Bionic nano material for tuberculosis targeted photodynamic therapy as well as preparation method and application of bionic nano material

Abstract

The invention discloses a bionic nano material for tuberculosis targeted photodynamic therapy as well as a preparation method and application of the bionic nano material. The biomimetic nano material is a nano vesicle structure (C-MV) formed by coating a hydrophobic region at the tail part of a phospholipid bimolecular layer on macrophage membrane vesicles with chlorine e6 with hydrophobic property. After the bionic nano material provided by the invention is administered in vivo, C-MV shows prolonged blood circulation and is accumulated to a tuberculosis lesion part, and the bionic nano material has effective targeting property and ultra-long retention period on tail granuloma of a mouse. Under irradiation of 660 nm laser, the photodynamic behavior of C-MV is activated, generation of a large amount of reactive oxygen species (ROS) is initiated, a killing effect on mycobacteria is generated, related injuries of observed tuberculosis lesion parts are relieved, pathological injuries of normal tissues cannot be caused, and accurate photodynamic therapy of tuberculosis is achieved. Meanwhile, the C-MV has satisfactory biological safety, so that the C-MV can be used for treating tuberculosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a bionic nanomaterial of a bionic macrophage membrane vesicle-coated photosensitizer dihydrochlorin e6 for targeted photodynamic therapy of tuberculosis, and a preparation method and application thereof. Background Art

[0002] Tuberculosis is a highly contagious disease that has become a major concern for global public health care due to its high morbidity and mortality rates. It is caused by infection with Mycobacterium tuberculosis and spreads very quickly. According to WHO data in 2022, it has infected about a quarter of the world's population and caused 1.3 million deaths. Currently, the anti-tuberculosis drugs isoniazid, rifampicin, pyrazinamide and ethambutol are ineffective in monotherapy for tuberculosis, leading to multidrug-resistant tuberculosis. According to the 2023 Global Tuberculosis Report, of all 10.6 million new tuberculosis cases worldwide, 410,000 (3.9%) were multidrug-resistant / rifampicin-resistant tuberculosis patients. The proportion of multidrug-resistant / rifampicin-resistant patients among new patients was 3.3%, and the proportion of multidrug-resistant / rifampicin-resistant patients among relapsed patients was 17%, which did not decrease compared with 2021, indicating that multidrug-resistant tuberculosis remains a security threat. Therefore, new therapeutic strategies are urgently needed to improve the efficacy of TB treatment and enhance the targeting of TB treatment without increasing the risk of antibiotic resistance. Photodynamic therapy (PDT) is such an approach.

[0003] Antibacterial photodynamic therapy has the characteristics of high antibacterial effect and low risk of drug resistance. PDT produces irreversible oxidative damage to bacteria, which is caused by the production of reactive oxygen species (ROS) triggered by the photosensitizer under laser irradiation. ROS can affect the integrity of bacterial lipids, proteins and DNA. Photodynamic therapy mediated by the photosensitizer chlorin e6 (Ce6) can kill tumor cells by driving the generation of reactive oxygen species (ROS). It is currently mainly used for cancer treatment and has not been used for tuberculosis treatment. At the same time, Ce6 has poor water solubility, adverse effects on healthy cells and lacks selectivity, which limits the clinical application of Ce6-mediated PDT. Summary of the invention

[0004] The purpose of the present invention is to overcome the above-mentioned defects and shortcomings in the prior art and to provide a bionic nanomaterial for targeted photodynamic therapy of tuberculosis.

[0005] The second object of the present invention is to provide a method for preparing the bionic nanomaterial.

[0006] The third object of the present invention is to provide the use of the bionic nanomaterial in preparing drugs for treating tuberculosis.

[0007] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0008] A biomimetic nanomaterial, the biomimetic nanomaterial is a nanovesicle (C-MV) formed by loading a photosensitizer dihydrochlorin e6 (Ce6) on a macrophage membrane vesicle (MV), wherein the photosensitizer dihydrochlorin e6 is coated inside the phospholipid bilayer of the macrophage membrane vesicle. That is, the hydrophobic side of the phospholipid bilayer of the macrophage membrane vesicle is coated with Ce6 molecules to form a sandwich structure.

