Isoliquiritigenin-plga nanoparticles, preparation method and application thereof

Abstract

The application provides a nano particle of isoliquiritigenin-PLGA, which is prepared by using an emulsification-solvent evaporation method with isoliquiritigenin and PLGA at a mass ratio of 1:12-18. The application also discloses a preparation method and application of the nano particle. The emulsification-solvent evaporation method is simple in process, has no pollution to the environment, and is suitable for large-scale production. The PLGA raw material used in the application has good biocompatibility, no toxicity, and good performance of capsule formation and film formation. The nano particle of isoliquiritigenin-PLGA prepared by the application enhances the solubility of isoliquiritigenin, improves the bioavailability and pharmacological activity of the drug, and enhances the protective effect on diabetic nephropathy.

Description

(I) Technical Field

[0001] This invention relates to isoliquiritigenin-PLGA nanoparticles, their preparation method, and applications, belonging to the field of pharmaceutical preparations. (II) Background Technology

[0002] Chronic kidney disease (CKD) is a public health problem causing serious morbidity and mortality. Diabetic nephropathy (DKD) is the leading cause of CKD, accounting for approximately half of the burden of end-stage renal disease (ESRD) in developed countries in the United States and many other developed and developing regions. In 2015, an estimated 415 million people worldwide had diabetes, and 5 million died from the disease, resulting in global health expenditures of US$673 billion. By 2040, the prevalence of diabetes is projected to increase to 642 million, with 30-40% of these individuals developing DKD.

[0003] Isoliquiritigenin (ISL) is a natural flavonoid compound isolated from licorice root. It is a functional food and medicinal plant widely used in Asia and Africa. The pharmacological and biological activities of isoliquiritigenin include anti-inflammatory, antioxidant, antibacterial, and anticancer effects. Recent studies have demonstrated that ISL can prevent diabetes-related diseases such as insulin sensitivity, diabetic neuropathy, and adipose tissue inflammation. However, isoliquiritigenin is a weak acid and is only soluble in less polar solvents such as ether and chloroform. This poor water solubility limits the effective development and utilization of its formulations. (III) Summary of the Invention

[0004] The technical objective of this invention is to provide isoliquiritigenin-PLGA nanoparticles and their preparation method, thereby enhancing the solubility of isoliquiritigenin and improving its bioavailability.

[0005] The technical solution adopted in this invention is:

[0006] An isoliquiritigenin-PLGA nanoparticle is prepared by emulsification-solvent evaporation method using isoliquiritigenin and PLGA at a mass ratio of 1:12-18, wherein the PLGA is a polylactic acid-glycolic acid copolymer.

[0007] The PLGA is PLGA 75:25, PLGA 60:40 or PLGA 50:50, preferably PLGA 75:25;

[0008] In the PLGA 75:25, PLGA 60:40, or PLGA 50:50 designations, the molar ratio of polylactic acid to glycolic acid in the PLGA is 75:25, 60:40, or 50:50, respectively. This is a well-known PLGA designation method in the art.

[0009] Furthermore, the mass ratio of isoliquiritigenin to PLGA is 1:12 to 1:18, preferably 1:15.

[0010] The isoliquiritigenin-PLGA nanoparticles of this invention can be prepared by the following method:

[0011] (1) Dissolve the drug isoliquiritin and PLGA in dichloromethane until they are completely dissolved to obtain an isoliquiritin-PLGA mixture;

[0012] (2) Add polyvinyl alcohol aqueous solution to isoliquiritigenin-PLGA mixture and then sonicate to obtain emulsion;

[0013] (3) The ultrasonically treated emulsion was added to a large amount of polyvinyl alcohol aqueous solution, stirred to evaporate the organic solvent, centrifuged, and freeze-dried to obtain isoliquiritigenin-PLGA nanoparticles.

[0014] This invention also provides a method for preparing the isoliquiritigenin-PLGA nanoparticles, the method comprising the following steps:

[0015] (1) Dissolve the drug isoliquiritin and polylactic acid-glycolic acid copolymer (PLGA) in dichloromethane until they are completely dissolved to obtain isoliquiritin-PLGA mixture;

[0016] (2) Add polyvinyl alcohol (PVA) aqueous solution to isoliquiritigenin-PLGA mixture and sonicate to obtain emulsion (O / W);

[0017] (3) The ultrasonically treated emulsion was added to a large amount of polyvinyl alcohol aqueous solution, stirred to evaporate the organic solvent, centrifuged, and freeze-dried to obtain isoliquiritigenin-PLGA nanoparticles.

