Phosphorus-containing dendrimer / antibacterial peptide composite material wrapped by exosome as well as preparation and application of phosphorus-containing dendrimer / antibacterial peptide composite material

Abstract

The invention relates to an exosome-coated phosphorus-containing dendrimer / antibacterial peptide composite material as well as preparation and application thereof. The exosome-coated phosphorus-containing dendrimer / antibacterial peptide composite material comprises a phosphorus-containing dendrimer, a peptide and an exosome. The preparation method disclosed by the invention has the characteristics of simplicity, high reaction process controllability, easiness in operation and separation and the like, the intracellular transmission efficiency and antibacterial activity of nisin can be improved, efficient removal of mycobacterium smegmatis is realized by virtue of an exosome-mediated host targeting function, and the preparation method has a good development prospect and application value in treatment of pulmonary tuberculosis patients.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to an exosome-encapsulated phosphorus-containing dendrimer / antimicrobial peptide composite material and its preparation and application. Background Art

[0002] Tuberculosis (TB) is a life-threatening chronic infectious disease caused by infection with Mycobacterium tuberculosis (M.tb), posing a serious threat to public health worldwide (Lancet Microbe 2023, 4, e20). According to the World Health Organization, 10 million people were diagnosed with TB in 2017, with a global economic burden of US$12 billion annually and projected to cost the global economy US$16.7 trillion by 2050 (Nat. Nanotechnol. 2021, 16, 369-384). Antibiotics such as isoniazid, rifampicin, pyrazinamide, ethambutol, and streptomycin are the main first-line anti-TB drugs in clinical practice. Currently, the clinical strategy for the treatment of TB is to use isoniazid, rifampicin, pyrazinamide, and ethambutol for 2 months, followed by a consolidation regimen of isoniazid and rifampicin for another 6 months. However, due to the poor solubility, instability in gastric acid, poor granuloma penetration, and poor host cell targeting of antibiotics, long-term and frequent combination dosing strategies are required to achieve optimal therapeutic effects. However, this can lead to the development of multidrug-resistant TB, and long-term treatment can lead to poor patient compliance, further complicating TB cure (PLoS One 2017, 12, e0172748).

[0003] Antimicrobial biomaterials, such as polycationic polymers, and biomaterial-assisted non-antibiotic therapies, such as bacteriophages, antimicrobial peptides (AMPs) and antimicrobial enzymes, have improved people's ability to treat antibiotic resistance and recurrent infections (Nat. Rev. Mater. 2022, 7, 39-54). Nisin is a natural polypeptide substance composed of 34 amino acid residues produced by Streptococcus lactis. It exerts its antibacterial effect by binding to the lipid II component of the bacteria to block the integrity of the bacterial cell wall (J. Chem. Inf. Model. 2024, 64, 7977-7986). However, Nisin is easily degraded by proteases in physiological environments or has difficulty crossing the cell membrane barrier to act on pathogens in host cells.

[0004] Among numerous nanocarriers, sodium phosphite- or hydroxyl-terminated phosphorus-containing dendrimers have been reported as promising protein delivery vehicles. They can complex with various proteins through various physical interactions and efficiently transfect into target cells (ACS Nano 2024, 18, 2195–2209), while also protecting the delivery targets from degradation by proteases (Biomaterials 2025, 316, 122999). In addition to their protein delivery properties, these phosphorus-containing dendrimers also exhibit a variety of inherent immunomodulatory activities, particularly anti-inflammatory activity. For example, sodium phosphite-terminated phosphorus-containing dendrimers have been used for anti-inflammatory treatments of acute lung injury and arthritis by inducing macrophage polarization toward the M2 phenotype and blocking the rapid translocation of nuclear factor kappa-B (NF-κB) from the cytoplasm to the nucleus (Theranostics 2022, 12, 3407–3419). Hydroxyl-modified phosphorus-containing dendrimers, while exhibiting immunomodulatory activity, can cross the damaged blood-brain barrier with their rich surface hydroxyl groups, achieving brain immune microenvironment regulation and functional behavioral recovery in Parkinson's and Alzheimer's diseases (Bioact.Mater.2024,38,45-54; Nano Today 2025,61,102664). Importantly, the introduction of cyclic amino compounds such as pyrrole, pyridine, and piperidine in the structure of phosphorus-containing dendrimers makes this polycationic polymer a new anti-tuberculosis drug, showing a certain inhibitory effect on attenuated Mycobacterium tuberculosis H37Ra, virulent Mycobacterium tuberculosis H37Rv and BCG (Biomacromolecules2021,22,2659-2675). Especially the 0th generation piperidine-terminated phosphorus-containing dendrimers (2G0 HCl ) still showed efficacy against single-drug resistant Mycobacterium tuberculosis H37Rv (resistant to rifampicin, isoniazid, ethambutol, or streptomycin). However, phosphorus-containing dendrimers alone were unable to penetrate the core of granulomas and had difficulty targeting host cells.

[0005] However, TB lesions are often accompanied by fibrosis, necrotic tissue, or caseous material. Nanoparticles may have difficulty effectively penetrating into the core of TB lesions due to size or surface charge limitations. How to enhance the ability of the complex to target granulomas and host cells is an urgent problem to be solved. Exosomes (Exo) are nanoscale membrane vesicles (30-200 nm in diameter) secreted by cells, which carry numerous active ingredients such as proteins, lipids, and nucleic acids. They are widely involved in intercellular communication, immune regulation, and inflammatory responses (Nat. Immunol. 2021, 22, 560-570). Exo has become an ideal choice for constructing biomimetic nanoplatforms due to its natural cellular uptake ability and inherent targeting properties. In addition, the natural composition of its lipid bilayer and the expression of multiple surface glycoproteins (such as prostaglandin F2 receptor negative regulator) can effectively enhance the delivery of drugs to neutrophils and macrophages (Sci. Adv. 2022, 8, eabj7002). However, it is far from enough to inhibit the excessive proliferation of intracellular Mycobacterium tuberculosis simply by improving the lysosomal autophagy pathway of host cells.