[0009] The cell membrane has a phospholipid bilayer structure, and the phospholipid bilayer is composed of two layers of phospholipid molecules, and the hydrophilic head of each layer of phospholipid molecules faces the water phase, while the hydrophobic tail faces the inside. That is, the phospholipid bimolecule has a hydrophilic head and a hydrophobic tail. The present invention shows that the photosensitizer dihydrochlorin e6 can be easily coated in the hydrophobic region inside the phospholipid bilayer of the macrophage membrane through physical action due to its hydrophobic properties, thereby combining the Ce6 molecule with the macrophage membrane to obtain a new type of nanovesicle C-MV. The hydrophobic tail region of the phospholipid bilayer of the macrophage membrane can provide a suitable environment for the photosensitizer dihydrochlorin e6, which also has hydrophobic properties. The biomimetic properties of the macrophage membrane vesicle protect the photosensitizer dihydrochlorin e6 from being cleared by the blood, so that the physiological stability of the biomimetic nanovesicle C-MV entering the blood circulation in the body can be maintained, while improving the biocompatibility of the photosensitizer dihydrochlorin e6. In addition, granuloma is a hallmark of tuberculosis, which responds to anti-mycobacterial immunity by recruiting immune cells (such as macrophages). The bionic nanomaterials formed by the macrophage membrane can be recruited to the inflammatory lesions, and the tuberculosis lesions are in an inflammatory environment. Therefore, the photosensitizer dihydrochlorin e6 coated with the macrophage membrane has the potential to target tuberculosis lesions and thus treat tuberculosis. The C-MV that reaches the tuberculosis site is activated by photodynamic behavior under laser irradiation, triggering the production of a large amount of reactive oxygen, causing the death of marine mycobacterium, reducing the degree of damage to the tail tuberculosis lesions without causing pathological damage to normal tissues, thereby achieving precise photodynamic therapy for tuberculosis.

[0010] Furthermore, the bionic nanomaterial has a spherical morphology, and an average diameter of 80 to 120 nm, preferably 100 nm.

[0011] Furthermore, the potential of the bionic nanomaterial is -25 to -23 mV, preferably 24.47±0.49 mV. Furthermore, the drug loading rate of dihydrochlorin e6 in the bionic nanomaterial is 3.9 to 4.1 wt%.

[0012] The present invention also provides a method for preparing any of the above-mentioned biomimetic nanomaterials, which comprises mixing macrophage cell membrane buffer with dihydrochlorin e6 solution, and incubating in the dark. Due to the hydrophobic interaction, the hydrophobic photosensitizer Ce6 is easily loaded into the hydrophobic region of the phospholipid bilayer of MV, thereby being coated to form C-MV.

[0013] Furthermore, the mass ratio of the macrophage cell membrane to dihydrochlorin e6 is 13-14:1.

[0014] Furthermore, the incubation time is 3 to 5 hours.

[0015] Furthermore, the macrophage cell membrane buffer is a solution formed by dispersing macrophage cell membrane in PBS buffer.

[0016] Furthermore, the dihydrochlorin e6 solution is a solution formed by dissolving dihydrochlorin e6 in DMSO.

[0017] Furthermore, the method for preparing the macrophage cell membrane is to collect macrophage cell sediments, break the cells in a homogenization buffer, remove cell debris by centrifugation, and obtain a light white sediment after centrifugation of the supernatant, which is the macrophage cell membrane.

[0018] Preferably, the buffer is a lysis buffer, and the buffer components are 10 mM KCl, 2 mM MgCl2, 15 mM Tris HCl, 100 mM sucrose and 20 mM phenylmethylsulfonyl fluoride.

[0019] Preferably, the centrifugation conditions for removing cell debris by centrifugation are 4° C., 6000 g, and 30 min.

[0020] Preferably, the supernatant is centrifuged at a speed of 150,000 g for 60 min.

[0021] Preferably, the macrophages are mouse macrophages, preferably mouse macrophages RAW264.7.

[0022] Preferably, the cells are broken by grinding, specifically grinding 20 to 30 times using a dounce homogenizer with a homogenization buffer.

[0023] Furthermore, after the light-proof incubation, the mixture is ultrafiltered, washed until the filtrate is colorless, and centrifuged to remove the solvent and free dihydrochlorin e6.

