Temperature-sensitive gel preparation entrapped with mycobacterium tuberculosis bacteriophage sustained-release microspheres and application of temperature-sensitive gel preparation

Abstract

The invention discloses a temperature-sensitive gel preparation entrapped with mycobacterium tuberculosis bacteriophage sustained-release microspheres and application of the temperature-sensitive gel preparation, and belongs to the technical field of biological medicine, the preparation comprises a temperature-sensitive gel matrix and targeted ROS sensitive sustained-release microspheres dispersed in the temperature-sensitive gel matrix; the targeted ROS sensitive sustained release microsphere comprises a mycobacterium tuberculosis phage, a composite targeted unit and an ROS sensitive monomer. According to the preparation disclosed by the invention, a composite targeting-ROS response drug release-temperature-sensitive retention three-in-one anti-multi-drug-resistant tuberculosis gel system is constructed, a closed loop of precise targeting aggregation-on-demand directional drug release-long-acting local effect is formed by the three components in a synergistic manner, efficient bacteriostasis, anti-inflammatory repair and medication safety are considered, and a reference scheme is provided for treatment of tuberculosis infection.

Description

Thermosensitive gel formulations encapsulating Mycobacterium tuberculosis phage sustained-release microspheres and their applications Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a thermosensitive gel formulation containing Mycobacterium tuberculosis phage sustained-release microspheres and its application. Background Technology

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis. It is highly contagious, difficult to treat, and has a high relapse rate, making it one of the key diseases controlled globally. TB treatment is lengthy and prone to drug resistance. Traditional anti-TB drugs, such as isoniazid and rifampin, have limited efficacy, with a cure rate of less than 60%. Furthermore, long-term medication can cause liver and kidney damage and gastrointestinal diseases. Therefore, TB treatment is challenging, and patient compliance is poor.

[0003] Mycobacterium tuberculosis phages are a class of viruses that specifically infect Mycobacterium tuberculosis and have wide applications in the diagnosis, treatment, and basic research of tuberculosis. Mycobacterium tuberculosis phages can specifically recognize specific structures such as lipoarabinomannan on the cell wall of Mycobacterium tuberculosis. Compared to traditional tuberculosis drugs, they do not damage human cells or other bacterial flora, have high safety, strong targeting, few side effects, and are less likely to induce drug resistance.

[0004] For example, in reference (1) YI GUO-HUA, YANG FAN, LABANI-MOTLAGH A, et al. Bacteriophage therapy for the treatment of Mycobacterium tuberculosis infections in humanized mice [J]. BioRxiv, 2023, 21., it was found that after injecting Mycobacterium tuberculosis phage DS-6A into mice infected with tuberculosis, the infection symptoms in the spleen and lungs of the mice were significantly reduced, indicating that Mycobacterium tuberculosis phage has the effect of killing infectious bacteria. Reference (2) LAPENKOVA MB, ALYAPKINAY S, VLADIMIRSKY M A. Bactericidal activity of liposomal form of lyticMycobacteriophage D29 in cell models of tuberculosis infection in vitro [J]. Bull Exp Biol Med, 2020, 169(3): 361-364. In this study, it was found that when the liposomal form of Mycobacterium tuberculosis phage exists in liposomal form, it has a more significant advantage in killing Mycobacterium tuberculosis.

[0005] However, natural Mycobacterium tuberculosis phages have significant drawbacks in clinical applications: on the one hand, phages are easily cleared by the immune system in vivo, making it difficult to form an effective drug concentration at the site of infection; on the other hand, Mycobacterium tuberculosis often parasitizes macrophages or forms fibrotic lesions, making it difficult for conventional drug administration methods to penetrate the lesion barrier, resulting in poor targeting and easy off-target release, leading to insufficient local concentration and poor treatment effect.

[0006] Based on this, the present invention designs a thermosensitive gel formulation that encapsulates Mycobacterium tuberculosis phage sustained-release microspheres. After encapsulating Mycobacterium tuberculosis phage, the drug is administered to form a sustained-release formulation, which allows the drug system to remain locally for a long time to achieve the therapeutic purpose. Summary of the Invention

[0007] To address the problems mentioned in the background section, this invention provides a thermosensitive gel formulation containing Mycobacterium tuberculosis phage sustained-release microspheres and its application.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a thermosensitive gel formulation encapsulating Mycobacterium tuberculosis phage sustained-release microspheres, comprising a thermosensitive gel matrix and ROS-sensitive sustained-release microspheres dispersed therein; the ROS-sensitive sustained-release microspheres comprise Mycobacterium tuberculosis phage, a composite targeting unit, and a ROS-sensitive monomer.

