An inhalable small molecule compound pulmonary targeting delivery system and its application in the protection against radiation-induced lung injury

Abstract

The present invention belongs to the field of biomedicine and specifically relates to an inhalable small molecule compound targeted lung delivery system and its application in protecting against radiation-induced lung injury. Based on the discovery that a small molecule compound having a molecular structure as represented by formula (I) exhibits excellent SOD and CAT enzyme activities and can protect against radiation-induced lung injury, the present invention has developed a probiotic-drug symbiotic delivery system based on Lactobacillus rhamnosus. The small molecule compound is loaded onto the surface of Lactobacillus rhamnosus and delivered directly to the alveolar cavity via aerosol inhalation, enhancing its targeting and bioavailability at the site of RILI. #imgabs0#(I).

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an inhalable small molecule compound lung-targeted delivery system and its application in protecting against radiation-induced lung injury. Background Art

[0002] Malignant tumors have the second highest mortality rate globally, second only to cardiovascular disease, and have become a major global health threat. Faced with this high incidence of cancer, radiotherapy plays a crucial role in cancer treatment, with cure rates reaching 40-45%. Despite its remarkable effectiveness in eliminating cancer cells, radiotherapy's damage to normal tissues, particularly radiation-induced lung injury (RILI), remains a major challenge. Minimizing damage to healthy tissues and improving treatment precision and safety are key goals of radiotherapy. Radiotherapy for thoracic tumors, particularly lung cancer, demonstrates a distinct dose-response relationship, with increasing irradiation doses associated with a decreasing risk of locoregional recurrence. However, increasing the dose of thoracic tumor radiotherapy is limited by normal tissue toxicity, with radiation-induced lung injury (RILI) being one of the most significant dose-limiting toxicities, occurring in 16.7% to 50.3%. Although improvements in precise planning, positioning technology, respiratory gating management, and image-guided techniques, along with the use of intensity-modulated radiotherapy (IMRT), stereotactic body radiotherapy (SBRT), and the development of new radioprotectants, have significantly reduced the risk of radiation pneumonitis, the incidence of symptomatic pneumonia remains at 9.4% to 28%. The occurrence of RILI during clinical diagnosis and treatment can interrupt chemotherapy, radiotherapy, immunotherapy, or targeted therapy for cancer patients, thereby impacting tumor control rates, prolonging hospital stays, and imposing a significant economic burden, making it a challenging issue for clinicians treating RILI.

[0003] The occurrence of RILI is primarily due to impaired barrier function between vascular endothelial cells and alveolar epithelial cells. In response to its complex and intertwined pathogenesis, relevant prevention and treatment research has emerged in an endless stream, including glucocorticoids, traditional Chinese medicine extracts, antioxidant therapies such as sulfhydryl compounds, antioxidant enzymes and analogs, and plant antioxidants. However, in reality, there is still a lack of specific and effective drugs other than glucocorticoids. Corticosteroids can reduce inflammatory responses and inhibit TNF-induced nitric oxide-mediated endothelial cell and lymphocyte toxicity. However, given the numerous side effects of glucocorticoids, such as secondary lung infections, increased blood sugar, peptic ulcers, Cushing's syndrome, obesity, and osteoporosis, corticosteroids are not suitable for the prevention of RP or long-term use. Therefore, there is an urgent need to develop effective methods for the prevention and treatment of RILI with minimal side effects.

[0004] Ensuring effective drug delivery in the body, controlled release in the lungs, and minimizing systemic toxicity have always been key areas of pharmaceutical research. This invention aims to optimize drug delivery systems to enhance drug targeting and bioavailability at the site of RILI, achieving precise drug release under specific conditions (e.g., enzyme activity, charge, pH, osmotic pressure, etc.). Summary of the Invention

[0005] To address the above problems, the present invention has developed a probiotic-drug symbiotic delivery system based on Lactobacillus rhamnosus, and combined it with a small molecule compound with excellent SOD and CAT enzyme activities and capable of protecting against radiation-induced lung injury caused by radiotherapy. The compound is loaded on the surface of Lactobacillus rhamnosus and directly delivered to the alveolar cavity via aerosol inhalation. This can enhance the targeting and bioavailability of the small molecule compound at the RILI site, and achieve precise release of the drug under specific conditions (such as enzyme activity, charge, pH, osmotic pressure, etc.).

