Nanoparticles for resisting Pseudomonas aeruginosa infection, preparation method and pulmonary inhalation preparation

Abstract

The present application belongs to the field of anti-bacterial infection technology, and in particular, relates to nanoparticles for resisting Pseudomonas aeruginosa infection, a preparation method, and a pulmonary inhalation preparation; the nanoparticles for resisting Pseudomonas aeruginosa infection provided by the present application can penetrate the mucus layer and enter the interior of the biofilm through the cationic nanoparticles, and improve the permeability of the outer membrane, thereby solving the technical problem in the prior art that antibacterial drugs such as cationic nanoparticles are difficult to reach the infection site of Pseudomonas aeruginosa and have low antibacterial effects.

Description

Technical Field

[0001] The present application belongs to the technical field of anti-bacterial infection, and in particular relates to nanoparticles for resisting Pseudomonas aeruginosa infection, a preparation method thereof, and a pulmonary inhalation preparation. Background Art

[0002] Pseudomonas aeruginosa (P. aeruginosa) is commonly found in the lungs of immunocompromised patients, especially those with cystic fibrosis (CF). The infection it causes is difficult to eradicate, prone to recurrence, and has a high mortality rate, posing a huge threat to public health.

[0003] Currently, antibiotics are commonly used in clinical treatment of Pseudomonas aeruginosa infection. Antibiotics such as penicillin can effectively act on Pseudomonas aeruginosa both in vivo and in vitro. However, the widespread use of antibiotics has led to the emergence of drug resistance in Pseudomonas aeruginosa. A second or multiple antibiotics can be used for combined treatment. However, as the treatment time of combined antibiotic treatment increases, the bacterial resistance rate will increase and the clearance rate will decrease. Therefore, it is necessary to provide a new means of treating Pseudomonas aeruginosa infection.

[0004] Although cationic nanoparticles have a high bactericidal effect on Pseudomonas aeruginosa infection, Pseudomonas aeruginosa infection is often accompanied by clinical symptoms such as abnormal increase in mucus secretion. The mucus layer becomes a barrier that prevents cationic nanoparticles from reaching the infection site. In addition, Pseudomonas aeruginosa also reduces the effectiveness of cationic nanoparticles in treating Pseudomonas aeruginosa infection by forming a biofilm. The outer membrane of Pseudomonas aeruginosa acts as a selective barrier to prevent the penetration of antimicrobial drugs. Summary of the invention

[0005] In view of this, the present application provides nanoparticles and preparation methods for resisting Pseudomonas aeruginosa infection and pulmonary inhalation preparations, which are used to solve the technical problems in the prior art that antibacterial drugs such as cationic nanoparticles are difficult to reach the infection site of Pseudomonas aeruginosa and have low antibacterial effects.

[0006] In a first aspect, the present application provides nanoparticles for resisting Pseudomonas aeruginosa infection, wherein the nanoparticles include composite nanoparticles and antibacterial drugs;

[0007] The composite nanoparticles include PEGylated ε-polylysine modified with dimethyl maleic anhydride, polycaprolactone-polyethylene glycol and polycaprolactone-polylysine;

[0008] The polycaprolactone-polyethylene glycol and polycaprolactone-polylysine are loaded with antibacterial drugs to construct cationic antibacterial nanoparticles;

[0009] The dimethylmaleic anhydride-modified PEGylated ε-polylysine coats the cationic antibacterial nanoparticles.

[0010] Preferably, the antibacterial drug includes azithromycin.

[0011] It should be noted that azithromycin can inhibit the quorum sensing system to downregulate the expression of virulence factors of Pseudomonas aeruginosa to exert an anti-virulence effect, prevent the recurrence of infection after bacterial eradication, and has the dual effects of anti-biofilm and anti-virulence.

[0012] Preferably, the antibacterial drugs also include but are not limited to ciprofloxacin, tobramycin, streptomycin, erythromycin or a combination thereof.

[0013] The second aspect of the present application provides a method for preparing nanoparticles for resisting Pseudomonas aeruginosa infection, the preparation method comprising the steps of:

[0014] Step S1, dissolving polycaprolactone ε-polylysine, polycaprolactone-polyethylene glycol and an antibacterial drug in an organic solvent, adding distilled water and stirring overnight;

[0015] Step S2: After stirring overnight, add PEGylated ε-polylysine modified with dimethylmaleic anhydride and stir to obtain nanoparticles that resist infection by Pseudomonas aeruginosa.

[0016] Preferably, in step S1, the preparation method of polycaprolactone ε-polylysine comprises: ultrasonically dispersing polycaprolactone-carboxyl, N-(3-(dimethylamino)-propyl)-N-ethylcarbodiimide and N-hydroxysuccinimide in a solvent, stirring overnight under a nitrogen atmosphere. Then, ε-polylysine is added and stirred overnight. The product is dialyzed in water and freeze-dried to obtain polycaprolactone ε-polylysine.

[0017] Preferably, in step S2, the preparation method of the dimethylmaleic anhydride modified PEGylated ε-polylysine comprises: dissolving ε-polylysine in phosphate buffer, adding polyethylene glycol-active ester and stirring overnight under nitrogen flow, adding dimethylmaleic anhydride, adjusting the pH value to greater than 7.0, stirring overnight, dialyzing in water, and lyophilizing to obtain dimethylmaleic anhydride modified PEGylated ε-polylysine.

[0018] Preferably, in step S1, the antibacterial drugs include but are not limited to azithromycin, ciprofloxacin, tobramycin, streptomycin, erythromycin or a combination thereof, and the mass ratio of polycaprolactone-polyethylene glycol to polycaprolactone-ε-polylysine is 1:1-16:1.

[0019] Preferably, in step S1, the solvent is tetrahydrofuran.

[0020] The third aspect of the present application provides a pulmonary inhalation preparation, which comprises the above-mentioned nanoparticles for resisting Pseudomonas aeruginosa infection and pharmaceutical excipients.

