Multifunctional nano-carrier as well as preparation method and application thereof

Abstract

The invention relates to the technical field of nano-carriers, and discloses a multifunctional nano-carrier as well as a preparation method and application thereof. The multifunctional nano-carrier comprises ginsenoside Rb1 and a cationic polymer, and the cationic polymer is one of poly (aminopropyl) biguanide (PAPB), chitosan (CS), polyacrylamide (PAM) or polyethyleneimine (PEI). The preparation method comprises the following steps: dispersing ginsenoside in normal saline, adding a cationic polymer solution, and carrying out ultrasonic treatment on the mixed solution to obtain the multifunctional nano-carrier. According to the multifunctional nano-carrier and the preparation method and application thereof, the multifunctional nano-carrier can specifically target macrophages, in a septicemia-related acute lung injury ALI model, macrophage-mediated Rb1 is promoted to be delivered to an inflammation part, inflammation is relieved, and the anti-inflammatory effect is achieved. The clearance rate of ALI related to blood infection and bacteria in a bacterial pneumonia model can be improved, and inflammation and immune response are relieved; the compound can be used for developing medicines for treating chronic inflammation.

Description

Technical Field

[0001] The present invention relates to the technical field of nano-carriers, and in particular to multifunctional nano-carriers, their preparation methods and applications. Background Art

[0002] Inflammatory diseases (IDs) include various tissue damages and dysfunctions caused by infections, immune disorders, and various physical or chemical factors. In particular, sepsis has had a severe impact globally. The lungs are particularly vulnerable during sepsis, and sepsis-induced acute lung injury (ALI) is one of the main causes of death. Bacterial pneumonia is a common consequence of bacterial infection, with a high incidence rate, and it has a particularly severe impact on vulnerable groups such as the elderly, children, and those with low immunity. Despite significant progress in diagnostic and treatment strategies over the past few decades, including methods for faster and more accurate diagnosis and management of IDs, traditional anti-inflammatory treatments are still hindered by limited efficacy and insufficient targeting. Therefore, the development of novel anti-inflammatory drug delivery systems is extremely urgent.

[0003] Chinese herbal medicines have a long and rich history of thousands of years in treating various diseases. Among medicinal plants, ginseng is particularly well-known for its extensive therapeutic effects, including anti-inflammatory and antioxidant properties. Ginsenoside Rb1 (Rb1) is the main active ingredient of ginseng and has attracted much attention due to its potent anti-inflammatory effects in multiple biological systems such as the central nervous system, digestive system, and cardiovascular system. Studies have shown that Rb1 can inhibit the activation of nuclear factor-κB (NF-κB) and reduce the release of inflammatory mediators, thereby alleviating inflammation. However, challenges related to their bioavailability and stability still exist, and these challenges need to be overcome to successfully apply them clinically.

[0004] To overcome the limitations of Rb1, recent studies have explored strategies to enhance its therapeutic potential. Rb1 contains a hydrophobic triterpenoid steroid aglycone and a hydrophilic glycan molecule, and these two molecules are amphiphilic, which is conducive to self-assembling into nanoparticles in an aqueous environment. Nanonization manufacturing can improve the solubility and stability of drugs, extend the circulation time of drugs, and enhance the ability of drugs to penetrate biological barriers. Previous studies have shown that nano-ginsenosides have higher bioavailability and pharmacodynamic effects compared to traditional formulations. In addition, the therapeutic potential of nano-Rb1 in treating diseases such as hepatitis and arthritis has also been explored. The Guo research group integrated Rb1 with mannose-modified azoaromatic hydrocarbons to create the Rb1@ManAC4A composition, which has triple-targeting efficacy in treating rheumatoid arthritis. However, the anti-inflammatory effect of mannose receptor-targeted nanoparticles is limited. The focus of the research of the present invention is to find a method that can make Rb1 more effective in treating IDs. Summary of the Invention

[0005] The object of the present invention is to provide a multifunctional nanocarrier and its preparation method and application. The multifunctional nanocarrier can specifically target macrophages. Not only in the sepsis-related acute lung injury (ALI) model, it can promote the delivery of Rb1 mediated by macrophages to the inflammatory site, relieve inflammation, but also improve the clearance rate of bacteria in the ALI model related to bloodstream infection and the bacterial pneumonia model, reduce inflammation and immune response; it can be used to develop drugs for treating chronic inflammation.

[0006] To achieve the above object, the present invention provides a multifunctional nanocarrier, which includes ginsenoside Rb1 and a cationic polymer. The cationic polymer is one of polyaminopropyl biguanide (PAPB), chitosan (CS), polyacrylamide (PAM) or polyethyleneimine (PEI).

[0007] Further, negatively charged ginsenoside Rb1 is located in the center, and the cationic polymer is electrostatically adsorbed on the surface of ginsenoside Rb1.

[0008] Further, the molar ratio of ginsenoside Rb1 to the cationic polymer is Rb1:cationic polymer = 5:(1 - 6).

[0009] Even further, the present invention also provides a preparation method of the above multifunctional nanocarrier, and the steps are as follows:

[0010] Disperse ginsenoside Rb1 in physiological saline to obtain a ginsenoside solution, then add the cationic polymer solution, and perform ultrasonic treatment on the mixed solution at 480 W for 5 minutes to obtain the multifunctional nanocarrier.

[0011] Further, the concentration of the ginsenoside solution is 1 mg / mL, and the molar ratio of ginsenoside Rb1 to the cationic polymer is Rb1:cationic polymer = 5:(1 - 6).

[0012] Even further, the present invention also provides the application of the above multifunctional nanocarrier in the preparation of drugs for IDs-targeted anti-inflammatory treatment.

[0013] Even further, the present invention also provides the application of the above multifunctional nanocarrier in the preparation of drugs for treating ALI caused by sepsis.

[0014] Even further, the present invention also provides the application of the above multifunctional nanocarrier in the preparation of drugs for treating bloodstream infection-related ALI caused by bacteria.

[0015] Even further, the present invention also provides the application of the above multifunctional nanocarrier in the preparation of drugs for treating bacterial pneumonia caused by bacteria.

[0016] Even further, the present invention also provides the application of the above multifunctional nanocarrier in the construction of an IDs-targeted anti-inflammatory treatment platform.

