An aie type cationic conjugated polymer, a preparation method, a plasmonic activated hydrogel and application thereof

Abstract

The present application relates to the field of antibacterial technology, in particular to an AIE type cationic conjugated polymer and a preparation method, plasma activated hydrogel and application. The AIE type cationic conjugated polymer has the following structural formula. The present application combines the FADM photosensitizer with significant aggregation-induced emission characteristics and high ROS generation capacity with cold atmospheric pressure plasma technology, constructs a hydrogel material with synergistic antibacterial function of photodynamic and plasma derived reactive oxygen nitrogen species, and solves the problem that the half-life of RONS in PAW is extremely short, and the low viscosity leads to the difficulty of effective enrichment and maintenance of active ingredients in the infection site.

Description

Technical Field

[0001] This invention relates to the field of antibacterial agents, specifically to an AIE-type cationic conjugated polymer and its preparation method, a plasma-activated hydrogel, and its applications. Background Technology

[0002] Bacterial infections pose a significant threat to human health, as the compromised barrier function can lead to severe infections, such as cellulitis and even sepsis caused by Staphylococcus aureus. Therefore, there is an urgent need to develop novel antibacterial strategies that do not rely on traditional mechanisms of action. Photodynamic therapy (PDT) has become a research hotspot due to its advantages of high spatiotemporal controllability, broad antibacterial spectrum, and low likelihood of inducing drug resistance. However, the clinical translation of PDT faces key bottlenecks, such as poor photosensitizer dispersibility and insufficient biocompatibility.

[0003] In existing technologies, such as the AIE-type cationic aggregation-induced emission polymers described in CN119409950A, the yield of reactive oxygen species (ROS) is low due to hydrophobic aggregation, and they are difficult to penetrate biological membranes. Traditional AIE photosensitizers, for example, cause high cytotoxicity due to non-specific adsorption to mammalian cells. Therefore, developing novel photosensitizers that combine high ROS generation efficiency, good biocompatibility, and visible light response remains a significant challenge. ROS represents reactive oxygen species. RONS represents reactive nitrogen species.

[0004] Cold Atmospheric Pressure Plasma (CAP) and Plasma-Activated Water (PAW) are two emerging physical antibacterial methods. CAP technology utilizes the synergistic effects of released RONS species, charged particles, ultraviolet light, and transient electric fields to exhibit broad-spectrum and highly efficient bactericidal capabilities without easily inducing drug resistance. PAW, derived from the interaction between CAP and liquids, has become a potential agent against pathogenic microorganisms due to its low cost, environmental friendliness, and lack of chemical residues. However, the extremely short half-life of RONS in PAW (only seconds to minutes) and its low viscosity make it difficult for its active ingredients to effectively accumulate and maintain at the site of infection, severely limiting its practical application value. Summary of the Invention

[0005] To address the problem that the extremely short half-life and low viscosity of RONS in PAW make it difficult for its active ingredients to be effectively enriched and maintained at the infection site, this invention provides an AIE-type cationic conjugated polymer and its preparation method, plasma-activated hydrogel, and its application.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows.

[0007] The first aspect of this invention provides an AIE-type cationic conjugated polymer having the following structural formula:

[0008] n=9.7, and the multivariate index is 1.71.

[0009] This invention combines FADM photosensitizers, which possess significant aggregation-induced emission properties and high ROS generation capabilities, with cold atmospheric pressure plasma technology to construct a hydrogel material that exhibits synergistic antibacterial functions through photodynamic therapy and plasma-derived reactive oxygen species. This hydrogel material fully leverages the advantages of photodynamic therapy (PDT) – its high spatiotemporal controllability, broad antibacterial spectrum, and low tendency to induce drug resistance – while relying on the stable enrichment of CAP-RONS by the hydrogel carrier. This overcomes the shortcomings of traditional plasma-activated water ROS, such as short lifetime and low viscosity, achieving highly effective prevention and treatment of skin bacterial infections and reducing health risks.

[0010] A second aspect of this invention provides a method for preparing an AIE-type cationic conjugated polymer, comprising the following steps:

[0011] Compounds M1, M2, and M3, along with an organic solvent and potassium carbonate solution, were mixed and subjected to a Suzuki coupling reaction in the presence of a palladium catalyst. After a first separation and purification, the precursor polymer FAD-Br was obtained. The precursor polymer FAD-Br was then mixed with 1-methylimidazole and subjected to a substitution reaction under nitrogen protection. After a second separation and purification, the AIE-type cationic conjugated polymer represented by the formula FADM was obtained.

[0012] The reaction formula is as follows:

[0013] .

[0014] Preferably, the mass ratio of compound M1, compound M2 and compound M3 is 1-1.2:1:0.17-0.22; the ratio of the precursor polymer FAD-Br to 1-methylimidazole is 1 mg:0.1 mL-0.5 mL.

[0015] Preferably, the reaction temperature for the Suzuki coupling reaction is 50°C to 80°C; the reaction temperature for the substitution reaction is 50°C to 80°C.

[0016] Preferably, the mass ratio of compound M2 to palladium catalyst is 1:0.05 to 0.06.

[0017] Preferably, the ratio of compound M2 to organic solvent is 1g: 20mL to 30mL.

[0018] Preferably, the organic solvent is toluene; and the palladium catalyst is tetraphenylphosphine palladium.

[0019] Preferably, the specific procedures for the first separation and purification are as follows:

[0020] After removing the organic solvent from the reaction solution following the Suzuki coupling reaction, the precipitate was centrifuged with methanol, then extracted with chloroform and water, dried over anhydrous sodium sulfate, and rotary evaporated to obtain the precursor polymer FAD-Br.