[0006] A search of domestic and foreign literature and patents has not yet found any reports on the use of exosome-encapsulated phosphorus-containing dendrimer / antimicrobial peptide nanocomplexes for the treatment of pulmonary tuberculosis. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide an exosome-encapsulated phosphorus-containing dendrimer / antimicrobial peptide composite material, as well as its preparation and application. The present invention utilizes the inflammatory tropism and active targeting function of exosomes to effectively penetrate the core of TB lesions and be targeted for phagocytosis by host cells. On the one hand, the dual antimicrobial activity and multiple antimicrobial mechanisms of the cationic phosphorus-containing dendrimer and antimicrobial peptide directly inhibit the proliferation of intracellular parasitic mycobacteria and prevent the development of drug-resistant strains. On the other hand, the contents carried by the infectious exosomes activate the phagosome-lysosome autophagy pathway, restarting the bacterial degradation function of the lysosomes and synergistically reducing the bacterial load in the host cells. Simultaneously, the macrophage M2 polarization properties of the phosphorus-containing dendrimers calm the inflammatory response activated during infection and antimicrobial treatment, addressing the lung damage caused by tuberculosis and achieving highly effective TB treatment mediated by a "host-directed, multimodal antimicrobial, and immune remodeling" strategy.

[0008] The present invention provides a phosphorus-containing dendrimer composite material, which comprises phosphorus-containing dendrimers, peptides, and exosomes; wherein the phosphorus-containing dendrimers and peptides are complexed to form a nanocomposite, and the surface of the nanocomposite is coated with exosomes.

[0009] The molecular formula of the phosphorus-containing dendrimer is C 336 H 576 N 87 O 66 P21 S 18 , structural formula

[0010]

[0011] The exosomes are one of infected exosomes and uninfected exosomes;

[0012] The peptide is an antimicrobial peptide, wherein the antimicrobial peptide is Nisin.

[0013] The present invention provides a method for preparing a phosphorus-containing dendrimer composite material, comprising:

[0014] (1) mixing a phosphorus-containing dendrimer, a peptide, and a solvent, and stirring at room temperature to obtain a phosphorus-containing dendrimer / peptide nanocomposite;

[0015] (2) Exosomes, phosphorus-containing dendrimer / peptide nanocomposites, and a solvent are mixed and ultrasonically treated to obtain a phosphorus-containing dendrimer composite material, i.e., an exosome-encapsulated phosphorus-containing dendrimer / antimicrobial peptide composite material.

[0016] Furthermore, in step (1), the phosphorus-containing dendrimer is added dropwise to the peptide solution, stirred at room temperature, and centrifuged; wherein the solvent of the peptide solution is water, wherein the water is ultrapure water.

[0017] Preferably, the molar ratio of the phosphorus-containing dendrimer to the peptide in step (1) is 0.5 to 6:1; the solvent is water, wherein the water is ultrapure water; wherein the peptide is Nisin; the molecular formula of the phosphorus-containing dendrimer is C 336 H 576 N 87 O 66 P 21 S 18 , molecular weight is 8118.36g / mol.

[0018] Preferably, the mixture is stirred at room temperature for 2 to 4 hours in step (1); centrifugation is performed after the stirring at room temperature in step (1); and the centrifugation process parameters are as follows: the complex solution is transferred to a 5000 to 10000 Da ultrafiltration centrifuge tube and centrifuged at 8000 to 12000 rpm for 5 to 15 minutes.

[0019] Preferably, the exosomes in step (2) are one of infected exosomes and uninfected exosomes.

[0020] Preferably, the infectious exosomes are exosomes secreted by macrophages infected with Mycobacterium smegmatis;

[0021] The preparation of exosomes secreted by macrophages infected with Mycobacterium smegmatis includes: culturing alveolar macrophages MH-S infected with Mycobacterium smegmatis M.sm with exosome culture medium, collecting cell culture fluid, and obtaining infectious exosomes after centrifugation.

[0022] In the preparation of exosomes secreted by macrophages infected with Mycobacterium smegmatis, the multiplicity of infection (MOI) of M.sm is 10; the culture time is 48 to 72 hours; the centrifugation process parameters are as follows: centrifugation at 300-500g for 5-10 minutes to remove cells, centrifugation at 3000-5000g for 10-15 minutes to remove cell debris, collecting the supernatant and filtering it through a 0.45μm filter membrane, and centrifuging it at 10000-120000g for 30-45 minutes. The supernatant is collected and centrifuged at 100000-120000g for 50-85 minutes, then the supernatant is discarded, the precipitate is resuspended in PBS and centrifuged at 100000-120000g for 50-85 minutes to collect the precipitate, and the temperature of all centrifugation steps is 4°C.

[0023] Furthermore, in step (2), the exosomes and the phosphorus-containing dendrimer / peptide nanocomplex are mixed, and the exosomes are reassembled on the surface of the phosphorus-containing dendrimer / peptide nanocomplex by an ultrasound-mediated dissociation-self-assembly process to obtain a phosphorus-containing dendrimer composite material.

[0024] Preferably, in step (2), the mass ratio of exosomes to phosphorus-containing dendrimers / peptide nanocomplexes is 0.05-0.2:1;

[0025] Preferably, the ultrasonic treatment in step (2) comprises performing an 8-10 second on and 8-10 second off cycle at 20-50 Hz, for 3-5 minutes, and for 3-5 cycles.

