Multifunctional nanocarriers, methods of making and using the same

Abstract

The present application relates to the technical field of nanocarrier, and discloses a multifunctional nanocarrier, a preparation method and application thereof.The multifunctional nanocarrier comprises ginsenoside Rb1 and a cationic polymer, and the cationic polymer is one of polyaminopropyl biguanide PAPB, chitosan CS, polyacrylamide PAM or polyethylenimine PEI.The preparation method is as follows: the ginsenoside is dispersed in physiological saline, a cationic polymer solution is added, and the mixed solution is ultrasonically treated to obtain the multifunctional nanocarrier.The multifunctional nanocarrier, the preparation method and application thereof can specifically target macrophages, can not only promote the macrophage-mediated delivery of Rb1 to the inflammation site in a sepsis-related acute lung injury (ALI) model, relieve inflammation, but also can improve the bacterial clearance rate in blood infection-related ALI and bacterial pneumonia models, reduce inflammation and immune response, and can be used for developing drugs for treating chronic inflammation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nanocarriers, in particular to a multifunctional nanocarrier and a preparation method and application thereof. BACKGROUND

[0002] Inflammatory diseases (IDs) include various tissue injuries and dysfunctions caused by infections, immune disorders and various physical or chemical factors, among which sepsis has caused serious impact worldwide. The lung is particularly vulnerable during sepsis, and sepsis-induced acute lung injury (ALI) is one of the leading causes of death. Bacterial pneumonia is a common consequence of bacterial infection, with a high incidence, especially affecting vulnerable groups such as the elderly, children and immunocompromised individuals. Despite significant progress in diagnostic and therapeutic strategies over the past few decades, including faster and more accurate methods for diagnosing and managing IDs, traditional anti-inflammatory treatments are still hindered by limited efficacy and insufficient targeting. Therefore, it is urgent to develop new anti-inflammatory drug delivery systems.

[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-regarded for its wide range of therapeutic effects, including anti-inflammatory and antioxidant properties. Ginsenoside Rb1 (Rb1) is the main active ingredient of ginseng, which 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-kappa B (NF-κB) and reduce the release of inflammatory mediators, thereby alleviating inflammation. However, challenges related to their bioavailability and stability still exist, which need to be overcome for their successful application in clinical settings.

[0004] To overcome the limitations of Rb1, recent studies have explored strategies to enhance its therapeutic potential. Rb1 contains a hydrophobic triterpenoid aglycone and a hydrophilic glycan molecule, both of which have amphiphilic properties, facilitating self-assembly into nanoparticles in aqueous environments. Nanofabrication can improve the solubility and stability of drugs, prolong the circulation time of drugs, and enhance the ability of drugs to penetrate biological barriers. Previous studies have shown that nanoginsenosides have higher bioavailability and efficacy compared to traditional formulations. In addition, the therapeutic potential of nano-Rb1 in treating diseases such as hepatitis and arthritis has also been explored. Guo's group integrated Rb1 with a mannose-modified azoarene aromatic hydrocarbon, creating 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 present application is to find a way to make Rb1 more effective in treating IDs. SUMMARY

[0005] The application aims to provide a multifunctional nanocarrier and a preparation method and application thereof, the multifunctional nanocarrier can specifically target macrophages, not only promotes the delivery of Rb1 to an inflammation site mediated by macrophages, relieves inflammation in an acute lung injury (ALI) model related to sepsis, but also can improve the clearance rate of bacteria in an ALI model related to blood infection and bacterial pneumonia caused by bacteria, and reduce inflammation and immune response, and can be used for developing a drug for treating chronic inflammation.

[0006] To achieve the above-mentioned purpose, the application provides a multifunctional nanocarrier, which comprises ginsenoside Rb1 and a cationic polymer, and the cationic polymer is one of polyaminopropyl biguanide (PAPB), chitosan (CS), polyacrylamide (PAM) or polyethyleneimine (PEI).

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

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

[0009] Further, the application also provides a preparation method of the multifunctional nanocarrier, and the steps are as follows:

[0010] The ginsenoside Rb1 is dispersed in physiological saline to obtain a ginsenoside solution, then a cationic polymer solution is added, and the mixed solution is subjected to 480W ultrasonic treatment for 5 minutes to obtain the multifunctional nanocarrier.

[0011] Further, 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).

[0012] Further, the application also provides an application of the multifunctional nanocarrier in preparing an Ids targeted anti-inflammatory treatment drug.

[0013] Further, the application also provides an application of the multifunctional nanocarrier in preparing an ALI drug caused by sepsis.

[0014] Further, the application also provides an application of the multifunctional nanocarrier in preparing an ALI drug related to blood infection caused by bacteria.

