Compound self-assembled nanogel based on natural active ingredients, preparation method and application thereof

Abstract

The application discloses a kind of based on natural active ingredient compound self-assembly nanogel and its preparation method and application, it is related to pharmaceutical preparation field.The nanogel of the application includes natural active molecule, stabilizer and gel matrix, component is reasonable, process is simple, and pharmacodynamic is remarkable.Based on natural active ingredient compound self-assembly nanogel has good synergistic antibacterial and anti-inflammatory activity, and the inhibition rate of intracellular staphylococcus aureus is 97.66%, can regulate macrophage proinflammatory phenotype (M1) to anti-inflammatory phenotype (M2) polarization during inflammation, inhibit the secretion of proinflammatory factor, increase the release of anti-inflammatory factor, and reduce inflammation.At the same time, the innovative combination of self-assembly nano and gel technology can reduce the aggregation of nanoparticle and increase its stability, improve the adhesion of natural active ingredient nanoparticle, promote the residence and absorption of drug in cavity mucosa.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, and particularly relates to a compound self-assembled nanogel based on natural active ingredients, its preparation method and application. Background Technology

[0002] The modernization of animal husbandry is accelerating, leading to increased stocking densities and a rise in various bacterial infections. Bacteria are widespread in the natural environment, and when an organism's immunity is weakened due to external factors, bacteria can easily invade, multiply, and cause infectious diseases. Although antibiotics are widely used in veterinary clinics due to their rapid action and significant antibacterial effects, recent years have seen drug residues and increased drug resistance resulting from excessive antibiotic use, posing a threat to human and animal health. Therefore, there is an urgent need to find new antibacterial molecules and develop multi-dimensional strategies to combat bacterial infections.

[0003] When bacteria invade the body and cause damage, local tissues recruit a large number of immune cells to fight the infection. In the early stages of inflammation, macrophages polarize into the classic M1 phenotype, secreting large amounts of pro-inflammatory cytokines to participate in the removal of pathogens. However, excessive accumulation of immune cells leads to narrowing of the lumen within the local tissue, hindering normal tissue function and causing changes in nutritional composition and physicochemical properties. In the late stages of inflammation, macrophages can inhibit the formation of the M2 phenotype and promote tissue repair. Due to the proliferation of large amounts of bacteria and the inflammatory response at the site of infection, macrophages tend to polarize into the M1 phenotype, while the ability to switch from the M1 to the M2 phenotype is reduced, resulting in a continuous influx and activation of pro-inflammatory factors, thus prolonging tissue recovery time. Therefore, an ideal antibacterial agent should have excellent antibacterial effects, avoid excessive activation of macrophages towards the pro-inflammatory M1 phenotype, and be able to regulate the shift of macrophages towards the anti-inflammatory M2 phenotype, thereby synergistically promoting tissue repair. Natural active ingredients are receiving increasing attention due to their excellent anti-inflammatory effects, low residue, low likelihood of developing drug resistance, few side effects, abundant sources, and ability to reverse drug resistance. Furthermore, compared to monotherapy, combining natural active ingredients with different antibacterial mechanisms can significantly improve antibacterial efficacy. However, natural active ingredients suffer from drawbacks such as low bioavailability, short half-life, and poor solubility, which greatly limit their efficacy and clinical application.

[0004] Self-assembled nanoparticles, leveraging their small size effect and high specific surface area, improve the formulation specifications of poorly soluble drugs, enhance bioavailability, and reduce the need for multiple clinical dosings, achieving cost reduction and efficiency enhancement, and contributing to maximizing drug efficacy. Combining self-assembled nanotechnology with sustained-release carrier systems can further protect drugs and delay drug release. Self-assembled nanogels are molecular gels based on nanoparticles, prepared by uniformly mixing nanoparticles with a gel matrix and other excipients. This reduces nanoparticle aggregation, increasing stability, improving the moisture retention and adhesion of nanoparticles, and promoting drug adhesion and absorption in cavitary mucosa. Compared to single natural active ingredients, the combined use of synergistic natural active ingredients shows more significant therapeutic effects on bacterial infections. Therefore, finding new synergistic formulations of natural active ingredients to improve the therapeutic effect of bacterial infections is urgently needed. Although traditional natural active ingredient compounds have made significant progress in the research of inflammation treatment, their duration of action at the site of infection is relatively short, and their antibacterial effect is poor, making it difficult to achieve ideal therapeutic effects. Therefore, natural active drug formulations with both antibacterial and immunomodulatory properties need further development. Summary of the Invention

[0005] Based on the preparation method and application scope of self-assembled nanogels, in order to enhance the synergistic antibacterial and anti-inflammatory effects of natural active ingredients and solve the problems of poor solubility and weak sustained-release ability of natural active molecules, this invention provides a compound self-assembled nanogel based on natural active ingredients, its preparation method and application.

[0006] To achieve the above objectives, the present invention provides a compound self-assembled nanogel based on natural active ingredients, wherein the raw materials, by mass percentage, include: 0.1-2.5% natural active molecules, 1.0-8.0% stabilizer, 2.0-7.0% gel matrix, and the balance being water;

[0007] The natural active molecule is composed of a first natural active molecule and a second natural active molecule, with a mass ratio of 2:1 between the first natural active molecule and the second natural active molecule.

[0008] Preferably, the self-assembled nanogel based on natural active ingredients comprises, by mass percentage: 1.2% natural active molecules, 3.0% stabilizer, 6.0% gel matrix, and the remainder being sterile water;

[0009] The natural active molecule is composed of a first natural active molecule and a second natural active molecule, with a mass ratio of 2:1 between the first natural active molecule and the second natural active molecule.

[0010] Preferably, the first natural active molecule is one of rhein, berberine, and coumarin;

[0011] The second natural active molecule is one of magnolol, pterostilbene, and glycyrrhizic acid monoammonium salt.

[0012] Preferably, the first natural active molecule is rhein (RHE), and the second natural active molecule is honokiol (HON).

[0013] Preferably, the stabilizer includes at least one of polyvinyl alcohol (PVA), poloxamer 188 (P188), and polyvinylpyrrolidone (PVP).

[0014] More preferably, the stabilizer is polyvinyl alcohol.

[0015] Preferably, the gel matrix comprises at least one of sodium alginate (SA) and poloxamer 407 (P407).

[0016] More preferably, the gel matrix is ​​sodium alginate and poloxamer 407, with a mass ratio of sodium alginate to poloxamer 407 of 1:3.

[0017] This invention provides a method for preparing the aforementioned compound self-assembled nanogel based on natural active ingredients, comprising the following steps:

[0018] (1) Weigh each raw material accurately according to the raw material ratio, and dissolve the natural active molecules in 0.03-0.06M sodium hydroxide solution according to the raw material ratio to obtain solution A;

[0019] (2) Dissolve the stabilizer in 0.03-0.06M hydrochloric acid solution according to the raw material ratio to obtain solution B;

[0020] (3) Add the solution B dropwise to the solution A while stirring. After the addition is complete, continue stirring for 1-8 hours. Then, use an ultrasonic breaker to prepare self-assembled nanoparticles.

[0021] (4) The self-assembled nanoparticles are added to the gel matrix and stirred to obtain a compound self-assembled nanogel based on natural active ingredients.

[0022] Preferably, in step (1), the natural active molecules are dissolved in a 0.05M sodium hydroxide solution according to the raw material ratio to obtain solution A.

