A kind of nanoparticle of cascade targeted co-delivery antituberculosis drug and its preparation method and application

Abstract

The application discloses a kind of nanoparticles for cascade targeted co-delivery antitubercular drugs and its preparation method and application.The biodegradable polymer is chemically modified in the application, so that its surface is simultaneously coupled with macrophage targeting ligand and aromatic boric acid derivative, and the nanoparticles with at least one water-soluble antitubercular drug and at least one fat-soluble antitubercular drug inside are obtained by using W / O / W double emulsion method; wherein, the aromatic boric acid derivative is oxidatively cleaved in the intracellular acidic or high active oxygen environment to trigger drug release, and can be combined with cis-diol structure on the cell wall of mycobacterium, to promote the enrichment of drug in intracellular bacteria local.The nanoparticles provided by the application can be applied in the preparation of drugs for treating pulmonary tuberculosis, non-tuberculous mycobacterial infection or macrophage intracellular pathogen infection.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a polylactic acid-glycolic acid copolymer (PLGA)-based nanoparticle (NDDS) with dual functions of mannose-mediated active targeting and phenylboronic acid responsiveness, particularly a multifunctional nanoparticle capable of simultaneously loading multiple anti-tuberculosis drugs and achieving highly efficient bactericidal activity within macrophages, its preparation method, and its use in the treatment of mycobacterial infections. Background Technology

[0002] Tuberculosis (TB) is a serious infectious disease caused by Mycobacterium tuberculosis, posing a significant threat to human health. Globally, there are over ten million new cases each year, making it one of the top ten causes of death worldwide. Drug-resistant tuberculosis (DR-TB), especially multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (ADR-TB), represents a major challenge to global TB control efforts and is one of the leading threats to global health. China has the third highest TB burden in the world and the second highest number of DR-TB cases globally, urgently requiring effective treatment of (drug-resistant) Mycobacterium tuberculosis.

[0003] Mycobacterium tuberculosis is a facultative intracellular parasite, meaning it can survive even after being engulfed by phagocytes. Therefore, phagocytes such as macrophages not only fail to eradicate the bacteria but also become breeding grounds for them, leading to recurrent infections. This is one of the key reasons why tuberculosis is difficult to cure. Currently, the treatment of (drug-resistant) tuberculosis requires the combined use of multiple drugs, repeated administration, and prolonged treatment, resulting in poor patient adherence.

[0004] In recent years, nanomedicine delivery systems have been widely explored for improving the efficacy of anti-tuberculosis treatment. Nanocarriers offer advantages such as high-dose drug loading, targeted drug delivery, and sustained drug release at the lesion site, thereby reducing drug dosage, frequency of administration, and treatment time, thus lowering toxic side effects. Furthermore, the small size and other nanoscale properties of the carriers hold promise for overcoming drug resistance. Among these, polylactic-co-glycolic acid copolymer (PLGA) has become one of the most commonly used carrier materials due to its good biocompatibility, biodegradability, and FDA approval history. However, most existing PLGA nanoparticles only possess passive targeting capabilities, lacking recognition and response mechanisms to the infection microenvironment. Moreover, most studies focus on single-drug delivery, failing to meet the needs of multi-drug synergistic therapy. In addition, some studies have attempted to achieve macrophage targeting through surface-modified glycoligands (such as mannose) or to introduce pH / ROS-sensitive bonds for stimulus-responsive release, but no reports have yet emerged of a comprehensive nanoplatform combining active targeting with microenvironment responsiveness, achieving co-loading of water-soluble and lipid-soluble drugs.

[0005] Therefore, developing a novel nanoparticle that can precisely target and infect macrophages and intelligently release multiple anti-tuberculosis drugs in an intracellular acidic and highly reactive oxygen species (ROS) environment is of great significance for improving efficacy, shortening treatment duration, and reducing toxicity. Summary of the Invention

[0006] To address the limitations of existing anti-tuberculosis nanomedicines in accurately responding to the acidic microenvironment of tuberculosis lesions and in eliminating bacteria within macrophages, this invention provides a cascaded targeted co-delivery nanoparticle for anti-tuberculosis drugs. The nanoparticles utilize a biodegradable polymer as a carrier, which is covalently coupled to an aromatic boric acid derivative via amide bonds. The aromatic boric acid derivative is then linked to a macrophage-targeting ligand via borate ester bonds. The carrier encapsulates at least one water-soluble and at least one lipid-soluble anti-tuberculosis drug. The aromatic boric acid derivatives undergo oxidative cleavage in a highly reactive oxygen species environment within the cell to trigger drug release, and can bind to the cis-diol structure on the cell wall of mycobacteria, promoting local accumulation of the drug in the intracellular bacteria.

