A synergistic anti-tuberculosis bionanoparticle based on a safe temperature threshold, and a preparation method and application thereof

Abstract

The application discloses a kind of based on safe temperature threshold's cooperative anti-tuberculosis bionanoparticle and its preparation method and application.Belong to tuberculosis diagnosis and treatment technical field, the bionanoparticle is the structure of core coated outer membrane, the outer membrane is the cell membrane of macrophage activated in advance by Mycobacterium marinum, the core is the aggregation luminescence photosensitizer I-BOD-TPA and under 808nm laser irradiation can produce stable photothermal to safe temperature threshold 42 ℃, and realize tuberculosis granuloma and M.tb dual targeting under safe temperature threshold, accurately act on infection site, reduce systemic toxic side effect.

Description

Technical Field

[0001] This invention belongs to the field of tuberculosis diagnosis and treatment technology, specifically relating to a synergistic anti-tuberculosis biomimetic nanoparticle based on a safe temperature threshold, its preparation method, and its application. Background Technology

[0002] Tuberculosis (TB) is a fatal chronic infectious disease caused by Mycobacterium tuberculosis (M.tb), primarily affecting the lungs (pulmonary tuberculosis) but also potentially impacting other organs throughout the body. Although TB ​​is preventable and treatable, it remains the world's leading infectious disease killer, posing a serious threat to global public health. The standard treatment for TB involves a combination of multiple antibiotics, including isoniazid, rifampin, pyrazinamide, and ethambutol hydrochloride, following the principles of "early, appropriate dosage, combination therapy, regular use, and complete treatment." However, these traditional antibiotics lack the ability to target specific lesions and pathogens, resulting in poor efficacy, high toxicity, and potential systemic toxicity. Long-term use can lead to decreased patient adherence. Furthermore, improper medication use and antibiotic pressure can easily lead to treatment failure and the spread of drug-resistant bacteria. Traditional high-temperature photothermal anti-tuberculosis treatments for tuberculosis use nanocarriers loaded with photosensitizers. These photosensitizers are passively or actively targeted and enriched at the tuberculous lesions. Under appropriate laser irradiation, temperatures exceeding 50°C are generated, directly killing Mycobacterium tuberculosis through high heat. However, this type of treatment only employs a single high-temperature photothermal mode, without incorporating anti-tuberculosis chemotherapy drugs, failing to synergistically activate the body's anti-tuberculosis immunity, lacking a safe temperature threshold control design, and lacking a biomimetic macrophage membrane targeting structure. Another approach is a biomimetic vesicle complex based on a pre-activated macrophage membrane, as disclosed in Chinese patent CN 115252778 A. This biomimetic vesicle complex has a membrane-encapsulated core structure, with its core being an aggregated luminescent photosensitizer complex and its membrane being a pre-activated macrophage membrane. This biomimetic vesicle complex can achieve dual targeting of specific pathogens within the lesion granuloma and pathogens within the granuloma, followed by targeted photothermal therapy (PDT) under near-infrared light irradiation, increasing efficacy, reducing treatment duration, and lowering drug toxicity. However, this method still has other technical defects. This application aims to provide a synergistic anti-tuberculosis biomimetic nanoparticle to solve the technical defects existing in the single treatment mode of high temperature photothermal therapy. Summary of the Invention

[0003] To address the technical challenges of lacking a synergistic anti-tuberculosis technology system that integrates photothermal, chemotherapy, and immunotherapy at a safe temperature threshold, and lacking targeted nanoparticles and complementary treatment methods adapted to this synergistic mechanism, this application proposes a biomimetic nanoparticle, IBT-Rif@M NPs, capable of synergistic anti-tuberculosis treatment. This is achieved by mixing a stable heat-generating photosensitizer with rifampin, followed by mixing with a pre-stimulated macrophage membrane. The specific technical solution is as follows:

[0004] The first objective of this invention is to provide an aggregation-emitting photosensitizer I-BOD-TPA, wherein the chemical structure of the photosensitizer I-BOD-TPA is as follows:

[0005] The aggregation-luminescent photosensitizer I-BOD-TPA can stably generate a photothermal effect within a safe temperature range of 42℃ under low-power laser.

[0006] The second objective of this invention is to provide a method for synthesizing the aggregation-luminescent photosensitizer I-BOD-TPA. The photosensitizer I-BOD-TPA is prepared stepwise from compound 1 as the starting material through aldol condensation, Michael addition, cyclization, BF2 coordination, and iodination. Specifically, the method includes the following steps:

[0007] Step 1, preparation of compound 3: Compound 1 and compound 2 were mixed, ethanol was added as a solvent, and then potassium hydroxide aqueous solution was added. The mixture was stirred at room temperature for 24 hours. After extraction, drying and column chromatography purification, compound 3 was obtained.

[0008] Step 2, preparation of compound 4: Compound 3 prepared in step 1 is mixed with nitromethane, and anhydrous ethanol is added as a solvent. The mixture is stirred at room temperature, and after extraction, drying, and column chromatography purification, compound 4 is obtained. The molar ratio of nitromethane to compound 3 is 5:1, and the amount of anhydrous ethanol used is 150 mL of anhydrous ethanol for every 9.06 mmol of compound 3.

[0009] Step 3, preparation of compound 5: Compound 4 prepared in step 2 was mixed with ammonium acetate, and n-butanol was added as a solvent. The mixture was heated to 100℃ and reacted at a constant temperature for 24 h to carry out an intramolecular cyclization reaction. After cooling, extraction, drying, and column chromatography purification, compound 5 was obtained. The molar ratio of ammonium acetate to compound 4 was 35:1, and the amount of n-butanol used was 100 mL of n-butanol for every 4.58 mmol of compound 4.

