Diosmetin nanostructure lipid carrier as well as preparation method and application thereof

Abstract

The invention discloses a diosmetin nanostructure lipid carrier, which is prepared from the following raw materials by weight: 5-10% of diosmetin; 50-70% of a lipid material; 8-20% of cholesterol modified by mannose; and 15-25% of an emulsifier, belonging to the field of biological medicines. By preparing the nanostructured lipid carrier, the stability of diosmetin is improved, the release speed of the drug in vivo is delayed, the blood concentration and bioavailability are improved, the lung targeting property of the drug is also improved, the cytotoxicity of the drug is reduced, and the nanostructured lipid carrier has a good application prospect in clinical treatment of non-small cell lung cancer.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a dioscorea lignin nanostructure lipid carrier and a preparation method and application thereof. Background Art

[0002] Diosmetin, chemically known as 3',5,7-trihydroxy-4'-methoxyflavone, is a natural flavonoid compound with a molecular formula of C 16 H 12 O 6 , the chemical formula is as follows. It is mainly found in natural medicines such as lemon, chrysanthemum, spearmint, spider incense, and fruits such as lemon and peanut. Dioscorea lignin appears in the form of yellow powder. Modern pharmacological research shows that dioscorea lignin has antioxidant, anti-infection, anti-shock and other effects, and is widely used in the medical field.

[0003]

[0004] Studies have shown that dianthus plays an anti-tumor role in non-small cell lung cancer (NSCLC), mainly by inhibiting the PI3K / Akt / GSK-3β pathway to reduce the stability of Nrf2 to induce the production of reactive oxygen species (ROS), thereby inducing cell apoptosis and enhancing the efficacy of paclitaxel against NSCL [1]. The results of Song et al. showed that dianthus can inhibit the proliferation and clone formation of HCC827 and A549 cells [2]. Chen et al. used non-small cell lung cancer cells, normal cell lines HLF-1 and BEAS-2B, and immunodeficient mice as models to study the inhibitory effect of dianthus on lung cancer and found that dianthus selectively induced apoptosis of NSCLC cells by destroying the accumulation of ROS in the PI3K / Akt / GSK-3β / Nrf2 pathway [3]. Peng Yufang et al. isolated and identified various flavonoid compounds such as geraniol from chrysanthemum and studied their anti-tumor activity. They found that geraniol could inhibit the proliferation of MDA-MB-231, A549 and SMMC-7721 cells and had no toxicity to normal cells[4].

[0005] [1] Kong Yuchi. Research progress on the mechanism of action of linalool in preventing and treating cancer[J]. Chinese Fruit and Vegetables, 2023, 43(09): 55-60. [2] SONG C, DENG S, HU H, et al. Diosmetin affects gene expression on human lung adenocarcinoma cells[J]. Journal ofOncology, 2022,20:5482148. [3] CHEN [4] Peng Yufang, Zou Wentao, Xu Chuanlian. Study on flavonoids in chrysanthemum and their inhibitory activity on human tumor cell proliferation[J]. Chinese Journal of Pharmaceutical Sciences, 2010, 45 (19): 1454-1459. However, geraniol has extremely poor water solubility and very low bioavailability after oral administration, and ordinary dosage forms are difficult to exert a therapeutic effect to achieve the purpose of clinical treatment of non-small cell lung cancer.

[0006] Nanostructured lipid carriers (NLCs) are an ideal carrier for pulmonary drug delivery and have many advantages. NLCs can achieve slow and controlled release of drugs in the lungs, thereby enhancing drug stability and improving the bioavailability of poorly soluble drugs. Most of the materials used to prepare these carriers are biodegradable and non-toxic when used in the lungs. NLCs have high structural stability. Due to their lipophilic nature and ability to adhere to the mucosa, they can reduce pulmonary ciliary movement and drug clearance by phagocytes, thereby prolonging the duration of drug action. This characteristic not only improves the therapeutic effect, but also improves patients' medication compliance. At the same time, NLCs have a certain degree of lung targeting, which can further improve the drug therapeutic index. After searching, no relevant reports on dioscorea lignin nanostructured lipid carriers have been found. Summary of the invention

[0007] The purpose of the present invention is to provide a dioscorea nanostructured lipid carrier to achieve the purpose of better treating non-small cell lung cancer.

[0008] To achieve the above object, the present invention uses the following technical solutions: A dioscorea nanostructured lipid carrier is prepared from the following raw materials in a weight ratio: Geranin 5-10% Lipid material 50-70% Mannose-modified cholesterol 8-20% Emulsifier 15-25%, The structural formula of the mannose-modified cholesterol is as follows: .