[0018] Furthermore, in step (1), the volume of dichloromethane used is generally 50-200 μL / mg based on the mass of isoglycyrrhizin, preferably 100 μL / mg.

[0019] In step (2), the polyvinyl alcohol aqueous solution refers to an aqueous solution with a PVA concentration of 2-3%.

[0020] Furthermore, the volume ratio of the polyvinyl alcohol aqueous solution to the dichloromethane used in step (1) is 1:4 to 5.

[0021] Furthermore, the ultrasound time in step (2) is preferably 30 to 60 seconds.

[0022] In step (3), the polyvinyl alcohol aqueous solution refers to an aqueous solution with a PVA concentration of 2-3%.

[0023] Furthermore, the volume ratio of the polyvinyl alcohol aqueous solution to the ultrasonically emulsion in step (3) is preferably 10-11:1;

[0024] The stirring time in step (3) is generally 4 to 6 hours; the stirring is generally done at a low speed, with a stirring rate of 400 to 500 r / min.

[0025] In step (3), the centrifugation speed is generally 4000-5000 rpm and the centrifugation time is generally 10-20 min.

[0026] In step (3), after centrifugation, the microspheres can be washed with water, collected, and freeze-dried to obtain isoliquiritigenin-PLGA nanoparticles.

[0027] This invention also provides the application of glycyrrhizin-PLGA nanoparticles in the preparation of antidiabetic drugs. Furthermore, they can be used to prepare antidiabetic nephropathy drugs.

[0028] The method for preparing isoliquiritigenin-PLGA nanoparticles provided by this invention employs an emulsification-solvent evaporation method. In this method, poorly soluble isoliquiritigenin and PLGA are dissolved in dichloromethane to form an organic phase. A small amount of PVA aqueous solution is added and ultrasonically emulsified (O / W). The emulsion is then added to a large amount of PVA aqueous solution and stirred at low speed to remove the organic solvent. The microspheres are then centrifuged, washed, collected, and freeze-dried to obtain isoliquiritigenin-PLGA nanoparticles.

[0029] The beneficial effects of this invention are mainly reflected in:

[0030] (1) The present invention adopts the emulsification-solvent evaporation method, which is relatively simple, does not pollute the environment, and is suitable for large-scale production.

[0031] (2) The poly(lactic-co-glycolic acid) copolymer (PLGA) used in this invention has good biocompatibility, is non-toxic, and has good encapsulation and film-forming properties.

[0032] (3) The present invention prepared isoliquiritin-PLGA nanoparticles, thereby enhancing the solubility of isoliquiritin, improving its bioavailability and pharmacological activity, and enhancing its protective effect against diabetic nephropathy. (iv) Description of the attached drawings

[0033] Figure 1 The in vitro dissolution curves of free isoliquiritigenin and isoliquiritigenin-PLGA nanoparticles are shown.

[0034] Figure 2 The pharmacokinetic curves are shown for male rats (200±20g) after oral administration of isoliquiritigenin nanoparticle solution and isoliquiritigenin solution (both administered at a dose of 100mg / kg isoliquiritigenin).

[0035] Figure 3 These are pathological photographs of kidney tissue sections from diabetic nephropathy mice after oral administration of isoliquiritigenin and isoliquiritigenin-PLGA nanoparticles, respectively. (V) Detailed Implementation

[0036] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are all commercially available.

[0037] Example 1

[0038] Isoliquiritin (2 mg) and PLGA 75:25 (30 mg) at a mass ratio of 1:15 were dissolved in 200 μL of dichloromethane to form an organic phase. The organic phase was then added dropwise to 800 μL of 2% PVA aqueous solution, followed by sonication for 30 s to form an O / W emulsion. The emulsion was then added dropwise to 10 mL of 2% PVA aqueous solution, and stirred at low speed for 4 h to remove the organic solvent, thus obtaining an isoliquiritin-PLGA nanoparticle suspension. The suspension was centrifuged at 4000 rpm for 15 min, washed three times with distilled water, and the nanoparticles were collected and freeze-dried to obtain isoliquiritin-PLGA nanoparticle lyophilized powder.

