A drug delivery system for targeting treatment of inflammatory diseases and its preparation method and application

Abstract

The application provides a drug delivery system for targeted treatment of inflammatory diseases and a preparation method and application thereof, and belongs to the technical field of biological drugs. The drug delivery system for targeted treatment of inflammatory diseases is M2 type macrophages phagocytizing drug-loaded nanoparticles, wherein the drug-loaded nanoparticles comprise a biodegradable high molecular material for coating drugs. The drug delivery system prepared by the application realizes lung targeted delivery through the natural inflammatory chemotaxis characteristics of M2 type macrophages, and in combination with the drug slow-release effect of the drug-loaded nanoparticles, the drug targeting and availability are significantly improved, so that the drug efficacy is improved, the biological safety is good, and a new effective means is provided for the clinical treatment of various inflammatory diseases including carbapenem-resistant Klebsiella pneumoniae (CRKP) pneumonia.

Description

Technical Field

[0001] This application belongs to the field of biopharmaceutical technology, specifically relating to a drug delivery system for targeted treatment of inflammatory diseases, its preparation method, and its application. Background Technology

[0002] Inflammatory diseases are characterized by multifactorial, progressive, systemic, and recurrent nature. They often exhibit chronic, progressive progression, with early symptoms often being subtle and easily overlooked. Once initiated, they are rarely reversible and can recur repeatedly, gradually worsening over time. Inflammation can manifest both locally and systemically, localized to specific tissues and organs such as joints, intestines, lungs, and blood vessels, or triggering a systemic inflammatory response through the circulatory system. Current clinical drug treatments for inflammatory diseases still face core bottlenecks such as insufficient targeting and low drug utilization. For example, tigecycline is an effective drug for carbapenem-resistant Klebsiella pneumoniae (CRKP) pneumonia, but its clinical application has several limitations: firstly, tigecycline has low oral bioavailability, requiring intravenous administration, and its targeting at the site of lung infection is poor, making it difficult to achieve effective drug concentrations; secondly, long-term or high-dose use of tigecycline can easily cause adverse reactions such as gastrointestinal reactions and liver damage, limiting its full clinical efficacy. Summary of the Invention

[0003] The purpose of this invention is to provide a drug delivery system for targeted treatment of inflammatory diseases, which can not only solve the problems of poor drug targeting and low utilization, but also control drug release to improve efficacy and reduce toxic side effects.

[0004] The present invention provides a drug delivery system for targeted treatment of inflammatory diseases, comprising M2 macrophages and drug-loaded nanoparticles located in the M2 macrophages, wherein the drug-loaded nanoparticles comprise a biodegradable polymer material for encapsulating the drug.

[0005] Preferably, the M2 macrophages are M2 macrophages that have undergone polarization-induced differentiation and matured and express the CD206 marker on their surface.

[0006] Preferably, the polarization induction reagents include 20-50 ng / mL IL-4 and 20-50 ng / mL IL-13.

[0007] Preferably, the drug-loaded nanoparticles contain at least one of the following drugs: tigecycline, doxycycline, levofloxacin, azithromycin, budesonide, tofacitinib, and carvacrol.

[0008] Preferably, the biodegradable polymeric material includes any one of the following copolymers: polylactic acid-glycolic acid copolymer, polyethylene glycol-polylactic acid copolymer, and polyethylene glycol-polylactic acid-glycolic acid copolymer.

[0009] Preferably, the drug-loaded nanoparticles have a particle size of 100~300nm, a zeta potential of -40mV~-20mV, a drug encapsulation efficiency of ≥70%, and a drug loading of 5%~15%.

[0010] This invention provides a method for preparing the drug delivery system for targeted treatment of inflammatory diseases, comprising the following steps: The drug and biodegradable polymer material are prepared into an oil phase, and sodium cholate solution is used as the aqueous phase. The oil phase and the aqueous phase are combined to form a proemulsion. The proemulsion is added to a fresh sodium cholate solution to remove the solvent from the oil phase. The precipitate is collected to obtain drug-loaded nanoparticles. The drug-loaded nanoparticles were mixed and incubated with M2 macrophages to remove the free drug-loaded nanoparticles, thus obtaining a drug delivery system.

[0011] Preferably, in the mixed incubation system, the ratio of drug-loaded nanoparticles to M2 macrophages is 1:100~300; The mass ratio of the drug to the biodegradable polymer in the oil phase is 1:5~20; The sodium cholate solution has a mass concentration of 1% to 3%, and the volume ratio of the oil phase to the water phase is 1:5 to 15.

