Mussel mucin-xylitol nasal spray as well as preparation method and application thereof

Abstract

The invention relates to a mussel mucin-xylitol nasal spray as well as a preparation method and application thereof. The nasal spray is used for treating allergic rhinitis. The spray takes mussel mucin and xylitol as core active components, the mussel mucin and the xylitol are compounded in a phosphate buffer solution according to a specific concentration, and a stable preparation is formed after sterilization. The retention time of xylitol in nasal mucosa is prolonged through the wet adhesiveness of MAP, meanwhile, the effects of repairing tight junction, inhibiting type 2 inflammation and resisting oxidation are synergistically achieved, and the defects that an existing medicine cannot synchronously improve AR double pathological mechanisms and is low in safety and short in action time are overcome. In-vitro cell experiments and in-vivo animal experiments prove that the spray disclosed by the invention can be used for remarkably improving the integrity of the nasal mucosa barrier and reducing the level of inflammatory factors, is high in safety and good in stability, is suitable for special crowds such as common AR patients and military personnel, and has important clinical transformation value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a mussel adhesive protein-xylitol nasal spray, its preparation method and application. Background Technology

[0002] Allergic rhinitis (AR) is a common disease characterized by chronic inflammation of the nasal mucosa. Its core pathological mechanisms include Th2 cell-mediated immune imbalance and impaired nasal mucosal epithelial barrier function. Statistics show that there are over 250 million AR patients in my country, with an even higher incidence rate among children, making it a significant public health challenge.

[0003] In the pathological process of AR, eosinophil infiltration, excessive mucus secretion, and downregulation of tight junction (TJ) proteins (such as ZO-1 and occludin) jointly damage the structural and functional integrity of the nasal mucosa. In addition, oxidative stress further aggravates the inflammatory response and epithelial damage by activating signaling pathways such as NF-κB, forming a vicious cycle of "inflammation-barrier destruction-oxidative stress".

[0004] Currently, first-line treatment for nasal allergy (AR) primarily involves nasal corticosteroids (such as mometasone furoate) and antihistamines, but these methods have significant limitations: First, long-term use of nasal corticosteroids can easily lead to nasal mucosal atrophy and increased risk of infection; second, approximately 29% of pediatric patients and 62% of adult patients experience inadequate symptom control, and existing medications cannot simultaneously achieve the dual goals of "inflammation suppression" and "barrier repair." Especially for special populations such as military personnel, who are chronically exposed to particulate matter and chemical irritants such as dust and smoke, their nasal mucosal barrier is more easily damaged, and conventional medications cannot meet their needs for both long-lasting efficacy and safety.

[0005] Mussel adhesive protein (MAP) is a type of natural protein with excellent wet adhesion and biocompatibility. Its core functional component, 3,4-dihydroxyphenylalanine (DOPA), can form a stable retention on the mucosal surface through strong adhesion mediated by catechol groups. It also has inherent anti-inflammatory, antioxidant and antibacterial activities, making it an ideal mucosal drug delivery carrier.

[0006] Xylitol, a natural pentose sugar alcohol, has been shown to upregulate tight junction protein expression and inhibit biofilm formation, and has seen initial applications in nasal care. However, in current technologies, both MAP and xylitol are used individually in related fields: MAP is mostly used in mucosal carrier design and has not been involved in AR treatment or synergistic combinations with xylitol; xylitol is only used for nasal irrigation, lacking verification of its anti-inflammatory mechanism and long-term retention capacity. No studies have yet combined the two to develop a dual-effect treatment product targeting the dual pathological mechanisms of "inflammation-barrier damage" in AR, nor have they addressed the technical challenges of existing drugs, such as low safety, short duration of action, and inability to simultaneously repair the barrier and inhibit inflammation. Summary of the Invention

[0007] The technical problems solved by this invention are: existing allergic rhinitis (AR) drugs can only intervene at a single target of "anti-inflammatory" or "barrier repair", and cannot simultaneously inhibit type 2 inflammation and rebuild the tight junction of the nasal mucosa; long-term use of glucocorticoids can easily lead to mucosal atrophy and high risk of infection; the duration of action is short (≤6 h) and multiple daily doses are required; and there is a lack of safe and long-acting solutions for people exposed to dust / chemical particles.

