Recombinant protein IL-17A multivesicular liposome as well as preparation method and application thereof

Abstract

The invention provides a recombinant protein IL-17A multivesicular liposome as well as a preparation method and application thereof, and belongs to the field of pharmaceutical preparations. The recombinant protein IL-17A multivesicular liposome is prepared from an oil phase, an inner water phase and an outer water phase, the oil phase consists of a lipid material and an organic solvent; the inner water phase consists of recombinant protein IL-17A and a sucrose water solution; and the outer water phase consists of a glucose aqueous solution and a lysine aqueous solution. The recombinant protein IL-17A multivesicular liposome is successfully prepared, the particle size of the recombinant protein IL-17A multivesicular liposome is small, the uniformity is good, the stability is good, the recombinant protein IL-17A can be stably and slowly released, and the slow release effect is achieved. A mouse depression model can be constructed by utilizing the recombinant protein IL-17A multivesicular liposome, and the recombinant protein IL-17A multivesicular liposome is used for related research of depression and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical preparations, and particularly relates to a recombinant protein IL-17A multivesicular liposome, a preparation method thereof, and uses thereof. Background Art

[0002] Interleukin 17A (IL-17A) is an important pro-inflammatory cytokine and belongs to the interleukin 17 family. IL-17A is mainly secreted by T helper cells 17 (Th17). In addition, γδ T cells, natural killer T (NKT) cells, CD8+ T cells, neutrophils, mast cells, and macrophages can also produce IL-17A. IL-17A plays an important role in a variety of inflammatory diseases, such as psoriasis, rheumatoid arthritis, and inflammatory bowel disease. At the same time, IL-17A is the main secreted product of Th17 cells and is involved in the pathogenesis of autoimmune diseases. In addition, IL-17A has a dual role in the tumor microenvironment, which can both promote tumor growth and inhibit tumors by regulating the immune response.

[0003] Recombinant protein IL-17A is IL-17A produced in vitro by genetic engineering technology and is mainly used in the following fields: (1) Basic research: used to study the mechanism of action of IL-17A in immune responses, such as inducing the production of antimicrobial peptides, cytokines, chemokines, and matrix metalloproteinases; (2) Disease model research: used to study the pathogenesis of autoimmune diseases (such as rheumatoid arthritis, psoriasis), infectious diseases, and tumors; (3) Drug development: used as a target for developing drugs such as anti-IL-17A monoclonal antibodies for the treatment of related diseases. Recombinant protein IL-17A has poor stability, and the dissolution and storage conditions are relatively harsh, so to a certain extent, it limits its application in experiments and clinics. It is very urgent to develop more forms of recombinant protein IL-17A for use to meet the requirements of applications in experiments and clinics.

[0004] Multivesicular liposomes (MVLs) are liposomes composed of multiple non-concentric vesicles. Their internal structure is similar to a honeycomb, with multiple aqueous compartments separated by lipid bilayers. Compared with traditional single-layer or few-layer liposomes, multivesicular liposomes have a larger internal aqueous phase volume and a higher drug encapsulation rate. Multivesicular liposomes usually have good biocompatibility and biodegradability and can be used for drug delivery.

[0005] However, multilamellar liposomes have the following problems: (1) Complex preparation process: The double emulsion method is one of the most commonly used methods for preparing multilamellar liposomes, but it has the characteristics of poor uniformity and stability of multilamellar liposomes, wide emulsion size distribution, and requires further centrifugation or sieving, resulting in waste of the original reagent and low utilization rate of the prepared emulsion; (2) Difficult particle size control: It is difficult to control the particle size of multilamellar liposomes below 10 μm, and the size distribution is wide, making it difficult to achieve uniformity of small sizes; (3) Insufficient stability: During the preparation process, the structure of multilamellar liposomes is easily damaged; (4) Although the internal aqueous phase of multilamellar liposomes has a large volume and a high encapsulation rate, burst release may occur during the drug release process; (5) The types of drugs loaded by multilamellar liposomes are limited. The above problems limit their wide application.