[0006] Specifically, in a first aspect, the present invention provides an inhalable small molecule compound lung-targeted delivery system, wherein the delivery system is a Lactobacillus rhamnosus with a small molecule compound loaded on its surface.

[0007] Furthermore, the molecular structure of the small molecule compound is shown in formula (I):

[0008]

[0009] (I).

[0010] Furthermore, the small molecule compound has SOD and CAT enzyme activities.

[0011] As used herein, SOD enzyme activity refers to superoxide dismutase (SOD) activity, and CAT enzyme activity refers to catalase (CAT) activity.

[0012] As used herein, the preparation method of the small molecule compound having the molecular structure shown in formula (I) can be prepared by a suitable method known in the art, for example, the method of the present invention can be used. Figure 1 Prepared according to the synthetic route shown in .

[0013] Furthermore, the Lactobacillus rhamnosus is a Lactobacillus rhamnosus GG (LGG) strain.

[0014] Furthermore, the delivery system is administered by aerosol inhalation.

[0015] In a second aspect, the present invention provides use of the inhalable small molecule compound lung-targeted delivery system as described herein in the preparation of a drug for protecting against radiation-induced lung injury.

[0016] In a third aspect, the present invention provides a method for preparing the inhalable small molecule compound lung-targeted delivery system as described herein, comprising the following steps:

[0017] (1) Dispersing small molecule compounds in phosphate buffer;

[0018] (2) resuspending the Lactobacillus rhamnosus into the suspension of the small molecule compound, fully mixing and incubating under shaking, so that the small molecule compound is adsorbed on the surface of the Lactobacillus rhamnosus, thereby obtaining Lactobacillus rhamnosus with the small molecule compound loaded on the surface.

[0019] Furthermore, the Lactobacillus rhamnosus is in the logarithmic growth phase.

[0020] Furthermore, the shaking incubation time in step (2) is 30 minutes to 1 hour.

[0021] Advantageous Effects of the Invention

[0022] Based on the discovery that a small molecule compound having a molecular structure as shown in Formula (I) (referred to in this specification and the accompanying drawings as Ru(bda)[Me-bpy]2[PF6]2 or simply RU) exhibits excellent SOD and CAT enzyme activities and can protect against radiation-induced lung injury, the present invention has developed a probiotic-drug symbiotic delivery system based on Lactobacillus rhamnosus (referred to in this specification and the accompanying drawings as LGG@Ru(bda)[Me-bpy]2[PF6]2 or simply LGG@RU). The small molecule compound is loaded on the surface of Lactobacillus rhamnosus and delivered directly to the alveolar cavity via aerosol inhalation, which can enhance the targeting and bioavailability of the small molecule compound at the site of RILI. Furthermore, after probiotic colonization, its extracellular polysaccharides (EPS) act as a biofilm to form a physical barrier, competitively inhibiting the proliferation of opportunistic pathogens, coordinating microbial interactions along the lung-gut axis, and maintaining lung microbial diversity. Bacterial lysis releases the drug, unleashing SOD and CAT enzyme activity, simultaneously activating local anti-inflammatory defenses (the IL-10 / STAT3 pathway) and immune regulation (M2 macrophage polarization). LGG@Ru(bda)[Me-bpy]2[PF6]2 also reduces proinflammatory metabolites and increases anti-inflammatory or repair-related metabolites, such as restoring short-chain fatty acid (SCFA) levels, regulating tryptophan-kynurenine metabolism, and improving lipid peroxidation. This targeted delivery system forms three major barriers: immune, physical, and metabolic, systematically enhancing the host's anti-inflammatory and tissue damage repair capabilities. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The molecular structure and synthesis process diagram of the Ru(bda)[Me-bpy]2[PF6]2 small molecule compound are shown.

[0024] Figure 2 The performance and characterization results of Ru(bda)[Me-bpy]2[PF6]2 and LGG@Ru(bda)[Me-bpy]2[PF6]2 are shown: (a) H NMR spectrum; (b) C NMR spectrum; (c) CAT enzyme activity determination; (d) SOD enzyme activity determination; (e) transmission electron microscopy (TEM) scanning and elemental mapping distribution (Mapping); (f) Zeta potential detection.