[0015] Further, the application also provides an application of the multifunctional nanocarrier in preparing a bacterial pneumonia drug caused by bacteria.

[0016] Further, the application also provides an application of the multifunctional nanocarrier in constructing an Ids targeted anti-inflammatory treatment platform. Further, the application also provides an application of the multifunctional nanocarrier in constructing an Ids targeted anti-inflammatory treatment platform.

[0017] The multifunctional nanocarrier, the preparation method and the application thereof have the advantages and positive effects that:

[0018] 1、In the present application, four kinds of cationic polymers with different ammonium groups are selected to modify Rb1 self-assembled bodies (GRb1 for short). The endocytosis of the modified GRb1 by macrophages is affected by factors such as surface zeta potential and hydration particle size. After screening, it is determined that GRb1 modified by PEI (GRb1@PEI) is the most effective formula. This preparation shows excellent anti-inflammatory effect in a mouse model of sepsis-related ALI induced by LPS, and can be used as a multifunctional nanocarrier for treating blood infection-related ALI caused by multiple drug-resistant (MDR) Klebsiella pneumoniae (Kp) or pneumonia caused by multiple drug-resistant Streptococcus pneumoniae (Sp) infection. The inflammation indicators of mice receiving GRb1@PEI treatment are almost completely normalized, which indicates that the degree of immune cell activation is reduced and has a potential immunomodulatory effect. These findings highlight the efficacy of cationically modified GRb1 and highlight its potential as a promising strategy for treating IDs.

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

[0020] 3、In a sepsis-related acute lung injury (ALI) model, the multifunctional nanocarrier promotes the delivery of Rb1 to the inflammation site mediated by macrophages, and relieves inflammation.

[0021] 4、In blood infection-related ALI and bacterial pneumonia models, the multifunctional nanocarrier can improve the clearance rate of bacteria, reduce inflammation and immune response, and therefore can be used for the development of drugs for treating chronic inflammation.

[0022] The technical solutions of the present application will be further described in detail below through the accompanying drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

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

[0024] Figure 2a Cell viability of MH-S cells treated with different concentrations of nano-Rb1 (n=3), b Cell viability of HUVECs cells treated with different concentrations of nano-Rb1 (n=3), c Fluorescence microscope images of MH-S cells treated with nano-Rb1, d Fluorescence microscope images of HUVECs cells treated with nano-Rb1, e PI positive cell rate of MH-S cells treated with nano-Rb1, f PI positive cell rate of HUVECs cells treated with nano-Rb1;

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

[0026] Figure 4 Transcriptome sequencing results of cells treated with GRb1 in the embodiments of the present application, wherein a and b are KEGG enrichment analysis, c and d are genomic enrichment analysis;

[0027] Figure 5 a Western blot analysis results of p-P65, b schematic diagram of TNF-α signaling pathway;

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

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

[0030] Figure 8 a is the experimental flow diagram of the Kp028 induced blood infection related ALI mouse model, b 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 PEI-PMB treatment group, a representative image of Kp028 smear in lung tissue homogenate (b-8), c is the total number of cells in the BALF of mice, d is the protein content in the BALF of mice, e is the IL-6 level in the BALF of mice, f is the TNF-α level in the BALF of mice, g is the IL-1β level in the BALF of mice (n=5), h is the level of inflammatory factor IL-6 in the lung tissue homogenate of mice, i is the level of inflammatory factor TNF-α in the lung tissue homogenate of mice, j is the level of inflammatory factor IL-1β in the lung tissue homogenate of mice (n=5), k is H&E staining of lung tissue (n=5), l is fluorescent staining of inflammatory factor IL-6 in lung tissue, m is fluorescent staining of inflammatory factor TNF-α in lung tissue, and n is MPO staining of lung tissue (n=5);

[0031] Figure 9Fig. 6a is a schematic diagram of the experimental procedure of the Sp-induced bacterial pneumonia mouse model, Fig. 6b is a representative image of the Sp smear of the lung tissue homogenate of the mice in each group: healthy group (b-1), Sp 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 PEI-PMB treatment group, Fig. 6c is the total number of cells in the BALF of the mice in each group, Fig. 6d is the protein content in the BALF of the mice in each group, Fig. 6e is the IL-6 level in the BALF of the mice in each group, Fig. 6f is the TNF-a level in the BALF of the mice in each group, Fig. 6g is the IL-1b level in the BALF of the mice in each group (n = 5), Fig. 6h is the IL-6 level in the lung tissue homogenate after treatment, Fig. 6i is the TNF-a level in the lung tissue homogenate after treatment, Fig. 6j is the IL-1b level in the lung tissue homogenate after treatment (n = 5), Fig. 6k is the H&E staining of the lung tissue (n = 5), Fig. 6l is the fluorescence staining of IL-6 in the lung tissue (n = 5), Fig. 6m is the MPO staining of the lung tissue (n = 5), and Fig. 6n is the evaluation of the M1 / M2 macrophage infiltration in the lung tissue (n = 5);

