Preparation of a plasma albumin delivery system loaded with cisplatin prodrug and use thereof

Abstract

The application discloses a preparation method of a plasma albumin delivery system loaded with cisplatin prodrug and application thereof. The preparation method of the plasma albumin delivery system loaded with cisplatin prodrug comprises the following steps: first, cisplatin is oxidized to prepare an intermediate product P1; then, pre-DDP with a similar structure of long-chain fatty acid is prepared through two-step reaction; then, a ligand-modified plasma albumin carrier M1A is synthesized; the two are mixed to realize non-covalent assembly combination; and finally, the albumin nano delivery system capable of targeting target cells with overexpression is obtained. The ligand-modified albumin nano delivery system prepared by the application can reduce the accumulation of the system in various metabolic organs, and further improve the safety of drug use. Meanwhile, the modified albumin carrier can not only circulate in the circulatory system for a long time, but also has the ability of recognizing and locating target cells, helps the pre-DDP carried to prolong the action time and precisely kill, improves the treatment effect, reduces the frequency of drug administration, and improves the compliance of patients.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the preparation and application of a plasma albumin delivery system loaded with cisplatin prodrug. Background Technology

[0002] Organ fibrosis is a group of progressive, chronic organic diseases that cause persistent and difficult-to-resolve suffering for patients. Taking pulmonary fibrosis as an example, it is characterized by irreversible scarring, epithelial-mesenchymal transition (EMT) imbalance, abnormal recruitment of immune cells, and massive accumulation of extracellular matrix in the lungs. This can lead to a continuous decline in lung function and may even be life-threatening. The development of pulmonary fibrosis is caused by external factors such as radiation, bacterial infection, smoking, and air pollution from environmental exposure. In addition, other diseases, especially lung-related diseases such as pulmonary hypertension, emphysema, and lung cancer, can also affect the development of pulmonary fibrosis. The etiology and pathogenesis of pulmonary fibrosis are not yet fully understood; pulmonary fibrosis of unknown etiology is also known as non-idiopathic pulmonary fibrosis.

[0003] The available medications for non-idiopathic pulmonary fibrosis (NPPF) are very limited. The use of corticosteroids (such as prednisone) in early mainstream pulmonary fibrosis treatments has been shown to be detrimental to patient prognosis and recovery. To date, only two drugs for treating NPPF have been approved by the FDA (Food and Drug Administration): pirfenidone and nintedanib. While they do offer some therapeutic benefit, they are far from curing NPPF, and they can cause numerous adverse reactions during long-term treatment, such as nausea, (photosensitive) rashes, indigestion, and diarrhea. Pulmonary fibrosis does not have a defined clinical stage; however, as the disease progresses, the physiological environment of the lesion area changes considerably, leading to reduced treatment effectiveness. To address this, researchers have employed various strategies in drug delivery regimens and system design to overcome this challenge. Multiple delivery systems, such as liposomes, micelles, and polymer nanoparticles, have been investigated. Among these drug delivery systems, nano / micro drug delivery systems are a research hotspot, as they can not only improve efficacy but also achieve sustained and controlled drug release, thus improving patient compliance. However, most drug delivery systems are designed to address the problem of "treating the symptoms but not the root cause," only slowing the progression of pulmonary fibrosis without reversing or curing damaged and fibrotic areas. Developing more effective treatments for pulmonary fibrosis is urgently needed.

[0004] To address these challenges, researchers need to develop a drug delivery system that blocks the progression of fibrosis at its source. One potentially effective strategy for designing delivery systems to address the complex and varied pathological processes of fibrosis is to precisely target drugs to specific reactive cells, such as macrophages and fibroblasts, and kill them before they reach the lesion area, thereby interrupting the progression of the disease and achieving a therapeutic effect. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, this invention provides a method for preparing a ligand-modified plasma albumin drug delivery system loaded with a cisplatin prodrug. The resulting drug-loaded albumin can circulate in the blood for a longer period. Simultaneously, the ligand-modified albumin enables the drug delivery system to more precisely target target cells in the circulatory system, while the albumin-loaded cisplatin prodrug can kill the target cells, interrupting disease progression and thus achieving better therapeutic effects. Furthermore, the ligand-loaded drug delivery system reduces accumulation in other organs, thus reducing drug toxicity.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of this invention provides a method for preparing a plasma albumin delivery system loaded with a cisplatin prodrug, comprising the following steps:

[0008] (1) Cisplatin is dissolved in water, hydrogen peroxide is added, the mixture is heated and stirred, and the reaction is carried out. The intermediate product P1 is then recrystallized to obtain intermediate product P1. Intermediate product P1 is dissolved in an organic solvent, succinic anhydride is added, and the reaction is stirred to obtain intermediate product P2. Intermediate product P2 and hexadecyl isocyanate are dissolved in an organic solvent, stirred, and purified to obtain cisplatin prodrug pre-DDP.

[0009] (2) Dissolve 3-mercaptopropionic acid, triethylamine and O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate in a solvent, stir, then add pleroxafer solution, and stir the reaction under a protective gas atmosphere to obtain ligand -SH;

[0010] (3) Take linker and / or Cy5-NHS-Et and mouse plasma albumin MSA and dissolve them in PBS. React under a protective gas atmosphere and in the dark. Dialyze to obtain MSA-l solution. Mix MSA-l solution with the ligand-SH and continue the reaction. Dialyze and dry to obtain MSA-l-ligand.

[0011] (4) The pre-DDP and the MSA-l-ligand were mixed and dissolved in PBS to obtain a plasma albumin delivery system loaded with cisplatin prodrug.

[0012] This invention oxidizes cisplatin to produce the intermediate product P1, then prepares the tetravalent P1 into pre-DDP with a structure similar to long-chain fatty acids through a two-step reaction. This pre-DDP is then non-covalently assembled with a ligand-modified plasma albumin carrier to obtain an albumin nanodelivery system capable of targeting overexpressing target cells. In this delivery system, the long-chain tetravalent cisplatin prodrug pre-DDP reduces its systemic toxicity through both valence state and carrier binding. Simultaneously, the modified albumin carrier not only circulates extensively in the circulatory system but also possesses the ability to recognize and locate target cells, helping the pre-DDP to prolong its duration of action and achieve precise killing. Furthermore, the ligand-modified albumin nanodelivery system obtained by this invention reduces systemic accumulation in various metabolic organs, further improving drug safety. The system designed in this invention can be applied to the treatment of various diseases involving target cells recruited through the circulatory system by modifying different ligands.

[0013] Preferably, in step (1), the organic solvent includes at least one of DMSO (dimethyl sulfoxide) and DMF (dimethylformamide).

[0014] Preferably, in step (1), the molar ratio of cisplatin to hydrogen peroxide is 1:(30-35).

[0015] Preferably, in step (1), the hydrogen peroxide is added slowly in three portions over two hours.

[0016] Preferably, in step (1), the temperature of heating and stirring after adding the hydrogen peroxide is 65-75°C, and the reaction time is 2-10h.

[0017] Preferably, in step (1), the molar ratio of the intermediate product P1 to succinic anhydride is 1:(0.85-1.2), and the stirring reaction time after adding succinic anhydride is 5-24h.

[0018] Preferably, in step (1), the molar ratio of the intermediate product P2 to hexadecyl isocyanate is 1:(2.5-3.5).

[0019] Preferably, in step (1), the specific purification method is as follows: filtration, removal of solvent under reduced pressure at 65°C, addition of diethyl ether to the residue, sonication, centrifugation, washing of the solid with dichloromethane and diethyl ether, and vacuum drying.

[0020] Preferably, in step (2), the stirring time after the 3-mercaptopropionic acid, triethylamine and O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate is dissolved in the solvent is 0.4-0.6 h, and the stirring time after adding the plexafor solution is 60-84 h.

[0021] Preferably, in step (2), the molar ratio of 3-mercaptopropionic acid, O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate, and plexafor in the plexafor solution is 1:(1-1.5):(0.8-1.2).

[0022] Preferably, in step (2), the solvent is anhydrous acetonitrile; the solvent of the plexafor solution is anhydrous acetonitrile.

