Drug-loaded nano particle Corr (at) PDA-PEG (at) CD11b as well as preparation method and application thereof

Abstract

The invention provides a drug-loaded nanoparticle Corr (at) PDA-PEG (at) CD11b and a preparation method and application thereof.The preparation method comprises the following steps that S1, a cordycepin solution and a PDA aqueous solution are mixed, so that cordycepin and PDA form a compound Corr (at) PDA through the pi-pi stacking effect; s2, carrying out PEG (Polyethylene Glycol) modification on the obtained compound Cor(at) PDA to prepare a compound Cor(at) PDA (at) PEG; and S3, dispersing the obtained Corr (at) PDA (at) PEG in a buffer solution, adding an anti-CD11b antibody, reacting for 24 hours, centrifuging, washing, collecting precipitate, and drying to obtain Corr (at) PDA-PEG (at) CD11b. The prepared drug-loaded nanoparticles Corr (at) PDA-PEG (at) CD11b provide a new thought and a new direction for treatment of sepsis-related lung injury, controlled release of cordycepin is realized through the characteristics of the PDA nanoparticles, the problem that cordycepin enters a body and is quickly oxidized and inactivated is avoided, the bioavailability of the cordycepin is improved, and the drug-loaded nanoparticles are suitable for clinical application. By means of immune cell surface specific antibodies and physiological characteristics, rapid chemotaxis recruitment is carried out at the early stage of acute inflammation, rapid and accurate drug targeting is achieved, and sepsis lung injury is relieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a drug-loaded nanoparticle Cor@PDA-PEG@CD11b and a preparation method and application thereof. Background Art

[0002] Sepsis is one of the common clinical critical illnesses. The third international consensus definition of sepsis and septic shock (Sepsis-3) released in 2016 updated sepsis as a life-threatening organ dysfunction caused by a dysregulated host response to infection. There are approximately 19 million sepsis patients worldwide each year. Global statistical analysis studies from 1990 to 2017 revealed that sepsis is the most common cause of hospital death, and its morbidity and mortality remain high, posing a major threat to global health. Current studies believe that the mechanism of tissue damage caused by sepsis is related to uncontrolled inflammatory response and oxidative stress, but its specific mechanism remains to be studied. If the condition of sepsis patients is not controlled in time, a large number of inflammatory cytokines will be released, forming an inflammatory cytokine storm and leading to immune dysfunction. Immune dysfunction further causes intestinal bacterial translocation, further exacerbating the infection and forming a vicious cycle, ultimately leading to excessive inflammatory response and multiple organ dysfunction and even failure. Therefore, early use of antibiotics and control of excessive inflammatory response are two key issues in the treatment of sepsis.

[0003] Sepsis is usually accompanied by multiple organ failure. Lung injury is one of the most common complications in the course of sepsis and can occur in the early stages of sepsis. Its in-hospital mortality rate is as high as 40-60%, which consumes medical resources extremely and seriously threatens human life and health. The lung is one of the most common infected and affected target organs of sepsis. 25% to 45% of sepsis patients suffer from acute lung injury (ALI), and its average mortality rate worldwide can reach 43%, which seriously affects the patient's prognosis. Sepsis 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. This has become a hot topic in medical research today. It is currently believed that a variety of pathogenic factors are involved in the occurrence and development of sepsis lung injury, such as pathogen infection and the release of inflammatory factors. The body's immune dysfunction and uncontrolled inflammatory response play an important role. In sepsis, a large number of inflammatory cells, such as macrophages and neutrophils, are recruited to the site of inflammatory damage. After overactivation, they release a large number of inflammatory factors and chemokines, further amplifying the inflammatory response, causing damage to the alveolar-capillary endothelial barrier function, tissue edema, neutrophil infiltration, and activation of the coagulation system. Sepsis causes lung inflammation that cannot subside and leads to the development of ALI, causing irreversible damage to the lungs. The mortality rate of sepsis-related ALI is higher than that of ALI caused by other reasons.

[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, leading to sepsis-related acute lung injury. ALI / ARDS is characterized by pulmonary edema due to increased permeability of the alveolar capillary barrier and subsequent impaired arterial oxygenation. Pulmonary edema, endothelial and epithelial damage are accompanied by an influx of neutrophils into the interstitium and bronchoalveolar spaces. Therefore, the activation and recruitment of neutrophils are thought to play a key role in the progression of ALI / ARDS. Neutrophils are the first cells recruited to the site of inflammation. This promotes pulmonary microvascular dysfunction, pulmonary thrombotic inflammation, and pulmonary endothelial cell damage. Despite a large number of research efforts to explore the pathogenesis and treatment of septic lung injury, including the use of extracorporeal membrane oxygenation, protective lung ventilation procedures, and drugs, effective treatments are still lacking. Summary of the invention

[0005] In view of the defects in the prior art, the purpose of the present invention is to provide a drug-loaded nanoparticle Cor@PDA-PEG@CD11b and a preparation method and application thereof.