[0010] Step four, preparation of BOD-TPA: Compound 5 prepared in step three is mixed with N,N-diisopropylethylamine, anhydrous dichloromethane is added as a solvent, nitrogen gas is purged, and the mixture is stirred at room temperature. After drying and purification by column chromatography, BOD-TPA is obtained. The molar ratio of N,N-diisopropylethylamine to compound 5 is 10:1, and the amount of anhydrous dichloromethane used is 100 mL of anhydrous dichloromethane for every 2.76 mmol of compound 5.

[0011] Step 5: Preparation of the target product photosensitizer I-BOD-TPA. The BOD-TPA prepared in step 4 is mixed with N-iodosuccinimide, and a mixed solvent of chloroform and acetic acid (the volume ratio of chloroform to acetic acid is 3:1) is added. Nitrogen gas is introduced for protection, and the temperature is raised to 30℃ and kept at a constant temperature for 10 hours. After extraction, drying, and column chromatography purification, the target product I-BOD-TPA is obtained.

[0012] The names and chemical structural formulas of compounds 1-5 are as follows:

[0013] Compound 1 is 4-(diphenylamino)benzaldehyde, and its structural formula is: ;

[0014] Compound 2 is acetophenone, and its structural formula is: ;

[0015] Compound 3 is (E)-3-(4-(diphenylamino)phenyl)-1-phenylprop-2-en-1-one, with the following structural formula: ;

[0016] The compound 4(E)-3-(4-(diphenylamino)phenyl)-1-phenyl-2-(nitromethyl)prop-1-one has the following structural formula: ;

[0017] Compound 5 is (Z)-4-(2-((3-(4-diphenylaminophenyl)-5-phenyl-1H-pyrrolo-2-yl)imino)-5-phenyl-2H-pyrrolo-3-yl)-N,N-diphenylaniline, with the following structural formula: .

[0018] The third objective of this invention is to provide an anti-tuberculosis nanoparticle, wherein the nanoparticle is a biomimetic macrophage membrane nanoparticle IBT-Rif@M NPs, which is constructed by encapsulating a pre-stimulated biomimetic macrophage membrane on the surface of a polylactic-co-glycolic acid copolymer (PLGA) core loaded with the aggregation-luminescent photosensitizer I-BOD-TPA and the anti-tuberculosis drug rif.

[0019] The fourth objective of this invention is to provide a method for preparing biomimetic macrophage membrane nanoparticles IBT-Rif@M NPs with anti-tuberculosis properties, specifically including the following steps:

[0020] S1, Pre-stimulated macrophage membrane extraction: Macrophages were activated by co-culturing Mycobacterium marineum with macrophage cell lines, the cells were resuspended in buffer, homogenized and disrupted, cell debris was removed by centrifugation, and the supernatant was collected by centrifugation to obtain the pre-stimulated cell membrane.

[0021] S2, photosensitizer and rifampin mixture: Dissolve polylactic acid-glycolic acid monomer PLGA in tetrahydrofuran, and add aggregation-luminescent photosensitizer. Mix with rifampin, quickly inject into PBS, stir in the dark to remove organic solvent, and obtain Nanoparticles;

[0022] S3, Obtain target particles IBT-Rif@M NPs: The pre-stimulated macrophage membrane extracted in S1 was combined with the IBT-Rif@M NPs prepared in S2. Nanoparticles were blended, ultrasonicated in a water bath, and then extruded stepwise through a liposome extruder to obtain a biomimetic vesicle complex. .

[0023] Furthermore, in S2, the weight ratio of the aggregation-luminescent photosensitizer I-BOD-TPA and rifampin is 1:1.

[0024] Furthermore, the protein concentration of the pre-stimulated macrophage membrane in S3 is 5.0 mg / ml, and the concentration of IBT-Rif nanoparticles is 5.0 mg / ml.

[0025] The fifth objective of this invention is to explore the application of anti-tuberculosis nanoparticles IBT-Rif@M NPs in the preparation of products for tuberculosis diagnosis, treatment, or integrated diagnosis and treatment.

[0026] Furthermore, the product is used under 808nm laser irradiation.

[0027] Furthermore, the nanoparticles are used in the study of the safe temperature threshold for anti-tuberculosis drugs, wherein the safe temperature threshold is 42°C.

[0028] A sixth objective of this invention is to provide a formulation for the diagnosis, treatment, or integrated diagnosis and treatment of tuberculosis, the formulation containing biomimetic macrophage membrane-derived anti-tuberculosis nanoparticles IBT-Rif@M NPs.

[0029] Furthermore, the formulation also includes pharmaceutically acceptable excipients.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] (1) Abandoning the traditional photothermal sterilization mode that requires high temperature above 50°C, the safe temperature threshold of 42°C is limited. While effectively eliminating Mycobacterium tuberculosis, it avoids damage to normal lung tissue, inflammatory response and immunosuppression caused by high temperature, thus greatly improving the safety of treatment.

[0032] (2) The photosensitizer I-BOD-TPA was synthesized, which can stably generate a photothermal effect within a safe temperature range of 42℃ under low-power laser, without the need for high-energy laser irradiation, further reducing photoradiation damage. The photosensitizer itself has extremely low toxicity, good compatibility with PLGA carriers and biomimetic macrophage membranes, high in vivo stability, and excellent biocompatibility. By using a pre-stimulated biomimetic macrophage membrane to encapsulate the nanocore, dual targeting of tuberculous granuloma and M.tb is achieved, solving the problems of weak targeting, wide systemic distribution, and low lesion utilization of traditional anti-tuberculosis drugs, precisely acting on the site of infection, and reducing systemic toxic side effects.

