Hyaluronic acid hydrogel xylitol nasal spray and preparation method thereof

Abstract

A preparation method of the hyaluronic acid hydrogel xylitol nasal spray comprises the following steps: step 1, taking purified water, and sequentially adding weighed buffer system raw materials into the purified water to prepare a buffer solution; and 2, slowly adding hyaluronic acid hydrogel into the buffer solution obtained in the step 1, uniformly stirring, then adding xylitol, and uniformly mixing to obtain the hyaluronic acid hydrogel xylitol. On the basis of the prepared nasal spray, hormone drugs can be further loaded to obtain a new nasal spray. The two nasal sprays prepared by the invention have good biological safety, and can be used for nursing and treating rhinitis and nasosinusitis for a long time. Meanwhile, the two nasal sprays both have the effects of inhibiting bacteria and preventing biological membranes of common pathogenic bacteria of paranasal sinuses from being formed, and compared with existing nasal sprays, the treatment effect is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the field of chemical pharmaceuticals, and particularly relates to a hyaluronic acid hydrogel xylitol nasal spray and a preparation method thereof. Background Art

[0002] Chronic rhinosinusitis (CRS) is a persistent inflammation that mainly affects the nasal cavity and sinus regions, significantly reducing the quality of life of patients. The pathological features of CRS include mucosal barrier dysfunction, infiltration of inflammatory cells, impaired ciliary clearance function, increased mucus secretion, and tissue remodeling.

[0003] The presence of biofilms is considered to be one of the important factors leading to the chronicity of rhinosinusitis. It not only increases the difficulty of treatment but may also exacerbate the symptoms of patients. For example, recurrence after nasal polypectomy may be related to the reduction of protective microbial communities and the increase in the number of pathogenic bacteria, which may lead to repeated bacterial infections and biofilm formation, thereby destroying the ciliary structure of the nasal mucosa and causing mucus accumulation. Research shows that the detection rate of bacterial biofilms in the sinus region of CRS patients is about 42%-80%. These biofilms can resist immune system attacks, antibiotic effects, and other treatment methods, and the drug resistance they produce is the main reason why many chronic infectious diseases are difficult to cure.

[0004] Common pathogens include Staphylococcus aureus (S. aureus), Streptococcus pneumoniae (S. pneumoniae), Pseudomonas aeruginosa (P. aeruginosa), and Haemophilus influenzae (H. influenzae). In particular, the biofilms formed by Staphylococcus aureus and the superantigens released by them are closely related to refractory CRS and may be the key driving factors for the progression of the disease.

[0005] Such biofilms can induce the nasal mucosa to secrete type 2 cytokines such as thymic stromal lymphopoietin (TSLP), interleukin-33 (IL-33), and IL-22, thereby causing problems such as airway epithelial damage, allergic reactions, increased mucus secretion, and airway remodeling, and are positively correlated with the high incidence rate and disease severity of eosinophilic inflammation.

[0006] Endoscopic sinus surgery is the gold standard for the treatment of refractory CRS, aiming to remove diseased tissues and improve the natural drainage and ventilation functions of the sinuses. However, the surgery cannot completely remove bacterial biofilms, and postoperative drug treatment for up to two years or longer is still required to remove the biofilms and promote mucosal recovery. Sinus irrigation and topical drug administration, as the preferred methods for the treatment of nasal diseases, have been considered an indispensable auxiliary means for surgery.

[0007] Due to their low systemic side effects, anti-inflammatory properties, and the ability to inhibit inflammatory gene transcription, intranasal corticosteroids have become the first-line treatment for CRS and can reduce the use of antibiotics. In the treatment of sinusitis, it is also of great significance to prolong the residence time of drugs on the local mucosa. However, due to the clearance effect of the nasal mucociliary transport system, traditional nasal sprays are difficult to stay on the nasal and sinus mucosa for a long time and usually only maintain a clearance half-life of about 15 minutes. Summary of the Invention

[0008] Technical problem solved by the present invention: Due to the clearance effect of the nasal mucociliary transport system, traditional nasal sprays are difficult to stay on the nasal and sinus mucosa for a long time and usually only maintain a clearance half-life of about 15 minutes.

[0009] In view of the technical problems existing in the prior art, the present invention designs a hyaluronic acid hydrogel xylitol nasal spray and its preparation method.

[0010] It should be noted that in the present invention, unless otherwise specified, the specific meaning of "including" involving compositional limitations and descriptions includes both the open-ended "including", "containing" and their similar meanings, as well as the closed-ended "consisting of" and their similar meanings.

