Background Art

[0002] Scutellaria baicalensis and Sophora flavescens are two traditional Chinese medicines with important medicinal values. In traditional Chinese medicine theory, Scutellaria baicalensis has the effects of clearing away heat and dampness, purging fire and detoxifying, and is commonly used to treat febrile diseases, damp - heat jaundice and other diseases. Sophora flavescens has the functions of clearing away heat and dampness, killing insects and promoting diuresis.

[0003] Baicalin is a flavonoid compound extracted from the dried roots of the Labiatae plant Scutellaria baicalensis, and has strong antibacterial, antiviral, antitumor, antioxidant and anti - inflammatory effects. For example: The research of Lai Zhen'an found that baicalin inhibits the proliferation, migration and invasion of uterine leiomyoma cells by inhibiting the PI3K / Akt signal. And the research of Li Lei found that baicalin alleviates chronic pulmonary infection of multidrug - resistant Pseudomonas aeruginosa by inhibiting the TLR4 / NF - κB pathway and significantly reduces lung tissue inflammatory damage.

[0004] Matrine is the main alkaloid component extracted from the traditional Chinese medicine Sophora flavescens, and has significant inhibitory effects on a variety of cancers, including lung cancer, breast cancer, liver cancer, gastric cancer, pancreatic cancer, prostate cancer, etc. It can also play an anti - inflammatory role by inhibiting the production of inflammatory factors (such as TNF - α) and regulating the oxidation - antioxidant balance. It also plays an anti - hepatic fibrosis role by reducing the expression of MCP - 1 in the liver and inhibiting the migration of monocytes to the liver during inflammation. In terms of pulmonary fibrosis, matrine plays an anti - pulmonary fibrosis role by regulating the oxidation - antioxidant balance, inhibiting the production of TNF - α, and reducing the content of MDA and HYP in lung tissue.

[0005] At present, there is no report on the self - assembled hydrogel of baicalin and matrine in the existing technology. Summary of the Invention

[0006] Object of the Invention: To solve the problems existing in the prior art, the first object of the present invention is to provide a baicalin - matrine self - assembled injectable hydrogel with excellent biocompatibility, low toxicity, good slow - release performance and capable of improving the solubility and therapeutic effect of drugs. The second object of the present invention is to provide a preparation method of the above - mentioned baicalin - matrine self - assembled injectable hydrogel.

[0007] Technical Solution: The baicalin - matrine self - assembled injectable hydrogel described in the present invention is composed of baicalin and matrine with a molar ratio of 2.5 - 6:1, and its microstructure is: a highly porous gel structure with obvious hydrogel characteristics.

[0008] Preferably, the molar ratio of baicalin to matrine is 3:1 - 6:1, more preferably 3:1.

[0009] Furthermore, the solid content of the baicalin - matrine self - assembled injectable hydrogel is > 18%.

[0010] The present invention provides a preparation method of the above - mentioned baicalin - matrine self - assembled injectable hydrogel, and the steps are as follows:

[0011] (1) Dissolve baicalin and matrine in a solvent to obtain a reaction solution, then add an alkali solution until baicalin is completely dissolved, and finally carry out heating and stirring, and cool after completion.

[0012] (2) Adjust the pH of the product obtained in step (1) to 3.5 - 5.5 with a strong acid solution to form a gel, and obtain the baicalin - matrine self - assembled injectable hydrogel.

[0013] Furthermore, in step (1), the solvent is water, the molar concentration of baicalin in the reaction solution is 0.4 - 0.6 mmol / ml; the conditions for heating and stirring are: stirring at 25 - 90 °C for more than 15 min, preferably 60 - 80 °C; the alkali solution is a sodium hydroxide solution with a concentration of 5 - 20%.

[0014] Furthermore, in step (2), the strong acid solution is a hydrochloric acid solution; the molar concentration is 0.1 - 6 mM.