[0024] Preferably, the ultrafiltration uses a molecular cut-off rate of 100 kDa.

[0025] The bionic nanomaterial C-MV provided by the present invention has a photodynamic effect. After in vivo administration, C-MV exhibits prolonged blood circulation and accumulates in the tuberculosis lesions. Under 660nm laser irradiation, the photodynamic behavior of C-MV is activated, triggering the massive generation of reactive oxygen species ROS, which has a killing effect on mycobacteria, alleviating the observed related damage to the tuberculosis lesions without causing pathological damage to normal tissues, thereby achieving precise photodynamic therapy for tuberculosis. The present invention's research shows that Ce6 is not only used for photodynamics to produce anti-mycobacterial effects, but also for fluorescent imaging indication of granulomas.

[0026] Therefore, the present invention also provides the use of any of the above-mentioned bionic nanomaterials in the preparation of drugs for treating tuberculosis.

[0027] Furthermore, the nano drug is used under 660nm laser irradiation to induce the generation of reactive oxygen species (ROS), thereby achieving the purpose of resisting the growth of tuberculosis bacteria in tuberculosis lesions.

[0028] The present invention also provides a therapeutic drug, which contains any of the above-mentioned bionic nanomaterials.

[0029] Furthermore, the drug also includes pharmaceutically acceptable excipients, preferably pharmaceutical excipients approved by the FDA.

[0030] The present invention also provides the use of the photosensitizer dihydrochlorin e6 in the preparation of tuberculosis treatment drugs or granuloma fluorescence imaging indicators.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The bionic nanomaterial of the present invention is a nanovesicle structure (C-MV) formed by coating the hydrophobic region of the tail of the phospholipid bilayer on the macrophage membrane vesicle (MV) with dihydrochlorin e6 (Ce6) having hydrophobic properties. After the bionic nanomaterial provided by the present invention is administered in vivo, C-MV shows prolonged blood circulation and accumulates to the site of tuberculosis lesions, and has effective targeting and ultra-long retention period for mouse tail granulomas. Under 660nm laser irradiation, the photodynamic behavior of C-MV is activated, triggering the massive generation of reactive oxygen ROS, which has a killing effect on mycobacteria, which reduces the observed related damage to the tuberculosis lesions without causing pathological damage to normal tissues, thereby achieving precise photodynamic therapy for tuberculosis. At the same time, C-MV has satisfactory biosafety, so C-MV can be used for the treatment of tuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1Characterization of C-MV nanoparticles. (a) Typical transmission electron microscopy images of MV and C-MV, respectively, with a scale bar of 50 nm; (b) Hydrodynamic diameters of MV and C-MV measured by dynamic light scattering; (b) Zeta potential of MV and C-MV, data are presented as mean ± SD (n = 3). (d) SDS-PAGE protein analysis of MV and C-MV stained with Coomassie Brilliant Blue; (e) UV-visible absorption spectra of MV, Ce6 and C-MV; (f) Fluorescence emission spectra of Ce6 and C-MV solutions; (g) Production of reactive oxygen species (ROS) by C-MV under 660 nm light.

[0034] Figure 2 The antibacterial activity of C-MV against marine mycobacteria in vitro. (a) Photo images of marine mycobacterium colonies with and without illumination; (b) The number of marine mycobacteria that survived under different treatments, (c) The number of marine mycobacteria that survived under different concentrations of C-MV under illumination conditions, (d) The number of marine mycobacteria that survived under different light doses, data are expressed as mean ± standard deviation (n = 3); (e) Fluorescence images of marine mycobacteria after different treatments, followed by staining with SYTO 9 (green channel) and PI (red channel) for 30 minutes, scale bar: 200 μm.

[0035] Figure 3 Characterization of the mouse granuloma model and the targeting effect of C-MV on granulomas. (a) Acid-fast staining of normal and (b) abnormal tissues in the tail. The black arrow marks the acid-fast-stained Mycobacterium marinum. Scale bar: 20 μm; (c) The targeting and retention of free Ce6, C-MV, and Ce6-extracellular vesicles (C-EV) in tail granulomas were analyzed using an animal imaging system. Mice in the C-MV group received a second injection 48 hours after the first injection and were imaged 24 hours after the second injection. (d) Schematic diagram of C-MV targeting granulomas.