[0012] The present invention provides the application of the drug delivery system for targeted treatment of inflammatory diseases or the drug delivery system for targeted treatment of inflammatory diseases prepared by the preparation method in the preparation of drugs for the prevention and / or treatment of inflammatory diseases.

[0013] The present invention provides a drug for treating inflammatory diseases, including the drug delivery system for targeted treatment of inflammatory diseases or the drug delivery system for targeted treatment of inflammatory diseases prepared by the preparation method.

[0014] This invention provides a drug delivery system for targeted therapy of inflammatory diseases, comprising M2 macrophages and drug-loaded nanoparticles located within the M2 macrophages. The drug delivery system provided by this invention uses M2 macrophages as delivery carriers, leveraging their natural chemotaxis towards sites of inflammation. These macrophages can actively recognize the inflammatory microenvironment induced by inflammation and migrate directionally to the lesion site, solving problems such as poor drug targeting, low bioavailability, easy drug resistance, and significant side effects. Simultaneously, the biodegradable nanoparticles, as drug carriers, can improve drug stability and control drug release. The two work synergistically to achieve highly efficient targeted therapy for CRKP pneumonia, improving efficacy and reducing toxic side effects.

[0015] Furthermore, the drug delivery system provided by this invention specifically defines the particle size, drug loading rate, and drug encapsulation rate of the drug-loaded nanoparticles. The appropriate particle size ensures efficient phagocytosis by M2 macrophages while preventing drug deposition in non-target tissues. Simultaneously, a high encapsulation rate (≥70%) ensures effective drug loading. After reaching the lesion, the drug-loaded macrophages can slowly release tigecycline through apoptosis or exocytosis, achieving a sustained high concentration distribution of the drug at the lesion site and effectively achieving the therapeutic goal.

[0016] Furthermore, the drug delivery system provided by this invention specifically specifies PLGA as the biodegradable polymer material. PLGA, as a carrier, encapsulates tigecycline, protecting the drug from enzymatic degradation in vivo and prolonging its half-life. Sodium cholate, as an emulsifier, exhibits superior biocompatibility compared to traditional polyvinyl alcohol, reducing the toxic side effects of the carrier material. Moreover, the drug delivery system achieves drug delivery through macrophage phagocytosis, reducing the direct stimulation of normal tissues by tigecycline and lowering the incidence of adverse reactions such as gastrointestinal reactions and liver damage. Attached Figure Description

[0017] Figure 1 Transmission electron microscopy image of tigecycline nanoparticles; Figure 2 The particle size distribution (A) and zeta potential (B) of tigecycline nanoparticles are shown. Figure 3 The in vitro drug release curve of tigecycline nanoparticles in Example 1; Figure 4 Image of CD206-positive cells after M2 macrophage induction by flow cytometry. Figure 5 Laser confocal microscopy image of M2 macrophages phagocytosing tigecycline nanoparticles; Figure 6 The image shows the targeted distribution of the drug delivery system in a CRKP pneumonia model mouse; where A represents the fluorescence intensity detection results in the mouse, and B represents the distribution of drug-loaded macrophages in various organs. Figure 7 The figure shows the effect of different drug administration groups on the bacterial load in the lungs of CRKP pneumonia model mice; where A represents the bacterial load in lung cells and B represents the statistical results of the bacterial load in lung cells. Figure 8 HE staining results of lung inflammation damage in CRKP pneumonia model mice in different drug administration groups. Detailed Implementation

[0018] The present invention provides a drug delivery system for targeted treatment of inflammatory diseases, comprising M2 macrophages and drug-loaded nanoparticles located in the M2 macrophages, wherein the drug-loaded nanoparticles comprise a biodegradable polymer material for encapsulating the drug.

[0019] The drug delivery system provided by this invention utilizes viable M2 macrophages that engulf drug-loaded nanoparticles. These M2 macrophages, acting as carriers, possess inflammatory chemotaxis properties, enabling active targeting of lung infection foci and precise drug delivery. Simultaneously, the living cells exhibit autonomous chemotaxis and migration capabilities, actively sensing chemical signals at the infection site and penetrating complex physiological barriers (such as the pulmonary blood-air barrier and inflamed tissue) to reach deeper into the lesion. Upon reaching the lesion, they produce and release high concentrations of antibacterial substances (such as hydrogen peroxide and antibacterial proteins) on demand (e.g., in response to bacterial stimulation), achieving a synergistic effect far exceeding the drug loading capacity.

