Preparation method and application of nasal spray protection hydrogel aiming at respiratory tract pathogens

Abstract

The invention relates to a preparation method and application of nasal spray protection hydrogel aiming at respiratory tract pathogens, and belongs to the technical field of biological medicines. The hydrogel specially used for protecting respiratory tract pathogens is designed, and the hydrogel has the core advantages of being good in biocompatibility, suitable for spraying mechanical properties (including the rapid gelling characteristic), convenient to use, excellent in protection performance and the like. The hydrogel material can be used as a loading tool of various drugs, and is suitable for new-generation pathogen infection resistant protection instruments and wound repair, so that the application range of the hydrogel material is widened, and a new way is provided for the fields of respiratory infectious disease prevention and treatment and biological medicine.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for preparing and applying a nasal spray protective hydrogel for respiratory pathogens. Background Technology

[0002] Respiratory viruses, due to their high mutation rate and cross-species transmission capabilities, frequently trigger global public health crises. Taking SARS-CoV-2 as an example, frequent mutations in the receptor-binding domain (RBD) of the spike protein (S protein) (such as Omicron subspecies BA.5 and XBB.1.5) allow neutralizing antibodies to escape, significantly weakening the clinical efficacy of existing drugs. Problems such as single-target antiviral drugs, frequent drug resistance, and the long development cycle and inability to cover newly emerging mutant strains of vaccines all significantly expose the limitations of traditional treatment methods. Existing drug prevention and treatment methods lack active antiviral function and cannot play their due role before the virus invades the body. Furthermore, traditional nasal sprays (such as antibody sprays) have short molecular half-lives (T0). 1 / 2 The virus's effectiveness is limited to less than 2 hours and cannot resist viral mutations, resulting in poor timeliness of protection (CN114470198A). Therefore, developing new antiviral technologies that combine long-term effectiveness with proactive defense is an urgent need to overcome the current challenges in prevention and control.

[0003] Aptamers, as a new generation of "chemical antibodies," have shown great potential in the antiviral field due to their high affinity, programmable sequences, and low-cost large-scale production. As an emerging antiviral strategy, aptamers can specifically bind to viral surface proteins, thereby preventing the virus from binding to host cells and inhibiting viral infection. However, free aptamers are easily degraded by enzymes in vivo, making it difficult to maintain long-term protective effects. Framework nucleic acid (FNA) nanotechnology can precisely protect aptamers through rigid three-dimensional structures, enhancing their resistance to enzymatic degradation and overcoming the deficiency of single-molecule aptamers being easily inactivated in physiological environments. More importantly, aptamers are naturally nucleic acid sequences, which can be precisely and controllably combined with nanostructures without any modification or chemical reaction, making the synthesis process more convenient and faster, and ensuring stable and consistent drug concentrations. This provides a new approach and solution for the current prevention and control of COVID-19 infection and transmission.

[0004] Furthermore, we noted that hydrogel materials possess excellent biocompatibility, biodegradability, tissue adhesion, and sustained-release properties, providing long-lasting protection for the nasal mucosa. Although nasal spray hydrogels have attracted widespread attention as potential drug carriers, capable of rapidly forming a protective barrier within the nasal cavity to prevent viral contact with host cells and thus exert antiviral effects, currently, truly suitable nasal spray protective hydrogels are very limited, thus necessitating further research. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing and applying a nasal spray protective hydrogel with a shorter gelation time, suitable for the nasal cavity, and effective against respiratory pathogens.

[0006] Using dynamic chemical bonds as the primary intermolecular crosslinking mechanism, the hydrogel preparation method of this invention functionalizes potential biomacromolecules, imbuing them with more reactive functional groups (amino or aldehyde groups). The prepolymer with these newly added functional groups reacts upon spraying onto specific sites, crosslinking to form a protective hydrogel barrier. This hydrogel barrier can load customized nanomedicines and exhibits mechanical properties similar to the surrounding tissue environment, along with good biocompatibility. The preparation process of the hydrogel of this invention requires no complex processes or harsh conditions, significantly reducing development costs. Its application is simple and suitable for large-scale production.

[0007] This invention utilizes Schiff base reactions and weak interactions (such as hydrogen bonds) to construct dynamic bonded hydrogels. By exploring the reaction ratio between amino and aldehyde groups, a spray-applied hydrogel with suitable physicochemical properties, ease of use, and enhanced protection was prepared. Furthermore, under physiological conditions, this type of dynamic bonded hydrogel can be rapidly degraded into non-toxic small molecules (glutamate, glucose, etc.) by enzymes (such as esterases and cellulases) or microorganisms in vivo, avoiding long-term retention in the body. More importantly, framework nucleic acid aptamers (A-FNAs) are also natural hydrophilic organic polymers carrying a negative charge, allowing them to firmly bind with positively charged hydrogel organic polymers without any modification. This innovative material fusion not only effectively solves the bottleneck problem of Aptamer's susceptibility to nuclease hydrolysis but also delays the clearance of nasal mucus, enabling the nasal mask to maintain its biological activity for a longer period. This invention utilizes the synergistic effect of the antiviral bioactivity of nucleic acid materials and the physical isolation of organic polymer hydrogels to develop an intranasal formulation that can be rapidly adapted to respiratory coronaviruses and has long-lasting protective function, providing a technical reserve for future responses to emerging respiratory infectious diseases.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] In a first aspect, the present invention provides a method for preparing a nasal spray protective hydrogel targeting respiratory pathogens. An amino-containing organic polymer is dissolved in a solvent to prepare a prepolymer solution one; an organic polymer containing sugar units and aldehyde groups is dissolved in a solvent to prepare a prepolymer solution two; prepolymer solution one and prepolymer solution two are mixed and reacted to obtain the nasal spray protective hydrogel; the solvent is water, physiological saline, or PBS buffer; the mass-volume fraction of the amino-containing organic polymer (e.g., γ-PGA-ADH) in the nasal spray protective hydrogel is 2-3%, and the mass-volume fraction of the organic polymer containing sugar units and aldehyde groups (e.g., CMC-CHO) in the nasal spray protective hydrogel is 4%. After prepolymer solution one and prepolymer solution two are sprayed into the nasal cavity, they are mixed and form a hydrogel barrier on the nasal mucosa surface through dynamic bonds and other weak interactions.