[0008] In view of the technical problems existing in the prior art, this invention designs a mussel adhesive protein-xylitol nasal spray, its preparation method, and its application. Through the synergistic effect of MAP and xylitol, it achieves a three-in-one effect of "repairing tight junctions + inhibiting inflammation + anti-oxidation," while also possessing excellent mucosal adhesion and safety, making it suitable for long-term treatment and special populations (such as children and military personnel).

[0009] It should be noted that, in this invention, unless otherwise specified, the specific meaning of "comprising" in relation to composition definition and description includes both open-ended meanings such as "comprising," "including," etc., and closed-ended meanings such as "composed of," etc., and similar meanings.

[0010] To solve the aforementioned technical problems, the present invention adopts the following solution: A method for preparing a mussel adhesive protein-xylitol nasal spray includes the following steps: Step 1: Dissolve xylitol in phosphate buffer to obtain a xylitol solution; Step 2: Add mussel adhesive protein to the xylitol solution described in Step 1 and stir to form a composite solution; Step 3: Adjust the pH of the composite solution obtained in Step 2, and filter to remove bacteria; Step 4: Irradiate the filtered and sterilized solution to obtain the nasal mussel adhesive protein-xylitol nasal spray.

[0011] Furthermore, in step 1, the concentration of the xylitol solution is 30-70 mg / mL.

[0012] Furthermore, in step 2, the concentration of mussel adhesive protein in the composite solution is 0.1-0.5 mg / mL.

[0013] Furthermore, the filtration sterilization in step 3 is performed by filtration sterilization through a 0.22μm filter membrane.

[0014] Furthermore, in step 4, irradiation sterilization is performed using... 60 The sterilization dose of Co-γ rays is 20-30 kGy, and the time is 20-40 min.

[0015] Furthermore, the preparation method of the mussel adhesive protein is as follows: recombinant Escherichia coli BL21(DE3) / pET-28a-MAP is used as the fermentation strain, and it is cultured in LB medium at 37°C until OD. 600 Expression was induced by 0.6-0.8 mM IPTG and cultured at 25°C for 16 h. The bacterial cells were collected by centrifugation, sonicated, purified by Ni-NTA affinity chromatography, and desalted by Superdex G-75 gel filtration chromatography to obtain the mussel adhesive protein.

[0016] Furthermore, the content of mussel adhesive protein DOPA is 16-20 mol; the content of mussel adhesive protein endotoxin is <0.1 EU / mg.

[0017] The present invention also discloses a mussel adhesive protein-xylitol nasal spray, which is prepared by the above-mentioned preparation method; The pH value of the spray is 5.8-6.2; the viscosity of the spray is 2.5-3.5 mPa·s.

[0018] In this invention, xylitol is dissolved in phosphate buffer at 37°C. The xylitol is pharmaceutical grade with a purity >99%.

[0019] In this invention, after adding mussel adhesive protein in step 2, it is necessary to stir to make the solution evenly mixed. Generally, stirring at room temperature for 25-40 minutes is required.

[0020] In this invention, step 3 typically involves adjusting the pH to 6.0-7.0 before performing filtration and sterilization.

[0021] The present invention also discloses the use of the above-mentioned mussel adhesive protein-xylitol nasal spray in the preparation of a medicament for the prevention and / or treatment of allergic rhinitis.

[0022] The drugs used in the above applications are used to repair tight junctions of the nasal mucosa epithelium and upregulate the expression of ZO-1 and occludin proteins; at the same time, they can be used to inhibit type 2 inflammatory responses and reduce the levels of one or more factors among IL-4, IL-5, IL-13, TNF-α, and IL-6.

[0023] The drugs used in the above applications are used to reduce oxidative stress levels in the nasal mucosa, increase the activity of superoxide dismutase and glutathione peroxidase, and reduce the levels of reactive oxygen species and malondialdehyde.

