Bergenin-lipid conjugate lipid nanoparticle and preparation method thereof

Abstract

The invention discloses a bergenin-lipid conjugate lipid nanoparticle and a preparation method of the bergenin-lipid conjugate lipid nanoparticle. Comprising a bergenin-lipid conjugate, a solid lipid material and a surfactant, wherein the ratio of lipid to medicine is 1: (2-10); the structural formula of the bergenin-lipid conjugate is as shown in the formula I. The bergenin-lipid conjugate lipid nanoparticles prepared by the invention are high in encapsulation efficiency, high in drug loading capacity, small in particle size, good in stability and high in bioavailability.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a bergenin-lipid conjugate lipid nanoparticle and a preparation method thereof. Background Art

[0002] Oral administration is the most widely used route of administration, and is also the safest and simplest route of administration. For patients, oral administration causes less discomfort, is usually less expensive, does not require professional medical staff to operate, and has no restrictions on the place of administration. It is the dosage form with the highest compliance. However, the solubility and permeability of drugs often limit the oral bioavailability of drugs, which is also the difficulty in the design of oral formulations.

[0003] Bergenin (BG) is an active ingredient of plants such as Bergenia chinensis and Astilbe chinensis of the Saxifragaceae family. Currently, many plant sources have been found. Domestic and foreign researchers have conducted extensive research on bergenin. Bergenin has good expectorant, antitussive, anti-inflammatory and analgesic effects. It is often used as an effective ingredient of antitussive and anti-inflammatory drugs in the clinic to treat acute and chronic tracheitis, duodenal ulcers and other diseases. It also has special effects on other respiratory diseases such as chronic obstructive pulmonary disease and bronchial asthma. In addition, the pharmacological activities of bergenin that have been discovered include anti-arrhythmic, anti-tumor, antioxidant, sleep promotion, metabolism regulation, liver and stomach protection, and certain immunomodulatory effects. Bergenin has significant pharmacological activity and has good development prospects. However, its low solubility and weak transmembrane transport capacity result in an oral bioavailability generally less than 5%, which greatly limits its efficacy and clinical application. The structure of bergenin is shown below.

[0004]

[0005] In order to improve the oral bioavailability of bergenin, its solubility and permeability need to be improved at the same time. Lipid-based nanoparticles (LBN) are a good choice. Lipid-based nanoparticles include liposomes, transfer bodies, solid lipid nanoparticles, etc. Among them, solid lipid nanoparticles (SLN) are composed of physiological lipids that remain solid at room temperature and body temperature. After digestion, the lipid material can form micelles with bile salts and other substances in the body to encapsulate drugs, improve the solubility of poorly soluble drugs, and promote their absorption. At the same time, solid lipid nanoparticles have a small particle size and are easy to adhere to the gastrointestinal mucosa, prolonging the absorption time and promoting absorption. Compared with other lipid delivery systems, solid lipid nanoparticles are low-cost, easy to prepare, non-toxic, and have long-term physical stability. Their stability is better than that of liposomes, making them good drug delivery carriers. Unfortunately, bergenin has poor compatibility with lipid carrier materials, low encapsulation rate, and a drug loading of only 4% to 5%, which limits its further development. Summary of the invention

[0006] In order to solve the problems of poor compatibility, low encapsulation efficiency, low drug loading, etc., the present invention is to provide a bergenin-lipid conjugate lipid nanoparticle (BG-lipid SLNs) and a preparation method thereof.

[0007] The present invention provides a bergenin-lipid conjugate lipid nanoparticle, comprising a bergenin-lipid conjugate, a solid lipid material, and a surfactant; wherein the lipid-drug ratio is 1:2-10; the structural formula of the bergenin-lipid conjugate is as shown in Formula I:

[0008]

[0009] In the present invention, the preparation method of the bergenin-lipid conjugate is prepared by referring to another patent application CN 118108732A of the inventor.

[0010] In some specific embodiments of the present invention, the drug-to-lipid ratio is 1:3.

[0011] In the present invention, the mass ratio of the surfactant to the bergenin-lipid conjugate is 500:90-325.

[0012] In some specific embodiments of the present invention, the solid lipid material comprises: one or more of cholesterol oleate, distearin, trilaurin, monostearin, stearic acid, glyceryl palmitostearate and behenic acid glyceryl.