[0010] This invention, through research, has discovered that a certain proportion of aqueous solutions containing amino groups such as γ-PGA-ADH and aqueous solutions containing sugar units and aldehyde groups such as CMC-CHO, when mixed, can form a hydrogel under physiological conditions, with a gelation time of 5-10 seconds, suitable for nasal spraying. In practical applications, the two aqueous solutions are sprayed separately using a sprayer and then mixed to form a hydrogel suitable for the nasal environment. The concentration ratio of amino groups such as γ-PGA-ADH to aldehyde groups such as CMC-CHO in the hydrogel is crucial; too high or too low a concentration may result in a hydrogel with a gelation time that is too fast or too slow, or a hydrogel that is too soft or too hard, unsuitable for nasal spraying. The hydrogel prepared by this invention has a storage modulus (G') of 100-500 Pa and a loss modulus (G”) of 50-200 Pa, possessing an elastic modulus adapted to the nasal mucus environment, making it suitable for nasal spraying.

[0011] In a preferred embodiment of the preparation method of the present invention, the molecular weight of the organic polymer containing amino groups is not greater than 100 kDa; and the molecular weight of the organic polymer containing sugar units and aldehyde groups is not less than 1000 kDa.

[0012] As a preferred embodiment of the preparation method of the present invention, the organic polymer with amino groups has a mass-volume fraction of 2% in the nasal spray protective hydrogel, and the organic polymer containing sugar units and aldehyde groups has a mass-volume fraction of 4% in the nasal spray protective hydrogel.

[0013] As a preferred embodiment of the preparation method of the present invention, the organic polymer with amino groups includes organic polymers with free amino groups or organic polymers with free carboxyl groups modified with amino groups; the organic polymer containing sugar units and aldehyde groups includes organic polymers containing sugar units and free aldehyde groups or organic polymers with sugar units and ortho-dihydroxy groups modified with aldehyde groups.

[0014] As a preferred embodiment of the preparation method described in this invention, the organic polymer containing amino groups includes at least one of the following: protein, polypeptide, amino polysaccharide, polyamino acid, glycosaminoglycan, polyvinyl alcohol, polyacid, polyester, pectin, polyaminoglucose, polyamino ketone, polyvinyl acid, polyaminothiazole, polyamide, nylon, polyvinyl alcohol, polyamino acrylate, polyamino amino acid salt, polyaminothiophene, polyaminonitroso, polyaminotetrahydropyridine, polyaminoethylene, etc.

[0015] In a preferred embodiment of the preparation method described in this invention, the polyamino acids include polyglutamic acid, polylysine, polyaspartic acid, polyglycine, polyaromatic amino acids, and polysulfone amino acids.

[0016] The glycosaminoglycans include hyaluronic acid, chitosan, and heparin;

[0017] The polyacids include polyacrylic acid, polylactic acid, polycarboxylic acid, polyvinyl alcohol, polyfuran acid, polyisofluoroolefin acid, polycarboxylic acid, polyaminobenzoic acid, polyaminoacrylic acid, polyaminocarboxylic acid, polyaminoacetic acid, polyaminochloroacetic acid, polyaminobutyric acid, and polylinolenic acid.

[0018] The polyester includes polyurethane, polyamino acid ester, polyamino acrylate, and polysulfate;

[0019] The polyamides include polyasparagine, polyglutamine, polyaminoamide, and polyaminoacrylamide.

[0020] In a preferred embodiment of the preparation method described in this invention, the polyamino acid is γ-polyglutamic acid (γ-PGA).

[0021] As a preferred embodiment of the preparation method described in this invention, the organic polymer containing sugar units and aldehyde groups includes at least one of the following: glucose, galactose, fructose, arabinose, xylose, mannose, mannitol, glucuronic acid, N-acetylglucosamine, N-acetylglucosamine, lactose, maltose, sucrose, hyaluronic acid, pectin, heparin, chondroitin sulfate, polylactic acid, polyglutamic acid, polymannan, ribose, deoxyribose, glucuronic acid, wood aldehyde, glyceraldehyde, glucosamine, triglycerides, fatty acids, polyesters, peptides, cellulose, and cellulose derivatives.

[0022] In a preferred embodiment of the preparation method described in this invention, the cellulose derivative is sodium carboxymethyl cellulose (CMC-Na).

[0023] In a preferred embodiment of the preparation method described in this invention, the amino modification is achieved by grafting amino groups using chemical means, including but not limited to reduction reactions (hydrogenation reduction, LiAlH4, NH3-BH3), amination reactions, amination reagents (aminosilanes, aminophosphates), esterification reactions, and biotransformation methods, or a combination of two or more of these methods. The aldehyde modification involves oxidizing an organic polymer containing ortho-dihydroxyl groups to impart an aldehyde group, including but not limited to oxidation reactions (CrO3, H2O2, KMnO4, NaIO4, pyridine chlorochromate, Dess-Martin oxidant), dehydration reactions (acid-catalyzed dehydration), acylation reactions (reaction with acyl chlorides), and biotransformation (enzymatic oxidation), or a combination of two or more of these methods.

[0024] As a preferred embodiment of the preparation method of the present invention, the preparation method of the nasal spray protective hydrogel includes the following steps:

[0025] S1. γ-polyglutamic acid (γ-PGA) is modified with amino groups by reacting it with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS) and adipic acid dihydrazide (ADH) to obtain amino-grafted γ-polyglutamic acid (γ-PGA-ADH).

[0026] S2. Sodium carboxymethyl cellulose (CMC-Na) is reacted with an oxidant to undergo aldehyde modification, resulting in aldehyde-modified carboxymethyl cellulose (CMC-CHO).

