Composition as well as preparation method and application thereof in acute lung injury

Abstract

The invention belongs to the technical field of medicines, and relates to Torin 1 albumin nanoparticles attached to the surfaces of red blood cells, a preparation method of the Torin 1 albumin nanoparticles and application of the Torin 1 albumin nanoparticles in acute lung injury. The preparation method comprises the following steps: self-assembling an indissolvable mTOR kinase inhibitor Torin1 and bovine serum albumin to form drug-loaded nanoparticles, homogenizing the drug-loaded nanoparticles, and attaching the drug-loaded nanoparticles to the surfaces of red blood cells to obtain RBC-Torin1-coated BSA. The system can improve the problem of poor solubility of Torin1, reduce nonspecific removal of nano-particles, improve distribution and enrichment of the nano-particles in lung tissues, and enhance the treatment effect on acute lung injury.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, and relates to a Torin1 albumin nanoparticle attached to the surface of red blood cells, its preparation method, and its application in acute lung injury. Background Technology

[0002] Acute lung injury (ALI) and its severe stage, acute respiratory distress syndrome (ARDS), are acute inflammatory lung injury syndromes induced by various factors such as infection, trauma, shock, and inhalation injury. ALI / ARDS has a rapid onset, rapid progression, and high mortality rate. Clinical treatment currently mainly relies on supportive measures such as mechanical ventilation, fluid management, control of the primary disease, and anti-infection treatment. Drug delivery strategies that effectively intervene in the inflammatory process still need further development.

[0003] Torin1 is a potent mammalian target of rapamycin (mTOR) kinase inhibitor with strong biological activity, showing potential applications in inflammation regulation, autophagy regulation, and tissue damage protection. Previous studies have demonstrated that Torin1 exhibits anti-inflammatory and tissue-protective effects in animal models of acute lung injury. However, Torin1 is a poorly soluble small molecule drug with low water solubility, resulting in low bioavailability under conventional administration conditions and difficulty in maintaining effective drug concentrations at lung lesion sites, thus limiting its further application.

[0004] To improve the solubility, stability, and in vivo distribution of poorly soluble drugs, nanomedicine delivery systems have been extensively studied. Among these, albumin nanoparticles are frequently used as delivery carriers for hydrophobic drugs due to their good biocompatibility, biodegradability, and strong drug loading capacity. In existing technologies, poorly soluble drugs are typically dissolved in an organic solvent and then mixed with an aqueous albumin solution. Non-covalent interactions, such as hydrophobic and electrostatic interactions, are then used to form drug-loaded albumin nanoparticles, thereby improving drug dispersibility and delivery performance. This approach can address the problem of poor drug solubility to some extent.

[0005] However, albumin nanoparticles, once in vivo, remain exogenous nanomaterials and are easily and rapidly recognized and cleared by the endothelial reticulum system, such as the liver and spleen. This results in a short circulation time, limiting the amount of drug that actually reaches the lung injury area and leading to insufficient lung targeting. Furthermore, the accumulation of nanoparticles in non-target organs may cause off-target distribution and potential safety issues. How to improve the poor solubility of Torin1 while further reducing the risk of rapid clearance of nanoparticles and increasing their accumulation efficiency in lung tissue remains a critical technical challenge that needs to be addressed with current technology.

[0006] Therefore, it is still necessary to provide a new drug delivery system that can improve the formulation performance of poorly soluble drugs such as Torin1, prolong the in vivo circulation time, reduce non-specific clearance, and improve the delivery efficiency of drugs to the site of lung injury, so as to meet the actual needs of acute lung injury treatment. Summary of the Invention

[0007] The present invention aims to at least partially solve one of the technical problems existing in the prior art. To this end, the present invention provides Torin1 albumin nanoparticles attached to the surface of red blood cells, a method for preparing the same, and their application in acute lung injury.

[0008] According to one aspect of the present invention, a drug-loaded albumin nanoparticle composition for attaching to the surface of red blood cells is provided, comprising red blood cells and drug-loaded albumin nanoparticles attached to the surface of the red blood cells; the drug-loaded albumin nanoparticles are formed by the self-assembly of the poorly soluble drug Torin1 and bovine serum albumin.

[0009] Preferably, the mass ratio of Torin1 to bovine serum albumin is 5% to 30%.

[0010] Preferably, the hydrated particle size of the drug-loaded albumin nanoparticles is 100–250 nm.