[0039] Example 2

[0040] Isoliquiritin (2 mg) and PLGA 60:40 (30 mg) at a mass ratio of 1:15 were dissolved in 200 μL of dichloromethane to form an organic phase. The organic phase was then added dropwise to 800 μL of 2% PVA aqueous solution, followed by sonication for 30 s to form an O / W emulsion. The emulsion was then added dropwise to 10 mL of 2% PVA aqueous solution, and the mixture was stirred at low speed for 4 h to remove the organic solvent, thus obtaining an isoliquiritin-PLGA nanoparticle suspension. The suspension was centrifuged at 4000 rpm for 15 min, washed three times with distilled water, and the nanoparticles were collected and freeze-dried to obtain isoliquiritin-PLGA nanoparticle lyophilized powder.

[0041] Example 3

[0042] Isoliquiritin (2 mg) and PLGA (50:50, 30 mg) at a mass ratio of 1:15 were dissolved in 200 μL of dichloromethane to form an organic phase. The organic phase was then added dropwise to 800 μL of 2% PVA aqueous solution, followed by sonication for 30 s to form an O / W emulsion. The emulsion was then added dropwise to 10 mL of 2% PVA aqueous solution, and the mixture was stirred at low speed for 4 h to remove the organic solvent, thus obtaining an isoliquiritin-PLGA nanoparticle suspension. The suspension was centrifuged at 4000 rpm for 15 min, washed three times with distilled water, and the nanoparticles were collected and freeze-dried to obtain isoliquiritin-PLGA nanoparticle lyophilized powder.

[0043] Example 4:

[0044] The preparation method of isoliquiritigenin-PLGA nanoparticles includes the following steps:

[0045] Isoliquiritin (2 mg) and PLGA 75:25 (24 mg) at a mass ratio of 1:12 were dissolved in 200 μL of dichloromethane to form an organic phase. The organic phase was then added dropwise to 800 μL of 2% PVA aqueous solution, followed by sonication for 30 s to form an O / W emulsion. The emulsion was then added dropwise to 10 mL of 2% PVA aqueous solution, and stirred at low speed for 4 h to remove the organic solvent, thus obtaining an isoliquiritin-PLGA nanoparticle suspension. The suspension was centrifuged at 4000 rpm for 15 min, washed three times with distilled water, and the nanoparticles were collected and freeze-dried to obtain isoliquiritin-PLGA nanoparticle lyophilized powder.

[0046] Example 5:

[0047] Isoliquiritin (2 mg) and PLGA 75:25 (30 mg) at a mass ratio of 1:15 were dissolved in 200 μL of dichloromethane to form an organic phase. The organic phase was then added dropwise to 800 μL of 2% PVA aqueous solution, followed by sonication for 30 s to form an O / W emulsion. The emulsion was then added dropwise to 10 mL of 2% PVA aqueous solution, and stirred at low speed for 4 h to remove the organic solvent, thus obtaining an isoliquiritin-PLGA nanoparticle suspension. The suspension was centrifuged at 4000 rpm for 15 min, washed three times with distilled water, and the nanoparticles were collected and freeze-dried to obtain isoliquiritin-PLGA nanoparticle lyophilized powder.

[0048] Example 6:

[0049] Isoliquiritin (2 mg) and PLGA 75:25 (36 mg) at a mass ratio of 1:18 were dissolved in 200 μL of dichloromethane to form an organic phase. The organic phase was then added dropwise to 800 μL of 2% PVA aqueous solution, followed by sonication for 30 s to form an O / W emulsion. The emulsion was then added dropwise to 10 mL of 2% PVA aqueous solution, and the mixture was stirred at low speed for 4 h to remove the organic solvent, thus obtaining an isoliquiritin-PLGA nanoparticle suspension. The suspension was centrifuged at 4000 rpm for 15 min, washed three times with distilled water, and the nanoparticles were collected and freeze-dried to obtain isoliquiritin-PLGA nanoparticle lyophilized powder.

[0050] Example 7

[0051] Performance testing: Characterization of isoliquiritigenin-PLGA nanoparticles.