[0020] In this invention, the M2 macrophages are preferably M2 macrophages that have undergone polarization-induced differentiation and matured and express the CD206 marker on their surface. The polarization-inducing reagent preferably comprises 20-50 ng / mL of IL-4 and 20-50 ng / mL of IL-13, but can be 25-45 ng / mL of IL-4 and 25-45 ng / mL of IL-13, or 30-40 ng / mL of IL-4 and 30-40 ng / mL of IL-13, or 35 ng / mL of IL-4 and 35 ng / mL of IL-13. The polarization induction method for M2 macrophages involves taking peripheral blood mononuclear cells or bone marrow mesenchymal stem cells, culturing them in DMEM medium containing 10% fetal bovine serum, adding IL-4 and IL-13, and inducing them for 5-7 days at 37°C and 5% CO2. After induction, the CD206 positivity rate was detected by flow cytometry to be ≥80%, ensuring that they have good inflammatory chemotaxis and targeting properties.

[0021] In this invention, the drug in the drug-loaded nanoparticles preferably includes tigecycline. Tigecycline is a glycylcycline antibiotic with good antibacterial activity against carbapenem-resistant Klebsiella pneumoniae (CRKP) and other multidrug-resistant Gram-negative bacteria. The drug also includes relevant active pharmaceutical ingredients for treating infectious inflammation, such as doxycycline, levofloxacin, azithromycin, budesonide, tofacitinib, and carvacrol. In this embodiment, to illustrate the good targeted therapeutic effect of the drug delivery system provided by this invention on inflammation, tigecycline is used as an example to treat CRKP-induced pneumonia, demonstrating the therapeutic effect of the drug delivery system.

[0022] In this invention, the biodegradable polymer material preferably comprises any one of the following copolymers: polylactic acid-glycolic acid copolymer, polyethylene glycol-polylactic acid copolymer, and polyethylene glycol-polylactic acid-glycolic acid copolymer. The biodegradable polymer material, as a carrier for the drug (tigecycline), can improve drug stability and control drug release. In this embodiment of the invention, tigecycline nanoparticles prepared from tigecycline and PLGA are used as an example to illustrate that, under pH conditions of 5.5–7.4, the drug release efficiency gradually decreases as the pH value increases.

[0023] In this invention, the drug-loaded nanoparticles have a particle size of 100-300 nm, a zeta potential of -40 mV to -20 mV, a drug encapsulation efficiency of ≥70%, and a drug loading of 5%-15%. The particle size range of the drug-loaded nanoparticles facilitates phagocytosis by M2 macrophages and avoids premature clearance by the mononuclear-macrophage system in vivo. The zeta potential of the drug-loaded nanoparticles, ranging from -40 mV to -20 mV, maintains the stability of the drug-loaded nanoparticle suspension; the drug encapsulation efficiency of ≥70% in the drug-loaded nanoparticles ensures effective drug loading and subsequent release efficiency.

[0024] This invention provides a method for preparing the drug delivery system for targeted treatment of inflammatory diseases, comprising the following steps: The drug and biodegradable polymer material are prepared into an oil phase, and sodium cholate solution is used as the aqueous phase. The oil phase and the aqueous phase are combined to form a pre-emulsion. The pre-emulsion is added to the sodium cholate solution, the solvent in the oil phase is removed, and the precipitate is collected to obtain drug-loaded nanoparticles. The drug-loaded nanoparticles were mixed and incubated with M2 macrophages to remove the free drug-loaded nanoparticles, thus obtaining a drug delivery system.

[0025] This invention prepares an oil phase from a drug and a biodegradable polymer material, uses a sodium cholate solution as an aqueous phase, and prepares a proemulsion by combining the oil phase and the aqueous phase. The proemulsion is then added to the sodium cholate solution to remove the solvent from the oil phase, and the precipitate is collected to obtain drug-loaded nanoparticles.

[0026] In this invention, the method for preparing an oil phase from a drug and a biodegradable polymer is preferably to mix the drug and the biodegradable polymer and then dissolve them in dichloromethane. The mass ratio of the drug to the biodegradable polymer in the oil phase is preferably 1:5~20, but can be 1:8~18, 1:10~15, or 1:1~14. The concentration of the drug in the dichloromethane is preferably 1~20 mg / mL, but can be 2~10 mg / mL.