[0017] The advantages and positive effects of the multifunctional nanocarrier, its preparation method and application described in the present invention are as follows:

[0018] 1. In the present invention, four cationic polymers with different ammonium groups were first selected to modify the Rb1 self-assembly (abbreviated as GRb1). The endocytosis of the modified GRb1 by macrophages is affected by factors such as surface zeta potential and hydrodynamic particle size. After screening, it was determined that PEI-modified GRb1 (GRb1@PEI) is the most effective formulation. This preparation showed excellent anti-inflammatory effects in a mouse model of LPS-induced sepsis-related ALI and can be used as a multifunctional nanocarrier for the treatment of bloodstream infection-related ALI caused by multidrug-resistant (MDR) Klebsiella pneumoniae (Kp) or pneumonia caused by multidrug-resistant Streptococcus pneumoniae (Sp). The inflammatory indicators of the mice treated with GRb1@PEI were almost completely normalized, indicating a reduced degree of immune cell activation and potential immunomodulatory effects. These findings emphasize the efficacy of cationic modification of GRb1 and highlight its potential as a promising strategy for the treatment of IDs.

[0019] 2. The multifunctional nanocarrier prepared in the present invention has high stability and can specifically target macrophages.

[0020] 3. In a sepsis-related acute lung injury ALI model, the multifunctional nanocarrier promotes macrophage-mediated delivery of Rb1 to the inflammatory site and alleviates inflammation.

[0021] 4. In bloodstream infection-related ALI and bacterial pneumonia models, the multifunctional nanocarrier can improve the bacterial clearance rate, reduce inflammation and immune responses, and thus can be used for the development of drugs for the treatment of chronic inflammation.

[0022] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and examples. Description of the Drawings

[0023] Figure 1 In which, a is the synthesis process of GRb1 and its cationic modifiers in the examples of the present invention, b is the representative TEM images of GRb1 and cationically modified GRb1, c is the DLS size distribution of nano-Rb1 before and after storage at room temperature for 72 hours, d is the ultraviolet-visible absorption spectrum of the nanoparticles, e is the Zeta potential of the nanoparticles before and after storage for 72 hours, f is the change in zeta potential of GRb1 and cationically modified GRb1 after storage for 72 hours; g is the image of nano-Rb1 in solution after preparation and after storage at room temperature for 72 hours;

[0024] Figure 2Where a is the cell viability of MH-S cells cultured with nano-Rb1 at different concentrations (n = 3), b is the cell viability of HUVECs cells cultured with nano-Rb1 at different concentrations (n = 3), c is the fluorescence microscope image of MH-S cells after culturing with nano-Rb1, d is the fluorescence microscope image of HUVECs cells after culturing with nano-Rb1, e is the PI positive cell rate of MH-S cells cultured with nano-Rb1, and f is the PI positive cell rate of HUVECs cells cultured with nano-Rb1;

[0025] Figure 3 Where a is the level of IL-6 in the supernatant of MH-S cells treated with nano-Rb1 (n = 3), and b is the level of TNF-α in the supernatant of MH-S cells treated with nano-Rb1 (n = 3);

[0026] Figure 4 This is the transcriptome sequencing result of GRb1-treated cells in the examples of the present invention, where a and b are KEGG enrichment analyses, and c and d are genomic enrichment analyses;

[0027] Figure 5 Where a is the Western blot analysis result of p-P65, and b is the schematic diagram of the TNF-α signaling pathway;

[0028] Figure 6 Where a is the confocal microscope image showing the cellular uptake of nano-Rb1 in Raw264.7 cells, b is the quantitative analysis of the red fluorescence intensity observed in the confocal image, c is the fluorescence distribution of Cy5.5-labeled nanoparticles in BALB / c mice 1 or 4 hours after injection (n = 4), and d is the targeting efficiency;

[0029] Figure 7In a, it is a schematic diagram of the experimental procedure for the LPS sepsis-related pneumonia model; in b, it is the wet / dry ratio of the mouse lungs indicating the degree of pulmonary edema (n = 5); in c, it is the total number of cells in the bronchoalveolar lavage fluid (BALF) of the mice; in d, it is the protein content in the BALF; in e, it is the level of the inflammatory factor IL-6 in the BALF; in f, it is the level of the inflammatory factor TNF-α in the BALF; in g, it is the level of the inflammatory factor IL-1β in the BALF, (n = 5); in h, it is the level of the inflammatory factor IL-6 in the serum; in i, it is the level of the inflammatory factor TNF-α in the serum; in j, it is the level of the inflammatory factor IL-6 in the lung tissue homogenate; in k, it is the level of the inflammatory factor TNF-α in the lung tissue homogenate; in l, it is the level of the inflammatory factor IL-1β in the lung tissue homogenate, (n = 5); in m, it is the hematoxylin and eosin (H&E) staining of the lung tissue (n = 5); in n, it is a representative fluorescence staining image of the inflammatory factor IL-6 in the lung tissue; in o, it is a representative fluorescence staining image of the inflammatory factor TNF-α in the lung tissue; in p, it is a representative fluorescence staining image of the inflammatory factor IL-1β in the lung tissue;

[0030] Figure 8 In a, it is a schematic diagram of the experimental procedure for the Kp028-induced bloodstream infection-related ALI mouse model; in b, it is the experimental grouping: healthy group (b-1), Kp028 model group (b-2), PMB treatment group (b-3), GRb1-PMB treatment group (b-4), GRb1@PAPB-PMB treatment group (b-5), GRb1@CS-PMB treatment group (b-6), GRb1@PAM-PMB treatment group (b-7), and a representative image of the smear of Kp028 in the lung tissue homogenate of the PEI-PMB treatment group (b-8); in c, it is the total number of cells in the BALF of the mice; in d, it is the protein content in the BALF of the mice; in e, it is the level of IL-6 in the BALF of the mice; in f, it is the level of TNF-α in the BALF of the mice; in g, it is the level of IL-1β in the BALF of the mice, (n = 5); in h, it is the level of the inflammatory factor IL-6 in the lung tissue homogenate of the mice; in i, it is the level of the inflammatory factor TNF-α in the lung tissue homogenate of the mice; in j, it is the level of the inflammatory factor IL-1β in the lung tissue homogenate of the mice, (n = 5); in k, it is the H&E staining of the lung tissue (n = 5); in l, it is the fluorescence staining of the inflammatory factor IL-6 in the lung tissue; in m, it is the fluorescence staining of the inflammatory factor TNF-α in the lung tissue; in n, it is the MPO staining of the lung tissue (n = 5);

