A self-assembled injectable hydrogel of baicalin-matrine and its preparation method

Abstract

This invention discloses a baicalin-matrine self-assembled injectable hydrogel and its preparation method, belonging to the field of medicinal chemistry. The above-mentioned self-assembled injectable hydrogel is composed of baicalin and matrine in a molar ratio of 2.5-6:1. The preparation steps are as follows: (1) Dissolve baicalin and matrine in a solvent to obtain a reaction solution, then add alkali solution until baicalin is completely dissolved, and finally heat and stir, and then cool; (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. This invention uses natural small molecule compounds to self-assemble and form an injectable hydrogel, which can exert its specificity, has the advantages of low toxicity and good biocompatibility, not only achieving the sustained release effect of bioactive ingredients, but also self-assembling into nanoparticles during the release process, which can not only improve drug solubility, but also enhance the therapeutic effect of the drug.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, and particularly relates to a baicalin-matrine self-assembled injectable hydrogel and its preparation method. Background Technology

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

[0003] Baicalin is a flavonoid compound extracted from the dried root of Scutellaria baicalensis, a plant in the Lamiaceae family. It possesses strong antibacterial, antiviral, antitumor, antioxidant, and anti-inflammatory effects. For example, Lai Zhen'an's research found that baicalin inhibits the proliferation, migration, and invasion of uterine leiomyomas by suppressing PI3K / Akt signaling. Li Lei's research found that baicalin alleviates chronic lung infection caused by multidrug-resistant Pseudomonas aeruginosa by inhibiting the TLR4 / NF-κB pathway and significantly reduces inflammatory damage to lung tissue.

[0004] Matrine is the main alkaloid extracted from the traditional Chinese medicine Sophora flavescens. It has significant inhibitory effects on various cancers, including lung cancer, breast cancer, liver cancer, stomach cancer, pancreatic cancer, and prostate cancer. It can also exert anti-inflammatory effects by inhibiting the production of inflammatory factors (such as TNF-α) and regulating the oxidation-antioxidant balance. Furthermore, it exerts anti-hepatic fibrosis effects by reducing the expression of MCP-1 in the liver and inhibiting the migration of monocytes to the liver during inflammation. Regarding pulmonary fibrosis, matrine exerts its anti-pulmonary fibrosis effect by regulating the oxidation-antioxidant balance, inhibiting TNF-α production, and reducing the content of MDA and HYP in lung tissue.

[0005] There are currently no reports on the self-assembled hydrogels of baicalin and matrine in existing technologies. Summary of the Invention

[0006] Purpose of the invention: In order to solve the problems existing in the prior art, the first purpose of the present invention is to provide a baicalin-matrine self-assembled injectable hydrogel with excellent biocompatibility, low toxicity, good sustained-release performance and the ability to improve drug solubility and therapeutic effect. The second purpose of the present invention is to provide a method for preparing the above-mentioned baicalin-matrine self-assembled injectable hydrogel.

[0007] Technical solution: The baicalin-matrine self-assembled injectable hydrogel of the present invention is composed of baicalin and matrine in a molar ratio of 2.5-6:1, and has a microstructure of 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] This invention provides a method for preparing the above-mentioned baicalin-matrine self-assembled injectable hydrogel, the steps of which are as follows:

[0011] (1) Dissolve baicalin and matrine in a solvent to obtain a reaction solution, then add alkali solution until baicalin is completely dissolved, and finally heat and stir, and then cool after the reaction is completed.

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

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

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

[0015] Invention Principle: The core of this invention lies in utilizing the molecular properties of two natural drugs, baicalin and matrine, to form a smart hydrogel with synergistic therapeutic functions through pH-responsive self-assembly. This invention utilizes electrostatic interactions and the negative charge (-O) of baicalin. - The positive charge of matrine (-NH3) + At a specific pH, ionic bonds are formed, serving as cross-linking points to construct a three-dimensional network. This invention utilizes pH adjustment to modulate the hydrophobic interaction of baicalin, enabling the hydrophobic aromatic ring of baicalin to form a stable gel with the hydrophobic structure of matrine via hydrogen bonds, synergistically achieving anti-inflammatory and tumor drug resistance reversal effects.

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

[0017] Figure 1 This is a photograph of a self-assembled injectable hydrogel containing baicalin and matrine.

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

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

[0020] Figure 4 The ITC values ​​for baicalin and matrine are shown.

