Injectable hydrogel for photothermally releasing nitric oxide against biofilm

Abstract

The present application provides an injectable hydrogel for photothermal release of nitric oxide antibiofilm, which solves the problem that the existing antibacterial hydrogel cannot completely eradicate biofilm infection. The present application selects degradable oxidized dextran (OD) and polylysine (EPL) with antibacterial properties as the matrix of the hydrogel, and loads the aldehyde-containing thermosensitive NO donor N-nitroso (4-aldehyde phenyl) -hydroxylamine ammonium (ACup) on the glucose amine modified polydopamine (PDG) and dopes it in the matrix of the hydrogel. The loading of the NO donor makes the hydrogel slowly release NO at physiological temperature, thereby promoting the dispersion of the biofilm; due to the presence of glucose amine modified polydopamine, the injectable hydrogel also has good photothermal performance, and the heat generated under the irradiation of near-infrared light not only has a certain photothermal sterilization performance, but also can promote the release of NO, and cooperate with the antibacterial peptide EPL to completely eradicate biofilm infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical nanomaterials technology, specifically relating to an injectable hydrogel that releases photothermal nitric oxide to resist biological membranes. Background Technology

[0002] Infections caused by pathogenic bacteria have long posed a threat to human health. Since the mid-20th century, a bacterial resistance crisis has rapidly emerged due to the overuse of antibiotics. Furthermore, bacteria always colonize and multiply in suitable areas, forming multicellular communities called biofilms. Bacteria living within biofilms are encased in their own extracellular matrix (EPS), which not only allows them to resist attacks from the host's immune response but also prevents the penetration of antimicrobial drugs. Therefore, biofilm formation further exacerbates bacterial resistance and leads to severe and recurrent infections. Due to the lack of effective treatments, biofilm-associated infections cause immense suffering and financial burden on patients. Therefore, the development of new treatments for bacterial biofilm infections is urgently needed.

[0003] As the effectiveness of traditional antibiotics in combating biofilm-related infections gradually diminishes, emerging treatment strategies, such as those utilizing cationic antimicrobial peptides, have attracted considerable research interest. However, due to the poor permeability of antimicrobial peptides within biofilms, complete eradication of biofilms is difficult. Therefore, to achieve optimal efficacy, antimicrobial peptides are often combined with photothermal therapy (PTT) to enhance their anti-biofilm efficacy. However, since the photothermal agents themselves also have poor permeability within biofilms, complete eradication is also challenging. To improve efficacy, PTT typically requires prolonged hyperthermia under high laser density, which inevitably damages nearby healthy tissue.

[0004] Therefore, it is necessary to find a method that can completely eradicate biofilms. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing antibacterial hydrogels in completely eradicating biofilm infection, and to provide an injectable hydrogel that releases nitric oxide photothermally to resist biofilm infection, thereby completely eliminating biofilm and inhibiting bacteria within the biofilm.

[0006] The concept of this invention:

[0007] EPS protection enables bacteria in biofilms to resist not only the host immune response but also the attack of exogenous antibacterial agents, thereby exacerbating bacterial resistance. Traditional antibacterial hydrogels cannot completely solve the problem of biofilm infection. This invention proposes to introduce an important gaseous signaling molecule—nitric oxide (NO). NO, as a gaseous messenger that can regulate important physiological and pathological processes, is effective at low concentrations (10⁻⁶ ppm). -12 -10 -9 NO in M ​​can dissipate biofilms by reducing extracellular polysaccharides in EPS, causing bacteria inside the biofilm to become planktonic bacteria that are more sensitive to antimicrobial agents. High concentrations (usually above 10) -6 NO (M) can cause DNA damage, lipid peroxidation, and membrane disruption through nitrification and oxidative stress, ultimately leading to bacterial death and enhancing its bactericidal ability. It is evident that the antibacterial ability of NO is dose-dependent, and it is also very suitable for antibacterial and anti-biofilm applications. However, NO also has certain shortcomings that limit its application in biofilms, namely, its relatively short half-life, inconvenient direct application, and difficulty in controlling the dosage.

[0008] Therefore, the research team of this invention designed an injectable hydrogel with photothermal controllable NO release, which is expected to slowly release low concentrations of NO at physiological temperature to dissipate biofilms. Once near-infrared light is applied, the hydrogel can accelerate the release of NO, coordinate antimicrobial peptides and PTT to completely eradicate the problem of biofilm infection, achieve efficient loading and controllable release of NO, and completely eradicate biofilm infection.

[0009] Based on the above inventive concept, and to achieve the above objectives, the technical solution provided by this invention is as follows:

[0010] An injectable hydrogel that releases NO via photothermal emission, characterized by:

[0011] The hydrogel uses biodegradable oxidized dextran (OD, i.e., oxidized natural polysaccharide) and antibacterial polylysine (EPL, i.e., natural cationic antimicrobial peptide) as the matrix, glucosamine-modified polydopamine nanoparticles (PDG) as the carrier, and N-nitroso(4-aldehydephenyl)-hydroxylamine ammonium (ACup) as the thermosensitive NO donor.

[0012] The thermosensitive NO donor is loaded onto the support via a Schiff base reaction, and then incorporated into the matrix of the hydrogel via a Schiff base reaction. Specifically, after the thermosensitive NO donor is loaded onto the support via a Schiff base reaction, it is blended with the polylysine and then crosslinked with oxidized dextran via a Schiff base reaction.

[0013] Furthermore, at physiological temperatures, it can slowly release NO, thereby dissipating the biofilm and transforming the bacteria within the biofilm into more sensitive planktonic bacteria; once near-infrared light stimulation is applied, the hydrogel can rapidly release NO, thus achieving excellent anti-biofilm effects in conjunction with photothermal therapy and chemotherapy.

[0014] The method for preparing the above-mentioned photothermal NO-releasing injectable hydrogel is characterized by including the following steps:

[0015] 1) Preparation of glucosamine-modified polydopamine nanoparticle solution loaded with a thermosensitive NO donor

[0016] The thermosensitive NO donor N-nitroso(4-aldehydephenyl)-hydroxyamine ammonium was dissolved in phosphate buffer solution to obtain a thermosensitive NO donor solution. Then, the thermosensitive NO donor solution was mixed with a glucosamine-modified polydopamine nanoparticle dispersion to obtain a glucosamine-modified polydopamine nanoparticle solution loaded with the thermosensitive NO donor (PDG / ACup).

[0017] 2) The glucosamine-modified polydopamine nanoparticle solution (PDG / ACup) with thermosensitive NO donor obtained in step 1) was mixed with a polylysine phosphate buffer solution with physiological pH (7.4) to obtain mixture A (EPL / PDG / ACup).

[0018] 3) The mixture A obtained in step 2) is mixed with the phosphate buffer solution of oxidized dextran and reacted to obtain an injectable hydrogel that releases NO through photothermal reaction (OD-EPL / PDG / ACup), wherein the volume ratio of the EPL / PDG / ACup mixture to the OD solution is 1:1.

[0019] In other words: First, the thermosensitive NO donor ACup solution and PDG nanoparticle dispersion are mixed to obtain PDG / ACup; then, it is added to an EPL solution at physiological pH to obtain EPL / PDG / ACup; at the same time, OD is dissolved in a phosphate buffer solution at physiological pH to obtain an OD phosphate buffer solution (referred to as OD solution); finally, equal volumes of EPL / PDG / ACup and OD solution are mixed to obtain an injectable hydrogel that releases NO, OD-EPL / PDG / ACup.

