Inhalable small molecule compound lung targeting delivery system and application thereof in radiation-induced lung injury protection

Abstract

The invention belongs to the field of biological medicine, and particularly relates to an inhalable small molecule compound lung targeting delivery system and application thereof in radiation-induced lung injury protection. The invention develops a probiotic-drug symbiotic delivery system based on lactobacillus rhamnosus on the basis of finding that a small molecular compound with a molecular structure as shown in a formula (I) has excellent SOD and CAT enzyme activity and can prevent radiation-induced lung injury caused by radiotherapy for the first time. Wherein the small molecule compound is loaded on the surface of lactobacillus rhamnosus and is directly delivered to the alveolar cavity through aerosol inhalation, so that the targeting property and bioavailability of the small molecule compound at the RILI part can be enhanced. # imgabs0 # (I)

Description

Technical Field

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

[0002] The mortality rate of malignant tumors ranks second globally, second only to cardiovascular diseases, and has become the most important health threat worldwide. Facing the challenge of the high incidence of tumors, radiotherapy occupies an important position in tumor treatment, with a cure rate of 40-45%. Although radiotherapy performs excellently in eliminating cancer cells, its damage to normal tissues, especially radiation-induced tissue damage problems such as radiation pneumonitis and radiation-induced heart disease, remains a difficult problem in radiotherapy. Reducing the damage to healthy tissues and improving the treatment accuracy and safety are the key goals that need to be considered in radiotherapy. There is an obvious dose-effect relationship in radiotherapy for chest tumors led by lung cancer. As the irradiation dose increases, the local recurrence risk decreases accordingly. However, the increase in the radiotherapy dose for chest tumors is limited by the toxicity of normal tissues. Among them, radiation-induced lung injury (RILI) is one of the most important dose-limiting toxicities, with an incidence rate of 16.7%-50.3%. Although currently, with the improvement of precise planning, positioning technology, respiratory gating management, image-guided technology, etc., and through the use of intensity-modulated radiotherapy (IMRT), stereotactic body radiotherapy (SBRT), and the research and development of new radioprotective agents, the risk of radiation pneumonitis has been significantly reduced clinically, but the probability of developing symptomatic pneumonia still accounts for 9.4%-28%. The occurrence of RILI in clinical diagnosis and treatment will lead to the interruption of the radiotherapy, chemotherapy, immunotherapy, or targeted therapy courses of tumor patients, thus affecting the tumor control rate, prolonging the hospital stay, causing a large disease economic burden, and becoming a thorny problem for clinicians to treat RILI.

[0003] The occurrence of RILI is mainly due to the impaired barrier functions of vascular endothelial cells and alveolar epithelial cells. In response to its complex and crosstalk pathogenesis, numerous related prevention and treatment studies have emerged, including glucocorticoids, traditional Chinese medicine extracts, antioxidant therapies such as sulfhydryl compounds, antioxidant enzymes and analogs, plant antioxidants, etc. However, in fact, there is still a lack of specific effective drugs except for glucocorticoids. Corticosteroids can reduce the inflammatory response and also inhibit the TNF-induced nitric oxide-mediated cytotoxicity of endothelial cells and lymphocytes. However, considering the many side effects of glucocorticoids, such as secondary pulmonary infection, elevated blood sugar, peptic ulcer, Cushing's syndrome, obesity, osteoporosis, etc., corticosteroids are not suitable for preventing RP or long-term use. Therefore, there is an urgent need to develop methods for effectively preventing and treating RILI with few side effects.

[0004] How to ensure the effective delivery of drugs in the body, controlled release in the lungs, and reduction of systemic toxicity has always been the focus of drug research. The present invention aims to continuously optimize the drug delivery system in order to enhance the targeting and bioavailability of drugs at the RILI site and achieve precise release of drugs under specific conditions (such as enzyme activity, charge, pH, osmotic pressure, etc.). Summary of the Invention

[0005] To solve 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 having excellent SOD and CAT enzyme activities and capable of protecting against radiation-induced lung injury caused by radiotherapy. It is loaded on the surface of Lactobacillus rhamnosus and directly delivered to the alveolar cavity by aerosol inhalation, which can enhance the targeting and bioavailability of the small molecule compound at the RILI site and achieve precise release of drugs under specific conditions (such as enzyme activity, charge, pH, osmotic pressure, etc.).