[0036] Figure 4 To evaluate the antimicrobial activity and biosafety of C-MV in vivo. (a) After or without C-MV treatment (2 mg / mL, 200 mW / cm 2 , 10min) after the mouse tail changes, scale bar: 10mm. C h-MV: High loading of Ce6 on macrophage membrane vesicles (MV) (loading rate 4.1wt%); (b) Quantitative analysis of visible tail lesions in mice, data are expressed as mean ± SD (n = 3); (c) Hemolysis detection of different concentrations of C-MV suspension under red blood cell incubation conditions, data are expressed as mean ± SD (n = 3). (d, e) Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of proinflammatory cytokines (TNF-α and IL-1β) in the serum of healthy mice after different treatments. Data are expressed as mean ± SD (n = 3); (f) Hematoxylin-eosin (H&E) staining of major organs (heart, liver, spleen, lung, kidney) of healthy mice treated with PBS or C-MV, scale bar: 100μm.

[0037] Figure 5 Schematic diagram of biomimetic nanoparticles used for macrophage membrane-guided tuberculosis granuloma-targeted photodynamic therapy. DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0039] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0040] Mycobacterium marinum (M.marinum) has a gene similarity of more than 95% with Mycobacterium tuberculosis, and is an excellent alternative model for studying Mycobacterium tuberculosis. In this manual, M.marinum is used as a surrogate bacteria for tuberculosis to carry out experiments, and the conclusions of each experiment are applicable to tuberculosis.

[0041] Chlorin e6 (Ce6) was purchased from Shanghai MacLean Biochemical Co., Ltd. Bicinchoninic acid (BCA) protein assay kit and 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Bio-Technology Co., Ltd. (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Waltham, USA). Michaelis 7H10 agar and oleic acid-albumin-glucose-catalase enrichment were purchased from BD DeFocus Laboratories (Sparks, USA). SYTO 9 / propidium iodide (PI) LIVE / DEAD BacLight kit was purchased from Thermo Fisher Scientific (Monza, Italy). Streptomycin and penicillin were purchased from Hyclone (Logan, USA). Mouse IL-1β and TNF-α ELISA kits were purchased from Dakoway Biotechnology Co., Ltd. (Shenzhen, China). Deionized water with a resistivity of 18 MΩ·cm (from Ultrapure Water System, Millipore) was used in all experiments. All other chemical reagents and solvents were of analytical grade.

[0042] The hydrodynamic size distribution was determined using a dynamic light scattering instrument (Zetasizer Nano-ZS, Malvern, UK). The UV-visible absorption spectrum was recorded using a spectrophotometer (Shimadzu UV-2600, Japan). The fluorescence intensity was measured using a microplate reader (Tecan Infinite 200PRO, Switzerland). Fluorescence images were captured using a fluorescence microscope (Carl Zeiss Axio Observer 7, Germany). In vitro fluorescence imaging was performed using a small animal imaging system (Bolu Teng Biotechnology Co., Ltd., China).

[0043] Example 1 Synthesis and Characterization of C-MV Nanoparticles

[0044] 1. Synthesis method

[0045] (1) Preparation of macrophage membrane vesicles: Mouse macrophage cell line RAW 264.7 was purchased from the American Type Culture Collection and used for macrophage membrane collection in the present invention. RAW264.7 cells were cultured in a DMEM medium containing 10% FBS and 1% penicillin / streptomycin at 37°C and a humidified incubator with 5% CO2. The method for preparing macrophage membranes was adapted from previous studies by the inventor's research group. The cells were gently blown off the bottom of the culture flask and washed three times with PBS by centrifugation (100g, 3 minutes). The cell pellet was then collected and ground 20 to 30 times in a homogenization buffer (10mM KCl, 2mM MgCl2, 15mM Tris-HCl, 100mM sucrose and 20mM phenylmethylsulfonyl fluoride) using a Dounce homogenizer. All subsequent centrifugation steps were performed at 4°C. After centrifugation at 6000 g for 30 min to remove large vesicles and small debris, the resulting supernatant was ultracentrifuged at 150000 g for 1 h to obtain macrophage membranes (MVs) as a pale white precipitate. Finally, the protein content of macrophage membranes was analyzed by standard BCA assay.

