Charge-switchable pH-sensitive lignin-based antibacterial nanoparticles, and preparation method and application thereof

Abstract

The application discloses charge-switchable pH-sensitive lignin-based nanoparticles and a preparation method and application thereof. First, polyallylamine rich in amine groups is grafted on lignin to obtain lignin-polyallylamine conjugates, then anhydride is grafted on the lignin-polyallylamine conjugates with positive charges, the unreacted amine groups are protected by anhydride, and lignin-polyallylamine-anhydride conjugates are obtained, and then, through a reverse solvent method, charge-switchable pH-sensitive lignin-based nanoparticles are obtained. The preparation method is convenient and efficient, the charge-switchable pH-sensitive lignin-based nanoparticles can be converted from negative electricity to positive electricity in a slightly acidic environment, the antibacterial activity of the lignin-based nanoparticles can be improved, and the increase of the positive electricity can enhance the inhibiting and cleaning effect on bacterial biofilms.

Description

Technical Field

[0001] This invention belongs to the field of lignin-based functional materials technology, specifically relating to a charge-convertible pH-sensitive lignin-based antibacterial nanoparticle, its preparation method, and its application. Background Technology

[0002] Infections caused by pathogenic bacteria have become a major cause of death, causing approximately 700,000 deaths worldwide each year. More than 60% of human infectious diseases can be attributed to bacterial biofilms, such as chronic bacterial prostatitis, chronic otitis media, and osteomyelitis. Biofilms and their surrounding environment are generally considered to be acidic and highly reducing. For example, the microenvironment of methicillin-resistant Staphylococcus aureus (MRSA) biofilms can have a pH of 5.5 or even lower. Utilizing the specific microenvironment of biofilms, it is urgent to design strategies to combat biofilm infections, especially those caused by drug-resistant bacteria. Site-specific drug delivery is an attractive strategy, offering higher drug concentrations and fewer side effects. However, drugs must overcome a series of barriers before reaching the bacteria, including airway mucus and the biofilm matrix. In recent studies, cationic nanoparticles have shown advantages in penetrating biofilms. In contrast, particle surfaces that maintain a neutral or slightly negative charge are more conducive to mucus penetration because they minimize undesirable interactions with mucins.

[0003] Lignin is the second largest biomass resource in the plant kingdom after cellulose. It is an amorphous biopolymer with a three-dimensional network structure formed by phenylpropane units linked together by ether and C-C bonds. Because lignin is rich in various active functional groups such as phenolic hydroxyl groups, alcoholic hydroxyl groups, methoxy groups, and benzene rings, it can participate in a variety of reactions including oxidation, reduction, hydrolysis, alcoholysis, acidolysis, photolysis, acylation, sulfonation, esterification, halogenation, nitration, and polycondensation, thus expanding its application areas. It is considered a natural antibacterial material with good bioactivity; however, the antibacterial effect relying solely on lignin's own bioactivity is limited. Patent CN115926206 A discloses lignin nanoparticles, their preparation method, and applications, relating to the field of antibacterial materials technology. This invention uses only lignin as a raw material. The lignin is fractionated using an organic solvent, achieving controlled distribution of molecular weight and chemical structure. The fractionated lignin is then dissolved in a mixture of organic solvent and water, and added dropwise to distilled water under stirring, or vice versa. After rotary evaporation and drying, lignin nanoparticles are obtained. This process exposes more active functional groups (phenolic hydroxyl and carboxyl groups), solving the problems of resource waste and low-value utilization caused by lignin's dispersibility and structural heterogeneity, as well as the issues of complex and costly preparation processes in existing lignin antibacterial materials, which often use composite materials. However, the antibacterial effects of these materials against lignin's inherent antibacterial activity are very limited. Chemically modifying lignin to obtain lignin-based composite antibacterial materials with stronger antibacterial bioactivity is an effective method. Patent CN 118435941 A discloses a method for preparing a lignin-silver nanocomposite antibacterial agent. By using a binary composite solvent obtained by combining water and a water-miscible organic solvent as the reaction solvent in the preparation process, the range of usable solvents is expanded from strongly polar solvents such as dimethylamide to weakly polar solvents such as tetrahydrofuran. This facilitates the synthesis of lignin-silver nanocomposite antibacterial agents, and the synthesized lignin-silver nanocomposite antibacterial agents have small particle size, good storage stability, good antibacterial activity, and low cytotoxicity. This solves the technical problem of the lack of a synthesis method for lignin-silver nanocomposite antibacterial agents in the prior art. Patent CN 111743862 A discloses a multi-bioactive modified lignin self-assembled drug-loaded nanomicelles and its preparation method. Lignin is prepared by acylation, esterification, and other reactions to prepare lignin-grafted bioactive compounds, which are then prepared into nanomicelles. These nanomicelles serve as excellent drug carriers and also possess multiple bioactivities of lignin and bioactive compounds.