[0009] Furthermore, the composite targeting unit includes a macrophage mannose receptor CD206 antibody and D-mannose.

[0010] Furthermore, the ROS-sensitive monomer is selected from acrylate monomers containing thioether bonds.

[0011] Furthermore, the thermosensitive gel matrix includes a PEG-PPG-PEG triblock copolymer.

[0012] Furthermore, in the PEG-PPG-PEG triblock copolymer, the PEG molecular weight is 1000-4000, the PPG molecular weight is 2000-6000, and the HLB value is 10-12.

[0013] Furthermore, the Mycobacterium tuberculosis phage is selected from phage D29 or phage TM4.

[0014] Furthermore, the preparation method of the temperature-sensitive gel formulation includes the following steps: (1) amplification and purification of bacteriophages: obtaining a titer ≥10 10 (2) Preparation of phage solution with PFU / mL: First, biodegradable polymer material and ROS-sensitive monomer are dissolved in an organic solvent to obtain an oil phase; then, composite targeting unit, crosslinking agent and phage solution are added to the oil phase system; then, sterile purified water is used as the aqueous phase, the oil phase and aqueous phase are mixed, and after emulsification, solidification, washing and freeze drying, phage-loaded ...

[0015] Furthermore, in step (2), the biodegradable polymer material is selected from PLGA, PLA, PBS, or PHB.

[0016] Furthermore, in step (2), the crosslinking agent is selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or its hydrochloride salt (EDC·HCl), and N-hydroxysuccinimide (NHS).

[0017] This invention also claims protection for the application of a thermosensitive gel formulation as described above, which can be used in anti-tuberculosis infection drugs.

[0018] This invention constructs a three-in-one anti-multidrug resistant tuberculosis gel formulation based on "composite targeting - ROS-responsive drug release - thermosensitive retention." It achieves highly efficient treatment through the synergistic effect of these three core units, with the following specific principles: First, the composite targeting unit identifies tuberculosis-infected lung tissue through multi-target synergistic recognition, solving the problems of off-target effects and insufficient enrichment associated with single-target therapy. This precisely guides the formulation to accumulate at the infection site, increasing local drug concentration. Second, the ROS-sensitive monomer responds to abnormally elevated reactive oxygen species (ROS) at the tuberculosis infection site, triggering targeted degradation and drug release, avoiding the side effects of systemic administration, while simultaneously overcoming the defense barrier of drug-resistant strains and enhancing antibacterial activity. Third, the thermosensitive matrix rapidly gels and solidifies at the physiological temperature of the lungs, achieving local retention of the formulation, prolonging the duration of drug action, and reducing drug loss. These three components synergistically form a closed loop of "precise targeted aggregation - on-demand targeted drug release - long-lasting local action," balancing highly effective antibacterial activity, anti-inflammatory repair, and medication safety.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The present invention achieves synergistic drug release by using a composite targeting unit combined with ROS sensitive monomers, which can more accurately achieve drug targeting and has a much higher antibacterial rate than traditional drugs such as isoniazid and rifampin.

[0020] (2) Compared with the shortcomings of single targeting units, such as easy off-target, low enrichment efficiency and large interference from the internal environment, the formulation of the present invention relies on the synergistic recognition of multiple targets, which greatly enhances the targeting specificity and affinity, avoids uneven local drug dosage, makes the phage enrichment multiple high, and accurately and stably aggregates at the site of infection, reducing systemic drug exposure and potential side effects.

[0021] (3) The formulation of the present invention can significantly reduce the level of serum inflammatory factors while effectively inhibiting bacteria, which can alleviate inflammatory damage and promote lung tissue repair. Combined with the local retention effect of the thermosensitive gel matrix, it enhances the overall therapeutic effect and safety. Detailed Implementation

[0022] A method for preparing a thermosensitive gel formulation containing Mycobacterium tuberculosis phage sustained-release microspheres includes the following steps: (1) Phage amplification and purification: Amplification is performed using Mycobacterium tuberculosis phage D29 or TM4 as the target phage. Both of these phages are broad-spectrum anti-Mycobacterium tuberculosis phages, suitable for infection scenarios caused by drug-resistant strains.