[0013] In some specific embodiments of the present invention, the solid lipid material is glyceryl distearate.

[0014] In some specific embodiments of the present invention, the surfactant includes one or more of phospholipids, Tween 80, Poloxamer 188, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, and surfactant OP.

[0015] In some specific embodiments of the present invention, the surfactant is Tween 80; further, it is 2% Tween 80.

[0016] The present invention provides a method for preparing the above-mentioned bergenin-lipid conjugate lipid nanoparticles, comprising the following steps:

[0017] (1) dropping the bergenin-lipid conjugate solution into the molten lipid material;

[0018] (2) adding a surfactant;

[0019] (3) Shearing to obtain colostrum;

[0020] (4) microfluidization high pressure homogenization, and cooling the obtained suspension in an ice bath to obtain bergenin-lipid conjugate lipid nanoparticles.

[0021] In some specific embodiments of the present invention, the shearing conditions are: 8000-12000 psi, shearing for 2-5 min.

[0022] In some specific embodiments of the present invention, the microfluidic high pressure homogenization is: 15000-35000psi, and is circulated 4-6 times.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The present invention adopts a melt-high pressure homogenization method to prepare bergenin-lipid conjugate lipid nanoparticles, the best lipid material is distearic acid glyceryl, the surfactant is 2% Tween 80, and the drug-lipid ratio is 1 / 3;

[0025] (2) The bergenin-lipid conjugate lipid nanoparticles prepared by the present invention have small particle size, uniform distribution, high encapsulation efficiency and drug loading capacity; the encapsulation rates of 6C-BG, 12C-BG, and 18C-BG lipid nanoparticles are 86.4%, 88.0%, and 99.4%, respectively, and the drug loading capacities are 21.6%, 22.0%, and 24.9%, respectively. The drug loading capacities of the three prodrugs converted into the original drug BG are 16.6%, 14.1%, and 13.7%, respectively, and the average particle sizes are 71.99±4.27, 75.90±2.05, and 61.80±0.48 nm, respectively.

[0026] (3) The relative bioavailability of 6C-BG, 12C-BG, and 18C-BG lipid nanoparticles relative to BG lipid nanoparticles were 239.87%, 864.05%, and 467.97%, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Differential scanning calorimetry analysis of 6C-BG and lipid materials (drug-to-lipid ratio is 1 / 3);

[0028] Figure 2 Differential scanning calorimetry analysis of 12C-BG and lipid materials (drug-to-lipid ratio is 1 / 3);

[0029] Figure 3 Differential scanning calorimetry analysis of 18C-BG and lipid materials (drug-to-lipid ratio is 1 / 3);

[0030] Figure 4 Differential scanning calorimetry analysis of 6C-BG solid lipid nanoparticles with different lipid ratios;

[0031] Figure 5Differential scanning calorimetry analysis of 12C-BG solid lipid nanoparticles with different lipid ratios;

[0032] Figure 6 Differential scanning calorimetry analysis of 18C-BG solid lipid nanoparticles with different lipid ratios;

[0033] Figure 7 Zeta potential distribution diagram of solid lipid nanoparticles (6C-BG (A), 12C-BG (B) and 18C-BG (C)) and Zeta potential of solid lipid nanoparticles (6C-BG (E), 12C-BG (F) and 18C-BG (G));

[0034] Figure 8 The stability of solid lipid nanoparticles in buffer and biologically relevant media (SGF, SIF, PBS, plasma) (the pH value of SGF is 1.2; the pH value of SIF is 6.8; the pH value of PBS is 7.4 (n=3));

[0035] Fig. 9 The curve showing the relationship between the concentration of bergenin in plasma and time (n=3). DETAILED DESCRIPTION

[0036] The technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Example 1 Preparation of Bergenin-Lipid Conjugate

[0038] Bergenin-lipid conjugates were synthesized by one-step reaction of BG with acyl chlorides (caproyl chloride, lauroyl chloride and stearoyl chloride) in anhydrous tetrahydrofuran, as shown below. That is, BG in tetrahydrofuran was reacted with acyl chlorides at room temperature; after 1.5 hours, the reaction was terminated by adding ice-cold HCl, the reaction mixture was further extracted with ethyl acetate, dried over anhydrous Na2SO4, and then concentrated under reduced pressure at 40°C, and purified by silica gel column chromatography to obtain 6C-BG, 12C-BG and 18C-BG.