[0027] In this invention, the mass concentration of the sodium cholate solution is preferably 1% to 3%, but can be 1.2% to 2.8%, 1.5% to 2.5%, or 2.0%. The sodium cholate, as an anionic amphiphilic surfactant, is compounded with biodegradable polymer materials. Through emulsification, it reduces the oil-water interfacial tension, inhibits nanoparticle aggregation, and obtains uniform particle size (mostly 50 to 300 nm). Furthermore, through surface modification, it imparts a negative charge to the nanoparticles, reducing non-specific adsorption and prolonging in vivo circulation. In addition, it enhances the solubility of hydrophobic drugs in the aqueous phase and improves drug loading efficiency by acting as a solubilizer.

[0028] In this invention, the volume ratio of the oil phase to the aqueous phase is preferably 1:5 to 15, and can also be 1:10. When preparing the primary emulsion, the oil phase is slowly added dropwise to the aqueous phase, and ultrasonic emulsification is performed for 15 to 25 minutes under ice bath conditions. The method for removing the solvent from the oil phase is preferably to magnetically stir at room temperature for 2 to 4 hours to allow the dichloromethane to fully evaporate. The method for collecting the precipitate is preferably to centrifuge the solvent-removed system, collect the precipitate, wash it 2-3 times with physiological saline to remove free drug and residual emulsifier, and then freeze-dry it.

[0029] After obtaining the drug-loaded nanoparticles, the present invention mixes and incubates the drug-loaded nanoparticles with M2 macrophages to remove the free drug-loaded nanoparticles, thereby obtaining a drug delivery system.

[0030] In this invention, the ratio of drug-loaded nanoparticles to M2 macrophages in the mixed incubation system is preferably 1:100-300, but can also be 1:120-280, 1:150-250, or 1:200. The preferred incubation conditions are: the drug-loaded nanoparticles and M2 macrophages mixture is inoculated into DMEM medium containing 10% fetal bovine serum and incubated at 37°C and 5% CO2 for 12-24 hours to allow the M2 macrophages to fully engulf the nanoparticles.

[0031] In this invention, before mixing and incubating, it is preferable to wash the induced M2 macrophages 2-3 times with physiological saline to remove free drugs and residual emulsifiers.

[0032] In this invention, the preferred method for removing free drug-loaded nanoparticles is to collect cells by low-speed centrifugation (1000 r / min, 5 min) after incubation, and wash them three times with PBS buffer to remove unphagocytosed free nanoparticles.

[0033] The present invention provides the application of the drug delivery system for targeted treatment of inflammatory diseases or the drug delivery system for targeted treatment of inflammatory diseases prepared by the preparation method in the preparation of drugs for the prevention and / or treatment of inflammatory diseases.

[0034] In this invention, when the drug is tigecycline, the prepared drug delivery system is used in drugs for the prevention and / or treatment of CRKP pneumonia.

[0035] In this invention, the drug is administered via intravenous injection, with a dosage of 3 mg / kg body weight based on tigecycline, administered once every 24 hours, and the treatment cycle consisting of 5 to 7 consecutive days.

[0036] The present invention provides a drug for treating inflammatory diseases, including the drug delivery system for targeted treatment of inflammatory diseases or the drug delivery system for targeted treatment of inflammatory diseases prepared by the preparation method.

[0037] The drug delivery system of this invention utilizes the inflammatory chemotactic properties of M2 macrophages to actively target lung infection foci, and combines this with the sustained-release effect of nanoparticles to enhance drug accumulation at the infection site. Results from the embodiments show that the drug delivery system can effectively target the lungs, significantly reducing the bacterial load in lung tissue and effectively treating CRKP pneumonia. The preparation method of this invention is simple and reproducible, and the delivery system exhibits strong targeting, high antibacterial activity, and good safety, providing a new strategy for the treatment of CRKP pneumonia and is suitable for the preparation of targeted therapeutic drugs.

[0038] In this embodiment of the invention, compared with the PBS group, the free tigecycline group, the tigecycline nanoparticle group, and the drug-loaded macrophage group significantly reduced the bacterial load in the lungs, and the bacterial load in the drug-loaded macrophage group was significantly lower than that in the free tigecycline group and the tigecycline nanoparticle group. Simultaneously, anti-inflammatory experiments showed that the alveolar structure in the lungs of the drug-loaded macrophage group remained intact, inflammatory cell infiltration was significantly reduced, and exudate essentially disappeared, indicating that it can effectively alleviate lung inflammatory damage caused by CRKP pneumonia. Furthermore, the drug delivery system exhibits good drug safety.