[0006] At present, there is no report on preparing recombinant protein IL-17A into the form of multilamellar liposomes. How to successfully prepare recombinant protein IL-17A into the form of multilamellar liposomes requires further research. Summary of the Invention

[0007] The object of the present invention is to provide a recombinant protein IL-17A multilamellar liposome, its preparation method and use.

[0008] The present invention provides a recombinant protein IL-17A multilamellar liposome, which is prepared from an oil phase, an inner aqueous phase and an outer aqueous phase; the oil phase is composed of a lipid material and an organic solvent; the inner aqueous phase is composed of recombinant protein IL-17A and a sucrose aqueous solution; the outer aqueous phase is composed of a glucose aqueous solution and a lysine aqueous solution.

[0009] Furthermore, the volume ratio of the oil phase, the inner aqueous phase and the outer aqueous phase is 1:(1 - 5):(1 - 5);

[0010] Preferably, the volume ratio of the oil phase, the inner aqueous phase and the outer aqueous phase is 1:1:2.

[0011] Furthermore, the lipid material is composed of soybean phospholipid, cholesterol, dipalmitoyl phosphatidylglycerol and glyceryl trioleate in a molar ratio of (3 - 5):(6 - 8):(1 - 2):(1 - 2);

[0012] Preferably, the lipid material is composed of soybean phospholipid, cholesterol, dipalmitoyl phosphatidylglycerol and glyceryl trioleate in a molar ratio of 4:7:1:1.

[0013] Furthermore, the organic solvent is a non-polar solvent;

[0014] Preferably, the organic solvent is dichloromethane, ethyl acetate, tetrahydrofuran or chloroform.

[0015] Further, the mass-volume ratio of the lipid material to the organic solvent is (0.01 - 0.05) g: 1 mL.

[0016] Further, the concentration of the recombinant protein IL-17A is 0.1 - 1 mg / mL;

[0017] Preferably, the concentration of the recombinant protein IL-17A is 0.4 mg / mL.

[0018] Further, the concentration of the sucrose aqueous solution is 1 - 10 wt%;

[0019] Preferably, the concentration of the sucrose aqueous solution is 4 wt%.

[0020] Further, the volume ratio of the glucose aqueous solution to the lysine aqueous solution is 1:1;

[0021] Preferably, the concentration of the glucose aqueous solution is 1 - 10 wt%, and the concentration of the lysine aqueous solution is 10 - 50 mmol / L;

[0022] More preferably, the concentration of the glucose aqueous solution is 3.2 wt%, and the concentration of the lysine aqueous solution is 40 mmol / L.

[0023] The present invention also provides a method for preparing the aforementioned recombinant protein IL-17A multi-lamellar liposomes, which comprises the following steps:

[0024] (1) Dissolve the lipid material in an organic solvent to form an oil phase;

[0025] (2) Dissolve the recombinant protein IL-17A in a sucrose aqueous solution to prepare an inner aqueous phase;

[0026] (3) Mix the glucose aqueous solution and the lysine aqueous solution evenly to obtain an outer aqueous phase;

[0027] (4) Add the inner aqueous phase to the oil phase, and emulsify to obtain a W / O type primary emulsion;

[0028] (5) Add the outer aqueous phase to the primary emulsion, and emulsify to obtain a W / O / W type multiple emulsion;

[0029] Preferably,

[0030] In step (4), the emulsification conditions are stirring at a speed of 3000 - 5000 rpm / min for 10 - 20 min;

[0031] and / or, in step (5), the emulsification conditions are stirring at a speed of 3000 - 5000 rpm / min for 1 - 5 min.

[0032] The present invention also provides the use of the aforementioned recombinant protein IL-17A multi-lamellar liposomes in the preparation of an animal model of depression.

[0033] The present invention has achieved the following beneficial effects:

[0034] The present invention successfully prepared a recombinant protein IL-17A multi-lamellar liposome. The recombinant protein IL-17A multi-lamellar liposome has a small particle size (below 10 μm), good homogeneity, and good stability, and can stably and slowly release the recombinant protein IL-17A, achieving a sustained release effect. Using this recombinant protein IL-17A multi-lamellar liposome, a mouse depression model can be constructed for research related to depression, and it has good application prospects.

