Drug-loaded nanoparticles Cor@PDA-PEG@CD11b, and a preparation method and application thereof

Abstract

The application provides a drug-loaded nanoparticle Cor@PDA-PEG@CD11b and a preparation method and application thereof, the preparation method comprises the following steps: S1, mixing cordycepin solution and PDA aqueous solution, so that the cordycepin and the PDA form a complex Cor@PDA through π-π stacking effect; S2, the obtained complex Cor@PDA is modified by PEG to prepare a complex Cor@PDA@PEG; S3, the obtained Cor@PDA@PEG is dispersed in a buffer solution, and an anti-CD11b antibody is added; after reacting for 24h, centrifugation, washing, collecting the precipitate, and drying, the Cor@PDA-PEG@CD11b is obtained. The drug-loaded nanoparticle Cor@PDA-PEG@CD11b prepared by the application provides a new idea and direction for the treatment of sepsis-related lung injury; through the characteristics of the PDA nanoparticles, the cordycepin is released in a controlled manner, and the problem that the cordycepin is rapidly oxidized and inactivated after entering the body is avoided, the bioavailability is improved, the specific antibodies on the surface of immune cells and the physiological characteristics are utilized, the immune cells are quickly and chemotactically recruited to the inflammatory injury site in the early stage of acute inflammation, the drug is quickly and accurately targeted, and the sepsis lung injury is reduced.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a drug-loaded nanoparticle Cor@PDA-PEG@CD11b, its preparation method, and its applications. Background Technology

[0002] Sepsis is a common and critical illness in clinical practice. The third international consensus definition of sepsis and septic shock (Sepsis-3), published in 2016, updated sepsis to a life-threatening organ dysfunction caused by a dysregulated host infection response. Globally, approximately 19 million people develop sepsis each year. A global statistical analysis from 1990 to 2017 revealed sepsis as the leading cause of in-hospital death, with persistently high morbidity and mortality rates, posing a significant threat to global health. Current research suggests that the mechanisms of tissue damage caused by sepsis are related to uncontrolled inflammatory responses and oxidative stress; however, the specific mechanisms remain to be investigated. If the condition of sepsis patients is not controlled in a timely manner, it will trigger the release of large amounts of inflammatory cytokines, forming an inflammatory cytokine storm that leads to immune dysfunction. This immune dysfunction further causes intestinal bacterial translocation, exacerbating the infection and creating a vicious cycle, ultimately leading to excessive inflammatory response and multiple organ dysfunction or even failure. Therefore, early use of antibiotics and controlling excessive inflammatory responses are two key issues in the treatment of sepsis.

[0003] Sepsis is often accompanied by multiple organ failure, and lung injury is one of the most common complications in the course of sepsis. It can occur in the early stages of sepsis, with an in-hospital mortality rate as high as 40-60%, consuming extremely high medical resources and seriously threatening human life and health. The lungs are one of the most common target organs for infection and involvement in sepsis, with 25% to 45% of sepsis patients developing acute lung injury (ALI). Its average mortality rate worldwide can reach 43%, seriously affecting patient prognosis. Septic lung injury accounts for 79% of the most common causes of all acute lung injuries, but the exact mechanism is unclear, and there is a lack of specific and effective prevention and treatment strategies, which has become a hot topic in current medical research. It is currently believed that multiple pathogenic factors are involved in the occurrence and development of sepsis-induced lung injury, such as pathogenic bacterial infection and the release of inflammatory factors, and the body's immune dysfunction and uncontrolled inflammatory response play important roles. In sepsis, a large number of inflammatory cells, such as macrophages and neutrophils, are recruited to sites of inflammatory damage. Overactivation of these cells leads to the release of numerous inflammatory factors and chemokines, further amplifying the inflammatory response and disrupting the alveolar-capillary endothelial barrier. This results in tissue edema, neutrophil infiltration, and activation of the coagulation system. Sepsis prevents the resolution of lung inflammation, leading to the development of atrial fibrillation (ALI) and causing irreversible lung damage. The mortality rate of sepsis-related ALI is higher than that of ALI caused by other factors.

[0004] Sepsis leads to the release of inflammatory cytokines, activation of immune cells, and damage to the pulmonary microvascular barrier and alveolar epithelial cells, resulting in the accumulation of protein-rich fluid in the alveoli and causing sepsis-related acute lung injury (ALI / ARDS). ALI / ARDS is characterized by pulmonary edema due to increased alveolar-capillary barrier permeability and subsequent impaired arterial oxygenation. Pulmonary edema, endothelial and epithelial damage are accompanied by neutrophil influx into the interstitium and bronchoalveolar space. Therefore, neutrophil activation and recruitment are considered to play a crucial role in the progression of ALI / ARDS. Neutrophils are among the first cells recruited to sites of inflammation. This promotes pulmonary microvascular dysfunction, pulmonary thrombotic inflammation, and pulmonary endothelial cell damage. Despite extensive research exploring the pathogenesis and treatment of septic lung injury, including extracorporeal membrane oxygenation (ECMO), protective lung ventilation, and drugs, effective treatments remain lacking. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a drug-loaded nanoparticle, Cor@PDA-PEG@CD11b, its preparation method, and its applications.

