Manganese-based Prussian blue-polyacrylic acid / polyvinyl alcohol hydrogel for preventing and treating allergic rhinitis

Abstract

The invention discloses manganese-based Prussian blue-polyacrylic acid / polyvinyl alcohol hydrogel for preventing and treating allergic rhinitis. Monodisperse manganese-based Prussian blue nanoparticles synthesized by taking polyacrylic acid as a template are stably immobilized in a polyacrylic acid / polyvinyl alcohol hydrogel matrix. The obtained composite hydrogel membrane has the following prominent advantages: the composite hydrogel membrane has excellent hydrogel characteristics, including controllable membrane thickness, good tissue adhesion and air permeability; reliable biocompatibility and safety are shown; allergen permeation is effectively blocked through a physical barrier effect; prussian blue is continuously released in the slow-release degradation process, active oxygen free radicals are remarkably removed, and the powerful anti-inflammatory effect is achieved. Due to the synergistic effect of double mechanisms of protection and treatment, the material has important clinical application value and development potential in the fields of prevention and treatment of allergic rhinitis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of nanocomposites and their applications, and particularly relates to a manganese-based Prussian blue-polyacrylic acid / polyvinyl alcohol hydrogel for preventing and treating allergic rhinitis. BACKGROUND

[0002] Allergic rhinitis (AR) is an immunoglobulin E-mediated type I hypersensitivity disease, which originates from chronic mucosal inflammation triggered by environmental allergens such as pollen, house dust mites (HDM) and animal dander, and is accompanied by asthma and chronic sinusitis and other complications. According to the data of "ARIA" (Allergic Rhinitis and its Impact on Asthma), the conservative estimate of AR patients worldwide is more than 500 million, and the first English version of AR diagnosis and treatment guidelines in China, "Chinese Allergic Rhinitis Allergy Diagnosis and Treatment Guidelines", proposed in 2018 that there are about 240 million AR patients in China, and the total social cost of simple drug treatment is as high as 174.9 billion US dollars per year, which is a huge burden to society. [Reference: Han De-min; Zhang Luo; Dong Zhen; Wang De-yun; Lin Xiaoping Allergic Rhinitis, 2007; Allergy 2008, 63 (s86), 8; Allergy Asthma Immunol. Res. 2018, 10 (4), 300.] AR has become a global health problem.

[0003] Current clinical treatment paradigms include allergen avoidance, pharmacotherapy and allergen-specific immunotherapy (AIT). However, complete avoidance of allergens is still unrealistic in clinical practice, and AIT also has limitations, including delayed treatment start (3-6 months), prolonged treatment time (3-5 years) and heavy financial burden. Current nasal drug delivery systems face many limitations, including mucosal irritation and short retention, requiring frequent dosing. Therefore, pharmacotherapy through nasal drug delivery has become the main intervention strategy, and the traditional nasal formulation has an unsatisfactory mucosal retention time, requiring frequent dosing, which makes us urgently need a new intranasal drug delivery system that combines drug sustained retention time with therapeutic effect. At the same time, if the treatment and the blocking of allergens can be combined, better therapeutic effect will be obtained. Based on the current treatment methods, the social needs cannot be met, and in view of the characteristics and pathogenesis of allergic rhinitis sensitization, new treatment strategies are urgently needed to solve the problem.