[0015] Principle of the invention: The core of the present invention is to utilize the molecular characteristics of two natural drugs, baicalin and matrine, to form an intelligent hydrogel with a synergistic therapeutic function through pH - responsive self - assembly. The present invention utilizes electrostatic interaction, and uses the negative charge (-O - ) of baicalin and the positive charge (-NH3 + ) of matrine to form ionic bonds at a specific pH as cross - linking points to construct a three - dimensional network. The present invention adjusts the hydrophobic interaction of baicalin through pH, forms a stable gel by hydrogen bonds between the hydrophobic aromatic ring of baicalin and the hydrophobic structure of matrine, and synergistically achieves the effects of anti - inflammation and reversing tumor drug resistance.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following remarkable effects: Based on the characteristics of traditional Chinese medicine active ingredients having low toxicity and multiple targets, the baicalin - matrine self - assembled injectable hydrogel prepared by the combined use of natural small - molecule compounds in the present invention has good sustained - release ability, can self - assemble into nanoparticles during release, and has good injectability and the effect of reversing tumor drug resistance. Description of the drawings

[0017] Figure 1 It is a physical picture of the baicalin - matrine self - assembled injectable hydrogel;

[0018] Figure 2 SEM image of baicalin - matrine self - assembled injectable hydrogel;

[0019] Figure 3 Infrared spectra of baicalin, matrine, baicalin - matrine self - assembled injectable hydrogel, and baicalin - matrine mixture (physical and mechanical mixture);

[0020] Figure 4 ITC spectra of baicalin and matrine;

[0021] Figure 5 Rheological property graph of baicalin - matrine self - assembled injectable hydrogel;

[0022] Figure 6 Injection force graph of baicalin - matrine self - assembled injectable hydrogel under different needle models;

[0023] Figure 7 Particle size graph during the release of baicalin and matrine. Specific implementation mode

[0024] The present invention will be further described in detail below in conjunction with examples and drawings.

[0025] Example 1: The detailed preparation process of the baicalin - matrine self - assembled injectable hydrogel provided in this example is as follows:

[0026] 1.1 Experimental materials and instruments

[0027] 1.1.1 Experimental materials

[0028] Baicalin and matrine, with a purity of ≥98%, were purchased from Bide Pharmatech. Beeswax, with a purity of ≥98%, was purchased from Bide Pharmatech. Squalane, with a purity of ≥95%, was purchased from Bide Pharmatech. Hydrochloric acid and sodium hydroxide, with a purity of ≥98%, were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0029] (2) Cell line

[0030] Mouse breast cancer cell doxorubicin - resistant strain (4T1 / DOX) was selected and donated by the research group of Professor Liu Runhui at East China University of Science and Technology.

[0031] (3) Cell culture reagents

[0032] 1640 medium, high - glucose DMEM medium, fetal bovine serum (FBS), and penicillin - streptomycin mixture (100×) were all purchased from Servicebio.

[0033] (4) Related index detection kits

[0034] Cell Counting Kit-8 (CCK8), purchased from APExBIO, was stored in a -20°C refrigerator and dissolved at room temperature in advance before use.

[0035] 1.1.2 Experimental Instruments

[0036] VS-1300L-U Clean Bench (Suzhou Antai Air Technology Co., Ltd.), carbon dioxide incubator (Thermo Scientific), Invitrogen hemocytometer, inverted fluorescence microscope (Leica, Germany), BioTek microplate reader, Thermo Nicolet iS5 Fourier transform infrared spectrometer, Nano ITC (TA Instruments, USA)

[0037] 1.2 Experimental Methods

[0038] 1.2.1 Preparation method of baicalin-matrine self-assembled injectable hydrogel: Weigh 506.3 mg of baicalin and 93.7 mg of matrine (the molar ratio of baicalin to matrine is 3:1), place them in a beaker, add 2.4 ml of purified water and stir. Then add 10% NaOH solution until baicalin is completely dissolved. Heat and stir at 60°C for 15 min. After cooling, adjust the pH to 4.5 with 0.5 mM hydrochloric acid to form a gel, and obtain the baicalin-matrine self-assembled injectable hydrogel.

[0039] 1.2.2 SEM:

[0040] After the hydrogel was prepared, it was pre-frozen at -80°C overnight, taken out and freeze-dried for 72 h. The freeze-dried gel was taken out, and the cross-section was fixed on a copper mesh with conductive glue and observed under a scanning electron microscope.

[0041] 1.2.3 Infrared test:

[0042] Baicalin powder, matrine powder, freeze-dried powder of hydrogel and mixed powder of baicalin and matrine were taken for infrared test respectively.

[0043] 1.3.4 ITC

[0044] The experimental group was: matrine titrating baicalin; the control group was: matrine titrating blank solvent (pure water).