[0039] The following detailed description, in conjunction with embodiments, illustrates a drug delivery system for targeted treatment of inflammatory diseases, its preparation method, and its application, but these should not be construed as limiting the scope of protection of this invention.

[0040] Example 1 Preparation and performance characterization of tigecycline nanoparticles 1 mg of tigecycline and 10 mg of PLGA (mass ratio 1:10) were weighed and dissolved together in 1 mL of dichloromethane (analytical grade, purity ≥99.5%). The solution was sonicated (150 W, 3 min) to obtain a homogeneous oil phase. Simultaneously, 0.05 g of sodium cholate was weighed and dissolved in 5 mL of deionized water. The solution was magnetically stirred (800 rpm) for 30 min to prepare a 1% sodium cholate solution as the aqueous phase. The oil phase was slowly added dropwise to the aqueous phase under magnetic stirring (400 rpm) (oil phase:water phase = 1:6, v / v). After the addition was complete, the solution was ultrasonically emulsified in an ice bath for 20 min (200 W, 3 s on, 2 s off) to form a milky white homogeneous primary emulsion. The primary emulsion was transferred to 5 mL of a 1% sodium cholate solution and magnetically stirred at room temperature for 3 h (500 rpm) to allow the dichloromethane to fully evaporate, resulting in a nanoparticle suspension. The suspension was centrifuged at 10,000 r / min for 30 min, the precipitate was collected, and washed three times with physiological saline (each centrifugation was performed under the same conditions) to remove free drug and residual emulsifier. The precipitate was then pre-frozen in an ultra-low temperature freezer at -80℃ for 2 h and freeze-dried for 24 h to obtain white powdered tigecycline nanoparticles.

[0041] A small amount of lyophilized nanoparticles was resuspended in deionized water to a concentration of 0.1 mg / mL, dropped onto a copper grid, stained with phosphotungstic acid, and then air-dried. The morphology was observed and photographed using a transmission electron microscope (TEM). Figure 1 The results showed that the prepared nanoparticles were spherical, with regular and uniform morphology, and no obvious aggregation. The particle size distribution and zeta potential of the above resuspension were detected using dynamic light scattering (DLS). Figure 2 The results showed that the average particle size of the nanoparticles was 197.0±0.2 nm, exhibiting a narrow distribution; the zeta potential was -36.4±1.08 mV, indicating good stability of the suspension.

[0042] High-performance liquid chromatography (HPLC) was used for detection. The chromatographic conditions were as follows: C18 column (4.6 mm × 250 mm, 5 μm), mobile phase: acetonitrile-0.1% trifluoroacetic acid aqueous solution (30:70 v / v), detection wavelength: 280 nm, column temperature: 30 ℃, flow rate: 1.0 mL / min, injection volume: 20 μL. 10 mg of lyophilized nanoparticles were accurately weighed, dissolved in 5 mL of methanol / acetonitrile (1:1 v / v), sonicated for 30 min, and centrifuged (12000 r / min, 10 min). The supernatant was filtered through a 0.22 μm filter membrane and analyzed by HPLC. The total drug amount was calculated based on the tigecycline standard curve. Separately, the same mass of nanoparticles was resuspended in 5 mL of physiological saline, centrifuged (10000 r / min, 30 min), and the free drug amount was determined from the supernatant. The encapsulation efficiency was calculated according to Formula I, and the drug loading rate was calculated according to Formula II.

[0043] Encapsulation rate = (total drug - amount of free drug) / total drug × 100%, Formula I.

[0044] Drug loading = (Total drug - Free drug) / Nanoparticle mass × 100% Formula II.

[0045] The results showed that the encapsulation efficiency of tigecycline was 70.9±1.2%, which met the design requirements.

[0046] Accurately weigh 20 mg of lyophilized nanoparticles, resuspend them in 2 mL of physiological saline, and place them in a dialysis bag with a molecular weight cutoff of 100 kDa. Seal the bag and place it in 50 mL of release medium (PBS with pH values ​​of 5.5, 6.5, and 7.4). Release the nanoparticles by shaking in a constant temperature shaker at 37 °C and 100 rpm. Take 5 mL samples at 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, and 72 h, and simultaneously add an equal volume of fresh release medium. Filter the samples through a 0.22 μm filter membrane, and detect the drug concentration using an ELISA reader. Calculate the cumulative release rate and plot the release curve. Figure 3 The results showed that the cumulative release rate of nanoparticles reached 79.8 ± 0.6% within 72 hours.