[0026] The phosphorus-containing dendrimer of the present invention is complexed with nisin to form a stable and uniform nanocomposite, and then exosomes secreted by macrophages infected with Mycobacterium smegmatis are extracted and assembled on the surface of the complex to form an exosome-encapsulated phosphorus-containing dendrimer / antimicrobial peptide nanocomposite; the molecular formula of the phosphorus-containing dendrimer is C 336 H 576 N 87 O 66 P 21 S 18 , with a molecular weight of 8118.36 g / mol. The present invention utilizes commercially available raw materials, a simple preparation method, highly controllable reaction processes, and easy operation and separation. It can improve the intracellular delivery efficiency and antibacterial activity of nisin, and achieve efficient clearance of Mycobacterium smegmatis through exosome-mediated host targeting. It has promising development prospects and application value in the treatment of pulmonary tuberculosis patients.

[0027] The present invention provides an application of any of the phosphorus-containing dendrimer composite materials in the preparation of antibacterial and anti-inflammatory drugs.

[0028] The present invention provides an application of any phosphorus-containing dendrimer composite material in preparing a drug for treating pulmonary tuberculosis.

[0029] (1) The M.sm strain was isolated and mixed with 146 / FITC-Nisin and 146 / FITC-Nisin@Exo. N 、146 / FITC-Nisin@Exo I The nanocomplex was mixed at room temperature for 5 min, and the adsorption of the material on the bacterial surface was observed by laser confocal microscopy (CLSM). (2) The M.sm strain was mixed with 146, Nisin, 146 / Nisin, 146 / Nisin@Exo N 、146 / Nisin@Exo I The nanocomplexes were incubated in a constant temperature shaker at 37°C for 12 h, and the colony forming units (CFU) of different treatment groups were quantified by the dilution coating plate method to evaluate the extracellular antibacterial ability of the nanomaterials.

[0030] (3) MH-S cells were plated in 96-well plates and cultured at 37°C and 5% CO2 for 24 h. The CCK-8 assay was used to evaluate the expression of 146, Nisin, 146 / Nisin, and 146 / Nisin@Exo. I Cytotoxicity of the nanocomplexes.

[0031] (4) MH-S cells were plated in 12-well plates and cultured at 37°C and 5% CO2 for 24 h. FITC-Nisin, 146 / FITC-Nisin, and 146 / FITC-Nisin@Exo were added 4 h after infection with M.sm. N 、146 / FITC-Nisin@Exo I The nanocomplexes were incubated with infected cells for 4 h, and the cells were collected, washed three times with PBS, and the phagocytic activity of the nanocomplexes was evaluated by flow cytometry.

[0032] (5) MH-S cells were plated in 12-well plates and cultured at 37°C in a 5% CO2 environment for 24 h. 146, Nisin, 146 / Nisin, and 146 / Nisin@Exo were added 4 h after infection with M.sm. N 、146 / Nisin@Exo I The nanocomplex was incubated with infected cells for 24 h. The cells were collected, washed three times with PBS, and lysed with 0.05% sodium dodecyl sulfate (SDS). The intracellular antibacterial activity of the nanomaterial was evaluated by the dilution plate method.

[0033] (6) MH-S cells were plated in 12-well plates and cultured at 37°C in a 5% CO2 environment for 24 h. 146, Nisin, 146 / Nisin, and 146 / Nisin@Exo were added 4 h after infection with M.sm. N 、146 / Nisin@Exo I The nanocomplexes were incubated with infected cells for 24 hours. The cells were then collected, stained with CD206 and CD86 fluorescent antibodies, washed with PBS, and the effects of the nanomaterials on macrophage polarization were analyzed by flow cytometry.

[0034] (7) 146 / Nisin, 146 / Nisin@Exo N 、146 / Nisin@Exo I Antibiotics were administered via pulmonary aerosol to the lungs of mice with M. sm-induced pulmonary tuberculosis. Model mice were treated with PBS as a control. Treatment was repeated every other day for a total of five times. After treatment, the mice were sacrificed and lung tissue was collected to prepare single-cell suspensions. The cells were lysed with SDS, diluted, and plated to measure colony-forming unit counts to evaluate the antimicrobial efficacy.

[0035] (8) After the in vivo treatment, the mice were sacrificed to obtain lung tissue for hematoxylin and eosin (H&E) staining to observe the changes in granulomas and damage recovery in the lung tissue.

[0036] (9) After the in vivo treatment, the bronchoalveolar lavage fluid (BALF) of the mice was collected and the expression of different inflammatory cytokines was detected by enzyme-linked immunosorbent assay (ELISA).

[0037] The present invention uses zeta potential and dynamic light scattering analysis (DLS), fluorescence spectroscopy, transmission electron microscopy (TEM), protein immunoblotting (WB), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and other methods to characterize the physical and chemical properties of the prepared nanomedicine. The bacterial targeting ability of the material is observed by CLSM; the extracellular antibacterial ability of the material is evaluated by the dilution plate method; the cytotoxicity of the material is evaluated using the CCK-8 method; the phagocytosis of the material by cells is detected by flow cytometry; the intracellular antibacterial ability of the material is evaluated by the dilution plate method; the effect of the material on macrophage typing is evaluated by flow cytometry; and finally, an in vivo pulmonary tuberculosis animal model is established. The antibacterial and anti-inflammatory therapeutic effects of the material on pulmonary tuberculosis are evaluated by counting the CFU of lung tissue, H&E staining, and ELISA.

[0038] Beneficial effects

[0039] (1) The process of the present invention is simple, the required reaction conditions are simple, the product is easy to operate and separate, and it has good development prospects.

[0040] (2) The exosome biomimetic nanomedicine prepared by the present invention has good cell compatibility, can enhance the host targeting ability of the antibacterial active ingredients (146 and Nisin) and enhance the intracellular delivery effect of Nisin, providing a new idea for the construction of safe and effective drug carriers.