[0027] S3. Grafted amino γ-polyglutamic acid (γ-PGA-ADH) is dissolved in a solvent to prepare prepolymer solution one; aldehyde-modified carboxymethyl cellulose (CMC-CHO) is dissolved in a solvent to prepare prepolymer solution two; prepolymer solution one and prepolymer solution two are mixed and reacted to obtain the nasal spray protective hydrogel.

[0028] The solvent is water, physiological saline, or PBS buffer; the grafted amino γ-polyglutamic acid (γ-PGA-ADH) has a mass-volume fraction of 2-3% in the nasal spray protective hydrogel, and the aldehyde-modified carboxymethyl cellulose (CMC-CHO) has a mass-volume fraction of 4%. After prepolymer solution one and prepolymer solution two are sprayed into the nasal cavity, they are mixed and form a hydrogel barrier on the nasal mucosa surface through dynamic bonding and intermolecular forces.

[0029] As a preferred embodiment of the preparation method of the present invention, the preparation method of the nasal spray protective hydrogel includes the following steps:

[0030] S1. An aqueous solution of γ-polyglutamic acid (γ-PGA) is mixed with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and reacted thoroughly. Then, N-hydroxysuccinimide (NHS) is added and mixed thoroughly. Next, adipate dihydrazide (ADH) is added and reacted thoroughly. The reaction solution is dialyzed to remove excess reactants. The dialysate is dried to obtain γ-polyglutamic acid grafted with amino groups (γ-PGA-ADH).

[0031] S2. React sodium carboxymethyl cellulose (CMC-Na) aqueous solution with NaIO4 until fully reacted. Dialyze the reaction solution to remove excess reactants and dry the dialysate to obtain aldehyde-modified carboxymethyl cellulose (CMC-CHO).

[0032] S3. Prepolymer solution one is prepared by dissolving amino-grafted γ-polyglutamic acid (γ-PGA-ADH) in a solvent; prepolymer solution two is prepared by dissolving aldehyde-modified carboxymethyl cellulose (CMC-CHO) in a solvent; prepolymer solution one and prepolymer solution two are mixed and reacted to obtain the nasal spray protective hydrogel; the solvent is water, physiological saline, or PBS buffer; the mass-volume fraction of amino-grafted γ-polyglutamic acid (γ-PGA-ADH) in the nasal spray protective hydrogel is 2-3%, and the mass-volume fraction of aldehyde-modified carboxymethyl cellulose (CMC-CHO) in the nasal spray protective hydrogel is 4%.

[0033] In a preferred embodiment of the preparation method described in this invention, the molar ratio of the carboxyl group of γ-polyglutamic acid to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS), and adipic acid dihydrazide (ADH) is 1:1:1:1; the molar ratio of sodium carboxymethyl cellulose to NaIO4 is 1:2. In step S3, the pH value of the water is 6.5-7.5.

[0034] In a preferred embodiment of the preparation method of the present invention, the γ-polyglutamic acid grafted with amino groups (γ-PGA-ADH) has a mass-volume fraction of 2% in the nasal spray protective hydrogel, and the aldehyde-modified carboxymethyl cellulose (CMC-CHO) has a mass-volume fraction of 4% in the nasal spray protective hydrogel.

[0035] Secondly, the present invention provides a nasal spray protective hydrogel prepared using the above-described preparation method.

[0036] Thirdly, the present invention provides the application of the above-mentioned nasal spray protective hydrogel in the preparation of medical devices, tissue engineering materials, biosensors or drug delivery carriers.

[0037] The nasal spray protective hydrogel of the present invention can be used as a drug carrier to combat respiratory pathogen infection; it can be used as a wound dressing in tissue engineering including hard and soft tissues; the tissues and organs to which the hydrogel can be applied include at least one of mucous membranes, skin, muscles, blood vessels, and brain.

[0038] Fourthly, the present invention provides a drug-loaded hydrogel containing the above-mentioned nasal spray protective hydrogel and a drug.

[0039] As a preferred embodiment of the drug-loaded hydrogel of the present invention, the drug includes one or a combination of two or more of the following: nanoparticles, miRNA, exosomes, liposomes, microvesicles, microspheres, framework nucleic acid aptamers, peptides, metal ions, metal-organic frameworks, traditional Chinese medicine extracts, and finished drugs.

[0040] In a preferred embodiment of the drug-loaded hydrogel of the present invention, the drug is a framework nucleic acid aptamer (A-FNA).

[0041] In a preferred embodiment of the drug-loaded hydrogel of the present invention, the framework nucleic acid aptamer includes a framework sequence, a linker sequence, and an aptamer sequence; the aptamer sequence is linked to the framework sequence through the linker sequence, and then assembled according to the base complementary pairing principle to form a tetrahedral framework nucleic acid aptamer (A-tFNA); the framework sequence includes four DNA strands, and the nucleotide sequences of the four DNA strands are shown below:

[0042] ssDNA A17: 5'-ACATTCCTAAGTCTGAAACATTACAGCTTGCTACACGAGAAGAGCCGCCATAGTA-3',

[0043] ssDNA B17: 5'-TATCACCAGGCAGTTGACAGTGTAGCAAGCTGTAATAGATGCGAGGGTCCAATAC-3',

[0044] ssDNAC17: 5'-TCAACTGCCTGGTGATAAAACGACACTACGTGGGAAT CTACTATGGCGGCTCTTC-3',

[0045] ssDNA D17: 5'-TTCAGACTTAGGAATGTGCTTCCCACGTAGTGTCGTTTGTATTGGACCCTCGCAT-3'.

[0046] The nucleotide sequence of the aptamer includes the three nucleotide sequences shown in S1-S3:

[0047] S1: 5'-CAGCACCGACCTTGTGCTTTGGGAGTGCTGGTCCAAGGGCGTT AATGGACA-3',

[0048] S2: 5'-ATCCAGAGTGACGCAGCATTTCATCGGGTCCAAAAGGGGCTGCTCGGGATTGCGGATATGGACACGT-3',

[0049] S3: 5'-CGCAGCACCCAAGAACAAGGACTGCTTAGGATTGCGATAGGTT CGG-3'.