[0031] Figure 9In a, it is a schematic diagram of the experimental procedure for the mouse model of Streptococcus pneumoniae (Sp)-induced bacterial pneumonia. In b, it is the experimental grouping: healthy group (b-1), Sp model group (b-2), polymyxin B (PMB) treatment group (b-3), ginsenoside Rb1-PMB treatment group (b-4), GRb1@PAPB-PMB treatment group (b-5), GRb1@CS-PMB treatment group (b-6), GRb1@PAM-PMB treatment group (b-7), and representative images of Sp smears in the lung tissue homogenates of mice in the PEI-PMB treatment group (b-8). In c, it is the total number of cells in the bronchoalveolar lavage fluid (BALF) of mice in each group. In d, it is the protein content in the BALF of mice in each group. In e, it is the interleukin-6 (IL-6) level in the BALF of mice in each group. In f, it is the tumor necrosis factor-α (TNF-α) level in the BALF of mice in each group. In g, it is the IL-1β level in the BALF of mice in each group (n = 5). In h, it is the level of the inflammatory factor IL-6 in the lung tissue homogenate after treatment. In i, it is the level of the inflammatory factor TNF-α in the lung tissue homogenate after treatment. In j, it is the level of the inflammatory factor IL-1β in the lung tissue homogenate after treatment, (n = 5). In k, it is the hematoxylin and eosin (H&E) staining of the lung tissue (n = 5). In l, it is the fluorescence staining of IL-6 in the lung tissue (n = 5). In m, it is the myeloperoxidase (MPO) staining of the lung tissue (n = 5). In n, it is the evaluation of macrophage M1 / M2 infiltration in the lung tissue (n = 5);

[0032] Figure 10 In a, it is the content of red blood cells (RBC) during the blood cell count of mice. In b, it is the content of white blood cells (WBC) during the blood cell count of mice. In c, it is the content of lymphocytes (Lymph) during the blood cell count of mice. In d, it is the content of granulocytes (Gran) during the blood cell count of mice. In e, it is the content of platelets (PLT) during the blood cell count of mice, (n = 5). In f, it is the level of alanine aminotransferase (ALT) during the blood biochemical analysis of mice. In g, it is the level of aspartate aminotransferase (AST) during the blood biochemical analysis of mice. In h, it is the level of blood urea nitrogen (BUN) during the blood biochemical analysis of mice. In i, it is the level of creatinine (CREA) during the blood biochemical analysis of mice. In j, it is the histological images of the heart, liver, spleen, lung, and kidney of mice 7 days after injecting PBS, GRb1, GRb1@PAPB, GRb1@CS, GRb1@PAM, and GRb1@PEI;

[0033] Figure 11 In a, it is the measured values of the particle size and zeta potential of GRb1-PAPB with different molar ratios. In b, it is the measured values of the particle size and zeta potential of GRb1-CS with different molar ratios. In c, it is the measured values of the particle size and zeta potential of GRb1-PAM with different molar ratios. In d, it is the measured values of the particle size and zeta potential of GRb1-PEI with different molar ratios;

[0034] Figure 12In this context, a represents the wet / dry ratio of the lung tissue in the pneumonia model induced by Kp028 after treatment with nano-Rb1, b represents the quantitative analysis of myeloperoxidase (MPO) in the representative immunofluorescence staining images of the lung tissue in the pneumonia model induced by Kp028, c represents the quantitative analysis of M1 macrophages in the representative immunofluorescence staining images of the lung tissue in the pneumonia model induced by Kp028, d represents the quantitative analysis of interleukin-6 (IL-6) in the representative immunofluorescence staining images of the lung tissue in the pneumonia model induced by Kp028, e represents the quantitative analysis of tumor necrosis factor-α (TNF-α) in the representative immunofluorescence staining images of the lung tissue in the pneumonia model induced by Kp028, f represents the quantitative analysis of interleukin-1β (IL-1β) in the representative immunofluorescence staining images of the lung tissue in the pneumonia model induced by Kp028, and the values are expressed as mean ± standard deviation (SD);

[0035] Figure 13 This is the fluorescence staining result (n = 5) of IL-1β and macrophage M1 / M2 subtypes in the lung tissue of the pneumonia model induced by Kp028 in the examples of the present invention. Detailed implementation mode

[0036] The technical solutions of the present invention will be further described below through the accompanying drawings and examples. Unless otherwise defined, the technical terms or scientific terms used in the present invention should have the ordinary meaning understood by those of ordinary skill in the field to which the present invention belongs. Based on the examples in the present invention, all other examples obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. The experimental methods without specific conditions noted in the following examples are usually determined according to national standards. The experimental instruments, equipment, and reagents not indicated in the following examples are all commercially available raw materials.

[0037] Unless otherwise defined or explained, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the methods of the present invention. It should be noted that, without conflict, the examples in the present invention and the features in the examples can be combined with each other.

[0038] Method steps of Example 1

[0039] 1.1 Preparation of nano-Rb1.

[0040] Dissolve 1 mg of Rb1 in 1 ml of physiological saline. Stir the solution thoroughly to evenly disperse Rb1. Subsequently, sonicate for 5 minutes at a power of 480 W to promote the formation of GRb1 nanoparticles.

[0041] Synthesize cation-modified GRb1 using a standardized method.

[0042] First, disperse 1 mg of Rb1 in 1 mL of physiological saline. Then, add 10 μL of cationic polymer solutions with the following concentrations: PAPB (17.1 mg mL -1 ), CS (5.8 mg mL -1 ), PAM (5.1 mg mL -1 ), and PEI (3.9 mg mL -1 ). Then, subject the mixture to ultrasonic treatment at 480 W for 5 minutes.

[0043] Synthesize GRb1 nanoparticles containing Cy5.5.

[0044] Mix Cy5.5-NH2 with GRb1 nanoparticles at a ratio of 1:10. The mixture is sonicated at room temperature and stirred overnight in the dark. Then, transfer the solution to an ultrafiltration tube with a molecular weight cut-off of 3000 Da. After centrifugation at 6000 rpm for 10 minutes, collect the upper layer solution for further use.

[0045] 1.2 Cell culture.

[0046] HUVECs and RAW264.7 macrophages are cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, v / v) and 1% penicillin-streptomycin solution (v / v). MH-S cells are maintained in complete RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, v / v) and 1% penicillin-streptomycin solution (v / v).

[0047] 1.3 Cell viability assessment.

[0048] Seed MH-S, RAW264.7, and HUVEC cells into 96-well plates at a density of 1×10 4 per well. After culturing for 24 hours, treat the cells with different concentrations of nano-Rb1. After culturing for another 24 hours, evaluate cell viability using the CCK-8 assay and measure the absorbance at a wavelength of 450 nm using a microplate reader.