[0023] Preferably, in step (3), the linker (Mal-PEG) n The molecular weight of -NHS is 1000-10000.

[0024] Preferably, in step (3), when the linker and mouse plasma albumin MSA are dissolved in PBS, the molar ratio of mouse plasma albumin MSA to linker is 1:(8-12).

[0025] Preferably, in step (3), when Cy5-NHS-Et and mouse plasma albumin MSA are dissolved in PBS, the molar ratio of mouse plasma albumin MSA to Cy5-NHS-Et is 1:(3-5).

[0026] Preferably, in step (3), when the linker, Cy5-NHS-Et and mouse plasma albumin MSA are dissolved in PBS, the molar ratio of mouse plasma albumin MSA, Cy5-NHS-Et and linker is 1:(3-5):(5-7).

[0027] Preferably, in step (3), the reaction time of linker and / or Cy5-NHS-Et and mouse plasma albumin MSA dissolved in PBS is 4-6 h; the reaction time of MSA-l solution and ligand-SH mixture is 5-24 h.

[0028] Preferably, in step (3), the molar ratio of MSA-l to ligand-SH in the MSA-l solution is 1:(2-4).

[0029] Preferably, in step (4), the molar ratio of pre-DDP to MSA-l-ligand is 1:(0.8-1.2).

[0030] A second aspect of the present invention provides a plasma albumin delivery system loaded with cisplatin prodrug, which is prepared by the method for preparing the plasma albumin delivery system loaded with cisplatin prodrug.

[0031] The third aspect of this invention provides the application of the method for preparing the plasma albumin delivery system loaded with cisplatin prodrug in the preparation of drugs for treating fibrotic diseases.

[0032] Preferably, the fibrotic disease is pulmonary fibrosis.

[0033] Compared with the prior art, the beneficial effects of the present invention are:

[0034] This invention discloses a method for preparing a ligand-modified plasma albumin delivery system for treating fibrosis, loaded with a cisplatin prodrug. First, cisplatin is oxidized to an intermediate product P1. Then, the tetravalent P1 is processed through a two-step reaction to prepare pre-DDP, which has a structure similar to long-chain fatty acids. Next, a ligand-modified plasma albumin carrier is synthesized. The two are then mixed and non-covalently assembled, finally yielding an albumin nanodelivery system capable of targeting overexpressing target cells. Compared to the common cytotoxic drug cisplatin, the synthesized tetravalent long-chain pre-DDP requires intracellular reduction to divalent platinum to exert its cytotoxic effect. Furthermore, its fatty acid-like structure allows it to utilize the transport function of plasma albumin, preventing the drug from indiscriminately killing normal cells, reducing systemic toxicity, and improving the safety of the delivery system. In addition, the ligand-modified albumin nanodelivery system prepared by this invention can reduce its accumulation in various metabolic organs, further improving drug safety. Simultaneously, the modified albumin carrier not only circulates extensively in the circulatory system but also possesses the ability to recognize and locate target cells, helping the pre-DDP carrier to prolong its duration of action and precisely kill cells, thereby improving therapeutic efficacy, reducing the frequency of administration, and increasing patient compliance. Therefore, the plasma albumin delivery system provided by this invention is an excellent delivery platform for the treatment of fibrotic diseases. Attached Figure Description

[0035] Figure 1 Quantitative graphs of pre-DDP concentration in blood at different time points within 7 days after administration of each delivery system;

[0036] Figure 2 Fluorescence images showing the retention and distribution of each dye-modified delivery system in the five internal organs (heart, liver, spleen, lungs, and kidneys);

[0037] Figure 3 A graph showing the percentage of fluorescence intensity retained in the liver for each dye-modified delivery system;

[0038] Figure 4 A quantitative graph showing the proportion of CXCR4+ cells in the blood of mice with pulmonary fibrosis treated by different delivery systems.

