Synthesis method of mangiferin-loaded lipidosome based on microfluidic technology, mangiferin-loaded lipidosome and application of mangiferin-loaded lipidosome

Abstract

The invention relates to the technical field of biological medicines, in particular to a synthesis method of a mangiferin-loaded liposome based on a microfluidic technology, the mangiferin-loaded liposome and application of the mangiferin-loaded liposome. The mangiferin-loaded liposome is synthesized by taking a mangiferin aqueous solution and a phospholipid-cholesterol organic solution as raw materials and using a micro-reactor with a micro-fluidic chip, so that high-flux and controllable synthesis of the liposome is realized, and the mangiferin-loaded liposome is suitable for industrial production and plays a positive role in treatment of arthritis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a synthesis method of mangiferin-loaded liposome based on microfluidic technology, mangiferin-loaded liposome and application. BACKGROUND

[0002] Osteoarthritis (OA) is a common degenerative joint disease, mainly characterized by joint cartilage degeneration, synovial inflammation and bone hyperplasia, leading to pain, swelling and joint dysfunction. Currently, the main methods for treating osteoarthritis in clinic include non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoid injection and joint cavity lubricant, but these methods have problems such as large side effects, limited efficacy or repeated administration.

[0003] Mangiferin: a natural polyphenolic compound with significant anti-inflammatory, antioxidant and cartilage protection effects. However, mangiferin has poor water solubility and low bioavailability, which limits its clinical application. Liposome: as an excellent drug delivery system, it can improve the solubility, stability and targeting of drugs, but the traditional liposome preparation methods (such as film hydration method, reverse evaporation method) have problems such as large batch-to-batch difference, low encapsulation efficiency and difficulty in large-scale production. Microfluidic technology: can precisely control the fluid mixing process, realize the continuous and controllable synthesis of liposomes, and improve the encapsulation efficiency and batch consistency. Therefore, the present application proposes a preparation method of mangiferin-loaded liposome based on microfluidic technology, which is used for the treatment of osteoarthritis.

[0004] However, the above methods have different defects: mangiferin has poor water solubility and low bioavailability, which affects its application in the treatment of osteoarthritis; traditional liposome preparation methods have large batch-to-batch difference and low encapsulation efficiency, which cannot meet the needs of industrial production; existing osteoarthritis treatment drugs have large side effects and limited efficacy, and there is an urgent need for safer and more effective delivery systems. SUMMARY

[0005] To solve the problems in the above background art, the purpose of the present application is to provide a synthesis method of mangiferin-loaded liposome based on microfluidic technology, mangiferin-loaded liposome and application, which is expected to solve the problem of preparing mangiferin as a drug for treating osteoarthritis.

[0006] The technical scheme is specifically as follows:

[0007] On the one hand, the present application provides a synthesis method of mangiferin-loaded liposome based on microfluidic technology, which comprises the following steps:

[0008] S100, taking solution A, the solution A being a mangiferin aqueous solution;

[0009] S200, taking solution B, which is a phospholipid-cholesterol organic solution;

[0010] S300, using a microreactor with a microfluidic chip to synthesize, the microreactor having at least three inlet ends, the solution A entering the microreactor through at least two of the inlet ends, the solution B entering the microreactor through at least one of the inlet ends to react to obtain a liposome solution, thereby obtaining a mangiferin-loaded liposome;

[0011] The flow rate of the solution A is 0.1-100 mL / min, and the flow rate ratio of the solution A to the solution B is 1-50:1.

[0012] As a preferred technical solution of the present application, the preparation method of the solution A is to dissolve mangiferin in water with a concentration of 0.1-100 mg / mL, heat to 35-95°C, and stir or ultrasonically treat for at least 1 min.

[0013] As a preferred technical solution of the present application, the preparation method of the solution B is to dissolve one or more of the phospholipid substances in anhydrous alcohol with a concentration of 0.1-200 mg / mL, selectively add cholesterols with a concentration of 0-80 mg / mL, heat to 35-80°C, and stir or ultrasonically treat for at least 1 min to obtain a mangiferin-loaded liposome.

[0014] As a preferred technical solution of the present application, in the step S300, the liposome solution is treated by ultrasonication for at least 1 min, and then one or more of the following steps is performed to obtain a mangiferin-loaded liposome: centrifugation, filtration, and freeze-drying.

[0015] As a preferred technical solution of the present application, the phospholipid substance is selected from one or more of the following: phosphatidylcholine (PC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and dipalmitoyl phosphatidylcholine (DPPC).

[0016] As a preferred technical solution of the present application, the microreactor has a spatial spiral microstructure.

[0017] As a preferred technical solution of the present application, the solution A enters the microreactor through two of the inlet ends, and the solution B enters the microreactor through one of the inlet ends.