[0039]

[0040] Example 2

[0041] Weigh 250 mg of 6C-BG, add 3 mL of tetrahydrofuran to dissolve, and obtain a 6C-BG solution. Take 750 mg of distearic acid glyceryl (ATO-5) and 25 mL of 2% Tween, place them in a 50 mL beaker and heat to 80°C and 75°C respectively. Drop the 6C-BG solution into the molten lipid material, continue heating and stirring until the solvent is completely removed. Pour the 75°C surfactant solution (aqueous phase) into the above-mentioned molten lipid (oil phase), and obtain colostrum by high shear (10,000 psi, 3 min). Immediately thereafter, the particle size is reduced by microfluidization high-pressure homogenization (20,000 psi, 5 cycles), and the resulting suspension is cooled in an ice bath to obtain 6C-BG ATO-5 (6C-BG 1:3). (The drug-lipid ratio is 1 / 3)

[0042] Example 3

[0043] Weigh 250 mg of 6C-BG, add 3 mL of tetrahydrofuran to dissolve, and obtain 6C-BG solution. Take 750 mg of behenic acid glyceryl ester (888ATO) and 25 mL of 2% Tween, place them in a 50 mL beaker and heat to 80°C and 75°C respectively. Drop the 6C-BG solution into the molten lipid material, continue heating and stirring until the solvent is completely removed. Pour the 75°C surfactant solution (aqueous phase) into the above-mentioned molten lipid (oil phase), and obtain colostrum by high shear (10,000 psi, 3 min). Immediately thereafter, the particle size is reduced by microfluidization high-pressure homogenization (20,000 psi, 5 cycles), and the resulting suspension is cooled in an ice bath to obtain 6C-BG 888ATO lipid nanoparticles. (The drug-lipid ratio is 1 / 3)

[0044] Example 4

[0045] Weigh 250 mg of 12C-BG, add 3 mL of tetrahydrofuran to dissolve, and obtain a 12C-BG solution. Take 750 mg of ATO-5 and 25 mL of 2% Tween, place them in a 50 mL beaker and heat to 80°C and 75°C respectively. Drop the 12C-BG solution into the molten lipid material, continue heating and stirring until the solvent is completely removed. Pour the 75°C surfactant solution (aqueous phase) into the above-mentioned molten lipid (oil phase), and obtain colostrum by high shear (10,000 psi, 3 min). Immediately thereafter, the particle size is reduced by microfluidization high-pressure homogenization (20,000 psi, 5 cycles), and the resulting suspension is cooled in an ice bath to obtain 12C-BG ATO-5 lipid nanoparticles. (The drug-lipid ratio is 1 / 3)

[0046] Example 5

[0047] Weigh 250 mg of 12C-BG, add 3 mL of tetrahydrofuran to dissolve, and obtain a 12C-BG solution. Take 750 mg of 888ATO and 25 mL of 2% Tween, place them in a 50 mL beaker and heat to 80°C and 75°C respectively. Drop the 12C-BG solution into the molten lipid material, continue heating and stirring until the solvent is completely removed. Pour the 75°C surfactant solution (aqueous phase) into the above-mentioned molten lipid (oil phase), and obtain colostrum by high shear (10,000 psi, 3 min). Immediately thereafter, the particle size is reduced by microfluidization high-pressure homogenization (20,000 psi, 5 cycles), and the resulting suspension is cooled in an ice bath to obtain 12C-BG 888ATO lipid nanoparticles. (The drug-lipid ratio is 1 / 3)

[0048] Example 6

[0049] Weigh 250 mg of 18C-BG, add 3 mL of tetrahydrofuran to dissolve, and obtain 18C-BG solution. Take 750 mg of ATO-5 and 25 mL of 2% Tween, place them in a 50 mL beaker and heat to 80°C and 75°C respectively. Drop the 18C-BG solution into the molten lipid material, continue heating and stirring until the solvent is completely removed. Pour the 75°C surfactant solution (aqueous phase) into the above molten lipid (oil phase), and obtain colostrum by high shear (10,000 psi, 3 min). Immediately thereafter, the particle size is reduced by microfluidization high-pressure homogenization (20,000 psi, 5 cycles), and the resulting suspension is cooled in an ice bath to obtain 18C-BG ATO-5 lipid nanoparticles. (The drug-lipid ratio is 1 / 3)