[0023] Preferably, in step (2), the stabilizer is dissolved in 0.05M hydrochloric acid solution according to the raw material ratio to obtain solution B.

[0024] Preferably, in step (3), the stirring speed is 500-1000 r / min, the dropping speed is 0.5-1 mL / min, the ultrasonic power is 140-200 W, and the ultrasonic time is 1-5 min. More preferably, the stirring speed is 700 r / min, the dropping speed is 0.75 mL / min, the ultrasonic power is 160 W, and the ultrasonic time is 3 min.

[0025] In step (4), the stirring speed is 200-1500 r / min and the stirring time is 5-15 h, more preferably the stirring speed is 300 r / min and the stirring time is 12 h.

[0026] Preferably, the preparation method of the self-assembled nanogel based on natural active ingredient compound includes the following steps:

[0027] (1) Dissolve 160 mg of rhein and 80 mg of magnolol in 10 mL of 0.05 M NaOH solution to obtain solution A;

[0028] (2) Dissolve 300 mg of polyvinyl alcohol in 10 mL of 0.05 M hydrochloric acid solution to obtain solution B;

[0029] (3) Add the solution B to the solution A at a titration rate of 0.75 mL / min while stirring at 700 r / min. After stirring for 6 h, sonicate at 160 W for 3 min to obtain self-assembled nanoparticles.

[0030] (4) Weigh 300 mg of sodium alginate and add it to the self-assembled nanoparticles. Stir continuously until completely dissolved. Add 900 mg of poloxamer 407 and stir continuously for 12 h at 300 r / min to obtain a compound self-assembled nanogel based on natural active ingredients.

[0031] This invention provides the application of the aforementioned compound self-assembled nanogel based on natural active ingredients in the preparation of drugs for treating bacterial infections.

[0032] This invention provides the application of the aforementioned compound self-assembled nanogel based on natural active ingredients in the preparation of drugs to alleviate or regulate bacterial infections and inflammatory diseases.

[0033] Compared with the prior art, the present invention has the following advantages and technical effects:

[0034] This invention utilizes the supramolecular interactions between molecules of natural active ingredients to develop a novel self-assembled nanogel based on natural active ingredient compounds. This nanogel effectively inhibits the growth of Staphylococcus aureus (S. aureus) at the application site. Furthermore, by modulating the phenotype of immune cells in tissues induced by S. aureus infection, it achieves multifaceted antibacterial and anti-inflammatory treatment at the infection site, providing an effective strategy to overcome the treatment challenges posed by S. aureus and other bacterial infections.

[0035] This invention utilizes a self-assembled nanogel of natural active ingredients, exhibiting excellent synergistic antibacterial and anti-inflammatory activity. It shows a 97.66% inhibition rate against intracellular Staphylococcus aureus and can regulate the polarization of macrophages from a pro-inflammatory phenotype (M1) to an anti-inflammatory phenotype (M2) during inflammation, inhibiting the secretion of pro-inflammatory factors, increasing the release of anti-inflammatory factors, and alleviating inflammation. Simultaneously, the innovative combination of self-assembled nanoparticles and gel technology reduces nanoparticle aggregation and increases their stability, improves the adhesion of natural active ingredient nanoparticles, and promotes drug retention and absorption in cavitary mucosa. Attached Figure Description

[0036] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0037] Figure 1 The diagram shows the molecular dynamics simulation of rhein and honokiol. A is the molecular simulation of the self-assembly process of RHE and HON monomer molecules, and B is the movement and distribution of rhein and honokiol molecules after water molecules are removed in the stable state. The dashed lines represent hydrogen bonds.

[0038] Figure 2 Scanning electron microscope images of self-assembled nanoparticles obtained by different preparation methods, where A is the one-step synthesis method, B is the nanoprecipitation method, and C is the hydrothermal method;

[0039] Figure 3 The in vitro release of self-assembled nanoparticles and self-assembled nanogels is shown in Figure A, which represents the rhein group, and Figure B represents the honokiol group.

[0040] Figure 4 To illustrate the inhibitory effect of self-assembled nanogels on intracellular bacterial infection, A represents the growth of intracellular bacteria in different groups; B represents the inhibitory efficiency of different groups on intracellular bacteria.

[0041] Figure 5 The effects of drug administration on macrophage inflammatory cytokines 24 hours after administration are shown in the following figures: the left figure shows the CD86 content level in different groups, the middle figure shows the iNOS content level in different groups, and the right figure shows the CD206 content level in different groups.

[0042] Figure 6 Tissue bacterial load and ocular lesions at different time points after drug administration in each group;

[0043] Figure 7 Bacterial load in breast tissue at different times after the end of drug administration in each group. A represents the bacterial load in different groups 3 days after the end of drug administration, and B represents the bacterial load in different groups 7 days after the end of drug administration.

[0044] Figure 8 Pathological sections of breast tissue from each group;

[0045] Figure 9 ELISA test results of breast tissue at different time points after the end of drug administration in each group. A represents the test results of different groups 3 days after the end of drug administration, and B represents the test results of different groups 7 days after the end of drug administration.

[0046] Figure 10 The appearance of the compound self-assembled nanogel based on natural active ingredients prepared under optimal conditions. Detailed Implementation

[0047] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0048] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0049] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0050] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0051] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0052] This invention uses one of the following natural active molecules as natural active ingredients: rhein, berberine, and coumarin, and one of the following: magnolol, pterostilbene, and glycyrrhizic acid monoammonium salt; one or more of the following: polyvinyl alcohol, poloxamer 188, and polyvinylpyrrolidone; and one or more of the following: sodium alginate and poloxamer 407. Self-assembled nanoparticles are prepared using supramolecular interactions between the natural active ingredients, and then these self-assembled nanoparticles are added to the gel matrix to obtain a compound self-assembled nanogel agent based on natural active ingredients, exhibiting both antibacterial and immunomodulatory effects. This invention screens rhein and magnolol, which have synergistic effects, and prepares self-assembled nanoparticles using a nanoprecipitation method, then gels them into a self-assembled nanogel agent. Nano-sizing increases cell membrane permeability and enhances intracellular residence time, thereby significantly improving the therapeutic and immunomodulatory effects of antibacterial drugs. Encapsulating the nanoparticles with gel further enhances drug efficacy, delays nanoparticle release, facilitates local administration, and reduces the frequency of clinical administration.

[0053] In this embodiment of the invention, rhein was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., berberine was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., coumarin was purchased from Yuanye Biotechnology Co., Ltd. (Shanghai), magnolol was purchased from Yuanye Biotechnology Co., Ltd. (Shanghai), pterostilbene was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., glycyrrhizic acid monoammonium salt was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., polyvinyl alcohol was purchased from Yuanye Biotechnology Co., Ltd. (Shanghai), poloxamer 188 was purchased from BASF GmbH (Germany), polyvinylpyrrolidone was purchased from Sinopharm Chemical Reagent Co., Ltd., and sodium alginate and poloxamer 407 were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0054] Example 1: Graded antibacterial concentrations of natural active molecule complex combinations and molecular dynamics simulation of the optimal combination

[0055] 1.1 A compound self-assembled nanogel based on natural active ingredients, including self-assembled nanoparticles and a gel matrix, wherein the combination of natural active molecules is shown in Table 1. The relationship between natural active molecules was determined by graded antimicrobial concentration (FIC). When the FIC index is ≤0.5, >0.5~1, >1~2, and >2, it represents synergistic, additive, unrelated, and antagonistic effects, respectively.