[0011] Preferably, the polydispersity index (PDI) of the drug-loaded albumin nanoparticles is not greater than 0.30.

[0012] Preferably, the red blood cells are mammalian red blood cells.

[0013] According to another aspect of the present invention, a method for preparing a drug-loaded albumin nanoparticle composition attached to the surface of red blood cells is provided, comprising the following steps: (1) dissolving Torin1 in dimethyl sulfoxide to obtain a Torin1 solution; (2) under stirring conditions, slowly adding the Torin1 solution dropwise to an aqueous solution of bovine serum albumin until the system becomes opalescent, and then continuing to stir so that Torin1 and bovine serum albumin self-assemble to form drug-loaded albumin nanoparticles; (3) homogenizing the drug-loaded albumin nanoparticles obtained in step (2); (4) ultrafiltration and washing of the homogenized nanoparticles to remove organic solvents, and concentrating to obtain Torin1@BSA nanoparticles; (5) mixing and incubating a red blood cell suspension with the Torin1@BSA nanoparticles obtained in step (4) so ​​that the Torin1@BSA nanoparticles attach to the surface of red blood cells; (6) removing the free Torin1@BSA nanoparticles to obtain a Torin1@BSA nanoparticle composition attached to the surface of red blood cells.

[0014] Preferably, in step (3), a high-pressure microjet is used for homogenization, with a homogenization pressure of 25,000 to 30,000 psi and a homogenization time of 5 to 8 min.

[0015] According to another aspect of the invention, the use of the above-described composition, or the composition prepared by the above-described method, in the preparation of a medicament for treating acute lung injury is provided.

[0016] Preferably, the composition is used to improve the distribution and enrichment of Torin1 in lung tissue.

[0017] Preferably, the composition is used to reduce inflammatory cell infiltration, congestion, alveolar wall thickening and / or tissue consolidation in acute lung injury.

[0018] This invention provides Torin1@BSA nanoparticles attached to the surface of erythrocytes and their application in acute lung injury. By using bovine serum albumin to self-assemble the poorly soluble drug Torin1 into nanoparticles, the problems of poor water solubility and insufficient dispersibility of Torin1 during direct administration can be improved. Furthermore, this invention attaches the Torin1@BSA nanoparticles to the surface of erythrocytes, improving the stability of the nanoparticles in vivo circulation and enhancing their distribution and accumulation in lung tissue of acute lung injury. Animal experiments show that Torin1@BSA nanoparticles attached to the surface of erythrocytes have a better protective effect on lung tissue than Torin1@BSA nanoparticles alone, manifested as reduced inflammatory cell infiltration and alleviated alveolar wall thickening. This invention can effectively improve the therapeutic effect of Torin1 on acute lung injury. Attached Figure Description

[0019] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:

[0020] Figure 1 This is a schematic diagram illustrating the preparation of albumin nanoparticles encapsulating the poorly soluble drug Torin1 in this invention. The diagram shows the process of slowly adding a DMSO solution of Torin1 to a BSA solution, followed by stirring for self-assembly, high-pressure microfluidic homogenization, ultrafiltration washing, and concentration to obtain Torin1@BSA nanoparticles.

[0021] Figure 2 The figures show the appearance of nanoparticle systems prepared by different ratios of Torin1 and BSA. The figures illustrate how the system gradually changes from a clear to an opalescent state as the Torin1 ratio increases, and how precipitation occurs when the drug ratio is too high.

[0022] Figure 3 The figure shows the particle size distribution of BSA nanoparticles prepared when the mass ratio of Torin1 to BSA is 10%. The figure indicates that the nanoparticles obtained under these conditions have a smaller particle size and a more concentrated distribution.

[0023] Figure 4 The figure shows the particle size distribution of BSA nanoparticles after high-pressure microfluidic homogenization under 10% Torin1 conditions. The figure demonstrates that the nanoparticle size was further reduced and the distribution became more uniform after homogenization.

[0024] Figure 5 This is a transmission electron microscope image of the Torin1@BSA nanoparticles. The image shows that the obtained nanoparticles have a well-defined nanoscale morphology.

[0025] Figure 6 This image shows the tissue distribution of RBC-Torin1@BSA nanoparticles in a mouse model of acute lung injury. The figure illustrates the fluorescence distribution of Cy7-labeled Torin1@BSA nanoparticles attached to the surface of erythrocytes in tissues such as the heart, liver, spleen, lung, kidney, and brain, with the most pronounced distribution observed in the lung tissue.