[0033] (3) It pioneered a triple synergistic effect of low-temperature photothermal, chemotherapy and immunity within a safe temperature range. Through safe low-temperature photothermal mediated by photosensitizer, it significantly enhances the sensitivity of M.tb to rifampin, achieving efficient sterilization under low-dose drug conditions, reducing the amount and duration of antibiotic use, and effectively reducing the risk of the generation and spread of drug-resistant strains.

[0034] (4) Safe low-temperature photothermal therapy can directionally induce macrophages in lesions to polarize to the M1 type, promote the release of pro-inflammatory and bactericidal factors such as TNF-α, IL-6, and IFN-γ, form a local pro-inflammatory immune microenvironment, achieve the dual effect of "direct bactericidal + immune defense", and enhance the body's own anti-tuberculosis ability. Attached Figure Description

[0035] Figure 1 This is a synthetic route diagram for the aggregation-luminescent photosensitizer I-BOD-TPA of the present invention.

[0036] Figure 2 This is a schematic diagram of the preparation process for IBT-Rif@M NPs.

[0037] Figure 3 A roadmap for IBT-Rif@M NPs-mediated pathogen targeting and cryophotothermal synergistic chemotherapy and immunotherapy for tuberculosis.

[0038] Figure 4 The photothermal effect of photosensitizer I-BOD-TPA is shown in Figure A; where: A represents the photothermal heating curves under different laser powers; and B represents the results of 5 cycles of photothermal stability testing.

[0039] Figure 5 The characterization results of IBT-Rif@M NPs are shown below, where: A is the hydration size and zeta potential diagram of IBT-Rif@M NPs; B is the transmission electron microscopy image of IBT-Rif@M NPs; and C is the particle size stability line plot of IBT-Rif@M NPs.

[0040] Figure 6To validate the macrophage targeting mechanism and immunophenotype of IBT-Rif@M NPs, where: A is the result of SDS-PAGE total protein electrophoresis; B is the result of Western Blot.

[0041] Figure 7 The image shows the photothermal effect of IBT-Rif@M NPs nanoparticles; where:

[0042] A represents the photothermal heating curves under different laser powers;

[0043] B represents the stability curve of a 6-cycle photothermal cycle.

[0044] Figure 8 The in vitro killing effect of IBT-Rif@M NPs nanoparticles on M.tb is shown in Figure 1, where: A is a CFU colony count plate; B is a CFU quantitative bar chart.

[0045] Figure 9 The expression levels of pro-inflammatory factors in macrophages after five different drug treatments plus and without laser treatment at 42℃ were shown.

[0046] A represents the expression level of TNF-α;

[0047] B represents the expression level of IL-6;

[0048] C represents the expression level of IL-1β;

[0049] D represents the expression level of IFN-γ.

[0050] Figure 10 The therapeutic effect of IBT-Rif@M NPs in a mouse model of pulmonary tuberculosis under a safe temperature threshold was evaluated, where:

[0051] A represents the CFU colony results under 5 treatment groups plus different treatment methods with and without laser;

[0052] B is a quantitative bar chart of CFU under 5 treatment groups and different treatment methods with and without laser;

[0053] C represents the level of the core pro-inflammatory cytokine IL-1β in the lungs under 5 treatment groups plus different treatment methods with and without laser;

[0054] D represents the level of the core pro-inflammatory cytokine IL-6 in the lungs under 5 treatment groups plus different treatment methods with and without laser.

[0055] E represents the acid-fast staining results of lung tissue under 5 treatment groups plus different treatment methods with and without laser;

[0056] F is a flow cytometry scatter plot used to show the expression ratio of CD86 in macrophages;

[0057] G represents the quantitative statistical results of CD86-positive macrophages.

[0058] Figure 11 The effects of different temperature gradients on cells, M. tb, and mice, where:

[0059] AB represents the temperature tolerance test results of normal cells. The top row shows RAW 264.7 (mouse macrophages, simulating immune cells in vivo); the bottom row shows A549 (human lung epithelial cells, simulating normal lung tissue cells).

[0060] CD represents the results of heat sensitivity tests on the attenuated strain H37Ra of Mycobacterium tuberculosis at different temperatures;

[0061] E represents the comparison of cell viability at different temperatures (blue bars represent RAW 264.7 macrophages; red bars represent H37Ra tuberculosis bacteria).

[0062] F represents the survival rate of mice at different temperatures;

[0063] J represents the photothermal response of mouse skin;

[0064] H represents the effect of different temperatures on lung tissue structure.

[0065] Figure 12 The safety assessment results of the nanoparticles IBT-Rif@M NPS of the present invention are as follows:

[0066] A represents the macrophage activity under the phototoxicity of RAW264.7 nanoparticles at various concentrations;

[0067] B represents macrophage activity under the influence of various concentrations of nanoparticles at the dark toxicity level of RAW264.7; C represents the results of biochemical index detection of liver and kidney function.

[0068] D represents the results of routine blood test.

[0069] Figure 13 This is a photograph of the aggregated luminescent photosensitizer I-BOD-TPA prepared in Example 1 of the present invention.

[0070] Figure 14 The image shows the hydrogen spectrum of the aggregation-luminescent photosensitizer I-BOD-TPA prepared in Example 1. Detailed Implementation

[0071] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings.