[0018] As a preferred technical solution of the present application, the flow rate of the solution A is 0.1-100 mL / min, and the preferred flow rate ratio of the solution A to the solution B is 5-20:1.

[0019] In another aspect, the present application provides a mangiferin-loaded liposome prepared by the above method.

[0020] In still another aspect, the application provides the use of the mangiferin-loaded liposome as described above in the preparation of a medicament for treating osteoarthritis.

[0021] By adopting the technical scheme described above, the application has the following beneficial effects:

[0022] The mangiferin is encapsulated into liposomes, thereby enhancing the anti-inflammatory and cartilage protection effects and improving the therapeutic effect. In addition, the liposomes can enhance the solubility and joint targeting of mangiferin, improve the drug delivery efficiency, and the microfluidic technology can realize high-throughput and controllable synthesis of the liposomes, which is suitable for industrial production. Compared with the traditional NSAIDs and hormone therapy, the mangiferin-loaded liposomes have better safety and sustained-release effect.

[0023] The technical scheme of the application will be further described in detail below through specific embodiments and examples. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Fig. 1 is a schematic diagram of the microfluidic synthesis process of the mangiferin-loaded liposome.

[0025] Figure 2 Fig. 2 is a transmission electron microscope (TEM) image of the mangiferin-loaded liposome, showing the particle size and morphology thereof.

[0026] Figure 3 Fig. 3 is a Micro-CT scanning 3D reconstruction model.

[0027] Figure 4 Fig. 4 is the HE staining and Ponceau O- fast green staining results. DETAILED DESCRIPTION

[0028] The technical scheme of the application will be further described in detail below through specific embodiments and examples.

[0029] Embodiment 1

[0030] As shown in Figure 1 , 50 mg of mangiferin was accurately weighed and dissolved in 10 mL of pure water, and then ultrasonically treated at 60℃ for 10 minutes.

[0031] POPC 100 mg and cholesterol 20 mg were dissolved in 10 mL of anhydrous ethanol, and then dissolved by stirring at 50℃.

[0032] The microfluidic chip (inner diameter 200 μm) was used to mix at a flow ratio of 10:1; 200 W ultrasonic treatment was performed for 10 minutes, and then filtered through a 0.22 μm filter membrane to obtain the mangiferin-loaded liposome, as shown in Figure 2

[0033] Embodiment 2 ​

[0034] Mangiferin 1000 mg was accurately weighed and dissolved in 10 mL of pure water, and ultrasonically treated at 60°C for 10 minutes.

[0035] DPPC 100 mg and cholesterol 10 mg were dissolved in 10 mL of anhydrous ethanol, and stirred to dissolve at 50°C.

[0036] The microfluidic chip (inner diameter 200 μm) was used to mix at a flow ratio of 10:1; 200 W ultrasonic treatment for 10 minutes, 0.22 μm filter membrane filtration, to obtain mangiferin-loaded liposomes.

[0037] Example 3

[0038] Mangiferin 1000 mg was accurately weighed and dissolved in 10 mL of pure water, and ultrasonically treated at 60°C for 10 minutes.

[0039] PC 200 mg was dissolved in 10 mL of anhydrous ethanol, and stirred to dissolve at 50°C.

[0040] The microfluidic chip (inner diameter 200 μm) was used to mix at a flow ratio of 20:1; 200 W ultrasonic treatment for 10 minutes, 0.22 μm filter membrane filtration, to obtain mangiferin-loaded liposomes.

[0041] Experimental Example: Mice Modeling and Treatment Effect

[0042] Modeling: The experimental mice were randomly assigned to three different research groups: blank control group, osteoarthritis model group, and mangiferin-loaded liposome treatment group, 8 in each group.

[0043] Blank control group (Control): No modeling operation was performed, and normal feeding and drinking water conditions were maintained, with 100 μl of normal saline being administered intragastrically daily during the 14-day administration period.

[0044] Osteoarthritis model group (Model): After one week of adaptive feeding of the mice, modeling surgery was performed. The mice were anesthetized by injecting 0.3 ml of tri-bromoethanol solution. After anesthesia, the experimental mice were disinfected with 75% alcohol, and a towel was laid on the knee joint area to ensure a sterile operating environment. The mouse knee joint on both sides was exposed with surgical scissors, and the anterior cruciate ligament was clearly exposed using an anterior medial incision. The mouse anterior cruciate ligament was completely cut using microscissors to cause loss of knee joint stability. After confirming that the mouse anterior cruciate ligament was completely cut, the joint cavity was flushed with normal saline to prevent infection. The joint cavity was then disinfected with 0.5% iodophor. Then the patella was reduced, and the incision was closed using absorbable sutures. After suturing, the surgical area was disinfected again with iodophor. Normal feeding and drinking water conditions were maintained, with 100 μl of normal saline being administered intragastrically daily during the 14-day administration period.