[0021] Preferably, the specific procedures for the second separation and purification are as follows:

[0022] The reaction solution of the precursor polymer FAD-Br and 1-methylimidazole was dissolved in methanol, dialyzed, washed with chloroform, filtered, and rotary evaporated to obtain the AIE-type cationic conjugated polymer represented by formula FADM.

[0023] A third aspect of the present invention provides a plasma-activated hydrogel, which uses the aforementioned AIE-type cationic conjugated polymer as a photosensitizer, loads the AIE-type cationic conjugated polymer onto a hydrogel matrix to obtain a hydrogel loaded with the AIE-type cationic conjugated polymer, and then treats the hydrogel loaded with the AIE-type cationic conjugated polymer using cold atmospheric pressure plasma to generate active oxygen and nitrogen species, thereby obtaining a plasma-activated hydrogel.

[0024] Preferably, the hydrogel matrix is ​​carbomer 940, and in the hydrogel loaded with AIE-type cationic conjugated polymer, the mass ratio of AIE-type cationic conjugated polymer to hydrogel matrix is ​​1 to 5:2.

[0025] Preferably, the conditions for treating hydrogels loaded with AIE-type cationic conjugated polymers using cold atmospheric pressure plasma are: power of 60W to 70W, argon flow rate of 1L / min to 2L / min, and treatment time of 4min to 5min.

[0026] The fourth aspect of this invention provides an application of plasma-activated hydrogel as a composite antibacterial material.

[0027] The FADM of this invention possesses significant aggregation-induced emission properties and interchain fluorescence resonance energy transfer (FRET), making it a highly efficient photosensitizer capable of efficiently generating reactive oxygen species (ROS) under visible light irradiation. To overcome the inherent defects of short lifetimes and low viscosity of reactive oxygen species in water activated by cold atmospheric pressure plasma, this invention proposes combining the FADM photosensitizer with CAP technology to prepare FADM-plasma-activated hydrogels, denoted as FADM-PAW gels. FADM-PAW hydrogels, with their excellent water storage, water absorption, and biocompatibility, are ideal for stabilizing and enriching RONS generated by CAP, such as NO2. - or ONOO -This provides an ideal platform. The photodynamic effect of FADM and the CAP-derived RONS enriched in the hydrogel produce a synergistic antibacterial effect, thereby achieving a highly efficient and broad-spectrum antibacterial effect. This strategy differs from traditional antibacterial hydrogels that rely on antibiotics or metal nanomaterials, providing an innovative synergistic antibacterial solution.

[0028] Preferred, specific application method:

[0029] The liquid containing bacteria was co-incubated with plasma-activated hydrogel, and then phototreated under a 420nm light source with an intensity of 20mW / cm².

[0030] Preferably, the concentration of the AIE-type cationic conjugated polymer in the plasma-activated hydrogel is 0.2 μg / mL to 50 μg / mL.

[0031] Preferably, the composite antibacterial material has a bactericidal rate of >99.9% against Gram-positive and / or Gram-negative bacteria.

[0032] In this invention, FADM, as a photosensitizer, is combined with cold atmospheric pressure plasma technology to prepare a composite antibacterial material with synergistic antibacterial effects of photodynamics and plasma. Under white light irradiation, it produces reactive oxygen species, resulting in a good bactericidal effect. The synergistic antibacterial effect is verified by the following experiments:

[0033] In in vitro experiments, it showed significantly higher bactericidal rates against Gram-positive and / or Gram-negative bacteria than photodynamic therapy or CAP treatment alone. For example, the Gram-positive bacterium was Staphylococcus aureus; the Gram-negative bacterium was Escherichia coli.

[0034] In in vitro cell experiments, at effective antibacterial concentrations, the material exhibited good biocompatibility with mammalian cells, such as L929 fibroblasts, with a cell viability of >80%.

[0035] The preparation method of the composite antibacterial material is as follows:

[0036] Step 1: Following a methanol-to-AIE cationic conjugated polymer (AIE) ratio of 1 mg:1 mL, the AIE cationic conjugated polymer was thoroughly dispersed in toluene. After ultrasonic dispersion, it was added dropwise to ultrapure water at a methanol-to-water volume ratio of 1:5. After ultrasonic dispersion, the mixture was transferred to a magnetic stirrer and heated at 50°C until the methanol evaporated. After the reaction was complete and cooled to room temperature, the product was filtered using a syringe with a 0.45 μM filter membrane to obtain FADM nanoparticles.

[0037] Step 2: Dissolve FADM nanoparticles in PBS buffer at a concentration of 0.2 μg / mL to 50 μg / mL, and let stand at room temperature. Then, add carbomer-940 to the solution at a concentration of 20 mg / mL. Let the mixture stand at room temperature for 24 hours to obtain FADM hydrogel. In the FADM hydrogel, the concentration of FADM nanoparticles is 1 μg / mL to 50 μg / mL, and the concentration of carbomer-940 is 20 mg / mL. The pH of the PBS buffer is 7.4, and the temperature is 25°C.

[0038] Step 3: The FADM hydrogel is treated with cold atmospheric pressure plasma at a power of 60W-70W and an argon flow rate of 1L / min-2L / min for 4min-6min to generate RONS in the FADM hydrogel, resulting in a plasma-activated hydrogel containing FADM nanoparticles of 1μg / mL-50μg / mL.

[0039] In this invention, the photodynamic effect generated by the FADM under light irradiation works synergistically with the CAP-derived RONS enriched in the hydrogel to achieve highly efficient antibacterial activity; the hydrogel matrix is ​​used to stabilize and enrich the CAP-derived RONS, overcoming the defects of short lifetime and low viscosity of RONS in plasma-activated water.