[0004] In summary, there is an urgent need to develop a lignin-based composite antibacterial material that possesses high antibacterial bioactivity, biocompatibility, and safety, and can successfully penetrate the mucus barrier and biofilm to reach the drug delivery site. Summary of the Invention

[0005] The purpose of this invention is to address the poor antibacterial effect of raw lignin on biofilms. A pH-sensitive, charge-convertible lignin-based antibacterial nanoparticle has been designed, which is a type of nanoparticle responsive to the microenvironment of biofilms. This invention grafts positively charged antibacterial active substances onto lignin, making its charge convertible, and then prepares it as a nanocarrier. This invention can improve the dispersibility of lignin, enhance its antibacterial activity, biocompatibility, and safety. Simultaneously, the pH-sensitive surface charge allows the nanoparticles to gradually penetrate mucus barriers and biofilms, directly reaching the drug delivery site, achieving a better antibacterial effect against biofilms.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing charge-convertible pH-sensitive lignin-based antibacterial nanoparticles includes the following steps:

[0008] S1. Dissolve polyallylamine (PAH) in dimethyl sulfoxide (DMSO) to obtain solution A; dissolve lignin in dimethyl sulfoxide to obtain solution B;

[0009] S2. Add solution B dropwise to solution A, then add carboxyl activator 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and catalyst triethylamine (TEA) to carry out the first reaction to obtain the first reaction solution; through this step, positively charged polyallylamine is grafted onto lignin via an amide reaction to increase the positive charge density of lignin, thus preparing a lignin-polyallylamine (LP) coupling compound.

[0010] S3. Add acid anhydride to the first reaction solution to carry out the second reaction, and then dialyze and freeze dry to obtain lignin-polyallylamine-acid anhydride (LPD) conjugate; by grafting acid anhydride onto the positively charged LP conjugate through this step, an acid-stimulated responsive LPD conjugate is obtained. On the one hand, it has a higher biocompatibility and negative charge, and on the other hand, it can undergo charge conversion to become positively charged in a slightly acidic environment.

[0011] S4. Dissolve the lignin-polyallylamine-anhydride coupling obtained in step S3 in dimethyl sulfoxide. Under stirring, add the resulting solution dropwise to water or add water dropwise to the resulting solution to obtain solution C. Dialyze the solution to obtain charge-convertible pH-sensitive lignin-based nanoparticles (LP-DNPs). The size of the prepared LP-DNPs is between 300 and 400 nm. The LPD coupling is prepared into nanoparticles using an antisolvent method to increase the dispersibility of the coupling.

[0012] In step S1, the polyallylamine is prepared by the following method: polyallylamine hydrochloride and sodium hydroxide are dissolved in water, dialyzed, and freeze-dried to obtain the product.

[0013] The amount of polyallylamine hydrochloride added is 10-80 g / L; the amount of sodium hydroxide added is 10-50 g / L; the dialysis uses a dialysis bag with a molecular weight cutoff of 15-20 kDa, uses water as the dialysis solution, and the dialysis time is 3-7 days; the freeze-drying is carried out at a temperature of -55 to -80°C for 2-3 days.

[0014] In step S1, the lignin is any one of alkali lignin, fractionated lignin, gramineous lignin, broadleaf lignin, coniferous lignin, and acid-precipitated lignin; the concentration of solution A is 4–100 g / L; and the concentration of solution B is 10–100 g / L.

[0015] In step S2, solution B is added dropwise to solution A to control the mass ratio of polyallylamine to lignin to be 1:1 to 1:20; the concentrations of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and triethylamine in the mixed solution of solution A and solution B are 1 to 10 g / L and 1 to 5 μL / mL, respectively.