[0009] Preferably, the weight ratio of the raw materials is: Geranin 5-8% Lipid material 60-70% Mannose-modified cholesterol 10-15% Emulsifier 15-20%.

[0010] Preferably, the lipid material consists of one or more of glyceryl monostearate, glyceryl monocaprylate, and lecithin.

[0011] Preferably, the emulsifier is Tween-80.

[0012] The present invention further provides a method for preparing the diosmetin nanostructured lipid carrier, comprising the following steps: dissolving diosmetin, lipid material, and mannose-modified cholesterol in ethanol as an oil phase; dissolving an emulsifier in water as an aqueous phase; adding the oil phase to the aqueous phase, heating and stirring until the ethanol is completely removed, and ultrasonicating after natural cooling to obtain the diosmetin nanostructured lipid carrier.

[0013] Wherein, the synthesis steps of the mannose-modified cholesterol are as follows: 1) Using DMF as solvent, DCC as dehydrating agent and DMAP as catalyst, Boc-protected γ-aminobutyric acid and cholesterol were subjected to esterification reaction, and cholesterol γ-aminobutyrate was obtained after removing the Boc protection.

[0014] 2) The ring-opened mannose undergoes a nucleophilic addition reaction with cholesterol γ-aminobutyrate, and the C=N double bond is reduced with sodium borohydride to obtain mannose-modified cholesterol.

[0015] The invention injects a liquid oil phase containing diosmetin into a water phase containing an emulsifier, forms oil-in-water type emulsion droplets under the action of the emulsifier, and disperses into nanoparticles under stirring and ultrasound. The method prepares nanoparticles by one-step emulsification, is not only simple to operate, but also can ensure the stability and encapsulation rate of diosmetin.

[0016] The beneficial effects of the present invention are: The invention improves the stability of dioscorea lignans, delays the release rate of the drug in the body, increases the blood concentration and bioavailability of the drug, significantly increases the content of the drug in the lungs, achieves the lung targeting of the drug, and thus enhances the therapeutic effect on non-small cell lung cancer. The invention also reduces the cytotoxicity of the drug and increases the safety of the drug.

[0017] In the process of preparing drugs for treating non-small cell lung cancer, the above-mentioned nanostructured lipid carrier (NLC) can be used, and excipients recognized in the pharmaceutical field can be introduced to prepare a drug form suitable for clinical treatment. For drugs with different dosage forms and administration methods, it is easy for those skilled in the art to select a suitable carrier material. According to a specific embodiment of the present invention, it is recommended to use an intravenous injection as the dosage form of the drug so that the drug can directly enter the blood circulation and take effect quickly. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the synthesis of mannose-modified cholesterol.

[0019] Figure 2 This is the particle size distribution diagram of the lignin nanostructured lipid carrier.

[0020] Figure 3 This is a transmission electron microscopy image of the lignin nanostructured lipid carrier.

[0021] Figure 4 This is the in vitro release curve of lignin nanostructured lipid carrier.

[0022] Figure 5 These are the cytotoxicity test results of dioscorea lignan nanostructured lipid carriers.

[0023] Figure 6 These are the results of the cellular uptake test of dioscorea lignan nanostructured lipid carriers.

[0024] Figure 7 These are the test results of the retention of lignin nanostructured lipid carrier in the lungs.

[0025] Figure 8 These are the results of measuring the blood drug concentration of dioscorea lignan nanostructured lipid carrier at different times.

[0026] Fig. 9 Effect of lignin nanostructured lipid carrier on tumor volume in mice. DETAILED DESCRIPTION

[0027] The present invention is described in detail below with reference to specific embodiments.

[0028] The preparation of the dioscorea nanostructured lipid carrier of the present invention mainly includes the following two steps: Step 1: Cholesterol is reacted with γ-aminobutyric acid and mannose to synthesize cholesterol-(γ-aminobutyric acid)-mannose. The chemical reactions involved in the synthesis are mainly esterification reaction and Schiff base reaction. The reaction principle can be found in Figure 1First, using DMF as solvent, DCC as dehydrating agent, and DMAP as catalyst, Boc (tert-butyloxycarbonyl) protected γ-aminobutyric acid was esterified with cholesterol, and cholesterol-(γ-aminobutyric acid) was obtained after removing the Boc protection. Then, the ring-opened mannose was subjected to nucleophilic addition reaction with cholesterol-(γ-aminobutyric acid), and the C=N double bond was reduced with sodium borohydride to obtain cholesterol-(γ-aminobutyric acid)-mannose.