[0072] Example 1: Synthesis of the aggregation-luminescent photosensitizer I-BOD-TPA

[0073] Table 1. Compounds for synthesizing the aggregation-luminescent photosensitizer I-BOD-TPA

[0074]

[0075] like Figure 1 As shown, the synthesis of the aggregation-luminescent photosensitizer I-BOD-TPA begins with 4-(diphenylamino)benzaldehyde (compound 1) as the starting material. The target product I-Bodipy (iodotriphenylaminofluoroboron dipyrrole derivative) is prepared stepwise via aldol condensation, Michael addition, cyclization, BF2 coordination, and iodination. The specific synthetic steps are as follows:

[0076] Step 1: Compound 1 (3.80 g, 14 mmol, 1.0 eq) was added to a 500 mL double-necked flask and stirred thoroughly to disperse and dissolve in ethanol (100 mL), with the solid partially dissolving. Potassium hydroxide (1.96 g, 35.20 mmol, 4.0 eq) was dissolved in water (10 mL), and the prepared KOH solution was added to the reaction system. Then, compound 2 (1.68 g, 14 mmol, 10 eq) was dissolved in 20 mL of ethanol. The ethanol solution of acetophenone was added dropwise to the reaction system, and the mixture was stirred at room temperature for 24 h. After the reaction was completed as monitored by TLC, the mixture was extracted with dichloromethane, washed three times with water, dried over anhydrous sodium sulfate, concentrated, and separated by chromatography to obtain the target product, yielding 5.20 g of an orange-yellow solid (compound 3), with a yield of 89.8%.

[0077] Step 2: Compound 3 (3.96 g, 9.06 mmol, 1.0 eq), nitromethane (2.76 g, 45.32 mmol, 5.0 eq), and sodium hydroxide (0.72 g, 18.12 mmol, 2.0 eq) were completely dissolved in anhydrous ethanol (150 mL). The reaction system was heated to reflux for 24 h. After the reaction was completed as monitored by TLC, most of the liquid was removed by rotary evaporation. 500 mL of ethyl acetate was added, and the mixture was washed three times with 500 mL of water. The organic phase was collected. The organic phase was dried over anhydrous sodium sulfate, and the filtrate was concentrated using a rotary evaporator. 3.58 g of orange solid (compound 4) was obtained, with a yield of 90.8%.

[0078] Step 3: In a 250 mL double-necked flask, compound 4 (2.00 g, 4.58 mmol, 1.0 eq) and ammonium acetate (12.34 g, 160.3 mmol, 35 eq) were dissolved in 100 mL n-butanol and reacted at 100 °C for 24 h. After the reaction was completed as monitored by TLC, the mixture was cooled to room temperature, the product was diluted with 500 mL of water, and extracted three times with 500 mL of dichloromethane. The organic phase was collected. The organic phase was dried over anhydrous sodium sulfate, and the filtrate was concentrated using a rotary evaporator to give 1.53 g of purple solid (compound 5), with a yield of 76.5%.

[0079] Step 4: In a 250 mL double-necked flask, compound 5 (2.16 g, 2.76 mmol, 1.0 eq) dissolved in 100 mL of anhydrous dichloromethane was added, followed by the addition of N,N-diisopropylethylamine (3.57 g, 27.60 mmol, 10 eq). Under nitrogen protection, boron trifluoride diethyl ether (3.92 g, 27.60 mmol, 10 eq) was added, and the reaction was carried out at room temperature for 24 h under nitrogen protection. After the reaction was completed as monitored by TLC, the product was diluted with 500 mL of water and extracted three times with 500 mL of dichloromethane. The organic phase was collected. The organic phase was dried over anhydrous sodium sulfate, and the filtrate was concentrated using a rotary evaporator to give 1.98 g of green solid (Bodipy), with a yield of 78%.

[0080] Step 5: In a 250 mL double-necked flask, add Bodipy (1.63 g, 2.00 mmol, 1.0 eq) and N-iodosuccinimide (1.12 g, 5.00 mmol, 2.5 eq) dissolved in 120 mL of a mixed solvent of chloroform and acetic acid (chloroform:acetic acid = 3:1). React at 30 °C for 10 h under nitrogen protection. After the reaction is completed as monitored by TLC, extract three times with 100 mL of saturated sodium thiosulfate and 200 mL of dichloromethane, and collect the organic phase. Dry the organic phase with anhydrous sodium sulfate, and concentrate the filtrate using a rotary evaporator to obtain 1.25 g of green solid I-Bodipy (i.e., the aggregation-luminescent photosensitizer I-BOD-TPA), with a yield of 57.7% (see [link to relevant documentation]). Figure 13 The product was subjected to nuclear magnetic resonance (NMR) to obtain the following results: (shown as a dark green solid powder). Figure 14 The hydrogen spectrum shown is similar to that of... Figure 1 The structures of the aggregated luminescent photosensitizer I-BOD-TPA shown are mutually corroborating, and there are no aliphatic hydrogens in the structure. , ), spectrum Except for the solvent peak, no sample peaks are visible, and they match each other; all the molecular hydrogen atoms are aromatic hydrogens, and they are all concentrated in the aromatic region. The large number of benzene rings and similar chemical environments result in overlapping peaks, consistent with the complex multi-peak phenomenon on the left side of the NMR spectrum.

[0081] Example 2: Verification of the photothermal effect of photosensitizer I-BOD-TPA

[0082] like Figure 4 The photothermal heating curves of I-BOD-TPA under different laser powers, shown in Figure A, indicate that the heating amplitude of I-BOD-TPA is positively correlated with the laser power.

[0083] Table 2 Heating parameters of I-BOD-TPA

[0084] 0.2W 26℃, only a slight increase in temperature 0.4 W 32℃ 0.6 W 38℃ 0.8 W 45℃ (reaching the upper limit of the safe temperature for normal cells) 1.0 W 50℃ (reaches the effective sterilization temperature for Mycobacterium tuberculosis)

[0085] like Figure 4 As shown in Figure A, all groups rapidly heated up and reached thermal equilibrium within 200–400 seconds, demonstrating that I-BOD-TPA has excellent photothermal conversion efficiency and can reach the required treatment temperature in a short time.