[0006] The first aspect of this invention provides a method for preparing drug-loaded nanoparticles Cor@PDA-PEG@CD11b, comprising the following steps:

[0007] S1. The cordycepin solution is mixed with the PDA aqueous solution, thereby forming the complex Cor@PDA through π-π stacking interaction between cordycepin and PDA;

[0008] S2. Modify the obtained Cor@PDA complex with PEG to obtain the Cor@PDA@PEG complex;

[0009] S3. Disperse the obtained Cor@PDA@PEG in buffer solution, add anti-CD11b antibody, react for 24 hours, centrifuge, wash, collect the precipitate, and dry to obtain Cor@PDA-PEG@CD11b.

[0010] In one embodiment of the present invention, in step S1, the concentration of the PDA aqueous solution is 2 mg mL⁻¹, the concentration of the cordycepin solution is 2 mg mL⁻¹, and the mixing volume ratio of the cordycepin solution to the PDA aqueous solution is 1:1.

[0011] In one embodiment of the present invention, in step S1, after mixing an equal volume of cordycepin solution and PDA aqueous solution, the mixture is stirred at room temperature for 24 hours, centrifuged, washed, and the precipitate is collected to obtain the complex Cor@PDA.

[0012] In one embodiment of the present invention, in step S2, the complex Cor@PDA is dispersed in double-distilled water, SH-PEG-streptavidin and NH4OH are added, the mixture is stirred at room temperature for 16-24 h, centrifuged, and the precipitate is collected to obtain the complex Cor@PDA@PEG.

[0013] In one embodiment of the present invention, in step S2, the mass ratio of the complex Cor@PDA, SH-PEG-streptavidin and NH4OH is 2:2:2.5.

[0014] In one embodiment of the present invention, in step S3, the buffer solution consists of the following: 1×PBS, 0.03% w / v bovine serum albumin (BSA) and 0.01% v / vt Tween-20.

[0015] In one embodiment of the present invention, in step S3, the mass ratio of Cor@PDA@PEG to anti-CD11b antibody is 5:1.

[0016] A second aspect of the present invention provides a drug-loaded nanoparticle, Cor@PDA-PEG@CD11b, which is prepared by the above-described preparation method.

[0017] A third aspect of the present invention provides the application of the above-mentioned drug-loaded nanoparticles Cor@PDA-PEG@CD11b in the preparation of drugs for treating lung injury.

[0018] In one embodiment of the present invention, the lung injury is acute lung injury due to sepsis.

[0019] In one embodiment of the present invention, the drug is an intravenous preparation.

[0020] Compared with the prior art, the embodiments of the present invention have the following beneficial effects:

[0021] 1. The method for preparing drug-loaded nanoparticles Cor@PDA-PEG@CD11b provided by this invention offers a new approach and direction for the treatment of sepsis-related lung injury. By utilizing the characteristics of PDA nanoparticles, controlled release of cordycepin is achieved, while avoiding the problem of rapid oxidation and inactivation of cordycepin after entering the body, thus improving its bioavailability. Utilizing specific antibodies on the surface of immune cells and their physiological characteristics, the drugs are rapidly recruited to the site of inflammatory damage in the early stage of acute inflammation, achieving rapid and precise drug targeting and alleviating sepsis-related lung injury.

[0022] 2. The drug-loaded nanoparticles Cor@PDA-PEG@CD11b provided by this invention can be used to prepare intravenous preparations for treating lung injury. They can improve the biosafety and pharmacological activity of cordycepin, and can reach the site of lung inflammation more quickly and conveniently via intravenous injection, reducing the production of pro-inflammatory factors and inflammatory cell infiltration. At the same time, they can prolong the drug retention time and adapt to the complex in vivo environment. The application of liposome nanomaterials and cordycepin significantly enhances its anti-inflammatory effect and better repairs lung damage in patients with sepsis.

[0023] 3. The drug-loaded nanoparticles Cor@PDA-PEG@CD11b provided by this invention, compared with the existing technology for utilizing cordycepin, avoid the liver and kidney toxicity caused by ADA inhibitor pentostatin, and ensure its biological safety while improving the pharmacological activity and bioavailability of cordycepin. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. In the drawings:

[0025] Figure 1 The drug loading efficiency of Cor in PDAs with different mass ratios (n=3) is expressed as mean ± standard deviation (SD);

[0026] Figure 2a This is a schematic diagram of the CPPC preparation process;

[0027] Figure 2b A representative TEM image of nanoparticles, scale bar: 500 nm;

[0028] Figure 2c For PDA, CP, and CPPC DLS sizes;

[0029] Figure 2d The zeta potential of the nanoparticles (n=3);

[0030] Figure 2e The image shows the SDS-PAGE analysis results of anti-CD11b antibody protein expression in CPCC.