[0016] In steps S2 and S3, the first or second reaction is carried out under the following conditions: stirring at 25–35°C for 24–48 hours.

[0017] In step S3, the acid anhydride is maleic anhydride or dimethylmaleic anhydride; the mass ratio of the acid anhydride to lignin is 1:10 to 1:150; the dialysis uses a dialysis bag with a molecular weight cutoff of 15 kDa, uses dimethyl sulfoxide as the dialysis solution, and the dialysis time is 3 to 5 days; the freeze-drying is carried out at a temperature of -55 to -80°C for 2 to 3 days.

[0018] In step S4, the stirring conditions are a rotation speed of 200-800 rpm, and the dropping time is controlled at 10-200 min; in solution C, the concentration of lignin-polyallylamine-anhydride coupling compound is 0.1-5 mg / mL; the volume ratio of dimethyl sulfoxide to water is 4:1-40; in the dialysis, the dialysis bag used has a molecular weight cutoff of 15-20 kDa, the dialysis solution used is water, and the dialysis time is 24-72 h.

[0019] The application of the charge-convertible pH-sensitive lignin-based nanoparticles in the preparation of antibacterial products.

[0020] The product is a drug or a drug carrier; the antibacterial agent is either antibacterial itself or an antibacterial biofilm.

[0021] The bacteria mentioned include Escherichia coli, Staphylococcus aureus, Monilia albican, Listeria monocytogenes, and Pseudomonas aeruginosa.

[0022] Beneficial effects:

[0023] (1) The preparation method of the present invention is simple, fast and environmentally friendly. Through an efficient, convenient and stable synthesis system, lignin is chemically coupled with polyallylamine and acid anhydride. The synthesized lignin-based nanoparticles can be stably and uniformly dispersed in water and have uniform particle size.

[0024] (2) The charge-convertible, pH-sensitive lignin-based antibacterial nanoparticles of this invention are stimuli-responsive nanoparticles to the microenvironment of biofilms. Positively charged antibacterial active substances are grafted onto lignin, making its surface charge convertible. This not only significantly enhances the antibacterial activity of lignin, but also, the pH-sensitive surface charge allows the nanoparticles to carry a positive charge in a slightly acidic biofilm environment, enabling them to better bind to negatively charged bacteria and gradually penetrate the mucus barrier and biofilm, directly reaching the drug delivery site for better antibacterial effects. When applied to the antibacterial field, they can effectively inhibit biofilm growth and have a scavenging effect on existing biofilms. Attached Figure Description

[0025] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0026] Figure 1 This is a particle size distribution diagram of the charge-convertible pH-sensitive lignin-based nanoparticles of the present invention;

[0027] Figure 2 The infrared spectra of the charge-convertible pH-sensitive lignin-based nanoparticles and lignin alone are shown in the figure. In the figure, LPD represents lignin-polyallylamine (LPD) coupling, LP represents lignin-polyallylamine coupling, and L represents lignin.

[0028] Figure 3 The pH-sensitive charge conversion property of the charge-convertible pH-sensitive lignin-based nanoparticles in Example 7;

[0029] Figure 4 The antibacterial activity of the charge-convertible pH-sensitive lignin-based nanoparticles in Example 8;

[0030] Figure 5 The images show the biofilm inhibition effect of the charge-convertible pH-sensitive lignin-based nanoparticles in Example 9 and the crystal violet staining of the biofilm at pH=5.

[0031] Figure 6 The images show the biofilm removal effect of the charge-convertible pH-sensitive lignin-based nanoparticles in Example 10 and the crystal violet staining of the biofilm at pH=5.

[0032] Figure 7 The in vitro release curve of the pH-sensitive lignin-based nanoparticles loaded with curcumin in Example 11 is shown. Detailed Implementation

[0033] The present invention will be further described below with reference to the embodiments described. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the present invention are within the scope of the present invention.

[0034] For any specific techniques or conditions not specified in the examples, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0035] Table 1 shows the elemental analysis of charge-convertible lignin-based nanoparticles and lignin alone. As can be seen from the table, the nitrogen content of lignin increased significantly after PAH modification, which further confirms the increase in lignin amino group content after modification.

[0036] Table 1. Elemental analysis of charge-convertible lignin-based nanoparticles and lignin alone.