[0047] Example 2 Induction and differentiation of M2 macrophages and verification of their purity The concentration of monocytes was adjusted to 1×10⁻⁶. 6 Cells were seeded at a density of 1 / mL into 6-well plates. Each well was then filled with 2 mL of DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin 100 U / mL + streptomycin 100 μg / mL. The plates were incubated at 37°C in a 5% CO2 incubator for 24 h. The supernatant and non-adherent cells were discarded, and the medium was replaced with fresh medium. IL-4 and IL-13 were added to a final concentration of 30 ng / mL for both cells. The plates were then cultured for another 6 days, with the medium replaced every 2 days with the same concentration of inducing factors.

[0048] Collect cells after induction culture, wash twice with PBS buffer, and adjust the concentration to 1×10⁻⁶. 6 Cells / mL were added, along with 5 μL each of F4 / 80-FITC fluorescent antibody and CD206-PE fluorescent antibody. The mixtures were incubated in the dark for 30 min, washed twice with PBS, and the positive expression rates of F4 / 80 and CD206 were detected by flow cytometry. Figure 4 The results showed that the CD206 positive cell rate was 80.7±0.72%, indicating that high-purity M2 macrophages were successfully induced and can be used as drug delivery carriers.

[0049] Example 3 Construction and phagocytic efficiency assay of tigecycline nanoparticles delivered by M2 macrophages The M2 macrophages induced in Example 2 were used at a rate of 1×10⁻⁶. 6 Tigecycline nanoparticles were seeded per well in 6-well plates, and 2 mL of DMEM medium containing 10% FBS was added. The plates were incubated for 24 h to allow complete adhesion. The tigecycline nanoparticles prepared in Example 1 were resuspended in fresh medium to a concentration of 3 × 10⁻⁶. 8 Cells were added to wells at a ratio of 1:300 (1 mL nanoparticle suspension per well) and incubated at 37°C with 5% CO2 for 18 h to allow macrophages to fully engulf the nanoparticles. After incubation, the cells were gently washed twice with PBS buffer to remove free nanoparticles from the surface, then digested with 0.25% trypsin (37°C, 5 min), centrifuged at low speed (1000 r / min, 5 min) to collect the cells, and washed three times with PBS to obtain M2-delivered tigecycline nanoparticles (drug-loaded macrophages).

[0050] The tigecycline nanoparticles prepared in Example 1 were labeled with the fluorescent dye coumarin 6 (Ce6) according to the instructions, and then co-incubated with M2 macrophages as described above. After 18 h of incubation, the supernatant was discarded, and the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 30 min, washed with PBS, stained with DAPI for 10 min (to stain the cell nuclei), washed again, mounted with anti-fluorescence quenching mounting medium, and observed and photographed under a confocal microscope. Figure 5 ).

[0051] The results showed that obvious green fluorescence (Ce6-labeled nanoparticles) appeared in the cytoplasm of M2 cells, and the fluorescence was evenly distributed, indicating that the nanoparticles were efficiently phagocytosed by macrophages; the statistical fluorescence positive cell rate was over 90%, proving that the drug delivery system was successfully constructed.

[0052] Example 4 In vitro antibacterial activity test The clinically isolated CRKP standard strain (ATCC BAA-1705) was selected and inoculated onto LB agar plates. It was incubated at 37°C for 18 hours. Single colonies were then picked and inoculated onto LB liquid medium, and cultured at 37°C with shaking at 200 rpm until the logarithmic growth phase (OD200). 600 =0.6~0.8), adjust the bacterial concentration to 1×10 using LB medium. 6 CFU / mL, for later use.

[0053] The CRKP strain was prepared as an inoculum, with a bacterial density of approximately 1 × 10⁻⁶ per well in a 96-well plate. 5 CFUs are divided into 4 groups, with 3 duplicates in each group: ① Control group (only bacterial suspension and culture medium were added); ② Free tigecycline group (tigecycline was added to the control group); ③ Tigecycline nanoparticle group (nanoparticles were added to the control group); ④ Drug-loaded macrophage group (drug-loaded macrophages were added to the control group).

[0054] Each test formulation was serially diluted (concentrations: 0, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 20 μg / mL, expressed as equivalent tigecycline content), and the 96-well plates were incubated at 37°C in a 5% CO2 incubator for 24 h. Colony counting was used. After incubation, 100 μL of bacterial culture from each group was serially diluted with physiological saline to a concentration of 10⁻⁶. 4 Take 100 μL of the diluted solution and spread it on LB agar plates. Incubate at 37°C for 24 h and then count the number of colonies (CFU).