[0011] In order to solve the above-mentioned existing technical problems, the present invention adopts the following solutions: The first technical solution A preparation method of a hyaluronic acid hydrogel xylitol nasal spray, comprising the following steps: Step 1: Take pure water, and sequentially add the weighed buffer system raw materials into the pure water to prepare a buffer solution; Step 2: Slowly add hyaluronic acid hydrogel to the buffer solution obtained in Step 1, stir evenly, and then add xylitol and mix evenly to prepare the hyaluronic acid hydrogel xylitol.

[0012] Further, the pH value of the buffer solution in Step 1 is 6-7.

[0013] Further, the molecular weight of the hyaluronic acid hydrogel in Step 2 is 500,000-1,000,000 Da; Further, the mass concentration of the hyaluronic acid hydrogel in Step 2 in pure water is 0.05-0.15%.

[0014] Further, the mass concentration of the xylitol in Step 2 in pure water is 3-7%.

[0015] In the present invention, the requirement for the buffer solution is to be able to adjust the pH value of the reaction system to 6-7 to ensure the effect of the prepared product.

[0016] In the present invention, the mass concentration mentioned is the mass / volume ratio (w / v).

[0017] It should be noted that in some specific embodiments of the present invention, the preferred buffer solution is a phosphate buffer solution, and the composition of the buffer system raw materials is sodium dihydrogen phosphate and disodium hydrogen phosphate, or potassium dihydrogen phosphate and dipotassium hydrogen phosphate.

[0018] The mass ratio of sodium dihydrogen phosphate and disodium hydrogen phosphate can be any ratio that maintains the pH value of the solution between 6 and 7.

[0019] The second technical solution A hyaluronic acid hydrogel xylitol nasal spray prepared by the above-mentioned preparation method.

[0020] In the present invention, the hyaluronic acid hydrogel xylitol nasal spray is abbreviated as: methyl nasal spray or xylitol solution.

[0021] The third technical solution A hyaluronic acid hydrogel xylitol nasal spray loaded with a hormonal drug, which is prepared by loading a hormonal drug into the hyaluronic acid hydrogel xylitol nasal spray described in claim 5, and the hormonal drug is mometasone furoate or budesonide or fluticasone propionate.

[0022] In the present invention, the hyaluronic acid hydrogel xylitol nasal spray loaded with a hormonal drug can be abbreviated as: ethyl nasal spray or xylitol solution with MF).

[0023] In the present invention, mometasone furoate is a topical glucocorticoid, and its main effects include: First, the anti-inflammatory effect: Mometasone furoate can reduce the release of inflammatory factors in the nasal mucosa and decrease the activity of allergy mediators such as histamine and leukotriene, thereby effectively reducing the inflammation of the nasal mucosa.

[0024] Second, the relief of nasal symptoms: Mometasone furoate can significantly relieve the symptoms of nasal congestion, runny nose, nasal itching, sneezing, etc. caused by allergic rhinitis, and at the same time can also improve the symptoms of eye itching, eye redness, tearing, etc.

[0025] Third, prevention and treatment: For patients with seasonal or perennial allergic rhinitis, mometasone furoate can also be used for prevention and treatment.

[0026] Fourth, high safety: Mometasone furoate does not cause systemic side effects at therapeutic doses, and its systemic bioavailability is extremely low, so the impact on the whole body is minimal.

[0027] In the present invention, Budesonide is a highly effective glucocorticoid, and its main role in nasal sprays is to relieve the inflammatory response of the nasal mucosa through its powerful anti-inflammatory and immunosuppressive effects. When Budesonide is used in the form of a nasal spray, it can directly act on the inflamed areas in the nasal cavity and exert the following effects: First, anti-inflammatory effect: Budesonide can inhibit the release of various inflammatory mediators, such as histamine, prostaglandins, leukotrienes, etc., thereby reducing the inflammatory response of the nasal mucosa.

[0028] Second, immunosuppression: It can reduce the activation and migration of inflammatory cells, lower the immune system's response to allergens, and thus relieve the symptoms of allergic rhinitis.

[0029] Third, symptom relief: Budesonide nasal spray is commonly used to treat allergic rhinitis and non-allergic rhinitis, and can effectively relieve symptoms such as nasal congestion, runny nose, sneezing, and nasal itching.

[0030] Fourth, local action: Since Budesonide is used locally in nasal sprays and has less systemic absorption, it reduces systemic side effects and makes long-term use safer.

[0031] In the present invention, Fluticasone propionate is a potent locally acting glucocorticoid. When it is used in the form of a nasal spray, it mainly exerts the following effects: First, anti-inflammatory effect: Fluticasone propionate can inhibit the release of inflammatory mediators, such as histamine, cytokines, and leukotrienes, etc., thereby reducing the inflammatory response of the nasal mucosa.