[0024] The drugs used in the above applications remain in the nasal mucosa for up to 24 hours.

[0025] The drugs used in the above applications have high safety and good stability, and are suitable for ordinary AR patients and special populations such as military personnel.

[0026] In this invention, the mechanism of action of the nasal spray is as follows: Inhibit type 2 inflammation: Reduce the levels of Th2 cytokines such as IL-4, IL-5, and IL-13, as well as pro-inflammatory factors such as TNF-α and IL-6 in the nasal mucosa and serum (ELISA showed that the concentrations of each factor were reduced by ≥30% compared with the AR model group). Antioxidant: Increases the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in nasal mucosa, and reduces the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) (activity assays showed that SOD activity was increased by ≥25% and ROS levels were reduced by ≥40% compared with the AR model group). Prolonged mucosal retention: The wet adhesiveness of MAP allows the spray to remain on the nasal mucosa for up to 24 hours (the xylitol residue was detected by HPLC-MS, and the xylitol concentration in the nasal mucosal eluent was still ≥5 μg / mL after 24 hours).

[0027] After being sterilized by irradiation, the spray maintains stable physicochemical properties within 6 months of storage at room temperature: viscosity remains at 3 mPa·s, and pH remains at 5.8.

[0028] This invention provides a mussel adhesive protein-xylitol nasal spray, its preparation method, and its application, which have the following beneficial effects: 1. This invention has a dual-effect synergistic innovation mechanism—for the first time, MAP and xylitol are combined. MAP prolongs the retention time of xylitol through adhesion, while exerting anti-inflammatory and antioxidant effects; xylitol focuses on repairing tight junctions. The two work synergistically to address the dual pathological mechanism of AR "inflammation-barrier damage", overcoming the limitations of existing single-target treatments.

[0029] 2. This invention has high safety: In vitro cell experiments show that the survival rate of human nasal epithelial cells (HNEpCs) of this spray is >85% at a concentration of 100 μg / mL, while the cell survival rate of existing nasal corticosteroids (such as mometasone furoate) drops to 62.3% at a concentration of 4 μg / mL, suggesting that it has higher safety and potentially reduces the risk of mucosal atrophy caused by long-term use, making it suitable for children and people who need long-term treatment.

[0030] 3. This invention has excellent stability and durability: its physicochemical properties remain stable for 6 months at room temperature, and it does not require cold chain transportation; the mucosal retention time reaches 24 hours, avoiding the loss of efficacy caused by mucociliary clearance and reducing the frequency of medication.

[0031] 4. The product of this invention has a wide range of applications: it is not only suitable for ordinary AR patients, but also meets the needs of military personnel and other people exposed to particulate matter / chemical irritants, ensuring respiratory health and work capacity by maintaining the integrity of the nasal mucosa. Attached Figure Description

[0032] Figure 1 : Adhesion and retention curve of the mussel adhesive protein-xylitol nasal spray prepared in Example 1 of this invention on the nasal mucosa of mice; Figure 2 : This is a schematic diagram of the establishment and treatment plan for an AR mouse model induced by mussel adhesive protein-xylitol nasal spray OVA prepared in Example 1 of the present invention; Figure 3 : The results of H&E staining of the nasal mucosa of AR mice treated with the mussel adhesive protein-xylitol nasal spray prepared in Example 1 of this invention; Figure 4 : This is a schematic diagram showing the expression and localization of ZO-1 and occludin proteins in the mussel adhesive protein-xylitol nasal spray prepared in Example 1 of the present invention, as revealed by immunofluorescence staining. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments and accompanying drawings: It should be noted that the MAP in this invention can be purchased commercially or prepared by recombinant microbial fermentation, and its DOPA content is required to be 18.2±1.5 mol% (HPLC detection), and its endotoxin level is <0.1 EU / mg; the xylitol is pharmaceutical grade with a purity >99% (Raman spectroscopy verification).