[0026] Figure 7 This is a quantitative analysis of the mean fluorescence intensity in the lung tissue of a mouse model of acute lung injury. The figure shows that the mean fluorescence intensity of RBC-Torin1@BSA nanoparticles in lung tissue is higher than that of Torin1@BSA nanoparticles without attached red blood cells.

[0027] Figure 8This figure shows the in vivo efficacy evaluation of albumin nanoparticles attached to the surface of red blood cells in a mouse model of acute lung injury. The figure also shows the HE staining results of lung tissue sections from each group after establishing the acute lung injury model via intratracheal instillation of LPS. The RBC-Torin1@BSA nanoparticle group showed milder inflammatory cell infiltration and alveolar wall thickening. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to the embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Equivalent substitutions or conventional optimizations made by those skilled in the art to the types of raw materials, process conditions, detection methods, dosing regimens, and application models without departing from the spirit and substance of the present invention should all fall within the scope of protection of the present invention.

[0029] Unless otherwise stated, the reagents, raw materials, experimental animals, and detection methods used in the embodiments of this invention can all be obtained or implemented through conventional means in the art. Experimental conditions not explicitly defined in the disclosure but which can be determined by those skilled in the art based on conventional knowledge can be selected and optimized using conventional methods.

[0030] This invention provides a biomimetic nanodelivery system for treating acute lung injury. The system uses the poorly soluble mTOR kinase inhibitor Torin1 as the therapeutic drug and bovine serum albumin (BSA) as the nanoparticle forming material. After self-assembly to form drug-loaded albumin nanoparticles, these nanoparticles are then attached to the surface of red blood cells, forming drug-loaded albumin nanoparticles attached to the red blood cell surface. This improves the distribution of Torin1 in lung tissue and enhances its therapeutic effect on acute lung injury.

[0031] In some embodiments of the present invention, the raw materials for the erythrocyte adsorption nanoparticle system may include: Torin1, at a mass fraction of 1% to 50%; bovine serum albumin, at a mass fraction of 50% to 95%; and erythrocyte suspension at a volume of 10 to 15 μL. Preferably, the mass ratio of Torin1 to BSA is 5% to 30%; more preferably, the mass ratio is 10%.

[0032] Example 1: Preparation of albumin nanoparticles loaded with Torin1 (Torin1@BSA)

[0033] This embodiment provides a method for preparing albumin nanoparticles loaded with the poorly soluble drug Torin1.

[0034] First, Torin1 is dissolved in dimethyl sulfoxide (DMSO) to obtain a DMSO solution of Torin1. Preferably, 1 mg of Torin1 is dissolved in 0.8 mL of DMSO. Separately, 20 mL of an aqueous solution of BSA with a concentration of 0.5 mg / mL is prepared.

[0035] Subsequently, under room temperature / ice bath stirring at 800 rpm, a DMSO solution of Torin1 was slowly added dropwise to an aqueous BSA solution. A tapping funnel, pipette, or syringe pump could be used for slow, uniform addition, with the addition time controlled between 0.2 and 0.4 minutes. As Torin1 was gradually added, the system gradually transitioned from a clear state to an opalescent state, indicating that Torin1 and BSA formed self-assembled nanoparticles through non-covalent interactions such as hydrophobic interactions. After the system achieved a stable opalescent state, stirring was continued for 2 hours to promote complete particle formation and improve system stability.

[0036] After self-assembly, the resulting nanoparticle suspension was transferred to a high-pressure microfluidic nanodispersor for homogenization and granulation. The homogenization pressure was controlled at 25,000–30,000 psi, and the homogenization cycle time was 5–8 min. High-pressure microfluidic treatment can further reduce the particle size and improve the uniformity of particle size distribution, thereby obtaining nanoparticle formulations with better dispersibility.

[0037] After homogenization, the resulting nanoparticle suspension was ultrafiltered using an ultrafiltration tube with a molecular weight cutoff of 100 kDa to remove free small molecules and residual organic solvents. Subsequently, deionized water (ddH2O) was added for washing to further remove DMSO. Preferably, the ultrafiltration centrifugation conditions were 4000 rpm for 20 min, with 2 mL of ddH2O added each time, and the washing was repeated three times. Ultrafiltration concentration was then continued until the final volume was 200–500 μL, yielding albumin nanoparticles encapsulated with Torin1, denoted as Torin1@BSA. The obtained Torin1@BSA nanoparticles can be stored at 4°C for later use.

