A drug-loaded liposome composite gel containing anti-inflammatory peptides and its preparation method and application

Abstract

The present invention provides a co-loaded drug liposome composite gel containing an anti-inflammatory polypeptide, and a preparation method and application thereof, and belongs to the field of medical technology. The amino acid sequence of the anti-inflammatory polypeptide of the present invention is shown in SEQ ID NO.1. The present invention encapsulates hydrophobic β-eucalyptol and atractylodesin in liposomes, and then prepares a composite gel with liposomes, berberine, oligofructose, anti-inflammatory polypeptide, and Pluronic F-127, and delivers them to the site of action together to achieve a synergistic effect. β-eucalyptol enhances the colonization ability of lactobacillus; atractylodesin promotes the expression of tight junction proteins and enhances the protective function of the vaginal epithelial barrier; the synergistic effect of anti-inflammatory polypeptides and traditional Chinese medicine monomers can accelerate the repair effect on the mucosa; berberine inhibits the growth of pathogens, and oligofructose promotes the proliferation of beneficial bacteria, further regulating the balance of vaginal microecology. The present invention has a good therapeutic effect on aerobic bacterial vaginitis and has a good market application prospect.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to a drug-co-loaded liposome composite gel containing an anti-inflammatory polypeptide, and a preparation method and application thereof. Background Art

[0002] Aerobic vaginitis (AV) is a vaginal inflammation caused by a decrease or absence of lactobacilli in the vagina and an increase in aerobic bacteria. There is currently no unified and recognized diagnostic standard for AV. The Donders scoring system is still the most widely reported in the literature and widely used in clinical practice in China. It is based on the analysis of microscopic wet mounts of vaginal secretions, including the presence of immature epithelial cells from the underlying vaginal mucosa, the level of lactic acid bacteria, the characteristics and number of inflammatory cells, and the characteristics of the microbial flora. The current consensus recommends clindamycin and cephalosporin antibiotics as first-line treatments for AV.

[0003] However, long-term use of antibiotics has obvious adverse reactions and is prone to cause the emergence of drug-resistant strains, increasing the difficulty of treatment and the chance of secondary infection. Traditional Chinese medicine is different from Western medicine antibacterial drugs, and drug resistance is relatively rare. However, Chinese medicine is slow to take effect when taken orally, and topical lotions are cumbersome to use, with low local drug concentration and long treatment cycle. In addition, most Chinese medicine monomers have poor solubility and low effective utilization.

[0004] In recent years, more and more biologically active sea cucumber peptides have been discovered, which have antioxidant, anti-fatigue, antihypertensive, antibacterial, anti-tumor, hypoglycemic, anti-inflammatory and other effects. Therefore, it is of great significance to prepare pharmaceutical dosage forms containing peptides, increase the solubility of hydrophobic drugs, improve the administration method, increase the retention time of drugs at the site of action, increase the local drug concentration, and shorten the treatment time. Summary of the invention

[0005] In view of the above problems in the prior art, the purpose of the present invention is to provide a drug-co-loaded liposome composite gel containing an anti-inflammatory polypeptide, and a preparation method and application thereof. The present invention delivers hydrophobic β-eucalyptol, atractylodesin, hydrophilic berberine, oligofructose, and anti-inflammatory polypeptide to the lesion site, and adds Pluronic F-127 to prepare a thermosensitive gel, which can respond to changes in ambient temperature, gel quickly, and enhance the adhesion of the drug, thereby enhancing the epithelial barrier protection and repair function, synergistic anti-inflammatory and antibacterial effects, improving aerobic bacterial vaginitis, and regulating the balance of vaginal microecology.

[0006] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

[0007] The present invention provides an anti-inflammatory polypeptide, the amino acid sequence of the anti-inflammatory polypeptide is shown in SEQ ID NO.1.

[0008] The present invention also provides a method for preparing a drug-loaded liposome composite gel containing an anti-inflammatory polypeptide, the preparation method comprising the following steps:

[0009] (1) dissolving β-eucalyptol, atractylodesin, egg yolk lecithin, and cholesterol in anhydrous ethanol, and the dissolved mixture is used as the oil phase; the oil phase is injected dropwise into the water phase formed by ultrapure water, and stirred to evaporate the ethanol;

[0010] (2) Add ultrapure water to make up to the initial volume of the aqueous phase, perform water bath sonication, and filter through a microporous membrane to obtain drug-loaded liposomes;

[0011] (3) The drug-loaded liposomes, berberine, oligofructose, anti-inflammatory peptides, and Pluronic F-127 are mixed to form a mixed solution, stirred evenly in an ice water bath, and allowed to stand at 4° C. to eliminate bubbles, thereby obtaining a liposome composite gel.