[0020] Further, in step 1), the preparation method of the thermosensitive NO donor N-nitroso(4-aldehydephenyl)-hydroxylamine ammonium (ACup) is as follows:

[0021] A1. Dissolve p-nitrobenzaldehyde in a methanol solution, then mix it with an aqueous solution of ammonium chloride to obtain a mixed solution; wherein the volume ratio of methanol to water is 5:1;

[0022] A2. Place the mixed solution obtained in A1 in an oil bath at 50-60℃, stir vigorously until it is evenly mixed, continue stirring vigorously and add excess Zn powder to react (stir for 3-4 hours at a stirring speed of 700 rpm), and monitor the reaction progress using thin-layer chromatography.

[0023] A3. After the reaction is complete (the reaction solution can also be quenched in ice water), the reaction solution is then filtered (specifically, a Bush funnel is used to filter out the byproduct Zn(OH)2 and unreacted zinc powder), and the filter cake is washed with ether (specifically, the solid residue is washed multiple times with ether). The filtrate and washing solution are combined to form a mixed solution.

[0024] A4. Use a separatory funnel to wash the blended solution obtained in A3 with diethyl ether (wash multiple times) to remove methanol and water, and dry the separated diethyl ether with anhydrous sodium sulfate to obtain an ether solution;

[0025] A5. Vigorously bubble ammonia gas through the ether solution obtained in A4 for 15-20 minutes, then add isobutyl nitrite in small batches over 15 minutes while maintaining cooling and vigorous bubbling of ammonia gas.

[0026] A6. After the reaction is complete, remove the diethyl ether by vacuum distillation to obtain the product N-nitroso(4-aldehydephenyl)-hydroxyamine ammonium (ACup). The product ACup should be sealed and protected from light and stored in a refrigerator at 0-4°C.

[0027] The molar ratio of p-nitrobenzaldehyde, ammonium chloride, zinc powder and isobutyl nitrite is 1:2:2:1.5.

[0028] Further, in step 1), the preparation method of the glucosamine-modified polydopamine nanoparticle dispersion is as follows:

[0029] B1. Under stirring (speed 2800 rpm, using a high-speed dispersion device IKA S 18N-19G), first add tris(hydroxymethyl)aminomethane and acetylglucosamine to ultrapure water, then add dopamine hydrochloride (the order of adding tris(hydroxymethyl)aminomethane and acetylglucosamine is not required, as long as dopamine hydrochloride is added last), and carry out an oxidative deposition copolymerization reaction. After the reaction is completed, centrifuge to obtain acetylglucosamine-modified polydopamine nanomaterial PDNG (centrifuge at a speed of 9000-10000 rpm for 15-10 min), freeze-dry, and store at room temperature in the dark.

[0030] Ultrapure water is used as a solvent; the molar ratio of ultrapure water, tris(hydroxymethyl)aminomethane, acetylglucosamine and dopamine hydrochloride is 11111∶2∶2.9~11.6∶5.8, with the optimal value being 11111∶2∶5.8∶5.8;

[0031] The ultrasonic high-speed dispersion time is 30-120 min, with the optimal value being 60 min, which can yield acetylglucosamine-modified polydopamine nanomaterials PDNG.

[0032] B2. Add concentrated hydrochloric acid to the aqueous solution of acetylglucosamine-modified polydopamine nanoparticles (PDNG) obtained in step 1), condense and reflux at 80–90°C (i.e., high-temperature acidic environment) for 50–70 min (optimal value is condensation and reflux at 85°C for 60 min); quench the reaction mixture in an ice bath, centrifuge, disperse in ultrapure water and freeze-dry to obtain PDG (i.e., deacetylate the obtained PDNG to obtain PDG); specifically, centrifuge at 9000–10000 rpm for 15–10 min, then resuspend twice by centrifugation with 1M PBS buffer, and finally disperse in ultrapure water and freeze-dry to obtain PDG; then repeatedly centrifuge and wash with phosphate buffer solution to obtain a neutral glucosamine-modified polydopamine nanoparticle dispersion;

[0033] The aqueous solution concentration of the PDNG is 1–2 mg / mL. -1 The optimal value is 1.5 mg / mL. -1 ;

[0034] The concentration of the concentrated hydrochloric acid is 12M.

[0035] Further, in step 2), the method for preparing the polylysine phosphate buffer solution with the physiological pH value is as follows:

[0036] Polylysine was dissolved in a phosphate buffer solution at room temperature, and the pH of the solution was adjusted to the physiological pH value (using 5M sodium hydroxide) to obtain a phosphate buffer solution of polylysine with a mass fraction of 10%.

[0037] Further, in step 3), the method for preparing the phosphate buffer solution of the oxidized dextran is as follows:

[0038] C1. At room temperature, dissolve dextran in ultrapure water (stir magnetically for at least 5 hours to dissolve) to obtain a dextran aqueous solution with a concentration of 10-20 g / L;

[0039] C2. At room temperature, sodium periodate is added to the dextran aqueous solution obtained in C1, and the oxidation reaction is carried out in the dark for 4-6 hours to obtain a mixed solution;

[0040] The molar ratio of sodium periodate to aldehyde groups in dextran is 0.2-0.8:1;

[0041] C3. The mixed solution obtained in C2 is placed in a dialysis bag and dialyzed at low temperature for 48-72 hours to obtain an aqueous solution of oxidized dextran; then freeze-dried to obtain oxidized dextran; within the above range, different molar ratios of sodium periodate to aldehyde groups in dextran can be used to obtain oxidized dextran ODs with different oxidation degrees. Under different oxidation degrees, the hydrogel formation time, stability, and degradation rate are different.

[0042] C4. Dissolve the oxidized dextran obtained in C3 in phosphate buffered saline (PBS) at a physiological pH of 7.4 to prepare a phosphate buffered saline solution with a mass fraction of 8% oxidized dextran.

[0043] In addition, the present invention also provides the application of the above-mentioned photothermal NO-releasing injectable hydrogel in the preparation of antibacterial and antibacterial drugs, in the preparation of drugs that dissipate biofilms, and in the preparation of drugs that promote the proliferation of fibroblasts and the migration of vascular endothelial cells.

[0044] The principle of this invention:

[0045] This invention selects biodegradable oxidized dextran (OD) and antibacterial polylysine (EPL) as the hydrogel matrix. Glucosamine-modified polydopamine (PDG) is loaded with N-nitroso(4-aldehydephenyl)-hydroxylamine ammonium (ACup), a thermosensitive NO donor containing aldehyde groups, and incorporated into the hydrogel matrix. Oxidized dextran is obtained by oxidizing the ortho-hydroxyl groups on natural dextran using sodium periodate, resulting in OD rich in aldehyde groups. Therefore, OD can dynamically crosslink with polylysine (EPL), which has a large number of amino groups, through Schiff base bonds, thus offering the advantage of injectability. Glucosamine-modified polydopamine (PDG) has abundant amino groups on its surface and can be loaded with a thermosensitive NO donor containing aldehyde groups via a Schiff base reaction. After blending with polylysine (EPL), it is incorporated into the injectable hydrogel matrix formed by oxidized dextran and polylysine through a Schiff base reaction with the aldehyde groups of oxidized dextran. The loading of the NO donor causes the hydrogel to slowly release NO at physiological temperatures, thereby promoting the dissipation of biofilms. Due to the presence of glucosamine-modified polydopamine, the injectable hydrogel also has good photothermal properties. The heat generated under near-infrared light irradiation not only has a certain photothermal bactericidal effect, but also promotes the release of NO, synergistically eliminating biofilm infection with the antimicrobial peptide EPL.