[0007] Furthermore, the biodegradable polymer is one of the aliphatic polyester polymers having terminal reactive functional groups; the aliphatic polyester polymer is polylactic acid-glycolic acid copolymer (PLGA), polylactic acid (PLA), or polycaprolactone (PCL); the terminal reactive functional group is a carboxyl group, an amino group, or a maleimide group.

[0008] Furthermore, the biodegradable polymer is PLGA; and the terminal reactive functional group is a carboxyl group.

[0009] Furthermore, the PLA or PCL includes block copolymers formed therefrom with other hydrophilic segments.

[0010] Furthermore, the macrophage-targeting ligand is one of mannose, fucose, galactose, or N-acetylglucosamine.

[0011] Furthermore, the macrophage targeting ligand is mannose, which is linked to the surface of the nanoparticles via amide bonds.

[0012] Furthermore, the aromatic boric acid derivative is one of phenylboronic acid or its substituted derivatives.

[0013] Furthermore, the aromatic boric acid derivative is one of phenylboronic acid, m-aminophenylboronic acid, 4-carboxyphenylboronic acid, 3-aminophenylboronic acid, or its ester derivatives, and is grafted onto the polymer backbone via borate ester bonds.

[0014] Furthermore, the loaded water-soluble and lipid-soluble antituberculosis drugs constitute a synergistic antibacterial combination, wherein: the water-soluble drug is used for rapid bactericidal action and tissue penetration, while the lipid-soluble drug is used for long-acting sustained release and accumulation within macrophages; or, the water-soluble drug inhibits protein synthesis, while the lipid-soluble drug interferes with cell wall / energy metabolism, achieving multi-target synergy. Further, the water-soluble antituberculosis drug is selected from at least one of levofloxacin hydrochloride, moxifloxacin hydrochloride, isoniazid, ethambutol, amikacin, streptomycin, or capreomycin; the lipid-soluble antituberculosis drug is selected from at least one of clofazimine, linezolid, bedaquiline, delamani, putomani, or rifabutin.

[0015] Furthermore, the water-soluble anti-tuberculosis drug is selected from levofloxacin hydrochloride or moxifloxacin hydrochloride; the lipid-soluble anti-tuberculosis drug is selected from clofazimine or linezolid.

[0016] Furthermore, the biodegradable polymer is selected from polylactic acid-glycolic acid copolymer (PLGA), the macrophage targeting ligand is mannose, the aromatic boric acid derivative is phenylboronic acid, and the nanoparticles have a drug encapsulation efficiency of 51.7% ± 3.7%, a particle size of 200-240 nm, and a PDI ≤ 0.21.

[0017] This invention also provides a method for preparing nanoparticles for cascaded targeted co-delivery of anti-tuberculosis drugs, comprising the following steps: (1) Chemically modify the biodegradable polymer so that the polymer is covalently coupled with aromatic boric acid derivatives through amide bonds, and the aromatic boric acid derivatives are linked to macrophage targeting ligands through borate ester bonds to obtain a modified carrier; (2) Using the W / O / W double emulsification method, at least one water-soluble anti-tuberculosis drug is encapsulated in the inner aqueous phase and at least one lipid-soluble anti-tuberculosis drug is dispersed in the oil phase, and W / O / W double emulsion is formed by two emulsifications; (3) Nanoparticles co-loaded with anti-tuberculosis drugs were obtained by solvent evaporation, centrifugation purification and freeze-drying.

[0018] Furthermore, the W / O / W dual emulsification method includes: (a) A lipid-soluble anti-tuberculosis drug and a functionalized polymer are dissolved in an organic solvent to form an oil phase; (b) Dissolving water-soluble antituberculosis drugs in water to form an internal aqueous phase; (c) The aqueous phase is added to the oil phase and ultrasonically emulsified under ice bath to form a W / O primary emulsion; (d) Add the W / O colostrum dropwise to an aqueous solution containing 0.5-2% (w / v) polyvinyl alcohol and then ultrasonically emulsify it again to form a W / O / W double emulsion.