[0012] Furthermore, in step (1), the mass ratio of the total mass of β-eucalyptol and atractylodesin to the mass of egg yolk lecithin and cholesterol is 1-3:40:5.

[0013] Furthermore, in the step (1), the mass ratio of β-cineole to atractylodesin is 1-2:1.

[0014] Furthermore, in step (1), the volume ratio of the oil phase to the water phase is 2:5, the stirring temperature is 55-60° C., and the stirring time is 1 h.

[0015] Furthermore, in step (2), the water bath ultrasonication time is 10 to 15 minutes.

[0016] Furthermore, in step (2), 0.45 μm and 0.22 μm microporous membranes are used for filtration three times respectively.

[0017] Furthermore, in the step (3), the concentration of berberine in the mixed solution is 0.8-1 mg / ml.

[0018] Furthermore, in step (3), the concentration of oligofructose in the mixed solution is 10-25 mg / ml.

[0019] Furthermore, in step (3), the concentration of the anti-inflammatory polypeptide in the mixed solution is 0.1-15 mg / ml.

[0020] Furthermore, in step (3), the mass percentage concentration of Pluronic F-127 is 20-30%.

[0021] The invention also provides the liposome composite gel prepared by the preparation method.

[0022] The present invention also provides the use of the liposome composite gel in the preparation of a drug for enhancing the protective and repairing function of the vaginal epithelial barrier and for synergistic anti-inflammatory and antibacterial effects.

[0023] Compared with the prior art, the advantages and technical effects of the present invention are as follows: the present invention encapsulates water-insoluble β-eucalyptol and atractylodesin in liposomes, and then loads them and water-soluble berberine, oligofructose, and anti-inflammatory polypeptides in a composite gel, and the prepared composite gel has extremely high bioavailability and biocompatibility, and the composite gel has temperature responsiveness, rapid gelation at body temperature, and enhanced drug adhesion. The co-loaded drug liposome composite gel containing polypeptides prepared by the present invention has a good therapeutic effect on aerobic bacterial vaginitis, wherein β-eucalyptol can promote the proliferation and migration of epithelial cells to repair the vaginal mucosal epithelium, enhance the colonization ability of lactobacillus, and further regulate the vaginal microenvironment; atractylodesin can enhance the integrity of the epithelial barrier by promoting the expression of tight junction proteins occludin, claudin-1, and zonula occludens-1; polypeptides and Chinese medicine monomers synergistically resist inflammation and accelerate the repair of mucosa; berberine also has a good inhibitory effect on pathogens, and oligofructose can promote the proliferation of lactobacillus. Moreover, the preparation method of the present invention is simple, easy to promote, and has good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results of β-eucalyptol and atractylodesin on the migration of Hacat cells;

[0025] Figure 2 The results of berberine's inhibition on Staphylococcus aureus ATCC6538;

[0026] Figure 3 The results of the mouse paw swelling experiment for anti-inflammatory peptides;

[0027] Figure 4 is the average particle size of Lip1 and Lip2;

[0028] Figure 5 is the gelation time of Lip1-Gel1, Lip1-Gel2, Lip2-Gel1, and Lip2-Gel2;

[0029] Figure 6 The migration results of Hacat cells by Lip1-Gel1, Lip1-Gel2, Lip2-Gel1, and Lip2-Gel2;

[0030] Figure 7 is the level of inflammatory factors in the vaginal tissue of rats after 8 days of treatment;

[0031] Figure 8The results of hematoxylin-eosin (HE) staining of the vaginal tissue of rats after 8 days of treatment. DETAILED DESCRIPTION

[0032] 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.