[0052] Encapsulation efficiency and drug loading determination:

[0053] From Examples 1-6, accurately measure 2.0 ml of isoliquiritigenin-PLGA nanoparticle suspension into an ultrafiltration centrifuge tube, centrifuge at 12500 rpm / min for 60 min, take an appropriate amount of the filtrate, and first determine the free isoliquiritigenin content (m2) using HPLC; accurately measure 2.0 ml of the nanoparticle suspension into a 50 ml volumetric flask, add acetonitrile to demulsify, dilute to volume with the mobile phase, filter through a 0.45 μm microporous membrane, take 2 ml of the filtrate into a 10 ml volumetric flask, dilute to volume with the mobile phase, and determine the total isoliquiritigenin content (m1). Encapsulation efficiency = [(m1-m2) / m1] × 100%, drug loading = [(m1-m2) / m1] × 100%. 总 ]×100%, where m 总 The total mass of isoglycyrrhizin and PLGA is given. After diluting the nano-suspension to 1 mg / mL, the particle size, PDI, and zeta potential were measured using a laser particle size analyzer (DLS).

[0054] Table 1 shows the effects of different PLGA types on the characterization of isoliquiritigenin-PLGA nanoparticles (encapsulation efficiency, drug loading, particle size, PDI, and Zeta potential).

[0055] Table 1

[0056]

[0057] Table 2 shows the effect of the mass ratio of isoliquiritigenin to PLGA on the characterization of isoliquiritigenin-PLGA nanoparticles.

[0058] Table 2

[0059]

[0060] Table 1 shows that when the PLGA ratio is 75:25, the encapsulation efficiency, drug loading, particle size, PDI, and Zeta potential are all superior to those of the 60:40 and 50:50 PLGA ratios. Table 2 shows that when the dosage of isoliquiritin is fixed at 2 mg, changing the ratio of isoliquiritin to PLGA to 1:15 results in superior encapsulation efficiency, drug loading, particle size, PDI, and Zeta potential compared to other ratios.

[0061] Example 8: In vitro drug release study:

[0062] Prepare 0.75% sodium dodecyl sulfate (SDS) and sonicate for 10 min as the dissolution medium. Take an appropriate amount of isoliquiritin-PLGA nanoparticle lyophilized powder, add 2 ml of dissolution medium, shake, and place in a dialysis bag (isoglycyrrhizin content 20 mg). Tie both ends of the dialysis bag tightly. Set the dissolution medium volume to 900 ml, the rotation speed to 100 rpm / min, and the temperature to 37 ± 1℃. Take a 3 ml sample at the set time point, and add another 3 ml of dissolution medium simultaneously. Calculate the drug release and plot the in vitro drug release curve. Plot the in vitro drug release curve of isoliquiritin raw material using the same method. The results are shown in [Figure Number]. Figure 1 .

[0063] from Figure 1 It can be seen that the cumulative release rate of isoliquiritin-PLGA nanoparticles at each time point is higher than that of the raw drug isoliquiritin, reaching 81.46% within 72 hours.

[0064] Example 9: Pharmacokinetic Study

[0065] Male rats (200±20g) were orally administered isoliquiritin nanoparticles and isoliquiritin suspension (both at a dose of 100mg / kg isoliquiritin). At 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, and 12 hours post-administration, rats were anesthetized with ether. 0.3mL of blood was collected from the fundus venous plexus and transferred to centrifuge tubes moistened with 1% heparin sodium solution. The samples were centrifuged at 4500rpm / min for 10min, and the supernatant plasma was collected and stored at -20℃. After thawing naturally at room temperature, 100μL of the frozen plasma sample was accurately measured and 2.5μg·mL⁻¹ was added. -1 40 μL of internal standard working solution was added, followed by 60 μL of methanol. The mixture was vortexed for 1 min, centrifuged at 12000 rpm for 10 min, and 20 μL of the supernatant was injected to detect the drug peak and internal standard peak. The results were substituted into the regression equation to calculate the blood drug concentration, and the drug-time curve was obtained. (See figure) Figure 2 The pharmacokinetic parameters are shown in Table 3. The isoliquiritigenin suspension was prepared by adding isoliquiritigenin to 3 mL of distilled water and shaking well.