[0037]

[0038] Example 1

[0039] Weigh 374 mg PAH·HCl and 200 mg NaOH and dissolve them in 5 mL of distilled water. Place the resulting solution in a dialysis bag (15 kDa) and dialyze it in distilled water for 3 days. Then freeze-dry it at -80℃ for 3 days to obtain PAH.

[0040] 20 mg of PAH was completely dissolved in 5 mL of DMSO and stirred at 25 °C. The solution was then poured into a three-necked round-bottom flask. Next, 100 mg of alkali lignin was completely dissolved in 10 mL of DMSO and added dropwise evenly to the flask. Finally, 20 mg of EDC and 15 μL of TEA were added to the flask, and the mixture was stirred at 25 °C for 24 h. After the reaction was complete, 2 mg of dimethyl maleic anhydride (DMMA) was added to the flask, and the mixture was stirred at 25 °C for another 24 h. After the reaction was complete, the product was placed in a dialysis bag (15 kDa) and dialyzed in DMSO for 3 days. The product was then freeze-dried at -80 °C for 3 days to obtain the LPD conjugate.

[0041] 150 mg of LPD conjugate dissolved in 15 mL of DMSO was added to 135 mL of distilled water at a stirring speed of 800 rpm at a rate of 10 mL / min, resulting in a final concentration of 1 mg / mL of LPD conjugate. The solution was then transferred to a dialysis bag (15 kDa) and dialyzed in distilled water for 72 h to remove DMSO. Finally, charge-convertible, pH-sensitive lignin-based nanoparticles were formed. Figure 1 The figure shows the particle size distribution of pH-sensitive lignin-based nanoparticles with charge conversion capability. The average particle size is 300–400 nm.

[0042] Figure 2 The image shows the infrared spectra of charge-convertible pH-sensitive lignin-based nanoparticles and lignin alone. In the figure, LPD represents lignin-polyallylamine-dimethylmaleic anhydride coupling, LP represents lignin-polyallylamine coupling, and L represents lignin. Infrared spectroscopy analysis confirmed that the LP coupling was synthesized by the reaction of the carboxyl group of lignin with the amino group of PAH. Figure 2 As shown, the stretching vibrations of the aliphatic and phenolic hydroxyl groups of lignin occur at 3410 cm⁻¹. -1 The CH stretching vibrations of lignin methyl and methylene groups correspond to 2935 cm⁻¹, respectively. -1 and 2840cm -1 1600, 1510 and 1430cm -1 The three absorption peaks at [location] correspond to the benzene ring structure of lignin. After reaction with PAH, hierarchical lignin exhibits [absorption peak] at 1704 cm⁻¹. -1 The original characteristic absorption peak of the carboxyl group was weakened. Further, by reacting DMMA with the remaining amine group of PAH to form an acid-sensitive chemical bond, the C=O absorption peak introduced by DMMA appeared at 1700 cm⁻¹. -1 .

[0043] Example 2

[0044] Weigh 374 mg PAH·HCl and 200 mg NaOH and dissolve them in 5 mL of distilled water. Place the resulting solution in a dialysis bag (20 kDa) and dialyze it in distilled water for 3 days. Then freeze-dry it at -80℃ for 3 days to obtain PAH.

[0045] 20 mg of PAH was completely dissolved in 5 mL of DMSO and stirred at 25 °C. The solution was then poured into a three-necked round-bottom flask. Next, 200 mg of alkali lignin was completely dissolved in 10 mL of DMSO and added dropwise evenly to the flask. Finally, 20 mg of EDC and 15 μL of TEA were added to the flask, and the mixture was stirred at 25 °C for 24 h. After the reaction was complete, 2 mg of dimethyl maleic anhydride (DMMA) was added to the flask, and the mixture was stirred at 25 °C for another 24 h. After the reaction was complete, the product was placed in a dialysis bag (15 kDa) and dialyzed in DMSO for 3 days. The product was then freeze-dried at -80 °C for 3 days to obtain the LPD conjugate.

[0046] 150 mg of LPD conjugate dissolved in 15 mL of DMSO was added to 135 mL of distilled water at a stirring speed of 800 rpm at a rate of 10 mL / min, resulting in a final concentration of 1 mg / mL of LPD conjugate. The solution was then transferred to a dialysis bag (20 kDa) and dialyzed in distilled water for 36 h to remove DMSO. Finally, charge-convertible, pH-sensitive lignin-based nanoparticles were formed.