[0034] In an embodiment of the present invention, the preparation method of mussel adhesive protein is as follows: Recombinant Escherichia coli BL21(DE3) / pET-28a-MAP was used as the fermentation strain and cultured in LB medium at 37°C until OD. 600Expression was induced by 0.6-0.8 mM IPTG and cultured at 25℃ for 16 h. Cells were collected by centrifugation, sonicated, purified by Ni-NTA affinity chromatography, and desalted by Superdex G-75 gel filtration chromatography to obtain the mussel adhesive protein. The DOPA content of the mussel adhesive protein was 16-20 mol%; the endotoxin content of the mussel adhesive protein was <0.1 EU / mg.

[0035] Example 1

[0036] Step 1: Dissolve 5g of xylitol in 100mL of phosphate buffer at 37℃ to obtain a xylitol solution with a xylitol concentration of 50 mg / mL; Step 2: Add 0.3g of the mussel adhesive protein prepared above to the xylitol solution, stir at room temperature for 30 min to form a composite solution, and the concentration of mussel adhesive protein is 0.3mg / mL; Step 3: Adjust the pH of the composite solution obtained in Step 2 to 6.0-7.0, and then filter it through a 0.22μm filter membrane for sterilization; Step 4: After dispensing, use 25kGy 60 After irradiating the filtered and sterilized solution with Co γ- for 30 minutes, the solution was sterilized by irradiation to obtain nasal mussel adhesive protein-xylitol nasal spray.

[0037] Example 2

[0038] Step 1: Dissolve 3g of xylitol in 100mL of phosphate buffer at 37℃ to obtain a xylitol solution with a concentration of 30 mg / mL; Step 2: Add 0.1g of the mussel adhesive protein prepared above to the xylitol solution, stir at room temperature for 25 min to form a composite solution, and the concentration of mussel adhesive protein is 0.1mg / mL; Step 3: Adjust the pH of the composite solution obtained in Step 2 to 6.0-7.0, and then filter it through a 0.22μm filter membrane for sterilization; Step 4: After dispensing, use 20kGy 60 The filtered and sterilized solution was irradiated with Co γ- for 40 minutes to obtain a nasal mussel adhesive protein-xylitol nasal spray.

[0039] In this embodiment, the finished product has a viscosity of 2.5 mPa·s and a pH of 6.0. Xylitol can still be detected after 24 hours, and the finished product has excellent anti-inflammatory and antioxidant effects.

[0040] Example 3

[0041] Step 1: Dissolve 7g of xylitol in 100mL of phosphate buffer at 37℃ to obtain a xylitol solution with a xylitol concentration of 70 mg / mL; Step 2: Add 0.5g of the mussel adhesive protein prepared above to the xylitol solution, stir at room temperature for 40 min to form a composite solution, and the concentration of mussel adhesive protein is 0.5mg / mL; Step 3: Adjust the pH of the composite solution obtained in Step 2 to 6.0-7.0, and then filter it through a 0.22μm filter membrane for sterilization; Step 4: After dispensing, use 30kGy 60 After irradiating the filtered and sterilized solution with Co γ- for 20 minutes, the solution was sterilized by irradiation to obtain nasal mussel adhesive protein-xylitol nasal spray.

[0042] In this embodiment, the finished product has a viscosity of 3.5 mPa·s and a pH of 6.2. Xylitol can still be detected after 24 hours, and the finished product has excellent anti-inflammatory and antioxidant effects.

[0043] Strains and reagents: pharmaceutical grade xylitol (from Sigma-Aldrich); Ni-NTA affinity chromatography column, Superdex G-75 gel column (from GE Healthcare); LPS (E. coli O111:B4, from Sigma); mometasone furoate (MF, from Merck); ZO-1 antibody (Proteintech, 68102-1-Ig), occludin antibody (Affinity, AF0120); ELISA kit (from Bioswamp); oxidative stress detection kit (from Beyotime).

[0044] Cells and animals: Human nasal epithelial cells (HNEpCs, purchased from ATCC); BALB / c mice (6-8 weeks old, weighing 18-22g, purchased from Shanghai Slack Laboratory Animal Co., Ltd.).