[0019] Furthermore, the organic solvent is one or more of dichloromethane, ethyl acetate, acetone, or tetrahydrofuran.

[0020] Furthermore, the emulsification in steps (c) and (d) is performed using an ultrasonic cell disruptor with a power of 80-150 W for 2-5 minutes.

[0021] The nanoparticles provided by this invention are used in the preparation of drugs for treating pulmonary tuberculosis, nontuberculous mycobacterial infection, or pathogen infection within macrophages.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) It is generally believed in the art that aromatic boric acid derivatives and their substituted derivatives are mainly used for glucose sensing. This invention is the first to use them in an anti-tuberculosis targeted system and achieve synergistic effect. The macrophage targeting ligand is combined with the pH / ROS responsiveness mediated by aromatic boric acid derivatives. The structural units that target bacterial cell walls and macrophages are sequentially modified at the ends of the amphiphilic polymer to construct a nanocarrier with a 'recognition-enrichment-response release' cascade effect, thereby achieving targeted killing of bacteria in macrophages, significantly improving the accumulation efficiency of drugs in the infection focus and the accuracy of intracellular release, enhancing the therapeutic effect, and reducing the distribution of drugs in non-target organs through the targeted delivery mechanism, reducing systemic toxic side effects, and significantly improving drug safety while ensuring efficacy.

[0023] (2) At the same time, it loads water-soluble and lipid-soluble anti-tuberculosis drugs with high encapsulation efficiency, avoids the physicochemical incompatibility problem in traditional compound preparations, realizes multi-drug synergistic delivery, improves treatment compliance and efficacy consistency. The prepared nanoparticles can significantly increase the concentration and retention time of drugs in infected macrophages, achieve long-term bactericidal effect, effectively inhibit the recurrence and drug resistance evolution of intracellular bacteria, and are especially suitable for the treatment of latent or chronic tuberculosis infection.

[0024] (3) The materials used in this invention are widely available, the synthesis steps are simple, the process is stable and reproducible, and it is suitable for large-scale production and future clinical translation applications. Furthermore, the PPM platform is not limited to specific drugs or pathogens and can be replaced by other hydrophilic and hydrophobic drugs to co-deliver multiple drugs in other combinations for the treatment of sensitive tuberculosis, single-drug resistant tuberculosis, multi-drug resistant tuberculosis and multidrug resistant tuberculosis. It has broad clinical application prospects and platform development potential. Attached Figure Description

[0025] Figure 1 This is the synthetic route diagram for the drug-loaded nanoparticle PLGA-PBA-MAN.

[0026] Figure 2 Structural characterization of PPM: (A) 1H NMR spectrum; (B) Fourier transform infrared spectrum.

[0027] Figure 3 The particle size and microstructure of the nanoparticles are shown in the images: (A) DLS image; (B) Transmission electron microscope image.

[0028] Figure 4 The drug release curves of drug-loaded nanoparticles under different conditions are shown: (A) (LVX+LNZ)@PPM NPs; (B) (MXF+CFZ)@PPM NPs.

[0029] Figure 5 For in vitro antibacterial effects: (A) Minimum inhibitory concentrations of LVX, LNZ, MXF, and CFZ; (B) Plate smear images of different samples against Mycobacterium smegma; (C) Inhibitory effects of (LVX+LNZ)@PPM NPs and (MXF+CFZ)@PPM NPs on different bacteria.

[0030] Figure 6 To demonstrate the effectiveness of in vitro anti-intracellular mycobacteria: (A) Photographs of intracellular Mycobacterium smegmatis treated under different conditions using the plate coating method; (B) CFU count of intracellular Mycobacterium smegmatis.

[0031] Figure 7 Evaluation of the ability of PPM NPs to target macrophages in vitro: (A) CLSM was used to observe the uptake of NR@PLGA NPs and NR@PPM NPs by RAW264.7 macrophages; (B) CLSM was used to observe the uptake of NR@PPM NPs by a mixed system of L929 fibroblasts and RAW264.7 macrophages; (C) TEM was used to observe the uptake of PPM NPs by Mycobacterium smegma and RAW264.7 macrophages.