[0039] Figure 5 Pathological staining images of lung tissue sections from mice with pulmonary fibrosis treated by each delivery system;

[0040] Figure 6 Quantitative graphs of pulmonary collagen deposition in mice with pulmonary fibrosis treated by different delivery systems;

[0041] Figure 7Ashcroft score plot for the degree of pulmonary fibrosis in mice treated with different delivery systems. Detailed Implementation

[0042] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0043] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0044] Example 1: Preparation of the MlApre-DDP plasma albumin nanodelivery system

[0045] First, pre-DDP is synthesized through a three-step reaction, as follows:

[0046] (1) Synthesis of cisplatin prodrug intermediate P1:

[0047] Cisplatin (50 mg, 0.17 mmol) was dissolved in 0.12 mL of water, stirred, and heated to 70 °C. Hydrogen peroxide (30 wt%, 0.54 mL, 5.4 mmol) was slowly added dropwise over two hours in three portions (0.18 mL each time). The reaction was continued for 3 hours to obtain a bright yellow solution. The solution was recrystallized at 4 °C, and the precipitate was washed successively with cold water, cold ethanol, and diethyl ether. The precipitate was then dried under vacuum to obtain intermediate P1.

[0048] (2) Synthesis of cisplatin prodrug intermediate P2:

[0049] P1 (25 mg, 0.075 mmol) was dissolved in 2 ml of DMSO (dimethyl sulfoxide) to prepare a suspension (P1 may be slightly in excess). Succinic anhydride (7.5 mg, 0.075 mmol) was added, and the reaction mixture was stirred overnight at room temperature. After vacuum drying, 1.25 ml of cold acetone was added to obtain a pale yellow solid precipitate. The precipitate was collected and washed successively with acetone and diethyl ether, and then vacuum dried to obtain intermediate P2.

[0050] (3) Synthesis of cisplatin prodrug pre-DDP:

[0051] P2 (20 mg, 0.047 mmol) and hexadecyl isocyanate (40 mg, 0.15 mmol) were dissolved in 2 mL of DMF (dimethylformamide). The reaction mixture was stirred overnight at room temperature to obtain a clear green solution. The solution was filtered, and the solvent was removed under reduced pressure at 65 °C. 2 mL of diethyl ether was added to the residue, and the mixture was sonicated for 1 min and centrifuged. The solid was washed with dichloromethane and diethyl ether, and dried under vacuum to obtain pre-DDP.

[0052] (4) Synthesis of AMD-SH:

[0053] 3-MPA (3-mercaptopropionic acid, 2.62 μl, 0.03 mmol) was dissolved in anhydrous acetonitrile (0.5 mL), and O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate (11.6 mg, 0.036 mmol) and triethylamine (8.4 μl, 0.06 mmol) were added. The mixture was stirred at room temperature under an argon atmosphere for 0.5 h. AMD3100 (Plorisaf, 20 mg, 0.03 mmol) was dissolved in anhydrous acetonitrile (1 mL), and the solution was added to the above reaction system. The mixture was stirred at room temperature under an Ar atmosphere for approximately 3 days until the reaction was complete, yielding AMD-SH.

[0054] (5) Synthesis of MlA:

[0055] Take MSA (mouse plasma albumin, 64.3 mg, 0.001 mmol) and linker (Mal-PEG). n -NHS, 10 mg, 0.01 mmol) was dissolved in PBS (1×, 2 ml, pH = 8.0) and reacted in the dark (37 °C, 400 rpm) under an argon atmosphere for 5 h at a reaction ratio of 1:10. The mixture was then dialyzed to obtain an MSA-l solution. The MSA-l solution and AMD-SH reaction system were mixed at a molar ratio of MSA-l:AMD-SH = 1:3, and the reaction was continued overnight. The mixture was then dialyzed and lyophilized to obtain MSA-l-AMD (MlA).

[0056] (6) Preparation of MlApre-DDP:

[0057] The nanodelivery system MlApre-DDP was prepared by dissolving pre-DDP and MlA in PBS solution at a molar ratio of 1:1 and mixing them evenly.