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

[0007] S1, mixing the cordycepin solution with the PDA aqueous solution, so that the cordycepin and PDA form a complex Cor@PDA through π-π stacking;

[0008] S2, performing PEG modification on the obtained complex Cor@PDA to obtain a complex Cor@PDA@PEG;

[0009] S3. Disperse the obtained Cor@PDA@PEG in buffer, 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, equal volumes of cordycepin solution and PDA aqueous solution are mixed, 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 reaction is stirred at room temperature for 16-24 hours, 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 composition is as follows: 1×PBS, 0.03% w / v bovine serum albumin (BSA) and 0.01% v / v 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] The second aspect of the present invention provides a drug-loaded nanoparticle Cor@PDA-PEG@CD11b, which is prepared by the above-mentioned preparation method.

[0017] The third aspect of the present invention provides the use 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 sepsis-induced acute lung injury.

[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 preparation method of the drug-loaded nanoparticles Cor@PDA-PEG@CD11b provided by the present invention and the prepared drug-loaded nanoparticles Cor@PDA-PEG@CD11b provide new ideas and directions for the treatment of sepsis-related lung injury. The controlled release of cordycepin is achieved through the characteristics of PDA nanoparticles, while also avoiding the problem of rapid oxidation and inactivation of cordycepin after entering the body, thereby improving its bioavailability. By utilizing the specific antibodies and physiological characteristics on the surface of immune cells, the cordycepin is rapidly chemotactically recruited to the site of inflammatory damage in the early stage of acute inflammation, thereby achieving rapid and precise targeting of drugs and alleviating sepsis-related lung injury.

[0022] 2. The drug-loaded nanoparticles Cor@PDA-PEG@CD11b provided by the present invention can be used to prepare intravenous preparations for the treatment of lung injury, which can improve the biosafety and pharmacological activity of cordycepin. Intravenous injection can reach the site of lung inflammation more quickly and conveniently, reduce the production of proinflammatory factors and inflammatory cell infiltration; at the same time, it can prolong the retention time of the drug and adapt to the complex in vivo environment. The application of liposome nanomaterials and cordycepin can significantly enhance its anti-inflammatory effect and better repair the lung damage of patients with sepsis.

[0023] 3. Compared with the prior art use of cordycepin drugs, the drug-loaded nanoparticles Cor@PDA-PEG@CD11b provided by the present invention avoid the liver and kidney toxicity caused by ADA inhibitor pentostatin, while improving the pharmacological activity and bioavailability of cordycepin and ensuring its biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. In the drawings:

[0025] Figure 1 is the drug loading efficiency of Cor in PDA with different mass ratios (n=3), and the values ​​are expressed as mean ± standard deviation (SD);

[0026] Figure 2a Schematic diagram of the CPPC preparation process;

[0027] Figure 2b Representative TEM images of nanoparticles, scale bar: 500 nm;

[0028] Figure 2c is the DLS size of PDA, CP, and CPPC;

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

[0030] Figure 2e This is the SDS-PAGE analysis result of the anti-CD11b antibody protein expression in CPPC;

[0031] Figure 2f UV-visible spectra of Cor, PDA, CP and CPPC;

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

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

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

[0035] Figure 2j The total antioxidant capacity results of PDA and CPPC are shown in the figure;

[0036] Figure 2k This is the result diagram of the scavenging ability of CPPC at different concentrations on hydroxyl free radicals;

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

[0038] Figure 2m This is the test result diagram 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 , 25 μg mL -1 , 50 μg mL -1 ) standard curve;

[0040] Figure 3b It is the representative curve of the reaction product of different concentrations of cordycepin and 0.1U mL-1ADA;

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

[0042] Figure 4a (a) The particle size of CPPC in PBS or PBS containing 10% FBS medium within 6 weeks;

[0043] Figure 4b (b) Zeta potential of CPPC in PBS or PBS containing 10% FBS medium for 6 weeks;

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

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

[0046] Figure 5c This is the result of counting the total number of cells in BALF;

[0047] Figure 5d The figure shows the results of protein (D) count in BALF (n=5);

[0048] Figure 5e is the level of IL-6 and TNF-α cytokines in BALF;

[0049] Figure 5f is the level of IL-6 and TNF-α cytokines in lung tissue;

[0050] Figure 5g is the level 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 score analysis;

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

[0054] Figure 5k for Figure 5j Quantitative analysis of the corresponding MPO immunohistochemical area;

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

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

[0057] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several changes and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0058] Example

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

[0060] S1, the concentration is 2mg mL -1 Cordycepin solution with a concentration of 2 mg mL-1 The PDA aqueous solution was mixed with equal volumes of the solution 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, and 2 mg of SH-PEG-streptavidin and 2.5 μL of NH 4 OH, stirred at room temperature for 16-24 h, PEG-modified, centrifuged at 13000 rpm for 15 min, and the precipitate was collected to obtain the complex Cor@PDA@PEG;

[0062] S3. Wash the obtained complex Cor@PDA@PEG with double distilled water for three times, disperse it in buffer, add anti-CD11b antibody, react for 24 hours, centrifuge at 13000 rpm for 15 minutes, wash it with PBS buffer for three times, collect the precipitate, and dry it to obtain drug-loaded nanoparticles Cor@PDA-PEG@CD11b.