[0033] Embodiment 1:

[0034] 1. Scratch experiment of β-eucalyptol and atractylodesin on Hacat cells

[0035] Hacat cells in the logarithmic growth phase were cultured for 10 6 Cells were inoculated into 6-well plates and cultured. A 10μl pipette tip was used to scratch vertically, and damaged cells were washed off with PBS. Different solutions were added according to the grouping: blank group (0.5% DMSO), β-eucalyptol group (3μg / ml), and atractylodesin group (1.75μg / ml), and cultured for 24h. Cell migration was photographed using an inverted microscope, and the scratch distance was calculated using Image-J.

[0036] The results are as follows Figure 1 As shown, compared with the blank group and the atractylodesin group, β-eucalyptol could significantly promote the migration of Hacat cells after 24 hours.

[0037] 2. Minimum inhibitory concentration of berberine against Staphylococcus aureus ATCC6538

[0038] The Staphylococcus aureus in the logarithmic growth phase was diluted with sterilized LB broth, and the OD value at a wavelength of 600 nm was measured by UV-visible spectrophotometer. The concentration of the bacterial solution was adjusted to 10 6 CFU / mL. In a sterile 96-well plate, 100 μl of each concentration of drug (82.5 μg / ml, 165 μg / ml, 330 μg / ml, 660 μg / ml) and 100 μl of the above-diluted bacterial solution were added to each well by the multiple microdilution method. Five parallel wells were set for each drug concentration. A blank control group and a positive control group were also set up in the experiment. The 96-well plate was placed in a constant temperature culture shaker at 37°C and 200 rpm for culture. After 24 hours, the absorbance value at a wavelength of 490 / 570 nm was measured using an ELISA reader. Inhibition rate (%) = (OD value of the positive control group - OD value of the drug group) / (OD value of the positive control group - OD value of the blank control group) × 100.

[0039] The results are as follows Figure 2 As shown, the minimum inhibitory concentration of berberine against Staphylococcus aureus is 82.5 μg / ml.

[0040] Example 2: Carrageenan-induced swelling of the paws of mice

[0041] The following peptides were selected from Chinese sea cucumber protein hydrolysate for testing:

[0042] Amino acid sequence of polypeptide KS1: YKGEKGSEGK (SEQ ID NO. 1);

[0043] The amino acid sequence of polypeptide KS2: GAPGDKGAPG (SEQ ID NO. 2);

[0044] The amino acid sequence of polypeptide KS3: GLPGGKGDDG (SEQ ID NO. 3);

[0045] The anti-inflammatory effects of the tested peptides KS1, KS2, and KS3 were evaluated by the mouse carrageenan paw swelling test. Kunming mice, 4 weeks old, 18-22g, were used. The mice were randomly divided into a blank group, a model group, a positive drug group (indomethacin 10 mg / kg), and a peptide group (50 mg / kg), with 10 mice in each group. The blank group and the model group were gavaged with the same volume of normal saline once a day for 5 consecutive days. One hour after the last gavage, 30 μl of normal saline was injected into the right hind paw of the blank group mice, and 30 μl of 1% carrageenan was injected into the model group, the positive drug group, and the peptide group. The thickness of the right hind paw of the mice was measured using a vernier caliper before inflammation and 1, 2, 3, 4, 5, and 6 hours after inflammation. The thickness of the right hind paw swelling (mm) = the thickness of the right hind paw after inflammation (mm) - the thickness of the right hind paw before inflammation (mm).

[0046] The results are as follows Figure 3 As shown, compared with the blank group, the swelling thickness of the mouse paws in other groups reached the highest peak within 2-3 hours. Compared with the model group, the peptide group showed different degrees of anti-inflammatory effects at 2-6 hours, among which the peptide KS1: YKGEKGSEGK (SEQ ID NO.1) had the best anti-inflammatory effect. Therefore, the following experiment selected the peptide KS1 for compounding with other components.

[0047] Example 3: Preparation of drug-loaded liposome composite gel containing polypeptide

[0048] 1. Preparation of drug-loaded liposome composite gel containing polypeptide

[0049] The polypeptide KS1 (SEQ ID NO.1) was synthesized by solid phase synthesis and stored at -20°C for future use. The method for preparing the drug-loaded liposome composite gel containing polypeptides provided by the present invention comprises the following steps:

[0050] (1) According to the mass ratio of β-euclidinol to atractylodisin of 1:1 and 2:1, weigh 3 mg of β-euclidinol and 3 mg of atractylodisin; 4 mg of β-euclidinol and 2 mg of atractylodisin respectively. According to the mass ratio of the total mass of β-euclidinol and atractylodisin mixed with egg yolk lecithin and cholesterol of 2:40:5, weigh 120 mg of egg yolk lecithin and 15 mg of cholesterol. Add 2 ml of anhydrous ethanol to each of them and solubilize them with ultrasound. Add them dropwise into 5 ml of ultrapure water and place them in a constant temperature magnetic stirrer at 55°C and stir for 1 hour to evaporate the ethanol.