[0031] Figure 2f The UV-Vis spectra of Cor, PDA, CP, and CPPC are shown.

[0032] Figure 2g FTIR spectra of COR, PDA, and CPPC;

[0033] Figure 2h Representative TEM images of CPPC in PBS buffer at pH 7.4, pH 6.5, and pH 5.5;

[0034] Figure 2i The graph shows the cumulative Cor release (%) of CPPC at pH 5.5, pH 6.5 and pH 7.4 (n=3);

[0035] Figure 2j The graph shows the total antioxidant capacity of PDA and CPPC.

[0036] Figure 2k The graph shows the scavenging capacity of different concentrations of CPPC against hydroxyl radicals.

[0037] Figure 2l The results of DPPH· scavenging ability of VC, PDA and CPC are shown in the figure;

[0038] Figure 2m The graph shows the test results of CPPC's anti-ADA enzyme activity.

[0039] Figure 3a For different concentrations of Cor (6.25 μg mL) -1 12.5 μg mL -1 25μg mL -1 50 μg mL -1 The standard curve of )

[0040] Figure 3b Representative curves showing the reaction products of different concentrations of cordycepin with 0.1 U mL-1 ADA;

[0041] Figure 3c For the merger Figure 3a and Figure 3b The curve image in the image;

[0042] Figure 4a Plot (a) showing the particle size of CPPC over 6 weeks in PBS or PBS containing 10% FBS.

[0043] Figure 4b The Zeta potentials (b) of CPC in PBS or PBS containing 10% FBS medium over 6 weeks;

[0044] Figure 5a A schematic diagram of the experimental procedure for a mouse model of LPS sepsis-induced acute lung injury (created using BioRender.com);

[0045] Figure 5b The wet / dry weight (W / D) ratio of the lungs after different treatments (n=5);

[0046] Figure 5c This is a graph showing the total cell count results in BALF;

[0047] Figure 5d The graph shows the counting results of protein (D) in BALF (n=5).

[0048] Figure 5e The levels of IL-6 and TNF-α cytokines in BALF;

[0049] Figure 5f The levels of IL-6 and TNF-α cytokines in lung tissue;

[0050] Figure 5g This refers to the levels of IL-6 and TNF-α cytokines in serum;

[0051] Figure 5h Representative H&E images of the lungs after different treatments;

[0052] Figure 5i for Figure 5h Corresponding lung injury scoring analysis;

[0053] Figure 5j MPO immunohistochemical staining images of lung tissue after different treatments;

[0054] Figure 5k for Figure 5j Corresponding MPO immunohistochemical area quantification analysis;

[0055] Figure 5l Representative images of IL-6 immunofluorescence staining in lung tissue after different treatments;

[0056] Figure 5m Representative images of IL-6 immunofluorescence staining in lung tissue after different treatments. Detailed Implementation

[0057] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0058] Example

[0059] This embodiment provides a method for preparing drug-loaded nanoparticles Cor@PDA-PEG@CD11b, characterized by the following steps:

[0060] S1, The concentration is 2 mg / mL -1 Cordycepin solution with a concentration of 2 mg / mL-1 Equal volumes of PDA aqueous solution were mixed and stirred at room temperature for 24 hours to allow cordycepin to form a complex with PDA through π-π stacking. The mixture was then centrifuged at 13,000 rpm for 15 minutes to remove any unbound cordycepin and washed three times with double-distilled water to obtain the complex Cor@PDA.

[0061] S2. The obtained complex Cor@PDA was dispersed in 10 mL of double-distilled water, 2 mg of SH-PEG-streptavidin and 2.5 μL of NH4OH were added, and the mixture was stirred at room temperature for 16-24 h to perform PEG modification. The precipitate was collected by centrifugation at 13000 rpm for 15 minutes to obtain the complex Cor@PDA@PEG.

[0062] S3. The obtained complex Cor@PDA@PEG was washed three times with double-distilled water, dispersed in buffer, and anti-CD11b antibody was added. After reacting for 24 hours, the mixture was centrifuged at 13000 rpm for 15 minutes, washed three times with PBS buffer, and the precipitate was collected and dried to obtain drug-loaded nanoparticles Cor@PDA-PEG@CD11b.

[0063] Specifically, the PDA (polydopamine) aqueous solution in step S1 of this embodiment is prepared through the following steps:

[0064] A. Take 40 mL of anhydrous ethanol using a graduated cylinder, mix it with 90 mL of ultrapure water, and add it to a flask. Add a sterilized magnetic stir bar to the flask, fix the flask on a shelf, and immerse the lower spherical part below the liquid surface of the constant temperature magnetic stirring water bath. Set the temperature to a constant 37°C, adjust the magnetic field strength so that the magnetic stir bar in the flask can stir the mixed liquid at an appropriate speed, and stir in a 37°C water bath for 0.5 h.

