A drug-loaded liposome complex containing polypeptide and its preparation method and application

Abstract

The invention discloses a drug-loaded liposome complex containing a polypeptide, a preparation method and an application thereof, and belongs to the technical field of medicine. The amino acid sequence of the polypeptide is shown in SEQ ID NO.1. The invention utilizes liposomes to encapsulate poorly soluble drugs tanshinone IIA and oroxylin B, and then compounds them with an angelica polysaccharide solution to prepare the drug complex. By encapsulating tanshinone IIA and oroxylin B with liposomes and dissolving and thickening angelica polysaccharide externally, the technical problems of poor solubility and difficulty in intra-articular injection of tanshinone IIA and oroxylin B are solved, and the obvious anti-inflammatory activity, cartilage repair and joint lubrication effects of the tanshinone IIA and oroxylin B can be exerted, and the preparation is convenient, and the tanshinone IIA and oroxylin B can be used to prepare drugs for treating or relieving inflammation and repairing cartilage, and the drug complex has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a drug-loaded liposome complex containing a polypeptide, and a preparation method and application thereof. Background Art

[0002] Osteoarthritis (OA) is an extremely common and disabling joint disease that causes cartilage degeneration, bone hyperplasia, and synovial inflammation. Due to the combined effects of the aging global population, increasing obesity, and increasing joint injuries, an estimated 2.5 billion people are currently affected worldwide, and the incidence of OA is increasing year by year. Given the aging of the population in my country, more and more patients will suffer from this disease, which will also impose a serious burden on the social economy and medical care. Among them, the common site of OA is the knee joint. According to the latest Global Burden of Disease (GBD) results, the global prevalence of osteoarthritis (OA) is 3.8%, which is the highest incidence of osteoarthritis. Research on the treatment of OA is imminent. At present, the pathogenesis of OA is not clear, but it is certain that OA is caused by multiple pathogenesis. Therefore, finding a means to effectively cure OA is a key scientific issue that needs to be solved urgently.

[0003] Tanshinone ⅡA and orchidinoside B are ingredients identified and screened from Salvia miltiorrhiza with good anti-inflammatory and cartilage repair functions. However, Tanshinone ⅡA and orchidinoside B are poorly soluble in water and are not convenient for joint cavity injection, which greatly affects their application. Summary of the invention

[0004] In view of the current status of arthritis treatment, the purpose of the present invention is to provide a drug-loaded liposome complex containing a polypeptide and its preparation method and application. The drug-loaded liposome complex has significant anti-inflammatory activity and cartilage repair effect. The technical solution of the present invention solves the problem of the poor solubility of tanshinone ⅡA and oroxylin B, and enhances the joint lubrication effect.

[0005] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a polypeptide, the amino acid sequence of the polypeptide is shown as SEQ ID NO.1.

[0007] The second aspect of the present invention provides a method for preparing a drug-loaded liposome complex containing a polypeptide, the preparation method comprising the following steps:

[0008] (1) Dissolve egg yolk lecithin, cholesterol, tanshinone ⅡA and oroxylin B in anhydrous ethanol;

[0009] (2) slowly injecting the mixture solution obtained in step (1) into ultrapure water, stirring and evaporating to remove ethanol;

[0010] (3) The solution obtained after rotary evaporation is supplemented with ultrapure water to make up the volume, then subjected to water bath sonication, and filtered through a membrane to obtain a liposome aqueous solution of tanshinone ⅡA and oroxylin B;

[0011] (4) Dissolving the polypeptide and angelica polysaccharide in ultrapure water to prepare angelica polysaccharide concentrate, adding the angelica polysaccharide concentrate to the liposome aqueous solution of tanshinone ⅡA and oroxylin B to obtain a drug-loaded liposome complex containing the polypeptide, tanshinone ⅡA, oroxylin B and angelica polysaccharide.

[0012] Furthermore, in step (1), the mass ratio of egg yolk lecithin, cholesterol, tanshinone ⅡA and oroxylin B is 100-130:10-30:1:1.