[0056] Table 1 Combination of Natural Active Molecules

[0057]

[0058] The specific method includes the following steps:

[0059] (1) Using 96-well sterile microplates, each drug was serially diluted with sterile LB broth to obtain 7 dilutions, namely 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL and 4 μg / mL;

[0060] (2) Take 50 μL of each and arrange them in rows and columns of the plate, then add 100 μL to the sterile microplate to dilute to 1.5 × 10⁻⁶. 6 The bacterial suspension was prepared at a concentration of CFU / mL, resulting in a final inoculum size of 1.5 × 10⁻⁶. 6 The minimum drug concentration at which no bacterial growth is achieved after overnight incubation (CFU / mL) is defined as the MIC.

[0061] (3) Calculate the FIC index of the combination drugs. FIC index = MIC (molecule A combined) / MIC (molecule A alone) + MIC (molecule B combined) / MIC (molecule B alone)

[0062] 1.2 FIC test results of each formulation combination

[0063] Table 2. Graded antibacterial concentration index of different combinations in Example 1

[0064]

[0065] As shown in Table 2, the combined use of rhein and magnolol exhibits an additive inhibitory effect on Staphylococcus aureus, achieving the goal of inhibiting the growth of Staphylococcus aureus at a drug ratio of 2:1 (16 μg / mL: 8 μg / mL).

[0066] 1.3 Molecular dynamics simulation of combined drug use with natural active molecules

[0067] 1.3.1 The interaction between rhein and magnolol molecules in aqueous solution was evaluated from the perspective of molecular dynamics simulation using Gromoacs 2020.4 software. The parameter settings are as follows:

[0068] (1) The force field for calculating intermolecular interactions was set to the GAFF force field, and the water molecule was modeled as SPCE; the size was 4.5×4.5×4.5nm. 3 A molecular system of RHE and HON was constructed in a water tank, and the density was set to 1.0 g / cm³. 3 ;

[0069] (2) The system was balanced under constant temperature (298K) and constant pressure (1atm) using the V-rescale automatic temperature control algorithm, and the energy of the initial configuration was minimized by the rapid descent method. The system was then briefly balanced under NPT integration to stabilize the system volume.

[0070] (3) Molecular dynamics simulations were performed under NVT with a time step of 2 fs and a total simulation time of 20 ns, and the trajectories were collected. Images of intermolecular interactions and binding sites were visualized using VMD software.

[0071] 1.3.2 Molecular dynamics simulation results of self-assembly of natural active molecules

[0072] Figure 1 Based on molecular dynamics simulations, the entire process takes 20 ns. Rhein and magnolol tend to stabilize around 15 ns. To better observe the intermolecular interaction state, Figure 1 Figure B shows the movement and distribution of rhein and magnolol molecules after water molecules are removed in a stable state. The dashed lines represent hydrogen bonds. Rhein and magnolol monomers are likely linked together by synergistic supramolecular interactions, including hydrogen bonds, π-π stacking, and hydrophobic interactions, to form self-assembled aggregates. Rhein and magnolol molecules form a layered structure through π-π stacking, which constitutes the basic framework of the self-assembled system. Subsequently, rhein and magnolol are connected by hydrogen bonds, embedding magnolol molecules into the gaps of the layered structure, making the system more stable.

[0073] Therefore, this invention uses rhein and magnolol as basic self-assembly units in a compound self-assembled nanogel based on natural active ingredients.

[0074] Example 2

[0075] This embodiment provides a self-assembled nanogel based on a natural active ingredient compound, comprising self-assembled nanoparticles and a gel matrix, wherein the self-assembled nanoparticles involve the following preparation method:

[0076] 2.1 Preparation of self-assembled nanoparticles by nanoprecipitation method

[0077] (1) Dissolve 160 mg of rhein and 80 mg of magnolol in 10 mL of 0.05 M sodium hydroxide solution to obtain solution A;

[0078] (2) Dissolve 200 mg of polyvinyl alcohol in 10 mL of 0.05 M hydrochloric acid solution to obtain solution B;

[0079] (3) Add the solution B to the solution A at a titration rate of 0.75 mL / min while stirring at 700 r / min. After stirring for 6 h, sonicate at 160 W for 3 min to obtain a self-assembled nanoparticle solution.

[0080] 2.2 One-step synthesis method for preparing self-assembled nanoparticles

[0081] (1) Dissolve 160 mg of rhein in 10 mL of 0.05 M sodium hydroxide solution to obtain solution A;

[0082] (2) Dissolve 80 mg of magnolol in 10 mL of 0.05 M sodium hydroxide solution to obtain solution B;

[0083] (3) Add solution B dropwise to solution A and stir continuously to obtain a self-assembled nanoparticle solution. The titration rate is 0.75 mL / min, the stirring rate is 700 r / min, and the stirring time is 6 h.

[0084] 2.3 Preparation of self-assembled nanoparticles by hydrothermal method

[0085] (1) Dissolve 160 mg of rhein and 80 mg of honokiol in 10 mL of dimethyl sulfoxide solution to obtain solution A;

[0086] (2) Add solution A dropwise to 10 mL of deionized water and stir continuously to obtain solution B. The specific conditions are: titration speed 0.75 mL / min, stirring speed 700 r / min, and stirring time 6 h.

[0087] (2) Stir solution B at 700 r / min at 80℃ for 60 min, then transfer it to a MW=3500 dialysis bag for dialysis for 6 h, and freeze dry to obtain self-assembled nanoparticles.

[0088] 2.4 Self-assembled nanoparticles obtained by different preparation methods were subjected to electron microscopy.

[0089] 2.4.1 Electron Microscopy Scanning Procedure:

[0090] (1) Dilute the products from the nanoprecipitation method and the one-step synthesis method by 1000 times.

[0091] (2) Take 10 μL of the diluted solution and drop it onto tin foil. Dry it by air drying, and then place the tin foil on the stage. Take a small amount of the hydrothermal product freeze-dried and place it on the stage.

[0092] (3) Conductivity treatment of the sample is performed by metal coating method.

[0093] 2.4.2 Electron Microscopy Results

[0094] Figure 2 The images show scanning electron microscope (SEM) images of the self-assembled nanoparticles obtained by different preparation methods in Example 2. To effectively construct the self-assembled system of rhein and magnolol, a one-step synthesis method, a nanoprecipitation method, and a hydrothermal method were used, respectively. The SEM results of the obtained products after appropriate dilution are shown below. Figure 2 As shown, the product prepared by the one-step synthesis method has a large number of nanofiber-like structures with irregular morphology (A); the product obtained by the nanoprecipitation method is spherical with regular morphology (B); the product obtained by the hydrothermal method has a layered structure with significant morphological differences (C).

[0095] Therefore, the nanoprecipitation method can produce particles with uniform morphology, making it suitable as a preparation method for subsequent construction of self-assembled nanoparticle systems.

[0096] Example 3

[0097] A self-assembled nanogel based on a compound of natural active ingredients, comprising self-assembled nanoparticles and a gel matrix, wherein the preparation method of the self-assembled nanoparticles involves the following processes:

[0098] 3.1 Design orthogonal experiments to optimize the prescription. The specific experimental factors and levels are shown in Table 3, and the specific experimental conditions are shown in Table 4.