[0032] Figure 10 Fig. 7a is the RBC content when the mice were subjected to blood cell counting, Fig. 7b is the WBC content when the mice were subjected to blood cell counting, Fig. 7c is the Lymph content when the mice were subjected to blood cell counting, Fig. 7d is the Gran content when the mice were subjected to blood cell counting, Fig. 7e is the PLT content when the mice were subjected to blood cell counting (n = 5), Fig. 7f is the ALT level when the mice were subjected to blood biochemical analysis, Fig. 7g is the AST level when the mice were subjected to blood biochemical analysis, Fig. 7h is the BUN level when the mice were subjected to blood biochemical analysis, Fig. 7i is the CREA level when the mice were subjected to blood biochemical analysis, Fig. 7j is the histological images of the heart, liver, spleen, lung, and kidney of the mice after injection of PBS, GRb1, GRb1@PAPB, GRb1@CS, GRb1@PAM, and GRb1@PEI for 7 days;

[0033] Figure 11 Fig. 8a is the particle size and zeta potential measurements of GRb1-PAPB at different molar ratios, Fig. 8b is the particle size and zeta potential measurements of GRb1-CS at different molar ratios, Fig. 8c is the particle size and zeta potential measurements of GRb1-PAM at different molar ratios, and Fig. 8d is the particle size and zeta potential measurements of GRb1-PEI at different molar ratios;

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

[0035] Figure 13 Fluorescence staining results of IL-1β and macrophage M1 / M2 subtypes in lung tissue of Kp028-induced pneumonia model in the embodiments of the present application (n=5). DETAILED DESCRIPTION

[0036] The technical solutions of the present application are further described below by means of the accompanying drawings and examples. Unless otherwise defined, the technical terms or scientific terms used in the present application shall have the usual meanings understood by those skilled in the art to which the present application belongs. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts shall fall within the scope of protection of the present application. The experimental methods in the following examples not otherwise specified are generally determined according to national standards. The experimental instruments, equipment and reagents in the following examples not otherwise specified are all commercially available raw materials.

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

[0038] Example 1 Method Steps

[0039] 1.1 Preparation of nano-Rb1.

[0040] Dissolve 1 mg of Rb1 in 1 ml of normal saline. Stir the solution thoroughly to disperse Rb1 uniformly. Then, treat with ultrasonic waves at a power of 480 W for 5 minutes to promote the formation of GRb1 nanoparticles.

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

[0042] First, 1 mg of Rbl was dispersed in 1 mL of normal saline. Then, 10 μL of cationic polymer solution was added, with concentrations as follows: 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 ). The mixture was then subjected to 480 W ultrasonication for 5 minutes.

[0043] Cy5.5-labeled GRb1 nanoparticles were synthesized.

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

[0045] 1.2 Cell culture.

[0046] HUVECs and RAW264.7 macrophages were cultured in high glucose Dulbecco's Modified Eagle Medium (DMEM) medium supplemented with 10% fetal bovine serum (FBS, v / v) and 1% penicillin-streptomycin solution (v / v). MH-S cells were 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] MH-S, RAW264.7, and HUVEC cells were seeded into 96-well plates at a density of 1 x 10 4 After 24 hours of incubation, the cells were treated with different concentrations of nanosized Rbl. After another 24 hours of incubation, cell viability was assessed using the CCK-8 detection method, and the absorbance was measured at 450 nm wavelength using a microplate reader.

[0049] 1.4 Live / dead cell staining.

[0050] MH-S, RAW264.7, and HUVEC cells were seeded into 96-well plates at a density of 1 x 10 5Cells were seeded at a density in 24-well plates. After a 24-hour stabilization period, nano-sized Rb1 was added and the plates were cultured for another 24 hours. At the end of the culture, cells were co-stained with calcein-AM and propidium iodide (PI). Cells were observed using a fluorescence microscope, and the PI positivity rate was quantified using ImageJ software.

[0051] 1.5 ELISA analysis of inflammatory factors in cells.

[0052] MH-S cells were distributed at a rate of 1 × 10⁶ cells per well. 5 Cells were seeded at a density of [missing information - likely a specific concentration] into 24-well plates. After 24 hours, cells were treated with 100 μM nano-sized Rb1 for 2 hours. Subsequently, [missing information - likely a specific concentration] 100 ng / mL [missing information - likely a specific concentration] was administered. -1 Cells were stimulated with lipopolysaccharide (LPS) for 24 hours. The cell supernatant was collected, centrifuged at 2000 rpm for 5 minutes, and then analyzed using an ELISA kit.