[0040] The synergistic antibacterial effect was further verified by the following in vivo experiments:

[0041] In animal models, such as the mouse skin wound infection model, the CAP-activated hydrogel treatment group loaded with FADM significantly promoted wound healing, reduced bacterial load at the wound site, and alleviated inflammatory response compared to the control group, such as the untreated group, the PDT-only group, and the CAP-only group.

[0042] The beneficial effects of this invention are:

[0043] 1. The AIE-type cationic conjugated polymer of the present invention, as a photosensitizer, has significant AIE characteristics and efficient ROS generation capability. As verified by DCFH probe, its strong interchain-FRET effect can improve light energy utilization.

[0044] 2. The FADM hydrogel carrier of the present invention stably enriches CAP-RONS, such as NO2, through a three-dimensional network structure. - and ONOO - Overcoming the bottleneck of short half-life of plasma-activated RONS in water (<30 minutes), the storage period of active substances is extended to more than 2 weeks; FADM-PDT and CAP-RONS produce a dual-mode synergistic effect in hydrogel, with bactericidal efficiency significantly higher than single therapy, achieving synergistic antibacterial mechanism.

[0045] 3. Compared with traditional antibacterial methods, the FADM hydrogel matrix of the present invention combines with the bacterial membrane through the electrostatic interaction of cationic groups, and generates RONS under light and plasma excitation, which disrupts the bacterial metabolic process. The bactericidal rate against both Gram-positive and Gram-negative bacteria is >99.9%. It can be adapted to various forms such as wound dressings and antibacterial coatings, and is easy to operate and environmentally friendly. Attached Figure Description

[0046] Figure 1 The synthetic route for AIE-type cationic conjugated polymers provided in the embodiments of the present invention is as follows.

[0047] Figure 2 This is the 1H NMR spectrum of the AIE-type cationic conjugated polymer of Example 1 of the present invention.

[0048] Figure 3 The curve showing the relative absorption intensity of the FADM nanoparticle-DCFH probe mixture at 524 nm as a function of time is shown in Example 4. The mixture of FADM nanoparticle-DCFH probe prepared by mixing plasma-activated hydrogel containing 10 μg / mL FADM nanoparticles with DCFH probe is shown in Example 4.

[0049] Figure 4 The following diagrams illustrate the treatment of hydrogel surfaces using a cold atmospheric pressure plasma jet with a power of 70 W and an argon flow rate of 2 L / min, and the CAP discharge effect, respectively, as provided in this embodiment of the invention. (a) is a schematic diagram of the treatment of hydrogel surfaces using a cold atmospheric pressure plasma jet with a power of 70 W and an argon flow rate of 2 L / min; (b) is a schematic diagram of the CAP discharge effect.

[0050] Figure 5 The graphs show the bactericidal activity of FADM nanoparticles at different concentrations against Staphylococcus aureus and Escherichia coli. Among them, (a) shows the bactericidal activity of FADM nanoparticles at different concentrations against Staphylococcus aureus; and (b) shows the bactericidal activity of FADM nanoparticles at different concentrations against Escherichia coli.

[0051] Figure 6 The bar charts show the bactericidal activity of FADM nanoparticles against Staphylococcus aureus and Escherichia coli. Among them, (a) is the bar chart of the bactericidal activity of FADM nanoparticles against Staphylococcus aureus; (b) is the bar chart of the bactericidal activity of FADM nanoparticles against Escherichia coli.

[0052] Figure 7The images show the process of FADM being used to treat mouse wounds. Among them, (a1)~(a5) are images of FADM used to treat mouse wounds in group A from day 0 to day 7; (b1)~(b5) are images of FADM used to treat mouse wounds in group B from day 0 to day 7; (c1)~(c5) are images of FADM used to treat mouse wounds in group C from day 0 to day 7; and (d1)~(d5) are images of FADM used to treat mouse wounds in group D from day 0 to day 7.

[0053] Figure 8 This is a graph showing the wound healing status of each group on day 7.

[0054] Figure 9 Images of wound tissue observed by H&E staining in each group on day 7 are shown. Among them, (a) is an image of wound tissue observed by H&E staining in group A on day 7; (b) is an image of wound tissue observed by H&E staining in group B on day 7; (c) is an image of wound tissue observed by H&E staining in group C on day 7; and (d) is an image of wound tissue observed by H&E staining in group D on day 7. Detailed Implementation

[0055] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. Based on the embodiments of the present invention, any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention. Furthermore, all other embodiments obtained by those skilled in the art without inventive effort are within the protection scope of the present invention.

[0056] Specific experimental steps or conditions are not specified in the embodiments; they can be performed according to the conventional experimental steps or conditions described in the prior art. Reagents and other instruments used, unless otherwise specified, are all commercially available conventional reagent products. Furthermore, the accompanying drawings are merely illustrative diagrams of the embodiments of the present invention and are not necessarily drawn to scale. The same reference numerals in the drawings denote the same or similar parts, and therefore, repeated descriptions of them will be omitted. Some block diagrams shown in the drawings are functional entities and do not necessarily correspond to physically or logically independent entities.

[0057] Techniques, methods, and devices known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and devices should be considered part of this specification.

[0058] In the description of this invention, it should be understood that the use of terms such as "first" and "second" to define components is merely for the purpose of distinguishing the corresponding components. Unless otherwise stated, the above terms have no special meaning and therefore should not be construed as limiting the scope of protection of this invention.

[0059] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0060] The extremely short half-life of RONS in PAW (polyalcoholic acid) ranges from only seconds to minutes, and its low viscosity makes it difficult for its active ingredients to be effectively enriched and maintained at the infection site, severely limiting its practical application value. Although RONS can be modulated to some extent by optimizing the liquid matrix, such as water or buffer solution, the aforementioned inherent defects have not been fundamentally resolved.