[0046] The advantages of this invention are:

[0047] 1. This invention prepares an injectable hydrogel that releases NO by loading a thermosensitive NO donor with aldehyde groups onto amino-rich glucosamine-modified polydopamine nanoparticles with good photothermal properties, and then doping it (through a Schiff base reaction with the aldehyde groups of oxyglucan) into an injectable hydrogel crosslinked with oxyglucan and polylysine. The hydrogel exhibits good photothermal properties; at physiological temperatures, it can slowly release NO. The relatively low level of NO released promotes the dissipation of biofilms, transforming drug-resistant bacteria within the biofilm into more sensitive planktonic bacteria. Once subjected to 10 minutes of near-infrared irradiation (808 nm, 1 W cm⁻¹), the NO release is further enhanced. -2 Hydrogels can accelerate the release of NO, meaning that under near-infrared light irradiation, the relatively high concentration of NO released can work synergistically with antimicrobial peptides and photothermal therapy to efficiently and thoroughly eradicate biofilms.

[0048] 2. The photothermal NO-releasing injectable hydrogel proposed in this invention not only has good degradability, injectability and photothermal properties, but also good biocompatibility and low hemolysis rate and cytotoxicity.

[0049] 3. In this invention, an aldehyde-containing nitric oxide donor, N-nitroso(4-aldehydephenyl)-hydroxylamine ammonium (ACup), is loaded onto glucosamine nanoparticles (PDG) modified with a large number of amino groups via a Schiff base reaction to obtain PDG / ACup. Simultaneously, oxidized dextran (OD) is prepared by oxidizing dextran with sodium periodate. Then, PDG / ACup is blended with the antimicrobial peptide polylysine (EPL), and then crosslinked with aldehyde-containing OD via a Schiff base reaction to obtain an injectable hydrogel that releases nitric oxide (OD-EPL / PDG / ACup). Attached Figure Description

[0050] Figure 1 This is a partial process flow diagram of the preparation of injectable hydrogels in this invention; wherein, a is the process flow of preparing oxidized dextran (OD) by oxidizing dextran with sodium periodate (NaIO4); b is the process flow of preparing N-nitroso(4-aldehydephenyl)-hydroxylamine ammonium (ACup); and c is the process flow of preparing glucosamine-modified polydopamine nanoparticles (PDG).

[0051] Figure 2 The diagram shows the mechanism of the preparation method of the present invention; wherein, a is the mechanism diagram of N-nitroso(4-aldehydephenyl)-hydroxyamine ammonium ACup loaded on glucosamine-modified polydopamine PDG nanoparticles; b is the mechanism diagram of the preparation of OD-EPL / PDG / ACup.

[0052] Figure 3This document presents confirmation and morphology images of some products generated during the preparation of the injectable hydrogel in this invention; where a is the 1H NMR spectrum of the prepared ACup. 1 b) is the actual oxidation degree of OD obtained by oxidation with different amounts of NaIO4; c) is the image of EPL and OD before and after gelation; d) is the gelation time of different hydrogels characterized by rheometer; e) is the scanning electron microscope image of OD-EPL, OD-EPL / PDG and OD-EPL / PDG / ACup.

[0053] Figure 4 The diagram shows the physical properties of the injectable hydrogel prepared in this invention; where a is the modulus change of different hydrogels with stress; b is the viscosity change of the hydrogel with shear rate; c is the remaining mass of different hydrogels at physiological pH with time; and d is the swelling rate of different hydrogels with time.

[0054] Figure 5 The graphs show the temperature test performance of the embodiments and comparative examples of the present invention; where a represents the OD-EPL / PDG temperature at 1W cm⁻¹. -2 Temperature change under 808nm near-infrared irradiation; b is OD-EPL / PDG at 1W cm⁻¹ -2 The temperature cycling curves under 808nm near-infrared light irradiation are shown in Figure 1; c represents the NO release from OD-EPL / PDG / ACup under NIR stimulation with and without switch on / off; d represents the NO release from OD-EPL / PDG / ACup at physiological temperature.

[0055] Figure 6 Crystal violet staining characterizes the biofilms of this invention after different hydrogel treatments; wherein, a is a methicillin-resistant Staphylococcus aureus (MRSA) biofilm; b is a tetracycline-resistant Escherichia coli (TREC) biofilm; and c is a Candida albicans (C. albicans) biofilm.

[0056] Figure 7 The figures show the antibacterial performance of the embodiments and comparative examples of the present invention; wherein, a represents the reduction in bacterial colony count in MRSA biofilms after different hydrogel treatments; b represents the reduction in bacterial colony count in TREC biofilms after different material treatments; c represents the reduction in bacterial colony count in C. albicans biofilms after different material treatments; and d represents the scanning electron microscope morphology of MRSA, TREC, and C. albicans before and after different material treatments.

[0057] Figure 8The images shown are related to the biocompatibility testing of the injectable hydrogel of the present invention; where a shows the dissolution of New Zealand rabbit erythrocytes after treatment with nanomaterials with different hydrogels; b shows the cell viability of mouse fibroblasts after different hydrogel treatments; and c shows the cell viability of mouse fibroblasts after different hydrogel treatments.

[0058] Figure 9 The figures shown are related to the in vitro migration test of umbilical vein endothelial cells (HUVECs) using the injectable hydrogel of the present invention; where a is the remaining scratch width of HUVECs after 36 hours of culture in the scratch assay; b is the statistical analysis of the remaining scratch width of HUVECs after 36 hours of culture in the scratch assay; c is the migration status of HUVECs after 1 day of culture in a Transwell chamber; and d is the quantitative statistical analysis of HUVECs after 1 day of culture in a Transwell chamber. Detailed Implementation

[0059] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments:

[0060] Example 1

[0061] 1) Preparation of OD2

[0062] Figure 1 In Figure 'a', the process flow diagram for OD is shown. The specific preparation steps for OD2 are as follows:

[0063] 2 g of dextran was dissolved in 100 mL of ultrapure water at room temperature. The resulting dextran aqueous solution was added to a 200 mL single-necked round-bottom flask. Then, 0.53 g of sodium periodate was added to the dextran aqueous solution. The reaction was carried out in the dark for 6 hours, dialyzed for 2 days, and lyophilized to obtain OD2. The molar ratio of aldehyde groups to sodium periodate in the dextran was 1:0.2.

[0064] The OD2 was then dissolved in phosphate buffer (PBS) at pH 7.4 to obtain an OD2 solution with a mass fraction of 8%.

[0065] 2) Preparation of EPL

[0066] 2g of EPL was dissolved in phosphate-buffered saline (PBS). The resulting EPL solution was adjusted to pH 7.4 with 5M sodium hydroxide, resulting in a final solution volume of 20mL. The mass fraction of the EPL solution was 10%.

[0067] There is no obvious chronological order between steps 1) and 2) above, and their order can be changed.

[0068] 3) Preparation of OD2-EPL hydrogel

[0069] 250 μL of OD2 (8% by mass) and 250 μL of EPL (10% by mass) were mixed to obtain OD2-EPL hydrogel.