[0021] It should be noted that the pulmonary inhalation preparation provided in the present application includes components such as PEGylated ε-polylysine, polycaprolactone-polyethylene glycol, polycaprolactone-polylysine and antibacterial drugs, among which ε-polylysine can be used as a drug carrier in the treatment of Pseudomonas aeruginosa bacterial infection in the lungs.

[0022] Preferably, the pulmonary inhalation preparation includes but is not limited to an inhalation aerosol, a dry powder inhaler, an inhalation spray or an inhalation solution.

[0023] In summary, the present application provides nanoparticles for resisting infection with Pseudomonas aeruginosa, a preparation method thereof, and a pulmonary inhalation preparation. The nanoparticles for resisting infection with Pseudomonas aeruginosa include composite nanoparticles and antibiotics. The composite nanoparticles include dimethylmaleic anhydride-modified PEGylated ε-polylysine, polycaprolactone-polyethylene glycol, polycaprolactone-polylysine, and antibacterial drugs to construct cationic antibacterial nanoparticles. The dimethylmaleic anhydride-modified PEGylated ε-polylysine modifies the cationic nanoparticles, which is beneficial to reduce the interaction between the nanoparticles and mucus proteins, so that the nanoparticles can be inhaled. The rice grains successfully penetrate the mucus layer and reach the Pseudomonas aeruginosa biofilm layer. After reaching the Pseudomonas aeruginosa biofilm layer, the nanoparticles remove the polyethylene glycol-modified ε-polylysine modified with dimethylmaleic anhydride, allowing them to smoothly penetrate into the interior of the biofilm and improve the anti-biofilm effect. After the polycaprolactone-modified ε-polylysine targets Pseudomonas aeruginosa, it releases antibacterial drugs to exert a bactericidal effect and improves the permeability of the outer membrane, thereby solving the technical problem in the prior art that antibacterial drugs such as cationic nanoparticles are difficult to reach the infection site of Pseudomonas aeruginosa and have a low antibacterial effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 This is a schematic diagram of the experimental results of testing the artificial sputum and biofilm permeability of nanoparticles against Pseudomonas aeruginosa infection in Experimental Example 1 of the present application;

[0026] Figure 2 This is a schematic diagram of the experimental results of testing the pH and lipase responsiveness of nanoparticles against Pseudomonas aeruginosa infection in Experimental Example 2 of this application;

[0027] Figure 3 This is a schematic diagram of the experimental results of testing the bacterial targeting, inner and outer membrane permeability, antibacterial activity and antibiotic resistance of nanoparticles against Pseudomonas aeruginosa infection in Experimental Example 3 of this application;

[0028] Figure 4 This is a schematic diagram of the experimental results of testing the anti-biofilm performance of nanoparticles against Pseudomonas aeruginosa infection in Experimental Example 4 of the present application;

[0029] Figure 5 This is a schematic diagram of the experimental results of testing the anti-virulence factor of nanoparticles against Pseudomonas aeruginosa infection in Experimental Example 5 of this application;

[0030] Figure 6 This is a schematic diagram of the in vivo distribution experimental results of nanoparticles tested against Pseudomonas aeruginosa infection in Experimental Example 6 of the present application;

[0031] Figure 7 This is a schematic diagram of the experimental results of the in vivo antibacterial effect of nanoparticles against Pseudomonas aeruginosa infection in Experimental Example 7 of the present application;

[0032] Figure 8 This is a schematic diagram of the experimental results of the safety evaluation of nanoparticles for resisting Pseudomonas aeruginosa infection in Experimental Example 8 of this application;

[0033] Fig. 9 Characterization of the nanoparticles against Pseudomonas aeruginosa infection prepared in Experimental Example 9 of the present application;

[0034] Figure 1 In the figure, (a and b) schematic diagram of SA-AZINPs or DA-AZI NPs penetrating artificial sputum (a) and penetration rate (b). (c) aggregation rate of SA-AZINPs or DA-AZI NPs after incubation in artificial sputum for 1 h, (df) laser confocal images of SA-AZI NPs or DA-AZINPs incubated with artificial sputum (d), and the total fluorescence intensity of nanoparticles in artificial sputum (e) and normalized fluorescence distribution (f), (h and g) laser confocal images of SA-AZI NPs or DA-AZI NPs incubated with biofilm (g), and the fluorescence intensity of nanoparticles detected in biofilm before and after washing (h), scale bar: 30 μm;

[0035] Figure 2 In the figure, (a and b) Malvern particle diameter and TEM images (a) and zeta potential (b) of SA-AZI NPs and DA-AZI NPs after incubation for 2 h at pH 7.4 or pH 6.5, scale bar: 200 nm, (c and d) Malvern particle size (c) and TEM image (d) of DA-AZI NPs after incubation for 8 h in PBS (pH 7.4) with or without lipase, scale bar: 200 nm, (e) cumulative release of SA-AZI or DA-AZI NPs under different conditions;

[0036] Figure 3Figure 2. (a) Confocal laser images of GFP-P. aeruginosa and P. aeruginosa incubated with Nile Red-labeled SA-AZI NPs or DA-AZINPs at pH 6.5. Scale bar: 20 μm. (b) Interaction between DA-AZI NPs and GFP-P. aeruginosa. (c and d) Outer membrane permeability (c) and inner membrane permeability (d) of P. aeruginosa after different treatments. (e and f) MIC (e) and MBC (f) of P. aeruginosa after different treatments. (g) Resistance development during serial passage of bacteria in the presence of sub-MIC concentrations of free AZI or DA-AZI NPs.

