Budesonide-loaded ginseng vesicle delivery system as well as construction method and application thereof

Abstract

The invention discloses a budesonide-loaded ginseng vesicle delivery system and a construction method and application thereof, and belongs to the technical field of plant-derived nano-drug delivery systems. According to the delivery system, nano-vesicles from ginseng are taken as carriers, and budesonide is entrapped to form a BUD-coated GDNPs compound. The delivery system can synergistically enhance the anti-inflammatory effect of budesonide, significantly alleviate airway inflammation of asthma model animals, reduce side effects such as blood glucose disorder and the like caused by high-dose budesonide, and has excellent biological safety. The delivery system provided by the invention can be used for clinical treatment of asthma, is especially suitable for patients who need to use glucocorticoid for a long time, solves the technical problems of low bioavailability and obvious side effects of traditional budesonide, and has important clinical application value and industrialization prospect.

Description

Technical Field

[0001] This invention relates to the field of plant-derived nanomedicine delivery systems, specifically to a ginseng vesicle delivery system loaded with budesonide, its construction method, and its application. Background Technology

[0002] Bronchial asthma is a common chronic respiratory disease characterized by airway inflammation, airway hyperresponsiveness, and reversible airflow limitation, and there is currently no complete cure. Inhaled corticosteroids (ICS) are the core medications for asthma treatment. Budesonide is widely used due to its significant anti-inflammatory effects, but long-term use has many limitations, such as systemic side effects including adrenal cortex suppression, osteoporosis, and blood sugar disorders, and local side effects such as oropharyngeal candidiasis. Furthermore, due to its poor water solubility, short half-life, and low bioavailability, high-dose administration is required, which exacerbates the risk of side effects.

[0003] Plant vesicles (PDNVs) are natural nanoparticles composed of lipid bilayers. They possess advantages such as good biocompatibility, non-immunogenicity, the ability to encapsulate both hydrophobic and hydrophilic drugs, and the ability to improve drug stability and solubility. Furthermore, they exhibit anti-inflammatory activity, making them a research hotspot for novel drug carriers. Ginseng, as a traditional Chinese medicine, possesses active ingredients (ginsenosides, polysaccharides, etc.) with various physiological activities including anti-inflammatory and immunomodulatory effects, showing great potential in the treatment of respiratory diseases. Currently, no novel nanodelivery systems using ginseng-derived nanovesicles (GDNPs) as carriers to encapsulate budesonide have been reported. Summary of the Invention

[0004] This invention provides a ginseng vesicle delivery system loaded with budesonide. By leveraging the carrier advantages of ginseng nanovesicles and the synergistic effect of budesonide's anti-inflammatory properties, it achieves enhanced efficacy and reduced side effects, aiming to solve the technical problems of low bioavailability and significant side effects in existing budesonide treatments for asthma.

[0005] The present invention achieves the above objectives through the following technical solutions: First, the present invention provides a ginseng vesicle delivery system loaded with budesonide, wherein the delivery system is a BUD@GDNPs complex formed by encapsulating budesonide in ginseng-derived nanovesicles as a carrier.

[0006] Second, the present invention provides the application of the above-mentioned ginseng vesicle delivery system loaded with budesonide in the preparation of drugs for treating bronchial asthma.

[0007] As a preferred embodiment of the present invention, the drug is administered by inhalation at a concentration of 0.1 mg / kg / 3 days.

[0008] As a preferred embodiment of the present invention, the drug is used to relieve airway inflammation and airway hyperresponsiveness caused by bronchial asthma.

[0009] Third, the present invention provides the application of the above-mentioned budesonide-loaded ginseng vesicle delivery system in the preparation of a drug to improve the side effects of glucocorticoids, wherein the glucocorticoid is budesonide, and the side effects caused by budesonide are manifested as blood sugar disorders, decreased bone density, and abnormal liver and kidney function.

[0010] As a preferred embodiment of the present invention, improving the side effects of budesonide specifically means improving the side effects of high-dose budesonide, wherein the concentration of high-dose budesonide is ≤2.15mg / kg / d.