[0025] Figure 3 Shown is the preparation of Lactobacillus rhamnosus-loaded RU and a schematic diagram of the mechanism of action.

[0026] Figure 4 Figure 3 shows the in vitro experimental results of lung radioprotection: representative ROS fluorescence images of BEAS-2B cells receiving different treatments (a); ROS peak detection diagram by flow cytometry (b); representative aggregates / monomers fluorescence staining images of JC-1 membrane potential detection (c); representative γ-H2AX fluorescence staining (d); representative Live / Dead fluorescence staining (e); representative flow cytometry detection of cell apoptosis (f); quantitative analysis of fluorescence intensity and apoptosis ratio of each group of BEAS-2B cells, the differences were statistically significant (g); cytoprotective mechanism of ROS clearance mediated by Ru(bda)[Me-bpy]2[PF6]2 (h).

[0027] Figure 5 The in vivo experimental results of lung radioprotection are shown: animal experimental plan after random grouping (a); chest hair loss of mice in different groups (b); mouse weight (c) and survival rate (d); lung tissue photos (e); colony formation of different tissue and organ homogenates after culture on MRS agar plates (f); detection of SOD, CAT, GSH, MDA and HYP content in lung tissue homogenates (g); HE staining of mouse lung tissue sections (h); Masson staining of lung tissue sections (i); HIF-α immunohistochemical staining (j); γ-H2AX immunofluorescence staining of lung tissue (k) and MPO immunofluorescence staining of lung tissue (l); in vitro fluorescence imaging of mouse tissue organ tubes (m). DETAILED DESCRIPTION

[0028] The present invention is further described below with reference to specific examples, which, however, are not intended to limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0029] Example 1: Synthesis of small molecule compound RU

[0030] Synthesis route such as Figure 1 shown.

[0031] 1. Weigh 1 g of RuCl3-xH2O and place it in a 50 mL flask. Then add 15 mL of dimethylsulfoxide (DMSO) and reflux for 5 minutes. Cool to room temperature and evaporate under reduced pressure. When a small amount of solvent remains, filter, wash with excess acetone, and vacuum dry to collect the yellow powder to obtain Ru(DMSO)4Cl2.

[0032] 2. Weigh 484 mg of Ru(DMSO)4Cl2 and 244 mg of H2BDA into a 100 mL two-necked flask. Add 40 mL of methanol and 0.8 mL of triethylamine. Reflux for 12 hours. Wash with excess methanol, acetone, and ether. Vacuum dry to collect the reddish-brown powder Ru(bda)(DMSO)2.

[0033] 3. Weigh 206.67 mg (0.1 mol) of [Me-Bpy]Cl and dissolve it in a certain amount of deionized water to form a saturated solution A. Weigh 736.24 mg of KPF6 and dissolve it in a certain amount of deionized water to form a saturated solution B. Rapidly add solution B to solution A to produce a precipitate. Filter, wash with 10 mL of water, and vacuum dry to collect the off-white solid [Me-bpy][PF6].

[0034] 4. Weigh 85 mg of pyridine-4-sulfonic acid and 100 mg of [Me-bpy][PF6] into a 50 mL flask. Add 15 mL of methanol and reflux for 12 h. Filter, collect the filtrate, and wash with excess methanol and ether to obtain a reddish-black solid. Dissolve the reddish-black solid in 80 mL of a 1:1 methanol / acetone mixture and rotary evaporation to 10 mL. Then, add 40 mL of methanol and evaporate until a precipitate forms. Filter, wash with 10 mL of methanol, and then with excess ether. Vacuum dry, and collect the reddish-black solid, Ru(bda)[Me-bpy]2[PF6]2.

[0035] Example 2: Characterization of the structure and enzymatic activity of Ru(bda)[Me-bpy]2[PF6]2 (RU)

[0036] The structure and composition of Ru(bda)[Me-bpy]2[PF6]2 were characterized in detail by nuclear magnetic resonance (NMR) technology. The hydrogen and carbon NMR spectra are shown in Figure 2. Figure 2 a and 2b, which confirm the synthesis of RU.