[0038] In this embodiment, BSA and Torin1 are self-assembled to form nanoparticles, which can improve the problems of poor water solubility and insufficient dispersibility of Torin1 for direct administration, and provide a basic formulation for subsequent red blood cell surface adhesion and lung delivery.

[0039] Figure 1This diagram illustrates the preparation of BSA nanoparticles encapsulating the poorly soluble drug Torin1. First, Torin1 was dissolved in DMSO. Then, the resulting solution was slowly added dropwise to an aqueous BSA solution with continuous stirring, allowing Torin1 and BSA to self-assemble into nanoparticles. Subsequently, the resulting particle system was transferred to a high-pressure microfluidic nanodispersor for homogenization. Finally, ultrafiltration, washing, and concentration steps were used to remove the organic solvent and obtain the Torin1@BSA nanoparticle formulation.

[0040] Example 2: Optimization of the synthesis process of albumin nanoparticles loaded with Torin1: Effect of the mass ratio of Torin1 to BSA on particle size.

[0041] To optimize the preparation process of Torin1@BSA nanoparticles, the effect of the mass ratio of Torin1 to BSA on the formation state and particle size of the nanoparticles was investigated.

[0042] Following the method in Example 1, the mass ratio of Torin1 to BSA was set to less than 1%, 5%, 10%, 20%, 30%, and 50%, respectively, while keeping other preparation conditions consistent. The changes in appearance and precipitation of the system under different mass ratios were observed, and the particle size and polydispersity index of the obtained nanoparticles were measured.

[0043] The results showed that as Torin1 was gradually added dropwise to the BSA solution, the system gradually changed from a transparent state to an opalescent state with increasing Torin1 content. When the mass ratio of Torin1 to BSA reached approximately 1%, the system began to exhibit opalescence, indicating the initiation of self-assembled nanoparticles. When the Torin1 content was less than 1%, no obvious opalescence was observed, indicating that nanoparticle formation was not significant. When the Torin1 content was between 5% and 30%, the system exhibited a uniform opalescent state, and no significant precipitation was observed after 2 hours of continued stirring, indicating that a relatively stable nanoparticle dispersion system could be formed within this range. When the Torin1 content was further increased to 50%, although the opalescence was enhanced, significant precipitation appeared in the system after 2 hours of continued stirring, indicating that an excessively high drug content was detrimental to the stability of the nanoparticle system.

[0044] The particle size and PDI were determined using dynamic light scattering (DLS). The results showed that when the mass ratio of Torin1 to BSA was 5%–30%, the resulting nanoparticles had a size of 150–250 nm, which was within a suitable range. Specifically, when the mass ratio of Torin1 to BSA was 10%, the hydrated particle size was the smallest (198.6 ± 4.1 nm), and the PDI was 0.18 ± 0.03. The particle size distribution is shown in the figure. Figure 3The above results indicate that, under the conditions of this invention, the preferred mass ratio of Torin1 to BSA is 5% to 30%, more preferably 10%, at which point the resulting nanoparticles possess both small particle size and good dispersibility.

[0045] Furthermore, it was found during the preparation process that the dropping rate of Torin1 had a significant impact on the formation of nanoparticles. To obtain nanoparticles with smaller particle size and more stable distribution, the Torin1 solution should be added to the BSA solution slowly and at a uniform rate to avoid excessively high local concentrations that could cause particle aggregation or drug precipitation.

[0046] Figure 2 The figures show the appearance of nanoparticle systems prepared by different ratios of Torin1 and BSA. As the proportion of Torin1 increases, the system gradually changes from a clear to an opalescent state; when the drug proportion is too high, the system stability decreases and precipitation occurs. Figure 3 The particle size distribution of BSA nanoparticles prepared when the mass ratio of Torin1 to BSA is 10% is shown in the figure. The results indicate that the nanoparticles prepared under these conditions have smaller particle sizes and more concentrated distribution.

[0047] Example 3: Optimization of the synthesis process of albumin nanoparticles loaded with Torin1: Effect of high-pressure microfluidic homogenization time on particle size and particle characterization

[0048] To further optimize the preparation process of Torin1@BSA nanoparticles and obtain smaller particle sizes while maintaining good dispersibility, this invention further investigated the effect of high-pressure microfluidic homogenization time on the particle size of nanoparticles.