[0086] Five more cycles of photothermal stability testing were conducted, and the results showed that ( Figure 4 B): During 5 cycles, the highest heating temperature of I-BOD-TPA was approximately 49-50℃, with almost no attenuation, and the cooling curves completely overlapped, demonstrating that it showed no significant photodegradation under repeated laser irradiation and that its photothermal performance was stable. This result proves that I-BOD-TPA can withstand multiple photothermal treatments and will not become ineffective due to repeated irradiation, making it suitable as a photosensitizer for clinical multiple photothermal treatments.

[0087] Example 3: Preparation of Synergistic Anti-Tuberculosis Biomimetic Nanoparticles Based on Safe Temperature Threshold

[0088] (1) Pre-stimulation of macrophage membranes: RAW 264.7 cells were cultured in T-175 flasks at a density of 90%. Cells were stimulated with Mycobacterium tumefaciens (OD=1) (MOI=10) for approximately 24 hours, followed by washing three times with PBS. Cells were collected, centrifuged (1,000 rpm, 5 min), and washed with PBS. Cells were resuspended in homogenization buffer containing 75 mM sucrose, 20 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 10 mM KCl, and a protease / phosphatase inhibitor (Pierce, ThermoFisher Scientific). The suspension was loaded into a Dounce homogenizer and homogenized 20 times. The suspension was centrifuged at 3,000 rpm for 10 min to remove cell debris. The supernatant was collected and centrifuged at 20,000 rpm for 30 min. The resulting supernatant was centrifuged at 100,000 rpm for 2 h. Finally, discard the supernatant and collect the pale white particles for subsequent experiments. Membrane protein content was quantified using the Pierce BCA assay kit (Life Technologies). Store at -80°C for extended periods.

[0089] (2) Preparation of biomimetic nanoparticles (IBT-Rif@M NPs): 10.0 mg of polylactic-co-glycolic acid monomer (PLGA, Sigma-Aldrich, USA) was dissolved in 1 ml of tetrahydrofuran (THF) solution. Then, 1.0 mg of the aggregation-luminescent photosensitizer I-BOD-TPA prepared in Example 1 and 1 mg of rifampin (Rif) were added to the solution. After mixing evenly, the mixture was quickly pipetted into 3 mL of PBS buffer using a 1 ml pipette. The mixture was stirred overnight at 300-1000 rpm under light-protected conditions with a magnetic stirrer to remove the THF solvent. The IBT-Rif nanoparticles were obtained. 5.0 mg / ml protein concentration of pre-stimulated macrophage membrane and 5.0 mg / ml IBT-Rif nanoparticles were mixed and then extruded through a liposome extruder for 5 min by sonication in a water bath. The mixture was then extruded sequentially through polycarbonate membranes with pore sizes of 800 nm, 400 nm, and 200 nm, 15-20 times each time. The final result is a biomimetic vesicle complex, namely the synergistic anti-tuberculosis biomimetic nanoparticles IBT-Rif@M NPs of this invention.

[0090] Example 4: Characterization of the IBT-Rif@M NPs prepared in Example 3 above.

[0091] (1) Zetasizer Nano nanoparticle size / zeta potential detector: The hydration particle size distribution and zeta potential values ​​of IBT-Rif@M NPs are shown in ( Figure 5As shown in A), the hydrated particle size of the biomimetic vesicle complex IBT-Rif@M NPs is approximately 132.8 nm; the Zeta potential results indicate that its surface is negatively charged, approximately -33.2 mV; the hydrated particle size monitoring results show that ( Figure 5 C), which can maintain essentially the same particle size for a long time in PBS buffer and healthy mouse serum, confirms its stability.

[0092] (2) Transmission electron microscopy (TEM): TEM images show the morphology and nanoscale size of IBT-Rif@M NPs. Results are referenced from [reference needed]. Figure 5 B. At a scale of 100 nm, the size of IBT-Rif@M NPs is about 100 nm, which is similar to the hydrated particle size result; its structure is a typical membrane-encapsulated spherical structure, which confirms that the macrophage membrane successfully coats the surface of IBT-RifNPs to form a complete IBT-Rif@M NPs structure.

[0093] (3) Western blot: SDS-PAGE was used to detect the total protein components of IBT-Rif@M NPs. Results are as follows. Figure 6 A. Compared to PLGA nanoparticles, IBT-Rif@M NPs exhibit a distinct protein composition consistent with that of pre-stimulated macrophage membranes, indicating successful macrophage membrane coating by IBT-Rif@M NPs. Western blotting was then used to detect whether IBT-Rif@M NPs carry relevant receptors. The Western blotting results showed ( Figure 6 (B) Compared to macrophages, the expression levels of related receptors such as CCR2, CD86, CD206, and TLR6 are higher, indicating that IBT-Rif@M NPs successfully retain the macrophage-targeting receptor CCR2 and can induce macrophages to highly express the M1-type marker CD86, demonstrating its excellent macrophage targeting and immune activation capabilities, providing a molecular basis for its in vivo targeted anti-tuberculosis therapy.