[0037] The guaiacol method was used to detect the catalase (CAT) activity of the materials; the xanthine oxidase method was used to determine the superoxide dismutase (SOD) activity. Figure 2 As shown in Figures 2c and 2d, RU has an extremely high clearance rate and a very fast clearance rate, indicating that RU is an extremely efficient biocatalyst, which is the first discovery of the present invention.

[0038] Example 3: Preparation of LGG@RU

[0039] The preparation method of Ru(bda)[Me-bpy]2[PF6]2 loaded Lactobacillus rhamnosus is as follows (refer to Figure 3 ):

[0040] LGG was cultured in 10 ml of MRS (de Man, Rogosa and Sharpe) broth and incubated in an anaerobic incubator at 37 °C for 24 h. The bacterial culture grew to the logarithmic growth phase, i.e., 1 × 10 9 CFU / ml (colony forming units per ml). LGG cells were separated from MRS broth solution (10 mL) by centrifugation at 6000 rpm, 4°C, for 5 min and washed 2-3 times with 1X pre-cooled phosphate-buffered saline (PBS) to remove residual MRS broth. LGG was then serially diluted to 1×10 7 The LGG cells were resuspended in a PBS solution containing Ru(bda)[Me-bpy]2[PF6]2 (1 mg / mL) and mixed thoroughly. The mixture was vortexed to form a uniform mixed solution and incubated on a shaker for 30 minutes. The LGG cells loaded with RU were collected by centrifugation. Zeta potential detection, transmission electron microscopy (TEM) scanning, and elemental mapping confirmed that LGG was successfully loaded with the small molecule drug RU. Figure 2 e and Figure 2 f).

[0041] Example 4: Application of LGG@RU in the protection against radiation-induced lung injury

[0042] Experimental Materials and Methods

[0043] 1. Cell lines: Human bronchial epithelial cells (BEAS-2B) and human embryonic lung fibroblasts (HELF) were cultured in DMEM (VivaCell) containing high-glucose, 0.5 mg / mL penicillin-streptomycin (Gibco), and 10% certified fetal bovine serum (VivaCell, Shanghai, China, C3840-0500).

[0044] 2. LGG culture: LGG was cultured in 10 ml of MRS (de Man, Rogosa and Sharpe) broth in an anaerobic incubator at 37°C for 24 hours until the bacterial culture reached the logarithmic growth phase, i.e., 1×10 9 CFU / ml (colony forming units per ml). Based on the CCK-8 assay results, the bacterial suspension was serially diluted with 1× sterile phosphate-buffered saline (PBS) to a final concentration of 1×10 7 CFU / mL, used for subsequent cell experiments.

[0045] 3. In vitro cell function protection experiment

[0046] 3.1. Detection of ROS clearance in cells after X-ray irradiation: BEAS-2B and HELF cells were collected during the growth phase, counted using a cell counting plate, and seeded into 6-well plates (15 × 10 4 / well) and cultured overnight. Cells were cultured in a 50 μg / mL (80 μg / mL) cell culture medium and grouped as follows: control group (Control), Ru(bda)[Me-bpy]2[PF6]2 (abbreviated as RU), LGG group, RT group, RT+Ru(bda)[Me-bpy]2[PF6]2 (abbreviated as RT+RU), and RT+LGG@Ru(bda)[Me-bpy]2[PF6]2 (abbreviated as RT+LGG@RU). The original cell culture medium was removed from each group and incubated with drug- or bacteria-containing culture medium for 2 hours. The cells in the RT, RT+RU, and RT+LGG@RU groups were then irradiated with 20 Gy of X-rays. Two hours after irradiation, the original culture medium was aspirated and serum-free culture medium containing DCFH-DA (1 mL / well) prepared according to the ROS detection kit instructions was added. The cells were incubated in a dark incubator for 20 minutes. After washing three times with serum-free culture medium, the ROS green fluorescence of the cells in each group was observed using a fluorescence microscope.

[0047] 3.2. Live / dead staining: Calcein (AM) / Propidium iodide (PI) staining: BEAS-2B and HELF cells were collected during the growth phase and seeded in 6-well plates (8 × 10 4Cells were cultured overnight in 4 wells (1 µl / well). Cell dosing and irradiation procedures were the same as above. 48 hours after irradiation, live cells were stained with 1 µl of AM solution in detection buffer, and dead cells were stained with 1 µl of PI solution. The captured cells in each cell group were then observed and counted using a fluorescence microscope.