[0006] Specifically, in the first aspect, the present invention provides an inhalable small molecule compound pulmonary targeting delivery system, and the delivery system is 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 the activity of superoxide dismutase (SOD), and CAT enzyme activity refers to the activity of catalase (CAT).

[0012] As used herein, 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, it can be prepared by the synthetic route shown in the present invention Figure 1 shown.

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

[0014] Furthermore, the administration method of the delivery system is aerosol inhalation.

[0015] In a second aspect, the present invention provides the use of the inhalable small molecule compound pulmonary targeting 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 an inhalable small molecule compound pulmonary targeting delivery system as described herein, which comprises the following steps:

[0017] (1) Disperse the small molecule compound in phosphate buffer solution;

[0018] (2) Resuspend Lactobacillus rhamnosus in the suspension of the small molecule compound, incubate with shaking after sufficient mixing, so that the small molecule compound is adsorbed on the surface of Lactobacillus rhamnosus, and Lactobacillus rhamnosus with the small molecule compound loaded on its surface is obtained.

[0019] Further, the Lactobacillus rhamnosus is the bacterial cells in the logarithmic growth phase.

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

[0021] Advantages of the Invention

[0022] Based on the first discovery that a small molecule compound with the molecular structure shown in formula (I) (referred to as Ru(bda)[Me-bpy]2[PF6]2 or simply RU in this specification and the accompanying drawings) has 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 as LGG@Ru(bda)[Me-bpy]2[PF6]2 or simply LGG@RU in this specification and the accompanying drawings). The small molecule compound is loaded on the surface of Lactobacillus rhamnosus and directly delivered to the alveolar cavity by aerosol inhalation, which can enhance the targeting and bioavailability of the small molecule compound at the RILI site. In addition, after the probiotics colonize, their exopolysaccharides (EPS) form a physical barrier as a biofilm, competitively inhibit the proliferation of opportunistic pathogens, coordinate the microbial interaction of the "lung-gut axis", and maintain the lung microbial diversity; the bacteria lysate releases the drug, exerts SOD and CAT enzyme activities, and synchronously activates local anti-inflammatory defense (IL-10 / STAT3 pathway) and immune regulation (M2 macrophage polarization); LGG@Ru(bda)[Me-bpy]2[PF6]2 can also reduce pro-inflammatory metabolites and increase anti-inflammatory or repair-related metabolites, such as restoring the level of short-chain fatty acids (SCFA), regulating the tryptophan-kynurenine metabolism, and improving lipid peroxidation. This targeting delivery system forms three major barriers of immunity, physics and metabolism, and systematically enhances the host's anti-inflammatory and tissue injury repair capabilities. Description of the Drawings

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

[0024] Figure 2 Shows the performance and characterization results of Ru(bda)[Me-bpy]2[PF6]2 and LGG@Ru(bda)[Me-bpy]2[PF6]2: (a) 1H NMR spectrum; (b) 13C NMR spectrum; (c) CAT enzyme activity assay; (d) SOD enzyme activity assay; (e) Transmission electron microscopy (TEM) scanning and elemental mapping distribution (Mapping); (f) Zeta potential detection.

[0025] Figure 3 Shows the schematic diagram of the preparation and mechanism of action of Lactobacillus rhamnosus loaded with RU.

[0026] Figure 4 Shows the in vitro experimental results of lung radiation protection: Representative ROS fluorescence images of BEAS-2B cells treated with different treatments (a); Flow cytometry detection of ROS peak graph (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 statistical analysis of fluorescence intensity and apoptosis ratio in each group of BEAS-2B cells, and the differences are statistically significant (g); The cell protection mechanism of ROS scavenging mediated by Ru(bda)[Me-bpy]2[PF6]2 (h).