[0032] Second, immunomodulation: It reduces the activation and infiltration of inflammatory cells, lowers the sensitivity of the immune system to allergens, and helps control the symptoms of allergic rhinitis.

[0033] Third, symptom relief: Fluticasone propionate nasal spray is used to treat seasonal allergic rhinitis and perennial allergic rhinitis, and can effectively relieve symptoms such as nasal congestion, runny nose, sneezing, and nasal itching.

[0034] Fourth, local high efficiency and low systemic absorption: Fluticasone propionate has a high anti-inflammatory effect when used locally, and at the same time, due to its low systemic absorption, it reduces the risk of systemic side effects and is suitable for long-term use.

[0035] The fourth technical solution

[0036] A preparation method of the above-mentioned hyaluronic acid hydrogel xylitol nasal spray loaded with hormonal drugs comprises the following steps: Step 1: Take pure water, add poloxamer 407 and hormonal drugs, and dissolve the hormonal drugs at 0 °C to prepare Liquid A. Step 2: Take pure water, add the weighed raw materials of the buffer system to the pure water in turn to prepare a buffer solution; slowly add hyaluronic acid hydrogel to the prepared buffer solution to obtain Liquid B. Step 3: Add Liquid A prepared in Step 1 to Liquid B prepared in Step 2. After mixing evenly, add xylitol and mix evenly to prepare a hyaluronic acid hydrogel xylitol nasal spray loaded with hormonal drugs.

[0037] Furthermore, the mass concentration of poloxamer 407 in pure water in Step 1 is 0.2 - 0.4%. Furthermore, the mass concentration of the hormonal drugs in pure water in Step 1 is 0.02 - 0.05%. Furthermore, the molecular weight of the hyaluronic acid hydrogel in Step 2 is 500,000 - 1,000,000 Da. Furthermore, the mass concentration of the hyaluronic acid hydrogel in pure water in Step 2 is 0.05 - 0.15%.

[0038] Furthermore, the pH value of the buffer solution in Step 2 is 6 - 7.

[0039] Furthermore, the mass concentration of xylitol in pure water in Step 3 is 1 - 5%.

[0040] Poloxamer is a non-ionic triblock copolymer composed of polyoxyethylene and polyoxypropylene, with a unique amphiphilic structure. It has good physical stability in the present invention. By forming micelle structures, it can increase the solubility of various drugs, prolong the residence time of drugs, prepare sustained-release or controlled-release preparations, and improve the solubility and absorption rate of drugs.

[0041] In the present invention, hyaluronic acid (HA) hydrogel (HA-gel) is of great significance: Firstly, the excellent mucoadhesive property and moisturizing effect of HA-gel can avoid side effects such as dehydration, increased nasal mucosa irritation, and nasal pain that may be caused by traditional nasal sprays.

[0042] Secondly, by slowly releasing the loaded drugs, HA-gel can maintain the same therapeutic effect at a lower initial dose and reduce systemic side effects.

[0043] Thirdly, nebulized hyaluronic acid has a good effect on promoting nasal mucosa healing after CRS surgery.

[0044] The negatively charged ion-containing hyaluronic acid hydrogel (HA-gel) has attracted much attention due to its properties similar to those of the natural extracellular matrix, as well as its functions such as inhibiting fibroplasia, hemostasis, isolating wounds, and promoting wound healing.

[0045] Fourthly, HA-gel also has good mucoadhesion, drug targeting, long-acting property, and safety, making it an ideal carrier for nasal spray administration.

[0046] In the present invention, the selection of xylitol is of great significance: Firstly, xylitol is a natural pentitol. Due to its inherent antibacterial and anti-adhesion properties, it can effectively reduce the ionic strength of the airway surface liquid, decrease the chloride concentration, and promote the decomposition of nasal crusts, which is particularly beneficial to CRS patients.

[0047] Secondly, nasal irrigation with xylitol is the only method that has a statistically significant reduction in the sinus symptom score after CRS surgery and has the highest level of evidence-based evidence.

[0048] In the present invention, the hyaluronic acid hydrogel HA-gel and xylitol have a synergistic effect: Firstly, HA-gel has extremely strong moisturizing ability and can form a hydrated film in the nasal cavity to reduce dryness and irritation. Xylitol also has moisturizing property, can adsorb moisture, enhance the moisturizing effect of HA-gel, further lubricate the nasal cavity, and relieve dryness and discomfort.

[0049] Secondly, HA-gel promotes cell migration and proliferation and helps repair damaged nasal mucosa. Xylitol has antibacterial and anti-inflammatory effects, reduces infection and inflammation, protects the mucosa, and enhances the repair effect.