[0065] B. Weigh 0.5g of dried dopamine hydrochloride powder using an electronic balance and place it in a flask; add 10mL of ultrapure water to dissolve it completely, then add it to the above mixed solution and maintain a water bath at 37℃;

[0066] C. Use a pH meter to measure the pH value of the mixture of dopamine hydrochloride and ethanol solution in the flask. According to the reading, add an appropriate amount of concentrated ammonia to adjust the pH of the system to 8.5±0.1. After pH adjustment, the liquid in the flask is observed to be clear and light brown. Maintain a water bath at 37°C and stir for 24 hours.

[0067] D. After 24 hours, the liquid in the flask was observed to be a uniform, clear, dark brown color with no obvious precipitate. Stirring was stopped and the magnetic stir bar removed. All the liquid was transferred to several 50 mL centrifuge tubes and centrifuged at 5000 g for 15 minutes at 4°C. This process was repeated 3-5 times. After each centrifugation, the liquid in the centrifuge tubes was observed to separate into two layers: a dark brown precipitate at the bottom and a light brown, translucent liquid at the top. The upper layer of liquid was removed using a 1000 μL pipette, and the liquid level was replenished with ultrapure water to balance the liquid level. The process was repeated until the upper layer of liquid was clear, colorless, and transparent.

[0068] E. After the final centrifugation, remove the supernatant using the method described above, add an appropriate amount of ultrapure water to resuspend the dark brown precipitate at the bottom, and obtain a homogeneous PDA aqueous solution. Adjust the solubility of the obtained PDA aqueous solution by 2 mg / mL. -1 .

[0069] In step S1, the cordycepin solution is prepared by mixing cordycepin powder and ultrapure water. The cordycepin powder is purchased from Sigma-Aldrich, catalog number C3394.

[0070] In this embodiment, PDA is generated through the self-polymerization of dopamine salt in an alkaline solution, using classic methods. Method 30 synthesized Cor. Cor was loaded into PDA nanoparticles via π-π stacking interactions to form (CP). The drug loading efficiency of the Cor@PDA complex obtained in step S2 was determined by UV-Vis spectroscopy, and was approximately 15.03%. Figure 1 ).

[0071] To improve the stability of the Cor@PDA complex, in this embodiment, the Cor@PDA complex is functionalized with thiol-terminated polyethylene glycol streptavidin (SH-PEG-streptavidin).

[0072] In this invention, SH-PEG-streptavidin can be selected from SH-PEG-streptavidin with product number Q-0276647 from Xi'an Qiyue Company, or it can be SH-PEG-streptavidin with product number Q-0276647 from Xi'an Qiyue Company; in this embodiment, SH-PEG-streptavidin is SH-PEG-streptavidin with product number Q-0276647 from Xi'an Qiyue Company.

[0073] In this embodiment, a drug nanodelivery platform Cor@PDA-PEG@CD11b (CPPC) was assembled through biotin-avidin interaction, and the particles were encapsulated with an anti-CD11b antibody to specifically target neutrophils. Figure 2a ).

[0074] Figure 2b Transmission electron microscope images of PDA, CP, and CPPC, by Figure 2b It can be seen that DA, CP, and CPCC have a spherical structure and are uniform in size.

[0075] The particle sizes of PDA, CP, and CPPC, determined by dynamic light scattering, are as follows: Figure 2c As shown, the results indicate that the hydrodynamic diameter of PDA is 276.60 ± 1.91 nm, while the diameter of CPPC increases to 347.30 ± 5.22 nm. The increase in nanoparticle size may be related to the modification with PEG and antibody proteins.

[0076] The Zeta potential was measured using the Zeta potential method, and the results are as follows: Figure 2d As shown, the surface potential of nanoparticles was tested and analyzed using a Bumalvin particle size analyzer, and the PDA was -11.94±0.29mV, which is consistent with the study by dynamic light scattering method. The CPPC was -25.07±1.82mV.

[0077] Protein coating was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The specific steps were as follows: Materials and antibodies were added to protein loading buffer, boiled for 5 min, and electrophoresed on a 7.5% sodium dodecyl sulfate-polyacrylamide gel (SDS-PEGA). The gel was then transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with blocking buffer, primary antibody was added and incubated overnight at 4°C, followed by incubation with secondary antibody at room temperature for 1 h. Finally, specific bands were observed using a femtosecond photochemiluminescence assay kit; the results showed that the antibody protein was clearly expressed on the CPPC. Figure 2e This confirms the successful modification of the antibody.

[0078] The synthesis of CPPC was further confirmed by UV-Vis spectroscopy. The UV absorption curves of different sample solutions were detected using a Shimadzu UV-Vis absorption spectrometer (Japan), and the results are as follows: Figure 2f As shown, the UV-Vis spectrum of Cor has a characteristic peak at 260 nm. The spectra of both CP and CPPC show this characteristic absorption peak at 260 nm, indicating the presence of Cor.