[0041] (3) The exosome biomimetic nanomedicine prepared by the present invention utilizes the inflammatory tropism of Exo to target the infected lesions, and triple blocks the survival pathway of intracellular mycobacteria through the synergistic antibacterial activity of Nisin and cationic phosphorus-containing dendrimers combined with the host cell phagosome-lysosome autophagy process activated by Exo; at the same time, with the help of phosphorus-containing dendrimer 146, macrophages are regulated to polarize toward the M2 phenotype, the immune microenvironment is reshaped to promote lung tissue repair, and the antibacterial and anti-inflammatory comprehensive treatment of pulmonary tuberculosis is achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 146 / Nisin@Exo prepared by the present invention I Schematic diagram of the synthesis of the nanocomplex and its treatment of tuberculosis;

[0043] Figure 2 The hydration kinetics size (A), polymerization dispersion index (B), and surface potential (C) of 146 / Nisin prepared at different 146 / Nisin molar ratios are shown;

[0044] Figure 3 Fluorescence emission spectra of FITC-Nisin at different 146 / Nisin molar ratios (A) and Nisin loading rate and encapsulation efficiency in the prepared 146 / Nisin complex (B);

[0045] Figure 4 Exo prepared by the present invention I (A)、146 / Nisin(B)、146 / Nisin@Exo I (C) TEM image;

[0046] Figure 5 Exo prepared by the present invention I 、146 / Nisin、146 / Nisin@Exo I SDS-PAGE images (A) and MH-S cells, Exo I 、146 / Nisin@Exo I Western blot images of TSG101, CD63, and Calnexin expression in the mouse model (B);

[0047] Figure 6 146 / FITC-Nisin and 146 / FITC-Nisin@Exo prepared by the present invention N 、146 / FITC-Nisin@Exo I CLSM images of colocalization with M.sm strains;

[0048] Figure 7 146, Nisin, 146 / Nisin, 146 / Nisin@Exo prepared by the present invention N and 146 / Nisin@Exo I Representative CFU graph (A) and quantitative results (B) after 12 h of co-incubation with M.sm strains;

[0049] Figure 8 Nisin (A) and 146, 146 / Nisin, 146 / Nisin@Exo prepared by the present invention I (B) Cell viability after 24 h of co-incubation with MH-S cells;

[0050] Figure 9 Nisin, 146 / Nisin, 146 / Nisin@Exo prepared by the present invention N and 146 / Nisin@Exo I Representative flow cytometry images (A) and fluorescence intensity quantification images (B) after 4 h of co-incubation with M.sm-infected MH-S cells;

[0051] Figure 10 146, Nisin, 146 / Nisin, 146 / Nisin@Exo prepared by the present invention N and 146 / Nisin@Exo I Representative CFU (A) and quantitative results (B) of intracellular M.sm content after co-incubation with M.sm-infected MH-S cells for 24 h; Figure 11 146, Nisin, 146 / Nisin, 146 / Nisin@Exo prepared by the present invention N and 146 / Nisin@Exo I Representative flow cytometric images of cell surface CD86 and CD206 expression levels after co-incubation with M.sm-infected MH-S cells for 24 h (A) and quantitative results of CD206 / CD86 (B);

[0052] Figure 12 The representative CFU (A) and quantitative results (B) of the M.sm content in the lung tissue of mice in each experimental group in Example 13;

[0053] Figure 13 Figure 14 shows the H&E staining results of lung tissues of mice in each experimental group (scale bar: 2 mm);

[0054] Figure 14 The expression levels of pro-inflammatory cytokines (AC) and anti-inflammatory cytokines (D) in the BALF of each experimental mouse in Example 15. DETAILED DESCRIPTION

[0055] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0056] Unless otherwise specified, all chemical reagents were used directly without further purification. Cationic phosphorus-containing dendrimer 146 was obtained from the research group of Professor J.P. Majoral at the French National Center for Scientific Research. Nisin was obtained from MedChemExpress. M.sm was obtained from Shanghai Pulmonary Hospital. MH-S cells (a mouse macrophage cell line) and RPMI-1640 complete medium were obtained from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. 7H9 and 7H10 bacterial culture media were obtained from Becton Dickinson. GOADC-Mycobacterium enrichment broth was obtained from Shanghai Jingnuo Biotechnology Co., Ltd. The CCK-8 kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd. C57BL / 6 mice were purchased from Shanghai Regen Biotechnology Co., Ltd. Antibodies such as Anti-CD86-PE and Anti-CD206-FITC were purchased from Thermo Fisher Scientific. Water with a resistivity greater than 18.2 MΩ.cm used in all experiments was purified using a laboratory water purification system (Cascada I, PALL, Beijing).

[0057] Example 1

[0058] The phosphorus-containing dendrimer 146 (400 μM) was added dropwise to the Nisin solution (100 μL, 200 μM) at different molar ratios (0.5:1, 1:1, 2:1, 4:1, 6:1) and the reaction system was supplemented to 1 mL with ultrapure water. After stirring at room temperature for 2 to 4 hours, the reaction mixture was transferred to an ultrafiltration centrifuge tube with a molecular weight cutoff of 5000 Da and centrifuged at 12000 rpm for 10 minutes to remove uncomplexed Nisin. The complex retained in the upper chamber of the ultrafiltration centrifuge tube was resuspended in 1 mL of ultrapure water to obtain the 146 / Nisin complex. First, the hydration kinetic size and surface potential of the 146 / Nisin complex at different feed ratios were detected by DLS. The results are shown in Figure 2. Figure 2 As shown in the figure, with the gradual addition of cationic phosphorus-containing dendrimer 146, the particle size of the 146 / Nisin complex gradually decreases and the surface potential gradually increases. When the molar ratio of 146 to Nisin is 4:1, the hydration kinetic size of the 146 / Nisin complex decreases from 425 nm for Nisin alone to 193.8 nm, and it has a minimum polydispersity coefficient (0.34), indicating that a more compact nanocomposite structure is formed between 146 and Nisin at this feed ratio.