[0029] Step 2: Using lignin, lipid material and cholesterol-(γ-aminobutyric acid)-mannose as raw materials, a conventional emulsification method is used to prepare lignin nanostructured lipid carrier.

[0030] In the present invention, γ-aminobutyric acid is an inhibitory neurotransmitter that exists naturally in the human body and has high biosafety. In addition, the raw material is simple and easy to obtain and the reaction conditions are mild. Cholesterol has good biocompatibility and membrane structure, which can improve the stability of lipid carriers and the drug encapsulation rate. Mannose can be recognized by active proteins on the alveolar surface and has a certain lung targeting. Therefore, the present invention uses γ-aminobutyric acid for bridging, connects mannose to the molecular structure of cholesterol, and adds the modified cholesterol to the nanostructured lipid carrier, so that geraniol can play a better role in the treatment of non-small cell lung cancer.

[0031] Example 1 Preparation prescription:

[0032] Preparation method: Add 911 mg of γ-aminobutyric acid protected with Boc (tert-butyloxycarbonyl) and 928 mg of DCC (dicyclohexylcarbodiimide) into a round-bottom flask, add a spoonful of DMAP (4-dimethylaminopyridine) and 25 mL of DMF (N,N-dimethylformamide), stir, add 1160 mg of cholesterol, stir, filter, add two times the volume of water and two times the volume of ethyl acetate to the filtrate for extraction, concentrate the oil phase volume to 2-3 mL, then add a silica gel column, elute with a mixture of ethyl acetate and ether, dry the eluate and dissolve it in dichloromethane, take four times the volume of trifluoroacetic acid and add stirring to remove the protection of Boc (tert-butyloxycarbonyl), and after the reaction is complete, evaporate it to obtain cholesterol-(γ-aminobutyric acid). 385 mg of mannose was dissolved in 2 mL of pH 4.0 acetic acid solution, stirred at 60 °C for 1 h to ring-open mannose, 9 times the volume of methanol of the acetic acid solution was added, stirred for 24 h, and then 88 mg of cholesterol-(γ-aminobutyric acid), 45 mg of NaBH 4 The mixture was put into the reaction system and stirred for 6 hours, then washed with water and filtered to obtain cholesterol-(γ-aminobutyric acid)-mannose.

[0033] Weigh 6 mg of Dios API, 40 mg of monostearate glyceryl, 10 mg of octanoic acid glyceryl, 15 mg of soy lecithin, and 13 mg of cholesterol-(γ-aminobutyric acid)-mannose into a beaker, add an appropriate amount of ethanol, and stir and dissolve in a constant temperature water bath magnetic stirrer at a temperature of 55-60°C as the oil phase; take 16 mg of Tween-80 and place it in a 25 mL beaker, add 20 mL of purified water and stir and dissolve as the water phase. While stirring, slowly inject the oil phase into the water phase, heat and stir until the ethanol is completely removed, then place it at room temperature to cool naturally, and finally ultrasonicate to obtain the product.

[0034] It was determined that the average particle size of the prepared nanostructured lipid carrier was 108.64 nm, the Zeta potential was -36.48 mV, and the drug encapsulation efficiency was 98.8%.

[0035] Example 2 Preparation prescription

[0036] Preparation method: Weigh 4 mg of Dios API, 37.6 mg of monostearate glyceryl, 9.4 mg of octanoic acid glyceryl, 8 mg of soy lecithin, and 17 mg of cholesterol-(γ-aminobutyric acid)-mannose into a beaker, add an appropriate amount of ethanol, and place in a constant temperature water bath magnetic stirrer at 55-60°C to stir and dissolve as the oil phase; take 24 mg of Tween-80 and place in a 25 mL beaker, add 20 mL of purified water and stir and dissolve as the water phase. While stirring, slowly inject the oil phase into the water phase, continue heating and stirring until the ethanol is completely removed, then place at room temperature to cool naturally, and finally ultrasonicate to obtain the product.

[0037] It was determined that the average particle size of the prepared nanostructured lipid carrier was 118.47 nm, the Zeta potential was -35.1 mV, and the drug encapsulation efficiency was 95.11%.