[0049] 1.4 Live / dead cell staining.

[0050] Seed MH-S, RAW264.7, and HUVEC cells into 96-well plates at a density of 1×10 5Inoculate cells at a density of [density value] into 24-well plates. After a 24-hour stationary phase, add nano-Rb1 and culture for another 24 hours. After the culture, co-stain the cells with calcein-AM and propidium iodide (PI). Observe using a fluorescence microscope and quantify the PI positive rate using ImageJ software.

[0051] 1.5 ELISA analysis of cell inflammatory factors.

[0052] Seed MH-S cells into 24-well plates at a density of 1×10[per well density value] per well. After 24 hours, treat the cells with nano-Rb1 at a concentration of 100 μM for 2 hours. Subsequently, stimulate the cells with 100 ng / mL 5 lipopolysaccharide (LPS) for 24 hours. Collect the cell supernatant, centrifuge at 2000 rpm for 5 minutes, take the supernatant and then analyze using an ELISA kit. -1

[0053] 1.6 RNA-Seq and transcriptome analysis.

[0054] Seed RAW264.7 cells into 6-well plates at a density of 1×10[per well density value] per well. After the cells grow stably for 24 hours, add GRb1 and culture for another 2 hours. Subsequently, stimulate the cells with 100 ng / mL 6 LPS for 24 hours. After the stimulation, remove the culture medium and quickly wash the cells once with PBS buffer. When extracting RNA, add 1 mL of TRIzol lysis buffer per 10 square centimeters of cells. Pipette the solution repeatedly with a 1 mL pipette tip until no cell clumps are visible and a non-viscous transparent liquid is formed. Then transfer the entire liquid to a RNase-free cryogenic tube and store at -80 °C. Subsequently, send the samples to Shanghai OE Biotech Co., Ltd. for analysis. -1

[0055] 1.7 In vitro targeting and cell response.

[0056] Seed RAW264.7 cells into 24-well plates at a density of 8×10[per well density value] per well. 5Cells were seeded at a density in a confocal dish and cultured for 24 hours. Subsequently, the cells were treated with nano-Rb1 labeled with Cy5.5 for 2 hours. After aspirating the culture medium, the cells were rinsed twice with PBS. When fixing, a 4% paraformaldehyde solution (PBS) was used to fix the cells at room temperature for 15 - 20 minutes. Then the cells were rinsed 2 - 3 times with PBS. To permeabilize the cells, a 0.5% Triton X-100 solution was used to soak the cells at room temperature for 10 minutes. Then the permeabilization solution was removed and the cells were rinsed again with PBS. The flavin was diluted in PBS at a ratio of 1:1000 and incubated with the cells overnight at 4°C in the dark. After rinsing three times with PBS, a DAPI solution was added and incubated at room temperature for 10 minutes. Finally, the DAPI solution was removed, and after rinsing three times with PBS again, 1 mL of PBS was added and the cells were observed under a confocal microscope.

[0057] 1.8 In vivo targeting.

[0058] Twenty BALB / c mice were randomly divided into 5 groups, with 4 mice in each group. A mouse model of LPS-induced ALI was established by intranasal injection of 20 μL of 2.5 mg / mL -1 LPS solution. One hour after administration, the mice were injected with nano-Rb1 via the tail vein, and each Rb1 was labeled with Cy5.5. In vivo imaging was performed using chemiluminescence imaging technology 1 hour and 4 hours after injection. Fluorescence was quantitatively analyzed to evaluate the targeting efficacy of various preparations.

[0059] 1.9 Animal experiments.

[0060] Male BALB / c mice, 6 - 8 weeks old (25 ± 2 g), were obtained from Vital River Laboratory Animal Technology Co., Ltd. The mice were housed in a controlled environment at a temperature of 25 ± 2°C and a humidity of 55%. All animal welfare and experimental procedures were approved by the Animal Care and Experimental Protocol Committee of Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine and complied with the established laboratory animal guidelines. Before the experiment, all mice were anesthetized by intraperitoneal injection of sodium pentobarbital.

[0061] 1.10 LPS-induced sepsis-related ALI model.

[0062] Sepsis was induced by intraperitoneal injection of 100 μL of 2.5 mg / mL -1 LPS solution. One hour after LPS administration, the mice received intravenous injection of various drug preparations. Twenty-four hours after intravenous injection, the mice were euthanized, and blood, BALF, and lung tissues were collected for treatment evaluation. The lung tissues were fixed, sectioned, stained with H&E and fluorescently stained for inflammatory factors, and then the results were quantified to evaluate the treatment effect.

[0063] 1.11 Kp028-induced bloodstream infection-related ALI model.

[0064] 100 μL of bacterial suspension (Kp028, 10 8 CFU mL -1 ) was injected into the tail vein of mice. One hour after infection, nano - Rb1 coated with PMB was injected via the tail vein. After 24 hours, the mice were sacrificed, and BALF and lung tissues were collected to evaluate the antibacterial and anti - inflammatory effects of the intervention. In addition, lung tissue homogenates were cultured on LB plates to determine the number of bacteria. The lung tissue homogenates were cultured on LB plates to determine the number of bacteria. In addition, histological analysis of lung tissues was performed, including evaluation of macrophage M1 / M2 polarization, myeloperoxidase levels, and inflammatory markers, and the results were quantified.

[0065] 1.12 Establishment of a bacterial pneumonia model induced by Sp.

[0066] A mouse model of bacterial pneumonia induced by Sp was established by intratracheal instillation of 25 μL of bacterial suspension (10 8 CFU mL -1 ). One hour after infection, various drug preparations were injected via the tail vein for therapeutic intervention. After 24 hours of treatment, the mice were sacrificed, and BALF and lung tissue homogenates were collected to evaluate the efficacy. In addition, lung tissue homogenates were cultured on blood agar plates to determine the number of bacteria. Then the lung tissues were fixed, sectioned, and stained with H&E, macrophage M1 / M2 infiltration, myeloperoxidase, and inflammatory cytokine fluorescence, and the results were quantified.

[0067] 1.13 In vivo biocompatibility evaluation.

[0068] To evaluate the in vivo biocompatibility of nano - Rb1, 30 mice were injected with PBS, GRb1, GRb1@PAPB, GRb1@CS, GRb1@PAM, and GRb1@PAI via the tail vein daily for one week. Blood samples were collected after 7 days for standard hematological and biochemical analyses. Major organs were collected for H&E staining to evaluate possible histopathological damage caused by the treatment.

[0069] 1.14 Statistical analysis.