[0032] Figure 8 To evaluate the ability of PPM NPs to target bacteria within macrophages in vitro. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0034] Example 1: Preparation of PPM NPs simultaneously encapsulated with clofazimine (CFZ) and moxifloxacin hydrochloride (MXF) according to Figure 1 The synthetic route for cascade-targeted co-delivery of multiple anti-tuberculosis drugs NDDS, and the specific preparation steps of the drug-loaded PLGA-PBA-MAN are as follows: (1) Preparation of phenylboronic acid-modified PLGA: 1.0 g of PLGA-COOH was dissolved in 10 mL of dimethyl sulfoxide (DMSO), and 240 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), 144 mg of N-hydroxysuccinimide (NHS), and 171 mg of m-aminophenylboronic acid (PBA) were added sequentially. The mixture was magnetically stirred at room temperature for 12 h. The resulting mixture was then transferred to a dialysis bag (Mw 3500) and dialyzed in ultrapure water for 3 days. After lyophilization, the intermediate product, phenylboronic acid-modified PLGA, was obtained, namely PLGA-PBA.

[0035] Among them, polylactic acid-glycolic acid copolymer PLGA-COOH, containing carboxyl terminus, serves as a polymer backbone material with good biocompatibility and degradability, and is used to construct nanoparticle cores; EDC can activate the carboxyl groups (-COOH) on PLGA to form active ester intermediates, promoting the reaction with amino groups to form amide bonds; NHS improves the efficiency and stability of the EDC reaction, forming more stable NHS-ester intermediates, preventing hydrolysis, and enhancing coupling yield; m-aminophenylboronic acid (PBA) contains primary amino groups (-NH2) and phenylboronic acid groups (-B(OH)2). Among them, -NH2 undergoes an amidation reaction with PLGA-COOH and is grafted onto PLGA; phenylboronic acid endows the material with ROS responsiveness (cleavage under high reactive oxygen environments) and potential pH sensitivity (reversible binding with cis-diols).

[0036] (2) Preparation of mannose-modified PLGA-PBA: 1.0 g of PLGA-PBA and 225 mg of mannose (MAN) were dissolved in 10 mL of DMSO and reacted magnetically at room temperature for 2 h. The resulting mixture was then transferred to a dialysis bag (Mw3500) and dialyzed in ultrapure water for 3 days. After lyophilization, the target product, mannose-modified PLGA-PBA, was obtained, namely PLGA-PBA-MAN, abbreviated as PPM.

[0037] The reaction between PLGA-PBA and mannose (MAN) is based on the reversible covalent bonding between the phenylboronic acid group and the cis-diol structure, forming a cyclic borate ester bond. PLGA-PBA has a terminal phenylboronic acid group (-B(OH)2), and the mannose molecule contains multiple ortho-cis-diol structures (-C(OH)-C(OH)-). In anhydrous or low-water-activity organic solvents (such as DMSO), the boron atom of the phenylboronic acid acts as a Lewis acid, coordinating with the oxygen atom of the cis-diol in mannose, removing one molecule of water, and forming a five- or six-membered cyclic borate ester.

[0038] Mannose is a specific ligand for the mannose receptor (MMR / CD206) on the surface of macrophages. Modification with this ligand allows for active targeting of Mycobacterium tuberculosis's primary host cells (alveolar macrophages). The ester bond formed between phenylboronic acid and diol is pH-sensitive, readily hydrolyzed in acidic environments, and ROS-sensitive; high concentrations of reactive oxygen species can oxidize and break the borate ester. At the site of tuberculosis infection (within macrophages), high ROS and an acidic environment can promote the breaking of this bond, enabling on-demand drug release or the release of the targeting ligand, thereby enhancing cellular uptake.

[0039] (3) Preparation of drug-loaded PLGA-PBA-MAN: To address the issue of low drug (especially lipid-soluble drugs) encapsulation efficiency in the preparation of anti-tuberculosis nanoparticles using the traditional emulsion-solvent evaporation method, this scheme investigates two factors through single-factor experiments: drug concentration and the volume ratio of the external aqueous phase to the promulgated emulsion (W2:PE). Specific conditions are shown in Table 1. The appearance morphology of the drug was observed, the average particle size and whether the particle size distribution was normal were detected, and the average drug loading, average encapsulation efficiency, and RSD value were calculated to examine the reproducibility of the formulation. The screening results are shown in Table 2.