[0099] Table 3 Specific experimental factors and levels

[0100]

[0101] Table 4. Orthogonal experimental design of formulation and process

[0102]

[0103]

[0104] 3.2 Solution preparation:

[0105] (1) Dissolve 100 mg of poloxamer 188 in 10 mL of 0.05 M hydrochloric acid solution to obtain solution A;

[0106] (2) Dissolve 300 mg of poloxamer 188 in 10 mL of 0.05 M hydrochloric acid solution to obtain solution B;

[0107] (3) Dissolve 600 mg of poloxamer 188 in 10 mL of 0.05 M hydrochloric acid solution to obtain solution C;

[0108] (4) Dissolve 100 mg of polyvinyl alcohol in 10 mL of 0.05 M hydrochloric acid solution to obtain solution D;

[0109] (5) Dissolve 300 mg of polyvinyl alcohol in 10 mL of 0.05 M hydrochloric acid solution to obtain solution E;

[0110] (6) Dissolve 600 mg of polyvinyl alcohol in 10 mL of 0.05 M hydrochloric acid solution to obtain solution F;

[0111] (7) Dissolve 100 mg of polyvinylpyrrolidone in 10 mL of 0.05 M hydrochloric acid solution to obtain solution G;

[0112] (8) Dissolve 300 mg of polyvinylpyrrolidone in 10 mL of 0.05 M hydrochloric acid solution to obtain solution H;

[0113] (9) Dissolve 600 mg of polyvinylpyrrolidone in 10 mL of 0.05 M hydrochloric acid solution to obtain solution I;

[0114] (10) Dissolve 160 mg of rhein and 80 mg of magnolol in 10 mL of 0.05 M sodium hydroxide solution to obtain solution J.

[0115] 3.3 Preparation of self-assembled nanoparticles under different conditions:

[0116] (1) Select the corresponding solution AI according to Table 4, and add the corresponding solution to solution J. The specific conditions are: titration speed 0.75 mL / min, stirring speed 700 r / min.

[0117] (2) According to Table 4, the ultrasound was performed for 1-5 minutes in different groups, followed by stirring at a speed of 700 r / min for 6 hours.

[0118] The specific experimental procedures are as follows:

[0119] (1) After the self-assembled nanoparticles were prepared, they were numbered 1-9 according to Table 4. 1 mL of each sample was taken, diluted 100 times with distilled water, and stored in 10 mL centrifuge tubes for later use. The particle size and polydispersity index were tested using a Malvern nanolaser particle size analyzer ZS90 at 25℃.

[0120] (2) The assembly rate of each group was determined by high performance liquid chromatography. The mobile phase was 0.1% phosphoric acid solution in phase A and methanol in phase B, with a volume ratio of 17:83. The detection wavelength was 254 nm. The injection volume was 20 μL. The flow rate was 1.0 mL / min.

[0121] 3.4 Results of Orthogonal Experiment Recipe Optimization

[0122] The optimal combination under different levels of conditions was selected using a comprehensive scoring method. The dispersion coefficient (Y1) and assembly rate (Y2) were used as evaluation indicators, and the weight coefficients of Y1 and Y2 were specified as 0.5. The maximum dispersion coefficient of 1 was defined as 0 points, and the minimum of 0.139 was defined as 50 points. For each increase of 1, 58.072 points were deducted [50 / (1-0.139)]. The minimum assembly rate of 53% was defined as 0 points, and the maximum of 95% was defined as 50 points. For each decrease of 1%, 0.407 points were deducted [50 / (53%-95%)]. The comprehensive formula was: Y3=50+119.0476×(Y1-0.95)+50-58.072×(Y2-0.139).

[0123] The scoring results of each group are shown in Table 5. Group 5 of the experiment had the highest Y3 under the preparation conditions and the highest comprehensive score. The corresponding experimental conditions were A2, B2, and C2 (type PVA, concentration 3wt%, ultrasonic time 3min).

[0124] Table 5 Scoring of Orthogonal Experiment Results for Each Group

[0125]

[0126] Example 4

[0127] A self-assembled nanogel based on a compound of natural active ingredients, comprising self-assembled nanoparticles and a gel matrix, wherein the gel matrix includes the following raw materials and viscosity and encapsulation efficiency tests:

[0128] 4.1 Gel matrix preparation and viscosity testing

[0129] 4.1.1 The mass ratio of the gel matrix is ​​shown in Table 6.

[0130] Table 6. Types and proportions of gel matrix preparation

[0131]

[0132] The preparation steps are as follows:

[0133] (1) Weigh 300 mg of sodium alginate and add it to 20 mL of distilled water. Stir continuously until completely dissolved. The specific conditions are: stirring speed 700 r / min and stirring time 3 h.

[0134] (2) Weigh 600 mg of poloxamer 407 and add it to the solution prepared in step (1). Stir continuously until a homogeneous state is obtained to obtain a gel matrix with a mass ratio of 1:2.

[0135] (3) Weigh 900 mg of poloxamer 407 and add it to the solution prepared in step (1). Stir continuously until a homogeneous state is obtained to obtain a gel matrix with a mass ratio of 1:3.

[0136] (4) Weigh 1500mg of poloxamer 407 and add it to the solution prepared in step (1). Stir continuously until a homogeneous state is obtained to obtain a gel matrix with a mass ratio of 1:5.

[0137] 4.1.2 Gel matrix viscosity test and results

[0138] The viscosity test methods for different proportions of gel matrix are as follows:

[0139] (1) Adjust the Shanghai Fangrui LV-SSR viscometer to the horizontal working state, select rotor No. 25 and screw it into the connecting screw, take 15mL of gel matrix with a mass ratio of 1:2 into a 50mL beaker, and measure and record the data;

[0140] (2) Adjust the Shanghai Fangrui LV-SSR viscometer to the horizontal working state, select rotor No. 25 and screw it into the connecting screw, take 15mL of gel matrix with a mass ratio of 1:3 into a 50mL beaker, and measure and record the data;

[0141] (3) Adjust the Shanghai Fangrui LV-SSR viscometer to the horizontal working state, select rotor No. 25 and screw it into the connecting screw, take 15mL of gel matrix with a mass ratio of 1:5 into a 50mL beaker, and measure and record the data.

[0142] The results are shown in Table 7.

[0143] Table 7 Absolute viscosity of gel matrix at different proportions

[0144]

[0145] As shown in Table 7, the viscosity of the gel matrix gradually increases with the change of the SA:P407 ratio, and the viscosity of the ungelled matrix is ​​relatively low when the mass ratio is 1:2. Subsequently, two schemes, 1:3 and 1:5, were selected as gel matrices for drug loading, and the optimal ratio was further screened by encapsulation efficiency.