[0050] The nucleotide sequence of the linker sequence includes tttttttttt. This framework aptamer (A-FNA) has been disclosed in patent CN118956881A.

[0051] As a preferred embodiment of the drug-loaded hydrogel of the present invention, the preparation method of the drug-loaded hydrogel is as follows: dissolve the drug in prepolymer solution one and / or prepolymer solution two of the hydrogel, and then mix them to obtain the drug-loaded hydrogel.

[0052] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0053] This invention designs a hydrogel specifically for the protection against respiratory pathogens. This hydrogel possesses core advantages such as good biocompatibility, suitable mechanical properties for spraying (including rapid gelation), ease of use, and excellent protective performance. It can serve as a loading tool for various drugs, making it suitable for next-generation anti-pathogen infection protective devices and wound repair, thereby broadening the application range of hydrogel materials and providing new avenues for the prevention and treatment of respiratory infectious diseases and the biomedical field. The preparation process of this hydrogel requires no complex processes or harsh conditions, significantly reducing development costs. Its simple application method makes it suitable for large-scale production. Attached Figure Description

[0054] Figure 1 The γ-PGA-ADH modification before and after in Example 1 of this invention 1 H NMR spectrum;

[0055] Figure 2 The CMC-CHO modification before and after in Example 1 of this invention 1 H NMR spectrum;

[0056] Figure 3 The images shown are the FT-IR spectra of P2C4 before and after the reaction in Example 1 of this invention.

[0057] Figure 4The storage modulus and loss modulus of hydrogels with different ratios under different time scans in Test Example 2 of this invention;

[0058] Figure 5 The results of storage modulus, loss modulus and shear modulus under different frequency scans after gelation of hydrogels with different ratios in Test Example 2 of the present invention are shown. A represents the results of storage modulus and loss modulus, and B represents the statistics of shear modulus.

[0059] Figure 6 The curve showing the viscosity of P2C4 as a function of shear rate in Test Example 2 of this invention;

[0060] Figure 7 The storage modulus and loss modulus of hydrogels 1-9 under different time scans in Example 3 of this invention;

[0061] Figure 8 The images shown are the hydrogel before and after gelation, the spraying state, a visual image of the hydrogel, and a SEM image in Test Example 3 of this invention. A represents the hydrogel before and after gelation, B represents the spraying state, C represents the visual image of the hydrogel, and D represents the SEM image with a low magnification of 400 μm and a high magnification of 100 μm.

[0062] Figure 9 The results of hydrogel degradation performance in Test Example 4 of this invention are shown in Figure A, which represents the state diagram of the hydrogel at different time points; and Figure B, which represents the hydrogel degradation curve.

[0063] Figure 10 The results of the in vitro biocompatibility test of the hydrogel in Test Example 5 of this invention are as follows: A represents HUVECs cell live / dead staining for 1 day; B represents HUVECs cell live / dead staining for 3 days; C represents ACE2-293T cell live / dead staining for 1 day; D represents ACE2-293T cell live / dead staining for 3 days; E represents HUVECs cell CCK-8 count OD value statistics for 1-3 days; F represents HUVECs cell viability statistics for 1-3 days; G represents ACE2-293T cell CCK-8 count OD value statistics for 1-3 days; H represents ACE2-293T cell viability statistics for 1-3 days.

[0064] Figure 11 The results of the in vivo biocompatibility test of P2C4-st17 in test example 5 of this invention are shown. A represents H&E staining of important organs (brain, heart, liver, spleen, lung, kidney); BG represents blood routine test statistics; HK represents blood biochemistry test statistics.

[0065] Figure 12The results of the in vitro antiviral efficacy test of P2C4-st17 in Test Example 6 of this invention are shown. A represents the fluorescence image of the in vitro antiviral efficacy of P2C4-st17 (scale bar: 100 μm); B represents the statistical graph of the in vitro antiviral efficacy of P2C4-st17.

[0066] Figure 13 The results of the in vivo antiviral efficacy test of P2C4-st17 in Test Example 6 of this invention are shown in Figure A, which represents the H&E staining image of lung tissue; Figure B represents the S protein and GFP fluorescence staining image of lung tissue; Figure C represents the S protein expression fluorescence intensity statistical graph; and Figure D represents the GFP expression fluorescence intensity statistical graph.

[0067] Figure 14 This is to demonstrate the drug retention effect of ST17 in Test Example 7 of this invention. Detailed Implementation

[0068] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0069] Unless otherwise specified, all other materials and reagents used in the examples are commercially available.

[0070] Example 1

[0071] A method for preparing a hydrogel includes the following steps:

[0072] S1. Amine Modification: A 10% (w / v) aqueous solution of γ-polyglutamic acid (γ-PGA, molecular weight 100 kDa) was continuously stirred. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) was added and dissolved, and the mixture was stirred at room temperature for 30 minutes. Then, N-hydroxysuccinimide (NHS) was added, and the mixture was stirred for 24 hours. Finally, adipic acid dihydrazide (ADH) was added, and the mixture was stirred for another 24 hours. The molar ratio of the carboxyl group of γ-PGA to EDC, NHS, and ADH was 1:1:1:1. After the reaction, the mixture was dialyzed in deionized water for three days using a dialysis bag with a molecular weight cutoff of 1 kDa to remove excess reactants. The dialysate was lyophilized to obtain amino-modified γ-polyglutamic acid (γ-PGA-ADH), i.e., an organic polymer with amino groups.

[0073] S2. Aldehyde Modification: NaIO4 aqueous solution was added dropwise to a 1% (w / v) sodium carboxymethyl cellulose (CMC-Na) aqueous solution, with a molar ratio of CMC-Na to NaIO4 of 1:2. After the reaction was complete, the mixture was dialyzed in deionized water for three days using a dialysis bag with a molecular weight cutoff of 10 kDa to remove excess reactants. The dialysate was then lyophilized to obtain an aldehyde-modified organic polymer containing sugar units and ortho-dihydroxyl groups (CMC-CHO), i.e., an organic polymer containing sugar units and aldehyde groups.