[0004] Skin covers the entire body and has important functions such as perspiration, temperature regulation, sensory detection, and prevention of fluid loss. Physical protection is one of the key functions of skin. Patients with allergic rhinitis have some damage to their nasal mucosa. Nasal mucosal epithelial cells form the first line of defense through tight junctions and the mucociliary clearance system, blocking allergens and pathogens. If nasal epithelial cells are damaged, the risk of attack from external pathogens and allergens increases. Among synthetic materials, hydrogels with a three-dimensional aqueous network have certain similarities to the structure and mechanical properties of natural skin. Due to their excellent biocompatibility / biodegradability, mechanical properties similar to natural tissues and organs, and environmental responsiveness, hydrogels have been widely used in many biomedical applications, including drug delivery, biosensors, and synthetic extracellular matrix (ECM) for tissue engineering and regenerative medicine. [ACS Macro Lett. 2020, 9, 512−524] Nanozymes have been gradually developed since Academician Yan Xiyun discovered in 2007 that magnetic nanoparticles have properties that mimic natural enzymes, and have been successfully applied in the biomedical field. [References: Nat. Nanotech. 2007, 2(9), 577; Chem. Soc. Rev. 2019, 48(4), 1004;] The development of Prussian blue (PB) nanozymes has attracted widespread attention because PB is an FDA-approved antidote for thallium and cesium poisoning, exhibiting significant multi-enzyme mimicry activity and the ability to effectively neutralize reactive oxygen species. [J. Am. Chem. Soc, 2016, 138, 5860-5865; Int. J. Mol. Sci. 2021, 22(11), 5993] To our knowledge, the excellent catalytic efficiency of nanomaterials stems from their large specific surface area. By altering their morphology and size, ultra-small PB analogs can be obtained, which can effectively improve their therapeutic performance. PB and PB analogs can act as multi-enzyme activities to scavenge reactive oxygen species and inhibit inflammation. PB exhibits neuroprotective effects in a Parkinson's disease (PD) model by inhibiting microglia pyroptosis and NLRP3-mediated dopaminergic neuronal degeneration. Therefore, designing and synthesizing skin-inspired hydrogels, combined with nanomaterials, to achieve both nasal mucosal protection and treatment of allergic rhinitis symptoms will be a significant challenge.

[0005] As can be seen from the above description, developing nasal hydrogels combined with nanomaterials is more beneficial for the treatment of AR. However, there are currently no reports on the use of Prussian blue nanoparticles combined with hydrogels for AR research. Therefore, developing hydrogel membranes containing therapeutic nanoparticles and allergen blocking properties is a challenging topic. Summary of the Invention

[0006] The purpose of this invention is to solve the above-mentioned problems and provide a manganese-based Prussian blue-polyacrylic acid / polyvinyl alcohol hydrogel for the prevention and treatment of allergic rhinitis.

[0007] Manganese-based Prussian blue-polyacrylic acid / polyvinyl alcohol hydrogel is prepared by the following method, including:

[0008] 1) Solution A: Add 0.65 mL of polyacrylic acid solution, 1-10 mmol of potassium citrate and 1-10 mmol of manganese sulfate monohydrate to 75 mL of deionized water and stir until dissolved.

[0009] 2) Solution B: Add 0.1~2 mmol of potassium ferrocyanide trihydrate and 25 mL of ethanol to 50 mL of deionized water and stir;

[0010] 3) Slowly add solution B to solution A in an oil bath at 55-65℃ and react for 12-24 hours. Centrifuge to remove the supernatant to obtain manganese-based Prussian blue nanoparticles.

[0011] 4) Add 1-3 mg of manganese-based Prussian blue nanoparticles to 50-500 μL of polyacrylic acid solution, dissolve by sonication, add 0.5-2 mL of polyvinyl alcohol solution, shake, and obtain nano-manganese-based Prussian blue composite hydrogel.

[0012] The concentration of the polyacrylic acid solution is 0.1~0.3 g / mL, and the concentration of the polyvinyl alcohol solution is 0.04~0.06 g / mL;

[0013] The concentration of the polyacrylic acid solution is 0.2 g / mL, and the concentration of the polyvinyl alcohol solution is 0.05 g / mL;

[0014] The potassium ferrocyanide trihydrate is 0.8 mmol;

[0015] 1 mL of the polyvinyl alcohol solution;

[0016] The potassium citrate content is 2 mmol;

[0017] The amount of manganese sulfate monohydrate mentioned in step 4) is 2 mmol;

[0018] Step 4) involves a polyacrylic acid solution of 100 μL and a polyvinyl alcohol solution of 1 mL.

[0019] The oil bath in step 2) is 60°C; after the shaking in step 4), it is dropped onto a glass slide and left to stand at 37°C for 1 hour.

[0020] Another object of the present invention is to provide the use of manganese-based Prussian blue-polyacrylic acid / polyvinyl alcohol hydrogel for the prevention and treatment of allergic rhinitis.

[0021] The application of the manganese-based Prussian blue-polyacrylic acid / polyvinyl alcohol hydrogel in the preparation of drugs for the prevention and treatment of allergic rhinitis.