[0023] Seed culture preparation: Take bacteriophage D29 or TM4 strain (refer to the methods described in the following literature to culture bacteriophage D29 and TM4. Reference [1] Fan Xiangyu. Systematic biological study of mycobacterial bacteriophages [D]. Southwest University, 2014; Reference [2] Peng Li, Chen Baowen, Luo Yongai, et al. Therapeutic effect of bacteriophage D29 on drug-resistant tuberculosis guinea pigs [J]. Journal of the Fourth Military Medical University, 2009, 30(17):1576-1579.), with logarithmic growth phase Mycobacterium tuberculosis H37Rv strain (existing standard strain) as host bacteria, inoculate in Middlebrook 7H9 liquid medium, and culture at 37℃ and 180r / min with shaking; D29 cultured for 24-30h, TM4 cultured for 28-36h, to obtain the corresponding bacteriophage seed culture, ensuring titer ≥10 8 PFU / mL; Amplification culture: Inoculate D29 or TM4 seed culture at a volume ratio of 1:80-1:100 into fresh Middlebrook 7H9 medium (formula same as seed culture), and amplify at 37℃ with shaking at 200 rpm. The amplification time for D29 is 30-36 h, and for TM4 it is 36-42 h. During this period, the titer is monitored every 4 h using the double-layer plate method to ensure that the titer steadily increases to 10. 9 -10 10 PFU / mL; Purification: The amplification buffer was centrifuged at 8000 r / min for 20 min at 4℃ to remove bacterial precipitate and impurities; the supernatant was filtered sequentially through 0.45 μm and 0.22 μm filters to remove residual bacterial fragments; purification was performed using the PEG 8000 precipitation method, and all buffers were incubated at 4℃ for 12 h; subsequently, the phage precipitate was collected by centrifugation at 12000 r / min for 30 min at 4℃ and dissolved in SM buffer (100 mmol / L NaCl, 8 mmol / L MgSO4·7H2O, 50 mmol / L Tris-HCl, pH 7.5; TM4 can be supplemented with 0.01% BSA to improve stability); Purity verification: The reconstituted phage solution was filtered through a 0.22 μm sterile filter for sterilization, and the titer was accurately determined using the double-layer plate method; the titer for D29 should be ≥10. 10 PFU / mL, TM4 must reach ≥1.2×10 10 PFU / mL, both with a purity ≥98%; after purification, the phage solution was stored at 4℃ protected from light for later use. The storage time for D29 was not more than 72h, and the storage time for TM4 was not more than 48h, to avoid activity decay.

[0024] (2) Preparation of ROS-sensitive sustained-release microspheres loaded with phages. First, raw material pretreatment: macrophage mannose receptor CD206 antibody (Beyotime AG2660) was selected, taken out from -20℃ and placed in 4℃ for slow thawing (thawing time 2-3h), and then aliquoted into single-use quantities (avoiding repeated freeze-thaw cycles); CD206 antibody and D-mannose (purchased from Shanghai Sinopharm Group, analytical grade) were weighed at a molar ratio of 1:(1.5-3) (preferably 1:2), and dissolved separately in sterile MES buffer (pH 5.0). The antibody solution concentration was controlled at 1mg / mL, and the D-mannose solution concentration was controlled at 0.05mol / L. The microspheres were stored at 4℃ for later use; biodegradable high-molecular-weight polymers were prepared. Weigh out the biodegradable polymer (PLGA, PLA, PBS, or PHB) and ROS-sensitive monomer (thioether-containing acrylate monomer or oxalate monomer) at a molar ratio of 1:(0.05-0.2), dissolve them in an organic solvent (dichloromethane, chloroform, or ethyl acetate, etc.), and prepare an oil phase solution with a concentration of 50 mg / mL. Take the purified D29 or TM4 phage solution, stir it at low speed at 4℃ until homogeneous, and set aside. The biodegradable polymer is carboxyl-terminated, the PLGA is carboxyl-terminated PLGA, and the lactic acid-glycolic acid molar ratio is 50:50 or 75:25. The ROS-sensitive monomer is an acrylate monomer containing thioether bonds, specifically selected from bis(2-acrylate oxyethyl). Thioethers, thiodiethylene glycol diacrylate, thiodiethylene glycol dimethacrylate, bis(4-(acryloyloxy)butyl) thioether, thiodipropionic acid diacrylate, ethyl 2-(2-pyridyldithio)methacrylate, 3-(methacryloyloxy)propyl thioether, or ethyl 2-(2-pyridyldithio)methacrylate.