[0050] Example 7

[0051] Weigh 250 mg of 18C-BG, add 3 mL of tetrahydrofuran to dissolve, and obtain 18C-BG solution. Take 750 mg of 888ATO and 25 mL of 2% Tween, place them in a 50 mL beaker and heat to 80°C and 75°C respectively. Drop the 18C-BG solution into the molten lipid material, continue heating and stirring until the solvent is completely removed. Pour the 75°C surfactant solution (aqueous phase) into the above molten lipid (oil phase), and obtain colostrum by high shear (10,000 psi, 3 min). Immediately thereafter, the particle size is reduced by microfluidization high-pressure homogenization (20,000 psi, 5 cycles), and the resulting suspension is cooled in an ice bath to obtain 18C-BG 888ATO lipid nanoparticles. (The drug-lipid ratio is 1 / 3)

[0052] Differential scanning calorimetry was used to analyze the lipid nanoparticles obtained in Examples 2 to 7. The results are as follows: Figure 1-3 As shown in the figure, 6C-BG, 12C-BG and 18C-BG are more compatible with ATO-5 than 888ATO.

[0053] Example 8

[0054] Weigh 325 mg of 6C-BG, add 3 mL of tetrahydrofuran to dissolve, and obtain a 6C-BG solution. Take 650 mg of ATO-5 and 25 mL of 2% Tween solution, place them in a 50 mL beaker and heat to 80°C and 75°C respectively. Drop the 6C-BG solution into the molten lipid material, continue heating and stirring until the solvent is completely removed. Pour the 75°C surfactant solution (aqueous phase) into the above-mentioned molten lipid (oil phase), and obtain colostrum by high shear (10,000 psi, 3 min). Immediately thereafter, the particle size is reduced by microfluidization high-pressure homogenization (20,000 psi, 5 cycles), and the resulting suspension is cooled in an ice bath to obtain 6C-BG 1:2 lipid nanoparticles. (The drug-lipid ratio is 1 / 2)

[0055] Example 9

[0056] Weigh 90 mg of 6C-BG, add 3 mL of tetrahydrofuran to dissolve, and obtain a 6C-BG solution. Take 900 mg of ATO-5 and 25 mL of 2% Tween, place them in a 50 mL beaker and heat to 80°C and 75°C respectively. Drop the 6C-BG solution into the molten lipid material, continue heating and stirring until the solvent is completely removed. Pour the 75°C surfactant solution (aqueous phase) into the above-mentioned molten lipid (oil phase), and obtain colostrum by high shear (10,000 psi, 3 min). Immediately thereafter, the particle size is reduced by microfluidization high-pressure homogenization (20,000 psi, 5 cycles), and the resulting suspension is cooled in an ice bath to obtain 6C-BG 1:10 lipid nanoparticles. (The drug-lipid ratio is 1 / 10)

[0057] Example 10

[0058] Weigh 325 mg of 12C-BG, add 3 mL of tetrahydrofuran to dissolve, and obtain a 12C-BG solution. Take 650 mg of ATO-5 and 25 mL of 2% Tween, place them in a 50 mL beaker and heat to 80 ° C and 75 ° C respectively. Drop the 12C-BG solution into the molten lipid material, continue heating and stirring until the solvent is completely removed. Pour the 75 ° C surfactant solution (aqueous phase) into the above molten lipid (oil phase), and obtain colostrum by high shear (10000 psi, 3 min). Immediately thereafter, the particle size is reduced by microfluidization high pressure homogenization (20000 psi, 5 cycles), and the resulting suspension is cooled in an ice bath to obtain 12C-BG 1:2 lipid nanoparticles (drug-to-lipid ratio is 1 / 2).