[0035] Obviously, based on the above content of the present invention, according to the common general technical knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0036] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Description of the Drawings

[0037] Figure 1 It is the characterization results of the physicochemical properties of the recombinant protein IL-17A multi-lamellar liposomes of the present invention: a is the 7-day sustained release result of the recombinant protein IL-17A multi-lamellar liposomes; b is the Coomassie brilliant blue staining result of the recombinant protein IL-17A multi-lamellar liposomes; c is the bright field microscope picture of the recombinant protein IL-17A multi-lamellar liposomes; d is the particle size distribution diagram of the recombinant protein IL-17A multi-lamellar liposomes.

[0038] Figure 2 It is the process and results of constructing a depression model with the recombinant protein IL-17A multi-lamellar liposomes of the present invention: a is the process and experimental method of constructing a depression model; b is the result of constructing a depression model. Detailed Description of the Invention

[0039] The raw materials and equipment used in the present invention are all known products, obtained by purchasing commercially available products.

[0040] Reagents: Recombinant protein IL-17A (MCE), soybean phospholipid (AR, Yuanye Bio-Technology Co., Ltd.), cholesterol (BR, Chengdu Kelong Chemical Co., Ltd.), dipalmitoyl phosphatidylglycerol (DPPG) (AR, Avitop Pharmaceutical Technology Co., Ltd.), triolein (99%, Shanghai Macklin Biochemical Co., Ltd.), glucose (AR, Shanghai Aladdin Biochemical Technology Co., Ltd.), sucrose (AR, Chengdu Kelong Chemical Co., Ltd.), lysine (98%, Shanghai Aladdin Biochemical Technology Co., Ltd.), dichloromethane (AR, Chengdu Kelong Chemical Co., Ltd.), sodium hydroxide (AR, 96%, Shanghai Aladdin Biochemical Technology Co., Ltd.), hydrochloric acid (AR, Chuandong Chemical Industry), normal saline (0.9%, Beijing Lanjie Technology Co., Ltd.).

[0041] Example 1: Preparation of Recombinant Protein IL-17A Multivesicular Liposomes

[0042] In this example, the complex emulsion method was used to prepare recombinant protein IL-17A multivesicular liposomes, which specifically included the following steps:

[0043] (1) Weigh 0.0801 g of soybean phospholipid, 0.0580 g of cholesterol, 0.0158 g of dipalmitoyl phosphatidylglycerol, and 0.0166 g of triolein according to the molar ratio of 4:7:1:1, dissolve them together in 5 mL of dichloromethane. After complete dissolution, slowly add 5 mL of a sucrose aqueous solution containing recombinant protein IL-17A (the concentration of recombinant protein IL-17A is 0.4 mg / mL), and perform emulsification. The emulsification conditions are 3000 rpm / min and mechanical stirring for 12 min to obtain the primary W / O emulsion. The preparation method of the sucrose aqueous solution is: weigh 4 g of sucrose and dissolve it in 100 mL of water to obtain it.

[0044] (2) Add the above primary emulsion to 10 mL of a mixed aqueous solution containing glucose and lysine, and perform emulsification. The emulsification conditions are 3000 rpm / min and mechanical stirring for 2 min to obtain the W / O / W double emulsion, which is the recombinant protein IL-17A multivesicular liposome. The organic solvent dichloromethane was removed by the combined use of gradient centrifugation and nitrogen blowing. The preparation method of the mixed aqueous solution containing glucose and lysine is: weigh 3.2 g of glucose and dissolve it in 100 mL of water to obtain a glucose aqueous solution with a concentration of 3.2 wt%, weigh 5.96 g of lysine and dissolve it in 100 mL of water to obtain a lysine aqueous solution with a concentration of 40 mmol / L, and mix the glucose aqueous solution and the lysine aqueous solution according to a volume ratio of 1:1 to obtain it.