[0037] Figure 4 In the figure, (a and b) agar plate images (a) and CFU (b) of biofilms treated with free AZI, SA-AZI NPs and DA-AZI NPs for 24 h, (c) laser confocal images of biofilms stained with SYTO9 / PI dye, scale bar: 30 μm. (d) SEM images of biofilms after different treatments, scale bar: 5 μm;

[0038] Figure 5 Middle, (ae) pyocyanin (a), siderophore (b), protease (c), elastase (d) and rhamnolipid (e) contents produced by P. aeruginosa after different treatments, (f) cell viability of bacteria after co-culture with RAW 264.7 cells after different treatments, (g) laser confocal images of bacteria treated with subinhibitory concentrations of AZI, SA-AZI NPs or DA-AZI NPs for 2 h, scale bar: 30 μm, (hi) concentration (h) and time dependence (i) of biofilm formation after different treatments;

[0039] Figure 6 Middle, (a and b) in vitro fluorescence imaging (a) and quantitative analysis (b) of major organs after inhalation of free DiR dye or DiR-labeled DA-AZI NPs, (c and d) immunofluorescence imaging (c) and colocalization analysis (d) of the lungs of infected mice 2 h after inhalation of DiR-labeled SA-AZI NPs or DA-AZINPs, scale bar: 60 μm;

[0040] Figure 7(ae) Contents of inflammatory-related cytokines such as IL-6 (a), IL-10 (b), TNF-α (c), MIP-2 (d) in BALF and PCT (e) in serum. (gf) Microscopic images of inflammatory cells in BALF (f) and quantitative analysis of the ratio of neutrophils to macrophages after different treatments (g), scale bar: 100 μm, (h) Flow cytometric analysis of neutrophils in BALF after different treatments, (i) Body weight curve of mice during the experiment, (j) Bacterial load of mice after different treatments (n=6), (k) Histological evaluation of H&E-stained lung sections, scale bar: 200 μm;

[0041] Figure 8 Middle, (ae) Changes of blood biochemical indexes of mice after different treatments, (f) H&E staining of main organs of mice after different treatments;

[0042] Fig. 9 In the figure, (a) H NMR spectrum of PCL-ε-PLL, (b) H NMR spectrum of PEG-PLL-DA, (c) H NMR spectrum of PEG-PLL-SA, (de) cytotoxicity study of PCL-ε-PLL on BEAS-2B cells (d) and RAW264.7 cells (e), (fg) antibacterial effect (f) and AZI loading (g) of cationic nanoparticles prepared with different ratios of PCL-PEG and PCL-PLL, (h) zeta potential of nanoparticles prepared with different ratios of PCL-ε-PLL and PEG-PLL-DA, (i) inhibitory effect of nanoparticles loaded with different antibiotics on biofilm formation. DETAILED DESCRIPTION

[0043] The present application provides nanoparticles for resisting Pseudomonas aeruginosa infection, a preparation method and a pulmonary inhalation preparation, which are used to solve the technical problem in the prior art that antibacterial drugs such as cationic nanoparticles are difficult to reach the infection site of Pseudomonas aeruginosa and have low antibacterial effect.

[0044] The technical solution of the present application will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0045] In the embodiments, the terms are as follows:

[0046] 1. P.aeruginosa: Pseudomonas aeruginosa

[0047] 2.EPS: Extracellular polymers

[0048] 3. AZI: Azithromycin

[0049] 4.LPS: Lipopolysaccharide

[0050] 5.DA: dimethyl maleic anhydride

[0051] 6. PCL-PLL: polycaprolactone ε-polylysine

[0052] 7. PCL-PEG: Polycaprolactone-polyethylene glycol

[0053] 8.PEG-PLL-DA: PEGylated ε-polylysine modified with dimethylmaleic anhydride

[0054] 9.DA-AZINPs: dimethylmaleic anhydride modified azithromycin-loaded antibacterial nanoparticles

[0055] 10.DA-CIPNPs: dimethylmaleic anhydride modified ciprofloxacin-loaded antibacterial nanoparticles

[0056] 11.DA-SMNPs: dimethylmaleic anhydride modified streptomycin-loaded antibacterial nanoparticles

[0057] 12.DA-TOB NPs: dimethylmaleic anhydride modified tobramycin-loaded antibacterial nanoparticles

[0058] 13.DA-EMNPs: dimethylmaleic anhydride modified erythromycin-loaded antibacterial nanoparticles

[0059] 14.SA-AZINPs: Succinic anhydride modified azithromycin-loaded antibacterial nanoparticles

[0060] 15.PLL-AZI NPs: Cationic antibacterial nanoparticles

[0061] 16. Zeta potential: Zeta potential

[0062] 17. CF: Cystic Fibrosis Patients

[0063] 18.SA: Succinic anhydride

[0064] 19.PBS: Phosphate buffered saline

[0065] 20.QS: Quorum Sensing System

[0066] 10. Beas-2B cell: human bronchial epithelial cells

[0067] 11.RAW264.7 cell: mouse mononuclear macrophage leukemia cell

[0068] 12.GFP-P.aeruginosa: Green fluorescent protein labeled Pseudomonas aeruginosa

[0069] 13.SYTO9 / PI: Live and dead bacteria dye

[0070] 14. Papp value: apparent permeability coefficient

[0071] 15.DMEM: high glucose medium

[0072] 16.MIC: Minimum inhibitory concentration. In the dilution method of microbial identification, the lowest drug concentration that completely inhibits bacterial growth in a test tube or a small well is the minimum inhibitory concentration.

[0073] 17.MBC: Minimum bactericidal concentration (MBC), which can kill bacteria in the culture medium (i.e. kill 99.9%

[0074] The lowest concentration of the test microorganism.