[0074] S3. Preparation of prepolymer solution: The amino-modified γ-polyglutamic acid (γ-PGA-ADH) from step S1 was prepared into a 4% (w / v) solution using water with a pH of 6.5-7.5 to obtain prepolymer solution one; the aldehyde-modified organic polymer (CMC-CHO) containing sugar units and ortho-dihydroxyl groups from step S2 was prepared into an 8% (w / v) solution to obtain prepolymer solution two.

[0075] S4. Using a sprayer, prepolymer liquid one and prepolymer liquid two from step S3 are sprayed into the nasal cavity at a volume ratio of 1:1. After waiting for 10 minutes, a hydrogel layer will form on the surface of the nasal mucosa. In this embodiment, the final concentration of γ-PGA-ADH is 2%, and the final concentration of CMC-CHO is 4%. This hydrogel is denoted as P2C4.

[0076] The solvent water in step S3 can also be replaced with physiological saline or PBS buffer.

[0077] Test Example 1

[0078] The hydrogel prepared in Example 1 and the raw materials were characterized and analyzed.

[0079] The proton nuclear magnetic resonance spectra of γ-PGA and γ-PGA-ADH before and after modification, as well as CMC-Na and CMC-CHO before and after modification, were analyzed using a nuclear magnetic resonance spectrometer.

[0080] Fourier transform infrared spectroscopy (FT-IR) was used to perform spectral analysis on γ-PGA-ADH, CMC-CHO and hydrogel (P2C4) before and after hydrogel formation.

[0081] Before and after γ-PGA-ADH modification 1 H NMR spectrum as shown Figure 1 As shown in the figure; the results show that ADH was grafted onto the γ-PGA molecular chain, and γ-PGA was successfully modified into γ-PGA-ADH with a grafting rate of approximately 66.52%.

[0082] Before and after CMC-CHO modification 1 H NMR spectrum as shown Figure 2 As shown in the figure; the results show that CMC-Na was successfully modified into CMC-CHO, with a grafting rate of approximately 78.25%.

[0083] The FT-IR spectra before and after the P2C4 reaction are as follows: Figure 3 As shown in the figure, the results indicate that the characteristic peaks of γ-PGA-ADH, CMC-CHO, and hydrogel can all appear in the constructed hydrogel spectrum, indicating that the hydrogel was successfully prepared.

[0084] Example 2

[0085] A method for preparing a hydrogel is the same as in Example 1, except that the ratio of the two prepolymer solutions is different.

[0086] A hydrogel was prepared by spraying a 4% (w / v) γ-PGA-ADH solution and a 4% (w / v) CMC-CHO solution at a volume ratio of 1:1 at 37°C. The final concentration of γ-PGA-ADH and the final concentration of CMC-CHO in the hydrogel were both 2%, denoted as P2C2.

[0087] A hydrogel was prepared by spraying a 6% (w / v) γ-PGA-ADH solution and an 8% (w / v) CMC-CHO solution at a volume ratio of 1:1 at 37°C. The final concentration of γ-PGA-ADH in the hydrogel was 3%, and the final concentration of CMC-CHO was 4%, denoted as P3C4.

[0088] A hydrogel was prepared by spraying a 10% (w / v) γ-PGA-ADH solution and an 8% (w / v) CMC-CHO solution at a volume ratio of 1:1 at 37°C. The final concentration of γ-PGA-ADH in the hydrogel was 5%, and the final concentration of CMC-CHO was 4%, denoted as P5C4.

[0089] Test Example 2

[0090] Elastic modulus statistics and rheological analysis were performed on the hydrogels with different ratios in Examples 1-2 using a Thermo Fisher HAAKE rotational rheometer.

[0091] The storage modulus and loss modulus of hydrogels with different ratios under different time scans are as follows: Figure 4 As shown in the results, at 37℃, P2C4 and P3C4 can begin to crosslink and form hydrogels within 5-10 seconds after the two prepolymers are mixed, while P2C2 has not yet shown significant crosslinking and formed a hydrogel within 10 seconds. P5C4 has completely crosslinked and formed a hydrogel within 5 seconds.

[0092] The results above indicate that the ratio of the two prepolymer solutions is crucial for the preparation of the hydrogel. If the ratio of γ-PGA-ADH solution to CMC-CHO solution is too low (P2C2), a gel barrier cannot be formed in a short time after spraying; if the ratio is too high (P5C4), the crosslinking speed is too fast, and there is a lack of time window for the spraying process (and mixing), making it unsuitable for nasal spraying.

[0093] The storage modulus, loss modulus, and shear modulus of hydrogels with different ratios after gelation at different frequencies are statistically analyzed as follows: Figure 5As shown, A represents the results of storage modulus and loss modulus, and B represents the statistics of shear modulus. The results show that in the frequency range of 1-10 rad / s, the shear modulus of P2C4 is about 100 Pa, and the shear modulus of P3C4 and P5C4 is about 1 kPa. In contrast, P2C4 has an elastic modulus (shear modulus) that is adapted to the nasal mucus environment.

[0094] The viscosity of P2C4 as a function of shear rate is shown in the curve. Figure 6 As shown in the figure, the results indicate that P2C4 exhibits shear-thinning properties.

[0095] In summary, under the same conditions, the ratio of the two components in the prepolymer directly affects the rheological properties of the hydrogel material. A better hydrogel is obtained when the mass-volume fraction of the γ-PGA-ADH solution is 4–6% and the mass-volume fraction of the CMC-CHO solution is 8%, mixed at a volume ratio of 1:1. Specifically, a better hydrogel is obtained when the mass ratio of γ-PGA-ADH to CMC-CHO is 2–3:4, the final concentration of γ-PGA-ADH in the hydrogel is 2–3%, and the final concentration of CMC-CHO is 4%. Furthermore, a better hydrogel is obtained when the mass-volume fraction of the γ-PGA-ADH solution is 4% and the mass-volume fraction of the CMC-CHO solution is 8%, i.e., the mass ratio of γ-PGA-ADH to CMC-CHO is 2:4, the final concentration of γ-PGA-ADH in the hydrogel is 2%, and the final concentration of CMC-CHO is 4%.