[0079] Furthermore, FTIR spectroscopy further confirmed the presence of Cor( ) in CPPC. Figure 2g (), showing Cor at 1662cm -1 The characteristic functional group C=C is present. This invention also investigated the pH-responsive behavior of CPPC and its ROS-mediated Cor release. TEM images showed that CPPC completely decomposed at pH 5.5, while retaining its intact form under physiological conditions (pH 7.4). Figure 2hThis indicates that PDA has high biodegradability in the acidic microenvironment of acute inflammation. For drug release studies, 1 mL of CPPC solution was placed in a dialysis bag (molecular cutoff: 1000 Da) and immersed in 50 mL of PBS at pH 5.5, 6.5, or 7.4, and maintained at 37°C. PBS samples (1 mL each) were collected at predetermined time intervals, and the cumulative release of Cor was quantified using UV-Vis absorption spectroscopy to generate drug release curves. UV-Vis spectroscopy further confirmed that CPPC released Cor faster and accumulated at pH 5.5 than under normal physiological conditions. Figure 2i ).

[0080] Because PDA possesses the property of scavenging reactive oxygen species, this invention uses 2,2'-azine-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radicals to evaluate the antioxidant capacity of CPPC. Total antioxidant capacity was determined using ABTS as a colorimetric reagent. Different concentrations (PDA dosage 25 μg / mL) were evaluated according to the manufacturer's instructions. -1 50 μg mL -1 100μgmL -1 200μgmL -1 500μg mL -1 The total antioxidant capacity of PDA and CPPC was compared. The results showed that the total antioxidant capacity increased with increasing PDA and CPPC concentrations, with no significant difference between the two. Figure 2j This indicates that antibody binding does not affect the antioxidant activity of CPPC. The scavenging activity of CPPC against ·OH was detected using methylene blue (MB) as a probe. A solution containing 5 μg / mL was prepared under light-protected conditions. -1 MB, 2mM H2O2, 1mM FeSO4 and different concentrations of CPPC (containing 0 μg / mL of PDA) -1 25μg mL -1 50 μg mL -1 100μgmL -1 200μg mL -1 The CPPC (concentrated hydroxyl radical) was dissolved in PBS buffer and allowed to stand for 20 min. After centrifugation to remove the nanoparticles, the absorbance peak of MB at 665 nm was recorded using UV-Vis spectroscopy. Methylene blue (MB) assays further confirmed that CPPC effectively scavenge hydroxyl radicals. Figure 2k The ability of CPPC to scavenge reactive nitrogen species (RNS) was detected using the 1,1-diphenyl-2-picrylhydrazine radical (DPPH·) method. A 0.1 mM DPPH working buffer (0.002 g DPPH dissolved in 50 mL ethanol) was prepared and stored protected from light. Samples were prepared from samples containing 100 μg / mL DPPH. -1The PDA consists of PDA and CPC solution, containing 0.5 mg / mL. -1 Ascorbic acid solution was used as a positive control. Sample group (S1) contained 100 μL of DPPH working buffer, and sample control group (S2) contained 100 μL of ethanol. Blank group (B1) contained 100 μL of PBS buffer mixed with an equal volume of ethanol; control group (C1) contained 100 μL of DPPH mixed with the same volume of PBS buffer. After incubation at room temperature in the dark for 0.5 h, the absorbance at 517 nm was measured using UV-Vis spectroscopy, and the average value was calculated to determine the DPPH scavenging rate. Scavenging rate (%) = ([(C1-B1)-(S1,S2)] / (C1-B1)) × 100%. The addition of CPC reduced the absorbance of DPPH at 517 nm, and its scavenging effect was comparable to that of the common antioxidant 1-ascorbic acid (AA). Figure 2l This indicates that CPPC has the ability to neutralize RNS generated during lung inflammation.

[0081] In this embodiment, to investigate the protective effect of CPPC against ADA-induced oxidative inactivation of cordycepin (Cor), the changes in Cor after the reaction of CPPC with ADA were observed using UV-Vis spectrophotometry. The specific steps are as follows: lyophilized adenosine deaminase (ADA) powder was dissolved in PBS buffer and mixed with free cordycepin solution and CPPC solution, to a concentration equivalent to 25 μg / mL. -1 Cordycepin. After the reaction, the CPPC and ADA mixture was centrifuged to separate and remove unreacted ADA. The resulting CPPC was washed three times with ddH2O. The reacted CPPC was exposed to PBS buffer at pH 5.5 to assess the release of cordycepin-loaded cordycepin. After centrifugation to remove the precipitate, the concentration of cordycepin in the supernatant was determined by UV-Vis spectroscopy. Figure 2m and Figure 3a , Figure 3b , Figure 3c As shown, 0.1 U of ADA effectively removed free Cor. However, no significant change was observed when the same concentration of ADA was applied to CPPC. UV-Vis spectroscopy showed that the maximum absorption peak of Cor released from CPPC was essentially consistent with that of free Cor, but slightly decreased due to minor losses during centrifugation. Stability testing was conducted by dissolving PDA and CPPC in PBS and PBS solutions containing 10% FBS for six weeks, with the particle size and Zeta potential of PDA and CPPC measured weekly using a Malvern particle size analyzer. The results confirmed (…). Figure 4a , Figure 4b CPPC nanoparticles remained stable for 6 weeks under physiological conditions.