[0045] Preparation and characterization of MAP MAP was prepared according to the preparation method of Example 1 above; HPLC analysis showed that the DOPA content was 18.5 mol%, and the endotoxin content detected by the Limulus amebocyte lysate (LAL) reagent method was 0.08 EU / mg, which met the requirements.

[0046] Performance and Characterization of the Mussel Adhesive Protein-Xylitol Nasal Spray Prepared in Example 1 of this Invention

[0047] The mussel adhesive protein-xylitol nasal spray prepared in Example 1 was used to test its physicochemical properties: viscosity 3.1 mPa·s, pH 5.8. After 6 months of storage at room temperature, the above parameters were 3.0 mPa·s and pH 5.8, respectively, with no significant changes, indicating good stability.

[0048] Experimental groups: control group (untreated), LPS model group (10 μg / mL LPS treated for 10 h), Mefp treatment group (LPS + the spray of this invention, final concentration 20 μg / mL), MF control group (LPS + 1 μg / mL MF).

[0049] result: (1) Western Blot: The expression levels of ZO-1 and occludin proteins in the Mefp treatment group increased by 52% and 48% respectively compared with the LPS model group, while the phosphorylation level of NF-κB decreased by 42%; (2) ELISA: The concentrations of IL-4, IL-13, and TNF-α in the Mefp treatment group were reduced by 38%, 40%, and 35%, respectively, compared with the LPS model group; (3) Oxidative stress detection: The SOD activity of the Mefp treatment group increased by 28% and the ROS level decreased by 47%, which was better than the MF control group.

[0050] This experiment demonstrates that the spray (Mefp) of this invention can comprehensively exert its protective, anti-inflammatory, and antioxidant effects on airway barrier through multiple targets and pathways, exhibiting a clear protective effect against LPS-induced airway epithelial damage. Particularly noteworthy is its superior effect in reducing ROS compared to the positive control group (MF), highlighting its unique antioxidant advantages and providing strong in vitro experimental evidence for its clinical application.

[0051] Thirdly, regarding in vivo animal experimental verification (ovalbumin OVA-induced AR mouse model, such as...) Figure 2 (As shown) Model building and grouping: Sensitization: On days 1, 8, and 15, mice were intraperitoneally injected with 200 μL of PBS containing 25 μg OVA and 2 mg aluminum hydroxide. Stimulation: From day 22 to 28, administer 20 μL of PBS containing 200 μg OVA via intranasal instillation daily; Groups: control group (physiological saline), AR model group (OVA sensitization + challenge + physiological saline), Mefp group (OVA sensitization + challenge + the spray of this invention, 2 sprays / nostril, twice a day), MF group (OVA sensitization + challenge + MF, 2 sprays / nostril, once a day), 12 animals in each group.

[0052] The results are as follows: (1) Symptom score: The number of sneezes (13.2±3.3 times) and the number of nasal frictions (9.1±3.2 times) in the Mefp group mice within 30 min were significantly lower than those in the AR model group (27.5±4.2 times and 21.2±5.3 times), and there was no statistical difference between the Mefp group and the MF group. (2) Pathological examination: The number of eosinophils infiltrating the nasal mucosa in the Mefp group (23.7±2.3 cells / HPF) and the number of goblet cells (14.3±3.2 cells / HPF) were significantly reduced compared with those in the AR model group (105.6±14.1 cells / HPF and 35.2±5.4 cells / HPF, respectively); the results are as follows. Figure 3 As shown; (3) Western Blot: The expression levels of ZO-1 and occludin proteins in the nasal mucosa of the Mefp group were increased by 60% and 55% respectively compared with those in the AR model group; Figure 4 As shown; (4) ELISA: The serum OVA-specific IgE concentration in the Mefp group was 42% lower than that in the AR model group, and the IL-5 and IL-13 concentrations in the nasal mucosa were 39% and 43% lower, respectively. The inhibitory effect of high-dose Mefp on TNF-α (reduction of 45%) was significantly better than that in the MF group (reduction of 32%).