[0022] This application provides a manganese-based Prussian blue-polyacrylic acid / polyvinyl alcohol hydrogel for the prevention and treatment of allergic rhinitis. Monodisperse manganese-based Prussian blue nanoparticles, synthesized using polyacrylic acid as a template, are stably immobilized within a polyacrylic acid / polyvinyl alcohol hydrogel matrix. The resulting composite hydrogel membrane possesses the following outstanding advantages: excellent hydrogel properties, including controllable membrane thickness, good tissue adhesion, and breathability; reliable biocompatibility and safety; effective blocking of allergen penetration through physical barrier action; and continuous release of Prussian blue during sustained-release degradation, significantly scavenging reactive oxygen species and exerting a potent anti-inflammatory effect. This synergistic effect of both protective and therapeutic mechanisms makes this material of significant clinical application value and development potential in the prevention and treatment of allergic rhinitis.

[0023] The present invention has the following advantages:

[0024] 1. The method for synthesizing nano-manganese-based Prussian blue composite hydrogel membranes according to this invention is simple and does not introduce other exogenous substances. In this reaction, polyacrylic acid, which induces the synthesis of manganese-based Prussian blue, is uniformly dispersed around the manganese-based Prussian blue and forms a hydrogel membrane with polyvinyl alcohol.

[0025] 2. The hydrogel membrane obtained by this invention can not only serve as a protective membrane against allergens, but also better remove reactive oxygen species (ROS) from inflamed areas, thus achieving the anti-inflammatory purpose of the material itself.

[0026] 3. The hydrogel membrane obtained by this invention possesses the advantages of hydrogels, providing an environment conducive to the repair of damaged nasal tissue. Furthermore, the manganese-based Prussian blue nanoparticles within it have a uniform, nanoscale particle size, which facilitates penetration through nasal mucus and faster entry into nasal mucosal epithelial cells. This achieves the goal of the material not only protecting against allergens and promoting repair but also possessing anti-inflammatory properties. Attached Figure Description

[0027] Figure 1 The images show transmission electron microscopy (TEM) images of nanoparticles in the hydrogel nanofilm prepared in this invention; and the scavenging of superoxide anions (O2·) by nanoparticles at different concentrations. - Multi-enzyme activity tests, including the scavenging effects of TMB colorimetric reaction and methylene blue colorimetric reaction on hydroxyl radicals (·OH);

[0028] Figure 2 SEM image of the freeze-dried hydrogel nanofilm prepared in this invention; FTIR results of the hydrogel film; solidification time of the hydrogel film at 37℃; test images and scanning electron microscope images of allergen protection;

[0029] Figure 3Evaluation of the therapeutic effect of manganese-based Prussian blue composite hydrogel membrane on mice with allergic rhinitis: percentage change in body weight of mice in each group during the experiment; number of times the mice rubbed their noses and sneezed; eosinophil (EOS) and erythrocyte (RBC) counts in the mice's blood routine examination; IgE level in the nasal lavage fluid of mice after drug administration; scan images of the mouse nasal mucosa. H&E stained sections of mouse nasal mucosa; PAS stained sections of mouse nasal mucosa. Detailed Implementation

[0030] Example 1:

[0031] 0.65 mL of polyacrylic acid solution (0.2 g / mL), 2 mmol of potassium citrate, and 2 mmol of manganese sulfate monohydrate were added to 75 mL of deionized water and stirred until dissolved to form solution A. 0.8 mmol of potassium ferrocyanide trihydrate and 25 mL of ethanol were added to 50 mL of deionized water and stirred to form solution B. Solution B was slowly added dropwise to solution A in a 60°C oil bath and reacted for 24 h. Manganese-based Prussian blue nanoparticles were obtained by centrifugation. 100 μL of polyacrylic acid solution (0.2 g / mL) was added to a solution containing 2 mg of manganese-based Prussian blue nanoparticles, and the mixture was dissolved by sonication. Then, 1 mL of polyvinyl alcohol solution (0.05 g / mL) was added, and the mixture was shaken to ensure sufficient contact, forming a nano-manganese-based Prussian blue composite hydrogel.