[0059] Embodiment 11

[0060] Weigh 90 mg of 12C-BG, add 3 mL of tetrahydrofuran to dissolve, and obtain a 12C-BG solution. Take 900 mg of ATO-5 and 25 mL of 2% Tween, place them in a 50 mL beaker and heat to 80 ° C and 75 ° C respectively. Drop the 12C-BG solution into the molten lipid material, continue heating and stirring until the solvent is completely removed. Pour the 75 ° C surfactant solution (aqueous phase) into the above molten lipid (oil phase), and obtain colostrum by high shear (10000 psi, 3 min). Immediately thereafter, the particle size is reduced by microfluidization high pressure homogenization (20000 psi, 5 cycles), and the resulting suspension is cooled in an ice bath to obtain 12C-BG 1:10 lipid nanoparticles (drug-to-lipid ratio is 1 / 10).

[0061] Example 12

[0062] Weigh 325 mg of 18C-BG, add 3 mL of tetrahydrofuran to dissolve, and obtain 18C-BG solution. Take 650 mg of ATO-5 and 25 mL of 2% Tween, place them in a 50 mL beaker and heat to 80 ° C and 75 ° C respectively. Drop the 18C-BG solution into the molten lipid material, continue heating and stirring until the solvent is completely removed. Pour the 75 ° C surfactant solution (aqueous phase) into the above molten lipid (oil phase), and obtain colostrum by high shear (10000 psi, 3 min). Then immediately reduce the particle size by microjet high pressure homogenization (20000 psi, 5 cycles), and cool the resulting suspension in an ice bath to obtain 18C-BG 1:2 lipid nanoparticles (drug-to-lipid ratio of 1 / 2).

[0063] Embodiment 13

[0064] Weigh 90 mg of 18C-BG, add 3 mL of tetrahydrofuran to dissolve, and obtain 18C-BG solution. Take 900 mg of ATO-5 and 25 mL of 2% Tween, place them in a 50 mL beaker and heat to 80 ° C and 75 ° C respectively. Drop the 18C-BG solution into the molten lipid material, continue heating and stirring until the solvent is completely removed. Pour the 75 ° C surfactant solution (aqueous phase) into the above molten lipid (oil phase), and obtain colostrum by high shear (10000 psi, 3 min). Then immediately reduce the particle size by microjet high pressure homogenization (20000 psi, 5 cycles), and cool the resulting suspension in an ice bath to obtain 18C-BG 1:10 lipid nanoparticles (drug-to-lipid ratio of 1 / 10).

[0065] Differential scanning calorimetry was used to analyze the lipid nanoparticles obtained in Examples 2, 4, 6, 8-13. The results are as follows: Figure 4-6As shown. 6C-BG is mostly compatible when the drug-lipid ratio is 1 / 3, but a small part is still not encapsulated. It is completely encapsulated when the drug-lipid ratio is 1 / 10. 12C-BG is mostly encapsulated when the drug-lipid ratio is 1 / 2, and is basically completely encapsulated when the drug-lipid ratio is 1 / 3 and 1 / 10. 18C-BG has the best compatibility with lipids and is completely encapsulated when the drug-lipid ratio is 1 / 2.

[0066] Embodiment 14

[0067] The 2% Tween 80 in Example 2 was replaced with 2% polyoxyethylene castor oil (EL) to prepare 6C-BG EL lipid nanoparticles.

[0068] Embodiment 15

[0069] The 2% Tween 80 in Example 2 was replaced with 2% vitamin E polyethylene glycol succinate (TPGS) to prepare 6C-BG TPGS lipid nanoparticles.

[0070] Example 16

[0071] The 2% Tween 80 in Example 4 was replaced with 2% polyoxyethylene castor oil (EL) to prepare 12C-BG EL lipid nanoparticles.

[0072] Embodiment 17

[0073] The 2% Tween 80 in Example 4 was replaced with 2% vitamin E polyethylene glycol succinate (TPGS) to prepare 12C-BG TPGS lipid nanoparticles.

[0074] Embodiment 18

[0075] The 2% Tween 80 in Example 6 was replaced with 2% polyoxyethylene castor oil (EL) to prepare 18C-BG EL lipid nanoparticles.

[0076] Embodiment 19

[0077] The 2% Tween 80 in Example 6 was replaced with 2% vitamin E polyethylene glycol succinate (TPGS) to prepare 18C-BG TPGS lipid nanoparticles.

[0078] Examples 2, 4, 6 and 14-19 are lipid nanoparticles prepared under different surfactants, and the results are shown in Tables 1-3.