[0013] Furthermore, in the step (1), the mass ratio of egg yolk lecithin, cholesterol, tanshinone ⅡA and oroxylin B is 125:25:1:1.

[0014] Furthermore, in step (2), the volume ratio of anhydrous ethanol to ultrapure water is 2:5.

[0015] Furthermore, in step (2), the stirring temperature is 55-60° C. and the rotation speed is 100 rpm.

[0016] Furthermore, in steps (3) and (4), the volume of the final composite solution is the same as the volume of ultrapure water before rotary evaporation.

[0017] Furthermore, in step (3), the water bath ultrasonication time is 15 to 20 minutes.

[0018] Furthermore, in step (3), the liposome solution of tanshinone ⅡA and oroxylin B is obtained by sequentially passing through a 450 nm filter membrane three times and a 220 nm filter membrane three times.

[0019] Furthermore, in step (4), the concentration of the polypeptide in the drug-loaded liposome complex is 0.2 mg / mL.

[0020] Furthermore, in step (4), the concentration of tanshinone ⅡA in the drug-loaded liposome complex is 0.2 mg / mL.

[0021] Furthermore, in step (4), the concentration of oroxylin B in the drug-loaded liposome complex is 0.1 mg / mL.

[0022] Furthermore, in the step (4), the concentration of Angelica polysaccharide in the drug-loaded liposome complex is 0.2 mg / mL.

[0023] Furthermore, in step (4), the concentrations of the polypeptide, tanshinone ⅡA, oroxylin B, and angelica polysaccharide in the complex are 0.2 mg / mL, 0.2 mg / mL, 0.1 mg / mL, and 0.2 mg / mL, respectively.

[0024] The third aspect of the present invention provides a drug-loaded liposome complex prepared by the preparation method.

[0025] The fourth aspect of the present invention provides the use of the drug-loaded liposome complex in the preparation of drugs for treating arthritis and cartilage repair.

[0026] Furthermore, the drug-loaded liposome complex dosage form is administered by injection.

[0027] Compared with the prior art, the advantages and technical effects of the present invention are: the liposome complex prepared by the present invention solves the problem of poor solubility of tanshinone ⅡA and orchidinoside B, realizes the intra-articular injection of tanshinone ⅡA and orchidinoside B, and at the same time, angelica polysaccharide can act as a thickener to increase the joint lubrication effect of the drug while exerting anti-inflammatory and cartilage repair effects. Tanshinone ⅡA and orchidinoside B can significantly inhibit the expression levels of IL-1β, TNF-α and iNOS in the tissues of inflammatory pain model mice and can reduce the expression of COX-2 and PGE2. Angelica polysaccharide is a component identified and screened from angelica with good anti-inflammatory and cartilage protection functions, which can inhibit the expression of LPS-induced M1 phenotype macrophage pro-inflammatory factors (IL-1B, TNF-α, IL-6). Angelica polysaccharide can upregulate the expression of M2 phenotype anti-inflammatory factors (IL-10, Arg-1), and at the same time, it can activate chondrocyte extracellular signal-regulated protein kinase 1 / 2 (ERK1 / 2) autophagy, significantly inhibit sodium nitroprusside (SNP)-induced cell apoptosis and enhance chondrocyte proliferation.

[0028] The present invention proves through experiments that the drug-loaded liposome complex can exert the anti-inflammatory and cartilage repair effects of polypeptide, tanshinone ⅡA, oroxylin B, and angelica polysaccharide; the liposome has good biocompatibility and degradability, and can exert the sustained release effect of tanshinone ⅡA and oroxylin B. Moreover, the preparation method of the drug-loaded liposome complex of the present invention is simple, and therefore, it has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The experimental results of mouse carrageenan foot swelling test for polypeptide;

[0030] Figure 2 is the average particle size of the drug-loaded liposome complex; A is the in vitro release curve; B is the particle size distribution curve;