[0050] Thirdly, HA-gel itself has a certain anti-inflammatory effect and can reduce nasal inflammation. Xylitol reduces nasal infection and inflammation by inhibiting bacterial growth and regulating immune responses, and synergistically enhances the anti-inflammatory effect with HA-gel.

[0051] Fourthly, HA-gel has good adhesiveness and prolongs the residence time of the drug in the nasal cavity. Xylitol enhances the adhesiveness of HA-gel, forms a more stable sustained-release system, and prolongs the moisturizing and repair effects.

[0052] Fifthly, HA-gel has good biocompatibility and is comfortable to use. Xylitol improves the use experience, reduces irritation, and enhances tolerance.

[0053] Based on the above theory, HA-gel is used as a carrier and effectively combined with Xylitol, or mometasone furoate (MF) (or budesonide, fluticasone propionate) is added to prepare a hyaluronic acid hydrogel xylitol nasal spray or a hyaluronic acid hydrogel xylitol nasal spray loaded with hormonal drugs, which can play a synergistic therapeutic role after CRS surgery.

[0054] The present invention provides a hyaluronic acid hydrogel xylitol nasal spray and its preparation method, which have the following beneficial effects: 1. The nasal spray prepared in the present invention has good biosafety and can be used for the nursing treatment of rhinitis and sinusitis for a long time. At the same time, both nasal sprays prepared in the present invention have the effects of inhibiting bacteria and preventing the formation of biofilms of common pathogenic bacteria in the sinuses, and the effect of nasal spray B is significantly improved compared with nasal spray A.

[0055] 2. The residence time of the nasal spray prepared in the present invention in the nasal mucosa is about 27.83 ± 1.03 minutes, which is significantly longer than the elimination half-life of about 15 minutes of ordinary nasal sprays. This shows that the xylitol nasal spray with hyaluronic acid hydrogel as the carrier should achieve better therapeutic effects when applied clinically than ordinary nasal sprays.

[0056] 3. The nasal spray prepared in the present invention has the feasibility of good large-scale production, with wide sources of raw materials, stable production processes, and low and controllable costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 : Schematic diagram of the CCK-8 test results of nasal sprays A and B prepared in Example 1 and Preparation Example 1 of the present invention; Figure 2 : Schematic diagram of the LDH content test results of nasal sprays A and B prepared in Example 1 and Preparation Example 1 of the present invention; Figure 3 : Schematic diagram of the qRT-PCR test results of nasal sprays A and B prepared in Example 1 and Preparation Example 1 of the present invention; Figure 4 : Schematic diagram of the antibacterial effect test results of nasal sprays A and B prepared in Example 1 and Preparation Example 1 of the present invention; Figure 5 : Schematic diagram of the biofilm prevention performance test results of nasal sprays A and B prepared in Example 1 and Preparation Example 1 of the present invention; Figure 6 : Scanning electron micrograph of the biofilm formation of Streptococcus pneumoniae in nasal spray A and B groups prepared in Example 1 and Preparation Example 1 of the present invention; Figure 7: SEM images of the formation of Staphylococcus aureus biofilms in the nasal sprays of Group A and Group B prepared in Example 1 and Preparation Example 1 of the present invention; Figure 8 : SEM images of the formation of Pseudomonas aeruginosa biofilms in the nasal sprays of Group A and Group B prepared in Example 1 and Preparation Example 1 of the present invention. Detailed implementation manners

[0058] The present invention will be further described below in conjunction with specific embodiments and the accompanying drawings: It should be noted that the hyaluronic acid hydrogel used in the present invention can be a commercially available standard product, and the xylitol used in the experiments is of medical grade.

[0059] Examples of the preparation method of the hyaluronic acid hydrogel xylitol nasal spray: Example

[0060] Step 1: Take 100 ml of purified water, add 0.05 g of sodium dihydrogen phosphate and 0.03 g of disodium hydrogen phosphate to prepare a buffer solution, and the pH value of the solution is about 6.90.

[0061] Step 2: Slowly add 0.1 g of hyaluronic acid hydrogel with a molecular weight of 800,000 Da to the buffer solution obtained in Step 1, stir evenly, and then add 5 g of xylitol and mix evenly to prepare the hyaluronic acid hydrogel xylitol. Example

[0062] Step 1: Take 100 ml of purified water, add 0.05 g of sodium dihydrogen phosphate and 0.01 g of disodium hydrogen phosphate to prepare a buffer solution, and the pH value of the solution is about 6.43.