[0048] 3.3. Mitochondrial membrane potential (JC-1) detection: BEAS-2B and HELF cells were collected during the growth phase and seeded in 6-well plates (8 × 10 4 Culture overnight in 100 mL / well of 4% flask (1 mL / well). Follow the same procedures for cell dosing and irradiation as above. Add 1 mL of JC-1 staining working solution and mix thoroughly. 24 hours after irradiation, incubate in a cell culture incubator at 37°C for 20 minutes. Aspirate the supernatant and wash twice with JC-1 staining buffer (1X). Then, add 2 mL of cell culture medium, which may contain serum and phenol red. Observe under a fluorescence microscope or confocal laser scanning microscopy.

[0049] 3.4. DNA Damage: BEAS-2B and HELF cells were harvested from the growth phase and seeded into 96-well plates (10,000 cells / well) for overnight culture. Cell dosing and irradiation procedures were the same as above. Within 6 hours after irradiation, cells were fixed with 4% paraformaldehyde and washed three times with washing buffer (5 minutes each). Immunostaining blocking buffer was added for 20 minutes at room temperature. After blocking, γ-H2AX rabbit monoclonal antibody was added, incubated at 4°C overnight, and then washed three times with washing buffer (5 minutes each). Anti-rabbit 488 was added and incubated at room temperature for 1 hour in the dark. Cell nuclei were counterstained with DAPI at room temperature. Cells were washed with washing buffer and examined under a fluorescence microscope.

[0050] 3.5. Flow cytometry of apoptosis (Annexin V-FITC): BEAS-2B and HELF cells were collected during the growth phase and seeded in 6-well plates (8 × 10 4 Cells were cultured overnight in 4 wells (100 μl / well). Cell dosing and irradiation procedures were the same as above. 48 hours after irradiation, the cell supernatant was collected into a centrifuge tube and an appropriate amount of trypsin was added to the cell culture medium to terminate digestion. The tubes were centrifuged at 1200 g for 5 minutes, the supernatant discarded, and the cells were collected. After washing with PBS and centrifugation, apoptosis detection reagents were added. The cells were then incubated at room temperature (20-25°C) in the dark for 10-20 minutes. Finally, flow cytometry was used to detect green fluorescence with Annexin V-FITC and red fluorescence with propidium iodide (PI).

[0051] 4. In vivo radiation-induced lung injury protection experiment

[0052] Animal Model: Male C57 mice (6-8 weeks old, 20-25 g) were purchased from GemPharmatech Co., Nanjing, China. All animal experimental procedures were performed in accordance with the Regulations on the Administration of Laboratory Animals approved by the State Council of the People's Republic of China. All mice were housed under specific pathogen-free (SPF) conditions (temperature ~22°C, humidity ~50%) with a 12 / 12 h dark / light cycle. After one week of housing, a reflex lung injury model was established in mice (single intrathoracic irradiation of 17 Gy). The specific grouping and testing procedures are as follows:

[0053] Mice were randomly divided into control, RU, LGG, RT, RT+RU, and RT+LGG@RU groups. Mice in the RU and RT+RU groups received nebulized RU 1 hour before radiotherapy, while mice in the LGG and RT+LGG@RU groups received nebulized LGG and LGG@RU, respectively, 1 hour before radiotherapy. Treatment continued every other day thereafter. The nebulized drug concentration for all four groups was 1 mg / mL. During treatment, the mice were observed every three days for dietary activity, chest hair removal, body weight, and survival. On day 30, the mice were euthanized, and lung tissue was collected and homogenized for analysis of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), malondialdehyde (MDA), and hydroxyproline (HYP) levels. The intact lung tissue obtained after dissection was fixed in 4% paraformaldehyde, paraffin-embedded sections were prepared, and pathological staining, scanning, and image analysis were performed. HE, Masson, immunohistochemistry (IHC), and immunofluorescence (IF) staining were used to assess differences in inflammatory structures, collagen deposition, hypoxia-inducible factor (HIF-α), DNA damage marker (γ-H2AX), and myeloperoxidase (MPO) between the treatment and control groups. Heart, liver, spleen, kidney, and lung tissues from the control and LGG groups were homogenized and diluted serially with PBS before being plated on MRS solid agar plates and incubated in an anaerobic incubator at 37°C for 48 hours. Furthermore, to quantitatively evaluate the biodistribution of LGG@RU, mice were treated with aerosolized Cy5.5-labeled RU and LGG@RU after irradiation. In vivo and ex vivo imaging of tissues and organs was performed at different time points. These in vivo studies demonstrated the feasibility, safety, and efficacy of Lactobacillus rhamnosus-delivered small molecule RU for the treatment of radiation-induced lung injury.