[0146] 4.2 Preparation and Encapsulation Efficiency Test of Self-Assembled Nanogels

[0147] 4.2.1 The preparation steps of the self-assembled nanogel are as follows:

[0148] (1) Dissolve 160 mg RHE and 80 mg HON in 10 mL of 0.05 M NaOH solution to obtain solution A;

[0149] (2) Dissolve 300 mg of polyvinyl alcohol in 10 mL of 0.05 M hydrochloric acid solution to obtain solution B;

[0150] (3) Add solution B to solution A under the following conditions: sonication time of 3 min, titration speed of 0.75 mL / min, stirring speed of 700 r / min, stirring for 6 h to obtain solution C;

[0151] (4) Weigh 300 mg of sodium alginate and add it to solution C obtained in step (3). Stir continuously until completely dissolved, then add 900 mg of poloxamer 407 and stir continuously until homogeneous to obtain a self-assembled nanogel agent based on natural active ingredients encapsulated in a gel matrix with a mass ratio of 1:3. The specific conditions are: stirring speed 300 r / min and stirring time 12 h;

[0152] (5) Weigh 300 mg of sodium alginate and add it to solution C obtained in step (3). Stir continuously until completely dissolved, then add 1500 mg of poloxamer 407. Continue stirring until homogeneous to obtain a 1:5 gel matrix-encapsulated self-assembled nanogel agent. The specific conditions are: stirring speed 300 r / min, stirring time 12 h;

[0153] (6) Accurately weigh 1g of the self-assembled nanogel based on natural active ingredients, disperse it in 20mL of methanol solution, shake it, and then sonicate it for 3min to disrupt the gel system. Then, take 1mL of the suspension, dilute it, and perform analysis to calculate the drug content in the gel. The encapsulation efficiency was tested using HPLC (detection conditions: mobile phase: A phase 0.1% phosphoric acid solution : B phase methanol = 17 : 83 (volume ratio); detection wavelength: 254nm; injection volume: 20μL; flow rate: 1.0mL / min). Encapsulation efficiency = (drug encapsulated in the formulation / total amount of encapsulated and unencapsulated drug in the formulation) × 100%.

[0154] 4.2.1 Results of encapsulation efficiency of self-assembled nanogels

[0155] When self-assembled nanoparticles are introduced into a gel matrix, the viscosity of the gel matrix is ​​too high when the mass ratio is 1:5, making it difficult for the nanoparticles to be uniformly dispersed in the gel matrix. Table 8 shows the encapsulation efficiency when the mass ratio of the gel matrix is ​​1:3. The final average encapsulation efficiency is 88.74±1.10%, which meets the target of more than 80% of the expected encapsulation efficiency. Moreover, the differences between the groups are small, indicating that the gel matrix encapsulates the nanoparticles relatively completely.

[0156] In summary, a 1:3 mass ratio of sodium alginate and poloxamer was selected for encapsulating self-assembled nanoparticles, which were then used in the preparation of self-assembled nanogels based on natural active ingredients and subsequently tested for sustained-release performance, antibacterial properties, and anti-inflammatory effects.

[0157] Table 8 Encapsulation efficiency of self-assembled nanoparticles

[0158]

[0159] Experimental Example 1: Preparation and sustained-release performance testing of self-assembled nanoparticles and self-assembled nanogels

[0160] 1.1 The preparation steps of the self-assembled nanogel assembly are as follows:

[0161] (1) Dissolve 160 mg of rhein and 80 mg of magnolol in 10 mL of 0.05 M NaOH solution to obtain solution A;

[0162] (2) Dissolve 300 mg of polyvinyl alcohol in 10 mL of 0.05 M hydrochloric acid solution to obtain solution B;

[0163] (3) Add solution B from step (2) to solution A from step (1) at a titration rate of 0.75 mL / min with a stirring speed of 700 r / min. After stirring for 6 h, sonicate at 160 W for 3 min to obtain a self-assembled nanoparticle solution.

[0164] (4) Weigh 300mg sodium alginate and add it to the self-assembled nano solution system obtained in step (3). Stir continuously until completely dissolved, add 900mg poloxamer 407, and stir continuously for 12h at 300r / min to obtain a compound self-assembled nano gel based on natural active ingredients. Adjust the volume to 20mL.

[0165] 1.2 The preparation steps of self-assembled nanoparticle groups are as follows:

[0166] (1) Dissolve 160 mg of rhein and 80 mg of magnolol in 10 mL of 0.05 M NaOH solution to obtain solution A;

[0167] (2) Dissolve 300 mg of polyvinyl alcohol in 10 mL of 0.05 M hydrochloric acid solution to obtain solution B;

[0168] (3) Add solution B from step (2) to solution A from step (1) at a titration rate of 0.75 mL / min with a stirring speed of 700 r / min. After stirring for 6 h, sonicate at 160 W for 3 min to obtain a self-assembled nanoparticle solution.

[0169] 1.3 The specific steps of the in vitro cumulative release test are as follows:

[0170] (1) The self-assembled nanogel group and the self-assembled nanoparticle group were added to the dialysis bag (MD: 3500) and dialyzed in 500 mL PBS solution (receiving solution) under the following conditions: 38±0.5℃ and 60 rpm r / min.

[0171] (2) Take 3 mL of sample from the receiving solution and add 3 mL of fresh receiving solution to maintain a constant volume. The test sample was repeated three times, and the concentration of the sample was measured by HPLC to plot the cumulative release curve.

[0172] 1.4 Results of in vitro cumulative release test

[0173] The in vitro release performance of each group is as follows Figure 3 As shown, the self-assembled nanoparticles were rapidly released within 4 hours, while after the introduction of the gel matrix, the drug was released slowly and continuously, taking 48 hours to be fully released, significantly prolonging the duration of drug action. Due to their advantages in particle size and surface area, nanoparticles are released rapidly in the medium. The effective encapsulation of the gel matrix allows for continuous release of the nanoparticles in the medium, prolonging the drug's residence time at the application site, effectively reducing the frequency of administration, and ensuring that the nanoparticles exert their full and sustained therapeutic effect.

[0174] Example 2: This invention is based on the application of a compound self-assembled nanogel of natural active ingredients in the treatment of bacterial infections, specifically including extracellular antibacterial activity testing and intracellular antibacterial activity testing.

[0175] 2.1 Preparation of each drug group

[0176] 2.1.1 The preparation steps of the self-assembled nanogel assembly (High-RHE / HON Gels) are as follows:

[0177] (1) Dissolve 160 mg of rhein and 80 mg of magnolol in 10 mL of 0.05 M NaOH solution to obtain solution A;

[0178] (2) Dissolve 300 mg of polyvinyl alcohol in 10 mL of 0.05 M hydrochloric acid solution to obtain solution B;

[0179] (3) Add solution B from step (2) to solution A from step (1) at a titration rate of 0.75 mL / min with a stirring speed of 700 r / min. After stirring for 6 h, sonicate at 160 W for 3 min to obtain a self-assembled nanoparticle solution.

[0180] (4) Weigh 300mg sodium alginate and add it to the self-assembled nano solution system obtained in step (3). Stir continuously until completely dissolved, add 900mg poloxamer 407, and stir continuously for 12h at 300r / min to obtain a compound self-assembled nano gel based on natural active ingredients. Adjust the volume to 20mL.

[0181] 2.1.2 The preparation steps of self-assembled nanoparticle groups (RHE / HON NPs) are as follows:

[0182] (1) Dissolve 160 mg of rhein and 80 mg of magnolol in 10 mL of 0.05 M NaOH solution to obtain solution A;

[0183] (2) Dissolve 300 mg of polyvinyl alcohol in 10 mL of 0.05 M hydrochloric acid solution to obtain solution B;

[0184] (3) Add solution B from step (2) to solution A from step (1) at a titration rate of 0.75 mL / min with a stirring speed of 700 r / min. After stirring for 6 h, use a 600 W ultrasonic disruptor with 40% power for 3 min to ultrasonically treat the solution; thus obtaining the self-assembled nanoparticle solution.