[0011] Fourth, the present invention provides a method for constructing a ginseng vesicle delivery system loaded with budesonide, comprising the following steps: (1) Ginseng was crushed and added to PBS buffer to prepare plant extract. The extract was purified by differential centrifugation and sucrose density gradient ultracentrifugation. The target band was collected to obtain GDNPs. (2) Budesonide was dissolved in an organic solvent and mixed with GDNPs suspension. After ultrasonic-assisted and gradient extrusion, the free drug was removed by centrifugation to obtain the BUD@GDNPs delivery system.

[0012] As a preferred embodiment of the present invention, in step (1), ginseng is crushed and added to PBS buffer to prepare plant extract. The mass ratio of ginseng crushed material to PBS buffer is 1:5-10. The mixture is centrifuged at 10000×g at 4℃ and the supernatant is collected. The supernatant is transferred and subjected to differential centrifugation at 2000×g and 10000×g in sequence, followed by ultracentrifugation at 100000×g, and the precipitate is collected.

[0013] As a preferred embodiment of the present invention, in step (1), the sucrose density gradient ultracentrifugation purification is to spread the precipitate on 15%, 30%, 45%, and 60% sucrose gradients respectively, and then ultracentrifuge at 150,000 × g for 1-2 hours.

[0014] As a preferred embodiment of the present invention, in step (2), the concentration of budesonide dissolved in the organic solvent is 1-10 mg / mL, and the concentration of GDNPs suspension is 0.05-1 mg / mL; the organic solvent is DMSO; after budesonide is dissolved in the organic solvent, it is mixed with GDNPs suspension at a mass ratio of 1:2-5, sonicated at 35-50 kHz for 1-10 min, and passed through polycarbonate membranes with pore sizes of 2 μm, 800 nm, and 400 nm sequentially at 4 °C, repeated 10 times, to obtain the BUD@GDNPs delivery system.

[0015] The beneficial effects of this invention are as follows: (1) The anti-inflammatory activity of ginseng nanovesicles and the synergistic effect of budesonide can significantly reduce airway inflammation in asthma and improve the therapeutic effect even at low doses; (2) By targeting and protecting the nanocarrier, the systemic exposure of budesonide is reduced, the side effects such as blood glucose disorder and bone loss caused by high-dose drug use are improved, and the safety of drug use is enhanced. (3) Ginseng nanovesicles are naturally derived, non-immunogenic, and do not damage the liver and kidney function or tissues of animals, making them suitable for long-term administration; (4) The extraction and packaging process does not require complex equipment, and can achieve large-scale production, with good industrialization prospects. Attached Figure Description

[0016] Figure 1 A flowchart of the extraction process for ginseng-derived nanovesicles (GDNPs); Figure 2 Characterization of ginseng-derived nanovesicles; (A) After ultrafiltration and sucrose gradient density centrifugation, two layers of yellow cake-like material were visible. Characterization confirmed that the second layer was the target vesicle; (B) The particle size of GDNPs was 255 nm; (C) The potential was -28.5 mV; (E, F) Spherical cup-shaped structure; Figure 3 Toxicity testing of GDNPs and BUD@GDNPs: (A) Dosing regimens of GDNPs and BUD@GDNPs in mice; (BD) Changes in body weight and serological indicators of liver and kidney function in mice treated with GDNPs and BUD@GDNPs; (E) Representative images of lung tissue from each treatment group after H&E staining and PAS staining; (F) Representative images of heart, liver, spleen and kidney sections from each treatment group after hematoxylin-eosin staining. Figure 4 To demonstrate that GDNPs synergistically enhance the anti-inflammatory effect of low-dose budesonide: (A) Schematic diagram of indoor dust mite sensitization / challenge experiment and intranasal administration of GDNPs, budesonide (BUD), and BUD@GDNPs; (BD) Total cell count, eosinophil count, and lymphocyte count in bronchoalveolar lavage fluid (BALF) of each group; (EG) Detection of IL-4, IL-5, and IL-13 levels in BALF; (HJ) Detection of IL-4, IL-5, and IL-13 mRNA expression levels in lung tissue; (K) Representative images of lung sections (H&E staining and PAS staining) of each treatment group; Figure 5To demonstrate the reversal effect of BUD@GDNPs on airway remodeling and epithelial damage in chronic asthmatic mice; (A) Representative immunofluorescence images of epithelial connexins in lung sections: ZO-1 (red), E-cadherin (green), and cell nuclei reverse-stained with DAPI (blue); (BC) Quantification of connexin fluorescence intensity; (D) Western blot analysis of epithelial connexins (ZO-1 and E-cadherin) in lung tissue; (E) Masson staining of lung airways, and representative immunohistochemical (IHC) staining images of α-SMA and collagen I expression; Figure 6 To improve budesonide-induced glucose homeostasis and liver and kidney injury, BUD@GDNPs were used. (A) Schematic diagram of budesonide and BUD@GDNPs in mice; (B) Changes in body weight of mice in each group; (C) Fasting blood glucose levels after 16 hours of fasting in the budesonide group and the BUD@GDNPs group; (DE) In-process glucose tolerance test (IPGTT) and relative area under the curve (AUC) in the budesonide group and the BUD@GDNPs group; (F) Relative expression levels of Pck1 and G6pc in mouse liver; (GH) Serological indicators of liver and kidney function. Figure 7 (A) 3D and 2D representative images of mouse femur; (BE) Statistical analysis of bone tissue parameters; (F) Representative HE images of mouse bone tissue. Detailed Implementation