[0032] Example 2:

[0033] 0.65 mL of polyacrylic acid solution (0.1 g / mL), 1 mmol of potassium citrate, and 10 mmol of manganese sulfate monohydrate were added to 75 mL of deionized water and stirred until dissolved to form solution A. 0.1 mmol of potassium ferrocyanide trihydrate and 25 mL of ethanol were added to 50 mL of deionized water and stirred to form solution B. Solution B was slowly added dropwise to solution A in a 55°C oil bath and reacted for 12 h. Manganese-based Prussian blue nanoparticles were obtained by centrifugation. 50 μL of polyacrylic acid solution (0.1 g / mL) containing 1.0 mg of manganese-based Prussian blue nanoparticles was added and dissolved by sonication. Then, 0.5 mL of polyvinyl alcohol solution (0.04 g / mL) was added and shaken to ensure full contact, resulting in the formation of a nano-manganese-based Prussian blue composite hydrogel.

[0034] Example 3:

[0035] 0.65 mL of polyacrylic acid solution (0.2 g / mL), 10 mmol of potassium citrate, and 1 mmol of manganese sulfate monohydrate were added to 75 mL of deionized water and stirred until dissolved to form solution A. 2.0 mmol of potassium ferrocyanide trihydrate and 25 mL of ethanol were added to 50 mL of deionized water and stirred to form solution B. Solution B was slowly added dropwise to solution A in a 65°C oil bath and reacted for 24 h. Manganese-based Prussian blue nanoparticles were obtained by centrifugation. 500 μL of polyacrylic acid solution (0.3 g / mL) was added to a solution containing 3 mg of manganese-based Prussian blue nanoparticles, and the mixture was dissolved by sonication. Then, 1.1 mL of polyvinyl alcohol solution (0.06 g / mL) was added, and the mixture was shaken to ensure full contact, resulting in a nano-manganese-based Prussian blue composite hydrogel.

[0036] The manganese-based Prussian blue nanoparticles prepared above were observed to be hexagonal by transmission electron microscopy (TEM), with a typical size of less than 100 nm (see...). Figure 1 a). As a rhinitis suppressant, antioxidant capacity is particularly important. Manganese-based Prussian blue nanoparticles exhibit concentration-dependent scavenging effects on reactive oxygen species such as superoxide anions and hydroxyl radicals, and can be used for scavenging reactive oxygen species and anti-inflammatory purposes (see...). Figure 1 bd).

[0037] The above-mentioned hydrogel film is formed and conforms to the porous network structure of hydrogel (see...). Figure 2 The hydrogel film can be completely formed in about 100 minutes when placed in an incubator at 37°C (see ac). Figure 2 d). Furthermore, it effectively prevents allergen mite irritant solution (HDM) from penetrating into the stained paper, a fact verified by scanning, demonstrating that the hydrogel film can act as an allergen protectant, preventing allergens from penetrating the hydrogel (see...). Figure 2 ef).

[0038] The in vivo inhibition of allergic rhinitis (AR) was achieved by nasally instilling 10 μL of high-density nasal discharge (HDM) containing 1 μg / mL into the nasal cavity of mice for three consecutive days. After 7 days of normal feeding, mice were stimulated by nasal instillation of 10 μL of HDM containing 10 μg / mL, along with PBS and manganese-based Prussian blue hydrogel. The control group consisted of normal mice, which only required PBS. This treatment was repeated for 7 days. During the experiment, changes in mouse weight and the frequency of typical AR symptoms such as nose scratching and sneezing were observed and recorded. Initially, it was observed that the weight of mice in each group remained relatively stable. The HDM group showed the least weight gain, while the control group showed stable weight gain, followed by the hydrogel nanofilm group (see...). Figure 3 a). By observing and comparing the number of times mice scratched their noses and sneezed in each group, it was determined that the HDM group had the most obvious symptoms, and after treatment, the hydrogel nanofilm group had the lowest frequency of nose scratching and sneezing, effectively alleviating AR symptoms (see...). Figure 3bc). On day 8, the mice were sacrificed, and blood and nasal mucosa were collected to observe the inhibitory effect on allergic rhinitis. Blood routine results showed that the manganese-based Prussian blue composite hydrogel film could alleviate allergic rhinitis, and the hydrogel film showed good blood safety in mice (see...). Figure 3 Enzyme-linked immunosorbent assay (ELISA) analysis of mouse nasal irrigation fluid showed that the hydrogel film significantly inhibited IgE production (see [details omitted]). Figure 3 f). SEM and H&E staining were used for histological analysis of the mouse nasal mucosa epithelium. The nasal mucosa tissue of mice without rhinitis was intact, with a regular and orderly ciliary structure. However, after HDM stimulation, significant damage to the nasal mucosa tissue was observed, with disordered ciliary clumps and extensive shedding. Among the treatment groups, the hydrogel nanofilm group showed the best recovery of nasal mucosa tissue and cilia (see f). Figure 3 g). Eosinophils are a type of white blood cell that typically plays a role in regulating allergic reactions. In nasal mucosa H&E staining, the area surrounding eosinophils is marked with a yellow arrow. A large number of eosinophils were observed in the HDM group, while their number significantly decreased after hydrogel membrane treatment (see [link]). Figure 3 h). We then performed PAS staining on the nasal mucosa tissue. Goblet cells were found in the PAS staining. Goblet cells are a special type of epithelial cell, and their number is often used as a diagnostic indicator of inflammation. They are marked with yellow arrows in the figure. HDM may induce HNECs to produce eosinophils and goblet cells; this condition was alleviated after the addition of the hydrogel film (see h). Figure 3 i).