[0053] 1.6 RNA-Seq and transcriptome analysis.

[0054] RAW264.7 cells were planted at a density of 1 × 10⁶ cells per well. 6 Cells were seeded at a density of [insert density here] in 6-well plates. After 24 hours of stable cell growth, GRb1 was added, and the cells were cultured for another 2 hours. Subsequently, 100 ng / mL [insert concentration here] was added. -1 Cells were stimulated with LPS for 24 hours. After stimulation, the culture medium was removed, and the cells were quickly washed once with PBS buffer. For RNA extraction, 1 mL of TRIzol lysis buffer was added per 10 square centimeters of cells. The solution was repeatedly transferred using a 1 mL pipette tip until no cell clumps were visible and a non-sticky, clear liquid was formed. The entire liquid was then transferred to an RNase-free, cryogenic tube and stored at -80°C. The samples were subsequently sent to Shanghai Ouyi Biotechnology Co., Ltd. for analysis.

[0055] 1.7 In vitro targeting and cellular response.

[0056] RAW264.7 cells were spaced at 8 × 10⁶ cells per well. 5The density seeded in confocal dishes and incubated for 24 hours. After that, the cells were treated with Cy5.5 labeled nano-Rb1 for 2 hours. After removing the culture medium, the cells were washed twice with PBS. For fixation, 4% paraformaldehyde solution (PBS) was used to fix the cells for 15-20 minutes at room temperature. Then the cells were washed with PBS for 2-3 times. For permeabilization of the cells, 0.5% Triton X-100 solution was used to soak the cells for 10 minutes at room temperature. Then the permeabilization solution was removed and the cells were washed with PBS again. The xanthophyll was diluted in PBS at a ratio of 1:1000 and incubated with the cells overnight at 4°C in the dark. After washing with PBS for three times, DAPI solution was added and incubated for 10 minutes at room temperature. Finally, the DAPI solution was removed and the cells were washed with PBS for three times before 1 mL PBS was added for observation under a confocal microscope.

[0057] 1.8 In vivo targeting.

[0058] Twenty BALB / c mice were randomly divided into 5 groups, 4 mice in each group. The 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. One hour and 4 hours after injection, in vivo imaging was performed using chemiluminescence imaging technology. The fluorescence was quantitatively analyzed to evaluate the targeting efficacy of various formulations.

[0059] 1.9 Animal experiments.

[0060] Six to eight-week-old male BALB / c mice (25 ± 2 g) were obtained from Vantianlihua Experimental Animal Technology Co., Ltd. The mice were housed in a controlled environment with 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 Program Committee of Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, and the established guidelines for experimental animals were followed. 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 formulations. Twenty-four hours after intravenous injection, the mice were euthanized, and blood, BALF, and lung tissue were collected for treatment evaluation. The lung tissue was fixed, sectioned, H&E stained, and inflammatory factor fluorescently stained, and the results were quantified to evaluate the treatment effect.

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

[0064] Mice were injected with 100 pL of bacterial suspension (Kp028, 10 8 CFU mL -1 ). One hour after infection, PMB-coated nanosized Rb1 was injected via the tail vein. After 24 hours, mice were sacrificed and BALF and lung tissue were collected to assess 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. Lung tissue homogenates were cultured on LB plates to determine the number of bacteria. In addition, lung tissue was also subjected to staining histological analysis, including assessment of macrophage M1 / M2 polarization, myeloperoxidase levels and inflammatory markers, and the results were quantified.

[0065] 1.12 Sp-induced bacterial pneumonia model.

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

[0067] 1.13 In vivo biocompatibility assessment.

[0068] To assess the in vivo biocompatibility of nanosized Rb1, 30 mice were injected daily via the tail vein with PBS, GRb1, GRb1@PAPB, GRb1@CS, GRb1@PAM and GRb1@PAI, for a week. After 7 days, blood samples were collected for standard hematological and biochemical analysis. Major organs were collected for H&E staining to assess possible histopathological damage caused by treatment.

[0069] 1.14 Statistical analysis.

[0070] All data are expressed as mean ± standard deviation (SD) and are derived from at least three independent experiments. Statistical significance between sample groups was assessed using one-way ANOVA and two-tailed unpaired t-test using GraphPad Prism 9.0 software. Levels of significance 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 Characterization of cationic modified GRb1.