[0027] Figure 5 Shows the in vivo experimental results of lung radiation protection: Animal experimental protocol after random grouping (a); Chest hair removal of mice in different groups (b); Mouse body weight (c) and survival rate (d); Lung tissue photos (e); Colony formation of different tissue homogenates after culturing on MRS agar plates (f); Detection of the contents of SOD, CAT, GSH, MDA and HYP 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); MPO immunofluorescence staining of lung tissue (l); Ex vivo fluorescence imaging of mouse tissues and organs (m). Detailed implementation methods

[0028] The present invention will be further described below in conjunction with specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0029] Example 1: Synthesis of Small Molecule Compound RU

[0030] The synthesis route is 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 dimethyl sulfoxide (DMSO), reflux for 5 minutes, cool to room temperature, and then perform vacuum distillation. After leaving a small amount of solvent, filter, wash with an excess of acetone, and dry in vacuo to collect the yellow powder to obtain Ru(DMSO)4Cl2.

[0032] 2. Weigh 484 mg of Ru(DMSO)4Cl2 and 244 mg of H2BDA, place them in a 100 mL two-necked flask, add 40 mL of methanol and 0.8 mL of triethylamine, reflux for 12 hours, wash with an excess of methanol, acetone, and ether, and dry in vacuo to collect the red-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 dry in vacuo 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], place them in a 50 mL flask, add 15 mL of methanol and reflux for 12 h. Filter and collect the filtrate. Wash with an excess of methanol and ether to obtain a red-black solid. Dissolve the red-black solid in 80 mL of a methanol / acetone mixed solution (1:1), rotary evaporate to 10 mL, then add 40 mL of methanol and evaporate until a precipitate precipitates. Filter, wash with 10 mL of methanol, and then wash with an excess of ether. Dry in vacuo to collect the red-black solid Ru(bda)[Me-bpy]2[PF6]2.

[0035] Example 2: Structure and Mimetic Enzyme Activity Characterization 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 1H NMR and 13C NMR spectra are shown in Figure 2 Figures 2a and 2b respectively, which confirmed the synthesis of RU.

[0037] The catalase (CAT) activity of the material was detected by the guaiacol method; the superoxide dismutase (SOD) activity was determined by the xanthine oxidase method. The results are shown in Figure 2 Figures 2c and 2d. RU has a very high scavenging rate and a very fast scavenging rate, indicating that RU is a highly efficient biocatalyst, which is the first discovery of the present invention.

[0038] Example 3: Preparation of LGG@RU

[0039] The preparation method of Lactobacillus rhamnosus loaded with Ru(bda)[Me-bpy]2[PF6]2 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 hours until the bacterial culture grew to the logarithmic growth phase, i.e., 1×10 9 CFU / ml (colony-forming units per milliliter). After centrifugation at 6000 rpm, 4 °C for 5 min, the LGG cells were separated from the MRS broth solution (10 mL), washed 2 - 3 times with 1X pre-cooled phosphate buffer saline (PBS) to remove the residual MRS broth. Then, the LGG was serially diluted to a concentration of 1×10 7 CFU / ml, and the centrifuged LGG cell pellet was resuspended in a PBS solution containing Ru(bda)[Me-bpy]2[PF6]2 (1 mg / mL) and mixed thoroughly. The mixture was vortexed thoroughly to form a homogeneous mixed solution and incubated on a shaker for 30 minutes. The LGG cells loaded with RU were collected by centrifugation. The successful loading of the small molecule drug RU onto LGG was verified by Zeta potential detection, transmission electron microscopy (TEM) scanning, and elemental mapping distribution (Mapping) ( Figure 2 Figures 2e and Figure 2 2f).

[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 lung bronchial epithelial cells (BEAS-2B) and human embryonic lung fibroblasts (HELF) were cultured using a medium containing high-glucose DMEM (VivaCell), 0.5 mg / mL penicillin-streptomycin (Gibco), and 10% certified fetal bovine serum (VivaCell, Shanghai, China, C3840-0500).

[0044] 2. LGG culture: LGG was incubated 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 milliliter). According to 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 for subsequent cell experiments.