[0031] Figure 3The electron microscopic morphology of the drug-loaded liposome complex; A is a 500nm scale microscope image; B is a 200nm scale microscope image;

[0032] Figure 4 The results of in vitro cell proliferation of peptides, tanshinone ⅡA, oroxylin B and angelica polysaccharide; A is the cell survival rate at 24 hours; B is the cell survival rate at 48 hours;

[0033] Group t+a: a composite solution containing polypeptide, tanshinone ⅡA, oroxylin B and angelica polysaccharide;

[0034] Group t: aqueous solution of polypeptide, tanshinone ⅡA, and oroxylin B;

[0035] Group a: Angelica polysaccharide;

[0036] Group k: blank liposome aqueous solution;

[0037] The concentrations of polypeptide, tanshinone ⅡA, oroxylin B and angelica polysaccharide in well 1 of group t+a, group t and group a were 0.2 mg / mL, 0.2 mg / mL, 0.1 mg / mL and 0.2 mg / mL, respectively; the concentrations of egg yolk lecithin and cholesterol in group k were 50 mg / mL and 12.5 mg / mL, respectively; 1-5 correspond to 5 wells, the concentration of well 2 was halved based on the concentration of well 1, and each well was halved based on the concentration of the previous well. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. The experimental methods in the following embodiments without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0039] Example 1: Screening of polypeptides

[0040] 1. Acquisition of peptides

[0041] According to the amino acid sequence of the polypeptide in the sea anemone (TLRACRAICRA, shown in SEQ ID NO.6), it was modified and then synthesized by solid phase synthesis. The anti-inflammatory and analgesic activities were screened using a carrageenan model experiment, thereby obtaining polypeptides SAP1, SAP2, SAP3, SAP4, and SAP5 with anti-inflammatory and analgesic activities.

[0042] 2. Determination of amino acid sequence:

[0043] The crude product of artificial solid phase synthesized polypeptide was purified by high performance liquid chromatography, and the purified polypeptides SAP1, SAP2, SAP3, SAP4, and SAP5 were subjected to Edman degradation method on a fully automatic protein sequencer to determine the complete amino acid sequences of polypeptides SAP1, SAP2, SAP3, SAP4, and SAP5. The results showed that the amino acid sequences of polypeptides SAP1, SAP2, SAP3, SAP4, and SAP5 were TLHQCRGICRA (SEQ ID NO.1), TLHQCRGNCRA (SEQ ID NO.2), TLHQCKGICRA (SEQ ID NO.3), TLHQARGICRA (SEQ ID NO.4), and TLHQFRGICRA (SEQ ID NO.5), respectively.

[0044] 3. Determination of anti-inflammatory and analgesic activity of analgesic peptides

[0045] In order to gain a deeper understanding of the anti-inflammatory and analgesic activities of polypeptides SAP1, SAP2, SAP3, SAP4, and SAP5, the present invention conducted a study using the classic carrageenan animal pain model, and the results showed that analgesic peptide SAP1 showed good anti-inflammatory and analgesic activities in the animal pain model. However, the anti-inflammatory effects of polypeptides SAP2, SAP3, SAP4, and SAP5 were not as good as those of polypeptide SAP1.

[0046] 4. Mouse carrageenan foot swelling experiment

[0047] The anti-inflammatory effects of peptides SAP1, SAP2, SAP3, SAP4, and SAP5 were evaluated by carrageenan-induced mouse foot swelling experiment. The mice were randomly divided into a normal group, a model group (carrageenan), a positive group (10 mg / kg indomethacin), and analgesic peptide group (50 mg / kg peptides SAP1, SAP2, SAP3, SAP4, and SAP5), with 6 mice in each group. The analgesic peptides were dissolved in sterile saline to a dilution concentration of 5 mg / ml. The mice in the normal group and model group were intraperitoneally injected with 200 μl of sterile saline, the mice in the analgesic peptide group were injected with 200 μl of 5 mg / ml peptide, and the mice in the positive group were injected with 200 μl of 10 mg / kg indomethacin; 30 min after intraperitoneal injection of the drugs, the right hind paws of the mice in the normal group were injected with 30 μl of saline, the model group were injected with 30 μl of 1% carrageenan, and the mice in the positive group, SAP1 group, SAP2 group, SAP3 group, SAP4 group, and SAP5 group were injected with 30 μl of 1% carrageenan, respectively. The thickness of the right hind toes of the mice was measured every hour since 1 hour.