[0096] Example 3

[0097] A method for preparing a hydrogel is the same as in Example 1, except that the solvent and concentration of the prepolymer solution are different. The solvent and concentration of the prepolymer solution are shown in Table 1 below.

[0098] Table 1

[0099]

[0100] The storage modulus and loss modulus of the hydrogels at different times were tested according to the method in Test Example 2. The storage modulus and loss modulus results of hydrogels 1-9 under different time scans are shown below. Figure 7 As shown in the results, the gelation time of hydrogels 1-9 was all over 50 seconds, which is relatively long. This indicates that reducing the concentration of the prepolymer solution or replacing the solvent of the prepolymer solution with an ethanol-water solution will significantly prolong the gelation time of the prepared hydrogels, making them unsuitable for nasal spraying.

[0101] Test Example 3

[0102] The state of the hydrogel in Example 1 before and after gelation was observed. After staining with Rhodamine B, it was sprayed and the sprayed state was observed. The microstructure of the hydrogel was observed using scanning electron microscopy (SEM).

[0103] The hydrogel's state before and after gelation, its spraying state, a visual image of the hydrogel, and SEM images are shown below. Figure 8 As shown, A represents the hydrogel before and after gelation, B represents the sprayed state, C represents a visual representation of the hydrogel, and D represents the SEM image with a low magnification of 400 μm and a high magnification of 100 μm. The results show that the hydrogel can be cross-linked into a transparent gel state by mixing with a prepolymer solution, which can be applied by a sprayer. The SEM images show that the drug-loaded hydrogel exhibits a three-dimensional porous network structure at the microscopic level.

[0104] Test Example 4

[0105] Example 1: Characterization of hydrogel degradation performance.

[0106] The hydrogel (P2C4) from Example 1 was immersed in PBS buffer at 37°C. The remaining hydrogel was extracted using a 70 μm filter at different time points and then lyophilized. The state and mass of the hydrogel at different times were recorded, and the corresponding curves were statistically analyzed.

[0107] The hydrogel degradation performance results are as follows Figure 9 As shown, A represents the state diagram of the hydrogel at different time points; B represents the degradation curve of the hydrogel; the results show that the hydrogel can disintegrate naturally within 48 hours.

[0108] Example 4

[0109] Preparation of drug-loaded hydrogels, wherein the drug is a framework nucleic acid aptamer (A-tFNA), which has been disclosed in patent CN118956881A. Specifically, the A-tFNA includes a framework sequence, a linker sequence, and an aptamer sequence; the aptamer sequence is linked to the framework sequence through the linker sequence, and then assembled into a tetrahedral framework nucleic acid aptamer according to the base complementary pairing principle; the framework sequence includes four DNA strands, and the nucleotide sequences of the four DNA strands are shown below:

[0110] ssDNAA17: 5'-ACATTCCTAAGTCTGAAACATTACAGCTTGCTACACGA GAAGAGCCGCCATAGTA-3',

[0111] ssDNAB17: 5'-TATCACCAGGCAGTTGACAGTGTAGCAAGCTGTAATAG ATGCGAGGGTCCAATAC-3',

[0112] ssDNAC17: 5'-TCAACTGCCTGGTGATAAAACGACACTACGTGGGAAT CTACTATGGCGGCTCTTC-3',

[0113] ssDNAD17: 5'-TTCAGACTTAGGAATGTGCTTCCCACGTAGTGTCGTTT GTATTGGACCCTCGCAT-3'.

[0114] The nucleotide sequence of the aptamer includes the three nucleotide sequences shown in S1-S3:

[0115] S1: 5'-CAGCACCGACCTTGTGCTTTGGGAGTGCTGGTCCAAGGGCGTT AATGGACA-3',

[0116] S2: 5'-ATCCAGAGTGACGCAGCATTTCATCGGGTCCAAAAGGGGCTGCTCGGGATTGCGGATATGGACACGT-3',

[0117] S3: 5'-CGCAGCACCCAAGAACAAGGACTGCTTAGGATTGCGATAGGTT CGG-3'.

[0118] The nucleotide sequence of the linking sequence includes tttttttttt.

[0119] The drug-loaded hydrogel was prepared as follows: Drug A-tFNA was dissolved in prepolymer solution one (γ-PGA-ADH prepolymer solution) and / or prepolymer solution two (CMC-CHO prepolymer solution) from Example 1. Then, prepolymer solution one and prepolymer solution two were sprayed at a 1:1 volume ratio to obtain the drug-loaded hydrogel. The final concentration of drug A-tFNA in the drug-loaded hydrogel was 500 nM. This drug-loaded hydrogel is designated P2C4-st17.

[0120] Test Example 5

[0121] Biocompatibility was evaluated in vitro and in vivo using both the hydrogel (P2C4) and the drug-loaded hydrogel (P2C4-st17). HUVECs and ACE2-293T cells were cultured using the hydrogel extract, and cell viability was assessed using cell viability and CCK-8 assays to evaluate the in vitro biocompatibility of P2C4-st17. P2C4-st17 was sprayed intranasally into mice and fed for 4 weeks. In vivo biocompatibility was evaluated using histological staining of vital organs (brain, heart, liver, spleen, lung, and kidney), complete blood count, and blood biochemistry.