[0063] Step 2: Slowly add 0.05 g of hyaluronic acid hydrogel with a molecular weight of 1,000,000 Da to the buffer solution obtained in Step 1, stir evenly, and then add 7 g of xylitol and mix evenly to prepare the hyaluronic acid hydrogel xylitol. Example

[0064] Step 1: Take 100 ml of purified water, add 0.06 g of potassium dihydrogen phosphate and 0.01 g of dipotassium hydrogen phosphate to prepare a buffer solution, and the pH value of the solution is about 6.32.

[0065] Step 2: Slowly add 0.15 g of hyaluronic acid hydrogel with a molecular weight of 500,000 Da to the buffer solution obtained in Step 1, stir evenly, and then add 3 g of xylitol and mix evenly to prepare the hyaluronic acid hydrogel xylitol.

[0066] Preparation examples of the preparation method of the hyaluronic acid hydrogel xylitol nasal spray loaded with hormonal drugs: Preparation Example 1 Step 1: Take 10 ml of pure water, add 0.3 g of poloxamer 407 and 40 mg of mometasone furoate, and dissolve the hormonal drug at 0 °C to prepare Liquid A; Step 2: Take 90 ml of pure water, add 0.05 g of sodium dihydrogen phosphate and 0.03 g of disodium hydrogen phosphate to prepare a buffer solution; slowly add 0.1 g of hyaluronic acid hydrogel with a molecular weight of 800,000 Da to the prepared buffer solution to obtain Liquid B; Step 3: Add Liquid A prepared in Step 1 to Liquid B prepared in Step 2, mix evenly, then add 5 g of xylitol and mix evenly to prepare a hyaluronic acid hydrogel xylitol nasal spray loaded with a hormonal drug.

[0067] Preparation Example 2 Step 1: Take 10 ml of pure water, add 0.2 g of poloxamer 407 and 20 mg of budesonide, and dissolve the hormonal drug at 0 °C to prepare Liquid A; Step 2: Take 90 ml of pure water, add 0.05 g of sodium dihydrogen phosphate and 0.01 g of disodium hydrogen phosphate to prepare a buffer solution; slowly add 0.05 g of hyaluronic acid hydrogel with a molecular weight of 1,500,000 Da to the prepared buffer solution to obtain Liquid B; Step 3: Add Liquid A prepared in Step 1 to Liquid B prepared in Step 2, mix evenly, then add 1 g of xylitol and mix evenly to prepare a hyaluronic acid hydrogel xylitol nasal spray loaded with a hormonal drug.

[0068] Preparation Example 3 Step 1: Take 10 ml of pure water, add 0.4 g of poloxamer 407 and 50 mg of fluticasone propionate, and dissolve the hormonal drug at 0 °C to prepare Liquid A; Step 2: Take 90 ml of pure water, add 0.06 g of potassium dihydrogen phosphate and 0.01 g of dipotassium hydrogen phosphate to prepare a buffer solution; slowly add 0.15 g of hyaluronic acid hydrogel with a molecular weight of 500,000 Da to the prepared buffer solution to obtain Liquid B; Step 3: Add Liquid A prepared in Step 1 to Liquid B prepared in Step 2, mix evenly, then add 4 g of xylitol and mix evenly to prepare a hyaluronic acid hydrogel xylitol nasal spray loaded with a hormonal drug.

[0069] In the following experimental property tests, the nasal sprays prepared in Example 1 (Nasal Spray A) and Preparation Example 1 (Nasal Spray B) of the present invention were used for determination.

[0070] First Group: Biocompatibility Experiment of Nasal Spray (1) Cell Culture Human nasal mucosa epithelial cells (HNEpC) (purchased from Wuhan Punosai Life Science Co., Ltd.: CP-H252) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C and 5% CO 2 2.

[0071] The experiments were performed using cells at passages 2 - 3. The cells were treated with 5 μg / mL of LPS for 10 h to establish an inflammatory cell model. LPS stands for Lipopolysaccharide A: Control group B: Group 1 (LPS) C: Group 2 (LPS + sterilized saline) D: Group 3 (LPS + Nasal Spray A) E: Group 4 (LPS + Nasal Spray B) (2) CCK-8 assay The purpose of the CCK-8 assay was to detect the cytotoxicity of the nasal sprays.

[0072] Cell viability was detected by CCK-8 assay at 24, 48, and 72 h.

[0073] The results were as Figure 1 shown: Compared with the Control group, the cell viability in Group 4, Group 1, Group 2, and Group 3 decreased significantly.

[0074] Compared with Group 1, there was no significant difference in cell viability between Group 2 and Group 3, while the cell viability in Group 4 increased significantly.

[0075] In Figure 1 , * indicates p < 0.05, ** indicates p < 0.01 vs Control group; & indicates p < 0.05 vs Group 1.