[0070] All data are presented as mean ± standard deviation (SD), and the data are from at least three independent experiments. One - way ANOVA and two - tailed unpaired t - tests were performed using GraphPad Prism 9.0 software to evaluate the statistical significance between sample groups. The significance levels are as follows: ns indicates no significant difference; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001.

[0071] Results and Discussion of Example 2

[0072] 2.1 Characteristics of cation-modified GRb1.

[0073] Four cationic polymers with different ammonium groups: polyaminopropyl biguanide (PAPB), chitosan (CS), polyacrylamide (PAM), and polyethyleneimine (PEI) were respectively prepared into GRb1@PAPB, GRb1@CS, GRb1@PAM, and GRb1@PEI. Since the surface of GRb1 is negatively charged, these cationic polymers can be electrostatically adsorbed onto the self-assembled nanoparticles, as Figure 1 shown in a of. To obtain stable nanoparticles, the optimal molar ratio of Rb1 to the cationic polymer was first investigated. Figure 11 The dynamic light scattering (DLS) and zeta potential results of the nanoparticles formed at different molar ratios are shown. Through comprehensive analysis, the following formulations were selected for further study: GRb1@PAPB (Rb1:PAPB = 5:6), GRb1@CS (Rb1:CS = 5:2), GRb1@PAM (Rb1:PAM = 5:4), and GRb1@PEI (Rb1:PEI = 1:1).

[0074] Transmission electron microscopy (TEM) images show that the GRb1 structure is uniformly dispersed and has a small particle size, approximately 10 nanometers. In contrast, the cation-modified GRb1 is much larger, with particle sizes ranging from 20 nanometers to 30 nanometers. Despite the increase in particle size, the cation-modified GRb1 still maintains a relatively regular circular shape and is uniformly dispersed ( Figure 1 as shown in b of). The results of DLS show that the hydrodynamic diameter of GRb1 is approximately 7 ± 3 nm. The hydrodynamic diameter of the cation-modified GRb1 is larger than that of GRb1. The average diameter of GRb1@PAPB is approximately 98 ± 12 nm, GRb1@CS is 92 ± 33 nm, GRb1@PAM is 92 ± 16 nm, and GRb1@PEI is 169 ± 44 nm ( Figure 1 as shown in c of). These results indicate that the outer polymer modification greatly increases the hydrodynamic diameter of GRb1. After storing at room temperature for 72 hours, the particle size did not change significantly, indicating that the modified GRb1 has good stability.

[0075] To confirm the successful cation modification, ultraviolet-visible absorption spectroscopy and zeta potential measurements were carried out. Compared with the physical addition of the absorption peaks of Rb1 with PAPB and Rb1 with PEI, the absorption peaks of GRb1@PAPB and GRb1@PEI changed significantly, indicating that PAPB and PEI interacted with Rb1 ( Figure 1 as shown in d of). Zeta potential measurement shows that GRb1 is negatively charged, with a zeta potential of approximately -6 ± 1.1 mV ( Figure 1In e). After polymer modification, the surface charge becomes positive. The charge values of GRb1@PAPB are about 21.2±2.1 mV, those of GRb1@CS are about 21.4±2.8 mV, those of GRb1@PAM are about 5.4±0.9 mV, and those of GRb1@PEI are about 14.9±1.1 mV. The change in zeta potential further confirms the successful modification of GRb1 by cationic polymers. Figure 1 Figure f shows the change in zeta potential of nano-Rb1 after storage at room temperature for 72 hours. The zeta potentials of GRb1, GRb1@PAPB, GRb1@CS, GRb1@PAM, and GRb1@PEI are -6.8±1.1 mV, 21.7±2.5 mV, 20.7±2.3 mV, 5.2±1.1 mV, and 14.8±1.5 mV, respectively. These values show no obvious differences compared with the newly prepared samples, indicating that nano-Rb1 maintains good stability. Figure 1 Figure g shows the images of nano-Rb1 in solution immediately after preparation and after storage at room temperature for 72 hours. The solution remains clear and transparent after 72 hours of storage, further confirming the stability of the nanoparticles. These results indicate that GRb1 nanoparticles modified with four different cationic polymers were successfully prepared using a simple self-assembly method, laying a solid foundation for further research on how cationic modification affects the targeted anti-inflammatory effect of Rb1.

[0076] 2.2 Biocompatibility and anti-inflammatory properties of nano-Rb1.

[0077] To evaluate the in vitro safety of these materials, cell viability assays were performed using the CCK-8 assay and live / dead staining. The cytotoxicity of nano-Rb1 was evaluated in MH-S mouse alveolar macrophages, Raw264.7 macrophages, and human umbilical vein endothelial cells (HUVECs). As Figure 2 shown in Figures a-b, the survival rates of cells treated with GRb1, GRb1@CS, GRb1@PAM, and GRb1@PEI remained above 85% at concentrations up to 200 μM. In contrast, GRb1@PAPB was cytotoxic to all three cell types at a concentration of 200 μM. Specifically, the survival rate of MH-S cells decreased by about 50%, that of Raw264.7 cells decreased by about 70%, and that of HUVEC cells decreased by about 40%. Based on these findings, a safe concentration range was determined for subsequent experiments. At a GRb1 concentration of 100 μM, the in vitro biocompatibility of nano-Rb1 was further evaluated using live / dead staining ( Figure 2In Figs. c-d). The results showed that GRb1 and the four cation-modified GRb1s had no significant effect on the cell viability and morphology of MH-S macrophages, Raw264.7 macrophages, or HUVECs. However, GRb1@PAPB showed slight cell death, as indicated by the increased red fluorescence in the images. Figure 2 The PI positive cell rate shown in Figs. e-f further confirmed this observation. In summary, at concentrations up to 200 μM, GRb1, GRb1@CS, GRb1@PAM, and GRb1@PEI showed good biocompatibility with all three cell types. However, GRb1@PAPB showed significant cytotoxicity at 200 μM, which may be due to the disruptive effect of the cationic component on the cell membrane.