[0055] The results showed that the bacterial count in the drug-loaded macrophage group was reduced to the lowest level (approximately 1.78 × 10⁻⁶). 8 The CFU / mL concentration was significantly lower than that of the free tigecycline group (approximately 7.3 × 10⁻⁶ CFU / mL). 8 CFU / mL) and tigecycline nanoparticles (approximately 3.52 × 10⁻⁶ CFU / mL) 8 The CFU / mL count indicates that the drug-loaded macrophages have stronger anti-CRKP activity in vitro.

[0056] Example 5 In vivo targeted experiments Female ICR mice aged 6-8 weeks were selected and anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg) after one week of acclimatization. 50 μL of the above-mentioned logarithmic growth phase CRKP bacterial culture (concentration 1×10⁻⁶) was then instilled nasally. 5 An acute CRKP pneumonia model was established using CFU / mL; the sham-operated group received only an equal volume of physiological saline. Twenty-four hours after modeling, blood was collected from the fundus venous plexus of mice, spread on LB agar plates, and cultured to confirm successful model establishment.

[0057] The drug-loaded macrophages prepared in Example 3 were treated with the near-infrared fluorescent dye DiR to obtain DiR-drug-loaded macrophages. Nine model mice were randomly divided into three groups (n=3 per group). Five hours after modeling, DiR-drug-loaded macrophages (3 mg / kg body weight based on tigecycline) were injected via the tail vein. The fluorescence intensity in the mice was detected using a small animal in vivo imaging system at 1, 4, 12, and 24 hours after administration. Figure 6 Mice were sacrificed 24 hours later, and heart, liver, spleen, lung, and kidney tissues were dissected and analyzed for fluorescence intensity. Figure 7 ).

[0058] The results showed that the fluorescence intensity in the lungs reached its peak 12 hours after drug administration, and was significantly higher than that in other tissues such as the heart, liver, spleen, and kidneys. The lungs maintained a high fluorescence intensity even after 24 hours, indicating that the drug-loaded macrophages had good lung-targeting aggregation ability and could accurately reach the lesion site of CRKP pneumonia.

[0059] Example 6 In vivo antibacterial efficacy and anti-inflammatory experiment Forty CRKP pneumonia mice that successfully modeled the disease were randomly divided into four groups (n=10 per group): ① saline control group; ② free tigecycline group (tail vein injection, dose 3 mg / kg, calculated as tigecycline); ③ tigecycline nanoparticle group (tail vein injection, dose 3 mg / kg, calculated as tigecycline); ④ drug-loaded macrophage group (tail vein injection, dose 3 mg / kg, calculated as tigecycline). All groups were administered the drug every 24 hours for 5 consecutive days.

[0060] After administration, mice were anesthetized and sacrificed. Lung tissue was aseptically harvested, and a 10% tissue homogenate was prepared using physiological saline, which was then serially diluted to 10%. 4 Double the volume, take 100 μL and spread it on LB agar plates. Incubate at 37°C for 24 h and then count the number of colonies. Figure 6 The results showed that the bacterial load in the lungs of the drug-loaded macrophage group was 1.3 × 10⁻⁶. 3 ±0.5×10 3 CFU / g was significantly lower than that of the free tigecycline group (3.5×10⁻⁶). 3 ±0.8×10 3 CFU / g) and tigecycline nanoparticle group (2.1×10 3 ±0.6×10 3 The CFU / g figure indicates that drug-loaded macrophages can effectively clear CRKP in vivo.

[0061] The above-mentioned lung tissue was taken, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and the pathological morphology of the lungs was observed under an optical microscope. Figure 8 The results showed that the lungs of mice in the saline control group exhibited significant alveolar structural damage, extensive inflammatory cell infiltration, and exudate. The inflammatory damage in the free tigecycline group and the nanoparticle group was reduced, but some alveolar dilation and inflammatory cell infiltration were still observed. The alveolar structure in the drug-loaded macrophage group remained intact, inflammatory cell infiltration was significantly reduced, and exudate was almost completely eliminated, indicating that it can effectively alleviate lung inflammatory damage caused by CRKP pneumonia.

[0062] Example 7 Safety evaluation experiment Twenty healthy ICR mice were randomly divided into a control group (saline) and a treatment group (drug-loaded macrophages, dose 5 mg / kg, calculated as tigecycline), with 10 mice in each group. The drug was administered via tail vein injection for 7 consecutive days. The mice's body weight, diet, and activity status were observed daily. No mice died or showed any abnormal behavior. After the drug administration was completed, there was no significant difference in body weight between the mice and the control group (P>0.05).