[0049] The system with a mass ratio of Torin1 to BSA of 10% in Example 2 was selected as the research object. Under the same conditions, the homogenization time of high-pressure microjets was changed, and the particle size and dispersion of nanoparticles after different homogenization times were compared.

[0050] Experimental results show that when the homogenization time is too short, the resulting nanoparticles have a relatively large particle size, making it difficult to achieve the desired nano-sizing effect; when the homogenization time is too long, it may lead to adverse situations such as drug leakage in the system. Therefore, considering particle size, dispersibility, and system stability, the optimal homogenization time for high-pressure microjets is finally determined to be 5–8 min.

[0051] Under the above-mentioned preferred conditions, the hydrated particle size of the homogenized Torin1@BSA nanoparticles decreased to 141.9±8.3 nm, and the PDI was 0.229±0.01. The particle size distribution is as follows: Figure 4 As shown in the figure. The results indicate that after high-pressure microfluidic granulation, the particle size of the obtained nanoparticles is further reduced while maintaining a good dispersion state, which is beneficial for subsequent in vivo delivery applications.

[0052] Furthermore, after negative staining the obtained BSA nanoparticles with phosphotungstic acid, they were observed using transmission electron microscopy (TEM). Figure 5 As shown. Preferably, the TEM testing conditions are an accelerating voltage of 100 kV and a count of no less than 20 particles. TEM results show that the Torin1@BSA nanoparticles exhibit a nanoscale morphology, with an average particle size of 56.9 ± 16.1 nm. TEM characterization further confirms that the method of the present invention can stably prepare nanoscale drug-loaded albumin particles.

[0053] Figure 4 The image shows the particle size distribution of BSA nanoparticles after high-pressure microfluidic homogenization under 10% Torin1 conditions. The results indicate that the particle size is further reduced after homogenization. Figure 5 The image shows a TEM image of the Torin1@BSA nanoparticles. The results show that the obtained particles have well-defined nanoscale morphological features.

[0054] Example 4: Preparation of Torin1@BSA nanoparticles attached to the surface of red blood cells

[0055] To further improve the stability of Torin1@BSA nanoparticles during in vivo circulation and their delivery efficiency to lung tissue, this invention attaches the Torin1@BSA nanoparticles to the surface of red blood cells to prepare drug-loaded albumin nanoparticles attached to the surface of red blood cells.

[0056] Specifically, 0.1 mL of whole blood from mice was collected, anticoagulated with EDTA, and red blood cells were separated. The cells were then washed three times with phosphate-buffered saline (PBS) to remove plasma components and other impurities. Preferably, the red blood cell separation conditions were centrifugation at 600 g for 7 min. The washed red blood cells were resuspended in 1 mL of PBS to obtain a red blood cell suspension.

[0057] Take 10–15 μL of the erythrocyte suspension, mix it with Torin1@BSA nanoparticles, and incubate at 37°C for 3 h to allow the Torin1@BSA nanoparticles to attach to the erythrocyte surface. During incubation, use a constant temperature water bath at 30°C, and gently mix every 30 min. After incubation, centrifuge at 1200 rpm for 5 min to remove free Torin1@BSA nanoparticles that are not attached to the erythrocyte surface. Collect the erythrocyte precipitate to obtain the Torin1@BSA nanoparticles attached to the erythrocyte surface, denoted as RBC-Torin1@BSA.

[0058] In some implementations, fluorescence observation can be used for auxiliary characterization to verify that the nanoparticles have adhered to the surface of red blood cells. The RBC-Torin1@BSA obtained by the above method can utilize the natural biocompatibility and immune escape characteristics of red blood cells to provide a biomimetic delivery platform for nanoparticles, thereby helping to prolong their residence time in the blood circulation and reduce their non-specific clearance in non-target organs such as the liver and spleen.

[0059] Example 5: Investigation of the lung targeting ability of Torin1@BSA nanoparticles attached to the surface of erythrocytes

[0060] To investigate the in vivo tissue distribution and lung targeting of the RBC-Torin1@BSA, Torin1@BSA nanoparticles were linked with the fluorescent dye Cy7 to prepare Cy7-labeled Torin1@BSA nanoparticles; further, Cy7-labeled Torin1@BSA nanoparticles were prepared on the surface of red blood cells according to the method in Example 4, namely Cy7-RBC-Torin1@BSA.