[0046] (2) Preparation of C-MVs: To prepare MVs loaded with Ce6, 200 μg of MVs (dispersed in 1 mL of PBS) were mixed with 15 μg of Ce6 (dissolved in 40 μL of DMSO) and then incubated with stirring in the dark for 4 h. During this process, MVs subsequently complexed with hydrophobic Ce6 in their membranes to obtain C-MVs. The mixture was then transferred to a 100 kDa ultrafiltration tube and washed several times with PBS until the filtrate was colorless, and DMSO and free Ce6 were removed by centrifugation. The solution after the first ultrafiltration was collected, and the drug loading efficiency of Ce6 was calculated according to the following formula: Drug loading efficiency (%) = (weight of Ce6 added - weight of Ce6 in the first ultrafiltration solution) / (weight of C-MV) × 100.

[0047] 2. Characterization methods

[0048] 1) The morphology and particle size of MVs and C-MVs were observed using a transmission electron microscope; the hydrodynamic size distribution of MVs and C-MVs was determined using a dynamic light scattering instrument; the zeta potential of MVs and C-MVs was determined using an electrophoresis method; the absorption and fluorescence spectra of C-MVs were determined using a spectrophotometer and an ELISA reader;

[0049] 2) Characterization of macrophage membranes on C-MVs: Membrane proteins were characterized and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Macrophage membranes and macrophage membrane-derived samples (C-MVs) were lysed with loading buffer and boiled at 100°C for 5 minutes. Then, equal amounts of protein samples were loaded onto SDS-PAGE gels at 80V for 0.5 hours to allow proteins to accumulate at the same starting point and then separated at 120V for 1.5 hours. Finally, the SDS-PAGE gels were stained with Coomassie Brilliant Blue and destained with deionized water for visualization.

[0050] (3) Reactive oxygen production of C-MV in solution: 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) is deacetylated to DCFH, which is further oxidized by reactive oxygen to the fluorescent compound DCF. C-MV with a Ce6 dose of 0.5 μg / mL was incubated with 5 μM DCFH and then incubated at 60 mW / cm 2 The samples were irradiated with 660 nm laser for different time periods (0, 1, 2, 3, 4, 5 minutes). The fluorescence intensity of DCF was monitored with an ELISA reader at excitation / emission wavelengths of 488 / 530 nm.

[0051] 3. Results

[0052] like Figure 1 As shown, transmission electron microscopy (TEM) showed that MVs and C-MVs exhibited a classic spherical morphology with an average diameter of approximately 100 nm ( Figure 1 This was further confirmed by dynamic light scattering ( Figure 1 b). The zeta potentials of MV and C-MV were -18.38±0.54 and -24.47±0.49 mV, respectively ( Figure 1 c). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) further showed that C-MV expressed macrophage membrane proteins ( Figure 1 d). The homologous homing effect mediated by these proteins may bring great therapeutic potential to C-MV targeting tuberculosis granulomas. The absorption and fluorescence spectra of C-MV show the typical absorption peak of Ce6 at 655nm ( Figure 1 e), showing the fluorescence peak of Ce6 at 664 nm ( Figure 1 f), indicating that Ce6 was successfully encapsulated into C-MVs. For tuberculosis treatment, the production of cytotoxic reactive oxygen species is essential for sterilization in photodynamic therapy. The reactive oxygen levels of C-MVs were carefully analyzed using DCFH-DA, and the green fluorescence intensity of the fluorescent compound DCF reflected the level of reactive oxygen. Under laser irradiation, the DCF fluorescence intensity of the C-MV group gradually increased with the increase of laser irradiation time ( Figure 1g), indicating that C-MV can produce significant reactive oxygen species. This produces a strong antibacterial effect to improve the efficacy of anti-tuberculosis treatment. It was calculated that the drug loading rate of dihydrochlorin e6 in the C-MV nanoparticles was 3.9wt%

[0053] Example 2 In vitro antibacterial activity test of C-MV against marine mycobacteria