[0073] Four cationic polymers with different ammonium groups, polyaminopropyl biguanide (PAPB), chitosan (CS), polyacrylamide (PAM) and polyethylenimine (PEI), were made into GRb1@PAPB, GRb1@CS, GRb1@PAM and GRb1@PEI, respectively. Since the surface of GRb1 is negatively charged, these cationic polymers can be electrostatically adsorbed onto the self-assembled nanoparticles, as shown in FIG. 1a. In order to obtain stable nanoparticles, the optimal molar ratio of Rb1 to cationic polymer was first studied. Figure 1 Figure 11 Dynamic light scattering (DLS) and zeta potential results of nanoparticles formed at different molar ratios are shown. After 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 of about 10 nm. In contrast, the cationically modified GRb1 is much larger, with a particle size ranging from 20 nm to 30 nm. Despite the increase in particle size, the cationically modified GRb1 still maintains a relatively regular circular shape and is uniformly dispersed (FIG. 1b). Figure 1 The results of DLS show that the hydrated particle size of GRb1 is about 7±3 nm. The hydrated particle size of the cationically modified GRb1 is larger than that of GRb1, with the average diameter of GRb1@PAPB being about 98±12 nm, GRb1@CS being 92±33 nm, GRb1@PAM being 92±16 nm, and GRb1@PEI being 169±44 nm (FIG. 1c). These results indicate that the outer layer of polymer modification greatly increases the hydrated particle size of GRb1. After 72 hours of storage at room temperature, there is no significant change in particle size, indicating that the modified GRb1 has good stability. Figure 1

[0075] In order to confirm the successful modification of cations, ultraviolet-visible absorption spectroscopy and zeta potential measurements were performed. The absorption peaks of GRb1@PAPB and GRb1@PEI have changed significantly compared to the physical addition of the absorption peaks of Rb1 and PAPB and Rb1 and PEI, indicating that PAPB and PEI have interacted with Rb1 (FIG. 1d). Zeta potential measurements show that GRb1 is negatively charged, with a zeta potential of about -6±1.1 mV (FIG. 1e). Figure 1 Figure 1 ​​​(e). After polymer modification, the surface charge becomes positive. The charge values ​​of GRb1@PAPB are approximately 21.2 ± 2.1 mV, GRb1@CS are approximately 21.4 ± 2.8 mV, GRb1@PAM are approximately 5.4 ± 0.9 mV, and GRb1@PEI are approximately 14.9 ± 1.1 mV. The change in zeta potential further confirms the successful modification of GRb1 by the cationic polymer. Figure 1 Figure f shows the zeta potential change 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 ​​are not significantly different from those of freshly prepared samples, indicating that nano-Rb1 maintains good stability. Figure 1 Image g shows images of nano-sized Rb1 in solution immediately after preparation and after 72 hours at room temperature. The solution remained clear and transparent after 72 hours, further confirming the stability of the nanoparticles. These results demonstrate the successful preparation of GRb1 nanoparticles modified with four different cationic polymers using a simple self-assembly method, laying a solid foundation for further research on how cationic modification affects the targeted anti-inflammatory effects of Rb1.

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

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

[0078] The anti-inflammatory potential of nano-sized Rb1 was evaluated in MH-S macrophages. Figure 3 As shown in Figures a and b, in LPS-induced macrophages, GRb1@PEI and GRb1@PAM, compared to GRb1, exhibited a more significant ability to reduce inflammatory cytokines (including IL-6 and TNF-α). Therefore, it is speculated that these cations may enhance the macrophage's phagocytic capacity for nanoparticles through electrostatic interactions 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 the p53 signaling pathway (e.g., Aifm2, Atm, Ccnd1, Ccnd3) and the TNF signaling pathway (e.g., Ccl20, Creb3l4, Creb5, Csf1, Csf2, Fos, Ifnb1, Nod2) and the NF-κΒ signaling pathway (e.g., Bcl10, Bcl2a1b, Gadd45b, Il1r1, Lck, Myd88, Rela) and the MAPK signaling pathway (e.g., Jun, Map3k5, Mknk1, Myd88, Nf1, Ptpn7, Rapgef2, Rasa1, Sos2) and the JAK-STAT signaling pathway (e.g., Crebbp, Csf2, Egf, Ep300, Ifna4, IL11, IL21r, IL7, Myc, Sos2), which are mainly related to inflammatory responses, confirming the anti-inflammatory function of GRb1 at the molecular level. Wiki pathway enrichment analysis showed that GRb1 can significantly down-regulate cytokines and inflammatory responses compared with LPS treatment. Notably, the TGF-beta signaling pathway (e.g., Crebbp, Egf, Ep300, Fos, Nfkb1) and the MAPK pathway (e.g., Bcl2l11, Bcl2l2, Casp4, Casp8, Irf1, Irf5, Jun, Map2k4, Nfkbie) closely related to apoptosis were significantly inhibited. In addition, down-regulation of the MAPK pathway was also observed. In addition, GSEA results showed up-regulation of genes related to the PPAR signaling pathway (e.g., Fabp4, Cd36, Scd2) and oxidoreductase activity (e.g., Ptges, Hmox1, Nos2) and DNA replication (e.g., Pold1, Mcm6, Pole2) and oxidative stress and redox pathways (e.g., Anpep, Gclm, G6pdx) and confirmed down-regulation of the MAPK and NK-κΒ signaling pathways. The results showed that GRb1 mainly exerts an anti-inflammatory effect by inhibiting the TNF signaling pathway, which is involved in inhibiting key inflammatory pathways, including the NF-κΒ, mitogen-activated protein kinase (MAPK), and JAK-STAT signaling pathways. Second, Rb1 can regulate apoptosis through the TGF-beta pathway. In addition, Rb1 can also affect metabolic processes and regulate various biological functions, including immune regulation, cell proliferation, and survival, through the PPAR signal. Figure 5As shown in Fig. a, Western blot analysis of p-P65 showed that p-P65 was significantly reduced in the GRb1, GRb1@PAM and GRb1@PEI treatment groups compared to the PBS treatment group. p-P65 was also slightly reduced in the GRb1@PAPB and GRb1@CS groups, confirming the inhibitory effect of Rb1 on the NF-κB pathway.