[0082] In summary, the drug-loaded nanoparticles Cor@PDA-PEG@CD11b prepared in this embodiment are CPPC nanoparticles with anti-ADA activity, which can achieve controlled release of Cor in a slightly acidic environment, ensuring their stability and effectiveness.

[0083] Experimental Case: This experimental case investigated the in vivo therapeutic effect of CPPC on acute lung injury caused by sepsis.

[0084] Using an LPS-induced acute lung injury mouse model, BALB / c mice were intraperitoneally injected with LPS (10 mg / kg). -1 One hour later, mice were intravenously injected with either nanoparticles or a control solution. Twenty-four hours later, mice were intraperitoneally injected with sodium pentobarbital 30 mg / kg. -1 Anesthesia was administered, and blood was collected by enucleation of the eyeballs. After blood collection, the mice were fixed in a dorsal position on the operating table. The skin was incised to expose the abdominal cavity, and the trachea was separated. The entire lungs were lavaged with pre-cooled 0.3 mL of physiological saline, held for 1 minute, and then slowly aspirated with a syringe. This process was repeated three times to collect bronchoalveolar lavage fluid (BALF). A trimmed syringe was filled with physiological saline and inserted into the left ventricle near the apex, at an angle roughly parallel to the midline of the heart, to inject saline for ventricular perfusion. After perfusion, lung tissue was harvested for subsequent analysis. Figure 5a In mice treated with CPPC, the lung wet / dry weight ratio was determined by harvesting the right upper lobe of the lung, removing connective tissue, washing with physiological saline, blotting dry with filter paper, weighing the wet weight, drying continuously in a 70°C oven for at least 72 hours, weighing and recording the dry weight of the lung tissue, and calculating the lung W / D value. The lung wet / dry weight ratio (a key marker of pulmonary edema) was significantly reduced. Figure 5b BLAF was centrifuged at 1000 rpm for 3 min in a low-temperature centrifuge. The supernatant was aliquoted and stored at -80℃. The precipitated cells were resuspended in 250 μL of physiological saline, mixed, and counted using an automated cell counter. The results showed that ( Figure 5c CPPC treatment significantly reduced the total cell count in BALF. Protein concentration in BALF was determined using the BCA method: Reagents A and B from the BCA kit were thoroughly mixed at a ratio of A:B = 50:1 to prepare the BCA working solution; protein standards were added sequentially to 96-well plates. The sample to be tested was added to each well (20 μL); 200 μL of BCA working solution was added to each well, and the plate was incubated at 37°C for 30 min; absorbance was measured at 562 nm using a microplate reader, a protein standard curve was plotted, and protein concentration was calculated. The results showed that ( Figure 5d CPC treatment resulted in a significant decrease in protein levels in BALF.

[0085] Mouse BLAF, lung tissue homogenate supernatant, and serum samples were collected and processed according to the ELISA kit instructions from Dakota Biotechnology Co., Ltd. The levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in mouse lung tissue homogenate and BLAF were detected. Specific operating steps: Before use, equilibrate the kit at room temperature for 30 min; Blank wells: Do not add samples, only add chromogenic reagents A and B and stop solution for zeroing; Standard wells: Add 100 μL of diluted standard to each well, followed by 50 μL of biotin antigen working solution; Zero wells: Add 100 μL of standard / sample dilution buffer. Then add 50 μL of biotin antigen working solution; Sample wells: Add 100 μL of sample, then add 50 μL of biotin antigen working solution; Gently shake, cover with sealing film, and incubate at 37°C for 90 min; Dilute the 50-fold concentrated washing buffer 50 times with distilled water for later use; First wash: Carefully remove the sealing film, discard the liquid, shake dry, fill each well with washing buffer, let stand for 60 seconds and then discard, repeat this 4 times, pat dry; Add 100 μL TMB to the zero well and standard well. Add the sample and product to the wells, gently shake, cover with sealing film, and incubate at 37°C for 30 min; Second wash: Carefully remove the sealing film, discard the liquid, shake dry, fill each well with washing buffer, let stand for 60 seconds and discard, repeat this 4 times, and pat dry; Color development: Add 100 μL of colorimetric reagent to each well, gently shake to mix, and develop at 37°C for 10 min; Termination: Add 100 μL of stop solution to each well to terminate the reaction; Measurement: Zero the instrument with the blank well and measure the absorbance of each well at 450 nm. The results showed that CPPC treatment significantly reduced the concentrations of pro-inflammatory factors IL-6 and TNF-α in BALF, lung tissue homogenate, and serum samples, showing superior efficacy compared to the PBS treatment group. Figure 5e , Figure 5f , Figure 5g These cytokines are known to be key contributors to the high mortality rates associated with cytokine storms and sepsis-induced acute lung injury. Overall, these findings highlight the potential of CPPCs to inhibit cytokine storms and mitigate lung injury in vivo.