[0070] Example 2

[0071] 1) Preparation of OD4

[0072] 2 g of dextran was dissolved in 100 mL of ultrapure water at room temperature. The resulting dextran aqueous solution was added to a 200 mL single-necked round-bottom flask. Then, 1.05 g of sodium periodate was added to the dextran aqueous solution. The reaction was carried out in the dark for 6 hours, dialyzed for 2 days, and lyophilized to obtain OD4. The molar ratio of aldehyde groups to sodium periodate in the dextran was 1:0.4.

[0073] The OD4 was then dissolved in phosphate buffer (PBS) at pH 7.4 to obtain an OD4 solution with a mass fraction of 8%.

[0074] 2) Preparation of EPL

[0075] 2g of EPL was dissolved in phosphate-buffered saline (PBS). The resulting EPL solution was adjusted to pH 7.4 with 5M sodium hydroxide, resulting in a final solution volume of 20mL. The mass fraction of the EPL solution was 10%.

[0076] There is no obvious chronological order between steps 1) and 2) above, and their order can be changed.

[0077] 3) Preparation of OD4-EPL

[0078] 250 μL of OD4 (8% by mass) and 250 μL of EPL (10% by mass) were mixed to obtain OD4-EPL hydrogel.

[0079] Example 3:

[0080] 1) Preparation of OD6

[0081] 2 g of dextran was dissolved in 100 mL of ultrapure water at room temperature. The resulting dextran aqueous solution was added to a 200 mL single-necked round-bottom flask. Then, 1.58 g of sodium periodate was added to the dextran aqueous solution. The reaction was carried out in the dark for 6 hours, dialyzed for 2 days, and lyophilized to obtain OD6. The molecular weight of the oxidized dextran was 500,000. The molar ratio of aldehyde groups to sodium periodate in the dextran was 1:0.6.

[0082] OD6 was then dissolved in phosphate buffer (PBS) at pH 7.4 to obtain an OD6 solution with a mass fraction of 8%.

[0083] 2) Preparation of ACP

[0084] Figure 1 Figure b shows the preparation flow chart of ACup. p-Nitrobenzaldehyde (211.7 mg, 1.4 mmol) was dissolved in methanol and then mixed with ammonium chloride aqueous solution (149.8 mg, 2.8 mmol), resulting in a final volume of 12 mL, with a methanol to H₂O volume ratio of 5:1. After stirring until homogeneous, Zn powder (0.18 g, 2.8 mmol) was added. The mixture was stirred at 50 °C for 3 h, and the reaction progress was monitored using thin-layer chromatography (TLC). After quenching the reaction with ice water, the byproduct Zn(OH)₂ and unreacted zinc powder were filtered off using a Bush funnel. The solid residue was then thoroughly washed three times with 20 mL of diethyl ether. The collected product and diethyl ether solution were mixed and washed three more times with diethyl ether using a separatory funnel. The separated diethyl ether was then dried with anhydrous sodium sulfate. The dried diethyl ether solution was cooled in an ice-salt bath mixture. Then, under ice bath conditions, ammonia gas was vigorously bubbled into the ether solution for 15 minutes. Subsequently, isobutyl nitrite (219 μL, 2.1 mmol, added in portions over 15 minutes) was added under ice bath and ammonia bubbling conditions. Finally, the mixture was extracted with ether, and the extract was distilled under reduced pressure to obtain the product Acup. Figure 3 'a' is ACup 1 H-NMR spectrum, 1 H NMR:δ H The results (500MHz, DMSO) showed 7.90 (d, 2H), 8.06 (d, 2H), and 9.94 (s, 1H), confirming the successful synthesis of ACup. 3.66 mg of ACup was weighed and dissolved in 1 mL of PBS to obtain a 20 mmol / L ACup solution.

[0085] 3) Preparation of PDG

[0086] Figure 1 C represents the PDG preparation process. 200 mL of deionized water was measured into a 500 mL beaker and dispersed at 2800 rpm. Then, 242 mg of tris(hydroxymethyl)aminomethane and 256 mg of acetylglucosamine were added sequentially to the beaker. After 5 min, 220 mg of dopamine hydrochloride was added to the above solution to initiate the reaction. The reaction was carried out at room temperature and 2800 rpm for 1 h. After the reaction was completed, the reaction solution was transferred to a 50 mL centrifuge tube, centrifuged at 10000 rpm, washed with deionized water, and freeze-dried to obtain PDNG.

[0087] 4 mL of a 1.5 mg / mL PDNG aqueous solution and 8 mL of 12 M concentrated hydrochloric acid were added to a 50 mL round-bottom flask and refluxed at 85 °C for 1 h. After the reaction was complete, the reaction solution was transferred to a 50 mL centrifuge tube and centrifuged at 9500 rpm for 15 min. The solution was then resuspended twice by centrifugation with 1 M PBS buffer. Finally, the PDG was dispersed in Milli-Q and freeze-dried to obtain PDG. PDG was then dispersed in PBS to prepare a 10 mg / mL PDG dispersion.

[0088] 4) Preparation of EPL

[0089] 2g of EPL was dissolved in PBS, and the resulting EPL solution was adjusted to pH 7.4 with 5M sodium hydroxide, resulting in a final solution volume of 20mL. The mass fraction of the EPL solution was 10%.

[0090] There is no obvious chronological order among the aforementioned steps 1)-4), and the order can be changed.

[0091] 5) Preparation of PDG (PDG / ACup) with a loaded thermistor NO donor (ACup)

[0092] Figure 2 'a' represents the mechanism of the interaction between PDG and ACup.

[0093] PDG / ACup was prepared by mixing 100 μL of PDG solution (10 mg / mL) and 1 mL of ACup solution (20 mmol / L).

[0094] 6) Preparation of EPL / PDG / ACup

[0095] The above PDG / ACup mixture was gradually added dropwise to 24 mL of the above EPL solution to obtain the EPL / PDG / ACup solution.

[0096] 7) Preparation of OD6-EPL, OD6-EPL / PDG, and NO-releasing anti-biofilm injectable hydrogel OD6-EPL / PDG / Acup

[0097] 250 μL of OD6 (8% by mass) and 250 μL of EPL (10% by mass) were mixed in equal proportions to obtain OD6-EPL hydrogel.

[0098] 100 μL of PDG solution (10 mg / mL) was gradually added dropwise to 25 mL of the above EPL solution to obtain an EPL / PDG solution; 250 μL of OD6 (mass fraction of 8%) and 250 μL of EPL / PDG solution were mixed in equal proportions to obtain an OD6-EPL / PDG hydrogel.

[0099] Figure 2 c represents the mechanism diagram of the OD6-EPL / PDG / ACup hydrogel. OD6-EPL / PDG / ACup hydrogel was obtained by mixing 250 μL of OD6 (8% by mass) and 250 μL of EPL / PDG / ACup in equal proportions.

[0100] Example 4:

[0101] 1) Preparation of OD8

[0102] 2 g of dextran was dissolved in 100 mL of ultrapure water at room temperature. The resulting dextran aqueous solution was added to a 200 mL single-necked round-bottom flask. Then, 2.11 g of sodium periodate was added to the dextran aqueous solution. The reaction was carried out in the dark for 6 hours, dialyzed for 2 days, and lyophilized to obtain OD8. The molar ratio of aldehyde groups to sodium periodate in the dextran was 1:0.8.