[0054] Experimental results

[0055] 1. In vitro cell function protection experiments: After verifying the multi-enzyme antioxidant activity of RU, the present invention cultured human bronchial epithelial cells (BEAS-2B) and human embryonic lung fibroblasts (HELF) in vitro and stimulated the cells to produce reactive oxygen species (ROS) under an X-ray system. After intervention with the synthesized RU drug, LGG, and LGG@RU, intracellular ROS levels were quantified using a reactive oxygen species fluorescent probe (DCFH-DA) using fluorescence microscopy and flow cytometry. RU's SOD and CAT enzyme activities were further verified using a series of experiments, including dead-alive fluorescence detection, DNA damage, cell cycle analysis, flow cytometry, cell senescence, and mitochondrial membrane potential. These results confirmed its ability to scavenge ROS, reduce intracellular DNA damage, inhibit lung endothelial cell apoptosis, and promote lung endothelial cell growth and repair. Furthermore, the results confirmed that LGG did not affect cell proliferation during cell culture.

[0056] The results of fluorescence and flow cytometry ROS detection showed that compared with the RT group, the RT+RU and RT+LGG@RU groups significantly reduced the generation of ROS and effectively alleviated the cellular oxidative stress state ( Figure 4 a, b). Mitochondrial membrane potential and DNA damage detection showed that the RT+RU and RT+LGG@RU groups significantly reduced mitochondrial damage compared with the RT group, and the expression of the DNA damage marker γ-H2AX was significantly decreased ( Figure 4 c, d). Live-dead AM / PI fluorescence staining showed that the red fluorescence signal of the RT+RU and RT+LGG@RU groups decreased, and their dead / live cell ratios were significantly reduced compared with the RT group ( Figure 4 e). Apoptosis flow cytometry showed that the apoptosis rate of cells in the RT group was significantly increased, while the apoptosis rate in the RT+RU and RT+LGG@RU groups was significantly decreased ( Figure 4 f). Comprehensive analysis of the quantitative fluorescence intensity and apoptosis rate of BEAS-2B cells ( Figure 4 g) showed that the radiation-induced ROS clearance and apoptotic cell death rate were statistically significantly different among the treatment groups, consistent with the cytoprotective mechanism of RU-mediated ROS clearance ( Figure 4 h). The above data indicate that RU can significantly reduce radiation-induced oxidative stress, cell apoptosis, and DNA damage. In vitro experiments also revealed that there was little difference in the protective effects of RT+RU and RT+LGG@RU on cells.

[0057] 2. In vivo lung radiation protection experiment: The animal experiment plan after randomization is as follows Figure 5 The experimental results showed that the fur of mice in the X-ray irradiation group was rough, while the fur of mice in the RT+LGG@RU group was smooth. In particular, after 3 weeks of RILI model induction, the X-ray group showed obvious chest hair loss compared with the other groups ( Figure 5b). In addition to coat condition and activity level, body weight also reflects the overall health of mice. After irradiation, the body weight of mice in the X-ray group showed a progressive decrease in the first week after irradiation and was significantly lower than that in the RT+RU and RT+LGG@RU groups ( Figure 5 c). In addition, compared with the RT+RU group, the RT+LGG@RU group showed more significant weight recovery after treatment. It is worth noting that the overall survival of the RT+LGG@RU combination treatment group was significantly prolonged compared with the other two treatment groups. The synergistic anti-inflammatory effect of LGG@RU in vivo was superior to that of RU alone ( Figure 5 d). 30 days after irradiation, gross morphological evaluation of the RT group showed patchy congestion with localized congestion and edema. In contrast, the lungs of the RT+LGG@RU treatment group remained uniformly pink and maintained good elasticity, showing a near-physiological tissue structure ( Figure 5 e). After serial dilution, the tissue homogenate was cultured on solid MRS agar plates. After 48 hours of anaerobic incubation at 37°C, it was observed that LGG colonies formed only in the lung tissue of mice treated with LGG aerosol inhalation, while LGG colonization was not detected in other tissues and organs, clearly confirming the lung targeting of the aerosol inhalation. Figure 5 f). The contents of SOD, CAT, and GSH in lung tissue homogenate were significantly increased in the RT+LGG@RU group and were superior to those in the RT+RU treatment group; the contents of MDA and HYP were significantly decreased in the RT+LGG@RU group, indicating that the damage to lung tissue after irradiation was less than that in the RT alone group ( Figure 5 g).