[0075] 18.DiR: A lipophilic, near-infrared fluorescent dye

[0076] 19.BALF: Bronchoalveolar lavage fluid

[0077] 20.H&E sections: Hematoxylin-eosin sections

[0078] 21.Wright Giemsa: Wright stain, used to distinguish immune cells

[0079] 22.IL-6: Interleukin-6

[0080] 23.MIP-2: Chemokine-2

[0081] 24.IL-10: Interleukin-10

[0082] 25.TNF-α: Tumor necrosis factor-α

[0083] 26.PCT: Procalcitonin

[0084] 27.AST: Aspartate aminotransferase

[0085] 28.ALT: Alanine aminotransferase

[0086] 29.BUN: urea nitrogen

[0087] 30.LDH: lactate dehydrogenase

[0088] 31.CRE: serum creatinine

[0089] Example 1

[0090] In view of the defects of cationic nanoparticles and other antibacterial drugs in the prior art that it is difficult to reach the infection site of Pseudomonas aeruginosa and the antibacterial effect is low, Example 1 of the present application provides a nanoparticle for resisting Pseudomonas aeruginosa infection, the nanoparticle comprising a composite nanoparticle and an antibacterial drug; the composite nanoparticle comprises PEGylated ε-polylysine modified with dimethylmaleic anhydride, polycaprolactone-polyethylene glycol and polycaprolactone-polylysine; polycaprolactone-polyethylene glycol and polycaprolactone-polylysine are loaded with antibacterial drugs to construct cationic antibacterial nanoparticles; and the PEGylated ε-polylysine modified with dimethylmaleic anhydride is coated with the cationic antibacterial nanoparticles.

[0091] Patients with Pseudomonas aeruginosa infection are often accompanied by clinical features such as damaged cilia, abnormal increase in mucus secretion, reduced ciliary transport, and reduced airway surface fluid. The mucus layer accumulated in the airways is the first barrier that prevents antibacterial drugs from reaching the site of infection. Effectively overcoming the abnormal mucus barrier in the airways of infected patients is the key to improving the antibacterial effect by allowing the drug to reach the site of infection in sufficient quantities. The nanoparticles for resisting Pseudomonas aeruginosa infection provided in the present application weaken the interaction between the mucus layer and the cationic nanoparticles, thereby easily passing through the mucus layer; the biofilm of Pseudomonas aeruginosa is a membrane-like microbial aggregate formed by bacteria producing a large amount of extracellular polymers (EPS) to wrap it. The dense biofilm reduces the penetration of antibacterial drugs, cuts off the supply of nutrients, downregulates bacterial metabolism, and greatly increases the internal bacterial mutation frequency and gene parallel transfer, thereby increasing bacterial resistance. The nanoparticles for resisting Pseudomonas aeruginosa infection provided in the present application can improve the drug resistance of bacteria by penetrating When passing through the mucus layer and reaching the biofilm location of Pseudomonas aeruginosa, the PEGylated ε-polylysine has excellent hydrophilicity, thereby increasing its ability to penetrate into the biofilm, and responding to the low pH microenvironment of the Pseudomonas aeruginosa biofilm layer, the negatively charged outer layer of the PEGylated ε-polylysine modified with dimethylmaleic anhydride is removed, and the charge is flipped to expose the polycaprolactone ε-polylysine loaded with antibiotics. The small-sized polycaprolactone ε-polylysine loaded with antibiotics can effectively penetrate into the biofilm, target Pseudomonas aeruginosa and release antibiotics in situ to exert a bactericidal effect; the outer membrane of Pseudomonas aeruginosa is an asymmetric bilayer of phospholipids and lipopolysaccharide (LPS), which contains porins that form β-barrel protein channels, forming a permeation barrier for hydrophilic and hydrophobic drugs. In addition, the low fluidity of lipopolysaccharide carbohydrates and the complex spatial structure of lipid A act as selective barriers to prevent the penetration of antibiotics. There are two kinds of pore proteins on the outer membrane. Antimicrobial drugs are transported into the body through relatively large pore proteins. However, due to congenital deficiency or gene mutation, the outer membrane of the bacteria lacks outer membrane proteins such as OprF, OprD, and OprH that transport antimicrobial drugs. The extremely low outer membrane permeability of P. aeruginosa limits the penetration of most antibiotics, and it has high inherent drug resistance. The nanoparticles against Pseudomonas aeruginosa infection provided in the present application can improve the outer membrane permeability and cooperate with antimicrobial drugs to exert antibacterial effects; thereby solving the technical problem in the prior art that antimicrobial drugs such as cationic nanoparticles are difficult to reach the infection site of Pseudomonas aeruginosa and have low antibacterial effects; the nanoparticles against Pseudomonas aeruginosa infection provided in Example 1 of the present application can be used for the treatment of Pseudomonas aeruginosa infection in vivo and in vitro.

[0092] Preferably, the antibacterial drug is azithromycin. The antibacterial drug azithromycin can inhibit the quorum sensing system to downregulate the expression of virulence factors of Pseudomonas aeruginosa to exert anti-virulence effects, thereby preventing recurrence of infection after bacterial eradication. Therefore, when the anti-Pseudomonas aeruginosa infection nanoparticles provided in the present application are loaded with the antibacterial drug azithromycin, they have the dual effects of anti-biofilm and anti-virulence.

[0093] Preferably, the nanoparticles for resisting Pseudomonas aeruginosa infection provided in the present application can also be loaded with other antibacterial drugs, which can synergistically exert antibacterial and anti-biofilm effects, thereby broadening the application scope of the nanoparticles; wherein the antibacterial drugs include but are not limited to ciprofloxacin, tobramycin, streptomycin, erythromycin or a combination thereof.

[0094] Example 2

[0095] Example 2 of the present application provides a method for preparing nanoparticles for resisting infection with Pseudomonas aeruginosa described in Example 1, the preparation method comprising the steps of preparing polycaprolactone-modified ε-polylysine, preparing PEGylated ε-polylysine modified with dimethylmaleic anhydride, and preparing nanoparticles for resisting infection with Pseudomonas aeruginosa.