[0094] (4) Photothermal effect of IBT-Rif@M NPs ( Figure 7 The effect of different photothermal powers on the photothermal efficiency of IBT-Rif@M NPs was investigated using an 808 nm laser. Specifically, an IBT-Rif@M NPs suspension (250 μg mL) was used. -1 Photosensitizer concentration) exposed to different power densities (0, 0.1, 0.3, 0.5, 0.7 W / cm²). -2 Temperature was measured under laser irradiation. Figure 7 A); then the IBT-Rif@M NPs suspension (250 μg mL⁻¹ photosensitizer concentration) was prepared at 0.6 w cm⁻¹. -2Under laser irradiation, the temperature was measured using an infrared thermal imager (UTI260B, NUI-T). Monitoring was stopped when the temperature almost reached its maximum, and then the 808nm laser was turned off. The temperature of the IBT-Rif@M NPs aqueous suspension gradually decreased until it approached the initial baseline temperature. The above heating-cooling process was repeated strictly with the same heating time, cooling time, and photothermal power for a total of 6 complete cycles. Figure 7 B). IBT-Rif@M NPs maintain high photothermal conversion performance even after multiple cycles.

[0095] Example 5: Investigating the optimal safe temperature for tuberculosis treatment

[0096] A temperature gradient of 37℃, 42℃, 45℃, 48℃, 52℃, 55℃, and 58℃ was set up. Macrophages (Raw264.7) and lung epithelial cells (A549) were selected as validation models (Raw264.7 simulates tuberculosis host cells, and A549 simulates normal tissue cells surrounding granulomas). Both cell types and Mycobacterium tuberculosis (H37Ra) were placed in different temperature environments (37℃, 42℃, 45℃, 48℃, 52℃, 55℃, and 58℃) ​​for 10 minutes, followed by incubation at 37℃. Cell viability was detected by CCK8 assay, and M.tb activity was detected by CFU assay. The results are as follows: Figure 11 B and Figure 11 As shown in Figure D, the survival rate of both cell types gradually decreases with increasing temperature: even at 45℃, the cell survival rate drops to 63%, and at 52℃, the cell death rate reaches as high as 86.3%. Figure 11 B); CFU detection of M.tb activity revealed that its survival rate decreased with increasing temperature, but complete eradication of H37Ra required a temperature of 58℃ ( Figure 11 CD). It is noteworthy that there is a significant difference in heat resistance between normal cells and M.tb. When Mycobacterium tuberculosis is killed by high temperatures, the survival rate of normal cells is less than 10% (CD). Figure 11 E), which directly limits the feasibility of high-temperature photothermal therapy for tuberculosis.

[0097] To assess the harmful effects of different temperatures on the body, mice intravenously injected with IBT-Rif@M NPS were subjected to 808nm laser lung irradiation (temperature range 37–58℃). Figure 11FJ). Results showed that at 37–45℃, mice showed no skin damage and did not struggle, indicating this temperature range was painless and safe; at 48℃, the skin turned slightly white but there was no struggle, suggesting this was the critical temperature for tissue protection; at 52℃, skin swelling appeared after 5 minutes of light exposure, and mice died within 6 hours after exposure, with a mortality rate of 26.7%; at 55℃, the skin turned white and wrinkled, and the mortality rate reached 73.3% within 6 hours after exposure; at 58℃, all mice died within 5 minutes with severe skin wrinkling, confirming that high temperatures caused irreversible and fatal damage to lung tissue. Pathological sections further verified the above results. Figure 11 H): ≤48℃: Lung tissue structure is intact, without inflammation or edema; ≥52℃: Alveolar epithelium is thickened, morphologically abnormal and vacuolated, and the degree of damage increases with increasing temperature.

[0098] Based on the dual validation of the above cell and mouse experiments, 42℃ can be selected as the optimal safe temperature for photothermal therapy. This temperature is strictly within the painless and safe temperature range of 37–45℃, and is the optimal temperature within the safe range that also has therapeutic potential. In cell experiments, after treatment at 42℃, both types of cells still maintained extremely high survival rates and showed no obvious cytotoxicity. In mouse experiments, after irradiation at 42℃, the mice's skin showed no damage or struggle, and their lung tissue structure remained intact without any pathological damage. This fully demonstrates the high safety of 42℃ for normal cells and the body, completely avoiding the risk of irreversible and fatal damage caused by high temperatures. Compared with 45℃, which is also within the safe range, 42℃ is safer and less stimulating to cells and the body. It is a gentler and more reliable safe temperature, which can fundamentally solve the core contradiction that "high-temperature sterilization will inevitably damage normal tissues." It has extremely high clinical translation potential, and subsequent experiments will all use 42℃ as the safe temperature threshold for anti-tuberculosis treatment for verification.

[0099] Example 6: Verification of the effect of IBT-Rif@M NPs on tuberculosis under a safe temperature threshold of 42℃

[0100] (1) Bactericidal ability of IBT-Rif@M NPs: After incubating H37Ra with PBS, Rif, IBT NPS, IBT-Rif NPs, and IBT-Rif@MNPs in vitro for 30 min, the bacteria were then treated with 0.3 mW / cm 2 After irradiation with an 808nm laser for 10 minutes, the temperature was maintained at 42℃. The bactericidal ability was determined using CFU plating counts, and the results are as follows: Figure 8As shown in Figure A, without light irradiation, PBS, IBT NPs, IBT-Rif NPs, and IBT-Rif@M NPs showed no significant killing effect on H37Ra. Under 808nm laser irradiation, compared with PBS buffer, IBT NPs, IBT-Rif NPs, and IBT-Rif@M NPs all significantly reduced the CFU of H37Ra, indicating that the killing effect depends on the core-loaded aggregation-luminescent photosensitizer IBT and rifampin. Furthermore, IBT-Rif@M NPs showed a more significant killing effect on *M. tb*, which is attributed to the specific targeting of *M. tb* by the macrophage membrane, bringing the biomimetic vesicle complex closer to the bacteria and enhancing the phototherapy effect. These results demonstrate that the IBT-Rif@M NPs biomimetic vesicles can significantly enhance the bactericidal effect of rifampin by specifically targeting *M. tb*, and effectively kill *M. tb* at a safe temperature of 42℃.