[0076] (3) Lactate Dehydrogenase (LDH) assay The results of the LDH content detected by the kit were as Figure 2 shown: Compared with the Control group, the LDH content in Group 1 and Group 2 increased significantly, while the LDH content in Group 3 decreased significantly; Compared with Group 1, there was no significant difference in the LDH content between Group 2, while the LDH content in Group 3 decreased significantly.

[0077] In Figure 2 it, ** indicates p < 0.01 vs Control group; ## indicates p < 0.05 vs Group 1.

[0078] (4)Detection by quantitative reverse transcription polymerase chain reaction (qRT-PCR) qRT-PCR was used to detect the mRNA expression level of the ciliated cell marker FOXJ1, and the results were as Figure 3 shown: Compared with the Control group, the FOXJ1 mRNA levels in Group 1, Group 2, and Group 3 were significantly decreased; Compared with Group 1, there was no significant difference in the FOXJ1 mRNA level in Group 2, and the FOXJ1 mRNA level in Group 3 was significantly increased.

[0079] In Figure 3 it, ** indicates p < 0.01 vs Control group; ## indicates p < 0.05 vs Group 1.

[0080] The results of the first group of studies above indicate that both nasal sprays A and B have no cytotoxicity to cilia, have good biocompatibility and safety, and can be used for nasal and sinus rehabilitation treatment for a long time.

[0081] The second group: Bacteriostatic effect of nasal spray and prevention of bacterial biofilm formation (1)Bacterial culture Strains of Staphylococcus aureus (S. aureus) (CICC 21600) and Pseudomonas aeruginosa (P. aeruginosa) (CICC 24649) were cultured in tryptic soy broth (TSB) in an incubator at 37 °C.

[0082] Strains of Streptococcus pneumoniae (S. pneumoniae) (CICC 24947) were cultured on Columbia blood agar plates in an incubator at 37 °C with 5% CO 2 2.

[0083] All work related to S. aureus, P. aeruginosa, and S. pneumoniae was carried out in a biosafety level 3 laboratory, and all experimental bacteria were purchased from the China Center for Industrial Culture Collection. Experimental grouping: Control group (sterilized PBS) Group 1: TSB + ampicillin and polymyxin B Group 2: TSB Group 3: Nasal spray A Group 4: Nasal spray B (2)Detection of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) The nasal spray stock solution was serially diluted two-fold (both Group A and Group B needed to be detected), and 2 mL of tryptic soy broth (TSB) containing the serially diluted nasal spray was prepared in a 5 mL culture tube, and then inoculated with S.aureus 、 P.arugino.sa and S. pneumoniae to an overnight culture with approximately 5 × 10 5 colony-forming units (CFU) / mL.

[0084] After incubation for 24 h, the growth of bacteria was observed. The lowest drug concentration in the tube that inhibited bacterial growth was defined as the MIC, and the lowest bactericidal concentration was defined as the MBC.

[0085] The specific operation is as follows: 21 g of dry powder of TSB tryptic soy broth medium was heated and dissolved in 1000 mL of purified water, and sterilized at 121℃ for 15 min. Take sterilized test tubes, number the test tubes, and dispense 2 mL of sterilized medium into the test tubes; According to the grouping, 2 mL of the nasal spray stock solution was added to the first test tube, mixed well, 2 mL of the mixed solution was aspirated and added to the second test tube and mixed evenly, and then 2 mL was aspirated and added to the third test tube and mixed evenly. The operation was carried out sequentially until the 8th test tube, and 2 mL was aspirated and discarded from the 8th test tube. At this time, the drug concentrations in the 1st - 11th tubes were 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 μg / mL in sequence. The 12th test tube was the control group.

[0086] The activated test strain was made into a bacterial suspension with a bacterial content of 5×10 5 CFU / mL with normal saline. 100 μL of the bacterial suspension was added to each of the 1st - 11th test tubes, and cultured at 35 - 37℃ for 24 h. Observe the growth situation. The lowest drug concentration in the tube that inhibits bacterial growth is defined as the MIC, and the lowest bactericidal concentration is defined as the MBC.

[0087] The MIC and MBC results of each group of S. aureus, P. aeruginosa and S. pneumoniae are shown in Table 1.

[0088] Among them, the MIC and MBC of each bacterium in the methyl nasal spray group were the highest, and the MIC and MBC of each bacterium in the ethyl nasal spray group were lower than those in the methyl nasal spray group.

[0089] The MIC and MBC of each bacterium in the negative control TSB + ampicillin and polymyxin B group were the lowest.