[0051] (2) Ultrapure water was added to make up to 5 ml, and the mixture was ultrasonicated in a water bath for 10 min. The mixture was filtered three times using 0.45 μm and 0.22 μm microporous membranes, respectively, to remove free drugs, thereby obtaining drug-loaded liposomes Lip1 and Lip2.

[0052] (3) The two drug-loaded liposomes, berberine (1 mg / ml), oligofructose (20 mg / ml), and 20% Pluronic F-127 were mixed with polypeptide KS1 (0.1 mg / ml and 15 mg / ml), respectively, stirred evenly in an ice-water bath, and allowed to stand at 4°C to eliminate bubbles, thereby obtaining polypeptide-containing co-drug-loaded liposome composite gels Lip1-Gel1, Lip1-Gel2, Lip2-Gel1, and Lip2-Gel2.

[0053] 2. Characterization of drug-loaded liposomes Lip1 and Lip2

[0054] The average particle size (Size), polydispersity index (PDI) and zeta potential of liposomes were determined by dynamic light scattering method using Malvern laser particle size analyzer.

[0055] The average particle size of drug-loaded liposomes is Figure 4 As shown: the average particle size of Lip1 is 135.9±1.061, and that of Lip2 is 138.7±1.801nm. The PDI of Lip1 is 0.172±0.006, and the zeta potential is -43.4±3.39. The PDI of Lip2 is 0.159±0.044, and the zeta potential is -40.5±0.451.

[0056] Example 4: Scratch test of liposome composite gel

[0057] 1. Gelation time of liposome composite gel

[0058] Place Lip1-Gel1, Lip1-Gel2, Lip2-Gel1, and Lip2-Gel2 in a 37°C water bath, and observe and record the gelation time.

[0059] The results are as follows Figure 5As shown, at 37°C, four gels containing different drug ratios can gel within one minute and reach a stable state.

[0060] 2. Scratch test of liposome composite gel on Hacat cells

[0061] Hacat cells in the logarithmic growth phase were cultured for 10 6 The cells were inoculated into 6-well plates and cultured. A 10μl pipette tip was used to scratch vertically, and the damaged cells were washed away with PBS. A blank group and a liposome composite gel group (Lip1-Gel1, Lip1-Gel2, Lip2-Gel1, Lip2-Gel2) were set up and cultured for 24 hours. Cell migration was photographed using an inverted microscope, and the scratch distance was calculated using Image-J. Cell migration rate = (mean distance between cells at 0h - mean distance between cells at 24h) / mean distance between cells at 0h.

[0062] The cell migration rate results were Figure 6 As shown, compared with the blank group, the liposome composite gel groups all had a certain migration effect on Hacat cells, and the composite gel containing Lip2 could significantly promote cell migration.

[0063] Example 5: Animal Experiment Verification

[0064] The experimental animals were SPF Wistar rats, female, 6 weeks old, 180-220g. The aerobic bacterial vaginitis rats infected with Staphylococcus aureus ATCC6538 in situ were used as the model. After the model was successfully established, they were randomly divided into 10 groups: model group, positive drug (clindamycin) group, and liposome composite gel group (Lip1-Gel1, Lip1-Gel2, Lip2-Gel1, Lip2-Gel2). 50μl PBS was injected into the vagina of the model group rats, and the other groups were given drugs once a day. After 8 days of treatment, the rats were anesthetized and killed, and their vaginal tissues were dissected and stored at -80℃ for the detection of inflammatory factor levels; some vaginal tissues were stored in paraformaldehyde, and HE staining was used to further evaluate the pathological changes of vaginal tissues in rats after treatment.