[0061] Cy7 labeling can be performed using covalent coupling, with a Cy7 to Torin1@BSA nanoparticle feed ratio of 1:100. Unbound free Cy7 is removed by ultrafiltration after labeling. To eliminate interference from free dye, it is recommended to include post-labeling purification steps and purification criteria in the final application.

[0062] Subsequently, Cy7-labeled Torin1@BSA nanoparticles and Cy7-RBC-Torin1@BSA were injected intravenously into normal mice and mice with an acute lung injury model, respectively. Preferably, the administration volume was 0.05 mL / mouse, and the dosage was 6 mg / kg based on Torin1. Mice were dissected at predetermined time points (2 h, 4 h, and 8 h) after administration, and major organs such as the heart, liver, spleen, lungs, kidneys, and brain were isolated. The fluorescence distribution in each tissue was observed using a small animal in vivo / ex vivo fluorescence imaging system, and the average fluorescence intensity in the lung tissue was quantitatively analyzed.

[0063] Experimental results showed that in the acute lung injury model, Torin1@BSA nanoparticles attached to the surface of erythrocytes exhibited the strongest fluorescence signal in lung tissue, indicating their highest distribution in the lung tissue. Furthermore, the distribution of Cy7-labeled Torin1@BSA nanoparticles attached to the surface of erythrocytes in lung tissue was significantly higher than that of Cy7-labeled Torin1@BSA nanoparticles not attached to erythrocytes. Mean quantitative fluorescence analysis of lung tissue further confirmed that RBC-Torin1@BSA has a higher lung tissue enrichment capacity in acute lung injury.

[0064] The above results demonstrate that attaching Torin1@BSA nanoparticles to the surface of erythrocytes can significantly improve the delivery efficiency of these nanoparticles to lung tissue in an acute lung injury model, thereby enhancing their lung targeting.

[0065] Figure 6 The figure shows the tissue distribution of RBC-Torin1@BSA nanoparticles in a mouse model of acute lung injury. The figure indicates that the fluorescence signal of Cy7-labeled Torin1@BSA nanoparticles attached to the surface of erythrocytes was the strongest, significantly higher than that of Cy7-labeled Torin1@BSA nanoparticles, suggesting a greater distribution of these nanoparticles in the lung tissue. Figure 7 This presents the quantitative analysis results of the mean fluorescence intensity in the lung tissue of a mouse model of acute lung injury. The results show that RBC-Torin1@BSA nanoparticles exhibited a higher mean fluorescence intensity in lung tissue compared to Torin1@BSA nanoparticles without attached erythrocytes, indicating that erythrocyte attachment can enhance the enrichment level of Torin1@BSA nanoparticles in lung tissue.

[0066] Example 6: In vivo efficacy study of Torin1@BSA nanoparticles attached to the surface of red blood cells

[0067] To evaluate the therapeutic effect of RBC-Torin1@BSA in acute lung injury, this invention further investigated its in vivo efficacy in a mouse model of acute lung injury.

[0068] Specifically, an acute lung injury model in mice was established by intratracheal instillation of lipopolysaccharide (LPS). Preferably, the dosage of LPS was 5 mg / kg, and the administration volume was 10 μL per mouse. Subsequent administration or tissue collection was performed 24 h after model establishment.

[0069] After modeling, the experimental animals were randomly divided into a normal control group, a model group, a Torin1 administration group, a Torin1@BSA nanoparticle administration group, and an RBC-Torin1@BSA nanoparticle administration group, with n=6 animals in each group. Each group was administered the corresponding formulation. Preferably, the administration route was tail vein injection, with a dosage of 6 mg / kg of Torin1, administered twice at 24-hour intervals. To ensure comparability between groups, the dosage equivalent of Torin1 in each of the Torin1, Torin1@BSA, and RBC-Torin1@BSA groups was preferably kept consistent.

[0070] Mice were sacrificed on day 2 after drug administration, and lung tissue was collected for fixation, paraffin embedding, sectioning, and hematoxylin-eosin (HE) staining. Preferably, the lung tissue fixative was 4% paraformaldehyde, the fixation time was 24 h, and the section thickness was 10 μm. Further semi-quantitative evaluation of indicators such as inflammatory cell infiltration, alveolar wall thickening, congestion, edema, and tissue consolidation can be performed using lung tissue injury scoring criteria.