Claims

1. Manganese-based Prussian blue-polyacrylic acid / polyvinyl alcohol hydrogel, prepared by the following method, including: 1) Solution A: Add 0.65 mL of polyacrylic acid solution, 1-10 mmol of potassium citrate and 1-10 mmol of manganese sulfate monohydrate to 75 mL of deionized water and stir until dissolved. 2.2) Solution B: Add 0.1~2 mmol of potassium ferrocyanide trihydrate and 25 mL of ethanol to 50 mL of deionized water and stir; 3) Slowly add solution B to solution A in an oil bath at 55-65℃ and react for 12-24 hours. Centrifuge to remove the supernatant to obtain manganese-based Prussian blue nanoparticles. 4) Add 1-3 mg of manganese-based Prussian blue nanoparticles to 50-500 μL of polyacrylic acid solution, dissolve by sonication, add 0.5-2 mL of polyvinyl alcohol solution, shake, and obtain nano-manganese-based Prussian blue composite hydrogel. The concentration of the polyacrylic acid solution is 0.1~0.3 g / mL, and the concentration of the polyvinyl alcohol solution is 0.04~0.06 g / mL.

3. The manganese-based Prussian blue-polyacrylic acid / polyvinyl alcohol hydrogel according to claim 1, characterized in that: The concentration of the polyacrylic acid solution is 0.2 g / mL, and the concentration of the polyvinyl alcohol solution is 0.05 g / mL.

4. The manganese-based Prussian blue-polyacrylic acid / polyvinyl alcohol hydrogel membrane according to claim 1, characterized in that: The potassium ferrocyanide trihydrate is 0.8 mmol.

5. The manganese-based Prussian blue-polyacrylic acid / polyvinyl alcohol hydrogel according to claim 1, 2, 3 or 4, characterized in that: 1 mL of the polyvinyl alcohol solution.

6. The manganese-based Prussian blue-polyacrylic acid / polyvinyl alcohol hydrogel according to claim 1, characterized in that: The potassium citrate content is 2 mmol.

7. The manganese-based Prussian blue-polyacrylic acid / polyvinyl alcohol hydrogel according to claim 1, characterized in that: The amount of manganese sulfate monohydrate mentioned in step 4) is 2 mmol.

8. The manganese-based Prussian blue-polyacrylic acid / polyvinyl alcohol hydrogel according to claim 1, characterized in that: Step 4) involves a polyacrylic acid solution of 100 μL and a polyvinyl alcohol solution of 1 mL.

9. The manganese-based Prussian blue-polyacrylic acid / polyvinyl alcohol hydrogel according to claim 1, characterized in that: The oil bath in step 2) is 60 ℃; after the shaking in step 4), it is dropped onto a glass slide and left to stand at 37 ℃ for 1 hour.

10. The use of the manganese-based Prussian blue-polyacrylic acid / polyvinyl alcohol hydrogel according to claim 1 in the preparation of drugs for the prevention and treatment of allergic rhinitis.

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