[0090] The above experimental results show that the ethyl nasal spray has good antibacterial and bactericidal effects, indicating that it can effectively inhibit and kill bacteria at a lower concentration and has excellent antibacterial activity. It can efficiently destroy the growth and survival mechanisms of bacteria and achieve significant antibacterial and bactericidal effects at a low concentration.

[0091] Table 1 MIC and MBC values of different strains

[0092] (3)Detection of antibacterial effect Prepare the nasal spray group A and group B solutions (for subsequent experiments at 5 times the MBC concentration), add them to a 96-well plate, add 100 μL of overnight cultures of Staphylococcus aureus, Pseudomonas aeruginosa, and Streptococcus pneumoniae and dilute them to 5×10 5 CFU / mL, add to each well, and statically culture in a humidified chamber at 37°C for 24 h.

[0093] Use sterilized phosphate-buffered saline (PBS) and ciprofloxacin (50 μg / mL) treatments as negative control and positive control respectively. After incubation, continuously dilute 10 μL of the sample in each well, and use a bacterial cell concentration meter to measure the viable bacteria CFU / mL.

[0094] The results are as Figure 4 shown: Compared with the Control group for strains such as S. aureus, P. aeruginosa, and S. pneumoniae, the CFU in the methyl nasal spray group decreased, but there was no significant difference, and the antibacterial effect was not obvious. The CFU in the ethyl nasal spray group and the ciprofloxacin group decreased significantly, and the antibacterial effect was enhanced.

[0095] (4)Biofilm formation experiment - spread plate Perform spread plate analysis on the above-mentioned incubated bacterial solution for 24 hours.

[0096] During spread plate analysis, serially dilute the bacterial suspension, perform gradient dilution 100-fold each time up to 1000-fold, spread it on tryptone soy agar (TSA), then incubate at 37°C, and count the colonies after 24 hours of incubation.

[0097] Biofilm prevention test - The biofilm prevention performance of the whole preparation was determined by the colony biofilm assay method.

[0098] The results are as Figure 5 shown: Compared with the Control group, the colony CFU of S. aureus, P. aeruginosa and S. pneumoniae decreased in the methyl nasal spray group, but there was no significant difference. The colony CFU in the ethyl nasal spray group and ciprofloxacin group decreased significantly.

[0099] (5)Biofilm formation assay - SEM detection 1) S. aureus, P. arugino.sa and S. pneumoniae strains were incubated overnight in tryptic soy broth (TSB) in an incubator at 37 °C.

[0100] 2) After centrifugation, the supernatant was discarded, and the bacteria were resuspended in TSB and the sample extract. The bacterial suspension was diluted to 1×106 CFU / mL for standby. A group of round glass cover slips with a diameter of 12 mm were autoclaved. The sterile glass cover slips were placed in a 24-well plate, and the diluted bacterial suspension (500 μL, 1×106 CFU / mL) was added to each well to immerse the glass cover slips, and incubated at 37 °C for 24 hours.

[0101] 3) After the cover slips were dried, the conductive tape was first adhered to the sample stage, then the powder sample was evenly sprinkled on the conductive tape, and then the unadhered sample was blown off with compressed air.

[0102] 4) The sample on the sample stage was placed in an ion sputtering instrument for coating. After coating, it was adhered to the edge of the slide and the stage with conductive glue to form a complete conductor between the coated surface and the stage.

[0103] 5) Electron microscopy observation: The accelerating voltage of the scanning electron microscope was set at 8 - 10 KV for observation and photography.

[0104] The results are as Figures 6 - 8 shown: Compared with the Control group, for the biofilm formation of bacteria such as S. aureus, P. aeruginosa and S. pneumoniae, it was clearly visible under magnification of 2000 times, 5000 times and 10000 times respectively in the scanning electron microscope. There was no significant difference in the colony biofilm formation in the methyl nasal spray group, while the colony biofilm formation in the ethyl nasal spray group and ciprofloxacin group decreased.

[0105] The results of the above second group of experimental studies indicate that both nasal sprays A and B have antibacterial effects and can prevent the formation of biofilms. Long-term application in the nasal cavity and paranasal sinuses may inhibit bacterial growth, prevent biofilm formation, and promote rehabilitation treatment.

[0106] Group 3: Testing the residence time of nasal sprays in the nasal cavity Ten healthy young students were recruited, with no history of chronic rhinitis and normal taste test results.

[0107] Method: Take 0.5 ml of nasal spray A and spray it into the middle nasal meatus under nasal endoscope monitoring. After 20 minutes, ask if the subject feels a sweet taste, and then ask every 5 minutes. Do this once a day for 3 days, and record the duration of the sweet taste.