[0045] The test concentration of matrine was 2 mM, and the test concentration of baicalin was 0.2 mM; the initial volume of matrine solution for titration was 50 μl (inhaled into the syringe), and the initial volume of baicalin solution for titration was 300 μl (added to the sample cell); 25 drops, 2 μl per drop, titration interval 120 s, temperature 25°C, stirring speed 350 r / min.

[0046] 1.3.5 Rheological test

[0047] Hydrogels, oleogels, and hybrid gels were used. Multiple experiments were conducted using a DHR-1 in oscillatory mode with 20 mm stainless-steel plates, 25 mm aluminum parallel plates, and 40 mm aluminum parallel plates. Finally, a 40 mm aluminum parallel plate with a 1000 μm gap was selected for rheological testing at room temperature of 25 °C.

[0048] 1.3.6. Injection Force Measurement

[0049] Measurement was carried out using a 1 ml syringe with a 0.45 μm needle. Hydrogels, oleogels, and hybrid gels were loaded into the syringe, and the maximum force required for injection was measured using the compression mode.

[0050] 1.3.7. Cytotoxicity Assay

[0051] Cell viability assay: The CCK8 kit was used to detect cell viability, and the OD value at 450 nM was measured using a microplate reader. Cell viability (%) = [(experimental well - blank well) / (control well - blank well)] × 100%; Graphpad Prism 8.0 software was used for plotting.

[0052] Low concentration group: By CCK8 method. Specifically, 4T1 / DOX cells (8×10 3 -1.2×10 4 cells / well) were seeded into 96-well plates and incubated overnight for 12 h. The supernatant was discarded, and then cell cultures were treated with final concentrations of 100 nM DOX + 200 μM baicalin, 100 nM DOX + 66.7 μM matrine, 100 nM DOX + 200 μM baicalin and 66.7 μM matrine for 72 h. The supernatant was discarded, CCK8 reagent was added, and incubation was continued for 0.5 - 1.5 h. The absorbance at 450 nm was measured using a microplate reader. Cell viability was expressed as a percentage of the control (untreated cells).

[0053] Medium concentration group: By CCK8 method. Specifically, 4T1 / DOX cells (8×10 3 -1.2×10 4 cells / well) were seeded into 96-well plates and incubated overnight for 12 h. The supernatant was discarded, and then cell cultures were treated with final concentrations of 1 μM DOX + 200 μM baicalin, 1 μM DOX + 66.7 μM matrine, 1 μM DOX + 200 μM baicalin and 66.7 μM matrine for 72 h. The supernatant was discarded, CCK8 reagent was added, and incubation was continued for 0.5 - 1.5 h. The absorbance at 450 nm was measured using a microplate reader. Cell viability was expressed as a percentage of the control (untreated cells).

[0054] High concentration group: By CCK8 method. Specifically, 4T1 / DOX cells (8×10 3 -1.2×104 Cells (at a density of -1 cells / well) were seeded into 96-well plates and incubated overnight for 12 h. The supernatant was discarded, and then cells were treated with a final concentration of 10 μM DOX + 200 μM baicalin, 10 μM DOX + 66.7 μM matrine, 10 μM DOX + 200 μM baicalin and 66.7 μM matrine for 72 h. The supernatant was discarded, CCK8 reagent was added, and the mixture was incubated for another 0.5 - 1.5 h. The absorbance at 450 nm was measured using a microplate reader. Cell viability was expressed as a percentage of the control (untreated cells).

[0055] 1.3.8. Release test

[0056] Twelve 1-ml hydrogels were prepared in 50-ml centrifuge tubes and divided into two groups, with 6 samples in each group. For the first group, 49.0 ml of 0.01 M PBS buffer solution with a pH of 7.2 - 7.4 was added, and for the second group, 49.0 ml of 0.01 M PBS buffer solution with a pH of 5.1 - 5.3 was added. The tubes were placed on a shaker at 37 °C with a shaking speed of 50 rpm. Sampling was carried out at 1, 3, 5, 7, 9, 11, 24, 36, 48, 60, and 72 hours, with 10 ml of sample taken each time and 10 ml of the corresponding buffer solution added to replenish. The absorbance of the samples at 380 nm was measured using a microplate reader, and the concentration and release rate were calculated using the standard linear curve of baicalin.