[0054] In order to reveal the antibacterial efficacy of C-MV, the present invention uses Mycobacterium marinum, which is a natural pathogen of poikilothermic vertebrates, closely related to Mycobacterium tuberculosis, causing tuberculosis-like infections in poikilothermic vertebrates, but rarely in humans. The viability, therapeutic effect and antibacterial mechanism of mycobacteria were evaluated using a standard colony forming unit (CFU) plate count method and a bacterial viability staining kit. The details are as follows:

[0055] 1. Methods

[0056] Marine mycobacteria were cultured in Michaelis 7H10 solid medium supplemented with 10% oleic acid-albumin-glucose-catalase enrichment. The marine mycobacteria suspension and C-MV were incubated in phosphate buffered saline (PBS) solution and illuminated with light (60 mW cm -2 , 10min) and no light. After culturing at 32°C for 12 hours, the treated marine mycobacterium suspension was taken out from each group and then diluted 1000 times with phosphate buffer. Thereafter, 50 μL of the diluted marine mycobacterium solution was evenly spread on a 7H10 plate and cultured at 32°C for about a week. The number of surviving marine mycobacterium colonies on each plate was counted and recorded. For the activity analysis of marine mycobacteria after different treatments, SYTO 9 / propidium iodide (PI) co-staining detection was performed according to the manufacturer's instructions.

[0057] 2. Results

[0058] The results are as follows Figure 2 As shown in the figure, laser or C-MV alone cannot effectively inhibit the proliferation of marine mycobacteria. However, under laser irradiation, the inhibitory effect of C-MV on mycobacterial colony formation was significantly enhanced ( Figure 2 a, 2b), indicating that C-MV exerted a strong photodynamic effect of a classic photosensitizer. In addition, C-MV showed significant antibacterial activity against Mycobacterium marinum, and was concentration- and light-dose-dependent ( Figure 2 c, 2d). The bacterial viability staining kit was used to further determine the bactericidal effect of C-MV on marine mycobacteria under laser irradiation, and fluorescence microscopy was used for imaging ( Figure 2e). Specifically, live bacteria with intact cell membranes were stained with SYTO 9 (green fluorescence), while dead cells with damaged membranes were labeled with propidium iodide (red fluorescence). Consistent with the CFU plate count results, almost all marine mycobacteria in the C-MV+laser group died, showing red fluorescence and severe cell membrane damage, showing the strongest antibacterial ability.

[0059] Example 3 Establishment and characterization of mouse granuloma model and C-MV targeting of granuloma

[0060] In order to study the tuberculosis targeting ability of C-MV in mammals, the present invention established a tuberculosis model by injecting marine mycobacterium into the tail vein of mice according to the previously reported method. Cell aggregates containing marine mycobacterium were formed in the tail granuloma, which had a strong similarity with human tuberculosis granuloma. The details are as follows:

[0061] 1. Methods

[0062] (1) Establishment of mouse granuloma model

[0063] Female C57BL / 6 mice (about 5 weeks old) were purchased from the Experimental Animal Center of Ningxia Medical University, and the experimental operation followed the requirements and regulations of the Animal Ethics Committee of Ningxia Medical University. A mature mouse tail granuloma model was constructed with reference to the previous research of the inventor's research group. In brief, marine mycobacteria were first cultured in 7H10 plate culture medium in a bacterial incubator. About a week later, the bacteria were scraped from the plate and resuspended with phosphate buffered saline (PBS). The uniform bacterial suspension was collected by centrifugation at 3500g for 10 minutes at 4°C, washed with phosphate buffered saline (PBS), and passed through the syringe needle several times. The optical density of the fresh bacterial suspension was measured at 600nm and adjusted to 2 with sterile phosphate buffered saline (PBS). Take 200μL of marine mycobacterium suspension for tail vein injection, and visible tail granulomas will form after about two weeks. The mouse tail was removed, fixed with 4% paraformaldehyde, embedded in paraffin, cut into sections, and then acid-fast stained.

[0064] (2) Study on the targeting effect of C-MV on granuloma

[0065] Mice with the established mouse tail granuloma model were intravenously injected with C-MV at a dose of 2 mg of dihydrochlorin e6 (Ce6) per kilogram of body weight. To monitor the biodistribution of C-MV, the main organs (including lungs, heart, liver, spleen and kidneys) and tails of mice infected with marine mycobacterium were removed 24 hours after injection and then imaged using a small animal fluorescence imaging system.