[0040] Table 1. Factors and levels of conditional experimental setup Table 2 Results of Conditional Experiments The optimal preparation steps for preparing PLGA nanoparticles co-loaded with multiple anti-tuberculosis drugs were ultimately determined through screening. Specifically: (1) Construction of oil phase and inner aqueous phase: 100 mg of PLGA-PBA-MAN and 40 mg of clofazimine (CFZ) were weighed and placed in a 10 mL EP tube. 2 mL of dichloromethane (DCM) was added to dissolve it completely, which served as the oil phase (O). DCM is volatile and can be used to solidify nanoparticles. 0.2 mL of moxifloxacin hydrochloride (MXF) aqueous solution (200 mg / mL) was added as the inner aqueous phase (W1). Among them, PLGA-PBA-MAN is a functionalized polymer carrier material that provides a nanoparticle framework; PBA imparts pH / ROS responsiveness; MAN enables macrophage targeting (mannose receptor-mediated endocytosis); CFZ is a lipid-soluble anti-tuberculosis drug that is soluble in the organic phase (DCM) and is encapsulated in the PLGA matrix; MXF is a water-soluble drug that needs to be encapsulated in the inner aqueous phase chamber by the W / O / W method to avoid direct contact with the organic phase, which could lead to degradation or leakage. This step achieves the co-encapsulation of hydrophilic and hydrophobic drugs, preventing drug degradation or leakage during the preparation process.

[0041] (2) Double emulsification: The mixture was placed in an ice-water bath and emulsified using an ultrasonic cell disruptor at a power of 120W for a total time of 3 min (3 s working time and 3 s intermittent time) to obtain a W / O primary emulsion. Then, the W / O primary emulsion was added to 40 mL of PVA solution (1%, w / v) in the external aqueous phase (W2) and emulsified again using an ultrasonic cell disruptor at a power of 120W for a total time of 3 min (3 s working time and 3 s intermittent time) to obtain a W / O / W secondary emulsion. PVA served as an external aqueous phase stabilizer, adsorbing onto the surface of the nanoparticles to reduce interfacial tension, prevent aggregation, and improve colloidal stability.

[0042] (3) The prepared emulsion was placed in a fume hood and magnetically stirred for 12 h to allow the DCM to fully evaporate and be removed, thereby solidifying the PLGA polymer chains and forming solid nanoparticle cores. The prepared emulsion was centrifuged (4 °C, 10000 rpm, 5 min), the supernatant was discarded, the nanoparticle precipitate was collected, and the precipitate was washed with deionized water to remove free drugs, PVA, and unencapsulated components. The centrifugation and washing process was repeated three times. The nanoparticles were then freeze-dried under vacuum to obtain nanocarriers (MXF+CFZ)@PPM NPs that simultaneously encapsulate two anti-tuberculosis drugs.

[0043] The encapsulation efficiency of the drug prepared by the above steps is increased to 51.7%±3.7%, the particle size is concentrated at 220±20 nm, the PDI≤0.21, and it can be stable in PBS buffer (pH 7.4) for at least 1 month.

[0044] Example 2: Preparation of PPM NPs simultaneously encapsulating levofloxacin hydrochloride (LVX) and linezolid (LNZ) PPM NPs simultaneously encapsulating levofloxacin hydrochloride (LVX) and linezolid (LNZ) were prepared using a method similar to that in Example 1, i.e., (LVX+LNZ)@PPM NPs. The difference from the preparation method of (MXF+CFZ)@PPM NPs is that 0.2 mL of levofloxacin hydrochloride (200 mg / mL) was used as the inner aqueous phase (W1); 100 mg of PLGA-COOH and 40 mg of linezolid were dissolved in 2 mL of DCM as the oil phase (O).

[0045] Example 3: Characterization of the structure, morphology, and drug release behavior of PPM (1) Adopt 1 The chemical structures of PLGA-PBA and PLGA-PBA-MAN were characterized by ¹H NMR (Avance III, Brookby Bayesian Ltd.) and FTIR (iS5, Thermo Fisher Scientific, Inc., USA), and the results are as follows: Figure 2As shown in the figure. The results show that the resonance peak at 10.0–10.2 ppm corresponds to the hydrogen atoms of the generated amide bond, the resonance peak at 7.0–8.5 ppm corresponds to the hydrogen atoms on the benzene ring, and the characteristic peak at 6.0–6.5 ppm corresponds to the characteristic peak of the hydrogen atom on the carbon at position 1 of D-mannose. In the FTIR spectrum, the characteristic peak at 1356 cm⁻¹ originates from the stretching vibration of the BO bond, the C=O stretching vibration peak of the amide bond is at 1650 cm⁻¹, and the broad peak at 3000–3500 cm⁻¹ is the stretching vibration peak of the NH bond.