[0059] Example 2

[0060] In order to further explore the interaction between phosphorus-containing dendrimers and Nisin, fluorescein isothiocyanate (FITC) and Nisin were mixed and stirred at room temperature in a molar ratio of (3:1) for 24 hours, and the mixed solution after the reaction was transferred to a 3000Da dialysis bag for 3 days to remove free FITC. The solution in the dialysis bag was collected and freeze-dried to obtain FITC-labeled Nisin (FITC-Nisin). 146 was added dropwise to the FITC-Nisin aqueous solution in accordance with the molar ratios (0.5:1, 1:1, 2:1, 4:1, 6:1) in Example 1, and the spectral change curves of FITC-Nisin at each feed ratio were monitored in real time by a fluorescence spectrometer to evaluate the complex behavior of 146 and Nisin. The results are shown in Figure 2. Figure 3 As shown in Figure A, the increase in the feed ratio of 146 leads to a gradual quenching of the fluorescence intensity of FITC-Nisin. This may be due to the aggregation of FITC-Nisin induced by 146, resulting in aggregation-induced quenching of the fluorescence signal. The unloaded Nisin was quantified by the fluorescence signal triggered by fluorescamine and the amino group of Nisin. At a molar ratio of 146 to Nisin of 4:1, the encapsulation efficiency and drug loading efficiency of Nisin in the 146 / Nisin complex reached 84.6% and 8.1%, respectively ( Figure 3B). Combined with the DLS data, a molar ratio of 4:1 between 146 and Nisin was selected as the optimal ratio for preparing the 146 / Nisin complex for subsequent material preparation and experiments.

[0061] Example 3

[0062] (1) MH-S cells were passaged into 75T cell culture flasks and cultured in a constant temperature incubator at 37°C and 5% CO2. When the cell density in the culture flask was above 70%, freshly cultured Mycobacterium smegmatis (bacteria / cell ratio = 10:1) was added to the culture flask to infect the cells for 4 hours. Then, the cells were washed three times with PBS to remove extracellular free bacteria. The infected cells were cultured with fresh exosome extraction medium for 48 to 72 hours. Finally, the cell culture medium was collected and subjected to density gradient centrifugation and ultracentrifugation at 4°C to extract exosomes.

[0063] (2) The cell culture medium collected in step (1) was centrifuged at 300g for 10 minutes to remove the cells in the precipitated portion. The supernatant was further centrifuged at 3000g for 10 minutes to remove the dead cells and cell debris in the precipitated portion. The collected supernatant was filtered through a filter membrane with a pore size of 0.45 μm, and then centrifuged at 10000g for 30 minutes to remove apoptotic bodies. The collected supernatant was centrifuged at 100000g for 75 minutes and then discarded. The precipitate was resuspended with PBS, and the precipitate was collected after centrifugation at 100000g for 75 minutes to obtain infectious exosomes (Exo I ).

[0064] (3) The Exo prepared in step (2) I The mixture was thoroughly mixed with the 146 / Nisin complex prepared in Example 1 at a mass ratio of 0.1:1. The mixture was then ultrasonically treated in an ice bath with a cycle of 10 s on, 10 s off, 20 Hz, and a duration of 5 min, repeated 3 times to obtain Exo I Encapsulated 146 / Nisin nanocomplex (146 / Nisin@Exo I The same procedure was used to extract exosomes from the culture medium of uninfected MH-S cells (Exo N ) and wrapped on the surface of 146 / Nisin complex to obtain the control material 146 / Nisin@Exo N .

[0065] Example 4

[0066] Take 146 / Nisin prepared in Example 1 and Exo prepared in Example 3 I and 146 / Nisin@Exo IThe hydration dynamics size and surface potential were characterized. The results are shown in Table 1. I The particle size of the nanocomposite is I After encapsulation, the surface potential increased from 193.8 nm of 146 / Nisin to 232.1 nm, and the surface potential decreased from 27.1 mV to -10.28 mV, which is close to that of the single Exo I (-12.8mV), which indicates that Exo I It was successfully coated on the surface of the 146 / Nisin composite and had good dispersibility (PDI=0.29).

[0067] Table 1 Hydration kinetic size, PDI and surface potential results of different materials

[0068] Sample Hydrodynamic size (nm) PDI Zetapotential(mV) <![CDATA[Exo I ]]> 223.7±6.5 0.24±0.06 -12.8±0.88 146 / Nisin 193.8±8.8 0.34±0.03 27.1±1.15 <![CDATA[146 / Nisin@Exo I ]]> 232.1±16.2 0.29±0.06 -10.28±1.61

[0069] Example 5

[0070] The 146 / Nisin prepared in Example 1 and the Exo I and 146 / Nisin@Exo I Prepare a sample with a concentration of 0.1 mg / mL. Drop 146 / Nisin onto the surface of a carbon-filmed copper mesh and let it dry at room temperature to obtain a sample. I and 146 / Nisin@Exo I After dripping onto the copper mesh, let it stand at room temperature for 10 to 15 minutes, gently remove the excess liquid and add 2% phosphotungstic acid (pH 6.5 to 7.0) for staining for 2 minutes. Remove the excess phosphotungstic acid and dry it to obtain the sample. The size and morphology of the sample were then examined using a JEM-2010F transmission electron microscope. The results are as follows: Figure 4 As shown in A, a single Exo I The 146 / Nisin nanocomplex showed a uniformly distributed spherical structure with an average size of 91.5 nm ( Figure 4 B) After Exo I After wrapping, 146 / Nisin@Exo I The size of the nanocomposite increased to 174.2 nm and exhibited a well-defined core-shell structure, further demonstrating that Exo I Successfully wrapped on 146 / Nisin surface ( Figure 4 C).