[0103] OD8 was then dissolved in phosphate buffer (PBS) at pH 7.4 to obtain an OD solution with a mass fraction of 8%.

[0104] 2) Preparation of EPL

[0105] 2g of EPL was dissolved in PBS, and the resulting EPL solution was adjusted to pH 7.4 with 5M sodium hydroxide, resulting in a final solution volume of 20mL. The mass fraction of the EPL solution was 10%.

[0106] 3) Preparation of OD8-EPL

[0107] 250 μL of OD8 (8% by mass) and 250 μL of EPL (10% by mass) were mixed to obtain OD8-EPL hydrogel.

[0108] Characterization of the gel-forming properties of hydrogels

[0109] Figure 3 b represents the oxidation degree of OD measured by potentiometric titration with hydroxylamine hydrochloride. The oxidation degrees of OD2, OD4, OD6, and OD8 are 18.7%, 37.4%, 56.6%, and 71.2%, respectively. This invention uses an inverted method to more intuitively observe the gelation phenomenon of the hydrogel. First, methylene blue dye is added to the solution of OD6, such as... Figure 3 As shown in Figure c, gelation is clearly observed after EPL and OD6 are mixed. The initial gelation process was monitored using time-scan rheological experiments. The critical point for sol-gel transition is when the storage modulus (G') exceeds the loss modulus (G”), as shown in Figure c. Figure 3As shown in Figure d, with increasing time, the G' of OD2-EPL, OD4-EPL, OD6-EPL, OD8-EPL, and OD6-EPL / PDG hydrogels gradually exceeded G', with gelation times of 91 s, 50 s, 32 s, 30 s, and 28 s, respectively. The gelation rate of the hydrogels accelerated with increasing oxidation degree, indicating that the crosslinking density of the OD-EPL hydrogel matrix gradually increased. However, when the oxidation degree increased from 56.6% to 71.2%, the gelation rate of OD8-EPL did not increase significantly; its gelation time was only slightly longer than that of OD6-EPL / PDG. The 6-EPL time decreased by 2 s, likely due to the saturation of the cross-linking reaction between the amino groups on EPL and the aldehyde groups on OD. Considering the ease of operation of injectable hydrogels in practical applications, OD6 was chosen as the optimal condition for hydrogel preparation. The gelation time of the OD6-EPL / PDG hydrogel was 28 s. Because PDG has abundant amino groups, PDG nanoparticles can also be cross-linked with OD via Schiff base reaction, enhancing the cross-linking density of the OD-EPL / PDG hydrogel. The morphology of the obtained hydrogels was observed using scanning electron microscopy, as shown below. Figure 3 As shown in Figure e, OD-EPL, OD-EPL / PDG, and OD-EPL / PDG / ACup all exhibit typical three-dimensional network porous morphology. It is evident that the addition of PDG and ACup did not alter the original morphology and structure of the hydrogel.

[0110] Furthermore, the mechanical behavior of the hydrogel was studied using a rheometer, and the critical points of the fluid and solid states of the matrix were determined by strain amplitude scanning experiments. The dynamic modulus of the OD-EPL hydrogel under high and low strain cycles was calculated. Figure 4 The strain amplitude scanning curves in section a show that all hydrogels exhibit G' / G”>10 within the linear viscoelastic domain, indicating the formation of a stable gel network. The rheological properties of hydrogels are closely related to frequency, such as... Figure 4 As shown in b, the viscosity of OD2-EPL, OD4-EPL, OD6-EPL, OD8-EPL and OD6-EPL / PDG hydrogels decreases with increasing shear rate, demonstrating the injectability of these hydrogels.

[0111] The in vitro degradation behavior of hydrogels prepared with different oxidation states (OD) in PBS solution at pH 7.4 was determined by measuring the weight loss of the hydrogels over a certain period of time. Figure 4As shown in Figure c, the OD2-EPL hydrogel completely degraded within 2 hours. This phenomenon may be due to the fewer crosslinking sites in the hydrogel prepared from low-oxidation OD, which accelerated the collapse of the internal network structure. The weights of the OD4-EPL, OD6-EPL, and OD8-EPL hydrogels gradually decreased within the first 12 hours, after which the degradation rate slowed down. After 72 hours, the weights of the OD4-EPL, OD6-EPL, and OD8-EPL hydrogels were 28%, 39%, and 55%, respectively. The dynamic swelling behavior of the OD / EPL hydrogels was investigated by lyophilizing them and then immersing them in PBS at pH 7.4. Figure 4 As shown in Figure d, all OD-EPL hydrogels swelled rapidly in PBS (pH = 7.4) and reached equilibrium within 1 min. OD2-EPL hydrogel exhibited the highest swelling rate, at 1208% ± 109%. The swelling rates of OD4-EPL, OD6-EPL, and OD8-EPL gradually decreased, at 946% ± 4%, 823% ± 8%, and 760% ± 25%, respectively. This is attributed to the increasing cross-linking density of the hydrogels with increasing oxidation. The swelling properties of the hydrogels facilitate the absorption and exudation of blood and tissue fluid at the wound site.

[0112] Photothermal property characterization of OD-EPL / PDG hydrogel

[0113] Polydopamine nanomaterials are excellent photothermal conversion agents. Therefore, the photothermal properties of OD-EPL / PDG were tested. 500 μL of OD-EPL / PDG hydrogels containing different concentrations of PDG nanoparticles were subjected to a 1.0 W cm⁻¹ laser beam at an 808 nm intensity. -2 Irradiation at a high power density for 10 minutes, with the temperature change of OD-EPL / PDG recorded every minute using a thermal imager, allows for the plotting of a temperature-time curve for the OD-EPL / PDG hydrogel. Figure 5 As shown in Figure a, the temperature of the OD-EPL / PDG hydrogel increases with increasing illumination time, and the temperature change exhibits a concentration-dependent relationship. When the concentration of PDG is 30 μg / mL... -1 At that time, the temperature of the OD-EPL / PDG hydrogel increased from 20.0℃ to 55.2℃, while the free phosphate-buffered saline (PBS) only increased slightly to 30.4℃. According to the heating-cooling curve (… Figure 5 (b) After three repeated heating and cooling cycles, the photothermal conversion capacity of the OD-EPL / PDG hydrogel did not decrease, indicating that the OD-EPL / PDG hydrogel has good cycling stability.

[0114] Determination of the kinetic properties of NO release from OD-EPL / PDG / ACup hydrogel

[0115] After incubating the OD-EPL / PDG / ACup hydrogel at 37°C for 0, 10, 20, 30, 40, 50, 60, 70, 80, and 90 minutes, NIR (1.0 W cm⁻¹) was measured without NIR. -2 After irradiation for 10 minutes, the released NO was measured using Griess reagent. Additionally, after incubating the OD-EPL / PDG / ACup hydrogel at 37°C for 1 to 36 hours, the NO released by the OD-EPL / PDG / ACup hydrogel without NIR irradiation was measured using Griess reagent. Figure 5 Figure c shows the on-demand NO release of the prepared OD-EPL / PDG / ACup hydrogel with and without NIR stimulation. Under NIR stimulation, the OD-EPL / PDG / ACup hydrogel rapidly heats up; the high temperature accelerates the decomposition of ACup, thus accelerating NO release. Without NIR irradiation, the OD-EPL / PDG / ACup hydrogel releases NO slowly. The release amount in the NIR-irradiated group is significantly greater than that in the non-NIR-irradiated group. This indicates that the OD-EPL / PDG / ACup hydrogel can release NO on demand under NIR stimulation. Figure 5 d represents the NO release of the prepared OD-EPL / PDG / ACup hydrogel over a prolonged period at 37°C without NIR irradiation. In the experimental group without NIR irradiation, the OD-EPL / PDG / ACup hydrogel exhibited a slow NO release characteristic with increasing incubation time, continuously releasing NO for up to 36 hours or even longer. Therefore, this indicates that the OD-EPL / PDG / ACup hydrogel slowly releases low concentrations of NO at physiological temperatures, while under NIR light stimulation, it thermally induces rapid NO release.