[0045] 3. In vitro cell function protection experiments

[0046] 3.1. Detection of ROS scavenging in cells after X-ray irradiation: BEAS-2B and HELF cells in the growth phase were collected, counted using a cell counting chamber, and then seeded in 6-well plates (15×10 4 / well) and cultured overnight. A cell medium containing RU (80 ug / mL) was prepared and grouped as follows: control group (Control), Ru(bda)[Me-bpy]2[PF6]2 (abbreviated as RU) group, LGG group, RT group, RT+Ru(bda)[Me-bpy]2[PF6]2 (abbreviated as RT+RU) group, and RT+LGG@Ru(bda)[Me-bpy]2[PF6]2 (abbreviated as RT+LGG@RU) group. The original cell medium was removed from each group of cells, and the drug-containing or bacteria-containing medium was added and incubated for 2 h. Subsequently, the RT, RT+RU, and RT+LGG@RU groups were irradiated with 20 Gy of X-rays. 2 h after the irradiation ended, the original medium was aspirated, and a serum-free medium containing DCFH-DA prepared according to the ROS detection kit instructions (1 mL / well) was added, and the cells were incubated in the incubator in the dark for 20 min. After washing the cells 3 times with serum-free medium, the green fluorescence of ROS in each group of cells was observed using a fluorescence microscope.

[0047] 3.2. Live / dead staining with calcein-AM / PI staining: BEAS-2B and HELF cells in the growth phase were collected and seeded in 6-well plates (8×10 4Cultured overnight in (the hole). The steps of cell administration and irradiation treatment are the same as above. After 48 h of culture at the end of irradiation, 1 μl of AM solution was used to stain live cells in the detection buffer, and 1 μl of PI solution was used to stain dead cells. Then, cells in each group were observed and counted by fluorescence microscopy.

[0048] 3.3. Detection of mitochondrial membrane potential (JC-1): BEAS-2B and HELF cells in the growth phase were collected and seeded in 6-well plates (8×10 4 / well) and cultured overnight. The steps of cell administration and irradiation treatment are the same as above. 1 ml of JC-1 staining working solution was added and mixed well. After 24 h of culture at the end of irradiation, the cells were incubated at 37 °C in a cell culture incubator for 20 minutes, then the supernatant was aspirated, and the cells were washed twice with JC-1 staining buffer (1X). Then, 2 ml of cell culture medium was added, and the culture medium could contain serum and phenol red. Finally, the cells were observed under a fluorescence microscope or a laser confocal microscope.

[0049] 3.4. DNA damage: BEAS-2B and HELF cells in the growth phase were collected and seeded in 96-well plates (10,000 / well) and cultured overnight. The steps of cell administration and irradiation treatment are the same as above. Within 6 h of culture at the end of irradiation, the cells were fixed with 4% paraformaldehyde and washed 3 times with washing solution for 5 min each time. Immunostaining blocking solution was added for blocking at room temperature for 20 min. After blocking, γ-H2AX rabbit monoclonal antibody was added, and the cells were incubated overnight at 4 °C and then washed 3 times with washing solution for 5 min each time. Anti-rabbit 488 was added and incubated in the dark at room temperature for 1 h, and then the cell nuclei were counterstained with DAPI staining solution at room temperature. After washing the cells with washing solution, they were placed under a fluorescence microscope for detection.

[0050] 3.5. Flow cytometry for apoptotic cells (Annexin V-FITC): BEAS-2B and HELF cells in the growth phase were collected and seeded in 6-well plates (8×10 4 / well) and cultured overnight. The steps of cell administration and irradiation treatment are the same as above. After 48 h of culture at the end of irradiation, the cell supernatant was collected into a centrifuge tube, and an appropriate amount of trypsin cell digestive solution was added. After adding the collected cell culture medium to terminate digestion, the cells were centrifuged at 1200 g for 5 minutes, the supernatant was discarded, the cells were collected, washed with PBS and centrifuged, and then apoptosis detection reagents were added respectively. Then, they were incubated in the dark at room temperature (20-25 °C) for 10-20 minutes. Finally, they were detected by flow cytometry. Annexin V-FITC was green fluorescence, and propidium iodide (PI) was red fluorescence.