[0079] Figure 5 Fig. b illustrates the activation process of the TNF pathway, which involves activated immune cells synthesizing and releasing TNF during inflammation. The released TNF binds to TNF receptors (TNFR) on the surface of target cells, forming a TNF-TNFR complex. This complex triggers a series of intracellular signaling 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 reduces the level of extracellular TNF, which is a key factor in reducing inflammation. Rb1 can also compete with TNF for binding to TNF receptors (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 inhibition disrupts downstream TNF signaling, which is crucial for initiating 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, it translocates to the nucleus to promote the transcription of inflammatory genes. Rb1 inhibits the activation of NF-κB by inhibiting upstream kinases such as inhibitor of kappa B kinase beta (IKKβ), leading to reduced 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 reduction in the degree of immune cell activation and have an immunomodulatory effect.

[0080] 2.3 Cation-modified GRb1 exhibits target-specificity in vitro and in vivo.

[0081] To further investigate the mechanism of the anti-inflammatory efficacy of cation-modified GRb1, experiments focusing on cellular uptake were conducted. Materials were labeled with Cy5.5 and co-cultured with Raw264.7 macrophages. Confocal microscopy was used to observe cellular uptake Figure 6In the middle a). Fluorescent imaging and quantitative analysis showed that GRb1@PAPB exhibited the highest red fluorescence, indicating the largest amount of cellular 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, which might be the reason for the enhanced endocytosis due to the strong electrostatic interaction with the macrophage membrane. Although the uptake rates of GRb1@PEI and GRb1@PAM were slightly different, the difference was small, which might be because the particle size of GRb1@PAM was smaller, partially offsetting the impact of the potential difference. Although the surface potential of GRb1@CS was similar to that of GRb1@PAPB, its cellular uptake was significantly lower. It was speculated that this difference might be caused by the intrinsic molecular structure of chitosan.

[0082] The relationship between cellular uptake and anti-inflammatory efficacy was further explored. Although GRb1@PAPB had the highest uptake 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 levels of cellular uptake. These findings suggest that enhancing cellular uptake is crucial for optimizing the anti-inflammatory properties of GRb1. To evaluate the inflammation targeting ability of cationic modified GRb1 in vivo, a LPS-induced ALI mouse model was used. The distribution of fluorescence was monitored using an in vivo imaging system (IVIS) to assess the targeting efficiency of each formulation in the lungs of ALI mice Figure 6 In the middle c). Statistical analysis of the percentage of lung fluorescence showed that cationic modified GRb1 formulations were more effective than GRb1 in targeting the inflamed lungs, with GRb1@PEI and GRb1@PAM having the highest targeting efficiency at 1 hour and 4 hours after tail vein injection Figure 6 In the middle d). The increase in targeting efficiency can be attributed to the increased uptake by macrophages. Higher cellular uptake allows macrophages to utilize their innate ability to target inflammatory sites, thereby improving the targeting of inflammation.