[0071] Example 6

[0072] To further verify 146 / Nisin@ExoI Exo I The successful encapsulation and structural integrity of Exo were verified by SDS-PAGE and WB, respectively. I Electrophoretic bands in the nanocomplex and the expression of exosome-related characteristic markers. Figure 5 As shown in A, 146 / Nisin@Exo I Show with Exo I The same electrophoresis bands were obtained, proving that Exo I Successfully coated on the surface of 146 / Nisin nanocomposite. WB test results are as follows Figure 5 As shown in B, TSG101 and CD63 are positive markers of exosomes. I and 146 / Nisin@Exo I The expression of calnexin in the endoplasmic reticulum was high in MH-S cells, while calnexin as an endoplasmic reticulum marker was only detected in MH-S cells. I It has the typical characteristics of exosomes, and the preparation process of the biomimetic nanocomposite does not affect the retention of the functional components of exosomes.

[0073] Example 7

[0074] To verify 146 / Nisin@Exo I For the targeting of M.sm, FITC-labeled Nisin was used to obtain 146 / FITC-Nisin and 146 / FITC-Nisin@Exo according to the above preparation method. N and 146 / FITC-Nisin@Exo I Nanocomplex. The three materials mentioned above (50 μL, [Nisin] = 1.25 μM) were mixed with M. sm bacterial suspension (50 μL, 1×10 7 CFU / mL) were mixed thoroughly at room temperature for 5 min. After washing with PBS, the co-localization of bacteria and materials was observed using CLSM. Figure 6 As shown in the figure, compared with the PBS group, only a small amount of 146 / FITC-Nisin was observed to be localized on the M.sm bacteria. N and 146 / FITC-Nisin@Exo I The affinity of 146 / Nisin@Exo to the bacterial surface showed a significantly higher green fluorescence signal than that of the 146 / Nisin-treated group, which indicated that the prepared 146 / Nisin@Exo I The nanocomplex is expected to exert enhanced antibacterial effects by targeting mycobacteria.

[0075] Example 8

[0076] To verify 146, Nisin, 146 / Nisin, 146 / Nisin@Exo I and 146 / Nisin@Exo N The extracellular antibacterial activity of 146, Nisin, 146 / Nisin, and 146 / Nisin@Exo I and 146 / Nisin@Exo N (

[146] =25μM, [Nisin]=6.25μM, 100μL) were added to the solution containing M.sm (5×10 6 CFU / mL, 0.9mL) bacterial suspension was incubated in a 7H9 medium at 37°C, 180 rpm constant temperature shaker for 12 hours. Then, each group of bacterial suspension was graded diluted with 7H9 medium, and 100μL of the diluted bacterial suspension was added to 7H10 agar plates for coating. After incubation in a 37°C constant temperature incubator for 48-72 hours, the number of CFU in different treatment groups was counted. The results are shown in Figure 2. Figure 7 As shown in Figure 1, both 146 and Nisin exhibited a certain degree of antibacterial activity. I The nanocomposite exhibits synergistic antibacterial activity of 146 and Nisin, and through the I The encapsulation enhances its adhesion to bacteria and shows a significantly enhanced ability to inhibit bacterial proliferation.

[0077] Example 9

[0078] Evaluation of 146, Nisin, 146 / Nisin, and 146 / Nisin@Exo using the CCK-8 method I Effect on MH-S cell viability. MH-S cells in the logarithmic growth phase were collected and 1×10 4 The cells were seeded into 96-well cell culture plates at a density of 10 cells per well and cultured at 37°C in a 5% CO2 environment for 24 hours. I The cells were incubated with fresh culture medium (146 concentrations were 0, 0.16, 0.31, 0.63, 1.25, 2.5, and 5 μM, and Nisin concentrations were 0, 0.04, 0.08, 0.16, 0.31, 0.63, and 1.25 μM, respectively) for 24 hours, the original culture medium was discarded, the cells were washed three times with PBS, and fresh serum-free culture medium containing 10% (v / v) CCK-8 was added and incubated in an incubator for another 2-3 hours. The absorbance of each well was measured at a wavelength of 450 nm using a multifunctional microplate reader. Cells treated with PBS were used as controls, and cell viability was recorded as 100%. The results are shown in Figure 2. Figure 8As shown in AB, within the experimental concentration range, various materials, especially 146 / Nisin@Exo I The viability of the treated MH-S cells was above 80%, indicating that the prepared biomimetic nanocomplex had good cell compatibility and could be used as a safe nanomedicine in subsequent experiments.

[0079] Example 10

[0080] M.sm-infected MH-S cells were used as a cell model to validate the 146 / Nisin@Exo I The host cell targeting efficiency of the complex was measured by collecting cells in the logarithmic growth phase and quantifying the effect of 1×10 5 The cells were seeded in a 12-well cell culture plate at a density of 10 cells per well and cultured at 37°C for 24 hours. Freshly cultured M.sm strains were added to the plate at a multiplicity of infection of 10 and infected for 4 hours. The cells were then washed with PBS to remove extracellular free M.sm and replaced with a medium containing Nisin, 146 / Nisin, 146 / Nisin@Exo. N and 146 / Nisin@Exo I Cells were incubated with fresh serum-free RPMI-1640 medium (using FITC-labeled Nisin, [Nisin] = 0.63 μM) for 4 h. The medium was discarded, and the cells were trypsinized, centrifuged, and collected. After washing three times with PBS, the intracellular fluorescence intensity was measured by flow cytometry. Figure 9 As shown in Figures AB, the 146 / Nisin nanocomplex can enhance the intracellular delivery efficiency of Nisin, which provides favorable conditions for Nisin to target pathogens within host cells. Because infected exosomes carry pathogen-associated antigens on their surface, they can specifically target Toll-like receptors on the surface of macrophages, thereby demonstrating optimal host cell targeting ability.