[0116] Testing of the dissipation biofilm performance of photothermal-releasing NO anti-biofilm hydrogels (using OD6 as an example).

[0117] The anti-biofilm properties of OD-EPL / PDG / ACup hydrogel were investigated using Gram-positive methicillin-resistant Staphylococcus aureus (MRSA), Gram-negative tetracycline-resistant Escherichia coli (TREC), and the fungus Candida albicans as representatives. First, 1-2 colonies were scraped with an inoculation loop and inoculated into TSB liquid culture medium. The culture was then incubated at 37°C in a shaker until the logarithmic growth phase (OD600 = 1). The bacterial culture was then diluted to 10⁻¹⁰ with TSB liquid culture medium. 7 CFU mL -1200 μL of diluted bacterial culture was placed in a 96-well plate and incubated at 37°C for 48 hours to allow a biofilm to form at the bottom of the plate. The TSB liquid culture medium was then slowly aspirated to remove airborne bacteria. Next, 100 μL of PBS, OD-EPL, OD-EPL / PDG, and OD-EPL / PDG / ACup hydrogel were slowly added to the wells, dividing the test group into NIR irradiated and non-NIR irradiated groups. After incubating at 37°C for 4 hours, the light-irradiated group was then subjected to 10 minutes of NIR irradiation (1W cm⁻¹). -2 The PBS and hydrogel in the wells were then slowly aspirated, and the biofilm in the wells was gently washed three times with PBS buffer. Afterwards, the biofilm content in different experimental groups was quantitatively characterized using crystal violet staining. The specific procedure was as follows: 100 μL of methanol was added to the wells to fix the biofilm. After 2 hours, the methanol was aspirated, and the wells were allowed to air dry in a biosafety cabinet. Then, 0.5% (w / w) crystal violet solution was added to stain the biofilm. After 15 minutes, excess crystal violet solution was aspirated, and the biofilm was gently washed with PBS buffer. The wells were then placed in a drying oven for 12 hours, and photographs were taken of different test groups. The biofilm was then destained using 30% (v / v) glacial acetic acid solution. After 30 minutes, 80 μL of the crystal violet destaining solution was placed in a multi-functional microplate detector to detect the UV absorbance at 590 nm. The absorbance value corresponds to the biofilm content.

[0118] like Figure 6 As shown in Figures a, b, and c, when biofilms were treated with PBS, OD-EPL, OD-EPL / PDG, and OD-EPL / PDG / ACup hydrogels, respectively, it was found that none of the experimental groups without NO loading exhibited biofilm dissipation performance. The OD-EPL / PDG+NIR test group showed some biofilm dissipation performance due to photothermal effects. OD-EPL / PDG / ACup and OD-EPL / PDG / ACup+NIR, which can release NO, showed excellent biofilm dissipation performance, with dissipation rates of 73.3% and 78.9% for MRSA biofilms, 62.0% and 77.4% for TREC biofilms, and 58.6% and 78.7% for C. albicans biofilms, respectively.

[0119] Anti-biofilming performance testing of photothermal NO-releasing injectable hydrogels (using OD6 as an example).

[0120] First, use an inoculation loop to scrape 1-2 colonies and inoculate them into TSB liquid culture medium. Incubate at 37°C in a shaker until the logarithmic phase (OD600 = 1). Dilute the bacterial culture with TSB liquid culture medium to a final concentration of 10. 7 cfu mL -1 200 μL of diluted bacterial culture was placed in a 96-well plate and incubated at 37°C for 48 hours to allow a biofilm to form at the bottom of the plate. The TSB liquid culture medium was then slowly aspirated to remove airborne bacteria. Next, 100 μL of PBS, OD-EPL, OD-EPL / PDG, and OD-EPL / PDG / ACup hydrogel were slowly added to the wells, dividing the test group into NIR irradiated and non-NIR irradiated groups. After incubating at 37°C for 4 hours, the light-irradiated group was then subjected to 10 minutes of NIR irradiation (1W cm⁻¹). -2 Subsequently, a 10-fold serial dilution was performed, and 5 μL of the diluted bacterial solution was placed on sterile solid LB medium and incubated at 37°C for 14 hours. The anti-biofilming effect of OD-EPL / PDG / ACup hydrogel on MRSA, TREC and C. albicans was evaluated by plate counting method.

[0121] The experimental results are as follows Figure 7As shown in Figures a, b, and c, the log reduction values ​​of OD-EPL and OD-EPL / PDG in the test groups without NIR irradiation were only 0.89±0.09 and 0.96±0.07 for MRSA, 1.74±0.05 and 1.89±0.18 for TREC, and 1.36±0.01 and 1.39±0.04 for C. albicans, respectively. Their bactericidal effect may originate from the antimicrobial peptide EPL. Since OD-EPL and OD-EPL / PDG lack the ability to dissipate biofilms, bacteria deep within the biofilm cannot be killed. Although PDG nanoparticles carry a positive charge, the low doping concentration of PDG means that their anti-biofilm ability is not significantly different from that of OD-EPL and OD-EPL / PDG. The OD-EPL / PDG / ACup hydrogel test group without NIR irradiation showed significantly improved anti-biofilm performance, with log reduction values ​​of 4.53±0.17, 4.06±0.08, and 3.00±0.14 for MRSA, TREC, and C. albicans, respectively. This is because the OD-EPL / PDG / ACup hydrogel can slowly release low levels of NO without NIR irradiation, thereby dissipating the biofilm and making the bacteria inside the biofilm more sensitive planktonic bacteria, thus enhancing the bactericidal ability of EPL. For the test group subjected to NIR irradiation, the anti-biofilm performance of OD-EPL / PDG+NIR was also significantly improved, with log reduction values ​​of 3.26±0.12, 2.98±0.14 and 2.52±0.02 for MRSA, TREC and C. albicans, respectively. According to the photothermal curve, 10 minutes of NIR irradiation can raise the temperature of OD-EPL / PDG hydrogel to 55.2℃. High temperature can deactivate the inherent bioactive matrix of biofilm (such as nucleic acids and proteins), thereby destroying the structure of biofilm and promoting the penetration of EPL, killing the protected bacteria. The OD-EPL / PDG / ACup+NIR hydrogel exhibited the best anti-biofilm ability among all test groups, with log reduction values ​​of 5.27±0.15, 4.16±0.04, and 3.56±0.04 against MRSA, TREC, and C. albicans, respectively. This is because, after 4 hours of incubation at 37°C, not only did the release of NO dissipate the biofilm, making the bacteria more susceptible to planktonic activity, but the increased temperature under NIR irradiation also enabled rapid NO release, enhancing the antimicrobial peptides and photothermal bactericidal properties. Overall, the photothermally NO-releasing OD-EPL / PDG / ACup hydrogel effectively combats MRSA, TREC, and C. albicans biofilms under NIR irradiation.