[0086] Lung tissue damage was observed using hematoxylin and eosin (H&E) staining. Twenty-four hours after model establishment, 30 mg / kg of sodium pentobarbital was injected intraperitoneally. -1Mice were anesthetized, and tissue from the right middle lobe of the lung was harvested and placed in pre-cooled physiological saline to remove residual blood. The tissue was then transferred to 10% formalin for fixation for 24 hours. After rinsing with running water, the tissue was dehydrated using a gradient of ethanol (concentrations of 30%, 50%, 80%, 95%, 95%, 100%, and 100%), cleared twice with xylene, and then embedded in paraffin to prepare paraffin sections with a thickness of 5 μm. These sections were then subjected to a series of treatments, including xylene dewaxing, descending-gradient ethanol hydration, hematoxylin and eosin staining, ascending-gradient ethanol dehydration, xylene clearing, and neutral resin mounting, to prepare pathological sections. Morphological changes in the lung tissue were observed using an optical microscope (200×). The results showed that LPS-treated mouse lung tissue exhibited significant structural damage, including varying degrees of interstitial edema, hemorrhage, necrosis, alveolar inflammatory cell infiltration, and alveolar wall thickening. In contrast, the lung tissue structure in the CPPC treatment group was clearer, with only mild edema and thickening of the alveolar walls, reduced immune cell infiltration, and significantly less lung damage compared to other groups. Figure 5h To provide a more objective assessment of lung injury severity, a standardized lung injury scoring system was used to quantify the degree of injury. The scoring criteria included pulmonary hemorrhage, edema, inflammatory cell infiltration, alveolar rupture, or hyaline membrane formation. Three fields of view were randomly selected from each slide for assessment, with scores ranging from 0 to 5 to represent different degrees of injury: 0 for no injury, 1 for mild injury, 2 for moderate injury, 3 for moderate to severe injury, 4 for severe injury, and 5 for extremely severe injury. Consistent with histological results, the CPC treatment group had the lowest lung injury score, at 1.2 ± 0.45. Figure 5i ).

[0087] The infiltration of leukocytes in lung tissue was observed by myeloperoxidase (MPO) immunohistochemical staining. Immunohistochemical staining was performed on paraffin sections following these steps: ① Antigen retrieval: Citrate antigen retrieval buffer (pH 6.0) was poured into a retrieval chamber and heated to boiling in a microwave oven. The sections were then placed in the retrieval chamber for antigen retrieval on high for 10 minutes. During this process, excessive evaporation of the buffer should be prevented, and the sections should not be allowed to dry out. ① After natural cooling, transfer the slide to PBS solution and wash on a destaining shaker for 5 minutes, repeating 3 times; ② Block endogenous peroxidase: transfer the slide to 3% hydrogen peroxide solution, incubate at room temperature in the dark for 25 minutes, place in PBS solution and wash on a destaining shaker for 5 minutes, repeating 3 times; ③ BSA blocking: gently shake the slide dry, draw a circle around the tissue with a histochemical pen, drop 3% BSA solution to cover the tissue in the circle, and block at room temperature for 30 minutes; ④ Primary antibody incubation: gently shake off the blocking solution, drop the primary antibody on the slide, add a small amount of water to the humidified chamber, place the slide horizontally in the humidified chamber, and incubate overnight at 4°C; ⑤ Secondary antibody incubation: transfer the slide to PBS solution and wash on a destaining shaker for 5 minutes, repeating 3 times. After gently shaking the slide dry, drop the secondary antibody of the corresponding species to the primary antibody into the circle and evenly cover the tissue, incubate at room temperature for 50 minutes; ⑥ DAB staining: place the slide in PBS solution and wash on a destaining shaker for 5 minutes, repeating 3 times. Gently shake the slide dry, drop freshly prepared AB staining solution into the circle, and observe the staining time under a microscope. A positive result is brownish-yellow. Rinse the slide with tap water to stop the staining process. ⑦ Counterstain cell nuclei: Counterstain with hematoxylin for about 3 minutes, rinse with tap water, differentiate with 1% hydrochloric acid alcohol for 3 seconds, rinse with tap water, return to blue with ammonia, and rinse with running water. ⑧ Dehydration and mounting: Transfer the slides sequentially to 75% ethanol for 6 minutes, 85% ethanol for 6 minutes, anhydrous ethanol for 16 minutes, anhydrous ethanol II for 6 minutes, and xylene for 15 minutes to dehydrate and clear, then air dry and mount with neutral resin. Observe the staining depth and area under a microscope and take pictures. MPO immunohistochemical staining images ( Figure 5j CPPC treatment significantly reduced MPO expression in the lungs. Quantitative analysis of immunohistochemical images was performed using ImageJ software. Figure 5k This indicates a significant reduction in neutrophil infiltration. This may be related to CPPC alleviating neutrophil inflammation and releasing Cor in lung tissue.