[0083] In summary, cationic modification of GRb1 affects the phagocytic ability of macrophages, thereby improving their targeting efficiency at inflammatory sites. Both GRb1@PEI and GRb1@PAM increase the uptake by macrophages, thereby improving their 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 for sepsis-related ALI, a LPS-induced sepsis-related ALI mouse model was established. Blood, bronchoalveolar lavage fluid (BALF), and lung tissue were collected after intravenous injection of the formulation to evaluate its therapeutic effect Figure 7In a) Lung tissues were processed using the weigh-dry-weigh method to calculate the wet-to-dry weight ratio for the assessment of pulmonary edema. As Figure 7 As shown in b), the untreated sepsis group had severe pulmonary edema. In contrast, the pulmonary edema was significantly alleviated after treatment with GRb1@PEI and GRb1@PAM, with GRb1@PEI reaching near normal levels. While GRb1 showed some therapeutic effect, the therapeutic effect of GRb1@PAPB and GRb1@CS was relatively poor, which is consistent with the anti-inflammatory effect observed in vitro Figure 3 In a, b) Likewise, the cell counts in BALF Figure 7 In c), total protein content Figure 7 In d) and pro-inflammatory cytokine levels, including IL-6, TNF-a and IL-1b Figure 7 In e-g) also showed the same trend. The levels of inflammatory factors in the serum reflect the ability of nano-Rb1 to alleviate systemic inflammation. Surprisingly, after treatment with GRb1@PEI, the levels of pro-inflammatory cytokines IL-6 and TNF-a in the serum Figure 7 In h-i) returned to normal. Similar decreases in inflammatory markers in lung homogenates Figure 7 In j-l) were observed after GRb1@PEI treatment. In summary, GRb1@PEI was the most effective, followed by GRb1@PAM, while GRb1@PAPB and GRb1@CS were less effective compared to GRb1.

[0086] Further histological examination of lung tissues from sepsis mice showed typical signs of pulmonary edema, including a significant thickening of the alveolar walls, and an increase in inflammatory cells in the PBS-treated sepsis group. Treatment with GRb1@PEI and GRb1@PAM significantly reduced lung injury, while GRb1, GRb1@PAPB and GRb1@CS did not have similar effects Figure 7 In m) Fluorescent staining images of inflammatory markers IL-6, TNF-a and IL-1b in lung tissues Figure 7 In n-p) further confirmed the reduction in inflammation and inflammatory factor levels after GRb1@PEI treatment. These results suggest that GRb1@PEI is a promising strategy for effectively alleviating systemic inflammation.

[0087] 2.5 Therapeutic potential of cationically modified GRb1 for Kp-induced blood infection-associated ALI.

[0088] In addition to inhibiting bacterial growth, controlling cytokine storm is also crucial for improving the prognosis of patients with infection-related endocrine diseases. Given its hydrophobic core and strong anti-inflammatory effect, GRb1@PEI can serve as a multifunctional nanocarrier for hydrophobic drugs such as antibiotics, providing a promising strategy for the treatment of bacterial inflammation. To evaluate its therapeutic potential, nanoRb1 was used in combination with polymyxin B (PMB) to treat Kp028-induced blood infection-related ALI in a mouse model. Mice received treatment by tail vein injection and the next day, bronchoalveolar lavage fluid (BALF) and lung tissue were collected from the mice Figure 8 a). Treatment with PMB or PMB in combination with nanoRb1 significantly reduced bacterial growth in lung homogenates. Notably, the cation-modified GRb1-PMB combination had a better antibacterial effect than GRb1-PMB, with the most significant antibacterial effect of GRb1@PEI-PMB Figure 8 b). This enhanced effect can be due to the ability of cationic polymers to disrupt bacterial cell membranes.

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

[0090] Myeloperoxidase (MPO) is an enzyme primarily found in neutrophils that plays a key role in the production of reactive oxygen species necessary for the inflammatory process. As Figure 12As shown in Fig. 11, MPO staining of lung tissue showed that neutrophil infiltration was significantly reduced in the GRb1@PEI-PMB group, indicating that GRb1@PEI-PMB can 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 subgroups is a key indicator of the severity and progression of inflammation. Figure 13 and Figure 9 The fluorescence staining of macrophages is shown, with M1 labeled in red and M2 labeled in green in lung tissue. The results show that in the GRb1@PEI-PMB group with the most significant efficacy, the dense population of M1 pro-inflammatory macrophages shifted to M2 anti-inflammatory macrophages. In summary, GRb1@PEI as a multifunctional nanocarrier can treat Kp-induced blood infection-related ALI.

[0091] 2.6 Therapeutic potential of cationic modified GRb1 for Sp-induced bacterial pneumonia.