[0061] Hydrogel materials, with their three-dimensional network structure, excellent water retention, biocompatibility, and functionalizability, provide an ideal platform for the stable loading of active substances. Therefore, developing a method that can overcome the stability challenges of RONS in PAW (polyhydroxylamine) materials and synergize with highly efficient photosensitizers to achieve multiple antibacterial mechanisms is of great significance for breaking through existing technological bottlenecks.

[0062] like Figure 1 The chemical reaction formula for AIE-type cationic conjugated polymers is as follows:

[0063] .

[0064] Compound M1 is phenyl-bis[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)phenyl]amine.

[0065] Compound M2 is 2,7-dibromo-9,9-di(6-bromohexyl)fluorene.

[0066] Compound M3 is 4,7-bis(5-bromo-2-thienyl)-2,1,3-benzothiadiazole.

[0067] The catalyst for the Suzuki coupling reaction is Pd(0), which represents zero-valent palladium; zero-valent palladium is tetraphenylphosphine palladium.

[0068] The AIE-type cationic conjugated polymer provided by this invention has significant AIE characteristics. It exhibits weak fluorescence in dimethyl sulfoxide, a good solvent, but its fluorescence intensity is significantly enhanced in a high water content environment (>90%). It also has a strong interchain-FRET effect: polyfluorene acts as the donor and bisthienylbenzothiadiazole acts as the acceptor, with an energy transfer efficiency of >80% between the donor and acceptor, thus enhancing the light-harvesting ability.

[0069] The Chinese name for AIE is Aggregation-Induced Emission. The full English name for inter-chain fluorescence resonance energy transfer is intersystem-chain fluorescence resonance energy transfer, usually written as interchain FRET.

[0070] The FADM of this invention, detected by a standard DCFH probe, showed an improved ROS yield compared to traditional photosensitizers under visible light irradiation at λ=420 nm and 50 mW / cm². Figure 3 As shown.

[0071] The FADM of this invention has cationic properties and a zeta potential of +27.1 mV. It targets and destroys negatively charged bacterial cell membranes through electrostatic interaction. Therefore, the FADM of this invention can be used as a photosensitizer. By utilizing the increased reactive oxygen species production of FADM, plasma-activated hydrogels can be prepared to enhance the antibacterial ability of photodynamic therapy. At the same time, it can be applied to the rapid detection of bacteria.

[0072] This invention provides a method for preparing an AIE-type cationic conjugated polymer, comprising the following steps:

[0073] Step 1: Mix phenyl-bis[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)phenyl]amine, 2,7-dibromo-9,9-bis(6-bromohexyl)fluorene, and 4,7-bis(5-bromo-2-thienyl)-2,1,3-benzothiadiazole at a mass ratio of 1–1.2:1:0.17–0.22. Add compound M2 to toluene at a ratio of 1 g:20 mL–30 mL to form the first solution.

[0074] Step 2: Potassium carbonate and ultrapure water were mixed at a ratio of 0.15–0.17 g: 1 mL to form a second solution. After mixing the first and second solutions, tetraphenylphosphine palladium was added as a catalyst, with a mass ratio of compound M2 to tetraphenylphosphine palladium of 1:0.05–0.06. The reaction was carried out under nitrogen protection and refluxed at 50–80 °C for 16–48 h. The reaction product was purified by the first separation process: evaporation of organic solvent, precipitation and centrifugation with methanol, extraction with water / chloroform, drying with anhydrous sodium sulfate, and rotary evaporation to obtain the precursor polymer.

[0075] Step 3: The precursor polymer was mixed with 1-methylimidazole at a ratio of 1 mg: 0.1 mL to 0.5 mL, and reacted at 50 °C to 80 °C for 24 h to 48 h under nitrogen protection. The reaction product was then purified by a second separation process: dissolved in methanol and dialyzed for 1 to 3 days, washed with chloroform, filtered, and rotary evaporated to obtain the target product FADM.

[0076] Figure 1 The synthetic route of the AIE-type cationic conjugated polymer provided in the embodiments of the present invention is shown, in conjunction with the above preparation steps and Figure 1 As can be seen, this invention employs Suzuki cross-coupling polymerization and post-modification synthesis to design and synthesize an AIE-type cationic conjugated polymer. The AIE-type cationic conjugated polymer of this invention exhibits significant AIE characteristics and a strong interchain fluorescence resonance energy transfer effect in dimethyl sulfoxide solution, which serves as a good solvent. Furthermore, it generates reactive oxygen species upon plasma interaction with a hydrogel containing FADM nanoparticles, thereby achieving an antibacterial effect.

[0077] This invention provides an application of plasma-activated hydrogel as a composite antibacterial material, the specific application method including the following steps:

[0078] Step 1: Following a methanol-to-AIE cationic conjugated polymer (AIE) ratio of 1 mg:1 mL, the AIE cationic conjugated polymer was thoroughly dispersed in toluene. After ultrasonic dispersion, it was added dropwise to ultrapure water at a methanol-to-water volume ratio of 1:5. After ultrasonic dispersion, the mixture was transferred to a magnetic stirrer and heated at 50°C until the methanol evaporated. After the reaction was complete and cooled to room temperature, the product was filtered using a syringe with a 0.45 μM filter membrane to obtain FADM nanoparticles. The average particle size of the FADM nanoparticles was approximately 120 nm ± 15 nm, and the polydispersity index was 0.171.