[0058] Example 2: Preparation of plasma albumin nanocarrier MlA-Cy5 pre-DDP

[0059] The preparation method is the same as in Example 1, except that:

[0060] (1) Preparation of MlA-Cy5 pre-DDP:

[0061] In Example 1, step (5) was modified as follows: MSA, Cy5-NHS-Et (Cy-NHS-ester) (CAS: 146368-14-1), and linker (Mal-PEGn-NHS) (CAS: 1325208-25-0) were dissolved in PBS (1×, 2 ml, pH = 8.0) at a molar ratio of 1:4:6. The mixture was reacted in the dark (37°C, 400 rpm) under an argon atmosphere for 5 h, followed by dialyzing and lyophilization to obtain MSA-l-Cy5. The MSA-l-Cy5 solution and the AMD-SH reaction system were mixed at a molar ratio of MSA-l-Cy5:AMD-SH = 1:3, and the reaction was continued overnight. The mixture was then dialyzed and lyophilized to obtain MlA-Cy5.

[0062] Example 3: Preparation of plasma albumin nanocarrier MSA-Cy5 pre-DDP

[0063] (2) Preparation of MSA-Cy5 pre-DDP:

[0064] In Example 1, step (5) is changed to: MSA and Cy5-NHS-Et (Cy-NHS-ester) (CAS: 146368-14-1) are dissolved in PBS (1×, 2 ml, pH=8.0) at a molar ratio of 1:4, and reacted in the dark (37℃, 400 rpm) under Ar atmosphere for 5 h, followed by dialyzing and lyophilization to obtain MSA-Cy5.

[0065] Experiment 1: Investigation of the metabolism of the plasma albumin nanodelivery system in mouse blood

[0066] In this experimental example, pre-DDP refers to cisplatin prodrug, MSApre-DDP refers to a plasma albumin nanodelivery system equipped with pre-DDP (prepared by mixing mouse plasma albumin MSA and pre-DDP in PBS solution at a molar ratio of 1:1 based on Example 1), and MlApre-DDP refers to a plasma albumin nanodelivery system modified with ligands and equipped with pre-DDP.

[0067] To evaluate the metabolism of the plasma albumin nanodelivery system in mouse blood, PBS solutions of Pre-DDP, MSAPre-DDP, and MSAPre-DDP were prepared and administered intravenously (10 nmol / kg) to mice (C57, 5 weeks old, male, ~20 g). Mice were sacrificed and blood was collected at 0.5 h, 1 h, 4 h, 8 h, 12 h, 24 h, 72 h, 120 h, and 168 h. 100 μl of whole blood diluted 5 times was taken and extracted by vortexing with 300 μl of octanol for 2 min. The supernatant was collected by centrifugation and analyzed by HPLC (Waters Alliance 2695). The concentration of pre-DDP in the mouse circulatory system was calculated based on the peak area.

[0068] like Figure 1 As shown, compared with pre-DDP, MSApre-DDP and MlApre-DDP administration resulted in higher pre-DDP concentrations in mouse blood within 7 days. This indicates that assembly using a plasma albumin carrier allows pre-DDP to remain in the circulatory system for a longer period.

[0069] Experiment 2: Investigation of the retention and distribution of the plasma albumin nanodelivery system in the five internal organs (heart, liver, spleen, lungs, and kidneys) of mice.

[0070] In this experimental example, MlA-Cy5 refers to a plasma albumin carrier modified with dye and ligand, MSA-Cy5 pre-DDP refers to a plasma albumin nanodelivery system modified with dye and assembled with pre-DDP, and MlA-Cy5 pre-DDP refers to a plasma albumin nanodelivery system modified with dye and ligand and assembled with pre-DDP.

[0071] To evaluate the retention and distribution of the nanodelivery system in various organs, PBS solutions and PBS solutions of MlA-Cy5, MSA-Cy5 Pre-DDP, and MlA-Cy5 Pre-DDP were prepared. Mice (C57, 5 weeks old, male, ~20g) were administered the drug intravenously (10 nmol / kg). Mice were sacrificed at 0.5h, 2h, 4h, 12h, 24h, and 48h, and their hearts, livers, spleens, lungs, and kidneys were removed. The residue of the nanodelivery system was observed using the Perkin Elmer small animal in vivo imaging system (Lumina XR Series III). After imaging with the small animal in vivo imaging system, quantitative fluorescence analysis was performed based on the ROI values ​​corresponding to the fluorescent regions. In addition, the fluorescence intensity of the nanodelivery system in the liver and kidneys at different time points (0.5, 2, 4, 12, 24, 48h) was analyzed using small animal in vivo imaging processing software (the software accompanying the imaging system).