[0185] 2.1.3 The preparation steps of the physical mixture (Mix) are as follows:

[0186] (1) Dissolve 160 mg RHE and 80 mg HON in 10 mL DMSO solution to obtain solution A;

[0187] (2) Add solution A from step (1) to 10 mL of distilled water to obtain a physical mixture.

[0188] 2.2 Extracellular antibacterial activity test of natural active drugs

[0189] 2.2.1 Materials and Methods

[0190] (1) Drugs: The three groups of natural drugs and their self-assembly systems prepared in 2.1.

[0191] (2) Experimental materials: LB broth, Staphylococcus aureus (ATCC29213).

[0192] (3) Instruments: alcohol lamp, 96-well plate, turbidimeter.

[0193] (4) Test methods:

[0194] Prepare a 96-well sterile culture plate. First, add 100 μL of culture medium to each row. Add 100 μL of the completely dissolved drug monomer to the first row and mix thoroughly with a pipette. Then transfer 100 μL to the second row, mix, and then transfer 100 μL to the third row, and so on until the eighth row. Discard the 100 μL. Then dilute to 1.5 × 10⁻⁶. 6 Add 100 μL of CFU / mL bacterial suspension to rows 1-10. At this point, the final drug concentrations in the culture plate from rows 1-8 are 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, and 2 μg / mL, respectively. Incubate at 37℃ for 24-36 hours. Column 11 is the positive control group (with added culture medium and bacterial suspension), and column 12 is the blank control group (culture medium only). The experiment is repeated 3 times. Judgment criteria: The minimum concentration in the clear wells is defined as the minimum inhibition concentration (MIC). For example, when all wells in row 1 containing the drug are confluent with bacteria, the MIC is greater than 256 μg / mL; when no bacteria grow in the wells in row 8 containing the drug, the MIC is less than 2 μg / mL. If skipped wells appear on the 96-well plate, the operation is incorrect and the experiment needs to be repeated.

[0195] 2.2.2 Results

[0196] The in vitro antibacterial effects of each group were evaluated using the micro-broth method, and the results are shown in Table 9. The MIC of the physical mixture group was 16 μg / mL. The self-assembled nanoparticle group further enhanced the antibacterial effect with a MIC of 8 μg / mL. The antibacterial effect of the self-assembled nanogel was similar to that of the self-assembled nanoparticles. The encapsulation of the gel matrix did not affect the antibacterial effect of the system.

[0197] Table 9 Antibacterial activity of each group

[0198]

[0199] 2.3 Intracellular antibacterial activity test of natural active drugs

[0200] 2.3.1 Materials and Methods

[0201] (1) Drugs: 2.1.1 self-assembled nanogel group (RHE / HON Gels), 2.1.3 physical mixture group (Mix), blank group (KB) without antibacterial drugs.

[0202] (2) Experimental materials: LB broth, Staphylococcus aureus (ATCC29213), gentamicin, MAC-T cells, Triton.

[0203] (3) Instruments: alcohol lamp, 96-well plate, turbidimeter, sterile ultra-clean workbench, constant temperature and humidity incubator, inverted microscope, pancreatic enzyme.

[0204] (4) Test methods:

[0205] MAC-T cells were observed under an inverted microscope. The experiment began when the cells reached 80%-90% confluence. The procedure was performed in a sterile laminar flow hood. The old culture medium was discarded, and the cells were washed twice with PBS at 37°C. 1 mL of trypsin was added, and the cells were digested at 37°C for 3 minutes. Then, 3 mL of complete culture medium was added to stop the digestion. After collecting the cells, the supernatant was removed by centrifugation, and the cells were resuspended and seeded into six-well plates. The cells were then cultured in a 37°C constant temperature and humidity incubator. 1 mL of 1.5 × 10⁶ cells / well was added to each well of the six-well plate. 6 CFU / mL bacterial suspension. After incubating in an incubator for 2 hours to allow bacterial invasion, wash with PBS, add 1 mL of 200 μg / mL gentamicin to kill extracellular bacteria, and allow the drug to act for 24 hours (see Table 10 for experimental drug groups; drug concentrations are expressed as RHE concentrations). Finally, lyse the cells with a solution containing 0.01 wt% Triton, then streak the cells onto plates for bacterial culture, and observe the colony count after 24 hours. Each drug group was performed in triplicate.

[0206] Table 10 Dosage regimens for each group in the intracellular antibacterial test

[0207]

[0208] 2.2.3 Results

[0209] like Figure 4 As shown, after 24 hours of drug treatment, the number of intracellular Staphylococcus aureus in each group decreased to varying degrees compared with the control group. The inhibitory efficiency of RHE / HON Gels against Staphylococcus aureus was significantly higher than that of the Mix group. The self-assembled nanosystem obtained by nanoprecipitation method further increased the antibacterial efficacy.

[0210] As shown in Table 11, the RHE / HON Gels group exhibited a 97.66% inhibition rate against intracellular Staphylococcus aureus, which was higher than that of the Mix group. The nano-sized drug facilitates its entry into cells, thereby clearing Staphylococcus aureus infection. This indicates that self-assembled nanogels can effectively inhibit intracellular Staphylococcus aureus infection.

[0211] Therefore, this invention further enhances the antibacterial efficacy based on a compound self-assembled nanogel of natural active ingredients, which can effectively eliminate intracellular and extracellular Staphylococcus aureus infections.

[0212] Table 11 Inhibition efficiency of each group against intracellular bacterial infection

[0213]

[0214] Experimental Example 3

[0215] The application of self-assembled nanogels based on natural active ingredients in alleviating or modulating lipopolysaccharide-mediated inflammatory diseases includes the following steps:

[0216] 3.1 Preparation of each group of drugs

[0217] 3.1.1 The preparation steps of rhein monotherapy (RHE) are as follows:

[0218] (1) Dissolve 160 mg RHE in 10 mL DMSO (dimethyl sulfoxide) solution to obtain solution A;

[0219] (2) Add solution A from step (1) to 10 mL of distilled water to obtain rhein single drug group.

[0220] 3.1.2 The preparation steps of honokiol monotherapy (HON) are as follows:

[0221] (1) Dissolve 80 mg HON in 10 mL DMSO solution to obtain solution A;

[0222] (2) Add solution A from step (1) to 10 mL of distilled water to obtain the magnolol single drug group.

[0223] 3.2 Establishment of a macrophage inflammation model and immunomodulatory experiments:

[0224] 3.2.1 Materials and Methods

[0225] (1) Drugs: self-assembled nanogel group prepared in 2.1.1, self-assembled nanoparticle group prepared in 2.1.2, physical mixture group prepared in 2.1.3, rhein monotherapy group prepared in 3.1.1, and honokiol monotherapy group prepared in 3.1.2.

[0226] (2) Experimental materials: macrophages, lipopolysaccharide, interleukin-4, culture medium.

[0227] (3) Instruments: alcohol lamp, 6-well plate, sterile ultra-clean workbench, constant temperature and humidity incubator, inverted microscope, sterile cell scraper.

[0228] Table 12 Immunomodulatory drug administration

[0229]

[0230] (4) Test methods:

[0231] Establishment of the inflammation model: LPS induced macrophage M1 polarization, and IL-4 induced macrophage M2 polarization. RAW 264.7 cells were observed under an inverted microscope. When the cells reached 80%-90% confluence, they were passaged. In a sterile laminar flow hood, the old culture medium was discarded, and the cells were washed twice with PBS at 37°C. Then, 4 mL of fresh complete culture medium was added. The cells were scraped off the culture surface with a sterile cell scraper, collected, centrifuged to remove the supernatant, resuspended, and seeded into 6-well plates. The cells were then cultured in a 37°C constant temperature and humidity incubator. When the cells reached 80%-90% confluence, inducing drugs (see Table 12 for the combination of inducing drugs) were added, and the cells were cultured in a 37°C constant temperature and humidity incubator for 12 h.