[0088] Select high-quality, uniformly thick tissue sections, dissolve them in paraffin, dewax them, and then quickly immerse the baked sections in xylene twice, 5 min each time. Next, perform gradient hydration: place them in 100%, 90%, 80%, and 70% ethanol for 5 min each, and then in distilled water for 5 min. For endogenous peroxidase blockade: add 3% hydrogen peroxide, incubate at room temperature in a humidified chamber for 10 min, and then in distilled water for 1 min. For antigen retrieval: place them in 1x... In CB, microwave at 80% power for 4 min, then at 40% power for 8 min, and cool to room temperature; permeabilize with 0.3% Triton at room temperature for 15 min; blocking: aspirate the fluid around the tissue, add 10% NGS, and place in a humidified chamber at room temperature for 2 hours; primary antibody: dilute the antibody according to the instructions; aspirate the fluid around the tissue, add the diluted primary antibody, and incubate overnight at 4°C; wash three times with 1xPBS, 5 min each time; secondary antibody: aspirate the fluid around the tissue, add the diluted fluorescent secondary antibody, incubate at room temperature in the dark for 1 h, and wash three times with PBS solution, 5 min each time; nucleus staining: add Hoechst fluorescent dye to label the cell nuclei, incubate at room temperature for 15 min, and wash twice with PBS solution, 5 min each time; mounting: add one drop of anti-fluorescence quenching mounting medium to the tissue, cover with a coverslip, and observe and photograph under a microscope. Immunofluorescence staining ( Figure 5l , Figure 5m The results showed that CPPC significantly reduced the levels of IL-6 and TNF-α pro-inflammatory factors in lung tissue.

[0089] In summary, the drug-loaded nanoparticles Cor@PDA-PEG@CD11b (CPPC) provided in this invention effectively inhibit cytokine storms, reduce lung tissue damage, and decrease inflammatory cell infiltration, thereby alleviating lung damage caused by sepsis.

[0090] Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. Unless otherwise specified, the embodiments and features described in this application can be arbitrarily combined with each other.

Claims

1. A method for preparing drug-loaded nanoparticles Cor@PDA-PEG@CD11b, characterized in that, Includes the following steps: S1. The cordycepin solution is mixed with the PDA aqueous solution, thereby forming the complex Cor@PDA through π-π stacking interaction between cordycepin and PDA; S2. Modify the obtained Cor@PDA complex with PEG to obtain the Cor@PDA@PEG complex; S3. Disperse the obtained Cor@PDA@PEG in buffer solution, add anti-CD11b antibody, react for 24 hours, centrifuge, wash, collect the precipitate, dry and obtain Cor@PDA-PEG@CD11b; In step S2, the complex Cor@PDA is dispersed in double-distilled water, SH-PEG-streptavidin and NH4OH are added, the mixture is stirred at room temperature for 16-24 h, centrifuged, and the precipitate is collected to obtain the complex Cor@PDA@PEG.

2. The method for preparing drug-loaded nanoparticles Cor@PDA-PEG@CD11b according to claim 1, characterized in that, The concentration of PDA aqueous solution in step S1 was 2 mg / mL -1 The concentration of cordycepin solution was 2 mg / mL -1 The volume ratio of cordycepin solution to PDA aqueous solution was 1:

1.

3. The method for preparing drug-loaded nanoparticles Cor@PDA-PEG@CD11b according to claim 2, characterized in that, In step S1, an equal volume of cordycepin solution was mixed with PDA aqueous solution, and the mixture was stirred at room temperature for 24 hours. After centrifugation, washing, and collection of the precipitate, the complex Cor@PDA was obtained.

4. The method for preparing drug-loaded nanoparticles Cor@PDA-PEG@CD11b according to claim 1, characterized in that, In step S2, the mass ratio of the complex Cor@PDA, SH-PEG-streptavidin, and NH4OH is 2:2:2.

5.

5. The method of claim 1, wherein the drug-loaded nanoparticles Cor@PDA-PEG@CD11b are prepared by, In step S3, the buffer composition is as follows: 1×PBS, 0.03% w / v bovine serum albumin (BSA) and 0.01% v / v Tween-20.

6. The method of claim 1, wherein the drug-loaded nanoparticles Cor@PDA-PEG@CD11b are prepared by, In step S3, the mass ratio of Cor@PDA@PEG to anti-CD11b antibody is 5:

1.

7. Drug-loaded nanoparticles Cor@PDA-PEG@CD11b, characterized in that, It is prepared by the preparation method according to any one of claims 1-6.

8. The use of the drug-loaded nanoparticles Cor@PDA-PEG@CD11b according to claim 7 in the preparation of drugs for treating lung injury.

9. Use according to claim 8, characterized in that, The lung injury described is acute lung injury due to sepsis.

10. Use according to claim 8, characterized in that, The drug is an intravenous preparation.

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