[0066] 2. Results

[0067] The results are as follows Figure 3 As shown in the figure, the mouse tail acid-fast staining experiment showed that compared with the normal group, the marine mycobacterium injection group had obvious granuloma structure formation, and mycobacteria were distributed around the granuloma cell clusters, confirming that the tail tuberculosis model was successfully established ( Figure 3 a, 3b). Fluorescence imaging was used to monitor the targeting effect of C-MV on the tail granulomas of mice infected with Mycobacterium marinum ( Figure 3 c). Compared with free Ce6, C-MV could be detected in the tail granuloma site 1 hour after injection. The accumulation of C-MV gradually increased over time, reaching a peak at 24 hours and was still retained after 48 hours, indicating that C-MV has effective targeting and ultra-long retention capabilities for mouse tail granulomas. After repeated administration of C-MV doses at 48 hours, the accumulation of C-MV improved in the next 24 hours. Due to the enhanced permeability and retention (EPR) effect, both C-EV (Ce6 loaded onto erythrocyte membrane vesicles) and C-MV nanoparticles were able to accumulate in tail granulomas, and C-MV nanoparticles showed enhanced accumulation due to macrophage membrane-guided tuberculosis homologous targeting ( Figure 3 d). This indicates that C-MV has an effective targeting effect on mouse tail granuloma and has an ultra-long retention period.

[0068] Example 4 C-MV in vivo antibacterial activity test

[0069] Encouraged by the good granuloma targeting and satisfactory in vitro antibacterial activity of C-MV, in vivo bactericidal experiments were conducted. A mouse tail tuberculosis model was established to evaluate the inhibitory effect of C-MV plus laser treatment on lesions. The details are as follows:

[0070] 1. Methods

[0071] After the establishment of mouse tail granulomas, mice with granulomas were intravenously injected with phosphate-buffered saline (PBS) and C-MV (200 μL, 2 mg / kg) every other day for a total of 9 treatments. 24 hours after the injection, the tail granuloma sites of mice infected with Mycobacterium marinum were illuminated (200 mW cm -2 At the end of the treatment, the tails of mice in different groups were photographed.

[0072] 2. Results

[0073] The results are as follows Figure 4 As shown, the tail of the phosphate-buffered saline (PBS) group gradually ruptured and was infiltrated with Mycobacterium marinum ( Figure 4 a). Compared with the PBS group, MV as a nanocarrier in the MV group had little effect on the injury of the mouse tail ( Figure 4 b). Due to the photodynamic therapy effect, Ce6 or Ce6-loaded groups (C-MV, Ch The tail injury of C-MV and C-EV mice was significantly improved. h The -MV group showed better tail injury repair than the C-MV group. Due to the homologous targeting effect of MV-guided granuloma, the tail scab and injury in the C-MV group were significantly better than those in the C-EV group. The above results further confirmed that the tail scab and injury in the C-MV group were significantly better than those in the Ce6 and other Ce6-loaded groups, which may be due to the MV-guided targeting effect on tuberculosis granulomas.

[0074] Example 5C-MV biosafety evaluation

[0075] Good biocompatibility is an important prerequisite for clinical application. Therefore, the in vivo biosafety of C-MV was studied by hemolysis test and hematoxylin-eosin (H&E) staining of major organ tissues.

[0076] The details are as follows:

[0077] 1. Methods:

[0078] (1) Blood compatibility test of C-MV. Water, phosphate buffered saline (PBS) and different concentrations of C-MV were incubated with fresh red blood cells for 3 hours. Subsequently, the mixtures were centrifuged at 860g for 5 minutes. The collected supernatants were analyzed using an ELISA reader at a wavelength of 540nm. The following formula was used to determine the hemolytic toxicity: Hemolysis rate (%) = (absorbance of macrophage vesicles - absorbance of phosphate buffer) / (absorbance of water - absorbance of phosphate buffer) × 100.

[0079] (2) Evaluation of inflammatory cytokine levels. Healthy mice were treated with C-MV nanoparticles (chlorin e6 dose: 2 mg / kg) or phosphate buffered saline (PBS) by intravenous injection every other day for a total of two treatments. Serum was collected and the expression of inflammatory cytokines including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) was detected by enzyme-linked immunosorbent assay (ELISA). Experimental procedures were performed according to the manufacturer's instructions.