[0045] Comparative Example 1: Preparation of Blank Liposomes

[0046] According to the method described in Example 1, except without adding recombinant protein IL-17A, blank liposomes were prepared.

[0047] The beneficial effects of the present invention are demonstrated by the following specific test examples.

[0048] Test Example 1. Verification, morphological observation and sustained-release effect study of the recombinant protein IL-17A multi-lamellar liposomes of the present invention

[0049] The recombinant protein IL-17A multi-lamellar liposomes involved in this test example were prepared according to the method described in Example 1, and the blank liposomes were prepared according to the method described in Comparative Example 1.

[0050] 1. Verification of recombinant protein IL-17A multi-lamellar liposomes by Coomassie Brilliant Blue staining

[0051] The Coomassie Brilliant Blue staining was used to verify whether the recombinant protein IL-17A multi-lamellar liposomes were successfully prepared. The specific method is as follows:

[0052] (1) Preparation of separating gel: (taking one plate as an example):

[0053] ddH 2 O: 1.6 ml; 30% Acr-Bis: 2.0 ml; 1.5 M Tris-HCl pH8.8: 1.3 ml; 10% SDS: 50 μl; 10% ammonium persulfate: 50 μl; TEMED: 2 μl.

[0054] (2) Add the separating gel to 1 cm below the lower edge of the comb teeth, then immediately add anhydrous ethanol to the upper edge of the glass plate, let it stand for 20 min, then pour out the anhydrous ethanol, and blot the residual liquid with filter paper.

[0055] (3) Preparation of stacking gel:

[0056] ddH 2 O: 2.1 ml; 30% Acr-Bis: 0.5 ml; 1 M Tris-HCl pH6.8: 0.38 ml; 10% SDS: 30 μl; 10% ammonium persulfate: 30 μl; TEMED: 3 μl.

[0057] (4) Add the stacking gel to the upper edge of the glass plate, immediately insert the sample loading comb, and let it stand for 20 min.

[0058] (5) Place the gel in the electrophoresis tank and load the samples:

[0059] Protein marker 2 μl + 1×loading buffer 7 μl

[0060] Recombinant protein IL-17A multi-lamellar liposomes 20 μL

[0061] Blank liposomes 20 μl.

[0062] (6) Electrophoresis: 80 V, 20 min; 120 V, 80 min.

[0063] (7) Cut the gel and place it in Coomassie Brilliant Blue staining solution, place it on a side-swing shaker and shake slowly, stain at room temperature for 2.5 h.

[0064] (8) Add an appropriate amount of Coomassie Brilliant Blue staining and decolorizing solution, place it on a side-swing shaker and shake slowly, decolorize at room temperature for 4 - 6 h, and change the decolorizing solution halfway (once every 2 h).

[0065] (9) Take pictures with a camera.

[0066] The results are as Figure 1 shown in Figure 1 b. As can be seen from Figure 1 b, the recombinant protein IL-17A multi-lamellar liposomes of the present invention were successfully prepared (

[0067] In b, it is multi-lamellar liposomes-recombinant IL17A).

[0068] Observe the morphology of the recombinant protein IL-17A multi-lamellar liposomes by bright-field microscopy, and at the same time detect the particle size of the recombinant protein IL-17A multi-lamellar liposomes by a Malvern particle size analyzer.

[0069] Figure 1 c shows the morphology of the recombinant protein IL-17A multi-lamellar liposomes under a 40× microscope ( Figure 1 In c, it is multi-lamellar liposomes-recombinant IL17A). The particle size of the multi-lamellar liposomes in the figure is about 2 - 5 μm. Figure 1 d shows the particle size results measured by a Malvern particle size analyzer. It can be seen that the particle size of the recombinant protein IL-17A multi-lamellar liposomes of the present invention is about 2.5 - 3 μm, and the uniformity is good. It shows that the recombinant protein IL-17A multi-lamellar liposomes with small particle size and good uniformity can be successfully prepared by the method of the present invention.