[0096] The preparation of polycaprolactone ε-polylysine includes ultrasonically dispersing polycaprolactone-carboxyl, N-(3-(dimethylamino)-propyl)-N-ethylcarbodiimide and N-hydroxysuccinimide in tetrahydrofuran, stirring overnight under a nitrogen atmosphere, then adding ε-polylysine and stirring overnight, dialyzing the product in water, and freeze-drying to obtain polycaprolactone ε-polylysine (PCL-ε-PLL);

[0097] The preparation of dimethylmaleic anhydride modified PEGylated ε-polylysine includes dissolving ε-polylysine in phosphate buffer, adding polyethylene glycol-active ester and stirring overnight under nitrogen flow, adding dimethylmaleic anhydride, adjusting the pH to be greater than 7.0, stirring overnight, dialyzing in water, and freeze-drying to obtain dimethylmaleic anhydride modified PEGylated ε-polylysine (PEG-PLL-DA);

[0098] The preparation of nanoparticles for resisting Pseudomonas aeruginosa infection includes dissolving polycaprolactone ε-polylysine, polycaprolactone-polyethylene glycol and antibacterial drugs in tetrahydrofuran, adding distilled water and stirring overnight, adding dimethylmaleic anhydride-modified polyethylene glycol ε-polylysine and stirring to obtain nanoparticles (DA-AZI NPs) for resisting Pseudomonas aeruginosa infection.

[0099] The crystal violet staining method was used to determine biofilm formation. The bacterial suspension was inoculated into a 96-well plate containing nanoparticles loaded with different antimicrobial drugs. After incubation for 24 h, the planktonic cells were removed by washing with PBS, and then stained with 0.1% crystal violet. Finally, 95% ethanol was added and the plate was incubated at OD 570Furthermore, the inhibitory effect on biofilm formation was evaluated by measuring the biofilm formation in the presence of different samples (1 / 8 MIC) for a predetermined time.

[0100] Characterization of the prepared products Fig. 9 As shown, H NMR spectra showed the successful synthesis of PCL-PLL, PEG-PLL-DA, and PEG-PLL-SA ( Fig. 9 ac), PCL-ε-PLL had no toxic effect on Beas-2B cells and RAW264.7 cells at a concentration of 1600 μg / mL ( Fig. 9 d, e), compared with other cationic materials, the cytotoxicity is lower, and the nanoparticles formed by the mass ratio of PCL-PEG and PEG-PLL between 1:1-16:1 have higher antibacterial ability, and the mass ratio between 4:1-16:1 has higher antibacterial drug loading capacity ( Fig. 9 f,g), so the optimal ratio was determined to be 4:1 for subsequent evaluation. PEG-PLL-DA has an effective charge shielding effect ( Fig. 9 h). DA-AZINPs has the strongest inhibitory effect on biofilm formation compared with other antibiotic nanoparticles ( Fig. 9 i). DA-AZI NPs are spheres with a diameter of 131 ± 4.2 nm ( Figure 2 a), With a slightly negatively charged surface at pH = 7.4, the size and charge of DA-AZI NPs are sufficient to penetrate mucus smoothly.

[0101] Example 3

[0102] Example 3 of the present application provides a pulmonary inhalation preparation, which includes the nanoparticles for resisting Pseudomonas aeruginosa infection described in Example 1 and pharmaceutically acceptable excipients.

[0103] The nanoparticles for resisting Pseudomonas aeruginosa infection in the pulmonary inhalation preparation provided in Example 3 of the present application have good biocompatibility, are degradable, and have no toxic side effects on the lungs, and can thus be used for antibacterial treatment of Pseudomonas aeruginosa in the lungs.

[0104] Example 4

[0105] Example 4 of the present application provides a nanoparticle (SA-AZI NPs) for resisting infection with Pseudomonas aeruginosa, wherein the negatively charged outer layer of the nanoparticle coated with polycaprolactone ε-polylysine is succinic anhydride-modified polyethylene glycol ε-polylysine, serving as a comparative example of the nanoparticle (DA-AZI NPs) for resisting infection with Pseudomonas aeruginosa described in Example 1.

[0106] The difference between the preparation method of succinic anhydride-modified PEGylated ε-polylysine and the preparation method in Example 2 is that succinic anhydride-modified PEGylated ε-polylysine is added during the preparation of nanoparticles for resisting Pseudomonas aeruginosa infection.

[0107] Experimental Example 1

[0108] Experimental Example 1 of the present application provides an experiment for testing the permeability of nanoparticles (SA-AZI NPs) against Pseudomonas aeruginosa infection and nanoparticles (DA-AZI NPs) against Pseudomonas aeruginosa infection in artificial sputum and biofilm.

[0109] The experimental process is as follows: after the Nile red-labeled nanoparticles are incubated in artificial sputum for 1 hour, the supernatant is collected, the fluorescence intensity of the supernatant and the initial nanoparticle solution is measured, and the aggregation rate of the nanoparticles is calculated. The Transwell system is used to measure the Papp value of the nanoparticles in artificial sputum. The artificial sputum is placed on a Transwell with a polycarbonate membrane, and PBS is added to the receiving pool. The nanoparticles are added to the surface of the artificial sputum and incubated in a shaker at 37°C. At the predetermined time point, the sample is taken out of the receiving pool, the fluorescence intensity is detected and the Papp value is calculated, and the penetration process of the nanoparticles in the artificial sputum is recorded using a laser confocal microscope.

[0110] Similar to artificial sputum, a GFP-P. aeruginosa flowing biofilm model was established, and laser confocal microscopy was used to record the penetration process of nanoparticles in the biofilm.

[0111] The experimental results showed that compared with the control group of nanoparticles, the aggregation rate of DA-AZI NPs in artificial sputum was lower, at 26.2% ( Figure 1 c). Their Papp values ​​are about 4.5 times that of PLL-AZINPs ( Figure 1 a, 1b), indicating that DA-AZI NPs have a stronger ability to penetrate mucus. Polyethylene glycol makes NPs invisible, which is beneficial to the penetration of mucus ( Figure 1 d). DA-AZINPs can penetrate artificial sputum and distribute evenly within 40 minutes, while PLL-AZINPs have poor penetration ability within 60 minutes and are accompanied by aggregation ( Figure 1 e,f). DA-AZI NPs penetrate biofilm faster and more widely than SA-AZINPs ( Figure 1 g). After PBS washing, no significant change in the fluorescence of DA-AZINPs was observed in the biofilm ( Figure 1 g). These results indicate that DA-AZI NPs can effectively cross the mucus and biofilm barriers to achieve long-term targeting of P. aeruginosa inside the biofilm.