[0058] HE pathological staining results showed that the inflammatory cell infiltration in the RT+LGG@RU and RT@RU groups was less than that in the RT group alone, with slight widening of the alveolar septa and a few alveolar fusions. The pulmonary inflammatory response was significantly milder than that in the RT group ( Figure 5 h); Masson staining showed that collagen fiber deposition was significantly reduced in the RT+LGG@RU and RT@RU groups ( Figure 5 i); IHC staining showed that HIF-α expression increased in the RT group ( Figure 5 j). IF staining showed that the expression levels of γ-H2AX and MPO were significantly increased in the RT group. On the contrary, their expression levels were decreased after RT+LGG@RU treatment and were better than those in the RT@RU group ( Figure 5 k, 5l). In vitro tissue imaging analysis of mice showed that Cy5.5-labeled RU and LGG@RU were mainly concentrated in the lungs 24 hours after aerosol inhalation, with only a small amount of fluorescence signal appearing in the liver. In addition, the fluorescence signal in the lung tissue of RT+LGG@RU mice was significantly stronger than that in the RT+RU group ( Figure 5 m), fully demonstrating that LGG can increase the targeted accumulation of drugs in lung tissue.

[0059] The above results all confirmed that the RT+LGG@RU group significantly reduced lung tissue damage after radiotherapy compared with the RT group, and all observation indicators were better than those of the RT+RU group, which fully demonstrated the targeted delivery effect of LR-loaded RU after aerosol inhalation to avoid "first-pass elimination", increased the utilization rate and duration of drug action, and exerted a better radiation protection effect in the body, indicating that the delivery system based on Lactobacillus rhamnosus can achieve effective delivery of RU in vivo, and can enhance the targeting and bioavailability of drugs at the site of radiation-induced lung injury.

[0060] It should be noted that the preferred embodiments of the present invention are given in the specification and drawings of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not intended to be additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features can be combined with each other to form various embodiments not listed above, which are all considered to be within the scope of the description of the present invention. Furthermore, it is obvious to those skilled in the art that improvements or changes can be made based on the above description, and all such improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. An inhalable small molecule compound lung-targeted delivery system, characterized in that: The delivery system is Lactobacillus rhamnosus with small molecule compounds loaded on its surface; The molecular structure of the small molecule compound is shown in formula (I): (I)。 2. The delivery system according to claim 1, wherein The small molecule compound has SOD and CAT enzyme activities.

3. The delivery system according to claim 1, wherein The Lactobacillus rhamnosus is the Lactobacillus rhamnosus GG strain.

4. The delivery system according to claim 1, wherein The delivery system is administered by inhalation.

5. Use of the inhalable small molecule compound lung-targeted delivery system according to any one of claims 1 to 4 in the preparation of a drug for protecting against radiation-induced lung injury.

6. A method for preparing an inhalable small molecule compound lung-targeted delivery system according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Dispersing small molecule compounds in phosphate buffer; (2) resuspending the Lactobacillus rhamnosus into the suspension of the small molecule compound, fully mixing and incubating under shaking, so that the small molecule compound is adsorbed on the surface of the Lactobacillus rhamnosus, thereby obtaining Lactobacillus rhamnosus with the small molecule compound loaded on the surface.

7. The preparation method according to claim 6, characterized in that The Lactobacillus rhamnosus is a bacterial body in the logarithmic growth phase.

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