[0025] Then, the composite targeting unit was incorporated into the phage loading: the pretreated CD206 antibody solution was mixed with D-mannose solution and slowly added dropwise to the above oil phase solution, while a cross-linking agent (specifically 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or EDC·HCl and N-hydroxysuccinimide (NHS), with a molar ratio of EDC or EDC·HCl to NHS of 1:1 and a final concentration of 5 mmol / L) was added. The mixture was magnetically stirred at 4°C and 150 r / min for 2 h to allow the CD206 antibody to be loaded onto the phage. 6. Antibody and D-mannose are covalently bound to the backbone of biodegradable polymer material to ensure that the total modification rate of the composite targeting unit is ≥85%; then, the prepared D29 or TM4 phage solution is slowly added and stirred continuously for 30 min to make the phage uniformly dispersed in the oil phase system and ensure the uniformity of the loading; through the specific binding of CD206 antibody to CD206 receptor on the surface of macrophage and the binding of D-mannose to the carbohydrate recognition domain of CD206, receptor-mediated endocytosis is triggered, so that the phage is taken up by macrophage and forms endosomes.

[0026] Subsequently, emulsification: using the above mixture as the oil phase and sterile purified water as the aqueous phase, add the aqueous phase at a volume ratio of oil phase:water phase = 1:5, and add emulsifier (Span 80 or Tween 80) to a final concentration of 0.5%-2%. w / v, placed in an ice bath environment (temperature ≤10℃), and subjected to intermittent ultrasonic emulsification (power 100-300W, 5s operation / 2s pause, duration 5-15min) to form a homogeneous emulsion; the ultrasonic power was controlled at 150-200W when encapsulating D29 and at 200-250W when encapsulating TM4, to adapt to the activity tolerance characteristics of both; then, solidification and washing: the emulsion was transferred to a rotary evaporator and rotary evaporated at 30℃ and 100r / min for 30min to remove organic solvents and solidify the microspheres; subsequently, it was centrifuged at 4℃ and 10000r / min for 15min, the microsphere precipitate was collected, and washed three times with sterile PBS buffer to remove unbound targeting units, emulsifiers, cross-linking agents, and free phages; finally, freeze-drying: the washed microspheres were resuspended in a small amount of sterile purified water, and a freeze-drying protectant (mannitol, final concentration 5%) was added. w / w (with an additional 0.05% sucrose added to encapsulate TM4 to improve activity retention), pre-frozen at -80℃ for 12h, then dried in a freeze dryer (vacuum ≤10Pa, temperature -50℃) for 24h to obtain loose targeted ROS-sensitive sustained-release microspheres; particle size was verified using dynamic light scattering (DLS) (room temperature, dispersion medium: sterile purified water) to ensure that the particle size was in the range of 100-500nm; the encapsulation efficiency was ≥85%, with D29 activity retention ≥95% and TM4 activity retention ≥94%; sealed and stored at 4℃ for later use, with a storage time not exceeding 1 month.

[0027] (3) Prepare the temperature-sensitive gel matrix solution. In a low temperature environment of 4-10℃ (temperature is controlled throughout to avoid premature phase change of the gel), slowly add the temperature-sensitive polymer (preferably PEG-PPG-PEG triblock copolymer (PEG molecular weight 1000-4000, PPG molecular weight 2000-6000, HLB value 10-12, purchased from Shanghai Zhenzhun Biotechnology Co., Ltd.) to sterile purified water, stir at low speed of 300-500r / min with magnetic stirring (avoid generating bubbles), and intermittently use ultrasonic to assist dissolution (50-100W, 30s each time, 2min interval), strictly control the ultrasonic parameters to prevent the polymer from thermally degrading. After the polymer is completely dissolved, a 15%-30% w / v gel matrix solution is formed. A sterile co-solvent (glycerol, propylene glycol, or PEG 400, single or in combination, final concentration 5%-15% w / w) is added proportionally and stirred for 10-15 min. Then, an antibacterial agent (benzalkonium chloride or phenylmercuric nitrate, final concentration 0.01%-0.1% w / w) is added, stirred until dissolved, and the pH is finely adjusted to 6.5-7.5. The solution is filtered through a 0.22 μm sterile membrane. The resulting gel matrix solution is clear and free of particles. It should be refrigerated at 4°C for use and stored for no more than 3 days to ensure system stability.