[0021] Figure 5 Rheological properties of the baicalin-matrine self-assembled injectable hydrogel;

[0022] Figure 6 Injection force diagrams of baicalin-matrine self-assembled injectable hydrogels under different needle sizes;

[0023] Figure 7 This is a particle size diagram showing the release process of baicalin and matrine. Detailed Implementation

[0024] The present invention will now be described in further detail with reference to the embodiments and accompanying 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, purity ≥98%, purchased from Bide Pharmaceutical. Beeswax, purity ≥98%, purchased from Bide Pharmaceutical. Squalane, purity ≥95%, purchased from Bide Pharmaceutical. Hydrochloric acid and sodium hydroxide, purity ≥98%, purchased from Sinopharm Group.

[0029] (2) Cell line

[0030] The doxorubicin-resistant mouse breast cancer cell line (4T1 / DOX) was selected and donated to Professor Liu Runhui's research group 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) Relevant indicator detection kit

[0034] Cell Counting Kit-8 (CCK8), purchased from APExBIO, stored at -20℃, dissolved at room temperature before use.

[0035] 1.1.2 Experimental Apparatus

[0036] VS-1300L-U clean bench (Suzhou Antai Air Technology Co., Ltd.), CO2 incubator (ThermoScientific), Invitrogen hemocytometer, inverted fluorescence microscope (Leica, Germany), BioTek microplate reader, ThermoNicolet iS5 infrared analyzer, 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 (molar ratio of baicalin to matrine is 3:1) and place them in a beaker. Add 2.4 ml of purified water and stir. Add 10% NaOH solution until baicalin is completely dissolved. Heat and stir at 60℃ for 15 min. After cooling, adjust the pH to 4.5 with 0.5 mM hydrochloric acid to form a gel and obtain baicalin-matrine self-assembled injectable hydrogel.

[0039] 1.2.2.SEM:

[0040] After the hydrogel was prepared, it was pre-frozen at -80℃ overnight, then freeze-dried for 72 hours. The freeze-dried gel was then removed, the cross-section was cut and fixed to a copper grid with conductive adhesive, and observed in a scanning electron microscope.

[0041] 1.2.3. Infrared testing:

[0042] Infrared spectroscopy was performed on baicalin powder, matrine powder, hydrogel freeze-dried powder, and a mixture of baicalin and matrine powder.

[0043] 1.3.4.ITC

[0044] The experimental group consisted of matrine titration with baicalin; the control group consisted of matrine titration with blank solvent (pure water).

[0045] The matrine was tested at a concentration of 2 mM, and the baicalin was tested at a concentration of 0.2 mM. The initial volume of the matrine solution was 50 μL (using a syringe), and the initial volume of the baicalin solution was 300 μL (added to the sample cell). 25 drops were added, each drop being 2 μL, with a titration interval of 120 s. The temperature was 25 °C, and the stirring speed was 350 r / min.

[0046] 1.3.5. Rheological Testing

[0047] Hydrogels, oleogels, and mixed gels were collected and tested using a DHR-1 in oscillation mode. Multiple experiments were conducted using 20mm stainless steel plates, 25mm aluminum parallel plates, and 40mm aluminum parallel plates. Finally, the 40mm aluminum parallel plate with a 1000µm gap was selected for rheological testing at room temperature (25℃).

[0048] 1.3.6. Injection Force Measurement

[0049] Using a 1ml syringe and a 0.45μm needle, the hydrogel, oleogel, and mixed gel were loaded into the syringe, and the maximum force required for injection was measured using compression mode.

[0050] 1.3.7. Cytotoxicity assay

[0051] Cell viability assay: Cell viability was detected using a CCK8 assay kit, and the OD value at 450 nM was measured using a microplate reader. Cell viability (%) = [(experimental wells - blank wells) / (control wells - blank wells)] × 100%; graphs were plotted using Graphpad Prism 8.0 software.

[0052] Low concentration group: determined by the CCK8 assay. Specifically, 4T1 / DOX cells (8 × 10⁻⁶) were used. 3 -1.2×10 4 Cells (100 cells / well) were seeded into 96-well plates and incubated overnight for 12 h. The supernatant was discarded. Cells were then treated with solutions of 100 nM DOX + 200 μM baicalin, 100 nM DOX + 66.7 μM matrine, and 100 nM DOX + 200 μM baicalin and 66.7 μM matrine for 72 h. The supernatant was discarded, and CCK8 reagent was added. Cells were incubated for another 0.5–1.5 h. The absorbance at 450 nm was measured using a microplate reader. Cell viability is expressed as a percentage of the control (untreated cells).

[0053] Medium concentration group: determined by the CCK8 assay. Specifically, 4T1 / DOX cells (8 × 10⁻⁶) were used. 3 -1.2×10 4 Cells (10 cells / well) were seeded into 96-well plates and incubated overnight for 12 h. The supernatant was discarded. Cells were then treated with solutions of 1 μM DOX + 200 μM baicalin, 1 μM DOX + 66.7 μM matrine, and 1 μM DOX + 200 μM baicalin and 66.7 μM matrine for 72 h. The supernatant was discarded, and CCK8 reagent was added. Cells were 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).