[0046] (2) The hydrated particle size of blank nanoparticles and drug-loaded nanoparticles was measured using a dynamic light scattering instrument (DLS, BeNano180Zeta, Dandong Better Instruments Co., Ltd.); their microstructure was observed using a transmission electron microscope. The results are as follows: Figure 3 As shown in the figure, the hydrated particle size of PPM NPs, (MXF+CFZ)@PPM NPs, and (LVX+LNZ)@PPM NPs is approximately 253 nm, and is unimodal. Their polydispersity index (PDI) is <0.2, indicating that the prepared nanoparticles have relatively uniform particle size. Their microstructure exhibits a spherical shell structure.

[0047] (3) The drug loading, encapsulation efficiency and drug release curve of drug-eluting nanoparticles under different preparation conditions were determined by high performance liquid chromatography (HPLC, LC-20A, Shimadzu Instruments (Suzhou) Co., Ltd.). Figure 4 The figures show the drug release curves of the drug-loaded nanoparticles under different conditions. The results show that the nanoparticles release more in an acidic environment at pH 5.5 compared to the release at pH 7.4. This indicates that the PPM NPs drug delivery system can release a large amount of drug in an acidic environment infected by bacteria, thereby increasing the local drug concentration for sterilization.

[0048] Example 4: Evaluation of the in vitro antibacterial properties of drug-loaded nanoparticles (1) Anti-extracellular bacteria First, the minimum inhibitory concentration (MIC) of different drugs against Mycobacterium smegmatis was determined: 100 μL of brain heart and brain extract (BHI) broth containing serially diluted compounds was added to 96-well plates; Mycobacterium smegmatis was diluted to 5 × 10⁻⁶. 6 CFU / mL was added to wells of a plate; after co-incubation with drug-loaded nanoparticles of different drug concentrations for 24 h, the absorbance (OD) at 600 nm of the culture was recorded using a microplate reader. 600 ).

[0049] In addition, *M. smegmatis* was treated with PBS (pH 7.4), LVX+LNZ, MXF+CFZ, PPM NPs, (LVX+LNZ)@PPM NPs, and (MXF+CFZ)@PPM NPs, respectively. The synergistic effect (FIC) of LVX+LNZ or MXF+CFZ was tested using a checkerboard method. The results of the antibacterial combination screening experiment showed that the combination of 0.5 μg / mL CFZ and 0.5 μg / mL MXF exhibited the best antibacterial effect, with a fractional inhibition concentration (FIC) of 0.625, indicating synergistic antibacterial activity. The FIC of the combination of 0.5 μg / mL LVX and 0.5 μg / mL LNZ was 1.0, showing an additive antibacterial effect. In subsequent experiments, the working concentration of both groups of combined drugs was set to 2 × MIC to ensure full antibacterial activity. The PLGA content in the blank PPM NPs was equivalent to the amount used in the drug-loaded group; after co-incubation at 37 °C for 36 h, M. smegmatis was collected and inoculated onto BHI solid medium plates to detect colony forming units (CFUs), and antibacterial activity was assessed by counting CFUs.

[0050] The results are as follows Figure 5 As shown, the drug-loaded nanoparticles exhibited antibacterial effects similar to those of free drugs, indicating that PPM encapsulation did not affect the antibacterial ability of existing antibacterial drugs, and bacteria could be eliminated through the release of the antibacterial drugs. Both (LVX+LNZ)@PPM NPs and (MXF+CFZ)@PPM NPs showed good antibacterial effects against different bacteria.

[0051] (2) Anti-intracellular bacteria First, an intracellular bacterial model was established. RAW264.7 macrophages were seeded in 24-well plates (5 × 10⁶ cells / well). 4 Cells / well were cultured for another 24 h to allow complete cell adhesion. *M. smegmatis* was seeded at a density of 25 times the number of cells per well. *M. smegmatis* was co-incubated with RAW264.7 macrophages for 2 h to ensure complete cell infection. The RAW264.7 macrophages were then washed three times with PBS, successfully establishing a macrophage model infected with *M. smegmatis*.