[0028] (4) Preparation of the final formulation: Under low temperature conditions of 4-10℃ (consistent with the storage temperature of the gel matrix to avoid phase change), slowly add the sustained-release microspheres prepared in step (2) to the gel matrix solution at a ratio of 5%-20% w / w, and stir at a low unidirectional speed of 200-300 r / min or gently stir manually to prevent microsphere aggregation. After stirring evenly, let stand for 5-10 min. If there is no precipitation, the dispersion is qualified. Adjust the pH to 6.5-7.5. Dispense into sterile bottles in a sterile operating table and store at 4℃. Avoid repeated freeze-thaw cycles and periodically sample to test phage activity.

[0029] The formulation obtained by this invention is a uniformly flowing liquid at 4-30℃, without stratification or precipitation, and can be drawn and injected using a sterile syringe. It transforms into a semi-solid gel within 3 minutes at 35-37℃. In vitro drug release performance: in a simulated ROS infection environment containing 100-200 μmol / L at 37℃, pH 5.8-6.4, the cumulative release rate of bacteriophage is 20%-40% at 24h, 50%-70% at 72h, and ≥85% at 168h. After refrigeration at 4℃ for 6 months, the bacteriophage activity retention rate is ≥85%, and the formulation shows no stratification or precipitation.

[0030] Examples 1-5 prepared thermosensitive gel formulations containing Mycobacterium tuberculosis phage sustained-release microspheres according to the proportions listed in Table 1 below and with reference to the preparation method described above.

[0031] Comparative Examples 1-3 were prepared according to the proportions listed in Table 1 below and with reference to the preparation method described above, using thermosensitive gel formulations containing Mycobacterium tuberculosis phage sustained-release microspheres.

[0032] Table 1. Raw material selection in each embodiment and comparative example.

[0033] To verify the efficacy of the formulations obtained in the above embodiments and comparative examples, the present invention conducted the following animal experiments.

[0034] BALB / c mice, aged 6-8 weeks and weighing 18-20g, purchased from Beijing Microton Lihua Laboratory Animal Technology Co., Ltd., were used. The mice were half male and half female, and the weight difference within the same batch did not exceed 2g. Following the method in CN114272364A, Mycobacterium tuberculosis strain H37Rv was injected into the tail vein of the mice to establish a mouse tuberculosis model.

[0035] The experiment included a blank control group, a model experimental group, a positive drug control group, an example control group, and a comparative drug administration group.

[0036] Among them: (1) Blank control group: 8 animals, no Mycobacterium tuberculosis infection, no drug intervention; (2) Model experimental group: 10 animals, only Mycobacterium tuberculosis infection was constructed, no drug intervention; (3) Positive drug control group: 8 animals, after successful model construction, conventional anti-tuberculosis drug intervention was given, isoniazid (50mg / kg) + rifampin (100mg / kg) were administered by gavage daily, with a gavage volume of 0.2mL / animal, for 4 consecutive weeks; (4) Example control group: 5 subgroups, 8 animals in each group, corresponding to the preparations obtained in Examples 1-5, and administered after successful infection model. Each subgroup was given a local lung injection of the corresponding example preparation, with a dose of 0.1mL / animal, once a week, for 4 consecutive weeks; (5) Comparative drug group: 3 subgroups, 8 animals in each group, corresponding to the preparations obtained in Comparatives 1-3, and administered after successful infection model. Each subgroup was given a local lung injection of the corresponding comparative preparation, with a dose of 0.1mL / animal, once a week, for 4 consecutive weeks.

[0037] Mice were euthanized by cervical dislocation, and whole blood was immediately collected by ocular blood sampling. The blood was placed in a sterile centrifuge tube, allowed to stand at room temperature for 30 minutes, and then centrifuged at 3000 r / min for 15 minutes at 4°C. The supernatant serum was collected, aliquoted into sterile EP tubes, and stored at -80°C for later use in the detection of inflammatory factors.