[0051] 4. In vivo protective experiment against radioactive lung injury

[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 carried out in accordance with the Regulations for 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. One week after housing, a mouse model of radiation-induced lung injury (single thoracic irradiation of 17 Gy) was established. The specific grouping and detection contents are as follows:

[0053] The mice were randomly divided into a Control group, an RU group, an LGG group, an RT group, an RT + RU group, and an RT + LGG@RU group. Mice in the RU group and the RT + RU group were given aerosol inhalation of RU 1 h before radiotherapy. Mice in the LGG group and the RT + LGG@RU group were given aerosol inhalation of LGG and LGG@RU respectively 1 h before radiotherapy, and then treated every other day. The concentration of the aerosol inhalation drug for the above four groups of mice was 1 mg / mL. During the treatment period, the diet, activities, chest hair loss, body weight, and survival of the mice were observed every 3 days. On the 30th day, the mice were euthanized, and the collected lung tissues were made into tissue homogenates for detecting the contents of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), malondialdehyde (MDA), and hydroxyproline (HYP) in the lung tissues. At the same time, the intact lung tissues obtained after dissection were fixed in 4% paraformaldehyde, paraffin sections were prepared, and pathological staining, section scanning, and image analysis were carried out. HE, Masson, immunohistochemistry (IHC), and immunofluorescence (IF) staining were used to observe the differences in inflammatory structures, collagen deposition, hypoxia-inducible factor (HIF-α), DNA damage marker (γ-H2AX), and myeloperoxidase (MPO) between the treatment group and the control group. At the same time, the heart, liver, spleen, kidneys, and lung tissues of mice in the Control and LGG groups were ground into tissue homogenates, serially diluted with PBS, and cultured on MRS solid agar plates in a 37ºC anaerobic incubator for 48 h. Further, in order to quantitatively evaluate the biodistribution of LGG@RU, Cy5.5-labeled RU and LGG@RU were aerosol inhaled into the mice after irradiation, and in vivo and ex vivo imaging analysis of the mice were carried out at different time points. Through the above in vivo related research contents, the feasibility, safety, and effectiveness of the small molecule drug RU delivered by Lactobacillus rhamnosus in the treatment of radiation-induced lung injury were explored.

[0054] Experimental results

[0055] 1. In vitro cell function protection experiment: After verifying the multi-enzyme-like antioxidant activity of RU, the present invention cultured human lung bronchial epithelial cells (BEAS-2B) and human embryonic lung fibroblasts (HELF) in vitro, and stimulated the cells to produce reactive oxygen species (ROS) under the X-ray system. After intervention with the synthesized RU drug, LGG, and LGG@RU, the intracellular ROS content was quantified using the reactive oxygen fluorescence probe (DCFH-DA) through a fluorescence microscope and a flow cytometer. Further, a series of experiments such as live-dead fluorescence detection, DNA damage, cell cycle, flow cytometry apoptosis, cell senescence, and mitochondrial membrane potential were used to verify the SOD and CAT enzyme activities of RU, which confirmed at multiple levels that it has the ability to scavenge ROS, reduce intracellular DNA damage, inhibit the apoptosis of lung endothelial cells, and promote the growth and repair of lung endothelial cells. And the results confirmed that culturing LGG in cells does not affect the proliferation activity of cells.