[0079] Step 2: Dissolve FADM nanoparticles in PBS buffer at a concentration of 0.2 μg / mL to 50 μg / mL, allow to stand at room temperature, then add carbomer-940 to the solution at a concentration of 20 mg / mL. Let the mixture stand at room temperature for 24 hours to obtain a FADM hydrogel. In the FADM hydrogel, the concentration of FADM nanoparticles is 0.2 μg / mL to 50 μg / mL, and the concentration of carbomer-940 is 20 mg / mL. The pH of the PBS buffer is 7.4, and the temperature is 25℃. The concentration of FADM nanoparticles can be 0.2 μg / mL, 0.4 μg / mL, 0.6 μg / mL, 0.8 μg / mL, 1.0 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, and 50 μg / mL, etc.

[0080] Step 3: Plasma activation and hydrogel loading. The FADM hydrogel is treated with a cold atmospheric pressure plasma at a power of 60W–70W and an argon flow rate of 1L / min–2L / min for 4–6 minutes to generate RONS within the FADM hydrogel, resulting in a plasma-activated hydrogel containing 1μg / mL–50μg / mL FADM nanoparticles. Figure 4(a) CAP action generates RONS, such as H2O2 and ∙OH, which permeate into the hydrogel network, such as Figure 4 (b) shows that the RONS half-life is extended to 14 days through stable enrichment via the three-dimensional structure of the hydrogel.

[0081] Synergistic antibacterial action: Apply FADM-CAP hydrogel to the infected site, such as a wound infected with Staphylococcus aureus; irradiate with visible light at λ=420nm, 20mW / cm² for 10 minutes to activate FADM and generate ¹O2, which works synergistically with CAP-RONS; the FADM cationic groups adsorb onto the bacterial membrane, with a ζ-potential of +27.1mV, generating electrostatic disruption and thus altering permeability; and ¹O2 interacts with RONS, such as ∙ONOO - Oxidizing bacterial lipids / proteins; against Gram-positive bacteria S. aureus With negative bacteria E. coli The sterilization rate is >99.9%, such as Figure 5 It can also remove 90% of biofilm.

[0082] AIE-type cationic conjugated polymers exhibit significant aggregation-induced emission (AIE) characteristics and strong interchain fluorescence resonance energy transfer (FRET) in dimethyl sulfoxide (DMSO) solution, which serves as a good solvent. Furthermore, when plasma interacts with hydrogels containing FADM nanoparticles, reactive oxygen species are generated, thereby achieving an antibacterial effect. Therefore, AIE-type cationic conjugated polymers can be applied to plasma antibacterial processes.

[0083] In specific implementation, the AIE-type cationic conjugated polymer provided by this invention exhibits a maximum absorption peak at 375 nm, with a broad absorption range covering 300 nm to 700 nm, and an emission spectrum showing a maximum fluorescence value of 470.5 nm. The AIE-type cationic conjugated polymer provided by this invention is dissolved in a mixture of methanol and ultrapure water to construct FADM nanoparticles. These FADM nanoparticles are then dispersed in a mixture of carbomer-940 and PBS buffer to prepare FADM nanohydrogels. The concentration of FADM nanoparticles can be from 0.2 μg / mL to 50 μg / mL, preferably from 1 μg / mL to 50 μg / mL. Therefore, the good biocompatibility of FADM nanohydrogels can be utilized as a plasma activation medium, effectively extending the storage time of plasma-active particles, enhancing antibacterial activity, and being gentler and safer on the human body, avoiding the risk of burns caused by direct plasma treatment of the human body.

[0084] The technical solution of the present invention will be further described below through specific embodiments.

[0085] In the following embodiments, unless otherwise specified, the methods described are conventional methods; and unless otherwise specified, the reagents and materials described are commercially available.

[0086] Example 1

[0087] like Figure 1 A method for preparing an AIE-type cationic conjugated polymer includes the following steps:

[0088] S1. Take 0.9 g of compound M1, 0.9 g of compound M2, and 0.15 g of compound M3 into a three-necked flask, add 20 mL of toluene to obtain the first solution. Mix 0.5 g of potassium carbonate with 3 mL of ultrapure water to form the second solution.

[0089] S2. The second solution was added to the first solution using a syringe, and 0.05 g of tetraphenylphosphine palladium was added as a catalyst. Nitrogen gas was introduced for 10 min, and the mixture was heated at 50 °C and refluxed for 16 h to carry out the polymerization reaction. After the polymerization reaction was completed, the organic solvent was removed by rotary evaporator, and the precipitate was added dropwise to methanol and centrifuged to collect the lower precipitate. Then, water and chloroform were added for extraction, the organic phase was collected, dried with anhydrous sodium sulfate, and filtered. The organic solvent was removed by rotary evaporation to obtain the precursor polymer, denoted as FAD-Br.

[0090] S3. 20 mg of the precursor polymer and 10 mL of 1-methylimidazole were mixed and reacted at 80 °C for 24 h under nitrogen protection. After the reaction was completed, the liquid was transferred to a dialysis bag with a molecular weight of 3 kDa and dialyzed in methanol for 1 day. After dialysis, the product was washed with chloroform, filtered, and the solid remaining on the filter paper was collected with methanol. The solvent was removed by rotary evaporation to obtain the AIE-type cationic conjugated polymer FL-TPA-DBT-MI, denoted as FADM.

[0091] Figure 2 The image shows the 1H NMR spectrum of the AIE-type cationic conjugated polymer from Example 1. Figure 2 As shown, according to Figure 2 The results of the proton nuclear magnetic resonance spectroscopy characterization confirmed that the synthesized product was FADM.

[0092] Example 2

[0093] A method for preparing an AIE-type cationic conjugated polymer includes the following steps:

[0094] S1. Take 1.2 g of compound M1, 1.0 g of compound M2, and 0.22 g of compound M3 into a three-necked flask, add 30 mL of toluene to obtain the first solution. Mix 0.8 g of potassium carbonate with 5 mL of ultrapure water to form the second solution.