[0047] Example 3

[0048] Weigh 250 mg PAH·HCl and 200 mg NaOH and dissolve them in 5 mL of distilled water. Place the resulting solution in a dialysis bag (15 kDa) and dialyze it in distilled water for 3 days. Then freeze-dry it at -80℃ for 3 days to obtain PAH.

[0049] 20 mg of PAH was completely dissolved in 5 mL of DMSO and stirred at 25 °C. The solution was then poured into a three-necked round-bottom flask. Next, 200 mg of alkali lignin was completely dissolved in 10 mL of DMSO and added dropwise evenly to the flask. Finally, 20 mg of EDC and 15 μL of TEA were added to the flask, and the mixture was stirred at 25 °C for 24 h. After the reaction was complete, 2 mg of dimethyl maleic anhydride (DMMA) was added to the flask, and the mixture was stirred at 25 °C for another 24 h. After the reaction was complete, the product was placed in a dialysis bag (15 kDa) and dialyzed in DMSO for 3 days. The product was then freeze-dried at -80 °C for 2 days to obtain the LPD conjugate.

[0050] 150 mg of LPD conjugate dissolved in 15 mL of DMSO was added to 135 mL of distilled water at a stirring speed of 800 rpm at a rate of 10 mL / min, resulting in a final concentration of 1 mg / mL of LPD conjugate. The solution was then transferred to a dialysis bag (15 kDa) and dialyzed in distilled water for 72 h to remove DMSO. Finally, charge-convertible, pH-sensitive lignin-based nanoparticles were formed.

[0051] Example 4

[0052] Weigh 250 mg PAH·HCl and 200 mg NaOH and dissolve them in 5 mL of distilled water. Place the resulting solution in a dialysis bag (15 kDa) and dialyze it in distilled water for 3 days. Then freeze-dry it at -80℃ for 3 days to obtain PAH.

[0053] 30 mg of PAH was completely dissolved in 5 mL of DMSO and stirred at 25 °C. The solution was then poured into a three-necked round-bottom flask. Next, 200 mg of alkali lignin was completely dissolved in 10 mL of DMSO and added dropwise evenly to the flask. Finally, 20 mg of EDC and 15 μL of TEA were added to the flask, and the mixture was stirred at 25 °C for 24 h. After the reaction was complete, 2 mg of dimethyl maleic anhydride (DMMA) was added to the flask, and the mixture was stirred at 25 °C for another 24 h. After the reaction was complete, the product was placed in a dialysis bag (15 kDa) and dialyzed in DMSO for 3 days. The product was then freeze-dried at -80 °C for 2 days to obtain the LPD conjugate.

[0054] 200 mg of LPD conjugate dissolved in 15 mL of DMSO was added to 135 mL of distilled water at a stirring speed of 800 rpm at a rate of 10 mL / min, resulting in a final concentration of 1.33 mg / mL for the LPD conjugate. The solution was then transferred to a dialysis bag (15 kDa) and dialyzed in distilled water for 72 h to remove DMSO. Finally, charge-convertible, pH-sensitive lignin-based nanoparticles were formed.

[0055] Example 5

[0056] Weigh 374 mg PAH·HCl and 200 mg NaOH and dissolve them in 5 mL of distilled water. Place the resulting solution in a dialysis bag (15 kDa) and dialyze it in distilled water for 3 days. Then freeze-dry it at -80℃ for 3 days to obtain PAH.

[0057] 30 mg of PAH was completely dissolved in 5 mL of DMSO and stirred at 25 °C. The solution was then poured into a three-necked round-bottom flask. Next, 200 mg of fractionated lignin was completely dissolved in 10 mL of DMSO and added dropwise evenly to the flask. Finally, 20 mg of EDC and 15 μL of TEA were added to the flask, and the mixture was stirred at 25 °C for 24 h. After the reaction was complete, 2 mg of dimethyl maleic anhydride (DMMA) was added to the flask, and the mixture was stirred at 25 °C for another 24 h. After the reaction was complete, the product was placed in a dialysis bag (15 kDa) and dialyzed in DMSO for 3 days. The product was then freeze-dried at -80 °C for 2 days to obtain the LPD conjugate.