[0101] (2) IBT-Rif@M NPs immune activation: The expression levels of TNF-α, IL-6, IL-1β, and IFN-γ in macrophages after treatment with different drugs were detected, and the results are as follows. Figure 9 As shown, only weak expression was observed in all non-light-exposed and light-exposed PBS and rifampicin groups, while higher levels of expression were observed in the light-exposed IBT NPs and IBT-Rif NPs groups, and abundant expression was observed in the IBT-Rif@M NPs group. These data indicate that mild photothermal therapy at 42℃ can significantly enhance the ability of macrophages to kill M.tb, and strongly induce the release of core pro-inflammatory / immune factors such as TNF-α, IL-6, IL-1β and IFN-γ from the lungs to form a pro-inflammatory microenvironment, thereby achieving efficient immune activation and synergistic anti-tuberculosis effects.

[0102] (3) Therapeutic effect of IBT-Rif@M NPs in a mouse model of pulmonary tuberculosis under safe temperature threshold: A mouse model of pulmonary tuberculosis was induced by H37Ra infection. After characteristic tuberculous granulomas formed in the lungs on day 21 of infection, mice were intravenously injected with 250 μL of PBS, Rif, IBT NPs, IBT-Rif NPs, and IBT-Rif@M NPs (protein concentration 50 mg / kg), respectively. In order to promote the killing of M. tb by rifampicin within a safe temperature of 42℃, 808nm laser (0.6 W / cm) was used or not used 24 hours after treatment. 2 Mice were irradiated with external pleural irradiation for 10 minutes, maintaining a temperature of 42°C throughout. On day 21 after each treatment, mice were sacrificed, and their lung tissue was harvested for colony formation assays. The collected lung tissue was evaluated for the expression of various pro-inflammatory cytokines (IL-6, TNF-α, and IFN-γ) to determine the extent of pulmonary inflammatory infiltration. Results are as follows: Figure 10As shown, after 808 nm laser irradiation, both IBT NPs and IBT-Rif NPs exhibited moderate bactericidal activity against H37Ra, with IBT-Rif@M NPs showing the strongest antibacterial effect, likely due to the inherent targeting ability of macrophage membranes. However, the PBS and rifampicin treatment groups treated with 808 nm laser irradiation showed no significant changes compared to the non-laser treatment groups. Furthermore, treatment with IBT-Rif@M NPs combined with 808 nm laser irradiation resulted in a significant reduction in the number of H37Ra colonies in lung tissue. This combined strategy demonstrated significantly higher antibacterial efficacy than antibiotic treatment alone. Acid-fast staining also yielded the same conclusion. Under 808 nm laser irradiation, the three nanoparticles exhibited varying degrees of pro-inflammatory effects, with IBT-Rif@MNPs showing the strongest pro-inflammatory activity, exceeding that of rifampicin treatment. In particular, the IBT-RIF@M NPs plus 808nm laser irradiation scheme has the strongest promoting effect on the expression of pro-inflammatory factors such as IL-1β and IL-6, further forming a bactericidal microenvironment.

[0103] In summary, intravenous injection of IBT-RIF@M NPs nanoparticles can precisely treat tuberculosis at a safe temperature threshold of 42°C, thus possessing great potential for clinical translation.

[0104] Example 6: Safety of IBT-Rif@M NPs in cells and mice at safe temperature thresholds

[0105] Currently, most photothermal or photodynamic therapy models only validate their dark toxicity, with few studies exploring the phototoxicity of these drugs. Given the good targeting and mild photothermal therapy application of our developed IBT-Rif@M NPs in tuberculosis, we comprehensively evaluated the phototoxicity and dark toxicity of IBT-Rif@M NPs through in vitro and in vivo experiments to verify its potential for clinical translation and practical application. First, after co-incubating IBT-Rif@M NPs with RAW 264.7 macrophage cells for 24 hours, they were irradiated with an 808nm laser, and the cell temperature was maintained at 42℃ by controlling the laser intensity. Cell viability was detected by CCK8 assay after 10 minutes. Figure 12 A), the results showed that macrophage activity was above 99% under the action of nanoparticles at all concentrations, indicating that photothermal therapy below 42℃ and nanoparticles at all concentrations had low cytotoxicity to cells, which is of great value for clinical applications. Dark toxicity was detected without 808nm laser irradiation. Figure 12 B). Next, healthy mice were intravenously injected with IBT-Rif@M NPs, and the mice were exposed to light or no light for 10 minutes on days 1, 3, 7, 17, 21, and 28. Blood samples were collected from the mice after day 28 for testing. Figure 12The biochemical test results of liver and kidney function in group C showed that there were no significant differences in the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), blood urea nitrogen (UREA), and creatinine (CR) between the two groups, proving that IBT-Rif@M NPs do not cause liver or kidney toxicity, thus solving the problem of liver damage side effects of traditional anti-tuberculosis drugs (such as rifampin); it also verified that the nanocarrier particles only accumulate in lesions and do not damage normal liver and kidney organs. For example... Figure 12 The blood routine indicators shown in Figure D indicate that there were no significant differences between the treatment group and the control group in all blood routine indicators, all of which were within the normal physiological range; this proves that IBT-Rif@M NPs does not affect the function of the blood system and has no side effects such as bone marrow suppression or coagulation abnormalities; this further verifies the systemic safety of the nano-formulation and provides a guarantee for long-term administration.