[0038] Example 3 Preparation prescription

[0039] Preparation method: Weigh 8 mg of Dios API, 46.4 mg of monostearate glyceryl, 11.6 mg of octanoic acid glyceryl, 10 mg of soy lecithin, and 9 mg of cholesterol-(γ-aminobutyric acid)-mannose into a beaker, add an appropriate amount of ethanol, and place in a constant temperature water bath magnetic stirrer at 55-60°C to stir and dissolve as the oil phase; take 15 mg of Tween-80 and place in a 25 mL beaker, add 20 mL of purified water and stir and dissolve as the water phase. While stirring, slowly inject the oil phase into the water phase, continue heating and stirring until the ethanol is completely removed, then place at room temperature to cool naturally, and finally ultrasonicate to obtain the product.

[0040] It was determined that the average particle size of the prepared nanostructured lipid carrier was 133.89 nm, the Zeta potential was -33.13 mV, and the drug encapsulation efficiency was 96.92%.

[0041] Test example Experiment 1: DLS measurement was performed using a laser nanoparticle size analyzer. 1 mL of the prepared diosmin nanostructured lipid carrier (hereinafter referred to as Man-Chol-Dios-NLCs) solution was diluted 3 times with ultrapure water, and its particle size was measured under a laser nanoparticle size analyzer. Particle size distribution is shown in Figure 2 .

[0042] Experiment 2: Take a small amount of Man-Chol-Dios-NLCs solution and drop it on the surface of the carbon-sprayed copper mesh, try to make the liquid cover the entire copper mesh, leave it for a suitable time to allow the nanoparticles to adsorb for 15 minutes, use 2.0% phosphotungstic acid to dye the nanoparticles, dry them for 10 minutes, and observe their morphology under a transmission electron microscope. The nanoparticles are evenly dispersed spherical or quasi-spherical, with uniform particle size and a complete and rounded surface ( Figure 3 ).

[0043] Experiment 3: The in vitro release curve of the drug was determined by dialysis method, with 1% Tween-Ringer solution as the in vitro release medium, the molecular weight of the dialysis bag was 3500 Daltons, and the drug release was simulated at 37±0.5℃ and 100r / min. The results showed that the cumulative release of diosmin suspension (Dio) was 80% in 24h, while the release of Man-Chol-Dios-NLCs was only 40% in 24h. The results showed that Man-Chol-Dios-NLCs prolonged the release time of the drug in vivo ( Figure 4 ).

[0044] Experiment 4: The live-dead cell test kit was used to detect the toxicity of diosgenin suspension (Dios), non-drug-loaded nanostructured lipid carriers (Man-Chol-NLCs), and drug-loaded nanostructured lipid carriers (Man-Chol-Dios-NLCs) to human A549 cells. A549 cells were seeded in a 96-well plate. When the cell concentration reached about 90%, culture media containing free Dios, Man-Chol-NLCs, and Man-Chol-Dios-NLCs were added respectively. After incubation for 4 hours, the culture box was removed, fixed with paraformaldehyde, and incubated with a DAPI solution at a concentration of 0.5μg / mL for 5 minutes. The cells were placed under a laser confocal microscope and photographed. The results showed that the cell proliferation after adding Man-Chol-NLCs was close to that of the blank control, indicating that the carrier is a safe material; the cell proliferation of free Dios and drug-loaded lipid carriers was significantly reduced, and the cell proliferation of drug-loaded lipid carriers was higher than that of free Dios ( Figure 5 ).

[0045] Experiment 5: A549 cells were seeded in a 6-well plate. When the cell concentration reached about 90%, Dios and Man-Chol-Dios-NLCs solutions were added respectively (n=3, Dios concentration was 90μg / mL). After administration, the cells were incubated for 6 hours, digested with trypsin, and the cell suspension was placed in a centrifuge tube. The old culture medium was discarded, and sodium dodecyl sulfate solution was added for incubation for 24 hours. The cells were ultrasonicated and centrifuged by a cell disruptor. The supernatant was taken for HPLC determination of Dios content, and the Dios uptake was calculated. The results showed that the uptake of Man-Chol-Dios-NLCs in A549 cells was much greater than that of free Dios ( Figure 6 ).

[0046] Experiment 6: Six ICR mice weighing about 25 g were randomly divided into two groups, with 3 mice in each group. The mice were injected with DID-labeled NLCs (blank NLCs without mannose-modified cholesterol) and Man-Chol-NLCs at a dose of 1 mg / kg DID through the tail vein. At different time points after administration, the mice were placed in a living imager to take living fluorescence photos. After 24 hours, the mice were killed, and the main organs of the mice were collected, rinsed with saline, fixed with fixative for 3 minutes, and the water was removed with filter paper and photographed. The results showed that 12 hours after tail vein injection, the fluorescence intensity in the lungs was: Man-Chol-NLCs>NLCs, indicating that the addition of mannose-(γ-aminobutyric acid)-cholesterol enhanced the lung targeting of the nanostructured lipid carrier ( Figure 7 ).