[0079] Table 1 Encapsulation efficiency and drug loading of 6C-BG lipid nanoparticles prepared by three surfactants

[0080]

[0081] Table 2 Encapsulation efficiency and drug loading of 12C-BG lipid nanoparticles prepared by three surfactants

[0082]

[0083] Table 3 Encapsulation efficiency and drug loading of 18C-BG lipid nanoparticles prepared by three surfactants

[0084]

[0085] Based on the results of Example 2 to Example 19, the best lipid material is glyceryl distearate; the surfactant is 2% Tween 80; and the drug-lipid ratio is 1:3.

[0086] Test Example 1

[0087] According to the optimized prescription in the example, 6C-BG, 12C-BG and 18C-BG lipid nanoparticles were prepared and the three lipid nanoparticles were tested.

[0088] 1 Experimental methods

[0089] 1.1 Determination of FA-BG lipid nanoparticle content

[0090] Accurately pipette 200 μL of each 6C-BG, 12C-BG and 18C-BG lipid nanoparticle suspension into a 10 mL volumetric flask, dilute with an appropriate amount of methanol, ultrasonically break the emulsion, shake well, and make up to volume with methanol. Centrifuge at 10,000 rpm for 10 min, take 1 mL of the supernatant into a centrifuge tube, add 1 mL of methanol, vortex mix, centrifuge at 10,000 rpm for 10 min, and take the supernatant for sampling.

[0091] 1.2 Determination of encapsulation efficiency

[0092] Accurately pipette 1.0 mL of each 6C-BG, 12C-BG, and 18C-BG lipid nanoparticle suspension, add 5 mL of pure water, take 1 mL of the dilution and filter it through a 0.22 μm microporous membrane, centrifuge at 8000 rpm for 10 min, take the supernatant and inject it for HPLC analysis, calculate the amount of free 6C-BG, 12C-BG, and 18C-BG, and record it as W F According to the results of FA-BG lipid nanoparticle content determination in 2.2.1, the total amount of 6C-BG, 12C-BG and 18C-BG was calculated and recorded as W T The entrapment efficiency (EE) of FA-BG lipid nanoparticles was calculated according to the following formula:

[0093]

[0094] 1.3 Determination of drug loading

[0095] The amount of free 6C-BG, 12C-BG and 18C-BG is recorded as W F Chapter 2.2.1.6FA-BG Lipid Nanoparticles The total amount of 6C-BG, 12C-BG and 18C-BG determined is recorded as W T , the amount of lipid material is recorded as W L The drug loading (DL) of FA-BG lipid nanoparticles was calculated according to the following formula:

[0096]

[0097] 1.4 Particle size and Zeta potential determination

[0098] Take 200 μL of 6C-BG, 12C-BG, and 18C-BG lipid nanoparticle suspensions respectively, add 800 μL of purified water, and mix well. Take 200 μL in the sample pool and use a laser particle size analyzer to detect the particle size, PDI, and Zeta potential of the lipid nanoparticles.

[0099] 1.5 In vitro stability and lipolysis.

[0100] In vitro lipolysis test under simulated fasting state: The lipolysis digestion solution (pH 7.5) is composed of maleate (50mM), NaCl (150mM), CaCl2 (5mM), sodium taurocholate (5mM), and lecithin (1.25mM). First, 1mL of BG-Lipid SLNs suspension was added to 9mL of lipolysis digestion solution, preheated to 37°C, stirred continuously, and pre-dropped NaOH solution (0.1M, flow rate 2mL / min) until the system pH was 7.5. 2mL of pancreatic enzyme solution (5000U / mL) was added to start the lipolysis reaction, and the temperature was kept at 37°C and magnetic stirring was used for 60min. As the lipolysis reaction occurred, the lipid material in the preparation was continuously degraded to release fatty acids. The pH titrator automatically sensed and gradually added NaOH solution to keep the system pH at around 7.5. When the cumulative consumption of NaOH no longer increased, an appropriate amount of 4-bromophenylboronic acid methanol solution was added to terminate the reaction. The lipolysis percentage was calculated according to the cumulative consumption of NaOH at different time points, and the lipolysis curve was drawn.