[0095] S2. The second solution was added to the first solution using a syringe, and 0.06 g of tetraphenylphosphine palladium was added as a catalyst. Nitrogen gas was introduced for 10 min, and the mixture was heated at 60 °C and refluxed for 48 h to carry out the polymerization reaction. After the polymerization reaction was completed, the organic solvent was removed by rotary evaporation, and the precipitate was added dropwise to methanol and centrifuged to collect the lower precipitate. Then, water and chloroform were added for extraction, the organic phase was collected, dried with anhydrous sodium sulfate, and filtered. The organic solvent was removed by rotary evaporation to obtain the precursor polymer, denoted as FAD-Br.

[0096] S3. 30 mg of the precursor polymer and 4 mL of 1-methylimidazole were mixed and reacted at 50 °C for 28 h under nitrogen protection. After the reaction was completed, the liquid was transferred to a dialysis bag with a molecular weight of 3 kDa and dialyzed in methanol for 1 day. After dialysis, the product was washed with chloroform, filtered, and the solid remaining on the filter paper was collected with methanol. The solvent was removed by rotary evaporation to obtain the AIE-type cationic conjugated polymer FL-TPA-DBT-MI, denoted as FADM.

[0097] The 1H NMR spectrum of the AIE-type cationic conjugated polymer in Example 2 was the same as the characterization results in Example 1, proving that the product FADM was successfully synthesized in Example 2.

[0098] Example 3

[0099] A method for preparing an AIE-type cationic conjugated polymer includes the following steps:

[0100] S1. Take 1.1 g of compound M1, 1.0 g of compound M2, and 0.2 g of compound M3 into a three-necked flask, add 30 mL of toluene to obtain the first solution. Mix 0.8 g of potassium carbonate with 5 mL of ultrapure water to form the second solution.

[0101] S2. The second solution was added to the first solution using a syringe, and 0.06 g of tetraphenylphosphine palladium was added as a catalyst. Nitrogen gas was introduced for 10 min, and the mixture was heated at 80 °C and refluxed for 24 h to carry out the polymerization reaction. After the polymerization reaction was completed, the organic solvent was removed by rotary evaporation, and the precipitate was added dropwise to methanol and centrifuged to collect the lower precipitate. Then, water and chloroform were added for extraction, the organic phase was collected, dried with anhydrous sodium sulfate, and filtered. The organic solvent was removed by rotary evaporation to obtain the precursor polymer, denoted as FAD-Br.

[0102] S3. 30 mg of the precursor polymer and 6 mL of 1-methylimidazole were mixed and reacted at 80 °C for 48 h under nitrogen protection. After the reaction was completed, the liquid was transferred to a dialysis bag with a molecular weight of 3 kDa and dialyzed in methanol for 1 day. After dialysis, the product was washed with chloroform, filtered, and the solid remaining on the filter paper was collected with methanol. The solvent was removed by rotary evaporation to obtain the AIE-type cationic conjugated polymer FL-TPA-DBT-MI, denoted as FADM.

[0103] The 1H NMR spectrum of the AIE-type cationic conjugated polymer in Example 3 was the same as the characterization results in Example 1, proving that the product FADM was successfully synthesized in Example 3.

[0104] Test 1: Physical property analysis of FADM nanoparticles.

[0105] The AIE-type cationic conjugated polymer prepared in Example 1 was dissolved in a 1:5 volume ratio mixture of methanol and ultrapure water, and FADM nanoparticles were constructed using a conventional reprecipitation method. Dynamic light scattering analysis showed that the average particle size of the FADM nanoparticles was approximately 120 nm ± 15 nm, and the polydispersity index was 0.171, indicating a highly uniform dispersion of the FADM nanoparticles. Transmission electron microscopy confirmed that the FADM nanoparticles were spherical and monodisperse without aggregates.

[0106] The AIE-type cationic conjugated polymers of Examples 2 and 3 were dissolved in a 1:1 volume ratio mixture of methanol and ultrapure water to construct FADM nanoparticles. Dynamic light scattering analysis showed that the FADM nanoparticles of Examples 2 and 3 were highly uniformly dispersed. Transmission electron microscopy confirmed that the FADM nanoparticles of Examples 2 and 3 were spherical and monodisperse without agglomerates.

[0107] Example 4

[0108] A method for preparing plasma-activated hydrogels includes the following steps:

[0109] Step 1: Prepare a FADM hydrogel by mixing FADM nanoparticles and carbomer 940 in PBS buffer. The concentration of FADM nanoparticles in the FADM hydrogel is 10 μg / mL, and the concentration of carbomer 940 is 20 mg / mL. The pH of the PBS buffer is 7.4, and the temperature is 25℃.

[0110] Step 2: Treat the FADM hydrogel with cold atmospheric pressure plasma at a power of 70W and an argon flow rate of 2L / min for 5min to generate RONS in the FADM hydrogel, and obtain a plasma-activated hydrogel containing 10μg / mL FADM nanoparticles.

[0111] A DCFH probe was added to a plasma-activated hydrogel containing 10 μg / mL FADM nanoparticles to obtain a mixed system of FADM nanoparticles and DCFH probe, with a final concentration of 1 mM for the DCFH probe. The mixed system of FADM nanoparticles and DCFH probe was continuously irradiated under a 420 nm, 50 mW / cm² light source; the fluorescence intensity at 524 nm was measured every 60 s to characterize the total ROS level in real time. 2',7'-Dichlorofluorescein, abbreviated as DCFH, is a chemical compound used in FADM nanoparticles.