[0070] 3. Study on the sustained-release effect of the recombinant protein IL-17A multi-lamellar liposomes

[0071] Take 500 μL of the recombinant protein IL-17A multi-lamellar liposomes prepared in Example 1 and transfer them to a 50 mL beaker containing 25 mL of normal saline. The beaker is incubated at a constant rotation of 150 rpm / min at 37 °C. Collect 3 samples at each time point (0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h), and measure the content of the recombinant protein in the supernatant after centrifugation at 600×g for 10 min, and measure the absorbance using a UV-visible spectrophotometer.

[0072] The results of the 7-day sustained release of recombinant protein IL-17A multi-lamellar liposomes are as follows Figure 1 shown in Figure 1 a. It can be seen from

[0073] a that the recombinant protein IL-17A multi-lamellar liposomes of the present invention can achieve a stable and slow release effect within one week.

[0074] The recombinant protein IL-17A multi-lamellar liposomes involved in this experimental example were prepared according to the method described in Example 1, and the blank liposomes were prepared according to the method described in Comparative Example 1.

[0075] 1. Method for constructing a depression model

[0076] (1) Male C57 mice at 8 weeks old were anesthetized with isoflurane gas and placed in a stereotaxic apparatus for the brain. A pulled glass electrode was fixed on the holder of the stereotaxic apparatus as an injection needle.

[0077] (2) The hair on the head of the mouse was cut off, disinfected with iodophor, the scalp was cut open and disinfected.

[0078] (3) After fixing the position and leveling, a hole was drilled at the position with parameters of AP - 3.3, ML ± 3.0.

[0079] (4) The glass electrode injection needle was used to aspirate 2 μl of recombinant protein IL-17A multi-lamellar liposomes or blank liposomes, and the recombinant protein IL-17A multi-lamellar liposomes ( Figure 2 in it is multi-lamellar liposomes - recombinant IL17A) or 333 nL of blank liposomes were respectively injected at the positions with parameters of AP - 3.3, ML 3.0, and depths DV - 3.5, - 2.5, - 1.5. Then, 333 nL of recombinant protein IL-17A multi-lamellar liposomes or blank liposomes were respectively injected at the positions with parameters of AP - 3.3, ML - 3.0, and depths DV - 3.5, - 2.5, - 1.5.

[0080] (5) The scalp was sutured, and the mouse was put back into the original cage for feeding.

[0081] 2. Method for detecting depressive phenotypes

[0082] (1) On the 6th day after the injection of recombinant protein IL-17A multi-lamellar liposomes or blank liposomes for positioning, an open field experiment was carried out. The mouse was placed in the center of a white open field box (44 cm long, 44 cm wide, 40 cm high). The mouse moved freely in the open field box, and a video was recorded for 5 minutes and 30 seconds. The first 30 seconds was a pre-experiment. After the experiment, the movement of the mouse in the last 5 minutes (total movement distance, ratio of distance in the central area to the total distance) was analyzed using EthoVision XT 13 software to clarify whether the mouse had motor dysfunction and anxiety-like behaviors.

[0083] (2) On the 7th day after the injection of recombinant protein IL-17A polycystic liposomes or blank liposomes by positioning injection, the forced swimming test was carried out to detect the despair-like behavior of mice and reflect the depressive state of mice. The diameter of the swimming bucket was 15 cm and the height was 30 cm. Water was placed to a height of 20 cm, and the water temperature was 24 ± 1 °C. The mice were placed in the swimming bucket, and the video was recorded for 6 minutes. The first 2 minutes were pre-experiments, and the movement of the mice (immobile time) in the last 4 minutes was analyzed by EthoVision XT 13 software. The longer the immobile time, the more representative the depressive-like phenotype.

[0084] 3. Experimental results

[0085] As Figure 2 shown: After intracerebral injection of the recombinant protein IL-17A polycystic liposomes of the present invention, the immobile time in the forced swimming test was longer than that of the mice in the blank liposome group, indicating that the mice injected with the recombinant protein IL-17A polycystic liposomes showed despair-like behavior, proving that the recombinant protein IL-17A polycystic liposomes of the present invention can promote depressive-like behavior and successfully establish a mouse depression model.