[0017] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0018] I. Materials 1.1 Extraction and Characterization of Ginseng Nanovesicles (GDNPs) (1) such as Figure 1 As shown, fresh ginseng was taken, washed, and pulverized using a tissue homogenizer. A homogenate was prepared by adding PBS buffer (pH=7.4) at a mass-to-volume ratio of 1:5. The homogenate was centrifuged at 10000×g for 15 min at 4℃, and the supernatant was collected. The supernatant was then transferred and subjected to differential centrifugation: sequentially at 2000×g and 10000×g to remove large particles, followed by ultracentrifugation at 100000×g at 4℃ for 40 min, and the precipitate was collected. The precipitate was resuspended in PBS and added to sucrose density gradient centrifuge tubes (15%, 30%, 45%, 60%, w / v). The tubes were ultracentrifuged at 150000×g for 2 h. The ginseng vesicle density was 1.13–1.19 g / mL, enriched at the 15-30% sucrose interface. Figure 2A), after removing sugar from the interface by ultrafiltration and passing it through a 0.45μm filter, high-purity GDNPs are obtained.

[0019] (2) According to DLS detection, the average particle size of GDNPs is 255 nm. Figure 2 B), the Zeta potential is -28.5mV ( Figure 2 C); TEM observation showed that GDNPs exhibited a spherical or near-spherical lipid bilayer structure with uniform particle size and good dispersibility. Figure 2 D, E).

[0020] 1.2 Construction of a Budesonide-Loaded Ginseng Vesicle Delivery System (BUD@GDNPs) (1) Accurately weigh 10 mg of budesonide (BUD), dissolve it in 1 mL of DMSO, and prepare a 10 mg / mL budesonide stock solution; (2) Take high-purity GDNPs to prepare a suspension (concentration 1 mg / mL), add budesonide stock solution, the mass ratio of budesonide stock solution to GDNPs suspension is 1:2, sonicate at 35 kHz for 1 min, 4℃; (3) The budesonide delivery system was obtained by passing the budesonide through polycarbonate membranes with pore sizes of 2μm, 800nm ​​and 400nm in sequence at 4℃, 10 times each. The budesonide loading rate was 35.2%, the particle size was 240-320nm, the PDI was <0.25, and the budesonide was embedded in the surface, inner and phospholipid bilayer of the vesicles. The ginseng vesicle surface protein activity retention rate was >85%.

[0021] 1.3 Experimental Mice After passing quarantine, male C57BL / 6J mice aged 6-8 weeks and weighing 18-22g were acclimatized for one week in an environment with a temperature of 22±2℃, humidity of 50%-70%, and a 12-hour light-dark cycle.