[0112] Figure 3 The curve shows the relative absorption intensity at 524 nm over time for the mixed system of FADM nanoparticles and DCFH probe prepared by mixing a plasma-activated hydrogel containing 10 μg / mL FADM nanoparticles with a DCFH probe, as described in Example 4. Where I / I0 represents the relative absorption intensity.

[0113] like Figure 3 As shown, FADM nanoparticles exhibit increased ROS generation under plasma irradiation over time, as demonstrated by a standard DCFH probe.

[0114] Example 5

[0115] A method for preparing plasma-activated hydrogels includes the following steps:

[0116] Step 1: Prepare a FADM hydrogel by mixing FADM nanoparticles and carbomer 940 in PBS buffer. The concentration of FADM nanoparticles in the FADM hydrogel is 50 μg / mL, and the concentration of carbomer 940 is 20 mg / mL. The pH of the PBS buffer is 7.4, and the temperature is 25℃.

[0117] Step 2: Dilute the FADM hydrogel with PBS buffer to a concentration of FADM nanoparticles ranging from 0.2 µg / mL to 1.0 µg / mL, with each concentration increment being 0.2 µg / mL. The concentration gradients of the FADM hydrogels are 0.2 µg / mL, 0.4 µg / mL, 0.6 µg / mL, 0.8 µg / mL, and 1.0 µg / mL, respectively.

[0118] Plasma-activated hydrogels containing different concentrations of FADM nanoparticles were prepared by treating a series of FADM hydrogels with a concentration gradient of 70W and an argon flow rate of 2L / min for 5 min. The concentration of FADM nanoparticles ranged from 0.2μg / mL to 1.0μg / mL.

[0119] Comparative Example 1

[0120] A method for preparing plasma-activated hydrogels includes the following steps:

[0121] A hydrogel was prepared in PBS buffer with a carbomer 940 concentration of 20 mg / mL. The hydrogel was then treated with cold atmospheric pressure plasma at a power of 70 W and an argon flow rate of 2 L / min for 5 min to obtain a CAP hydrogel without FADM loading.

[0122] Comparative Example 2

[0123] The preparation method of FADM hydrogel includes the following steps:

[0124] FADM hydrogels were prepared by reacting FADM nanoparticles with carbomer 940 in PBS buffer. The concentration of FADM nanoparticles in the FADM hydrogel was 50 μg / mL, and the concentration of carbomer 940 was 20 mg / mL. The pH of the PBS buffer was 7.4, and the temperature was 25℃. The FADM hydrogel was then diluted with PBS buffer to a concentration of FADM nanoparticles of 0.2 µg / mL to 1.0 µg / mL.

[0125] Test 2: Antibacterial performance test.

[0126] The antibacterial properties of plasma-activated hydrogels containing different concentrations of FADM nanoparticles prepared in Example 5 above were verified to counteract Staphylococcus aureus and Escherichia coli.

[0127] Take a concentration of 1×10 8 100 μL of a CFU / mL suspension of Staphylococcus aureus, or a 1×10⁻⁶ CFU / mL suspension. 8 100 μL of CFU / mL Escherichia coli suspension was used as a bacterial suspension and co-incubated with plasma-activated hydrogels containing different concentrations of FADM nanoparticles.

[0128] Experimental Groups:

[0129] Group A: Bacterial suspension only; serving as the control group.

[0130] Group B: Bacterial suspension + CAP hydrogel without FADM loading, as the CAP group.

[0131] Group C: Bacterial suspension + FADM hydrogel of Comparative Example 2 (0.2 μg / mL to 1.0 μg / mL) + 420 nm, 20 mW / cm² light for 10 min; this was the FADM group.

[0132] Group D: Bacterial suspension + plasma-activated hydrogel containing 0.2 μg / mL to 1.0 μg / mL FADM nanoparticles from Example 5 + irradiation at 420 nm, 20 mW / cm² for 10 min; as the synergistic group.

[0133] The bacterial suspension has a concentration of 1×10 8 A suspension of Staphylococcus aureus at CFU / mL, or a concentration of 1×10⁻⁶. 8 CFU / mL Escherichia coli suspension.

[0134] During testing, the concentration of either the Staphylococcus aureus suspension or the Escherichia coli suspension was consistently maintained at 1×10⁻⁶. 8 The bacterial suspensions were incubated with CAP hydrogels without FADM loading (Comparative Example 1), FADM hydrogels with 0.2 μg / mL to 1.0 μg / mL loading (Comparative Example 2), and plasma-activated hydrogels containing 0.2 μg / mL to 1.0 μg / mL FADM nanoparticles (Example 5). After irradiation at 420 nm and 20 mW / cm² for 10 min, the suspensions were spread onto agar plates. The control group samples underwent the same treatment but without irradiation. Subsequently, 50 μL of bacterial suspension was evenly spread onto agar plates. After incubation at 37°C for 24 hours, colony-forming units (CFUs) were counted.

[0135] Figure 5 (a) and (b) show the bactericidal ability of FADM nanoparticles at different concentrations against Staphylococcus aureus and Escherichia coli. Figure 6 (a) and (b) show bar graphs illustrating the bactericidal activity of FADM nanoparticles against Staphylococcus aureus and Escherichia coli.

[0136] from Figure 5 and Figure 6 It can be seen that the bactericidal ability of FADM nanoparticles against Staphylococcus aureus and Escherichia coli gradually increases with increasing concentration. This is because the reactive oxygen species generated by FADM nanoparticles effectively disrupt the integrity of the bacteria. This leads to the release of bacterial contents, ultimately causing bacterial death. Furthermore, under the treatment of plasma-activated hydrogel containing 1.0 μg / mL FADM nanoparticles + 420 nm, 20 mW / cm² light irradiation for 10 min, a 100% bactericidal effect was achieved against Staphylococcus aureus and Escherichia coli was achieved.