[0065] The results of inflammatory factor levels in rat vaginal tissue are as follows Figure 7 As shown in the figure, compared with the model group, the positive drug group and the gel group can reduce the content of proinflammatory cytokines TNF-α and IL-6, playing an anti-inflammatory role. In the liposome composite gel group, the regulatory effect of Gel2 group is better than that of Gel1 group.

[0066] The pathological changes of vaginal tissue in rats after treatment were as follows Figure 8As shown in the figure, a large number of inflammatory cells infiltrated and tissue epithelial mucosa sloughed in the model group. In the positive drug group, the inflammatory cells decreased, and the thickness of the stratified squamous epithelium was also significantly thinned. In the liposome composite gel group, the inflammatory cells decreased, and the density of the stratified squamous epithelium was significantly thickened compared with the positive drug group.

[0067] In summary, the liposome composite gel prepared by the present invention has a good therapeutic effect on aerobic bacterial vaginitis, can enhance the protection and repair function of the vaginal epithelial barrier, reduce inflammatory response, inhibit the abnormal increase of pathogens, promote the proliferation of beneficial bacteria, and regulate the vaginal flora microenvironment. Among them, Lip2-Gel2 is superior to other components in repairing mucosal epithelium and inhibiting inflammatory response, indicating that β-eudesmol has a repairing effect on vaginal mucosal epithelium, and the polypeptide and the Chinese medicine monomer synergistically resist inflammation, enhance the repair ability, improve the colonization ability of lactobacillus, and further regulate the vaginal microecological balance.

[0068] The present invention loads hydrophobic β-eucalyptol and atractylodesin, and hydrophilic berberine, oligofructose, and polypeptides into a liposome composite gel, which responds to changes in ambient temperature and rapidly gels. The prepared polypeptide-containing co-drug-loaded liposome composite gel is verified to have the effects of enhancing epithelial barrier protection and repair functions, and synergistic anti-inflammatory and antibacterial effects through scratch experiments on Hacat cells, antibacterial experiments on Staphylococcus aureus, and animal experiments on a rat model of aerobic bacterial vaginitis, thereby laying a foundation for the research and development of clinical drugs.

[0069] 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. An anti-inflammatory polypeptide, characterized in that The amino acid sequence of the anti-inflammatory polypeptide is shown in SEQ ID NO.

1.

2. A method for preparing a drug-loaded liposome composite gel containing the anti-inflammatory polypeptide according to claim 1, characterized in that: The preparation method comprises the following steps: (1) dissolving β-eucalyptol, atractylodesin, egg yolk lecithin, and cholesterol in anhydrous ethanol, and using the dissolved mixture as an oil phase; injecting the mixture dropwise into an aqueous phase formed by ultrapure water, and stirring to volatilize the ethanol; the mass ratio of the total mass of β-eucalyptol and atractylodesin to the mass ratio of egg yolk lecithin and cholesterol is 2:40:5; and the mass ratio of β-eucalyptol to atractylodesin is 1-2:1; (2) Add ultrapure water to make up to the initial volume of the aqueous phase, perform water bath sonication, and filter through a microporous membrane to obtain drug-loaded liposomes; (3) The drug-loaded liposomes, berberine, oligofructose, anti-inflammatory peptides, and Pluronic F-127 are mixed to form a mixed solution, stirred evenly in an ice water bath, and allowed to stand to eliminate bubbles, thereby obtaining a liposome composite gel.

3. The preparation method according to claim 2, characterized in that: In the step (1), the volume ratio of the oil phase to the water phase is 2:5, the stirring temperature is 55-60° C., and the stirring time is 1 h.

4. The preparation method according to claim 2, characterized in that: In the step (3), the concentration of berberine in the mixed solution is 1 mg / ml.

5. The preparation method according to claim 2, characterized in that: In the step (3), the concentration of oligofructose in the mixed solution is 20 mg / ml, and the mass concentration of Pluronic F-127 is 20%.

6. The preparation method according to claim 2, characterized in that: In the step (3), the concentration of the anti-inflammatory polypeptide in the mixed solution is 0.1-15 mg / ml.

7. The liposome composite gel prepared by the preparation method according to any one of claims 2 to 6.

8. Use of the liposome composite gel according to claim 7 in the preparation of a drug for enhancing the protective and repairing function of the vaginal epithelial barrier, synergistic anti-inflammatory effects, and inhibiting Staphylococcus aureus.

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