[0108] The test results of the residence time of nasal sprays in the nasal cavity are shown in Table 2 below: Table 2

[0109] From the above three groups of experiments, it can be seen that: 1. Nasal sprays A and B have good biosafety and can be used for the long-term care and treatment of rhinitis and sinusitis. At the same time, both nasal sprays have antibacterial effects and can prevent the formation of biofilms by common pathogenic bacteria in the sinuses. Compared with nasal spray A, the effect of nasal spray B is significantly improved.

[0110] 2. The residence time in the nasal mucosa is approximately 27.83 ± 1.03 minutes, which is significantly longer than the elimination half-life of about 15 minutes of ordinary nasal sprays. This shows that the xylitol nasal spray with hyaluronic acid hydrogel as the carrier should achieve better therapeutic effects when applied clinically than ordinary nasal sprays.

[0111] Verified by experiments, the two nasal sprays prepared in the examples and preparation examples of the present invention both have good biosafety, have antibacterial and biofilm formation prevention effects, and the nasal clearance time in clinical trials is significantly extended. The nasal sprays prepared by the present invention have significantly enhanced clinical effects.

[0112] The present invention has been described exemplarily above in combination with the embodiments and the drawings. Obviously, the implementation of the present invention is not limited by the above methods. As long as various improvements are made by adopting the method concept and technical solution of the present invention, or the concept and technical solution of the present invention are directly applied to other occasions without improvement, they are all within the protection scope of the present invention.

Claims

1. A method for preparing a hyaluronic acid hydrogel xylitol nasal spray, characterized in that: The steps include: Step 1: Take purified water, and add the weighed buffer system raw materials into the purified water in sequence to prepare a buffer solution; Step 2: slowly add the hyaluronic acid hydrogel to the buffer solution obtained in step 1, stir evenly, then add xylitol, mix evenly, and prepare the hyaluronic acid hydrogel xylitol.

2. The method for preparing the hyaluronic acid hydrogel xylitol nasal spray according to claim 1, characterized in that: The pH value of the buffer solution described in step 1 is 6-7.

3. The method for preparing the hyaluronic acid hydrogel xylitol nasal spray according to claim 1, characterized in that: The molecular weight of the hyaluronic acid hydrogel described in step 2 is 500,000-1,000,000 Da; The mass concentration of the hyaluronic acid hydrogel in pure water described in step 2 is 0.05-0.15%.

4. The method for preparing the hyaluronic acid hydrogel xylitol nasal spray according to claim 1, characterized in that: The mass concentration of xylitol in pure water described in step 2 is 3-7%.

5. A hyaluronic acid hydrogel xylitol nasal spray prepared according to the preparation method according to any one of claims 1 to 4.

6. A hyaluronic acid hydrogel xylitol nasal spray loaded with hormone drugs, characterized in that: The nasal spray is prepared by loading hormone drugs on the hyaluronic acid hydrogel xylitol nasal spray as claimed in claim 5, wherein the hormone drugs are mometasone furoate, budesonide or fluticasone propionate.

7. A method for preparing the hyaluronic acid hydrogel xylitol nasal spray loaded with hormone drugs according to claim 6, characterized in that The following steps are included: Step 1: Take purified water, add poloxamer 407 and hormone drugs, and dissolve the hormone drugs at 0°C to prepare liquid A; Step 2: Take purified water, add the weighed raw materials of the buffer system into the purified water in sequence to prepare a buffer solution; slowly add the hyaluronic acid hydrogel into the prepared buffer solution to prepare liquid B; Step 3: Add the liquid A prepared in step 1 to the liquid B prepared in step 2, mix well, then add xylitol, mix well, and prepare a hyaluronic acid hydrogel xylitol nasal spray loaded with hormone drugs.

8. The method for preparing the hyaluronic acid hydrogel xylitol nasal spray loaded with hormone drugs according to claim 7, characterized in that: The mass concentration of poloxamer 407 in pure water described in step 1 is 0.2-0.4%; The mass concentration of the hormone drug described in step 1 in pure water is 0.02-0.05%.

9. The method for preparing the hyaluronic acid hydrogel xylitol nasal spray loaded with hormone drugs according to claim 7, characterized in that: The molecular weight of the hyaluronic acid hydrogel described in step 2 is 500,000-1,000,000 Da; The mass concentration of the hyaluronic acid hydrogel in pure water described in step 2 is 0.05-0.15%; The pH value of the buffer solution described in step 2 is 6-7.

10. The method for preparing the hyaluronic acid hydrogel xylitol nasal spray loaded with hormone drugs according to claim 7, characterized in that: The mass concentration of xylitol in pure water described in step 3 is 1-5%.

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