[0078] The anti-inflammatory potential of nano-Rb1 was evaluated in MH-S macrophages. As Figure 3 shown in Figs. a, b, in LPS-induced macrophages, GRb1@PEI and GRb1@PAM had a more obvious ability to reduce inflammatory cytokines (including IL-6 and TNF-α) compared with GRb1. Therefore, it was speculated that these cations might enhance the phagocytosis ability of macrophages for nanoparticles through the electrostatic interaction between the cations and the cell membrane. To further investigate the anti-inflammatory mechanism of Rb1, transcriptome sequencing was performed on LPS-stimulated macrophages treated with GRb1 ( Figure 4)。A total of 2779 differentially expressed genes were found between the LPS group and the GRb1 group, including 1232 down-regulated genes and 1547 up-regulated genes. Gene Ontology (GO) enrichment analysis showed that the differentially expressed genes were significantly enriched in various biological processes, such as biological processes related to apoptosis, p53 signaling pathway (such as Aifm2, Atm, Ccnd1, Ccnd3), TNF signaling pathway (such as Ccl20, Creb3l4, Creb5, Csf1, Csf2, Fos, Ifnb1, Nod2), NF-κB signaling pathway (such as Bcl10, Bcl2a1b, Gadd45b, Il1r1, Lck, Myd88, Rela), MAPK signaling pathway (such as Jun, Map3k5, Mknk1, Myd88, Nf1, Ptpn7, Rapgef2, Rasa1, Sos2) and JAK-STAT signaling pathway (such as Crebbp, Csf2, Egf, Ep300, Ifna4, IL11, IL21r, IL7, Myc, Sos2), which were mainly related to the inflammatory response, confirming the anti-inflammatory function of GRb1 at the molecular level. WikiPathway enrichment analysis showed that GRb1 could significantly down-regulate cytokines and inflammatory responses compared with LPS treatment. Notably, the TGF-beta signaling pathway closely related to apoptosis (such as Crebbp, Egf, Ep300, Fos, Nfkb1) (such as Bcl2l11, Bcl2l2, Casp4, Casp8, Irf1, Irf5, Jun, Map2k4, Nfkbie) was significantly inhibited. In addition, a down-regulation of the MAPK pathway was also observed. In addition, the GSEA results showed that genes related to the PPAR signaling pathway (such as Fabp4, Cd36, Scd2), oxidoreductase activity (such as Ptges, Hmox1, Nos2), DNA replication (such as Pold1, Mcm6, Pole2), oxidative stress and redox pathways (such as Anpep, Gclm, G6pdx) were up-regulated, and the down-regulation of the MAPK and NK-κB signaling pathways was confirmed. The results showed that GRb1 mainly exerted its anti-inflammatory effect by inhibiting the TNF signaling pathway, and the TNF signaling pathway was involved in inhibiting key inflammatory pathways, including the NF-κB, mitogen-activated protein kinase (MAPK) and JAK-STAT signaling pathways. Secondly, Rb1 could regulate apoptosis through the TGF-β pathway. In addition, Rb1 could also affect the metabolic process and regulate various biological functions through the PPAR signaling, including immune regulation, cell proliferation and survival. Such as Figure 5As shown in a, Western blot analysis of p-P65 showed that p-P65 in the GRb1, GRb1@PAM, and GRb1@PEI treatment groups was significantly decreased compared with that in the PBS treatment group. p-P65 in the GRb1@PAPB and GRb1@CS groups also decreased slightly, confirming the inhibitory effect of Rb1 on the NF-κB pathway.

[0079] Figure 5 Panel b shows the activation process of the TNF pathway, which involves the synthesis and release of TNF by activated immune cells during inflammation. The released TNF binds to the TNF receptor (TNFR) on the surface of target cells, forming a TNF-TNFR complex. This complex triggers a series of intracellular signal transduction events, including the activation of the NF-κB and MAPK pathways, leading to the expression of inflammatory genes and the production of inflammatory cytokines. Rb1 exerts an anti-inflammatory effect by inhibiting the TNF signaling pathway and related cascades through multiple mechanisms. First, Rb1 can reduce the expression of the TNF gene in activated immune cells, thereby reducing the synthesis and release of TNF. This decreases the extracellular TNF level, which is a key factor in alleviating inflammation. Rb1 can also compete with TNF and bind to the TNF receptor (TNFR) on target cells. By occupying the TNFR binding site, Rb1 prevents TNF from binding to its receptor, thereby blocking the formation of the TNF-TNFR complex. This inhibitory effect disrupts downstream TNF signaling, which is crucial for triggering inflammation. By blocking the formation of the TNF-TNFR complex, Rb1 interferes with the activation of key intracellular pathways such as NF-κB and MAPK, which regulate the expression of inflammatory genes and the production of cytokines. NF-κB is a core regulator of inflammation and, when activated, translocates to the nucleus to promote the transcription of inflammatory genes. Rb1 inhibits the activation of NF-κB by suppressing upstream kinases such as inhibitor of κB kinase β (IKKβ), resulting in a decrease in gene expression. Similarly, Rb1 can also inhibit the MAPK pathway, which controls processes such as cell proliferation, differentiation, and apoptosis. By inhibiting the activation of MAPK, Rb1 can reduce the production of inflammatory cytokines and inhibit cell proliferation, which may indicate a reduced degree of activation of immune cells and an immunomodulatory effect.

[0080] 2.3 Cationically modified GRb1 exhibits targeted specificity in vitro and in vivo.

[0081] To further investigate the mechanism of the anti-inflammatory efficacy of cationically modified GRb1, experiments focusing on cellular uptake were conducted. The materials were labeled with Cy5.5 and co-cultured with Raw264.7 macrophages. Cellular uptake was observed using a confocal microscope ( Figure 6In a). Fluorescence imaging and quantitative analysis showed that GRb1@PAPB exhibited the highest red fluorescence, indicating the largest cell uptake, followed by GRb1@PEI and GRb1@PAM. In contrast, the endocytosis of GRb1@CS was the weakest. Notably, GRb1@PAPB exhibited the highest zeta potential, and its strong electrostatic interaction with the macrophage membrane might be the reason for the enhanced endocytosis. Although the absorption rates of GRb1@PEI and GRb1@PAM were slightly different, the difference was very small, which might be because the smaller particle size of GRb1@PAM partially offset the effect of the potential difference. Although the surface potential of GRb1@CS was similar to that of GRb1@PAPB, its cell uptake was significantly lower. It was speculated that this difference might be caused by the inherent molecular structure of chitosan.