[0080] (3) In vivo biocompatibility of C-MV. Healthy mice were treated with C-MV nanoparticles (chlorin e6 dose: 2 mg / kg) or phosphate buffered saline (PBS) by intravenous injection. Seven days after injection, the mice were killed, and the main organs (spleen, heart, kidney, liver, and lung) were removed and stained with hematoxylin-eosin (H&E) for histological examination.

[0081] 2. Results

[0082] The results are as follows Figure 4 As shown, the in vitro compatibility of C-MVs was evaluated by hemolysis assay ( Figure 4 c). Even at a concentration as high as 0.4 mg / mL, the hemolytic rate of C-MV was less than 5%, indicating that C-MV has good blood compatibility. The immune response to C-MV was studied by evaluating the levels of serum proinflammatory cytokines after two repeated administrations of C-MV to healthy mice. The concentrations of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in serum were monitored using enzyme-linked immunosorbent assay (ELISA) ( Figure 4 d, 4e). The expression levels of proinflammatory cytokines in the C-MV group were similar to those in the PBS group, indicating that C-MV nanoparticles did not induce excessive immune responses. In addition, hematoxylin-eosin (H&E) staining of major organ tissues was performed to investigate the in vivo biosafety of C-MV ( Figure 4 f). No tissue damage or inflammation was observed after administration of C-MV nanoparticles, indicating that C-MV nanoparticles do not cause significant tissue damage or inflammation when administered in vivo.

[0083] In summary, the above results indicate that the C-MV designed in the present invention has precise granuloma targeting ability, significant Mycobacterium marinum killing effect and good biocompatibility, and can be used for the treatment of tuberculosis.

[0084] In short, if Figure 5 As shown, the present invention has developed a biomimetic nanovesicle C-MV with effective photodynamic therapy efficacy for the treatment of tuberculosis. A universal photosensitizer Ce6 is used not only for photodynamic antibacterial effects, but also for fluorescent imaging indication of granulomas. In addition, macrophage membrane-related targeting may be used for diseases other than tuberculosis, such as monkeypox, atherosclerosis, cancer and rheumatoid arthritis. Ce6 and macrophage membranes are combined to prepare a new type of nanovesicle. In vitro and in vivo, C-MV exhibited prolonged blood circulation, negligible systemic toxicity, significant granuloma targeting and effective pathogen killing. Overall, the biomimetic C-MV demonstrates a new paradigm and highlights its great potential in clinical tuberculosis treatment.

[0085] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A bionic nanomaterial, characterized in that: The nanovesicle is formed by loading the photosensitizer dihydrochlorin e6 on the macrophage membrane vesicle; the photosensitizer dihydrochlorin e6 is coated inside the phospholipid bilayer of the macrophage membrane vesicle.

2. The bionic nanomaterial according to claim 1, characterized in that: The bionic nano material has a spherical shape and an average diameter of 80 to 120 nm.

3. The bionic nanomaterial according to claim 1, characterized in that: The potential of the bionic nano material is -25 to -23 mV.

4. The method for preparing the bionic nanomaterial according to any one of claims 1 to 3, characterized in that: The macrophage cell membrane buffer solution and the dihydrochlorin e6 solution are mixed evenly, and incubated in a dark place to obtain the product.

5. The preparation method according to claim 4, characterized in that: The method for preparing the macrophage cell membrane comprises collecting macrophage cell sediments, breaking the cells in a homogenization buffer, removing cell debris by centrifugation, and obtaining a light white sediment after centrifugation of the supernatant, which is the macrophage cell membrane.

6. The preparation method according to claim 4, characterized in that: The mass ratio of the macrophage cell membrane to dihydrochlorin e6 is 13-14:

1.

7. Use of the bionic nanomaterial according to any one of claims 1 to 3 in the preparation of drugs for treating tuberculosis.

8. The use according to claim 7, characterized in that: The nano drug is used under 660nm laser irradiation.

9. A therapeutic drug, characterized in that: Contains the bionic nanomaterial according to any one of claims 1 to 3.

10. The drug according to claim 9, characterized in that: The drug also includes pharmaceutically acceptable excipients.

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