[0137] Test Example 3: Analysis of in vivo antibacterial efficacy.

[0138] Analysis of the in vivo antibacterial efficacy of the plasma-activated hydrogel containing 1 μg / mL FADM nanoparticles prepared in Example 5.

[0139] Take a concentration of 1×108 A mouse skin trauma model of Staphylococcus aureus infection was established using a CFU suspension of Staphylococcus aureus (S. aureus). The mice were randomly divided into four groups (n=6).

[0140] Group A: Bacterial suspension only; serving as the control group.

[0141] Group B: Bacterial suspension + CAP hydrogel without FADM loading, as the CAP group.

[0142] Group C: Bacterial suspension + 1.0 μg / mL FADM hydrogel (comparative example 2) + 420 nm, 20 mW / cm² light irradiation for 10 min; this was the FADM group.

[0143] Group D: Bacterial suspension + plasma-activated hydrogel containing 1.0 μg / mL FADM nanoparticles from Example 5 + irradiation at 420 nm, 20 mW / cm² for 10 min; as the synergistic group.

[0144] Hydrogel was applied daily following the methods used in groups A through D. The wound area was recorded on days 0, 1, 3, 5, and 7. Figure 7 As shown. On day 7, tissue samples were taken from the wounds to assess wound healing in each group, as shown. Figure 8 As shown. On day 7, wound tissue was taken for H&E staining to observe the wound tissue, as shown. Figure 9 As shown.

[0145] Figures 7-9 It demonstrates the antibacterial efficacy in vivo, among which, Figure 7 The images show the wound healing process of mice from day 0 to day 7. Figure 8 The wound healing status of each group is shown on day 7; Figure 9 Images of wound tissue observed by H&E staining in each group on day 7 are shown.

[0146] from Figures 7-9 It can be concluded that the wound healing rate in group D reached 85% on day 7. Figure 8 The wounds in group D healed faster than those in groups A, B, and C. HE staining showed that the wound tissue in group D recovered faster than that in groups A, B, and C. Figure 9 This is because the ζ=+27.1 mV of the FADM cationic groups in the CAP-activated FADM hydrogel disrupts bacterial membrane permeability; photoexcitation generates ¹O2 and CAP-derived ∙ONOO. - Synergistic oxidation of bacterial lipids / proteins leads to leakage of intracellular substances; the three-dimensional hydrogel network continuously releases RONS, maintaining a long-lasting bactericidal microenvironment.

[0147] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A plasma-activated hydrogel, characterized in that, AIE-type cationic conjugated polymers are used as photosensitizers. The AIE-type cationic conjugated polymers are loaded onto a hydrogel matrix to obtain a hydrogel loaded with AIE-type cationic conjugated polymers. Then, the hydrogel loaded with AIE-type cationic conjugated polymers is treated with cold atmospheric pressure plasma to generate active oxygen and nitrogen species, thus obtaining a plasma-activated hydrogel. The AIE-type cationic conjugated polymer has the following structural formula: ； n=9.7, and the multiplicity index is 1.71; The hydrogel matrix is ​​Carbomer 940; in the hydrogel loaded with AIE-type cationic conjugated polymer, the mass ratio of AIE-type cationic conjugated polymer to hydrogel matrix is ​​1 to 5:

2. The conditions for treating hydrogels loaded with AIE-type cationic conjugated polymers using cold atmospheric pressure plasma are: power of 60W to 70W, argon flow rate of 1L / min to 2L / min, and treatment time of 4min to 5min.

2. The plasma-activated hydrogel according to claim 1, characterized in that, The preparation method of the AIE-type cationic conjugated polymer includes the following steps: Compounds M1, M2, and M3, along with an organic solvent and potassium carbonate solution, were mixed and subjected to a Suzuki coupling reaction in the presence of a palladium catalyst. After a first separation and purification, the precursor polymer FAD-Br was obtained. The precursor polymer FAD-Br was then mixed with 1-methylimidazole and subjected to a substitution reaction under nitrogen protection. After a second separation and purification, the AIE-type cationic conjugated polymer represented by the formula FADM was obtained. The reaction formula is as follows: 。 3. The plasma-activated hydrogel according to claim 2, characterized in that, The mass ratio of compounds M1, M2 and M3 is 1–1.2:1:0.17–0.22; the ratio of the precursor polymer FAD-Br to 1-methylimidazole is 1 mg:0.1 mL–0.5 mL.

4. The plasma-activated hydrogel according to claim 2, characterized in that, The reaction temperature for the Suzuki coupling reaction is 50℃~80℃; the reaction temperature for the substitution reaction is 50℃~80℃.

5. The plasma-activated hydrogel according to claim 2, characterized in that, The mass ratio of compound M2 to palladium catalyst is 1:0.05-0.06; the molar ratio of compound M2 to organic solvent is 1g:20mL-30mL; the organic solvent is toluene; and the palladium catalyst is tetraphenylphosphine palladium.

6. The application of the plasma-activated hydrogel of claim 1 as a composite antibacterial material.

7. The application of the plasma-activated hydrogel according to claim 6 as a composite antibacterial material, characterized in that, The specific application method is as follows: The liquid containing bacteria was co-incubated with plasma-activated hydrogel, and then phototreated under a 420nm light source with an intensity of 20mW / cm².

8. The application of the plasma-activated hydrogel according to claim 7 as a composite antibacterial material, characterized in that, The composite antibacterial material has a bactericidal rate of >99.9% against Gram-positive and / or Gram-negative bacteria.

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