[0058] 200 mg of LPD conjugate dissolved in 15 mL of DMSO was added to 135 mL of distilled water at a stirring speed of 800 rpm at a rate of 10 mL / min, resulting in a final concentration of 1.33 mg / mL for the LPD conjugate. The solution was then transferred to a dialysis bag (15 kDa) and dialyzed in distilled water for 72 h to remove DMSO. Finally, charge-convertible, pH-sensitive lignin-based nanoparticles were formed.

[0059] Example 6

[0060] Weigh 374 mg PAH·HCl and 200 mg NaOH and dissolve them in 5 mL of distilled water. Place the resulting solution in a dialysis bag (15 kDa) and dialyze it in distilled water for 3 days. Then freeze-dry it at -80℃ for 3 days to obtain PAH.

[0061] 30 mg of PAH was completely dissolved in 5 mL of DMSO and stirred at 25 °C. The solution was then poured into a three-necked round-bottom flask. Next, 100 mg of fractionated lignin was completely dissolved in 10 mL of DMSO and added dropwise evenly to the flask. Finally, 20 mg of EDC and 15 μL of TEA were added to the flask, and the mixture was stirred at 25 °C for 24 h. After the reaction was complete, 2 mg of dimethyl maleic anhydride (DMMA) was added to the flask, and the mixture was stirred at 25 °C for another 24 h. After the reaction was complete, the product was placed in a dialysis bag (15 kDa) and dialyzed in DMSO for 3 days. The product was then freeze-dried at -80 °C for 2 days to obtain the LPD conjugate.

[0062] 200 mg of LPD conjugate dissolved in 15 mL of DMSO was added to 135 mL of distilled water at a stirring speed of 800 rpm at a rate of 10 mL / min, resulting in a final concentration of 1.33 mg / mL for the LPD conjugate. The solution was then transferred to a dialysis bag (15 kDa) and dialyzed in distilled water for 72 h to remove DMSO. Finally, charge-convertible, pH-sensitive lignin-based nanoparticles were formed.

[0063] Example 7

[0064] Taking the lignin-based nanoparticles prepared in Example 1 as an example, the charge conversion properties of the lignin-based nanoparticles of the present invention were evaluated. The pH values ​​of the nanoparticles dispersed in the aqueous solution were adjusted to 7 and 5, respectively, and the nanoparticles were incubated at 37°C and 200 rpm for 180 min. The changes in the zeta potential on the surface of the nanoparticles under the two pH conditions were measured simultaneously. Figure 3 pH-sensitive charge conversion properties of pH-sensitive lignin-based nanoparticles for charge conversion, from Figure 3 As can be seen, when nanoparticles are in an environment with pH=5, their charge changes from negative to positive after 10 minutes. However, in a neutral environment with pH=7, they remain negatively charged.

[0065] Example 8

[0066] The nanoparticle dispersion prepared in Example 1 was concentrated to 15 mg / mL by rotary evaporation, and then diluted with LB liquid medium at pH 5 to concentrations of 0, 0.625, 1.25, and 2.5 mg / mL, respectively. Escherichia coli was then inoculated into the above medium and incubated for 2 hours. The bacterial suspension was then removed from each medium and diluted 10... 9 The lignin-based nanoparticles were coated onto LB agar plates and incubated at 37°C until colonies grew. Colony counting was then performed to test the short-term antibacterial effect of the nanoparticles. The above describes the construction process for the pH=5 experimental group. A pH=7 control group was also constructed using the same procedures as the pH=5 experimental group. Figure 4 The figure shows the antibacterial activity of charge-convertible lignin-based nanoparticles. As the concentration of nanoparticles increases, in a slightly acidic environment, the surface charge of the nanoparticles converts to positive charge, inhibiting the growth of more *E. coli*. When the concentration is 2.5 mg / mL, the antibacterial rate can reach 100%. However, in a neutral environment, the antibacterial effect is not significant because the nanoparticles remain negatively charged.