[0101] Take 5 mL of two stability solutions in different media, add the bergenin conjugate monomer solution to the first part, and add the SLNs suspension to the second part. Place in a 37°C constant temperature water bath oscillator, take samples at 1, 2, 4, 8, and 12 hours, centrifuge for 10 minutes (12,000 rpm), take the supernatant, and determine the content of the corresponding conjugate by HPLC.

[0102] PBS with pH 7.4: Take 1.36 g of potassium dihydrogen phosphate, add 79 mL of 0.1 M sodium hydroxide solution, dilute to 200 mL with purified water, shake well, and you have it.

[0103] Artificial simulated gastric fluid (SGF): Take 23.4 mL of concentrated hydrochloric acid and add 100 mL of water to prepare dilute hydrochloric acid. Take 1.64 mL of the above dilute hydrochloric acid, add about 80 mL of water and 1.0 g of pepsin, mix well, and dilute with water to 100 mL.

[0104] Artificial simulated intestinal fluid (SIF): Take 0.68g of potassium dihydrogen phosphate, add 50mL of water to dissolve it, adjust the pH value to 6.8 with 0.1M sodium hydroxide solution, take 1.0g of pancreatic enzyme, add appropriate amount of water to dissolve it, mix the two, dilute with water to 100mL, shake well, and you have it.

[0105] Plasma: Blood was collected from the abdominal aorta of rats and centrifuged at 500 rpm for 5 min to obtain the upper plasma.

[0106] 1.5 Bioavailability

[0107] The oral bioavailability of bergenin-lipid conjugate lipid nanoparticles was evaluated by monitoring BG in rat plasma, with bergenin lipid nanoparticles as a control.

[0108] All animal study protocols were reviewed and approved by the Animal Care and Use Committee of the School of Pharmacy, Fudan University.

[0109] 2 Experimental results

[0110] 2.1 Determination of FA-BG lipid nanoparticle content

[0111] The drug contents in 6C-BG, 12C-BG and 18C-BG lipid nanoparticles were measured respectively, and the contents of 6C-BG, 12C-BG and 18C-BG in the three nanoparticles were calculated to be 8.92, 8.80 and 9.92 mg / mL respectively.

[0112] 2.2 Encapsulation efficiency and drug loading

[0113] As shown in Table 4, the encapsulation efficiency of 6C-BG, 12C-BG, and 18C-BG lipid nanoparticles was 86.4%, 88.0%, and 99.4%, respectively. The drug loading of the three groups of lipid nanoparticles was 21.6%, 22.0%, and 24.9%, respectively. The drug loading of the three prodrugs converted into the original drug BG was 16.6%, 14.1%, and 13.7%, respectively. The encapsulation efficiency and drug loading of bergenin lipid nanoparticles were 30.0% and 7.50%, respectively.

[0114] Table 4 Encapsulation efficiency and drug loading of 6C-BG, 12C-BG, and 18C-BG lipid nanoparticles

[0115]

[0116] 2.3 Particle size, Zeta potential and morphology observation

[0117] The particle size, PDI and Zeta potential of the three groups of FA-BG solid lipid nanoparticles are shown in Table 5; the average particle sizes of 6C-BG, 12C-BG and 18C-BG solid lipid nanoparticles were 71.99±4.27, 75.90±2.05, and 61.80±0.48 nm, respectively, and the PDIs were 0.082±0.033, 0.20±0.02, and 0.19±0.004, respectively, and the Zeta potentials were 3.02±0.36, -6.33±0.48, and -2.77±0.086 mV, respectively.

[0118] Table 5 Particle size, PDI and Zeta potential of three groups of nanoparticles

[0119]

[0120] The particle size distribution of 6C-BG, 12C-BG and 18C-BG solid lipid nanoparticles is as follows Figure 7 As shown, 6C-BG has the narrowest particle size distribution and the most uniform nanoparticle size. The surface potentials of the three FA-BG lipid nanoparticles are close to neutrality.