[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). The 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 level of the DNA damage marker γ-H2AX decreased significantly ( Figure 4 c, d). The live-dead AM / PI fluorescence staining showed that the red fluorescence signal in the RT+RU and RT+LGG@RU groups decreased, and the ratio of dead / live cells was significantly reduced compared with the RT group ( Figure 4 e). Apoptosis flow cytometry detection found that the apoptosis rate of the RT group was significantly increased, and the apoptosis ratio of 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 there were significant statistical differences in radiation-induced ROS scavenging and the proportion of apoptotic cell death among the treatment groups, which was consistent with the cell protection mechanism of RU-mediated ROS scavenging ( Figure 4 h). The above data indicated that RU could significantly reduce radiation-induced oxidative stress, apoptosis, and DNA damage, and at the same time, it was found in vitro experiments 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 protocol after random grouping is as Figure 5 shown in a. The experimental results showed that the fur of the mice in the X-ray irradiation group was rough, while the fur of the mice in the RT+LGG@RU group was smooth. Especially 3 weeks after the induction of the RILI model, obvious chest hair loss occurred in the X-ray group compared with other groups ( Figure 5b). In addition to fur condition and activity level, body weight also reflects the overall health status of mice. After irradiation, the body weight of mice in the X-ray group showed a progressive decrease in the first week after irradiation, which 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 body weight recovery after treatment in the RT+LGG@RU group was more obvious. It is worth noting that compared with the other two treatment groups, the overall survival period of the RT+LGG@RU combined treatment group was significantly prolonged, and LGG@RU showed a better synergistic anti-inflammatory effect in vivo than the single RU drug treatment ( Figure 5 d). After 30 days of irradiation, the gross morphological evaluation of the RT group showed patchy congestion accompanied by local 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). Through serial dilution, the tissue homogenate was cultured on solid MRS agar plates. After incubation in an anaerobic incubator at 37 °C for 48 hours, it was observed that LGG colonies formed single colonies only in the lung tissues of mice after LGG aerosol inhalation treatment, and no LGG colonization was detected in other tissue organs, clearly confirming the lung targeting of aerosolization ( Figure 5 f). The contents of SOD, CAT, and GSH in the lung tissue homogenate were significantly increased in the RT+LGG@RU group and were better than those in the RT+RU treatment group; the contents of MDA and HYP were significantly decreased in the RT+LGG@RU group, showing that the damage to the lung tissue after irradiation was lower than that in the single RT group ( Figure 5 g).

[0058] The HE pathological staining results showed that the infiltration of inflammatory cells in the RT+LGG@RU and RT@RU groups was less than that in the single RT group, with slight widening of the alveolar septum and a little alveolar fusion, and the pulmonary inflammatory reaction was significantly lighter than that in the RT group ( Figure 5 h); The Masson staining showed that the deposition of collagen fibers was significantly reduced in the RT+LGG@RU and RT@RU groups ( Figure 5 i); The IHC staining showed that the expression of HIF-α increased in the RT group ( Figure 5 j). The 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 decreased after RT+LGG@RU treatment and were better than those in the RT@RU group ( Figure 5 k, 5l). The imaging analysis of mouse ex vivo tissue organs showed that Cy5.5-labeled RU and LGG@RU mainly concentrated in the lungs 24 hours after aerosol inhalation, with only a small amount of fluorescence signal appearing in the liver, and the fluorescence signal in the lung tissues of RT+LGG@RU mice was significantly stronger than that in the RT+RU group ( Figure 5 m), fully indicating that LGG can increase the targeted accumulation of drugs in the lung tissue.

[0059] All of the above results confirmed that the RT+LGG@RU group significantly alleviated the lung tissue injury after radiotherapy compared with the RT group, and all the observed indexes were better than those of the RT+RU group, which fully reflected the targeted delivery effect of LR-loaded RU aerosol inhalation to avoid "first-pass elimination", increased the drug utilization rate and the duration of action, and exerted a better radioprotective effect in vivo, indicating that the delivery system based on Lactobacillus rhamnosus could achieve the effective delivery of RU in vivo and enhance the targeting and bioavailability of the drug at the site of radioactive lung injury.

[0060] It should be noted that the description and drawings of the present invention give preferred embodiments 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 additional limitations to the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosed content of the present invention more thorough and comprehensive. Moreover, the above technical features continue to be combined with each other to form various embodiments not listed above, which are all regarded as the scope described in the description of the present invention; further, for those of ordinary skill in the art, improvements or transformations can be made according to the above description, and all these improvements and transformations should fall within the protection scope of the appended claims of the present invention.

Claims

1. An inhalable small molecule compound pulmonary 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 Lactobacillus rhamnosus GG strain.

4. The delivery system according to claim 1, wherein The administration method of the delivery system is aerosol inhalation.

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

6. A preparation method of an inhalable small molecule compound pulmonary targeting delivery system according to any one of claims 1-4, characterized in that, Comprising the following steps: (1) Disperse the small molecule compound in phosphate buffer solution; (2) Resuspend Lactobacillus rhamnosus in the suspension of the small molecule compound, incubate under oscillation after sufficient mixing, so that the small molecule compound is adsorbed on the surface of Lactobacillus rhamnosus, and obtain Lactobacillus rhamnosus with small molecule compounds loaded on its surface.

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

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