[0067] Example 9

[0068] Dilute the Staphylococcus aureus culture to OD using LB liquid medium at pH 5. 600To achieve a concentration of 0.05, the nanoparticle dispersion prepared in Example 1 was concentrated to 15 mg / mL by rotary evaporation. The concentration of the nanoparticles was then adjusted using LB liquid medium at pH 5. The adjusted nanoparticle dispersion was then added to the diluted bacterial solution, resulting in nanoparticle concentrations of 0, 0.3, 0.6, 1.25, and 2.5 mg / mL. The bacterial solution was then inoculated into 96-well plates and incubated at 37°C for 24 h. The biofilm at the bottom of the 96-well plates was then washed with PBS. Subsequently, the plates were stained with 0.1% crystal violet aqueous solution (1 g / L) for 30 min. After staining, the plates were washed with PBS until the supernatant was colorless. Finally, 200 μL of 95% ethanol aqueous solution was added to dissolve the crystal violet, and the absorbance was measured at 570 nm. The absorbance, representing the crystal violet content, indicated the amount of biofilm formed. The above describes the construction process for the pH 5 experimental group. A control group at pH 7 was also constructed, following the same procedures as the pH 5 experimental group. Figure 5 Images show the biofilm inhibition effect of charge-convertible lignin-based nanoparticles and crystal violet staining of the biofilm at pH 5. Figure 5 The results showed that in a slightly acidic environment (pH=5), the nanoparticles became positively charged after charge conversion, and their inhibitory effect on biofilms was better than in neutral conditions, with an inhibition rate of up to 44%.

[0069] Example 10

[0070] Dilute the Staphylococcus aureus bacterial suspension to OD. 600 The concentration was 0.05 mg / mL, and the nanoparticles were inoculated into 96-well plates and incubated at 37°C for 24 hours. The nanoparticle dispersion prepared in Example 1 was concentrated to 15 mg / mL by rotary evaporation, and the concentration of nanoparticles was adjusted with LB liquid medium at pH 5. The adjusted nanoparticle dispersion was then added to the bacterial culture in the 96-well plates that had been incubated for 24 hours, so that the concentrations of nanoparticles in the medium in the 96-well plates were 0, 0.3, 0.6, 1.25, and 2.5 mg / mL, respectively, and then co-incubated for another 24 hours. After incubation, the biofilm at the bottom of the 96-well plates was rinsed with PBS. Subsequently, the plates were stained with 0.1% crystal violet for 30 minutes. After staining, the plates were rinsed with PBS until the supernatant was colorless. Finally, 200 μL of 95% ethanol was added to dissolve the crystal violet, and the absorbance was measured at 570 nm. The absorbance represents the amount of crystal violet, indicating the amount of biofilm. The above describes the construction process of the pH 5 experimental group. At the same time, a control group at pH 7 was constructed, and the operation steps were the same as those for the pH 5 experimental group. Figure 6 This image shows the biofilm scavenging effect of charge-convertible lignin-based nanoparticles. Figure 6The results show that the nanoparticles can achieve a clearance rate of up to 76% in a neutral environment and up to 82% in a slightly acidic environment. Experimental results demonstrate that the nanoparticles of this invention can effectively remove biofilms, and their removal effect on biofilms is superior in an acidic environment compared to a neutral environment, with a clearance rate approximately 1.1 times that in a neutral environment.

[0071] Example 11

[0072] 100 mg curcumin and 200 mg LPD were mixed thoroughly in 10 mL DMSO to obtain a homogeneous solution. Using an antisolvent method, the solution was added to 90 mL of distilled water at a rate of 10 mL / min with a stirring speed of 800 rpm to prepare curcumin-loaded LP-DNPs. The solution was transferred to a dialysis bag (15 kDa) and dialyzed in distilled water for 72 h to remove DMSO and free curcumin. Finally, charge-convertible pH-sensitive lignin-based nanoparticles (LP-DCNPs) loaded with curcumin were formed, with a drug encapsulation efficiency of 48%. The drug release behavior of curcumin-loaded lignin nanoparticles under simulated slightly acidic in vivo conditions was studied using dialysis. 1 mL of LP-DCNPs dispersion with a concentration of 1 mg / mL was added to a dialysis bag (MWCO = 500 Da). The dialysis bag was then immersed in 50 mL of 0.01 M PBS (pH 5.0) release medium and shaken at 37 °C and 200 rpm / min in a constant temperature shaker. 2 mL of PBS release medium was collected as samples at 0 h, 20 min, 40 min, 2 h, 3 h, 10 h, and 24 h, and an equal volume of fresh PBS release medium was added to each sample. The absorbance of the samples was measured at 428 nm using a UV spectrophotometer, and the concentration of curcumin in the samples was calculated using the standard curve. The in vitro release experiment was repeated three times in parallel. Figure 7 This is the in vitro release curve of the pH-sensitive lignin-based nanoparticles loaded with curcumin in this embodiment. Figure 7 It can be seen that when the sustained-release time is 10 hours, the cumulative release of curcumin reaches its maximum of 29.4%, and the release remains basically unchanged at 24 hours.