[0052] PBS, LVX+LNZ, MXF+CFZ, PPM NPs, (LVX+LNZ)@PPM NPs, and (MXF+CFZ)@PPM NPs were co-incubated with RAW264.7 macrophages infected with *M. smegmatis* for 12, 24, and 48 h, respectively. Then, RAW264.7 macrophages were lysed with 1.0 mL of PBS containing 0.1% (v / v) Triton X-100 to obtain an intracellular bacterial suspension. This suspension was diluted to 10 mL with PBS and spread in triplicate onto BHI agar plates. After 36 h, colony-forming units were counted to evaluate the inhibitory effect of each group of samples on intracellular mycobacteria.

[0053] The results are as follows Figure 6 As shown, compared with free drugs, (LVX+LNZ)@PPM NPs and (MXF+CFZ)@PPM NPs exhibited better antibacterial activity against intracellular bacteria in macrophages. The free drug group showed good antibacterial activity in the short term, but the number of bacteria gradually increased with prolonged culture time. In contrast, the (LVX+LNZ)@PPM NPs and (MXF+CFZ)@PPM NPs groups showed good antibacterial effects at different time points. This is mainly because free drugs rely on passive diffusion to enter cells, which is inefficient and easily expelled by efflux pumps. PPM NPs, on the other hand, are efficiently taken up by macrophages through mannose receptor-mediated endocytosis. The phenylboronic acid structure cleaves responsively in the high ROS and acidic environment of lysosomes, promoting drug release and achieving targeted enrichment + stimulus-response release, thereby improving intracellular bactericidal efficiency.

[0054] Example 5: Evaluation of the ability of PPM NPs to target macrophages in vitro RAW264.7 macrophages were seeded in confocal culture dishes (2 × 10⁶ cells / mL). 5 In cells / well, 2 mL of complete culture medium was added, and the cells were cultured at 37 °C and 5% CO2 for 24 h. NR@PLGA NPs and NR@PPM NPs (10 μg / mL NR) were added and co-incubated for a certain time (1 h, 2 h), respectively. The cells were then washed three times with PBS, and fixed with 4% paraformaldehyde for 20 min. Finally, the cell nuclei were stained with diamidine phenylindole (DAPI) (5–10 min), and the uptake of nanoparticles by RAW264.7 macrophages was observed using a confocal laser scanning microscope (CLSM, FV3000, Olympus, Japan). Results are as follows: Figure 7As shown in Figure A, the unmodified NR@PLGA NPs group cells did not show obvious fluorescence, while the modified NR@PPM NPs were present in the cytoplasm of RAW264.7 cells, indicating that MAN modification enhances the macrophages' ability to take up nanoparticles.

[0055] NR@PPM NPs were co-incubated with L929 and RAW264.7 cells for 2 h, and fluorescence images were observed using CLSM. Results are as follows: Figure 7 As shown in Figure B, in the mixed system of RAW264.7 and L929 co-culture, the nanoparticles clearly aggregated in the cytoplasm of RAW264.7, indicating that PPM NPs have good macrophage targeting ability.

[0056] Example 6 Evaluation of the ability of PPM NPs to target bacteria within macrophages in vitro RAW264.7 macrophages infected with FITC-labeled Mycobacterium smegmatis were obtained and then co-cultured with NR@PLGA NPs and NR@PPM NPs for 2 h. Confocal images and colocalization fluorescence intensity distribution of FITC-M. smegmatis within the cells were observed using CLSM. (See attached image.) Figure 8 As shown, FITC-M. smegmatis successfully infected RAW264.7 macrophages and was located in the cytoplasm. The red fluorescence of NR@PPM NPs almost completely overlapped with the green fluorescence of intracellular M. smegmatis, exhibiting bright yellow fluorescence in the merged field of view. These results indicate that the constructed PPM NPs showed stronger cell association and higher co-localization in M. smegmatis-infected macrophages, suggesting that they may be preferentially taken up by activated / infected macrophages, thereby achieving passive / active synergistic targeting of the infection microenvironment.

[0057] Although the above embodiments have described the present invention and its implementation in detail, it should be noted that for those skilled in the art, any changes, modifications, substitutions, combinations, simplifications, etc., made to the corresponding conditions without departing from the technical principles of the present invention should be considered as equivalent substitutions, and these improvements should also be considered within the scope of protection of the present invention.