[0122] 1. The in vitro experimental methods are as follows:

[0123] (1) The biocompatibility of the hydrogel was evaluated using an animal cell viability / toxicity assay kit. 1 mL of sterile PBS (pH = 7.4) buffer was added to a 1.5 mL microcentrifuge tube. Then, under light-protected conditions, 1 μL of 4 mM Calcein-AM solution A was added to label live cells, and 2 μL of 2 mM Ethidium homodimer (EthD-III) solution B was added to label dead cells. The mixture was then gently pipetted to mix. Human umbilical vein endothelial cells (HUVECs) and ACE2-expressing human embryonic kidney cells (ACE2-293T) were seeded at a density of 2 × 10⁶ cells / mL. 4 Cells were transferred to 24-well plates and cultured for a specific period (1 to 3 days) using P2C4 and P2C4-st17 extraction solutions (obtained by pre-soaking the corresponding group of hydrogels in complete culture medium for 24 hours, with 1g of hydrogel soaked in 40mL of culture medium). Cell viability was then observed by staining to assess cell survival in each group under hydrogel extraction conditions. Cells were washed three times with sterile PBS for approximately 5 minutes each time before each staining to remove residual culture medium and cell debris. After the final wash, the PBS buffer was aspirated, and the prepared viability staining solution was added to cover the bottom surface. Cells were incubated at room temperature in the dark for 5-10 minutes, then transferred and observed using a fluorescence microscope.

[0124] (2) HUVECs and ACE2-293T cells were seeded into 48-well plates, approximately 5000 cells per well. Cells were cultured using the hydrogel from Example 1 and the drug-loaded hydrogel from Example 4, respectively. CCK-8 cell viability was measured at specific time points (1 to 3 days). First, the old culture medium was aspirated and discarded. Then, the cells in the wells were washed three times with PBS. Next, the basal culture medium and CCK-8 detection solution were mixed at a ratio of 10:1 (V / V) and added to the 48-well plates. A blank control group was also included. The plates were incubated at 37℃ with 5% CO2 for 30 min–1 h, during which time the color of the liquid in the wells was carefully observed. Once the color change was visible to the naked eye, samples were transferred to 96-well plates, 100 μL per well. Cell viability was measured using a microplate reader, and the absorbance (OD value) at 450 nm was recorded. Cell viability was calculated based on the results.

[0125] 2. The in vivo experimental methods are as follows:

[0126] BALB / c mice aged 8-12 weeks were anesthetized, and the drug-loaded hydrogel (P2C4-st17) of Example 4 was pre-coated into the nasal cavity. The mice were observed and fed for 4 weeks, and the survival of the experimental animals was recorded and survival curves were plotted. After 4 weeks, blood biochemistry and routine blood tests were performed on the surviving animals, and histological staining of important organs (brain, heart, liver, spleen, lung, and kidney) was performed. The results were compared with those of healthy mice to assess the biosafety of the implanted component in the experimental animals.

[0127] 3. Results

[0128] (1) Results of in vitro experiments

[0129] The results of in vitro biosafety tests on hydrogels are as follows: Figure 10 As shown, A represents HUVECs cell live / dead staining for 1 day; B represents HUVECs cell live / dead staining for 3 days; C represents ACE2-293T cells live / dead staining for 1 day; D represents ACE2-293T cells live / dead staining for 3 days; E represents HUVECs cell CCK-8 count OD value statistics for 1-3 days; F represents HUVECs cell viability statistics for 1-3 days; G represents ACE2-293T cells CCK-8 count OD value statistics for 1-3 days; H represents ACE2-293T cells viability statistics for 1-3 days. The results show that P2C4 and P2C4-st17 have good biocompatibility in vitro.

[0130] (2) Results of in vivo experiments

[0131] The in vivo biosafety test results of P2C4-st17 are as follows: Figure 11 As shown, A represents H&E staining of important organs (brain, heart, liver, spleen, lung, kidney); BG represents blood routine test statistics; HK represents blood biochemistry test statistics. The results show that P2C4-st17 has good biocompatibility in vivo.

[0132] In summary, the hydrogel and drug-loaded hydrogel of the present invention have good biocompatibility.

[0133] Test Example 6

[0134] Evaluation of the in vitro and in vivo anti-coronavirus efficacy of drug-loaded hydrogels and control drugs. The efficacy of drug-loaded hydrogels and control drugs against SARS-CoV-2 pseudovirus (B.1.1.529) infection was evaluated in vitro and in vivo. The in vitro anti-coronavirus efficacy was assessed by inoculating B.1.1.529 pseudovirus into Transwell chambers pre-coated with drug-loaded hydrogel and culturing ACE2-293T cells protected by the pseudovirus, and by observing the expression of green fluorescent protein in the cells. The in vivo anti-coronavirus efficacy was assessed by inducing B.1.1.529 pseudovirus infection in the nasal cavity of mice and by histological staining and immunofluorescence staining of lung tissue.

[0135] 1. The in vitro experimental methods are as follows:

[0136] PBS, A-tFNA(st17), P2C4, and P2C4-st17 were pre-coated onto Transwell chambers and then placed in 24-well plates, with 2 × 10⁶ cells per well. 410 ACE2-293T cells. After the cells have spread to the bottom, 10 B.1.1.529 pseudoviruses carrying green fluorescent protein are inoculated. 5 CFU / well was inserted into each group of Transwell chambers, and after 48 h of culture, the expression of green fluorescent protein in the cells at the bottom of the wells was observed under a fluorescence microscope. The fluorescence density was counted to evaluate the antiviral binding effect of each component on the cells.

[0137] 2. The in vivo experimental methods are as follows:

[0138] A model was established in 8-10 week old BALB / c mice. After anesthetizing the animals, physiological saline, st17, P2C4, and P2C4-st17 were pre-coated into the nasal cavity, respectively. Then, the same dose of B.1.1.529 pseudovirus fluid was administered into the nasal cavity. Subsequently, lung and brain tissues were collected at specific time points for histological sections and immunofluorescence staining to observe inflammatory cell infiltration in the lungs, viral S protein localization, and green fluorescent protein expression to evaluate its efficacy against coronavirus infection in animals.

[0139] 3. Results

[0140] (1) Results of in vitro experiments

[0141] The results of the in vitro antiviral efficacy test of P2C4-st17 are as follows: Figure 12 As shown, A represents the fluorescence image of the in vitro antiviral efficacy of P2C4-st17 (scale bar: 100 μm); B represents the statistical graph of the in vitro antiviral efficacy of P2C4-st17. The results show that st17, P2C4, and P2C4-st17 can all protect cells against B.1.1.529 pseudovirus infection in vitro. P2C4-st17 is more effective than P2C4, and the hydrogel is more effective than st17.