[0081] Example 11

[0082] M.sm-infected MH-S cells were used as a cell model to validate the 146 / Nisin@Exo I The intracellular antibacterial activity of the complex was investigated. Cells in the logarithmic growth phase were collected and 1×10 5 The cells were seeded at a density of 10 cells per well in a 12-well cell culture plate and cultured at 5% CO2 and 37°C for 24 hours. Freshly cultured M.sm strains were added to the plate at a multiplicity of infection of 10 and infected for 4 hours. The cells were then washed with PBS to remove extracellular free M.sm and replaced with 146, Nisin, 146 / Nisin, 146 / Nisin@Exo N and 146 / Nisin@ExoI The cells were incubated with fresh RPMI-1640 medium (

[146] = 2.5 μM, [Nisin] = 0.63 μM) for 24 hours. The culture medium was discarded, the cells were digested with trypsin, centrifuged, and collected. After washing three times with PBS, 100 μL of SDS lysis buffer (0.05%) was added to lyse the cells for 5 minutes. After gradient dilution of the cell lysate, 100 μL was spread on a 7H10 agar plate and placed in a 37°C constant temperature incubator for 48 to 72 hours. The number of CFU in different treatment groups was counted. The 146 / Nisin nanocomplex showed an intracellular antibacterial therapeutic effect that was superior to that of Nisin alone by enhancing the intracellular delivery efficiency of Nisin and synergizing the antibacterial activity of the carrier itself. Importantly, 146 / Nisin@Exo N The CFU count of the treated group was lower than that of the 146 / Nisin treated group, which may be due to the homologous targeting mediated by macrophage-derived exosomes to enhance the phagocytic performance of host cells to the material. Similarly, in addition to the homologous targeting effect, the pathogens carried by the infectious exosomes can further recognize the Toll-like receptors on the surface of macrophages, so 146 / Nisin@Exo I The treatment group had the lowest intracellular CFU number ( Figure 10 These data indicate that the prepared 146 / Nisin@Exo I It can target host macrophages and effectively inhibit the proliferation of intracellular pathogens, providing important guarantees for the in vivo treatment of pulmonary tuberculosis.

[0083] Example 12

[0084] M.sm-infected MH-S cells were used as a cell model to validate the 146 / Nisin@Exo I The macrophage polarization ability of the complex was investigated. Cells in the logarithmic growth phase were collected and 1×10 5 The cells were seeded in a 12-well cell culture plate at a density of 10 cells per well and cultured at 37°C for 24 hours. Freshly cultured M.sm strains were added to the plate at a multiplicity of infection of 10 and infected for 4 hours. The cells were then washed with PBS to remove extracellular free M.sm and replaced with PBS containing 146, Nisin, 146 / Nisin, 146 / Nisin@Exo. N and 146 / Nisin@Exo IThe cells were incubated with fresh RPMI-1640 medium (

[146] = 2.5 μM, [Nisin] = 0.63 μM) for 24 hours. The culture medium was discarded, the cells were digested with trypsin, centrifuged, and collected. The cell pellet was resuspended with 300 μL PBS. Anti-CD206-FITC and Anti-CD86-PE antibodies were added to the cells and incubated with the cells at 4°C in the dark for 30 minutes. After washing three times with PBS to remove the antibodies that were not bound to the cells, the cell pellet was resuspended with 300 μL PBS and transferred to a flow cytometer. The effect of the nanomedicine on macrophage polarization was evaluated by detecting the changes in the expression levels of CD86 and CD206 in the cells. The results are shown in Figure 2. Figure 11 As shown in Figure 2, M.sm infection leads to the polarization of macrophage phenotype toward M1, which may be because the lipopolysaccharide components on the bacterial surface activate the inflammatory signaling pathway in macrophages. N and 146 / Nisin@Exo I Treatment can significantly induce the polarization of M1 phenotype macrophages to M2 phenotype. This immunomodulatory activity is mainly due to the presence of phosphorus-containing dendrimer 146, because Nisin alone has no significant contribution to macrophage polarization.

[0085] Example 13

[0086] All animal experiments were conducted in strict accordance with the standards of the Animal Care Association. 7 CFU / mouse) was administered by nasal drops to the lungs of four-week-old male C57BL / 6J mice once a day. After 14 days, a TB mouse model with obvious granulomatous pathological characteristics was successfully established. The TB mouse model was divided into 5 groups, PBS, 146 / Nisin, 146 / Nisin@Exo N 、46 / Nisin@Exo I(100 μL, [146 / Nisin] = 10 mg / kg), Antibiotics (100 μL, including the following antibiotic combination: [Rifampicin] = 2 mg / kg; [Isoniazid] = 10 mg / kg; [Pyrazinamide] = 20 mg / kg; [Ethambutol Hydrochloride] = 15 mg / kg) were injected into the lung tissue through a pulmonary nebulizer. After 5 treatments (once every other day), the antibacterial and anti-inflammatory therapeutic effects were evaluated. After the treatment, the mice were sacrificed, and the lung tissues of the mice in the different treatment groups were collected and ground in PBS buffer solution. The cell pellets were collected by centrifugation, and the red blood cells were lysed to obtain a single cell suspension. 1 mL of 0.05% SDS was added to the cell pellet of each group to lyse the cells for 15 minutes, and the bacterial pellet released from the cell lysis was collected by centrifugation (5000 rpm, 10 minutes). The precipitate was resuspended in 1 mL of 7H9 medium, and 100 μL was spread on 7H10 agar plates after gradient dilution. The plates were then incubated at 37°C for 72 hours and the number of CFU was counted to evaluate the in vivo antibacterial therapeutic effects of different materials. Figure 12 As shown, 146 / Nisin@Exo N The number of CFU in the lung tissue of the treatment group was significantly lower than that of the 146 / Nisin treatment group. This is because the exosome encapsulation gave the material excellent host cell localization ability, thereby enhancing the antibacterial activity of 146 / Nisin. N Compared to 146 / Nisin@Exo I The ability to reduce the bacterial load in the lungs of tuberculosis mice was further enhanced, which was due to the fact that the infectious exosomes had a better targeting ability to host cells than normal exosomes, and the infectious exosomes could activate the phagosome-lysosome autophagy program in the host cells to exert the ability of lysosomes to degrade bacteria. It is worth noting that since antibiotics alone have difficulty penetrating into the core of the lesion and have poor targeting to host cells, 146 / Nisin@Exo I The antibacterial ability of the bionic nanomedicine is significantly higher than that of the antibiotic treatment group, which makes the bionic nanomedicine prepared by the present invention promising to become an antibiotic substitute for the treatment of pulmonary tuberculosis, thereby avoiding the drug resistance problem caused by excessive use of antibiotics.