[0082] The relationship between cell absorption and anti-inflammatory efficacy was further explored. Although GRb1@PAPB had the highest absorption rate, its anti-inflammatory effect was not ideal, which might be due to its inherent toxicity. In contrast, the anti-inflammatory effects of GRb1@PAI, GRb1@PAM, and GRb1@CS were consistent with their respective cell absorption levels. These findings indicated that enhancing cell uptake was crucial for optimizing the anti-inflammatory properties of GRb1. To evaluate the inflammatory targeting ability of cationically modified GRb1 in vivo, an LPS-induced ALI mouse model was used. The fluorescence distribution was monitored using an in vivo imaging system (IVIS) ( Figure 6 In c). Statistical analysis of the percentage of fluorescence in the lungs showed that the cationically modified GRb1 formulations targeted the inflamed lungs more effectively than GRb1, and GRb1@PEI and GRb1@PAM had the highest targeting efficiency 1 hour and 4 hours after tail vein injection ( Figure 6 In d). The increased targeting efficiency could be attributed to the increased macrophage uptake. The higher cell uptake enabled macrophages to utilize their innate ability to target the inflammatory site, thus improving the inflammatory targeting.

[0083] In summary, the cationic modification of GRb1 affected the phagocytic ability of macrophages, thus improving its targeting efficiency at the inflammatory site. Both GRb1@PEI and GRb1@PAM increased the macrophage uptake, thus improving the targeting ability in vivo.

[0084] 2.4 Therapeutic effect of nanosized Rb1 on sepsis-related ALI.

[0085] Sepsis-related ALI is a severe systemic inflammation. To evaluate the therapeutic potential of nanosized Rb1 on sepsis-related ALI, an LPS-induced sepsis-related ALI mouse model was established. Blood, bronchoalveolar lavage fluid (BALF), and lung tissues were collected after intravenous injection of the formulation to evaluate its therapeutic effect ( Figure 7a). The lung tissues were processed using the weighing-drying-weighing method, and the wet-to-dry weight ratio was calculated to evaluate pulmonary edema. As Figure 7 shown in b), pulmonary edema was severe in the untreated sepsis group. In contrast, after treatment with GRb1@PEI and GRb1@PAM, pulmonary edema was significantly alleviated, and GRb1@PEI reached a level close to normal. Although GRb1 showed some therapeutic effects, the therapeutic effects of GRb1@PAPB and GRb1@CS were relatively poor, which was consistent with the anti-inflammatory effects observed in vitro ( Figure 3 a, b). Similarly, the cell count in BALF ( Figure 7 c), total protein content ( Figure 7 d), and pro-inflammatory cytokine levels (including IL-6, TNF-α, and IL-1β) ( Figure 7 e-g) also showed the same trend. The levels of inflammatory factors in serum reflected the ability of nano-Rb1 to relieve systemic inflammation. Surprisingly, after treatment with GRb1@PEI, the levels of pro-inflammatory cytokines IL-6 and TNF-α in serum ( Figure 7 h-i) returned to normal. After treatment with GRb1@PEI, a similar decrease was also observed in the inflammatory markers in lung tissue homogenates ( Figure 7 j-l). In summary, compared with GRb1, GRb1@PEI had the best therapeutic effect, followed by GRb1@PAM, while GRb1@PAPB and GRb1@CS had relatively poor therapeutic effects.

[0086] Further histological examination of the lung tissues of septic mice showed typical signs of pulmonary edema, including obvious thickening of the alveolar wall and increased inflammatory cells in the sepsis group treated with PBS. Treatment with GRb1@PEI and GRb1@PAM could significantly reduce lung injury, while GRb1, GRb1@PAPB, and GRb1@CS had no similar effects ( Figure 7 m). Fluorescent staining images of inflammatory markers such as IL-6, TNF-α, and IL-1β in lung tissues ( Figure 7 n-p) further confirmed the reduction of inflammation and inflammatory factor levels after treatment with GRb1@PEI. These results indicate that GRb1@PEI is a promising strategy for effectively relieving systemic inflammation.

[0087] 2.5 Therapeutic potential of cation-modified GRb1 against Kp-induced bloodstream infection-related ALI.

[0088] In addition to inhibiting bacterial growth, controlling cytokine storms is also crucial for improving the prognosis of patients with infection-related endocrine diseases. Given its hydrophobic core and potent anti-inflammatory effects, GRb1@PEI can serve as a multifunctional nanocarrier for hydrophobic drugs such as antibiotics, providing a promising strategy for treating bacterial inflammation. To evaluate its therapeutic potential, nano-Rb1 was used in combination with polymyxin B (PMB) to treat Kp028-induced bloodstream infection-related ALI in a mouse model. Mice were treated via tail vein injection, and bronchoalveolar lavage fluid (BALF) and lung tissues were collected on the following day ( Figure 8 in a). Treatment with PMB or the combination of PMB and nano-Rb1 significantly reduced bacterial growth in lung tissue homogenates. Notably, compared with GRb1-PMB, the cation-modified GRb1-PMB combination had better antibacterial effects, with GRb1@PEI-PMB showing the most significant antibacterial effect ( Figure 8 in b). This enhanced effect may be due to the ability of the cationic polymer to disrupt bacterial cell membranes.

[0089] The therapeutic effect on pulmonary edema was evaluated using the lung wet-to-dry weight ratio. Both GRb1@PEI-PMB and GRb1@PAM-PMB restored pulmonary edema to normal levels, with GRb1@PEI-PMB significantly reducing the total cell count in BALF ( Figure 8 in c), total protein content ( Figure 8 in d), and pro-inflammatory cytokine levels ( Figure 8 in e-g), including IL-6, TNF-α, and IL-1β in both BALF and lung tissue homogenates ( Figure 8 in h-j). Histological analysis, including hematoxylin and eosin (H&E) staining and related immunofluorescence, showed obvious pulmonary edema, thickening of the alveolar wall, and infiltration of inflammatory cells in the lungs infected with Kp028 ( Figure 8 in k). Both GRb1@PAM-PMB and GRb1@PEI-PMB significantly alleviated pulmonary inflammation, with GRb1@PEI-PMB showing the most obvious improvement, bringing lung histology close to normal levels. IL-6 ( Figure 8 in l) and TNF-α ( Figure 8 in m) confirmed the good therapeutic effect of GRb1@PEI-PMB on pulmonary inflammation.

[0090] Myeloperoxidase (MPO) is an enzyme mainly present in neutrophils and plays a key role in generating reactive oxygen species necessary for the inflammatory process. As Figure 8As shown in n, MPO staining of lung tissue showed that neutrophil infiltration in the GRb1@PEI-PMB group was significantly reduced, indicating that GRb1@PEI-PMB could reduce the generation of reactive oxygen species during the inflammatory process. Macrophages are mainly divided into two phenotypes: pro-inflammatory M1 and anti-inflammatory M2. The infiltration of these macrophage subsets is a key indicator of the severity and progression of inflammation. Figure 12 and Figure 13 Fluorescent staining of macrophages was shown. In lung tissue, M1 was labeled red and M2 was labeled green. The results showed that in the GRb1@PEI-PMB group with the most significant efficacy, the dense clusters of M1 pro-inflammatory macrophages shifted to M2 anti-inflammatory macrophages. In summary, GRb1@PEI, as a multifunctional nanocarrier, can treat ALI related to bloodstream infection caused by Kp.