[0142] (2) Results of in vivo experiments

[0143] The results of the in vivo antiviral efficacy test of P2C4-st17 are as follows: Figure 13 As shown in the figures, A represents H&E staining images of lung tissue; B represents S protein and GFP fluorescence staining images of lung tissue; C represents a statistical graph of S protein expression fluorescence intensity; and D represents a statistical graph of GFP expression fluorescence intensity. The results showed that st17, P2C4, and P2C4-st17 could all resist B.1.1.529 pseudovirus infection in vivo. Among them, P2C4-st17 was more effective than P2C4, and P2C4 was more effective than st17.

[0144] In summary, the hydrogel and drug-loaded hydrogel of the present invention can resist SARS-CoV-2 pseudovirus infection both in vivo and in vitro.

[0145] Test Example 7

[0146] The long-term antiviral protective effect of drug-loaded hydrogels in mice was investigated. Saline and P2C4 were used as delivery systems for A-tFNA (st17) (st17 and P2C4-st17, respectively). The effects of different delivery systems on drug retention were evaluated using the following methods:

[0147] St17 (Cy5 modified) of the same concentration was sprayed onto the nasal mucosa of mice using physiological saline and P2C4, respectively, and the fluorescence signal in the nasal cavity was observed using an in vivo imaging device at specific time points.

[0148] The drug retention effect of ST17 is as follows: Figure 14 As shown in the figure. The results showed that P2C4 was able to record the fluorescence signal of st17 in the nasal cavity at longer time points. This indicates that, compared with saline, hydrogel can effectively prolong the action time of A-tFNA in the nasal cavity.

[0149] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a nasal spray protective hydrogel against respiratory pathogens, characterized in that, An organic polymer containing amino groups is dissolved in a solvent to prepare a prepolymer solution one; an organic polymer containing sugar units and aldehyde groups is dissolved in a solvent to prepare a prepolymer solution two; the prepolymer solution one and the prepolymer solution two are mixed and reacted to obtain the nasal spray protective hydrogel. The solvent is water, physiological saline, or PBS buffer. The amino-containing organic polymer has a mass-volume fraction of 2-3% in the nasal spray protective hydrogel. The organic polymer containing sugar units and aldehyde groups has a mass-volume fraction of 4% in the nasal spray protective hydrogel.

2. The preparation method according to claim 1, characterized in that, The organic polymer containing amino groups has a molecular weight of not more than 100 kDa; the organic polymer containing sugar units and aldehyde groups has a molecular weight of not less than 1000 kDa.

3. The preparation method according to claim 1 or 2, characterized in that, The organic polymer containing amino groups includes organic polymers with free amino groups or amino-modified organic polymers with free carboxyl groups; the organic polymers containing sugar units and aldehyde groups include organic polymers containing sugar units and free aldehyde groups or aldehyde-modified organic polymers containing sugar units and ortho-dihydroxy groups.

4. The preparation method according to claim 3, characterized in that, The organic polymers containing amino groups include at least one of the following: proteins, polypeptides, amino polysaccharides, polyamino acids, glycosaminoglycans, polyvinyl alcohol, polyacids, polyesters, pectin, polyglucosamine, polyaminoketones, polyvinyl acid, polyaminothiazoles, polyamides, nylon, polyvinyl alcohol salts, polyamino acrylates, polyamino acid salts, polyaminothiophene, polyaminonitroso, polyaminotetrahydropyridine, or polyaminoethylene. And / or, the organic polymer containing sugar units and aldehyde groups includes at least one of the following: glucose, galactose, fructose, arabinose, xylose, mannose, mannitol, glucuronic acid, N-acetylglucosamine, N-acetylglucosamine, lactose, maltose, sucrose, hyaluronic acid, pectin, heparin, chondroitin sulfate, polylactic acid, polyglutamic acid, polymannan, ribose, deoxyribose, glucuronic acid, wood aldehyde, glyceraldehyde, glucosamine, triglycerides, fatty acids, polyesters, peptides, cellulose, or cellulose derivatives.

5. The preparation method according to claim 4, characterized in that, The polyamino acid is γ-polyglutamic acid; and / or, the cellulose derivative is sodium carboxymethyl cellulose.

6. The preparation method according to claim 5, characterized in that, The preparation method of the nasal spray protective hydrogel includes the following steps: S1. γ-polyglutamic acid is modified with amino groups by reacting it with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide and adipate dihydrazide to obtain amino-grafted γ-polyglutamic acid. S2. Sodium carboxymethyl cellulose is reacted with an oxidant to modify the aldehyde group, thereby obtaining aldehyde-modified carboxymethyl cellulose; S3. Dissolve γ-polyglutamic acid grafted with amino groups in a solvent to prepare prepolymer solution one; dissolve aldehyde-modified carboxymethyl cellulose in a solvent to prepare prepolymer solution two; mix and react prepolymer solution one and prepolymer solution two to obtain the nasal spray protective hydrogel. The solvent is water, physiological saline, or PBS buffer; the mass-volume fraction of the grafted amino γ-polyglutamic acid in the nasal spray protective hydrogel is 2-3%, and the mass-volume fraction of the aldehyde-modified carboxymethyl cellulose in the nasal spray protective hydrogel is 4%.

7. A nasal spray protective hydrogel prepared by the preparation method according to any one of claims 1 to 6.

8. The use of the nasal spray protective hydrogel of claim 7 in the preparation of medical devices, tissue engineering materials, biosensors or drug delivery carriers.

9. A drug-loaded hydrogel, characterized in that, It contains the nasal spray protective hydrogel of claim 7 and the drug.

10. The drug-loaded hydrogel as described in claim 9, characterized in that, The drug comprises one or a combination of two or more of the following: nanoparticles, miRNA, exosomes, liposomes, microvesicles, microspheres, framework nucleic acid aptamers, peptides, metal ions, metal-organic frameworks, traditional Chinese medicine extracts, or finished drugs.

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