[0045] The liposome loaded with mangiferin prepared in Example 1 was used to treat the group, and the osteoarthritis model was constructed according to the above method.

[0046] After the model was successfully constructed, the liposome loaded with mangiferin was administered to the mice in the treatment group by gavage at a concentration of 640 mg / ml, 200 μl per day, for 14 days, and the time point of each administration was ensured to be consistent.

[0047] While the mice in each group were gavaged for 8 weeks, the mice were placed on an animal treadmill for high-intensity running at a speed of 6 m / min for 30 min per day. After 8 weeks of treatment, the knee joints of the mice in each group were collected, and the excess bone and soft tissue around the knee joints were removed. Then the knee joints were fixed in a 4% paraformaldehyde solution for 48 h.

[0048] The Micro-CT device uses a high-resolution three-dimensional imaging technology based on X-ray imaging, which can scan and three-dimensionally reconstruct the internal structure of a small sample such as biological tissue with high precision without damaging the sample. For example, Figure 3 The Micro-CT scanning and 3D reconstruction model showed that the articular cartilage surface of the mice in the blank control group was smooth and complete, and there was no osteophyte; the articular cartilage of the mice in the model group was damaged in a large area, and a large amount of osteophyte was generated; the articular cartilage of the mice in the treatment group was repaired, and the osteophyte was reduced.

[0049] For example, Figure 4 HE staining and Ponceau O- fast green staining of the sections showed that the articular cartilage surface of the mice in the blank control group was smooth and complete, and the cells in each layer were arranged in an orderly and uniform manner. The articular cartilage surface of the mice in the model group was thin and concave, and the chondrocytes were significantly reduced, and the cells in each layer were arranged in disorder. The articular cartilage surface of the mice in the treatment group was repaired to some extent, and the number of chondrocytes was increased compared with the model group, and the arrangement was more orderly. The results showed that the liposome loaded with mangiferin could alleviate the symptoms of the osteoarthritis mouse model and inhibit the cartilage damage of the knee joint to some extent.

[0050] Further, the liposome loaded with mangiferin prepared in Examples 2 and 3 was used to perform the above experiment, and the conclusion was the same as above, and also had a therapeutic effect.

[0051] The purpose, technical solutions and beneficial effects of the present application are further described in detail, and it should be understood that the above is only a specific embodiment of the present application and does not limit the protection scope of the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for synthesizing mangiferin-loaded liposomes based on microfluidic technology, characterized in that, Includes the following steps: S100, take solution A, wherein solution A is an aqueous solution of mangiferin; S200, take solution B, wherein solution B is a phospholipid-cholesterol organic solution; S300, using a microreactor with a microfluidic chip for synthesis, the microreactor having at least three inlet ends, the solution A entering the microreactor through at least two of the inlet ends, and the solution B entering the microreactor through at least one of the inlet ends to react and prepare a liposome solution, thereby preparing mango-loaded glycoside liposomes; The flow rate of solution A is 0.1–100 mL / min, and the flow rate ratio of solution A to solution B is 1–50:

1.

2. The synthesis method according to claim 1, characterized in that, The method for preparing solution A is to dissolve mangiferin in water at a concentration of 0.1–100 mg / mL, heat to 35–95°C, and stir or sonicate for at least 1 min.

3. The synthesis method according to claim 1, characterized in that, The method for preparing solution B is to dissolve one or more phospholipids in anhydrous ethanol at a concentration of 0.1–200 mg / mL, selectively add cholesterol at a concentration of 0–80 mg / mL, heat to 35–80°C, and stir or sonicate for at least 1 min to obtain mango-loaded liposomes.

4. The synthesis method according to claim 1, characterized in that, In step S300, the liposome solution is subjected to ultrasonic treatment for at least 1 minute, followed by one or more steps including centrifugation, filtration, and freeze-drying to obtain mango-loaded glycoside liposomes.

5. The synthesis method according to claim 3, characterized in that, The phospholipids are selected from one or more of phosphatidylcholine (PC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and dipalmitoylphosphatidylcholine (DPPC).

6. The synthesis method according to claim 1, characterized in that, The microreactor has a spatial helical microstructure.

7. The synthesis method according to claim 1, characterized in that, Solution A enters the microreactor through both inlet ends, and solution B enters the microreactor through one inlet end.

8. The synthesis method according to claim 1, characterized in that, The flow rate of solution A is 0.1 to 100 mL / min, and the preferred flow rate ratio of solution A to solution B is 5 to 20:

1.

9. A liposome loaded with mangiferin prepared according to any one of claims 1 to 8.

10. The application of the mangiferin-loaded liposomes according to claim 9, characterized in that, It is used in the preparation of drugs for treating osteoarthritis.

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