[0092] Sp is the main cause of pneumonia and other respiratory tract infections, and thus becomes 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 a). Compared with the PBS control group, the growth rate of bacteria in the lung homogenate of mice treated with ampicillin (AMP) formulation was significantly reduced. Notably, the cationic modified GRb1 group showed a synergistic bactericidal effect compared with the AMP group, thereby reducing bacterial growth. Among them, GRb1@PEI showed the most significant bactericidal effect Figure 9 b). In terms of inflammation suppression, GRb1@PEI-AMP can restore lung edema to normal levels. The total cell count Figure 9 c) and total protein content Figure 9 d) in BALF further confirmed that GRb1@PEI-AMP is the most effective treatment. In addition, GRb1@PEI-AMP restored the levels of inflammatory factors IL-6, TNF-a and IL-1b in BALF and lung tissue homogenate to normal levels Figure 9 e-j).

[0093] Pathological analysis of model lung tissue Figure 9k) showed that PBS-treated group exhibited significant cell proliferation and structural damage, indicating severe lung injury. Compared with PBS group, AMP group and GRb1-AMP group showed slight improvement, but GRb1@PAPB-AMP group and GRb1@CS-AMP group showed little or even no improvement. In contrast, the therapeutic effect of GRb1@PEI-AMP group was the most significant, followed by GRb1@PAM-AMP. These results confirmed that GRb1@PEI could enhance the therapeutic effect of AMP in the treatment of bacterial pneumonia. Further analysis of key inflammatory markers in lung tissue, including IL-6, TNF-a and IL-1b, as well as MPO staining and infiltration of M1 / M2 macrophages, confirmed the inhibitory effect of GRb1@PEI-AMP on the inflammatory storm caused by bacterial infection Figure 10 l-n). In summary, cationically modified GRb1 can enhance the anti-inflammatory properties of Rb1, alleviate the release of cytokines, and possibly inhibit the immune response triggered by cytokines. In addition, it can also serve as a carrier for hydrophobic antibiotics, improving the targeting of antibiotics in antibacterial therapy. These combined effects suggest that GRb1@PEI can be a promising immunoprotective therapeutic agent for the treatment of various bacterial inflammatory diseases.

[0094] 2.7 In vivo biological safety of cationically 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 through the tail vein at therapeutic doses. As Figure 10 a-e, no significant statistical differences were found between the control group and each group receiving GRb1 or its cationically modified forms in terms of blood cell counts, including red blood cells, white blood cells, lymphocytes, granulocytes and platelets. Blood biochemical analysis Figure 10 f-i) showed that there were 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 ​ j) showed that there were no significant differences in H&E staining patterns of the heart, liver, spleen, lung and kidney. In summary, cationically modified GRb1, including GRb1@PEI, has good in vivo biological safety and broad clinical application prospects.

[0096] Therefore, the application adopts the multifunctional nanocarrier, the preparation method and the application, the multifunctional nanocarrier can specifically target macrophages, not only in sepsis-related acute lung injury (ALI) model, promote macrophage-mediated Rb1 delivery to the inflammation site, relieve inflammation, but also can improve the clearance rate of bacteria in blood infection-related ALI and bacterial pneumonia model, reduce inflammation and immune response; can be used for developing drugs for treating chronic inflammation.

[0097] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit them, although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that: it can still modify or equivalently replace the technical solutions of the present application, and these modifications or equivalent replacements also cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. Multifunctional nanocarriers, characterized in that: The ginsenoside Rb1 and the cationic polymer are included, and the cationic polymer is polyacrylamide (PAM) or polyethyleneimine (PEI); The negatively charged ginsenoside Rb1 is located at the center of the multifunctional nanocarrier, and the cationic polymer is electrostatically adsorbed on the surface of the ginsenoside Rb1.

2. The multifunctional nanocarrier of claim 1, wherein: The molar ratio of the ginsenoside Rb1 to the cationic polymer is Rb1: cationic polymer = 5: (1-6).

3. The method for preparing the multifunctional nanocarrier as described in claim 1 or 2, characterized in that, The steps are as follows: The ginsenoside Rb1 is dispersed in physiological saline to obtain a ginsenoside solution, and then a cationic polymer solution is added, and the mixed solution is subjected to 480W ultrasonic treatment for 5 minutes to obtain the multifunctional nanocarrier.

4. The method for preparing the multifunctional nanocarrier according to claim 3, characterized in that: The concentration of the ginsenoside solution is 1mg / mL, and the molar ratio of the ginsenoside Rb1 to the cationic polymer is Rb1: cationic polymer = 5: (1-6).

5. Use of the multifunctional nanocarrier according to claim 1 or 2 in the preparation of a drug for treating acute lung injury (ALI) caused by sepsis.

6. Use of the multifunctional nanocarrier according to claim 1 or 2 in the preparation of a drug for treating acute lung injury (ALI) related to blood infection caused by multi-drug resistant Klebsiella pneumoniae.

7. Use of the multifunctional nanocarrier according to claim 1 or 2 in the preparation of a drug for treating bacterial pneumonia caused by Streptococcus pneumoniae.

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