[0054] High concentration group: Measured using the CCK8 assay. Specifically, 4T1 / DOX cells (8 × 10⁻⁶) were used. 3 -1.2×104 Cells (10 cells / well) were seeded into 96-well plates and incubated overnight for 12 h. The supernatant was discarded. Cells were then treated with solutions of 10 μM DOX + 200 μM baicalin, 10 μM DOX + 66.7 μM matrine, and 10 μM DOX + 200 μM baicalin and 66.7 μM matrine for 72 h. The supernatant was discarded, and CCK8 reagent was added. Cells were incubated for another 0.5–1.5 h. Absorbance at 450 nm was measured using a microplate reader. Cell viability is expressed as a percentage of the control (untreated cells).

[0055] 1.3.8. Release Test

[0056] Twelve 1ml hydrogels were prepared in 50ml centrifuge tubes and divided into two groups of six. One group was treated with 49.0ml of 0.01M PBS buffer solution (pH 7.2-7.4), and the other group was treated with 49.0ml of 0.01M PBS buffer solution (pH 5.1-5.3). The tubes were placed on a shaker at 37℃ and 50rpm. Samples were taken at 1, 3, 5, 7, 9, 11, 24, 36, 48, 60, and 72 hours, with 10ml of sample taken each time and 10ml of buffer solution added accordingly. The absorbance of the samples at 380nm 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 testing

[0058] Take a sample from 1.3.8 for particle size testing.

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

[0060] Depend on Figure 1 It is known that the baicalin-matrine self-assembled injectable hydrogel exhibits a gel state at room temperature and does not flow when inverted. From Figure 2 It is known that the baicalin-matrine self-assembled injectable hydrogel has a highly porous structure, which is a typical state of gel.

[0061] Depend on Figure 3 The infrared spectrum of the baicalin-matrine self-assembled injectable hydrogel shows that, compared with the simple mixture, the baicalin / matrine gel has the same γ-COC-(1065cm⁻¹) as the baicalin monomer. -1 The characteristic of baicalin-matrine gels, compared to monomers, shows a shift towards lower wavenumber regions. This is due to the electrostatic interaction between the carboxyl group and the quaternary ammonium nitrogen atom, leading to C=O delocalization and a weakening of the chemical bond force constant. Compared to the simple mixture, the baicalin-matrine gel has a 1727 cm⁻¹ diameter. -1 The peak shifts towards the lower wavenumber region, proving that baicalin and matrine successfully self-assembled.

[0062] The binding constants of baicalin and matrine were determined using ITC. Figure 4 It is evident that the titration of baicalin with matrine involves an exothermic heat change, suggesting the presence of a binding reaction. Kd is less than 1, indicating a reversible reaction. ΔH and ΔS are less than 0, demonstrating that the reaction proceeds spontaneously at 95℃. The binding of baicalin and matrine is a spontaneous exothermic reaction with a chemical binding constant of 4.31 × 10⁻⁶. -4 M.

[0063] Figure 5 The rheological properties of the baicalin-matrine self-assembled injectable self-assembled hydrogel were shown. The linear viscoelastic region of the baicalin / matrine self-assembled hydrogel was determined to be 0.001%-0.1% based on the oscillatory strain change. Frequency scanning at different temperatures showed that the hydrogel exhibited the properties of an elastic colloid at both 25℃ and 37℃.

[0064] Figure 6 The results show the injection force required for injecting the gel using different needles. The baicalin-matrine self-assembled injectable hydrogel exhibits excellent injectability, with injection force significantly lower than the clinically injectable 20N across various needle sizes.

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

Claims

1. A baicalin-matrine self-assembled injectable hydrogel, characterized in that, The self-assembled injectable hydrogel is formed by the self-assembly of baicalin and matrine in a molar ratio of 2.5-6:1 under pH conditions of 3.5-5.

5. It has a highly porous microstructure and exhibits obvious hydrogel characteristics.

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

1.

3. The baicalin-matrine self-assembled injectable hydrogel according to claim 1, characterized in that, 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 alkali solution until baicalin is completely dissolved, and finally heat and stir, and then cool after the reaction is completed; (2) The pH of the product obtained in step (1) was adjusted to 3.5-5.5 by using hydrochloric acid solution with a molar concentration of 0.1-6 mM to form a gel, and baicalin-matrine self-assembled injectable hydrogel was obtained.

5. The preparation method according to claim 4, characterized in that, In step (1), the molar concentration of 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 heating and stirring conditions are: stirring at 25-90℃ for more than 15 minutes.

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

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