[0063] After drug administration, blood was collected from the orbital sinus of mice, and serum was separated by centrifugation. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum creatinine (Scr), and blood urea nitrogen (BUN) were measured using an automated biochemical analyzer. Simultaneously, heart, liver, spleen, lung, and kidney tissues were dissected and observed for pathological morphology after HE staining. Results showed no significant differences in ALT, AST, Scr, and BUN levels between the drug-treated group and the control group. P >0.05); no cellular degeneration, necrosis or other damaging changes were observed in any of the tissue pathological sections, indicating that the drug delivery system of the present invention has good safety and no obvious toxic side effects.

[0064] The above examples demonstrate that the M2 macrophage-delivered tigecycline nanoparticles prepared in this invention achieve targeted lung delivery through the natural inflammatory chemotactic properties of M2 macrophages. Combined with the sustained-release effect of PLGA nanoparticles, this significantly enhances the anti-CRKP activity and anti-inflammatory effect of tigecycline, while also exhibiting good biocompatibility, providing a new and effective means for the clinical treatment of CRKP pneumonia.

[0065] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A drug delivery system for targeted treatment of inflammatory diseases, characterized in that, It includes M2 macrophages and drug-loaded nanoparticles located within the M2 macrophages, the drug-loaded nanoparticles comprising biodegradable polymeric materials for encapsulating drugs.

2. The drug delivery system for targeted treatment of inflammatory diseases according to claim 1, characterized in that, The M2 macrophages are M2 macrophages that have been differentiated and matured through polarization induction and express the CD206 marker on their surface.

3. The drug delivery system for targeted treatment of inflammatory diseases according to claim 2, characterized in that, The polarization induction reagents include IL-4 at 20-50 ng / mL and IL-13 at 20-50 ng / mL.

4. The drug delivery system for targeted treatment of inflammatory diseases according to claim 1, characterized in that, The drug-loaded nanoparticles contain at least one of the following drugs: tigecycline, doxycycline, levofloxacin, azithromycin, budesonide, tofacitinib, and carvacrol.

5. The drug delivery system for targeted treatment of inflammatory diseases according to claim 1, characterized in that, The biodegradable polymer material includes any one of the following: polylactic acid-glycolic acid copolymer, polyethylene glycol-polylactic acid copolymer, and polyethylene glycol-polylactic acid-glycolic acid copolymer.

6. The drug delivery system for targeted therapy of inflammatory diseases according to any one of claims 1 to 5, characterized in that, The drug-loaded nanoparticles have a particle size of 100~300nm, a zeta potential of -40mV~-20mV, a drug encapsulation efficiency of ≥70%, and a drug loading of 5%~15%.

7. A method for preparing a drug delivery system for targeted treatment of inflammatory diseases according to any one of claims 1 to 6, characterized in that, Includes the following steps: The drug and biodegradable polymer material are used to prepare an oil phase, and sodium cholate solution is used as an aqueous phase. The oil phase and the aqueous phase are combined to form a proemulsion. The proemulsion is added to a fresh sodium cholate solution to remove the solvent from the oil phase. The precipitate is collected to obtain drug-loaded nanoparticles. The drug-loaded nanoparticles were mixed and incubated with M2 macrophages to remove the free drug-loaded nanoparticles, thus obtaining a drug delivery system.

8. The preparation method according to claim 7, characterized in that, In the mixed incubation system, the ratio of drug-loaded nanoparticles to M2 macrophages is 1:100~300; The mass ratio of the drug to the biodegradable polymer in the oil phase is 1:5~20; The sodium cholate solution has a mass concentration of 1% to 3%, and the volume ratio of the oil phase to the water phase is 1:5 to 15.

9. The use of the drug delivery system for targeted treatment of inflammatory diseases according to any one of claims 1 to 6, or the drug delivery system for targeted treatment of inflammatory diseases prepared by the preparation method according to claim 7 or 8, in the preparation of medicaments for the prevention and / or treatment of inflammatory diseases.

10. A drug for treating inflammatory diseases, characterized in that, The drug delivery system for targeted treatment of inflammatory diseases as described in any one of claims 1 to 6, or the drug delivery system for targeted treatment of inflammatory diseases prepared by the preparation method described in claim 7 or 8.

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