[0072] like Figure 2 , Figure 3 As shown, compared with MSA-Cy5, MlA-Cy5 accumulates at lower levels in metabolic organs such as the liver and kidneys, and can return to normal levels within 12 hours (fluorescence intensity is no different from the PBS control group). Furthermore, the assembly of pre-DDP does not affect the accumulation tendency of the albumin carrier. Therefore, the modified MlA-Cy5 pre-DDP can reduce the toxicity of the drug to the metabolic organs of mice.

[0073] Experiment 3: Investigation of the targeting and killing effect of the plasma albumin nanodelivery system on CXCR4-positive cells in the blood of pulmonary fibrosis mice.

[0074] In this experimental case, all groups except the Health group underwent disease modeling. DDP refers to cisplatin, pre-DDP refers to cisplatin prodrug, MSA pre-DDP refers to a plasma albumin nanodelivery system equipped with pre-DDP, MlA refers to ligand-modified plasma albumin carrier, and MlA pre-DDP refers to a plasma albumin nanodelivery system modified with ligand and equipped with pre-DDP.

[0075] To evaluate the targeting and killing effect of the plasma albumin nanodelivery system on CXCR4-positive cells in the blood of mice with pulmonary fibrosis, pulmonary fibrosis was first induced in the experimental animals: mice (C57, 5 weeks old, male, ~20g) were anesthetized by intraperitoneal injection with 5% chloral hydrate saline solution, and then bleomycin (3.5 mg / kg) was administered intratracheally using a lung aerosol injector to establish a mouse model of pulmonary fibrosis. After modeling, PBS, DDP, Pre-DDP, MSAPre-DDP, MLA, and MLAPre-DDP (10 nmol / kg) were administered intravenously on days 14 and 21, and the mice were followed up for 28 days. On day 28, mice were sacrificed and blood was collected. Mononuclear cells were obtained from the mouse blood using the Ficoll separation method. The specific method is as follows: The mouse blood collected in the blood collection tube was diluted to 3 ml with diluent. 3 ml of separation solution was added to a 15 ml centrifuge tube. The diluted blood was carefully added to the separation solution, and centrifuged to separate the layers (450 g, 15 min). The centrifuged cell layer (1 ml) was centrifuged again (1000 rpm, 5 min), and the supernatant was discarded. The cells were resuspended in 500 μl PBS, and fluorescent dye labeled CXCR4 was added. The cells were incubated on ice for 30 min, and then centrifuged again (1000 rpm, 5 min) to discard the supernatant. The washing steps of adding 500 μl of washing solution to resuspend and centrifuging to discard the supernatant were repeated twice. Finally, the cell solution resuspended in 500 μl PBS was filtered through a cell filter, and the cell data were recorded and analyzed using a flow cytometer (Beckman Coulter CytoFLEX).

[0076] like Figure 4As shown, compared with other groups, the MlApre-DDP group had the lowest level of CXCR4+ cells in the blood of mice. This indicates that MlApre-DDP can precisely kill CXCR4+ fibroblasts recruited in the circulatory system.

[0077] Experiment 4: Investigation of the therapeutic effect of the plasma albumin nanodelivery system on mice with pulmonary fibrosis

[0078] In this experimental case, all groups except the Health group underwent disease modeling. DDP refers to cisplatin, pre-DDP refers to cisplatin prodrug, MSA pre-DDP refers to a plasma albumin nanodelivery system equipped with pre-DDP, MlA refers to ligand-modified plasma albumin carrier, and MlA pre-DDP refers to a plasma albumin nanodelivery system modified with ligand and equipped with pre-DDP.