[0086] In summary, the present invention successfully prepared a recombinant protein IL-17A polycystic liposome. The recombinant protein IL-17A polycystic liposome has a small particle size (below 10 μm), good homogeneity, and good stability, and can stably and slowly release the recombinant protein IL-17A, achieving a sustained-release effect. Using this recombinant protein IL-17A polycystic liposome, a mouse depression model can be established for research related to depression, and it has good application prospects.

Claims

1. A recombinant protein IL-17A multivesicular liposome, characterized in that: It is prepared from an oil phase, an inner aqueous phase and an outer aqueous phase; the oil phase is composed of lipid materials and organic solvents; the inner aqueous phase is composed of recombinant protein IL-17A and a sucrose aqueous solution; and the outer aqueous phase is composed of a glucose aqueous solution and a lysine aqueous solution.

2. The recombinant protein IL-17A multivesicular liposome according to claim 1, characterized in that: The volume ratio of the oil phase, the inner water phase and the outer water phase is 1:(1-5):(1-5); Preferably, the volume ratio of the oil phase, the inner water phase and the outer water phase is 1:1:

2.

3. The recombinant protein IL-17A multivesicular liposome according to claim 1 or 2, characterized in that: The lipid material is composed of soybean lecithin, cholesterol, dipalmitoylphosphatidylglycerol and triolein in a molar ratio of (3-5):(6-8):(1-2):(1-2); Preferably, the lipid material consists of soybean lecithin, cholesterol, dipalmitoylphosphatidylglycerol and triolein in a molar ratio of 4:7:1:

1.

4. The recombinant protein IL-17A multivesicular liposome according to claim 1 or 2, characterized in that: The organic solvent is a non-polar solvent; Preferably, the organic solvent is dichloromethane, ethyl acetate, tetrahydrofuran or chloroform.

5. The recombinant protein IL-17A multivesicular liposome according to claim 1 or 2, characterized in that: The mass volume ratio of the lipid material and the organic solvent is (0.01-0.05) g:1 mL.

6. The recombinant protein IL-17A multivesicular liposome according to claim 1 or 2, characterized in that: The concentration of the recombinant protein IL-17A is 0.1-1 mg / mL; Preferably, the concentration of the recombinant protein IL-17A is 0.4 mg / mL.

7. The recombinant protein IL-17A multivesicular liposome according to claim 1 or 2, characterized in that: The concentration of the sucrose aqueous solution is 1 to 10 wt %; Preferably, the concentration of the sucrose aqueous solution is 4 wt %.

8. The recombinant protein IL-17A multivesicular liposome according to claim 1 or 2, characterized in that: The volume ratio of the glucose aqueous solution to the lysine aqueous solution is 1:1; Preferably, the concentration of the glucose aqueous solution is 1 to 10 wt %, and the concentration of the lysine aqueous solution is 10 to 50 mmol / L; More preferably, the concentration of the aqueous glucose solution is 3.2 wt %, and the concentration of the aqueous lysine solution is 40 mmol / L.

9. A method for preparing the recombinant protein IL-17A multivesicular liposomes according to any one of claims 1 to 8, characterized in that: It includes the following steps: (1) dissolving the lipid material in an organic solvent as an oil phase; (2) dissolving the recombinant protein IL-17A in a sucrose aqueous solution to prepare an inner aqueous phase; (3) mixing the glucose aqueous solution and the lysine aqueous solution uniformly to obtain an external aqueous phase; (4) adding the internal water phase to the oil phase and emulsifying to obtain W / O type colostrum; (5) adding an external water phase to the colostrum and emulsifying to obtain a W / O / W type emulsion; Preferably, In step (4), the emulsification condition is stirring at a rotation speed of 3000 to 5000 rpm / min for 10 to 20 min; And / or, in step (5), the emulsification condition is stirring at a rotation speed of 3000 to 5000 rpm / min for 1 to 5 min.

10. Use of the recombinant protein IL-17A multivesicular liposome according to any one of claims 1 to 8 in preparing an animal model of depression.

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