Claims

1. A cascaded targeted co-delivery nanoparticle for anti-tuberculosis drugs, characterized in that, The nanoparticles are carried by a biodegradable polymer, which is covalently coupled to an aromatic boric acid derivative via an amide bond, and the aromatic boric acid derivative is linked to a macrophage targeting ligand via a borate ester bond. The carrier encapsulates at least one water-soluble anti-tuberculosis drug and at least one lipid-soluble anti-tuberculosis drug. The aromatic boric acid derivatives can undergo oxidative cleavage in an acidic and highly reactive oxygen species environment within cells to trigger drug release, and can bind to the cis-diol structure on the cell wall of mycobacteria to promote local accumulation of the drug in the intracellular bacteria.

2. The nanoparticles according to claim 1, characterized in that, The biodegradable polymer is one of the aliphatic polyester polymers with terminal reactive functional groups; the aliphatic polyester polymer is polylactic acid-glycolic acid copolymer (PLGA), polylactic acid (PLA) or polycaprolactone (PCL); the terminal reactive functional group is a carboxyl group, an amino group or a maleimide group.

3. The nanoparticles according to claim 1, characterized in that, The macrophage-targeting ligand is one of mannose, fucose, galactose, or N-acetylglucosamine.

4. The nanoparticles according to claim 1, characterized in that, The aromatic boric acid derivative is one of phenylboronic acid or its substituted derivatives.

5. The nanoparticles according to claim 1, characterized in that, The loaded water-soluble and lipid-soluble anti-tuberculosis drugs form a synergistic antibacterial combination, wherein: the water-soluble drugs are used for rapid sterilization and tissue penetration, and the lipid-soluble drugs are used for long-term sustained release and accumulation in macrophages; or, the water-soluble drugs inhibit protein synthesis, and the lipid-soluble drugs interfere with cell wall / energy metabolism, achieving multi-target synergy.

6. The nanoparticles according to claim 1, characterized in that, The water-soluble anti-tuberculosis drug is selected from at least one of levofloxacin hydrochloride, moxifloxacin hydrochloride, isoniazid, ethambutol, amikacin, streptomycin, or capreomycin; the lipid-soluble anti-tuberculosis drug is selected from at least one of clofazimine, linezolid, bedaquiline, delamani, putomani, or rifabutin.

7. The nanoparticles according to claim 1, characterized in that, The biodegradable polymer is selected from polylactic acid-glycolic acid copolymer (PLGA), the macrophage targeting ligand is mannose, the aromatic boric acid derivative is phenylboronic acid, and the nanoparticles have a drug encapsulation efficiency of 51.7% ± 3.7%, a particle size of 200-240 nm, and a PDI ≤ 0.

21.

8. A method for preparing nanoparticles for cascaded targeted co-delivery of anti-tuberculosis drugs as described in any one of claims 1-7, characterized in that, Includes the following steps: (1) Chemically modify the biodegradable polymer so that the polymer is covalently coupled with aromatic boric acid derivatives through amide bonds, and the aromatic boric acid derivatives are linked to macrophage targeting ligands through borate ester bonds to obtain a modified carrier; (2) Using the W / O / W double emulsification method, at least one water-soluble anti-tuberculosis drug is encapsulated in the inner aqueous phase and at least one lipid-soluble anti-tuberculosis drug is dispersed in the oil phase, and W / O / W double emulsion is formed by two emulsifications; (3) Nanoparticles co-loaded with anti-tuberculosis drugs were obtained by solvent evaporation, centrifugation purification and freeze-drying.

9. The method according to claim 8, characterized in that, The W / O / W dual emulsification method includes: (a) A lipid-soluble anti-tuberculosis drug and a functionalized polymer are dissolved in an organic solvent to form an oil phase; (b) Dissolving water-soluble antituberculosis drugs in water to form an internal aqueous phase; (c) The aqueous phase is added to the oil phase and ultrasonically emulsified under ice bath to form a W / O primary emulsion; (d) Add the W / O colostrum dropwise to an aqueous solution containing 0.5-2% (w / v) polyvinyl alcohol, and then ultrasonically emulsify it again to form a W / O / W double emulsion; The organic solvent in step (a) is one or more of dichloromethane, ethyl acetate, acetone or tetrahydrofuran; the emulsification in steps (c) and (d) is performed using an ultrasonic cell disruptor with a power of 80-150 W for 2-5 min.

10. The nanoparticles according to claims 1-9, characterized in that, It is used in the preparation of drugs for treating pulmonary tuberculosis, nontuberculous mycobacterial infections, or intracellular pathogen infections in macrophages.

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