[0073] This invention provides a method for preparing and applying charge-convertible pH-sensitive lignin-based nanoparticles. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for preparing charge switchable, pH sensitive lignin-based antibacterial nanoparticles, characterized by, The method comprises the following steps: S1. Dissolving polyallylamine in dimethyl sulfoxide to obtain solution A; Dissolving lignin in dimethyl sulfoxide to obtain solution B; S2. Adding solution B dropwise to solution A, and then adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and triethylamine to carry out a first reaction to obtain a first reaction solution; S3. Adding an acid anhydride to the first reaction solution to carry out a second reaction, and then performing dialysis and freeze-drying to obtain a lignin-polyallylamine-acid anhydride conjugate; S4. Dissolving the lignin-polyallylamine-acid anhydride conjugate obtained in step S3 in dimethyl sulfoxide, and then adding the obtained solution dropwise to water or adding water dropwise to the obtained solution under stirring to obtain solution C, and then performing dialysis to obtain charge-switchable pH-sensitive lignin-based nanoparticles.

2. The production method according to claim 1, characterized by, In step S1, the polyallylamine is prepared by dissolving polyallylamine hydrochloride and sodium hydroxide in water, dialysis, and freeze-drying.

3. The preparation method according to claim 2, characterized in that, The addition amount of the polyallylamine hydrochloride is 10-80 g / L; the addition amount of the sodium hydroxide is 10-50 g / L; the dialysis is performed using a dialysis bag with a molecular weight cut-off of 15-20 kDa and water as the dialysate for 3-7 days; and the freeze-drying is performed at a temperature of-55 to-80 ℃ for 2-3 days.

4. The method of claim 1, wherein, In step S1, the lignin is any one of alkali lignin, fractionated lignin, gramineous lignin, broadleaf wood lignin, coniferous wood lignin, and acid precipitated lignin; the concentration of solution A is 4-100 g / L; and the concentration of solution B is 10-100 g / L.

5. The preparation method according to claim 1, characterized in that, In step S2, solution B is added dropwise to solution A to control the mass ratio of polyallylamine to lignin to be 1:1-1:20; the concentrations of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and triethylamine in the mixed solution of solution A and solution B are 1-10 g / L and 1-5 μL / mL, respectively.

6. The method of claim 1, wherein, In steps S2 and S3, the first reaction or the second reaction is performed under the following conditions: stirring at 25-35 ℃ for 24-48 h.

7. The preparation method according to claim 1, characterized in that, In step S3, the acid anhydride is maleic anhydride or dimethyl maleic anhydride; the mass ratio of the acid anhydride to lignin is 1:10-1:150; the dialysis is performed using a dialysis bag with a molecular weight cut-off of 15 kDa and dimethyl sulfoxide as the dialysate for 3-5 days; and the freeze-drying is performed at a temperature of-55 to-80 ℃ for 2-3 days.

8. The method of claim 1, wherein, In step S4, the stirring condition is a rotation speed of 200-800 rpm, the dropping is controlled in a time of 10-200 min; in the solution C, the concentration of the lignin-polyallylamine-acid anhydride conjugate is 0.1-5 mg / mL; the volume ratio of dimethyl sulfoxide to water is 4:1-40; the dialysis uses a dialysis bag with a molecular weight cut-off of 15-20 kDa, uses water as the dialysate, and the dialysis time is 24-72 h.

9. The charge-switchable pH-sensitive lignin-based nanoparticles prepared by the preparation method in any one of claims 1-8.

10. The use of the charge-switchable pH-sensitive lignin-based nanoparticles in claim 9 in the preparation of antibacterial products.

11. Use according to claim 10, characterized in that, The product is a pharmaceutical product or a pharmaceutical carrier; the antibacterial is antibacterial itself or antibacterial biofilm.

12. The use according to claim 10, characterized in that, The bacteria include Escherichia coli, Staphylococcus aureus, Candida albicans, Listeria and Pseudomonas aeruginosa.

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