[0022] II. Methods 2.1 Biosafety Testing of the BUD@GDNPs Delivery System C57BL / 6J mice were randomly divided into a blank control group (Control), a BUD@GDNPs group, and a GDNPs group, with 3 mice in each group. The BUD@GDNPs group received BUD@GDNPs suspension at a dose of 0.1 mg / kg, while the control group received an equal volume of PBS. Administration was once daily for 28 consecutive days. Figure 3 A). Mice were weighed starting from day 1, and sacrificed after 28 days. Specimens were collected for H&E, PAS staining, and other tests.

[0023] The results showed that there was no significant difference in body weight change among the three groups of mice over 28 days (P>0.05). Figure 3B); Liver and kidney function indicators (CREA, BUN) were all within the normal range. Figure 3 CD); HE staining of the heart, liver, spleen, kidneys, and lung tissue showed no abnormalities ( Figure 3 (EF), indicating that BUD@GDNPs have good biocompatibility.

[0024] 2.2 Anti-inflammatory effect test of BUD@GDNPs delivery system Twenty C57BL / 6J mice from the same batch were divided into five groups. The control group (Mice) received a normal diet without any treatment. The remaining groups were given HDM extract to induce a mouse asthma model. HDM was administered via intraperitoneal injection on days 0 and 7, and then via nasal drops daily starting on day 8. The treatment regimen was as follows: once every three days from day 8 until day 21. Figure 4 A)

[0025] The HDM-induced mouse asthma model was established. After the adaptation period, the mice were observed daily. During the modeling period, the main symptoms of HDM mice were rapid breathing and nasal flaring, which worsened after activity. In severe cases, paradoxical chest and abdominal movements were observed. After dissection, HE staining showed a large number of inflammatory cells infiltrating the peribronchial and pulmonary interstitial tissues, mainly eosinophils and lymphocytes. PAS staining showed airway epithelial cell shedding and disorder, goblet cell proliferation, increased mucus secretion and airway obstruction. MASSON staining showed airway wall thickening, collagen fiber deposition, and airway smooth muscle hyperplasia. These findings were consistent with the description of an HDM-induced mouse asthma model, indicating that the experimental modeling was successful.

[0026] On day 22, medication was stopped in each treatment group, mice were sacrificed, serum was extracted, and a portion was used for subsequent routine blood tests. Mice were anesthetized and sacrificed, and lung tissue, bronchoalveolar lavage fluid, and other specimens were collected for HE, PAS staining, MASSON and other tests.

[0027] The results showed that HE, PAS, and MASSON staining of lung tissue from mice in the BUD@GDNPs group significantly reduced airway inflammation. Figure 4 H, Figure 5 E), the total cell count, eosinophil count, and lymphocyte count in the bronchoalveolar lavage fluid were significantly lower than those in the model group and the BUD group (P<0.05). Figure 4 BD); IL-4, IL-5, and IL-13 mRNA expression levels were significantly downregulated (P<0.05) Figure 4 EG); ZO-1 and E-cadherin protein expression levels were significantly higher in the BUD group than in the BUD group (P<0.05). Figure 5This indicates that BUD@GDNPs can synergistically enhance the anti-inflammatory effect of budesonide and repair the airway barrier.

[0028] It can be concluded that ginseng vesicles encapsulating budesonide (BUD@GDNPs) can alleviate airway inflammation in HDM-induced asthmatic mice.

[0029] 2.3 The effect of the BUD@GDNPs delivery system on improving the side effects of high-dose budesonide Twelve C57BL / 6J mice were randomly divided into a control group, a high-dose BUD group, and a high-dose BUD@GDNPs group, with four mice in each group. The high-dose BUD group received budesonide at a dose of 2.15 mg / kg, the high-dose BUD@GDNPs group received budesonide at a dose of 2.15 mg / kg, and the control group received an equal volume of PBS. Administration was once daily for 56 consecutive days. Figure 6 A). Mice were weighed starting from day 1, and sacrificed after 56 days. Specimens were collected for subsequent measurements of blood glucose, IPGTT, Pck1 and G6pc, and tibia and femur micro-CT.