[0112] Experimental Example 2

[0113] Experimental Example 2 of the present application provides an in vitro drug release test of nanoparticles against Pseudomonas aeruginosa infection.

[0114] The experimental process is as follows: the particle size and zeta potential of DA-AZINPs after incubation in PBS with different pH values ​​and with or without lipase are measured using a particle size analyzer, the morphology of the nanoparticles is observed using a transmission electron microscope, and the in vitro drug release of the nanoparticles is measured in buffer solutions with different pH values ​​and with or without lipase.

[0115] The experimental results show that the particle size of DA-AZI NPs decreases in an acidic microenvironment with a pH of 6.5 ( Figure 2 a), the zeta potential changes from -3.29 mV to 11.2 mV ( Figure 2 b). In the lipase-rich medium, the size of the cationic nanoparticles increased significantly, reaching 224.7 ± 32 nm ( Figure 2 c), with fragments ( Figure 2 d). When the pH value is 7.4, the drug leakage of DA-AZI NPs is limited under physiological conditions. In the presence of lipase, DA-AZI NPs release more than 70% of AZI within 24 h, and up to 80.2% after 48 h of incubation ( Figure 2 e).

[0116] Experimental Example 3

[0117] Experimental Example 3 of the present application provides experiments to test the bacterial targeting, inner and outer membrane permeability, antibacterial activity and antibiotic resistance of nanoparticles against Pseudomonas aeruginosa infection.

[0118] The experimental process is as follows: the nanoparticles are labeled with Nile red, washed with PBS three times to remove the culture medium, and then incubated with the nanoparticles at pH 6.5 for 15 minutes. Then, the bacterial suspension is washed away and the images are taken using laser confocal microscopy.

[0119] The outer membrane permeability was measured using the NPN probe. The bacterial suspension was pre-incubated with the NPN probe for 5 min, and then free AZI or a nanoparticle solution at pH 6.5 was added, and the NPN fluorescence was measured using a microtiter plate reader to measure the outer membrane permeability.

[0120] The bacterial inner membrane permeability was measured based on the amount of intracellular ATP released. 600 = 0.4, and samples were collected after incubation with free AZI or nanoparticles and detected using an ATP detection kit.

[0121] MIC and MBC were determined by broth microdilution and dilution plate count methods. Bacteria were inoculated in 96-well plates. Then, LB broth containing two-fold serial dilution samples was placed in each well of the plate. After incubation at 37°C for 24 h, the OD 600 The MIC was determined. Subsequently, the suspension without obvious bacterial growth was plated on LB agar plates, and the lowest concentration for colony formation was the MBC.

[0122] Antibiotic resistance test was performed under sublethal dose treatment. P. aeruginosa was subcultured every 24 h in LB broth containing 1 / 2 MIC concentration of free AZI or nanoparticles. The MIC value of bacteria after each subculture was recorded to evaluate the development of antibiotic resistance.

[0123] The experimental results showed that DA-AZINPs had a strong interaction with P. aeruginosa ( Figure 3 a, b), indicating that the nanoparticles can target and adhere to the bacterial outer membrane. DA-AZI NPs showed similar permeability to PLL-AZI NPs, enabling NPN to penetrate the outer membrane and produce strong fluorescence ( Figure 3 c). DA-AZI NPs also caused significant damage to the bacterial inner membrane, resulting in higher permeability to ATP ( Figure 3 d). Except for the positive control, no damage to eukaryotic cells was observed in the other groups ( Figure 3 h), indicating that DA-AZI NPs can selectively penetrate the inner and outer membranes of bacteria without damaging the cells. DA-AZI NPs have strong antibacterial activity, with MIC and MBC 2 and 4 times lower than those of AZI, respectively ( Figure 3 e,f). This is related to the increased bacterial membrane permeability and enhanced antibiotic absorption after DA-AZI NPs treatment.

[0124] Experimental Example 4

[0125] Experimental Example 4 of the present application provides an experiment to test the anti-Pseudomonas aeruginosa biofilm situation of nanoparticles against Pseudomonas aeruginosa infection.

[0126] The experimental process is as follows: the bacterial suspension was inoculated into a 96-well plate and cultured at 37°C for 24 hours. The plate was then washed with PBS to remove planktonic bacteria, and then the biofilm was incubated with free AZI or nanoparticles at pH 6.5. The biofilm was then dispersed by ultrasonic vortexing, and the number of surviving bacteria in the biofilm was determined by the dilution plate count method.

[0127] In the CLSM experiment, the biofilm was cultured in a flow medium and treated with free AZI or NPs at a concentration of 256 μg / mL for 24 h, then stained with SYTO9 / PI for 15 min and observed using a laser confocal microscope.

[0128] The morphology of the biofilm was further observed using a scanning electron microscope. Sterile microscope slides were placed in a 12-well plate, and each well of the plate was inoculated with a bacterial suspension to form a biofilm on the slide. After treatment with AZI or nanoparticles (256 μg / mL), they were fixed overnight with 2.5% glutaraldehyde and then dehydrated with ethanol solutions in sequence. Finally, the slides were dried with flowing carbon dioxide, sprayed with gold, and observed using a scanning electron microscope.

[0129] The cytotoxicity of DA-AZINPs was tested on BEAS-2B cells. Rabbit erythrocytes were used for hemolysis experiments. The erythrocyte suspension was incubated with an equal volume of SA-AZINPs or DA-AZINPs at 37°C for 1, 2, 3, and 4 hours. The negative and positive controls were PBS and 0.5% Triton X-100, respectively. The supernatant was centrifuged at 1000g for 5 minutes at 4°C, and the absorbance was measured at 576nm to calculate the hemolysis rate.