[0038] Then, the mice were aseptically dissected, and the bilateral lung tissue and mediastinal lymph nodes were separated, removing surface connective tissue and bloodstains. 0.05g of left lung tissue and 0.03g of mediastinal lymph nodes were taken and placed in a sterile tissue homogenizer in an ice bath. 0.5mL of sterile physiological saline was added, and the mixture was thoroughly homogenized to prepare a tissue suspension for colony counting and phage enrichment detection. A small piece of right lung tissue (approximately 0.1cm × 0.1cm) was taken and fixed in 4% paraformaldehyde solution for pathological damage detection.

[0039] Test items: (1) Colony count of lung tissue and lymph nodes (to assess antibacterial effect). The tissue homogenate suspension was serially diluted 10-fold (dilution 1-10). 4 100 μL of each dilution was evenly spread onto 7H11 solid medium plates containing 10% ADC supplementation solution, with three replicates for each dilution. The plates were incubated at 37°C and 5% CO2 for 21 days. The number of colonies (CFU) on the plates was counted, and the number of colonies per gram of tissue (CFU / g) was calculated. The inhibition rate of each group was compared as follows: (number of colonies in the model control group - number of colonies in the experimental group) / number of colonies in the model control group × 100%.

[0040] (2) Phage enrichment fold detection (evaluation of targeting effect). The above tissue homogenate suspension was centrifuged at 4℃ and 10000 r / min for 20 min. The supernatant was collected, and the phage titer (PFU / mL) was detected using the double-layer agar plate method. Enrichment fold = phage titer in the experimental group tissue / initial phage titer of the preparation in the example. The differences in enrichment ability among the groups were compared to verify the effect of the composite targeting unit.

[0041] (3) Detection of serum inflammatory factors (to assess anti-inflammatory effect). The levels of TNF-α and IL-6 in serum were detected by ELISA, and the procedure was strictly followed according to the kit instructions. ① Reagent equilibration: Remove the kit from 2-8℃ and equilibrate at room temperature for 15-30 min; ② Sample addition: Add no sample or reagent to the blank wells, only the chromogenic agent and stop solution; add 50 μL of standard to the standard wells, add 40 μL of serum sample + 10 μL of antibody working solution to the sample wells, and then add 50 μL of streptavidin-HRP to each well, and incubate at 37℃ for 60 min; ③ Plate washing: Wash the ELISA plate 5 times with 30-fold diluted washing buffer, letting it stand for 30 seconds each time before discarding the liquid and patting it dry; ④ Color development: Add 50 μL of chromogenic agent A and 50 μL of chromogenic agent B to each well, and develop the color at 37℃ in the dark for 15 min; ⑤ Termination and reading: Add 50 μL of stop solution to each well, zero the instrument with the blank well within 10 min, and measure the OD value of each well at a wavelength of 450 nm. Calculate the serum TNF-α and IL-6 concentrations according to the standard curve.

[0042] (4) Lung tissue pathological damage score (to assess the effect of tissue repair).

[0043] Sample processing: Lung tissue fixed with 4% paraformaldehyde was perfused and rinsed under physiological pressure, then routinely dehydrated in a gradient, embedded in paraffin, and 5μm serial sections were prepared for hematoxylin-eosin (HE) staining. The staining quality should be such that the cell nuclei are blue-purple, the cytoplasm and interstitial spaces are pink, and there is no staining blurring or detachment.

[0044] Observation guidelines: A blinded assessment method was adopted, which was performed independently by two pathology professionals. At least 20 non-overlapping high-power fields of view were randomly selected under an optical microscope at 400x magnification (the distance between each field of view is ≥1 field width, and the alveoli account for ≥50% of each field of view) to avoid field duplication or bias.

[0045] Scoring Dimensions and Standards: The scoring is based on three core dimensions: alveolar structural integrity, degree of inflammatory cell infiltration, and tissue necrosis and exudate. The total score is 0-10 (0 points for no damage, 10 points for the most severe damage). The specific standards are as follows: Alveolar structural integrity (0-3 points): 0 points = completely intact structure, no expansion or collapse of alveolar cavities; 1 point = mild damage, ≤20% of alveolar cavities are expanded, collapsed, or the septa are slightly thickened; 2 points = moderate damage, 21%-50% of alveolar structure is disordered, and the septa are significantly thickened; 3 points = severe damage, >50% of alveolar structure is lost, and the septa are significantly thickened or fused.