[0030] The results showed that the body weight of mice in the high-dose BUD group gradually decreased. Figure 6 B), fasting blood glucose levels were significantly elevated in mice in the high-dose BUD group (B), Figure 6 C), the area under the IPGTT curve increases ( Figure 6 DE, upregulation of liver gluconeogenesis markers Pck1 and G6pc mRNA expression ( Figure 6 F); while the high-dose BUD@GDNPs group showed significant improvement in liver and kidney function indicators (P<0.05). Figure 6 GH), and femoral micro-CT scan showed no significant decrease in bone mineral density (GH). Figure 7 This indicates that GDNPs can effectively reduce the side effects of high-dose budesonide.

[0031] It can be concluded that ginseng vesicles encapsulating high doses (2.15 mg / kg) of budesonide (BUD@GDNPs) can improve the side effects caused by budesonide, including glycemic disorders, decreased bone density, and abnormal liver and kidney function.

[0032] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A ginseng vesicular delivery system loaded with budesonide, characterized in that, The delivery system is a BUD@GDNPs complex formed by nanovesicles from ginseng as carriers, loading budesonide.

2. The ginseng nanovesicle delivery system loading budesonide of claim 1 in the preparation of a drug for treating bronchial asthma.

3. Use according to claim 2, characterized in that, The drug is administered by inhalation, and the administration concentration is 0.1 mg / kg / 3d.

4. Use according to claim 2, characterized in that, The drug is used to reduce airway inflammation and airway hyperresponsiveness caused by bronchial asthma.

5. Use of the budesonide-loaded ginseng vesicular delivery system according to claim 1 in the preparation of a drug for improving side effects of glucocorticoids, wherein the drug is a drug for improving side effects of glucocorticoids. The glucocorticoid is budesonide, and the side effects caused by the budesonide are blood glucose disorder, decreased bone mineral density, and abnormal liver and kidney function.

6. Use according to claim 5, characterized in that, Improving the side effects of budesonide specifically refers to improving the side effects of high-dose budesonide, and the concentration of the high-dose budesonide is ≤2.15 mg / kg / d.

7. The method for constructing the budesonide-loaded ginseng vesicle delivery system according to claim 1, wherein, The method comprises the following steps: (1) crushing ginseng and adding PBS buffer to prepare a plant extract, and then sequentially performing differential centrifugation and sucrose density gradient ultracentrifugation purification to collect a target band to obtain GDNPs; (2) dissolving budesonide in an organic solvent, mixing the budesonide with a GDNPs suspension, and then performing ultrasonic assistance and gradient extrusion, and then removing free drugs by centrifugation to obtain a BUD@GDNPs delivery system.

8. The method of constructing the budesonide-loaded ginseng vesicle delivery system according to claim 7, wherein, In step (1), the crushed ginseng is added to PBS buffer to prepare a plant extract, and the mass ratio of the crushed ginseng to the PBS buffer is 1:5-10, and the supernatant is obtained by centrifugation at 10000×g at 4°C; the supernatant is transferred and sequentially subjected to differential centrifugation at 2000×g and 10000×g, and then subjected to ultracentrifugation at 100000×g to collect the precipitate.

9. The method of constructing the budesonide-loaded ginseng vesicular delivery system according to claim 7, wherein, In step (1), the sucrose density gradient ultracentrifugation purification is that the precipitate is respectively laid on 15%, 30%, 45%, and 60% sucrose gradients, and ultracentrifugation is performed at 150000×g for 1-2h.

10. The method of constructing the budesonide-loaded ginseng vesicle delivery system according to claim 7, wherein, In step (2), the concentration of the budesonide dissolved in the organic solvent is 1-10 mg / mL, and the concentration of the GDNPs suspension is 0.05-1 mg / mL; The organic solvent is DMSO; The budesonide dissolved in the organic solvent is mixed with the GDNPs suspension at a mass ratio of 1:2-5, ultrasonic treatment is performed at 35-50 kHz for 1-10 min, and the BUD@GDNPs delivery system is obtained by sequentially passing through polycarbonate membranes with pore sizes of 2 μm, 800 nm, and 400 nm at 4°C for 10 times.

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