[0130] The experimental results showed that compared with planktonic bacteria, DA-AZINPs had the highest bacterial removal rate ( Figure 4 a, b). DA-AZINPs showed significant eradication effect on Pseudomonas aeruginosa biofilm, and most of the bacteria were inactivated after 24 h of incubation ( Figure 4 c). After treatment with DA-AZI NPs, some bacteria shrank and showed membrane deformation and rupture ( Figure 4 d). DA-AZI NPs have no cytotoxic and hemolytic activity at the concentrations used to eradicate biofilms ( Figure 4 e,f).

[0131] Experimental Example 5

[0132] Experimental Example 5 of the present application provides an experiment to test the anti-virulence factor effect of nanoparticles against Pseudomonas aeruginosa infection.

[0133] The experimental process is as follows: after the bacteria are incubated with free AZI or nanoparticles at pH 6.5 for 24 hours, the supernatant is collected to detect virulence factors.

[0134] Pyocyanin and siderophore assay: The culture supernatant was diluted 3-fold with Tris–HCl and the OD 405 The supernatant was mixed with 500 μL of chloroform and then 300 μL of 5 mM HCl was added. 520 Measure the absorbance.

[0135] Protease and elastase activity assay: Azocasein and Congo red (ECR) were used as substrates to evaluate protease and elastase activities, respectively. For protease, the culture supernatant was incubated with an equal volume of 1% azocasein at 37°C for 1 h, and 7% glacial perchloric acid was added. After centrifugation, 10 M sodium hydroxide was added to the supernatant and the OD was 0. 430 For elastase, the culture supernatant was incubated with ECR buffer at 37°C for 3 h. Insoluble ECR was then removed and the OD 495 Measure the absorbance at

[0136] Rhamnolipid assay: Extract the supernatant twice with ether. Evaporate the ether fraction to dryness and resuspend in distilled water. Add 1.6% methanol and 70% sulfuric acid and incubate at 80°C for 30 minutes. 421 Measure the absorbance.

[0137] RAW264.7 cells were used to measure the toxicity of P. aeruginosa culture media after different treatments. RAW264.7 cells cultured in 96-well plates were added to different treatment media and incubated for 1 hour. Subsequently, DMEM was added to each well and incubated for 24 hours to measure cell viability.

[0138] Bacterial adhesion test was performed on BEAS-2B cells. BEAS-2B cells were added to a 24-well confocal plate and incubated for 24 hours. Hoechst staining was performed for 30 minutes, and fluorescently labeled cells were infected with GFP-P.aeruginosa. Finally, the cells were washed with PBS and observed under a laser confocal microscope.

[0139] Biofilm formation was determined by crystal violet staining. The bacterial suspension was inoculated into a 96-well plate in the presence of free AZI or nanoparticles. After incubation at 37°C for 24 h, the planktonic cells were removed by washing with PBS and stained with 0.1% crystal violet for 30 min. Finally, 200 μL of 95% ethanol was added and the plate was plated at OD 570 Furthermore, by measuring the biofilm formation in the presence of different samples (1 / 4 MIC) for a predetermined time, the time-dependent inhibitory effect on biofilm formation was evaluated.

[0140] The results showed that, except for rhamnolipid, all virulence factors were reduced after incubation with free AZI or nanoparticles at sub-inhibitory concentrations, and the virulence factor levels were the lowest after DA-AZINPs treatment ( Figure 5 ae). The cell viability of the DA-AZI NPs-treated group remained above 80%, indicating that DA-AZI NPs can protect host cells from damage by virulence factors to a certain extent ( Figure 5 f). The inhibitory effect of DA-AZINPs on the adhesion of P. aeruginosa was stronger than that of other groups ( Figure 5 g), indicating that DA-AZI NPs can effectively inhibit the recurrence of P. aeruginosa infection. DA-AZINPs showed concentration- and time-dependent inhibitory activity on biofilm formation, with the highest inhibition rate, inhibiting about 90% of biofilm formation under 1 / 4MIC conditions ( Figure 5 h,i).

[0141] Experimental Example 6

[0142] Experimental Example 6 of the present application provides an in vivo distribution experiment of nanoparticles to test the resistance to Pseudomonas aeruginosa infection.

[0143] The experimental process was as follows: the mouse P. aeruginosa chronic lung infection model was established using the agar bead embedding method, and the bacterial infection amount per mouse was 1×10 7 CFU. After establishing the chronic lung infection model, DiR-labeled DA-AZI NPs and free DiR dye were aerosolized into infected mice. The fluorescence signals of major organs were measured at 0, 12, and 24 h after administration.

[0144] To compare the distribution of nanoparticles in infected lungs, mice were sacrificed 2 hours after administration. Lungs were removed, rinsed with PBS, and immediately stored at -80°C. Frozen tissue sections were stained with DAPI. Sections were imaged using a laser confocal microscope.

[0145] The results showed that DA-AZI NPs exhibited prolonged lung retention and lower systemic exposure compared with free DiR dye ( Figure 6 a,b). Immunofluorescence imaging ( Figure 6 c) shows that Pseudomonas aeruginosa in the DA-AZI NPs group has a stronger co-localization relationship with the nanoparticles, which is confirmed by the Pearson coefficient ( Figure 6 d), indicating that DA-AZI NPs can reach and accumulate at the infection site better than SA-AZINPs.

[0146] Experimental Example 7

[0147] Experimental Example 7 of the present application provides an experiment to test the in vivo antibacterial effect of nanoparticles against Pseudomonas aeruginosa infection.

[0148] The experimental process was as follows: 5 days after the mice were infected, free AZI, SA-AZI and DA-AZI NPs were aerosolized and inhaled into the infected mice at a dose of 15 mg / kg (equivalent to AZI) once a day for 2 consecutive days, and the mice were observed for 1 day after drug withdrawal. 8 days after infection, a part of the mice were taken to collect BALF. After that, the infected lung homogenate was collected and plated. The bacterial load in the lungs of infected mice was expressed as the total number of bacteria in the lung homogenate and BALF. The remaining BALF was centrifuged for the analysis of cytokines and inflammatory cells. ELISA kits were used to detect cytokines IL-6, MIP-2, IL-10 and TNF-α. Neutrophils were labeled with CD11b and Ly6G antibodies, and inflammatory cells were detected using flow cytometry and Wright-Giemsa stain. At the same time, ELISA kits were used to detect the content of PCT in serum. Lung histology was performed after treatment, and the lungs of mice that were not lavaged were taken for H&E staining.