[0122] To further investigate the bactericidal mechanism of OD-EPL / PDG / ACup hydrogel, this invention used scanning electron microscopy to characterize the bacterial morphology of five test groups treated with PBS, OD-EPL, OD-EPL / PDG, and OD-EPL / PDG / ACup hydrogels. The characterization results are as follows: Figure 7 As shown in d, compared with the PBS control group, the OD-EPL / PDG / ACup, OD-EPL / PDG+NIR, and OD-EPL / PDG / ACup+NIR test groups caused significant damage to the bacterial cell membrane, resulting in cell wall disruption. Particularly in the OD-EPL / PDG / ACup+NIR experimental group, MRSA and TREC bacteria exhibited a shrunken morphology. The degree of bacterial damage in the OD-EPL and OD-EPL / PDG test groups was less severe. This indicates that the OD-EPL / PDG / ACup hydrogel, under NIR irradiation, can kill bacteria by disrupting the integrity of the bacterial cell wall, consistent with the bactericidal effects of antimicrobial peptides, NO, and photothermal stimulation. Antimicrobial peptides can induce bacterial death by disrupting cell membrane structure, altering cell membrane permeability, and inducing the outflow of intracellular solutions. NO can induce bacterial death through oxidative and nitrifying damage to microbial proteins, DNA, and membrane structures. High temperatures can physically disrupt the membrane structure of bacteria, inactivating their inherent bioactive matrix, such as nucleic acids and proteins, thereby promoting bacterial death.

[0123] Blood compatibility assay of photothermal-released NO anti-biofilm hydrogel (using OD6 as an example).

[0124] The biocompatibility of different hydrogels with hemoglobin was assessed after co-culturing them with hemoglobin. 5 mL of fresh blood was collected from the ears of healthy New Zealand rabbits, and heparin sodium was added to prevent clotting. The blood was then allowed to stand for 2 hours. The blood was washed with Tris-NaCl solution (pH 7.2-7.4) by centrifugation (1000 rpm × 10 min) until the supernatant was clear. The supernatant was discarded, and the lower layer of red blood cells (RBCs) was preserved. The RBCs were diluted to a volume fraction of 5% with Tris-NaCl solution. 100 μL of PBS, OD-EPL, OD-EPL / PDG, and OD-EPL / PDG / ACup hydrogels were added to each well of a 96-well plate, respectively. Physiological saline and 0.1% Triton X-100 were used as negative and positive controls, respectively. 100 μL of 5% red blood cell solution was then added. The plates were co-cultured in a light-protected, temperature-controlled shaker for 4 hours. After centrifugation, 80 μL of the supernatant was transferred to a new 96-well plate, and the absorbance of the supernatant at 545 nm was measured using a multi-functional microplate reader. The hemolysis rate was then calculated using the following formula.

[0125]

[0126] Figure 8 As can be seen from a, compared with the positive control, OD-EPL, OD-EPL / PDG and OD-EPL / PDG / ACup hydrogels all have a hemolysis rate of less than 5%, indicating that OD-EPL, OD-EPL / PDG and OD-EPL / PDG / ACup hydrogels have good blood compatibility.

[0127] Cytotoxicity assay of photothermal NO-releasing anti-biofilm hydrogel (using OD6 as an example).

[0128] Fibroblasts (NIH3T3) were resuscitated in high-glucose medium (DMEM + 10% newborn calf serum + 1% penicillin antibiotics). After the cells were in good condition in a 37°C, 5% CO2 incubator, they were passaged. Once the cells reached the third passage and were stable, they were seeded into 96-well plates at a density of 4000 cells / well and incubated for 24 hours. 200 μL of OD-EPL, OD-EPL / PDG, and OD-EPL / PDG / ACup hydrogels were placed in sterilized 24-well plates and sterilized by irradiation with UV light for 45 minutes in a biosafety cabinet to prevent cell contamination. After washing the hydrogels with cell culture medium, 2 mL of cell culture medium was added and the plates were soaked for 24 hours. Finally, culture media containing OD-EPL, OD-EPL / PDG, and OD-EPL / PDG / ACup hydrogel extracts were added to the cell wells, respectively. A corresponding control group was also set up, with cells cultured in media without hydrogel extracts. Each group had three replicates. The 96-well plates were incubated in a cell culture incubator for 24 h and 48 h, respectively. After incubation, the old culture medium was discarded, and 110 μL of medium containing 10% amaranth was added to each well, and the plates were incubated in the dark for 4 h. Finally, the fluorescence value of the solution was detected using a microplate reader (excitation wavelength 530 nm, emission wavelength 590 nm), and the cell viability was calculated based on the fluorescence value.

[0129]

[0130] Figure 8As shown in b, OD-EPL, OD-EPL / PDG, and OD-EPL / PDG / ACup hydrogels did not exhibit significant toxicity to NIH3T3 cells after 24 hours of culture. Among them, the OD-EPL / PDG / ACup hydrogel demonstrated the ability to promote cell growth due to NO production. After 24 hours of culture with the extracts of OD-EPL, OD-EPL / PDG, and OD-EPL / PDG / ACup hydrogels, the cell viability rates were 91.20±2.96%, 91.40±12.04%, and 102.70±6.72%, respectively; after 48 hours of culture, the cell viability rates were 101.66±3.13%, 104.04±7.93%, and 106.97±3.18%, respectively. Figure 8 As can be seen from graph c, compared with the control group, the NIH3T3 cells treated with hydrogel remained in a healthy, extended state. Therefore, this indicates that the hydrogel has good cell compatibility.

[0131] Cell migration assay of photothermal NO-resistant biofilm hydrogel (using OD6 as an example).

[0132] The migration ability of endothelial cells is an important factor in promoting wound healing. To verify the potential of the photothermal-releasing NO membrane hydrogel prepared in this invention to promote chronic wound healing, the migration ability of HUVECs was evaluated by scratch assay and Transwell assay. HUVECs were resuscitated with high-glucose medium (DMEM + 10% fetal bovine serum + 1% penicillin-dextrose antibody). After the cells were in good condition in a 37°C, 5% CO2 incubator, they were passaged until the cells reached the third generation and were stable. In the scratch assay, HUVECs were seeded at a density of 10,000 cells per well in a 24-well plate. After a certain period of culture, the cells were observed under a microscope. Once the cells had filled the wells, a straight line of equal width was drawn in the center of each well using a 200 μL pipette tip. After washing away the floating cells with PBS, culture medium containing different hydrogel extracts was added and the cells were cultured again. The width of the scratch was then observed and photographed under an optical microscope at specific time intervals. When performing cell migration experiments using Transwell chambers, cells are seeded in the upper layer of the chamber and serum-free culture medium is added. The lower layer of the chamber contains normal culture medium with different scaffolds. After culturing for 1 day, the cells are fixed with paraformaldehyde and the cells in the upper layer of the chamber are wiped off with cotton swabs. The cells in the lower layer are stained with crystal violet and the number of migrating cells is observed and photographed under an optical microscope.