[0053] This experiment demonstrates that the spray (Mefp) of this invention, in an animal model of allergic rhinitis, can exert a comprehensive therapeutic effect through multiple pathways, including repairing the nasal mucosal barrier, inhibiting IgE production, reducing eosinophil infiltration, and downregulating various inflammatory factors, effectively relieving allergy symptoms and controlling nasal inflammation. Its potential to inhibit certain inflammatory factors (such as TNF-α) is superior to existing standard drugs, providing solid in vivo experimental evidence for its clinical application in the treatment of allergic rhinitis.

[0054] Healthy mice (n=3) were given 20 μL of the spray of this invention or PBS intranasally. Nasal irrigation fluid was collected at 0, 1, 3, 6, 12 and 24 h, and xylitol concentration was detected by HPLC-MS.

[0055] The results are as follows Figure 1 The results showed that the xylitol concentration in the irrigation fluid of the spray group was still 8.3±1.2μg / mL after 24 h, while xylitol was undetectable in the PBS group after 6 h, proving that the spray has excellent mucosal retention.

[0056] The present invention has been described above by way of example with reference to the embodiments and accompanying drawings. Obviously, the implementation of the present invention is not limited to the above-described manner. Any improvements made by adopting the inventive concept and technical solution of the present invention, or the direct application of the inventive concept and technical solution of the present invention to other occasions without modification, are all within the protection scope of the present invention.

Claims

1. A method of preparing a Mytilus mucus-xylitol nasal spray, characterized in that, The method comprises the following steps: Step 1: dissolving xylitol in phosphate buffer to obtain a xylitol solution; Step 2: adding mussel mucin to the xylitol solution obtained in step 1 to form a complex solution; Step 3: adjusting the pH of the complex solution obtained in step 2 and filtering to remove bacteria; Step 4: irradiation sterilization of the solution after filtering to remove bacteria to obtain the mussel mucin-xylitol nasal spray.

2. The method of preparing a mussel myoglobin-xylosol nasal spray according to claim 1, characterized in that: In step 1, the concentration of the xylitol solution is 30-70 mg / mL.

3. The method of preparing a mussel myoglobin-xylitol nasal spray according to claim 1, characterized in that: In step 2, the concentration of the mussel mucin in the complex solution is 0.1-0.5 mg / mL.

4. The method of preparing a mussel myoglobin-xylosol nasal spray according to claim 1, characterized in that: In step 3, the filtering to remove bacteria is performed by filtering through a 0.22 μm filter membrane.

5. The method of preparing a mussel myoglobin-xylosol nasal spray of claim 1, characterized by: In the step 4, the irradiation sterilization uses 60 Co-γ ray, the irradiation sterilization dose is 20-30 kGy, and the time is 20-40 min.

6. The method of preparing a mussel myoglobin-xylosol nasal spray of claim 1, characterized by: The preparation method of the mussel mucin is as follows: recombinant Escherichia coli BL21 (DE3) / pET-28a-MAP is used as a fermentation strain, LB culture medium is cultured at 37 DEG C until OD 600 =0.6-0.8, 0.5mM IPTG is used for inducing expression, and culture is carried out at 25 DEG C for 16 hours; the bacterial body is collected by centrifugation, is broken by ultrasonic, and is purified by Ni-NTA affinity chromatography, desalination is carried out by Superdex G-75 gel filtration chromatography, and the mussel mucin is obtained.

7. A method of preparing a mussel myoglobin-xylitol nasal spray according to claim 6, characterised in that: The mussel mucin has a DOPA content of 16-20 mol%, and an endotoxin content of <0.1 EU / mg.

8. A mussel mucin-xylitol nasal spray prepared according to the preparation method of any one of claims 1-7, wherein the pH of the spray is 5.8-6.2, and the viscosity of the spray is 2.5-3.5 mPa·s.

9. Use of the mussel mucin-xylitol nasal spray of any one of claims 1-7 in the preparation of a medicament for preventing and / or treating allergic rhinitis.

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