[0149] The results showed that the lowest levels of pro-inflammatory cytokines including IL-6, TNF-α, and MIP-2 were observed in mice treated with DA-AZI NPs compared with the control group ( Figure 7 ac). However, the different treatments had no effect on the production of the anti-inflammatory cytokine IL-10 ( Figure 7 d). In addition, after DA-AZI NPs treatment, the PCT content in serum, which reflects the degree of systemic infection and inflammation, was also significantly reduced ( Figure 7 e). The total number of inflammatory cells and the neutrophil / macrophage ratio decreased in the DA-AZI NPs group ( Figure 7 f,g). Flow cytometry analysis also showed that neutrophil infiltration was reduced to 12.0% after DA-AZINPs treatment ( Figure 7 h). The weight of mice decreased significantly after inoculation of bacteria, but the weight of each group recovered, indicating that the mice had developed chronic lung infection with Pseudomonas aeruginosa ( Figure 7 i). The greatest decrease in bacterial load was observed in the lungs of mice treated with DA-AZI NPs, exceeding 2 orders of magnitude ( Figure 7 j). Analysis of lung tissues of control mice showed severe lung lesions, obvious inflammatory infiltration, and significant thickening of alveolar septa. The lung structure of DA-AZINPs-treated mice remained relatively normal, with no obvious signs of inflammatory infiltration and alveolar damage, suggesting that lung damage was restored after eradication of the biofilm ( Figure 7 k).

[0150] Experimental Example 8

[0151] Experimental Example 8 of the present application provides a safety evaluation experiment for testing resistance to Pseudomonas aeruginosa infection.

[0152] The experimental process is as follows: the main organs of the mice in Implementation Case 7 were sliced ​​for histological examination. Blood was collected and analyzed using ELISA kits for blood biochemical indicators, including AST, ALT, BUN, LDH and CRE.

[0153] The experimental results showed that the blood biochemical indexes of mice were all within the normal range, indicating that DA-AZINPs had no adverse effects on liver and kidney function ( Figure 8 ae). Histological examination showed that the tissue structures of major organs such as heart, liver, spleen, and kidney remained normal without obvious lesions, proving that DA-AZI NPs have good biocompatibility in vivo ( Figure 8 f).

[0154] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A nanoparticle for resisting Pseudomonas aeruginosa infection, characterized in that: The nanoparticles shown include composite nanoparticles and antimicrobial drugs; The composite nanoparticles include PEGylated ε-polylysine modified with dimethyl maleic anhydride, polycaprolactone-polyethylene glycol and polycaprolactone-polylysine; The polycaprolactone-polyethylene glycol and polycaprolactone-polylysine are loaded with antibacterial drugs to construct cationic antibacterial nanoparticles; The dimethylmaleic anhydride-modified PEGylated ε-polylysine coats the cationic antibacterial nanoparticles.

2. The nanoparticles for resisting Pseudomonas aeruginosa infection according to claim 1, characterized in that: The antibacterial drugs include but are not limited to azithromycin, ciprofloxacin, tobramycin, streptomycin, erythromycin or a combination thereof.

3. A method for preparing nanoparticles for resisting infection by Pseudomonas aeruginosa according to any one of claims 1 to 2, characterized in that: The preparation method comprises the steps of: Step S1, dissolving polycaprolactone ε-polylysine, polycaprolactone-polyethylene glycol and an antibacterial drug in an organic solvent, adding distilled water and stirring overnight; Step S2: After stirring overnight, add PEGylated ε-polylysine modified with dimethylmaleic anhydride and stir to obtain nanoparticles that resist infection by Pseudomonas aeruginosa.

4. The method for preparing nanoparticles for resisting infection by Pseudomonas aeruginosa according to claim 3, characterized in that: In step S1, the preparation method of polycaprolactone ε-polylysine includes: ultrasonically dispersing polycaprolactone-carboxyl, N-(3-(dimethylamino)-propyl)-N-ethylcarbodiimide and N-hydroxysuccinimide in a solvent, stirring overnight under a nitrogen atmosphere, adding ε-polylysine and stirring overnight, dialyzing the product in water, and freeze-drying to obtain polycaprolactone ε-polylysine.

5. The method for preparing nanoparticles for resisting Pseudomonas aeruginosa infection according to claim 4, characterized in that: In step S2, the preparation method of the dimethylmaleic anhydride modified PEGylated ε-polylysine comprises: dissolving ε-polylysine in phosphate buffer, adding polyethylene glycol-active ester and stirring overnight under nitrogen flow, adding dimethylmaleic anhydride, adjusting the pH to greater than 7.0, reacting overnight, dialyzing in water, and freeze-drying to obtain dimethylmaleic anhydride modified PEGylated ε-polylysine.

6. The method for preparing nanoparticles for resisting Pseudomonas aeruginosa infection according to claim 4, characterized in that: In step S1, the antibacterial drugs include but are not limited to azithromycin, ciprofloxacin, tobramycin, streptomycin, erythromycin or a combination thereof, and the mass ratio of polycaprolactone-polyethylene glycol to polycaprolactone-polylysine is 1:1-16:

1.

7. A preparation for pulmonary inhalation, characterized in that: The pulmonary inhalation preparation comprises the nanoparticles for resisting Pseudomonas aeruginosa infection according to any one of claims 1 to 2 and pharmaceutical excipients.

8. A pulmonary inhalation preparation according to claim 7, characterized in that: The pulmonary inhalation formulations include, but are not limited to, inhalation aerosols, dry powder inhalers, inhalation sprays or inhalation solutions.

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