[0057] 1.3.9. Particle size test

[0058] The samples from 1.3.8 were taken for particle size testing.

[0059] The above test results are shown in Figures 1 - 7 .

[0060] As can be seen from Figure 1 , the self-assembled injectable hydrogel of baicalin-matrine presents a gel state at room temperature and does not flow when inverted. As can be seen from Figure 2 , the self-assembled injectable hydrogel of baicalin-matrine has a highly porous structure, which is a typical state of the gel.

[0061] As can be seen from Figure 3 , the infrared spectrum of the self-assembled injectable hydrogel of baicalin-matrine shows that, compared with the simple mixture, the baicalin / matrine gel has the same characteristic of γ-C-O-C- (1065 cm -1 ) as the baicalin monomer. Compared with the monomer, the peak of the baicalin-matrine gel shifts to the low wavenumber region, which is due to the electrostatic interaction between the carboxyl group and the quaternary ammonium nitrogen atom, resulting in the delocalization of C=O and the weakening of the chemical bond force constant. Compared with the simple mixture, the peak at 1727 cm -1 of the baicalin-matrine gel shifts to the low wavenumber region. This proves the successful self-assembly of baicalin and matrine.

[0062] The binding constant of baicalin and matrine was measured by ITC. It can be seen from Figure 4 that there is a heat change (exothermic) during the titration of matrine with baicalin, indicating the existence of binding. The Kd is less than 1, and the reaction is reversible. ΔH and ΔS are less than 0. Through calculation, the reaction is spontaneous at 95 °C. The binding of baicalin and matrine is a spontaneous exothermic reaction, and the chemical binding constant is 4.31×10 -4 M.

[0063] Figure 5 shows the rheological properties of the baicalin - matrine self - assembled injectable hydrogel. According to the change of oscillatory strain, the linear viscoelastic region of the baicalin / matrine self - assembled hydrogel is determined to be 0.001% - 0.1%. Frequency scanning at different temperatures shows that both at 25 °C and 37 °C, it exhibits the properties of an elastic colloid.

[0064] Figure 6 shows the injection force required to inject the gel with different needles. The injection force of the baicalin - matrine self - assembled injectable hydrogel with different needle models is much lower than the clinically injectable 20 N, indicating good injectability.

[0065] During the gel release process, the baicalin - matrine self - assembled injectable hydrogel can self - assemble into nanoparticles again, and the trend of particle size change is as follows Figure 7 shown.

Claims

1. A baicalin - matrine self - assembled injectable hydrogel, characterized in that, The self-assembled injectable hydrogel is composed of baicalin and matrine with a molar ratio of 2.5-6:1, has a highly porous microstructure, and has obvious hydrogel characteristics.

2. The baicalin - matrine self - assembled injectable hydrogel according to claim 1, wherein The molar ratio of the baicalin to the matrine is 3:1-6:

1.

3. The baicalin - matrine self - assembled injectable hydrogel according to claim 1, wherein The solid content of the baicalin-matrine self-assembled injectable hydrogel is >18%.

4. A method for preparing the baicalin - matrine self - assembled injectable hydrogel according to claim 1, characterized in that, The steps are as follows: (1) Dissolve baicalin and matrine in a solvent to obtain a reaction solution, then add an alkali solution until the baicalin is completely dissolved, and finally carry out heating and stirring, and cool after completion. (2) Adjust the pH of the product obtained in step (1) to 3.5-5.5 with a strong acid solution to form a gel, and prepare the baicalin-matrine self-assembled injectable hydrogel.

5. The preparation method according to claim 4, characterized in that, In step (1), the molar concentration of the baicalin in the reaction solution is 0.4-0.6 mmol / ml.

6. The preparation method according to claim 4, characterized in that, In step (1), the solvent is water.

7. The preparation method according to claim 4, characterized in that, In step (1), the conditions for the heating and stirring are: stirring at 25-90 °C for more than 15 min.

8. The preparation method according to claim 4, characterized in that, In step (1), the alkali solution is a sodium hydroxide solution with a concentration of 5-20%.

9. The preparation method according to claim 4, characterized in that, In step (2), the strong acid solution is a hydrochloric acid solution.

10. The preparation method according to claim 9, wherein, The molar concentration of the hydrochloric acid solution is 0.1-6 mM.

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