[0121] 2.4 In vitro stability and lipolysis

[0122] like Figure 8 As shown, among the four different media (SIF, SGF, PBS, plasma), the effects were most significant in SIF and plasma, with stronger stability in SGF than in PBS. Alkalinity is another trigger for BG-lipid hydrolysis; in SIF and plasma, 6C-BG was completely hydrolyzed from the first hour; while 12C-BG was more stable than 6C-BG, and the prodrug could not be detected in plasma after 4 hours. 18C-BG showed the highest stability due to its longest chain length and greatest steric hindrance, and it lasted for more than 12 hours even in SIF and plasma. It can be clearly observed that encapsulating lipid prodrugs in SLNs can further enhance their stability in different media. Traditional lipolysis studies performed by alkaline compensation further confirmed that all three BG-lipid SLNs could be completely degraded within 50 minutes. The longer the chain of the BG-lipid, the slower the degradation rate. At 10 minutes, about 6%, 7%, and 14% of SLNs remained in the 6C-, 12C-, and 18C-BG groups, respectively.

[0123] 2.5 Bioavailability

[0124] Table 6 and Fig. 9The pharmacokinetic parameters and plasma BG concentration-time curves of various SLNs with BG SLNs as reference are shown. The BG exposure level in the blood circulation of the control group (BG SLNs) was much lower than that of the other BG-lipid SLNs groups. max and C max showed an upward trend (T max 0.25, 0.42 and 0.5 hours respectively; C max The 12C- and 18C-BG SLNs had the longest circulation time in the blood and were still detectable after 24 hours, while the BG and 6C-BG groups were undetectable after 10 and 12 hours, respectively. The relative bioavailability (RBA) of 6C-, 12C-, and 18C-SLNs relative to BG SLNs was 239.87%, 864.05%, and 467.97%, respectively.

[0125] Table 6 Pharmacokinetic parameters of bergenin after oral administration of 6C-, 12C- and 18C-BG solid lipid nanoparticles (SLNs) and BG SLNs as control to rats (n=3).

[0126]

[0127] In summary, the present invention adopts the melt-high pressure homogenization method to prepare FA-BG lipid nanoparticles, the best lipid material is distearic acid glyceryl, the surfactant is 2% Tween 80, and the drug-lipid ratio is 1 / 3. According to the prescription, three groups of FA-BG lipid nanoparticles with smaller particle size, uniform distribution, highest encapsulation rate and drug loading, and best stability are prepared. The drug loading of the three groups of lipid nanoparticles converted into the original drug BG is 16.6%, 14.1%, and 13.7%, respectively, and the average particle size is 71.99±4.27, 75.90±2.05, and 61.80±0.48nm, respectively.

[0128] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A bergenin-lipid conjugate lipid nanoparticle, characterized in that: It includes a bergenin-lipid conjugate, a solid lipid material, and a surfactant; wherein the lipid-drug ratio is 1:2-10; the structural formula of the bergenin-lipid conjugate is as shown in Formula I:

2. The lipid nanoparticle according to claim 1, characterized in that The drug-lipid ratio is 1:

3.

3. The lipid nanoparticle according to claim 1, characterized in that The mass ratio of the surfactant to the bergenin-lipid conjugate is 500:90-325.

4. The lipid nanoparticle according to claim 1, characterized in that The solid lipid material comprises one or more of cholesterol oleate, distearin, trilaurin, monostearin, stearic acid, glyceryl palmitostearate and behenic acid glyceryl.

5. The lipid nanoparticle according to claim 4, characterized in that The solid lipid material is glyceryl distearate.

6. The lipid nanoparticle according to claim 1, characterized in that The surfactant includes one or more of phospholipids, Tween 80, Poloxamer 188, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, and surfactant OP.

7. The lipid nanoparticle according to claim 6, characterized in that The surfactant is Tween 80; further, it is 2% Tween 80.

8. The method for preparing the bergenin-lipid conjugate lipid nanoparticles according to claims 1-7, characterized in that: The steps include: (1) dropping the bergenin-lipid conjugate solution into the molten lipid material; (2) adding a surfactant; (3) Shearing to obtain colostrum; (4) microfluidization high pressure homogenization, and cooling the obtained suspension in an ice bath to obtain bergenin-lipid conjugate lipid nanoparticles.

9. The method according to claim 7, characterized in that: The shearing conditions are: 8000-12000psi, shearing for 2-5min.

10. The method according to claim 7, characterized in that The microfluidic high pressure homogenization is: 15000-35000psi, and is circulated 4-6 times.

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