[0079] To evaluate the therapeutic effect of the plasma albumin nanodelivery system on pulmonary fibrosis in mice, the modeling and drug administration regimens were the same as in Experiment 3. The difference was that on day 28, mice were sacrificed, lungs were harvested, fixed in 4% paraformaldehyde fixative, embedded in paraffin, and sectioned. The sections were then stained using three staining kits (Thermo Fisher Scientific) according to their respective instructions: H&E, Mason rubes, and Sirius red. Pathological findings were observed and photographs were taken. Additionally, collagen deposition was quantified based on Mason rubesiology results (ImageJ), and three researchers performed Ashcroft scoring based on H&E rubesiology results under a double-blind procedure.

[0080] like Figure 5 As shown, in the PBS-treated group of the pulmonary fibrosis model, the alveolar spaces were widened, fibrotic exudation within the alveolar cavities was significant, and collagen fiber infiltration was severe. Compared with other groups, the MlApre-DDP group showed the least obvious pathological features in the lungs and the lowest degree of pulmonary fibrosis. Figure 6 , Figure 7 As shown, the mice in the MlApre-DDP group had the lowest lung collagen levels and the least degree of lung lesions. This indicates that MlApre-DDP has the best therapeutic effect on pulmonary fibrosis.

[0081] In summary, the MlApre-DDP plasma albumin nanodelivery system can remain in the circulatory system for a long time, reducing the accumulation in metabolic organs while precisely killing targeted fibroblasts, thus demonstrating excellent therapeutic effects on pulmonary fibrosis.

[0082] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. The application of a plasma albumin delivery system loaded with cisplatin prodrug in the preparation of drugs for treating pulmonary fibrosis, characterized in that, The method for preparing the plasma albumin delivery system loaded with cisplatin prodrug includes the following steps: (1) Cisplatin is dissolved in water, hydrogen peroxide is added, the mixture is heated and stirred, and the reaction is carried out. The intermediate product P1 is then recrystallized to obtain intermediate product P1. Intermediate product P1 is dissolved in an organic solvent, succinic anhydride is added, and the mixture is stirred to obtain intermediate product P2. Intermediate product P2 and hexadecyl isocyanate are dissolved in an organic solvent, stirred, and purified to obtain cisplatin prodrug pre-DDP. (2) Dissolve 3-mercaptopropionic acid, triethylamine and O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate in a solvent, stir, then add plerusore solution, and stir the reaction under a protective gas atmosphere to obtain ligand -SH; (3) Dissolve the linker and mouse plasma albumin MSA in PBS, or dissolve the linker, Cy5-NHS-Et, and mouse plasma albumin MSA in PBS, react under a protective atmosphere and in the dark, and dialyze to obtain an MSA-l solution; mix the MSA-l solution with the ligand-SH, continue the reaction, dialyze and dry to obtain the MSA-l-ligand; the linker is Mal-PEG. n -NHS, the Mal-PEG n -The molecular weight of NHS is 1000-10000; (4) The pre-DDP and the MSA-l-ligand are mixed and dissolved in PBS to obtain a plasma albumin delivery system loaded with cisplatin prodrug.

2. The application according to claim 1, characterized in that, In step (2), the stirring time after the 3-mercaptopropionic acid, triethylamine and O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate are dissolved in the solvent is 0.4-0.6 h, and the stirring time after adding the plexafor solution is 60-84 h.

3. The application according to claim 2, characterized in that, In step (2), the molar ratio of 3-mercaptopropionic acid, O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate, and plexafor in the plexafor solution is 1:(1-1.5):(0.8-1.2).

4. The application according to claim 1, characterized in that, In step (3), the linker and mouse plasma albumin MSA are dissolved in PBS, and the molar ratio of mouse plasma albumin MSA to linker is 1:(8-12).

5. The application according to claim 1, characterized in that, In step (3), the linker, Cy5-NHS-Et and mouse plasma albumin MSA are dissolved in PBS, and the molar ratio of mouse plasma albumin MSA, Cy5-NHS-Et and linker is 1:(3-5):(5-7).

6. The application according to claim 1, characterized in that, In step (3), the molar ratio of MSA-l to ligand-SH in the MSA-l solution is 1:(2-4).

7. The application according to claim 1, characterized in that, In step (4), the molar ratio of pre-DDP to MSA-l-ligand is 1:(0.8-1.2).

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