[0047] Experiment 7: Fifteen healthy SD rats were selected and adaptively fed for three days. They were randomly divided into three groups and fasted for 12 hours before the experiment, but were not allowed to drink water. After weighing, Dios, Dios-NLCs, and Man-Chol-Dios-NLCs were injected into the tail vein at 3 mg / kg. Blood was collected from the tail vein at 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12, and 24 hours after administration and placed in heparinized plastic centrifuge tubes. After processing, the blood drug concentrations at different times were measured. The results showed that the blood drug concentration of Man-Chol-Dios-NLCs in rats was higher than that of Dios and Dios-NLCs ( Figure 8 ).

[0048] Experiment 8: Take A549 cells with good growth status and dilute them with saline to a density of 8×10 6 After the tumors were grown to 85 mm, 200 μL was taken and inoculated subcutaneously on the right side of male SPFBALB / c mice aged 5-6 weeks. 3 The modeling was completed at 3:30 pm. On the third day after inoculation, the tumor volume of the mice grew to 85 mm 3 ; Nine mice were divided into three groups, with three mice in each group; they were injected with normal saline, Dios suspension (3 mg / kg) and Man-Chol-Dios-NLCs (3 mg / kg) respectively, and the drugs were slowly injected into the tumor at three points. The drugs were divided into four doses, once every three days, and the mice were killed by cervical dislocation on the 16th day. From the first dose, the long diameter (L) and short diameter (D) of the tumor were measured and recorded with a vernier caliper every day, the hair around the mouse tumor was cleaned, and the tumor volume (V) was calculated by the formula V=0.5*(L*D2). The results showed that the anti-tumor effect of Man-Chol-Dios-NLCs in rats was higher than that of Dios ( Fig. 9 ).

Claims

1. A dioscorea nanostructured lipid carrier, characterized in that Made from the following raw materials in weight ratio: Geranin 5-10% Lipid material 50-70% Mannose-modified cholesterol 8-20% Emulsifier 15-25%, The structural formula of the mannose-modified cholesterol is as follows: 。 2. The dioscorea nanostructured lipid carrier according to claim 1, characterized in that: The weight ratio of the raw materials is: Geranin 5-8% Lipid material 60-70% Mannose-modified cholesterol 10-15% Emulsifier 15-20%.

3. The dioscorea nanostructured lipid carrier according to claim 1, characterized in that: The lipid material is composed of one or more of glyceryl monostearate, glyceryl caprylate and lecithin.

4. The dioscorea nanostructured lipid carrier according to claim 1, characterized in that: The emulsifier is Tween-80.

5. The method for preparing the dioscorea nanostructured lipid carrier according to any one of claims 1 to 4, characterized in that The following steps are involved: Dissolve lignin, lipid material and mannose-modified cholesterol in ethanol as the oil phase; dissolve the emulsifier in water as the aqueous phase; add the oil phase into the aqueous phase, heat and stir until the ethanol is completely removed, cool naturally and then perform ultrasound to obtain the lignin nanostructure lipid carrier.

6. The method for preparing the dioscorea lignin nanostructure lipid carrier according to claim 5, characterized in that: The synthesis steps of the mannose-modified cholesterol are as follows: 1) Using DMF as solvent, DCC as dehydrating agent and DMAP as catalyst, Boc-protected γ-aminobutyric acid and cholesterol were subjected to esterification reaction, and cholesterol γ-aminobutyrate was obtained after removing the Boc protection; 2) The ring-opened mannose undergoes a nucleophilic addition reaction with cholesterol γ-aminobutyrate, and the C=N double bond is reduced with sodium borohydride to obtain mannose-modified cholesterol.

7. Use of the dioscorea nanostructured lipid carrier according to any one of claims 1 to 4 in the preparation of a drug for treating non-small cell lung cancer.

8. A drug for treating non-small cell lung cancer, comprising the dioscorea lignan nanostructure lipid carrier according to any one of claims 1 to 4.

9. The drug for treating non-small cell lung cancer as claimed in claim 8, further comprising a pharmaceutically acceptable carrier.

10. The drug for treating non-small cell lung cancer as claimed in claim 8, which is an intravenous injection.

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