[0071] HE staining results showed that after LPS modeling, the lung tissue of the model group showed obvious inflammatory damage, manifested as massive infiltration of inflammatory cells, tissue congestion, alveolar cavity shrinkage, alveolar wall thickening and severe tissue consolidation, indicating that the acute lung injury model was successfully established.

[0072] Compared with the model group, the pathological damage to lung tissue in the Torin1@BSA nanoparticle administration group was reduced. Some inflammatory cell infiltration, reduced congestion, milder alveolar wall thickening, and improved tissue consolidation were observed, indicating that Torin1 delivered by albumin nanoparticles has a certain alleviating effect on acute lung injury.

[0073] Furthermore, in the RBC-Torin1@BSA nanoparticle administration group, only mild inflammatory cell infiltration and mild alveolar wall thickening were observed in the lung tissue, and the overall pathological damage was further reduced, showing a better improvement effect than the Torin1@BSA nanoparticle administration group. These results indicate that attaching Torin1@BSA nanoparticles to the surface of erythrocytes can further improve their effective delivery in lung tissue, thereby enhancing their therapeutic effect on acute lung injury.

[0074] Figure 8 This study presents the in vivo efficacy results of albumin nanoparticles attached to the surface of erythrocytes. An acute lung injury model was established in mice using intratracheal instillation of LPS. Torin1, Torin1@BSA nanoparticles, and RBC-Torin1@BSA nanoparticles were administered, and lung tissue sections were stained with hematoxylin and eosin (HE). The results showed that the RBC-Torin1@BSA nanoparticle group exhibited milder inflammatory cell infiltration and alveolar wall thickening compared to the Torin1@BSA nanoparticle group, suggesting a superior ameliorative effect on acute lung injury.

[0075] The following examples are provided to help those skilled in the art better understand the present invention. It should be noted that the following examples are not intended to limit the scope of protection claimed by the present invention, but are merely illustrative. Unless otherwise specified, the raw materials, reagents, or devices mentioned in the following examples are commercially available or obtained through known existing methods.

Claims

1. A red blood cell surface-attached drug-loaded albumin nanoparticle composition, characterized in that, The red blood cells and drug-loaded albumin nanoparticles attached to the surface of the red blood cells; the drug-loaded albumin nanoparticles are formed by self-assembly of poorly soluble drug Torin1 and bovine serum albumin.

2. The composition of claim 1, wherein, The mass ratio of the Torin1 to the bovine serum albumin is 5% to 30%.

3. The composition of claim 1, wherein, The hydrated particle size of the drug-loaded albumin nanoparticles is 100 to 250 nm.

4. The composition of claim 1, wherein, The polydispersity index (PDI) of the drug-loaded albumin nanoparticles is not greater than 0.

30.

5. The composition of claim 1, wherein, The red blood cells are mammalian red blood cells.

6. A method of preparing the red blood cell surface-attached drug-loaded albumin nanoparticle composition of any one of claims 1 to 5, characterized in that, The method comprises the following steps: (1) dissolving Torin1 in dimethyl sulfoxide to obtain a Torin1 solution; (2) slowly adding the Torin1 solution to an aqueous bovine serum albumin solution under stirring until the system appears opalescent, and then continuing to stir to allow the Torin1 and the bovine serum albumin to self-assemble to form drug-loaded albumin nanoparticles; (3) homogenizing the drug-loaded albumin nanoparticles obtained in step (2); (4) ultrafiltrating and washing the homogenized nanoparticles to remove organic solvents, and concentrating to obtain Torin1@BSA nanoparticles; (5) mixing a red blood cell suspension with the Torin1@BSA nanoparticles obtained in step (4) and incubating to allow the Torin1@BSA nanoparticles to attach to the surface of the red blood cells; (6) removing free Torin1@BSA nanoparticles to obtain a Torin1@BSA nanoparticle composition attached to the surface of the red blood cells.

7. The preparation method according to claim 6, characterized in that, In step (3), high-pressure microfluidization is used for homogenization, the pressure for homogenization is 25000 to 30000 psi, and the homogenization time is 5 to 8 min.

8. The composition of any one of claims 1 to 5, or the composition prepared according to the method of claim 6 or 7, for use in the preparation of a medicament for treating acute lung injury.

9. Use according to claim 8, characterized in that, The composition is used for improving the distribution and enrichment of Torin1 in lung tissue.

10. Use according to claim 8, characterized in that, The composition is used for reducing inflammatory cell infiltration, hyperemia, alveolar wall thickening and / or tissue consolidation in acute lung injury.

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