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

[0064] A. Take 40mL of anhydrous ethanol with a measuring cylinder, mix it with 90mL of ultrapure water and add it to the flask. Add a sterilized magnetic stirring bar to the flask, fix the flask on the rack, and immerse the lower spherical part below the liquid level of the constant temperature magnetic stirring water bath. Set the temperature to a constant 37℃, adjust the magnetic field strength so that the magnetic stirring bar in the flask stirs the mixed liquid at an appropriate speed, and place it in a 37℃ water bath for 0.5h;

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

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

[0067] D. After 24 hours, the liquid in the flask is a uniform, clear dark brown color with no obvious precipitation. Stop stirring and remove the magnetic stirrer, transfer all the liquid to several 50mL centrifuge tubes, centrifuge at 4℃5000g for 15 minutes, repeat 3-5 times; after each centrifugation, the liquid in the centrifuge tube can be seen to be stratified, with the lower layer being a dark brown precipitate and the upper layer being a light brown translucent liquid; use a 1000μL pipette to suck out the upper layer of liquid, and add ultrapure water to level the liquid surface, and continue the next centrifugation until the upper layer of liquid is clear, colorless and transparent;

[0068] E. After the last centrifugation, remove the upper layer of liquid using the above method, add an appropriate amount of ultrapure water to resuspend the dark brown precipitate at the bottom to obtain a uniform PDA aqueous solution. Adjust the solubility of the obtained PDA aqueous solution to 2 mg mL -1 .

[0069] In step S1, the cordycepin solution is prepared by preparing cordycepin powder and ultrapure water. The cordycepin powder is purchased from Sigma-Aldrich with a product number of C3394.

[0070] In this embodiment, PDA is self-polymerized by dopamine salt in alkaline solution using the classic Cor was loaded into PDA nanoparticles through π-π stacking to form (CP). The composite Cor@PDA obtained in step S2 was measured by UV-visible spectroscopy, and the drug loading efficiency was about 15.03% ( Figure 1 ).

[0071] In order to improve the stability of the complex Cor@PDA, in this example, the complex Cor@PDA was functionalized with thiol-terminated polyethylene glycol streptavidin (SH-PEG-streptavidin).

[0072] Among them, the SH-PEG-streptavidin in the present invention can be selected from SH-PEG-streptavidin with the item number Q-0276647 of Xi'an Qi Yue Company, or it can be SH-PEG-streptavidin with the item number Q-0276647 of Xi'an Qi Yue Company; the SH-PEG-streptavidin in this embodiment is SH-PEG-streptavidin with the item number Q-0276647 of Xi'an Qi Yue Company.

[0073] In this example, a drug nano-delivery platform Cor@PDA-PEG@CD11b (CPPC) was assembled through biotin-avidin interaction and the particles were coated with anti-CD11b antibodies to specifically target neutrophils ( Figure 2a ).

[0074] Figure 2b Transmission electron micrographs of PDA, CP and CPPC. Figure 2b It can be seen that DA, CP and CPPC are spherical structures with uniform sizes.

[0075] The particle sizes of PDA, CP and CPPC were determined by dynamic light scattering. Figure 2c As shown in the figure, the hydrodynamic diameter of PDA was 276.60±1.91nm, and the diameter of CPPC increased to 347.30±5.22nm. The increase in the particle size of the nanoparticles may be related to the modification of PEG and antibody protein.

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

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

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

[0079] In addition, FTIR spectroscopy further confirmed the presence of Cor( Figure 2g ), showing that Cor at 1662cm -1 The characteristic functional group C=C at . The pH response behavior of CPPC and its ROS-mediated Cor release were also studied in the present invention. TEM images showed that CPPC was completely decomposed at pH 5.5, while it had a complete morphology under physiological conditions (pH 7.4) ( Figure 2h), indicating that PDA is highly biodegradable 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-visible absorption spectroscopy to generate drug release curves. UV-visible spectroscopy further confirmed that CPPC released Cor faster and in larger amounts at pH 5.5 than under normal physiological conditions ( Figure 2i ).