[0232] Table 13 Macrophage phenotype induction doses

[0233]

[0234] Immunomodulation: Cells were induced to develop the M1 or M2 phenotype by adding an inducer for 12 hours, followed by a 12-hour treatment with the drug combination. The dosing regimen is shown in Table 12 (- indicates the addition of complete culture medium). After drug administration, the culture medium was discarded, and cells were washed three times with PBS. 200 μL of protein lysis buffer was added to each well and incubated for 5 minutes. Cells were then scraped clean from each well and placed in 1.5 mL centrifuge tubes, centrifuged at 12,000 rpm for 10 minutes at 4°C, and the supernatant was collected for protein assay. The protein concentration was normalized to 7 mg / mL using a BCA kit. Subsequently, the proteins from each group were used for ELISA detection.

[0235] 3.2.3 Test Results

[0236] Macrophages influence the development and progression of inflammation, and their phenotype plays a crucial role in immune regulation. In the early stages of inflammation, macrophages undergo classical activation to the M1 phenotype, simultaneously secreting large amounts of pro-inflammatory cytokines to participate in pathogen removal. In the later stages of inflammation, M2 phenotype macrophages can suppress inflammation by inducing high endocytic clearance of mannosylated ligands and reducing pro-inflammatory cytokine secretion, thus promoting tissue remodeling. An ideal breast infusion agent should possess antibacterial capabilities to prevent excessive activation of M1 macrophages and regulate macrophage shift towards the M2 phenotype, thereby synergistically promoting mastitis healing. A cellular inflammation model was established using macrophages. The process of macrophage polarization towards the M1 phenotype was quantified by the levels of M1 cell markers CD86 and iNOS; the process was also quantified by the level of the M2 cell marker CD206.

[0237] like Figure 5 As shown, the levels of the M1 phenotypic marker CD86 and the pro-inflammatory factor iNOS were significantly increased under LPS stimulation, while the level of the M2 phenotypic marker CD206 decreased. In the presence of LPS, co-treatment with various drugs reduced the expression of M1 markers and increased the level of M2 markers. The RHE / HON Gels group showed the strongest ability to regulate macrophage polarization towards the M2 phenotype. Therefore, the self-assembled nanogels prepared in this study can regulate immune function by affecting M1 / M2 macrophage polarization, inhibiting the expression of pro-inflammatory genes, and promoting the expression of anti-inflammatory genes.

[0238] Test Example 4

[0239] The application of self-assembled nanogels based on natural active ingredients in a mouse bacterial infection model includes the following steps:

[0240] 4.1 Preparation of each group of drugs

[0241] 4.1.1 The preparation steps of the low-dose gel group (Low-RHE / HON Gels) are as follows:

[0242] (1) Dissolve 80 mg of rhein and 40 mg of magnolol in 10 mL of 0.05 M NaOH solution to obtain solution A;

[0243] (2) Dissolve 300 mg of polyvinyl alcohol in 10 mL of 0.05 M hydrochloric acid solution to obtain solution B;

[0244] (3) Add solution B from step (2) to solution A from step (1) at a titration rate of 0.75 mL / min with a stirring speed of 700 r / min. After stirring for 6 h, sonicate at 160 W for 3 min to obtain a self-assembled nanoparticle solution.

[0245] (4) Weigh 300 mg of sodium alginate and add it to the self-assembled nanosolution system obtained in step (3). Stir continuously until completely dissolved, add 900 mg of poloxamer 407, and stir continuously for 12 h at 300 r / min to obtain a compound self-assembled nanogel based on natural active ingredients. Adjust the volume to 20 mL. 4.2 Materials and Methods

[0246] (1) Drugs: self-assembled nanogel group (High-RHE / HON Gels group) prepared in 2.1.1, self-assembled nanoparticle group (RHE / HON NPs group) prepared in 2.1.2, physical mixing group (Mix group) prepared in 2.1.3, and low-dose gel group (Low-RHE / HON Gels) prepared in 4.1.1.

[0247] (2) Experimental animals: 30 SPF-grade Kunming mice, housed separately in the Experimental Animal Center of Huazhong Agricultural University.

[0248] (3) Experimental materials: Staphylococcus aureus (ATCC29213), alcohol, electronic balance, paraformaldehyde fixative.

[0249] (4) Instruments: alcohol lamp, 6-well plate, sterile ultra-clean workbench, constant temperature and humidity incubator, inverted microscope, tissue grinder.

[0250] (5) Test methods:

[0251] Establishment of a bacterial inflammation model in mice: The experiment was conducted under strict adherence to animal welfare principles, ensuring suitable temperature, sufficient feed, and free access to water. Thirty healthy lactating female mice were randomly divided into five groups (C, M1, M2, M3, and M4), with six mice in each group. Two hours before inoculation, the lactating mice were separated from their pups, anesthetized with ether, and then immobilized. The skin of the fourth pair of mammary glands was disinfected with 75% (v / v) alcohol. The nipples were then gently grasped with forceps, and a microsyringe was inserted along the nipple duct to a depth of approximately 2-3 mm. While keeping the syringe still, 1.5 × 10⁻⁶ ml of bacterial solution was slowly injected. 7 CFU / mL.

[0252] Mouse Treatment Experiment: The experiment was conducted under strict adherence to animal welfare principles, ensuring suitable temperature, sufficient feed, and free access to water. In the control group, each female mouse received an injection of 0.1 mL of sterile saline into each side of the fourth pair of mammary glands. The experimental groups M1, M2, M3, M4, and M5 represented the model group, the physical mixture group (Mix group), the self-assembled nanoparticle group (RHE / HON NPs group), the low-dose gel group (Low-RHE / HON Gels group), and the high-dose gel group (High-RHE / HON Gels group), respectively. Specifically, lactating female mice were separated from their pups 2 hours before inoculation, anesthetized with ether, and then immobilized. The skin of the fourth pair of mammary glands was disinfected with 75% alcohol. The nipples were then gently grasped with forceps, and a microsyringe was inserted along the nipple duct to a depth of approximately 2-3 mm. The syringe was kept stationary while the drug was slowly injected. Injections were administered to the mammary region at 24-hour intervals, for a total of 7 injections. Clinical and mammary gland symptoms in mice were recorded daily during the administration period. Three mice from each group were sacrificed three days after the last administration, and the remaining mice were sacrificed seven days later. One side of the mammary gland tissue was fixed in 10% (v / v) formaldehyde solution for later preparation of histopathological sections. The other side was frozen at -80°C for later bacterial counting and cytokine assays.

[0253] 4.3 Results

[0254] 4.3.1 Therapeutic effects of self-assembled nanogels composed of natural active molecules

[0255] Breast tissue lesions and bacterial load, as shown Figure 6 As shown. Three days after administration, tissue lesions were alleviated. The Mix group, RHE / HON NPs group, Low-RHE / HON Gels group, and High-RHE / HON Gels group all showed significant differences compared to the model group, with the High-RHE / HON Gels group exhibiting the strongest clearance ability of Staphylococcus aureus in tissues. With increasing treatment duration, the number of bacterial colonies in the Mix group, RHE / HON NPs group, Low-RHE / HON Gels group, and High-RHE / HON Gels mouse tissues decreased to varying degrees. The High-RHE / HON Gels group showed the best treatment effect and the most significant decrease in bacterial colony count, while the physical mixing group showed the worst treatment effect. Figure 7 ).