[0087] Example 14

[0088] After the treatment, the mice in the different treatment groups were euthanized and their lung tissues were collected. After immersion in tissue fixative for 24 hours, the pathological changes of the lung tissues were analyzed by H&E staining. Figure 13 As shown in the figure, the lung tissue of TB model mice (PBS group) showed obvious lung injury pathological features such as alveolar wall thickening, fibrosis, a large number of inflammatory cell infiltration, large-area alveolar structural destruction, and a high number of granuloma structures.I After treatment, there were only a few inflammatory damage areas in the lung tissue of mice, and the number of granulomas was significantly reduced, indicating that 146 / Nisin@Exo I It can not only reduce the pathogen content in the lung tissue of tuberculosis mice, but also improve the lung damage caused by mycobacterium infection, providing a powerful antibacterial / anti-inflammatory treatment tool for tuberculosis.

[0089] Example 15

[0090] After treatment, BALF of mice in different treatment groups were collected, and the expression levels of different inflammatory cytokines were detected by ELISA to further evaluate the anti-inflammatory therapeutic effect of the material. N The M2 polarization of macrophages by phosphorus-containing dendrimers can inhibit the expression of pro-inflammatory cytokines such as TNF-α, IL-6, and IFN-γ in BALF to a certain extent. Figure 14 AC). However, the expression levels of related pro-inflammatory cytokines in BALF of tuberculosis mice treated with Antibiotics were also significantly lower than those of model mice (PBS group). This may be because antibiotic treatment partially downregulated the bacterial load in the lungs, thereby inhibiting the immune response activated during pathogen infection. It is worth noting that 146 / Nisin@Exo I The expression level of pro-inflammatory cytokines in the treatment group was the lowest, and the expression of anti-inflammatory cytokine IL-10 in BALF was significantly increased ( Figure 14 D) This is thanks to 146 / Nisin@Exo I The 146 / Nisin@Exo prepared by the present invention has high antibacterial and anti-inflammatory activities. I It integrates multiple functions such as "host targeting-efficient antibacterial-tissue repair" and achieved efficient antibacterial / anti-inflammatory therapeutic effects in the M.sm-induced tuberculosis mouse model that are better than the combination of four antibiotics.

Claims

1. A phosphorus-containing dendrimer composite material, characterized in that: The phosphorus-containing dendrimer composite material comprises phosphorus-containing dendrimers, peptides, and exosomes; wherein the phosphorus-containing dendrimers and peptides are complexed to form a nanocomposite, and the surface of the nanocomposite wraps the exosomes.

2. The phosphorus-containing dendrimer composite material according to claim 1, characterized in that: The molecular formula of the phosphorus-containing dendrimer is C 336 H 576 N 87 O 66 P 21 S 18 , structural formula The exosomes are one of infected exosomes and uninfected exosomes; the peptide is an antimicrobial peptide, wherein the antimicrobial peptide is nisin.

3. A method for preparing a phosphorus-containing dendrimer composite material, comprising: (1) mixing a phosphorus-containing dendrimer, a peptide, and a solvent, and stirring at room temperature to obtain a phosphorus-containing dendrimer / peptide nanocomposite; (2) Exosomes, phosphorus-containing dendrimer / peptide nanocomposites, and a solvent are mixed and ultrasonically treated to obtain a phosphorus-containing dendrimer composite material.

4. The preparation method according to claim 3, characterized in that In step (1), the molar ratio of the phosphorus-containing dendrimer to the peptide is 0.5 to 6:1; the solvent is water; The peptide in step (1) is Nisin; the molecular formula of the phosphorus-containing dendrimer is C 336 H 576 N 87 O 66 P 21 S 18 .

5. The preparation method according to claim 3, characterized in that: In the step (1), stirring is performed at room temperature for 2 to 4 hours; and after the stirring at room temperature in the step (1) is completed, centrifugation is performed.

6. The preparation method according to claim 3, characterized in that: The exosomes in step (2) are either infected exosomes or uninfected exosomes.

7. The preparation method according to claim 6, characterized in that: The infectious exosomes are exosomes secreted by macrophages infected with Mycobacterium smegmatis; The preparation of exosomes secreted by macrophages infected with Mycobacterium smegmatis includes: culturing alveolar macrophages MH-S infected with Mycobacterium smegmatis M.sm with exosome culture medium, collecting cell culture fluid, and obtaining infectious exosomes after centrifugation.

8. The preparation method according to claim 3, characterized in that: In step (2), the mass ratio of exosomes to phosphorus-containing dendrimers / peptide nanocomplexes is 0.05-0.2:1; The ultrasonic treatment in step (2) includes performing an 8-10 second on and 8-10 second off cycle at 20-50 Hz for 3-5 minutes, and repeating the cycle 3-5 times.

9. Use of the phosphorus-containing dendrimer composite material according to any one of claims 1 to 2 in the preparation of a drug for treating pulmonary tuberculosis.

10. Use of the phosphorus-containing dendrimer composite material according to any one of claims 1 to 2 in the preparation of antibacterial and anti-inflammatory drugs.

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