[0106] In summary, the results indicate that IBT-Rif@M NPs, with their mild photothermal effect (effectively eliminating Mycobacterium tuberculosis at a safe temperature threshold of 42℃ while avoiding damage to normal lung tissue, inflammatory response, and immunosuppression caused by high temperatures), exhibit extremely low toxicity and high biocompatibility. In vitro cell experiments showed no significant toxicity to normal cells, and high survival rates were maintained even at high concentrations. In vivo safety experiments showed that it did not damage liver or kidney function, did not affect the blood system, and had no significant systemic toxic side effects. It perfectly solves the pain points of traditional anti-tuberculosis drugs (rifampin), such as hepatotoxicity and severe side effects, while retaining antibacterial efficacy. This demonstrates that IBT-Rif@M NPs not only effectively treat tuberculosis but also possess good biocompatibility, making it an ideal candidate drug component for the optical diagnosis and treatment of tuberculosis.

Claims

1. A synergistic anti-tuberculosis biomimetic nanoparticle based on a safe temperature threshold, characterized in that, The anti-tuberculosis nanoparticles are biomimetic macrophage membrane nanoparticles IBT-Rif@M NPS, which are constructed by encapsulating a pre-stimulated biomimetic macrophage membrane on the surface of a polylactic-co-glycolic acid monomer PLGA core loaded with the aggregation-luminescent photosensitizer I-BOD-TPA and the anti-tuberculosis drug rifampin Rif.

2. A photosensitizer I-BOD-TPA that exhibits aggregation and luminescence, characterized in that, The structure of the I-BOD-TPA is as follows: ； The aggregation-luminescent photosensitizer I-BOD-TPA can stably generate a photothermal effect within a safe temperature range of 42°C under low-power laser conditions.

3. A method for preparing the aggregation-emitting photosensitizer I-BOD-TPA according to claim 2, characterized in that, The photosensitizer I-BOD-TPA is prepared stepwise from compound 1 as the starting material through aldol condensation, Michael addition, cyclization, BF2 coordination and iodination reaction, specifically including the following steps: Step 1, preparation of compound 3: Compound 1 and compound 2 were mixed, ethanol was added as a solvent, and then potassium hydroxide aqueous solution was added. The mixture was stirred at room temperature for 24 hours. After extraction, drying and column chromatography purification, compound 3 was obtained. Step 2, preparation of compound 4: Compound 3 prepared in step 1 is mixed with nitromethane, anhydrous ethanol is added as a solvent, the mixture is stirred at room temperature, and after extraction, drying and column chromatography purification, compound 4 is obtained. Step 3, preparation of compound 5: Compound 4 prepared in step 2 is mixed with ammonium acetate, n-butanol is added as solvent, the temperature is raised to 100℃ and kept at a constant temperature for 24 h to carry out intramolecular cyclization reaction, and after cooling, extraction, drying and column chromatography purification, compound 5 is obtained. Step 4, preparation of BOD-TPA: Compound 5 prepared in step 3 is mixed with N,N-diisopropylethylamine, anhydrous dichloromethane is added as a solvent, nitrogen gas is introduced for protection, the reaction is stirred at room temperature, and after drying and column chromatography purification, BOD-TPA is obtained. Step 5, preparation of the target product I-BOD-TPA: The BOD-TPA prepared in step 4 is mixed with N-iodosuccinimide, and a mixed solvent of chloroform and acetic acid is added. The volume ratio of chloroform to acetic acid is 3:

1. Nitrogen gas is introduced for protection, and the temperature is raised to 30°C and kept at a constant temperature for 10 hours. After extraction, drying, and column chromatography purification, the target product I-BOD-TPA is obtained. The names and chemical structural formulas of compounds 1-5 are as follows: The structural formula of compound 1 is: ; The structural formula of compound 2 is: ; The structural formula of compound 3 is as follows: ; The structural formula of compound 4 is: ; The structural formula of compound 5 is as follows: .

4. A method for preparing anti-tuberculosis biomimetic nanoparticles according to claim 1, characterized in that, Includes the following steps: S1, Pre-stimulated macrophage membrane extraction: Macrophages were activated by co-culturing Mycobacterium marineum with macrophage cell lines, the cells were resuspended in buffer, homogenized and disrupted, cell debris was removed by centrifugation, and the supernatant was collected by centrifugation to obtain the pre-stimulated cell membrane. S2, photosensitizer and rifampin mixture: Dissolve polylactic acid-glycolic acid monomer PLGA in tetrahydrofuran, and add the aggregation-emitting photosensitizer as described in claim 2. Mix with rifampin, quickly inject into PBS, stir in the dark to remove organic solvent, and obtain... ; S3, Obtain the target particle IBT-Rif@M NPS: Combine the pre-stimulated macrophage membrane extracted in S1 with the NPS prepared in S2. The mixture was blended, ultrasonicated in a water bath, and then extruded stepwise using a liposome extruder to obtain a biomimetic vesicle complex. .

5. The method according to claim 4, characterized in that, The weight ratio of the aggregation-luminescent photosensitizer I-BOD-TPA and rifampicin Rif in S2 is 1:

1.

6. The method according to claim 4, characterized in that, The protein concentration of the pre-stimulated macrophage membrane in S3 is 5.0 mg / ml, and the concentration of IBT-Rif NPs is 5.0 mg / ml.

7. The application of nanoparticles prepared by the method according to claim 4 in the preparation of tuberculosis diagnosis, treatment or integrated diagnostic and therapeutic products.

8. The application according to claim 7, characterized in that, The product is used under 808nm laser irradiation.

9. The application according to claim 7, characterized in that, The nanoparticles are used in the study of the safe temperature threshold for anti-tuberculosis drugs, wherein the safe temperature threshold is 42°C.

10. A preparation for the diagnosis, treatment, or integrated diagnosis and treatment of tuberculosis, characterized in that, It contains the synergistic anti-tuberculosis biomimetic nanoparticles as described in claim 1.

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