[0133] like Figure 9 As shown in Figure a, after 36 hours of culture, cells in all groups migrated to the central scratch site to some extent. The scratches in the OD-EPL / PDG / ACup group, which can release NO, almost completely disappeared, while the other groups still had obvious gaps. Figure 9 Quantitative assessment of the wound healing rate in sample b showed that the wound healing rate of the OD-EPL / PDG / ACup hydrogel group was 94.2%. This confirmed the promoting effect of NO release on cell migration ability. In cell migration experiments conducted in Transwell chambers, cells were able to migrate to the lower layer of the chamber under the chemotactic effect of the culture medium. The cell migration results after 24 hours of culture were as follows: Figure 9 As shown in c, it can be seen that the number of cells migrating in the OD-EPL / PDG / ACup hydrogel group, which can release NO, is much higher than that in the control group, OD-EPL, and OD-EPL / PDG hydrogel groups. Figure 9 Quantitative evaluation in d also demonstrated that the OD-EPL / PDG / ACup hydrogel group can promote endothelial cell migration, which is expected to be used to promote wound healing.

[0134] It is evident that the photothermal NO-releasing injectable hydrogel prepared in this invention can accelerate NO release, coordinate antimicrobial peptides and PTT to completely eradicate biofilm infection, achieve efficient NO loading and controllable release, and completely eradicate biofilm infection.

[0135] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the scope of the technology disclosed in the present invention, and such modifications or substitutions should all be covered within the scope of protection of the present invention.

Claims

1. An injectable hydrogel that releases NO via photothermal emission, characterized in that: Using oxidized dextran and polylysine as the matrix of the hydrogel, and glucosamine-modified polydopamine nanoparticles as the carrier, with... N -Nitrosino(4-aldehydephenyl)-hydroxyamine ammonium is a thermosensitive NO donor; The carrier is loaded with the thermosensitive NO donor via a Schiff base reaction, then blended with polylysine, and then dynamically crosslinked with oxidized dextran by forming Schiff base bonds, ultimately yielding an injectable hydrogel that releases NO through photothermal reaction.

2. The injectable hydrogel that releases NO via photothermal emission according to claim 1, characterized in that: It can slowly release NO at physiological temperature; and can rapidly release NO under near-infrared light irradiation.

3. The method for preparing the photothermal NO-releasing injectable hydrogel according to claim 1, characterized in that, Includes the following steps: 1) Preparation of glucosamine-modified polydopamine nanoparticle solutions supported on thermosensitive NO donors Thermosensitive NO donor N -Nitrosino(4-aldehydephenyl)-hydroxyamine ammonium was dissolved in phosphate buffer solution to obtain a thermosensitive NO donor solution. Then, the thermosensitive NO donor solution was mixed with a glucosamine-modified polydopamine nanoparticle dispersion to obtain a glucosamine-modified polydopamine nanoparticle solution loaded with a thermosensitive NO donor. 2) The solution of glucosamine-modified polydopamine nanoparticles loaded with thermosensitive NO donor obtained in step 1) is mixed with a phosphate buffer solution of polylysine at physiological pH to obtain mixture A; 3) Mix the mixture A obtained in step 2) with the phosphate buffer solution of oxidized dextran and react to obtain an injectable hydrogel that releases NO through photothermal reaction.

4. The method for preparing the photothermally releasing NO injectable hydrogel according to claim 3, characterized in that: In step 1), the thermosensitive NO donor N The preparation method of -nitroso(4-aldehydephenyl)-hydroxyamine ammonium is as follows: A1. Dissolve p-nitrobenzaldehyde in a methanol solution, then mix it with an aqueous solution of ammonium chloride to obtain a mixed solution; A2. Place the mixed solution obtained in A1 in an oil bath at 50-60 ℃, stir and mix evenly, continue stirring and add excess Zn powder to carry out the reaction; A3. After the reaction is complete, filter the reaction solution and wash the filter cake with ether. Combine the filtrate and washing solution to form a mixed solution. A4. Use a separatory funnel to wash the blended solution obtained in A3 with diethyl ether to remove methanol and water, and dry the separated diethyl ether with anhydrous sodium sulfate to obtain an ether solution; A5. Bubbling ammonia gas through the ether solution obtained in A4 for 15-20 minutes, followed by the addition of isobutyl nitrite in batches over 15 minutes, while maintaining the cooling ammonia gas bubbling. A6. After the reaction is complete, remove the diethyl ether by vacuum distillation to obtain the product. N -Nitrosino(4-aldehydephenyl)-hydroxyamineammonium.

5. The method for preparing the injectable hydrogel that releases NO via photothermal emission according to claim 4, characterized in that, In step 1), the preparation method of the glucosamine-modified polydopamine nanoparticle dispersion is as follows: B1. Under stirring, tris(hydroxymethyl)aminomethane and acetylglucosamine were first added to ultrapure water, followed by dopamine hydrochloride, to carry out an oxidative deposition copolymerization reaction. After the reaction was completed, acetylglucosamine-modified polydopamine nanoparticles (PDNG) were obtained by centrifugation, freeze-dried, and stored at room temperature in the dark. The molar ratio of tris(hydroxymethyl)aminomethane, acetylglucosamine, and dopamine hydrochloride is 2:2.9 to 11.6:5.

8. B2. Add concentrated hydrochloric acid to the aqueous solution of acetylglucosamine-modified polydopamine nanoparticles (PDNG) obtained in step 1), condense and reflux at 80-90 °C for 50-70 min, quench the reaction mixture in an ice bath, centrifuge, disperse in ultrapure water and freeze dry to obtain PDG, and then repeatedly centrifuge and wash with phosphate buffer solution to obtain a neutral glucosamine-modified polydopamine nanoparticle dispersion. The aqueous solution concentration of PDNG is 1-2 mg / mL. -1 .

6. The method for preparing the photothermally releasing NO injectable hydrogel according to claim 5, characterized in that: In step 2), the method for preparing the polylysine phosphate buffer solution with the physiological pH value is as follows: Polylysine was dissolved in a phosphate buffer solution at room temperature, and the pH of the solution was adjusted to the physiological pH value to obtain a phosphate buffer solution of polylysine with a mass fraction of 10%.

7. The method for preparing the photothermally releasing NO injectable hydrogel according to claim 6, characterized in that: In step 3), the method for preparing the phosphate buffer solution of the oxidized dextran is as follows: C1. At room temperature, dextran is dissolved in ultrapure water to obtain a dextran aqueous solution with a concentration of 10-20 g / L; C2. At room temperature, sodium periodate is added to the dextran aqueous solution obtained in step C1, and the reaction is carried out in the dark for 4-6 hours to obtain a mixed solution; The molar ratio of sodium periodate to aldehyde groups in dextran is 0.2-0.8:1; C3. The mixed solution obtained in C2 was placed in a dialysis bag and dialyzed for 48-72 h to obtain an aqueous solution of oxidized dextran, which was then freeze-dried to obtain oxidized dextran. C4. Dissolve the oxidized dextran obtained in C3 in a phosphate buffer solution with a physiological pH value to prepare a phosphate buffer solution with a mass fraction of 8% oxidized dextran.

8. The use of the photothermal NO-releasing injectable hydrogel of claim 1 in the preparation of antibacterial drugs.

9. The use of the photothermal NO-releasing injectable hydrogel of claim 1 in the preparation of a drug for dissipating bacterial biofilms.

10. The use of the photothermal NO-releasing injectable hydrogel of claim 1 in the preparation of a drug that promotes fibroblast proliferation and vascular endothelial cell migration.

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