[0256] 4.3.2 Pathological and Histological Changes

[0257] Breast tissue pathological observation, such as Figure 8As shown. In the model group, the mammary tissue showed an increase in glands and ducts; occasional small necrotic foci, a large amount of cell necrosis, nuclear fragmentation, and eosinophilic material (black arrows); a small number of ductal epithelial cells necrotized and sloughed off (blue arrows); a large amount of connective tissue hyperplasia, increased collagen fibers, accompanied by a large amount of lymphocyte infiltration (green arrows).

[0258] The Mix group had few ducts and acini, but abundant adipocytes and connective tissue; occasional small calcifications were visible (black arrows); a large amount of lymphocyte infiltration was visible (blue arrows); and a small amount of eosinophilic material was visible in the ducts (green arrows).

[0259] The Low-RHE / HON Gels group had fewer ducts and acini, but abundant adipocytes and connective tissue; a large amount of lymphocyte infiltration was observed (blue arrow); a small amount of eosinophilic material was observed in the ducts (black arrow).

[0260] In the High-RHE / HON Gels group, the breast tissue showed extensive ductal dilation (black arrow); a small number of ductal epithelial cells necrotized and sloughed off (blue arrow); large areas of connective tissue hyperplasia, increased collagen fibers, and extensive lymphocyte infiltration (green arrow).

[0261] The RHE / HON NPs group had fewer ducts and acini, but abundant adipocytes and connective tissue; a large amount of lymphocyte infiltration was observed (blue arrow); a small amount of eosinophilic material was observed in the ducts (black arrow).

[0262] 4.3.3 Immunomodulatory effects of self-assembled nanogels composed of natural active molecules

[0263] Bacterial infection leads to the release of inflammatory factors in the mammary gland, thereby causing inflammation. Macrophage phenotype plays an important role in the immune regulation process. In this experiment, the levels of CD86, CD206, iNOS, IL-6, and IL-10 in mouse mammary tissue were detected by ELISA at 3 and 7 days after drug administration. The results are as follows: Figure 9As shown: 3 days after drug administration, compared with the model group, the expression levels of macrophage M1 phenotypic markers CD86 and iNOS in the Mix group, Low-RHE / HON Gels, High-RHE / HON Gels, and RHE / HON NPs group were significantly decreased, and the IL-6 level was also significantly decreased. The expression level of M2 phenotypic marker CD206 was significantly increased, and the IL-10 level was significantly increased in all groups. Among the groups, the expression level of iNOS was lowest in High-RHE / HON Gels, with no significant difference among the other drug groups; among the groups, the expression level of CD86 was lowest in High-RHE / HON Gels, followed by RHE / HON NPs, with no significant difference among the other drug groups; among the groups, the expression level of CD206 was highest in High-RHE / HON Gels, followed by RHE / HON NPs, with significant differences among all drug groups. Among all groups, High-RHE / HON had the lowest IL-6 expression level; among all groups, High-RHE / HON Gels had the highest IL-10 expression level, followed by Low-RHE / HON Gels.

[0264] Seven days after administration, the self-assembled nanogel showed a higher degree of inhibition of the M1 phenotype and promotion of the M2 phenotype. The self-assembled nanogel with excellent antibacterial and anti-inflammatory properties plays a decisive role in the cure of bacterial inflammation. Although the pro-inflammatory behavior is beneficial to inhibiting bacterial proliferation in the early stage of inflammation, it affects the normal function of tissue repair. The self-assembled nanogel with both antibacterial and anti-inflammatory capabilities is more beneficial to the entire inflammation treatment process.

[0265] In summary, based on the optimal formulation of self-assembled nanogels made from natural active ingredients, such as... Figure 10 As shown, it is orange-yellow in color, has good fluidity, and moderate viscosity. This invention is based on the excellent antibacterial and immunomodulatory properties of a compound self-assembled nanogel of natural active ingredients. It can kill surrounding bacteria, regulate the immune microenvironment, and subsequently promote tissue healing through the release of anti-inflammatory cytokines.

[0266] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A compound self-assembled nanogel based on natural active ingredients, characterized in that, The raw materials include, in terms of mass percentage, 0.1-2.5% of natural active molecules, 1.0-8.0% of stabilizers, 2.0-7.0% of gel matrix, and the balance of water; The natural active molecules are composed of a first natural active molecule and a second natural active molecule, and the mass ratio of the first natural active molecule to the second natural active molecule is 2:1; The first natural active molecule is rhein, and the second natural active molecule is honokiol; The stabilizers include at least one of polyvinyl alcohol, poloxamer 188, and polyvinylpyrrolidone; The preparation method of the natural active ingredient compound-based self-assembled nanogel agent includes the following steps: (1) accurately weighing each raw material according to the raw material ratio, dissolving the natural active molecules in a sodium hydroxide solution according to the raw material ratio to obtain solution A; (2) dissolving the stabilizers in a hydrochloric acid solution according to the raw material ratio to obtain solution B; (3) adding the solution B dropwise into the solution A while stirring, continuously stirring for 1-8 h after the dropwise addition is completed, and then ultrasonically preparing self-assembled nanoparticles; (4) adding the self-assembled nanoparticles into the gel matrix and stirring to prepare the natural active ingredient compound-based self-assembled nanogel agent.

2. The compound self-assembled nanogel based on natural active ingredients according to claim 1, characterized in that, The gel matrix includes at least one of sodium alginate and poloxamer 407.

3. The compound self-assembled nanogel based on natural active ingredients according to claim 2, characterized in that, The gel matrix is sodium alginate and poloxamer 407, and the mass ratio of sodium alginate to poloxamer 407 is 1:

3.

4. A method for preparing the self-assembled nanogel based on natural active ingredients according to any one of claims 1-3, characterized in that, The preparation method includes the following steps: (1) accurately weighing each raw material according to the raw material ratio, dissolving the natural active molecules in a sodium hydroxide solution according to the raw material ratio to obtain solution A; (2) dissolving the stabilizers in a hydrochloric acid solution according to the raw material ratio to obtain solution B; (3) adding the solution B dropwise into the solution A while stirring, continuously stirring for 1-8 h after the dropwise addition is completed, and then ultrasonically preparing self-assembled nanoparticles; (4) adding the self-assembled nanoparticles into the gel matrix and stirring to prepare the natural active ingredient compound-based self-assembled nanogel agent.

5. The preparation method according to claim 4, characterized in that, In step (3), the stirring speed is 500-1000 r / min, the dropwise addition speed is 0.5-1 mL / min, the ultrasonic power is 140-200 W, and the ultrasonic time is 1-5 min; In step (4), the stirring speed is 200-1500 r / min, and the stirring time is 5-15 h.

6. The natural active ingredient compound-based self-assembled nanogel agent according to any one of claims 1-3 for use in the preparation of a medicament for treating bacterial infections.

7. The natural active ingredient compound-based self-assembled nanogel agent according to any one of claims 1-3 for use in the preparation of a medicament for reducing or regulating inflammatory diseases caused by bacterial infections.

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