[0066] Table 3: Summary Table of Pharmacokinetic Trial Parameters

[0067]

[0068] C max Peak concentration T at peak time max Time to peak blood drug concentration

[0069] T 1 / 2 Blood concentration half-life AUC 0→t Area of ​​the plasma drug concentration-time curve at each time point

[0070] AUC 0→∞ Area of ​​the plasma drug concentration-time curve

[0071] As can be seen from Table 3, the area under the plasma drug concentration-time curve (AUC), i.e. bioavailability, of the isoliquiritigenin-PLGA nanoparticles prepared in this invention is significantly improved.

[0072] from Figure 2 It can be seen that at each time point, the therapeutic blood concentration of isoliquiritigenin-PLGA nanoparticles was higher than that of the raw drug, indicating that it is easier to achieve therapeutic concentrations after being made into nanoparticles.

[0073] Therefore, oral administration of isoliquiritigenin nanoparticles can significantly promote the absorption of isoliquiritigenin, increase the blood concentration of isoliquiritigenin, and improve the bioavailability of the drug.

[0074] Example 10: Experiment on the treatment of diabetic nephropathy with isoliquiritigenin-PLGA nanoparticles:

[0075] Mice were randomly divided into two groups: db / db mice treated with isoliquiritigenin (db / db-ISL, n=7) and db / db mice treated with isoliquiritigenin-PLGA nanoparticles (db / db-ISL-PLGA, n=7). The db / db-ISL and db / db-ISL-PLGA groups were administered orally at 20 mg / kg ISL solution or ISL-PLGA solution every two days. The treatment lasted 12 weeks, and all animals were sacrificed after anesthesia. Kidney tissue was collected and preserved at -80°C for further analysis. Kidney tissue was fixed in 4% paraformaldehyde solution, embedded in paraffin, and sectioned into 5 μm sections. The sections were then dehydrated and stained with HE and PAS. To assess histopathological damage, images of the sections were captured and examined under an optical microscope (×400 magnification; Nikon, Japan). After dewaxing and rehydration, paraffin sections (5 μm) of the kidney specimens were stained with Sirius Red according to the manufacturer's instructions to assess type IV collagen deposition. Results are shown in [Figure number missing]. Figure 3 .

[0076] The early pathological change in diabetic nephropathy is mesangial expansion, mainly due to extracellular matrix deposition and mesangial cell proliferation. There is a widespread increase in mesangial matrix in the glomeruli, which is eosinophilic and PAS-positive. Pathologically, this manifests as glomerular mesangial cell proliferation, basement membrane thickening, and glomerular mesangial expansion. As the disease progresses, renal fibrosis occurs.

[0077] from Figure 3HE staining showed that isoliquiritigenin-PLGA nanoparticle treatment inhibited mesangial cell and matrix proliferation, with better efficacy than the raw material isoliquiritigenin. PAS staining showed fewer PAS-positive areas after isoliquiritigenin-PLGA nanoparticle treatment. Sirius Red staining showed fewer collagen fibers stained red in the isoliquiritigenin-PLGA nanoparticle treatment group.

[0078] Therefore, the experiment showed that isoliquiritigenin-PLGA nanoparticles had a significant protective effect against diabetic nephropathy in mice after oral administration.

[0079] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing isoliquiritigenin-PLGA nanoparticles, characterized in that, The method includes the following steps: (1) Dissolve the drug isoliquiritin and PLGA in dichloromethane until they are completely dissolved to obtain an isoliquiritin-PLGA mixture. (2) Add polyvinyl alcohol aqueous solution to isoliquiritigenin-PLGA mixture and sonicate to obtain emulsion; (3) The ultrasonically treated emulsion was added to a large amount of polyvinyl alcohol aqueous solution, stirred to evaporate the organic solvent, centrifuged, and freeze-dried to obtain isoliquiritigen-PLGA nanoparticles. In step (1), the PLGA is PLGA 75:25, the mass ratio of isoliquiritigenin to PLGA is 1:15, and the volume of dichloromethane used is 50~200μL / mg based on the mass of isoliquiritigenin. In step (2), the polyvinyl alcohol aqueous solution refers to an aqueous solution with a PVA concentration of 2-3%, the volume ratio of the polyvinyl alcohol aqueous solution to the dichloromethane is 1:(4-5), and the volume ratio of the polyvinyl alcohol aqueous solution to the ultrasonically emulsion is (10-11):

1.

2. The application of isoliquiritigenin-PLGA nanoparticles prepared by the method according to claim 1 in the preparation of antidiabetic drugs.

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