[0091] 2.6 Therapeutic potential of cation-modified GRb1 against Sp-induced bacterial pneumonia.

[0092] Sp is the main cause of pneumonia and other respiratory infections, and thus has become a key target for the study of inflammation-related diseases. To further explore the therapeutic potential of GRb1@PEI, its effect on Sp-induced pneumonia in mice was studied ( Figure 9 in a). Compared with the PBS control group, the bacterial growth rate in the lung homogenates of mice treated with the ampicillin (AMP) preparation was significantly reduced. Notably, the cation-modified GRb1 group showed a synergistic bactericidal effect compared with the AMP group, thus reducing bacterial growth. Among them, the bactericidal effect of GRb1@PEI was the most significant ( Figure 9 in b). In terms of inflammation inhibition, GRb1@PEI-AMP could restore pulmonary edema to normal levels. The total cell count in BALF ( Figure 9 in c) and the total protein content ( Figure 9 in d) further confirmed that GRb1@PEI-AMP was the most effective treatment method. In addition, GRb1@PEI-AMP restored the levels of inflammatory factors IL-6, TNF-α, and IL-1β in BALF and lung tissue homogenates to normal levels ( Figure 9 in e-j).

[0093] Pathological analysis of the model lung tissue ( Figure 9Figure k) shows that the PBS treatment group exhibited significant cell proliferation and structural damage, indicating severe lung injury. Compared with the PBS group, the AMP group and the GRb1-AMP group showed slight improvement, but the GRb1@PAPB-AMP group and the GRb1@CS-AMP group showed little or no improvement. In contrast, the GRb1@PEI-AMP group had the most significant therapeutic effect, followed by GRb1@PAM-AMP. These results confirmed that GRb1@PEI could enhance the efficacy of AMP in the treatment of bacterial pneumonia. Further analysis of key inflammatory markers in lung tissue, including IL-6, TNF-α, and IL-1β, as well as MPO staining and infiltration of M1 / M2 macrophages, all confirmed the inhibitory effect of GRb1@PEI-AMP on the inflammatory storm caused by bacterial infection ( Figure 9 Figures l-n). In summary, cation-modified GRb1 can enhance the anti-inflammatory properties of Rb1, relieve the release of cytokines, and potentially inhibit cytokine-induced immune responses. In addition, it can also serve as a carrier for hydrophobic antibiotics, improving the targeting of antibiotics in antibacterial therapy. These combined effects indicate that GRb1@PEI could be a promising immunoprotective therapeutic agent for the treatment of various bacterial inflammatory diseases.

[0094] 2.7 In vivo biosafety of cation-modified GRb1.

[0095] To evaluate the clinical potential of GRb1@PEI, healthy BALB / c mice were injected with GRb1, GRb1@PAPB, GRb1@CS, GRb1@PAM, and GRb1@PEI via the tail vein at a therapeutic dose. As Figure 10 shown in Figures a-e, no significant statistical differences were found in blood cell counts (including red blood cells, white blood cells, lymphocytes, granulocytes, and platelets) between the control group and the groups treated with GRb1 or its cation-modified forms. Blood biochemical analysis ( Figure 10 Figures f-i) showed no significant changes in alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), or creatinine (CREA) levels between the control group and the treatment groups, indicating no obvious acute liver or kidney toxicity. In addition, histological examination of major organs ( Figure 10 Figure j) showed no significant differences in the H&E staining patterns of the heart, liver, spleen, lung, and kidney. In summary, cation-modified GRb1 (including GRb1@PEI) has good in vivo biosafety and broad clinical application prospects.

[0096] Therefore, the present invention adopts the above-mentioned multifunctional nanocarrier and its preparation method and application. The multifunctional nanocarrier can specifically target macrophages. Not only in the sepsis-related acute lung injury (ALI) model, it can promote the delivery of Rb1 mediated by macrophages to the inflammatory site and relieve inflammation, but also can improve the clearance rate of bacteria in the ALI and bacterial pneumonia models related to bloodstream infection, and reduce inflammation and immune response; it can be used to develop drugs for treating chronic inflammation.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions of the present invention or make equivalent replacements, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Multifunctional nanocarrier, characterized in that: It includes ginsenoside Rb1 and a cationic polymer, and the cationic polymer is one of polyaminopropyl biguanide PAPB, chitosan CS, polyacrylamide PAM or polyethyleneimine PEI.

2. The multifunctional nanocarrier according to claim 1, wherein: Negatively charged ginsenoside Rb1 is located in the center, and the cationic polymer is electrostatically adsorbed on the surface of ginsenoside Rb1.

3. The multifunctional nanocarrier according to claim 1, characterized in that: The molar ratio of ginsenoside Rb1 to the cationic polymer is Rb1:cationic polymer = 5:(1 - 6).

4. The preparation method of the multifunctional nanocarrier according to any one of claims 1-3, characterized in that, The steps are as follows: Disperse ginsenoside Rb1 in physiological saline to obtain a ginsenoside solution, then add the cationic polymer solution, and perform ultrasonic treatment on the mixed solution at 480W for 5 minutes to obtain a multifunctional nanocarrier.

5. The preparation method of the multifunctional nanocarrier according to claim 4, characterized in that: The concentration of the ginsenoside solution is 1mg / mL, and the molar ratio of ginsenoside Rb1 to the cationic polymer is Rb1:cationic polymer = 5:(1 - 6).

6. The application of the multifunctional nanocarrier according to any one of claims 1 - 3 in the preparation of an IDs-targeted anti-inflammatory therapeutic drug.

7. The application of the multifunctional nanocarrier according to any one of claims 1 - 3 in the preparation of a drug for treating ALI caused by sepsis.

8. The application of the multifunctional nanocarrier according to any one of claims 1 - 3 in the preparation of a drug for treating ALI related to bloodstream infection caused by bacteria.

9. The application of the multifunctional nanocarrier according to any one of claims 1 - 3 in the preparation of a drug for treating bacterial pneumonia caused by bacteria.

10. The application of the multifunctional nanocarrier according to any one of claims 1 - 3 in the construction of an IDs-targeted anti-inflammatory therapeutic platform.

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