[0046] Degree of inflammatory cell infiltration (0-4 points): 0 points = no inflammatory cell infiltration; 1 point = slight infiltration, ≤10 inflammatory cells per high-power field, limited to the interstitium; 2 points = moderate infiltration, 11-30 inflammatory cells per high-power field, involving the interstitium and alveolar walls; 3 points = severe infiltration, 31-50 inflammatory cells per high-power field, diffusely distributed in the interstitium and alveolar spaces; 4 points = very severe infiltration, >50 inflammatory cells per high-power field, forming inflammatory foci or clusters.

[0047] Tissue necrosis and exudate (0-3 points): 0 points = no necrosis and exudate; 1 point = mild, with a small amount of protein exudate and no obvious necrotic foci; 2 points = moderate, with scattered small necrotic foci (≤10% of the field of view) and a large amount of exudate in the alveolar cavities; 3 points = severe, with large areas of necrotic foci (>10% of the field of view), exudate filling most of the alveolar cavities, accompanied by tissue disintegration.

[0048] Result determination: When the difference between the scores of two evaluators is ≤1 point, the average score is taken as the final score; when the difference is >1 point, a third professional will review the score and the average of the three scores will be taken to ensure the objectivity of the result.

[0049] All test data were analyzed using statistical software (such as SPSS). The t-test or ANOVA was used for comparisons between groups. P < 0.05 was considered statistically significant. Results are expressed as "mean".

[0050] The test results are shown in Table 2.

[0051] Table 2 Test Results

[0052] In summary, the results of this experiment indicate that the targeted formulation has a significant antibacterial effect against Mycobacterium tuberculosis, with an inhibition rate of 89.8%-91.7% and a targeted enrichment fold of 4.1-4.5. Serum inflammatory factor levels and lung tissue pathological scores were significantly better than those of the positive drug control group and the comparative group. This demonstrates that the formulation achieves highly efficient antibacterial activity, targeted enrichment, and lung tissue protection through the synergistic action of the targeting unit, ROS-sensitive monomer, and temperature-sensitive matrix, providing a reference treatment for tuberculosis infection with good safety and efficacy.

[0053] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A thermosensitive gel formulation encapsulating Mycobacterium tuberculosis phage sustained-release microspheres, characterized in that: It includes a thermosensitive gel matrix and ROS-sensitive sustained-release microspheres dispersed therein; the ROS-sensitive sustained-release microspheres include Mycobacterium tuberculosis phage, a composite targeting unit and a ROS-sensitive monomer.

2. The thermosensitive gel formulation as described in claim 1, characterized in that: The composite targeting unit includes a macrophage mannose receptor CD206 antibody and D-mannose.

3. The thermosensitive gel formulation as described in claim 1, characterized in that: The ROS-sensitive monomer is selected from acrylate monomers containing thioether bonds.

4. The thermosensitive gel formulation as described in claim 1, characterized in that: The thermosensitive gel matrix includes a PEG-PPG-PEG triblock copolymer.

5. The thermosensitive gel formulation as described in claim 4, characterized in that: The PEG-PPG-PEG triblock copolymer has a molecular weight of PEG of 1000-4000, a molecular weight of PPG of 2000-6000, and an HLB value of 10-12.

6. The thermosensitive gel formulation as described in claim 1, characterized in that: The Mycobacterium tuberculosis phage is selected from phage D29 or phage TM4.

7. The thermosensitive gel formulation as described in claim 1, characterized in that, The preparation method of the thermosensitive gel formulation includes the following steps: (1) amplification and purification of bacteriophages: obtaining a titer ≥10 10 (2) Preparation of phage solution with PFU / mL: First, biodegradable polymer material and ROS-sensitive monomer are dissolved in an organic solvent to obtain an oil phase; then, composite targeting unit, crosslinking agent and phage solution are added to the oil phase system; then, sterile purified water is used as the aqueous phase, the oil phase and aqueous phase are mixed, and after emulsification, solidification, washing and freeze drying, phage-loaded ...

8. The thermosensitive gel formulation as described in claim 7, characterized in that: In step (2), the biodegradable polymer material is selected from PLGA, PLA, PBS, or PHB.

9. The thermosensitive gel formulation as described in claim 7, characterized in that: In step (2), the crosslinking agent is selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or its hydrochloride, and N-hydroxysuccinimide.

10. The application of a thermosensitive gel formulation as described in any one of claims 1-9, characterized in that: The thermosensitive gel formulation can be used in anti-tuberculosis infection drugs.

Images