[0048] The experimental results are as follows Figure 1As shown in the figure, comparison of the data of the control group and the model group showed that the carrageenan mouse model was successfully established. Both the model group and the positive group reached the peak of swelling at 3 h. Compared with the model group, the analgesic peptides had different degrees of inhibitory effects on inflammation at 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h. Among them, the anti-inflammatory effects of SAP2, SAP3, SAP4, and SAP5 were not as good as the anti-inflammatory effect of polypeptide SAP1.

[0049] Example 2: Preparation of a composite solution of tanshinone ⅡA, oroxylin B, and angelica polysaccharide

[0050] 1. Preparation of a composite solution containing tanshinone ⅡA, oroxylin B and angelica polysaccharide

[0051] The preparation method of the composite solution comprises the following steps:

[0052] (1) Dissolve egg yolk lecithin, cholesterol, tanshinone ⅡA and oroxylin B in anhydrous ethanol; the mass ratio of egg yolk lecithin, cholesterol, tanshinone ⅡA and oroxylin B is 125:25:1:1;

[0053] (2) slowly injecting the mixed solution in step (1) into ultrapure water, wherein the volume ratio of anhydrous ethanol to ultrapure water is 2:5; placing the solution in a constant temperature magnetic stirrer and stirring to evaporate the solution to remove the ethanol;

[0054] (3) After rotary evaporation, the sample solution was obtained, and ultrapure water was added to make up to the initial volume. Then, the solution was ultrasonicated in a water bath for 15-20 min, and the solution was filtered through a 450 nm filter three times and a 220 nm filter three times to obtain a liposome aqueous solution of tanshinone ⅡA and oroxylin B;

[0055] (4) Dissolving the polypeptide (TLHQCRGICRA) and Angelica polysaccharide in ultrapure water to prepare Angelica polysaccharide concentrate, adding the Angelica polysaccharide concentrate to the liposome aqueous solution of Tanshinone ⅡA and Oroxyloside B, to obtain a composite solution containing polypeptide, Tanshinone ⅡA, Oroxyloside B, and Angelica polysaccharide, wherein the concentrations of polypeptide, Tanshinone ⅡA, Oroxyloside B, and Angelica polysaccharide in the composite solution are 0.2 mg / mL, 0.2 mg / mL, 0.1 mg / mL, and 0.2 mg / mL, respectively. The volume of the final composite solution is the same as the volume of ultrapure water before rotary evaporation.

[0056] 2. Characterization of composite solution

[0057] Particle size and potential detection Before the measurement, the composite solution sample was diluted 10 times with distilled water, and the particle size, polydispersity index (PDI) and Zeta potential of the liposomes were measured using a Malvern particle size analyzer.

[0058] The results are as follows Figure 2As shown, the average particle size of the liposomes was 141±2.203d.nm, the PDI was 0.152±0.044, and the zeta potential was -39.8±0.758.

[0059] Liposome morphology characterization Liposomes were diluted 10 times with distilled water, and the sample was dropped onto a copper grid. Uranyl acetate was added to the sample for staining for 3 minutes. The excess liquid was absorbed by filter paper and dried. TEM photos were taken for observation. Figure 3 As shown, the liposome particle size observed under the microscope is 180nm~220nm.

[0060] Example 3: Cell proliferation experiment with polypeptide, tanshinone ⅡA, oroxylin B, and angelica polysaccharide

[0061] In order to further understand the cytotoxicity and anti-inflammatory activity of the related components of the composite solution, Tanshinone ⅡA, Oroxyloside B, and Angelica polysaccharide, the present invention uses the MTT method to verify the cytotoxicity and lipopolysaccharide (LPS)-induced macrophage RAW264.7. The results showed that Tanshinone ⅡA, Oroxyloside B, and Angelica polysaccharide showed good cell proliferation effects, among which Angelica polysaccharide cell proliferation effect was better than Tanshinone ⅡA and Oroxyloside B, as described in detail as follows:

[0062] Rat chondrocytes (ATDC5) in the logarithmic growth phase were plated, and a composite solution containing peptide, tanshinone ⅡA, oroxylin B and angelica polysaccharide (t+a), an aqueous solution of peptide, tanshinone ⅡA and oroxylin B (t), an angelica polysaccharide aqueous solution (a), and a blank liposome aqueous solution (k) were prepared. The cell proliferation effect of the drug was measured using a 96-well plate using the isoproportional dilution method. 100 µL of drugs of different concentrations were drawn into a 96-well plate, and each drug and each concentration were repeated in 5 wells; the concentration of each drug was halved in 5 wells in turn; and finally, the cells were placed in a 37°C incubator and cultured for 24h and 72h.

[0063] like Figure 4 As shown, at 24 h, the composite solution made of polypeptide, tanshinone ⅡA, oroxylin B and angelica polysaccharide had obvious cell proliferation activity, and the proliferation effect of 72 cells was reduced.

[0064] The present invention provides a compound made of tanshinone ⅡA and orchidinoside B liposomes compounded with angelica polysaccharide. The present invention first prepares a tanshinone ⅡA and orchidinoside B liposome aqueous solution, solves the limitations of tanshinone ⅡA and orchidinoside B being poorly soluble and difficult to inject into the joint cavity, can exert their obvious anti-inflammatory activity, cartilage repair and joint lubrication effects, and the preparation method is convenient, can be used to prepare drugs for treating or relieving inflammation and repairing cartilage, and has good application prospects.

[0065] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A polypeptide, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. A method for preparing a drug-loaded liposome complex containing the polypeptide according to claim 1, characterized in that: The preparation method comprises the following steps: (1) dissolving egg yolk lecithin, cholesterol, tanshinone ⅡA and oroxylin B in anhydrous ethanol; the mass ratio of the egg yolk lecithin, cholesterol, tanshinone ⅡA and oroxylin B is 100-130:10-30:1:1; (2) slowly injecting the mixture solution obtained in step (1) into ultrapure water, stirring and evaporating to remove ethanol; the volume ratio of anhydrous ethanol in step (1) to ultrapure water in step (2) is 2:5; (3) The solution obtained after rotary evaporation is supplemented with ultrapure water to the initial volume, then subjected to water bath sonication and filtered through a filter membrane to obtain a liposome aqueous solution of tanshinone ⅡA and oroxylin B; (4) Dissolving the polypeptide and Angelica polysaccharide in ultrapure water to prepare Angelica polysaccharide concentrate, adding the Angelica polysaccharide concentrate to the tanshinone ⅡA and orchidinoside B liposome aqueous solution to obtain a drug-loaded liposome complex containing the polypeptide, tanshinone ⅡA, orchidinoside B, and Angelica polysaccharide; the concentration of the polypeptide in the drug-loaded liposome complex is 0.2 mg / mL; the concentration of tanshinone ⅡA in the drug-loaded liposome complex is 0.2 mg / mL; the concentration of orchidinoside B in the drug-loaded liposome complex is 0.1 mg / mL; and the concentration of Angelica polysaccharide in the drug-loaded liposome complex is 0.2 mg / mL.

3. The drug-loaded liposome complex prepared by the preparation method according to claim 2.

4. Use of the drug-loaded liposome complex according to claim 3 in the preparation of drugs for treating arthritis and cartilage repair.

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