[0080] Since PDA has the property of scavenging reactive oxygen species, 2,2'-azine-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) free radical was used in the present invention to evaluate the antioxidant capacity of CPPC. The total antioxidant capacity was determined using ABTS as a colorimetric reagent. According to the manufacturer's instructions, different concentrations (PDA dose of 25 μg mL -1 , 50 μg mL -1 , 100μgmL -1 , 200μgmL -1 , 500 μg mL -1 )Total antioxidant capacity of PDA and CPPC. The results showed that the total antioxidant capacity increased with the increase of PDA and CPPC concentrations, and there was no significant difference between the two ( Figure 2j ), indicating that antibody binding does not affect the antioxidant activity of CPPC. Methylene blue (MB) was used as a probe to detect the scavenging activity of CPPC against ·OH. -1 MB, 2mM H2O2, 1mM FeSO4 and different concentrations of CPPC (containing PDA in an amount of 0 μg mL -1 , 25 μg mL -1 , 50 μg mL -1 , 100μgmL -1 , 200 μg mL -1 CPPC) 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 by UV-visible spectroscopy. Methylene blue (MB) test further confirmed that CPPC effectively scavenged hydroxyl free radicals ( Figure 2k ). The 1,1-diphenyl-2-picryl-hydrazine free radical (DPPH·) method was used to detect the ability of CPPC to scavenge reactive nitrogen (RNS). Prepare 0.1mM DPPH working buffer (0.002g DPPH dissolved in 50mL ethanol) and store in the dark. The sample concentration was 100μg mL -1PDA and CPPC solution composition, containing 0.5 mg mL -1 Ascorbic acid solution was used as a positive control. The sample group (S1) contained 100 μL DPPH working buffer, and the sample control group (S2) contained 100 μL ethanol. The blank group (B1) contained 100 μL PBS buffer mixed with an equal volume of ethanol; control group (C1): 100 μL 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 determined by UV-visible spectroscopy and the average value was calculated to determine the DPPH clearance rate. Clearance rate (%) = ([(C1-B1)-(S1,S2)] / (C1-B1))×100%. The addition of CPPC reduced the absorbance of DPPH at 517 nm, and its scavenging effect was comparable to that of the common antioxidant l-ascorbic acid (AA) ( Figure 2l ). This suggests that CPPC has the ability to neutralize RNS produced during lung inflammation.

[0081] In this example, in order to explore the protective effect of CPPC on the oxidative inactivation of cordycepin (Cor) induced by ADA, the changes of Cor after the reaction of CPPC and ADA were observed by UV-visible spectrophotometry. The specific steps are as follows: freeze-dried adenosine deaminase (ADA) powder was dissolved in PBS buffer, mixed with free cordycepin solution and CPPC solution, and the concentration was equivalent to 25 μg mL -1 After the reaction, the mixture of CPPC and ADA was centrifuged to separate and remove the unreacted ADA. 2 The resulting CPPC was washed three times. The reacted CPPC was exposed to PBS buffer at pH 5.5 to evaluate the release of loaded cordycepin. After centrifugation to remove the precipitate, the cordycepin concentration in the supernatant was determined by UV-visible spectroscopy, as shown in Figure 2m and Figure 3a , Figure 3b , Figure 3c As shown, 0.1U of ADA effectively removed free Cor. However, when the same concentration of ADA acted on CPPC, no obvious changes were observed. The UV-visible spectrum showed that the maximum absorption peak of Cor released from CPPC was basically consistent with that of free Cor, but it decreased slightly due to a small amount of loss during the centrifugation process. The stability test was conducted by dissolving PDA and CPPC in PBS and PBS solution containing 10% FBS for six weeks, and the particle size and Zeta potential of PDA and CPPC were detected by Malvern particle size analyzer every other week. The results confirmed that ( Figure 4a , Figure 4b ), CPPC nanoparticles remained stable under physiological conditions for 6 weeks.

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

[0083] Experimental Example In this experiment, the in vivo therapeutic effect of CPPC on acute lung injury caused by sepsis was studied.

[0084] The LPS-induced acute lung injury mouse model was used. BALB / c mice were intraperitoneally injected with LPS (10 mg kg -1 ), and 1 hour later, the nanoparticles or control solution were intravenously injected. 24 hours later, the mice were intraperitoneally injected with sodium pentobarbital 30 mg kg -1 Anesthetize, remove the eyeballs and draw blood; fix the mouse dorsally on the operating table after blood drawing, cut the skin, expose the abdominal cavity, separate the trachea, and lavage the whole lung with 0.3mL pre-cooled saline. After staying for 1 minute, slowly withdraw the blood with a syringe, repeat three times, and collect bronchoalveolar lavage fluid (BALF); use a trimmed syringe filled with saline to pierce the left ventricle close to the apex at an angle roughly parallel to the left and right midlines of the heart, and inject saline for ventricular perfusion. After the perfusion is completed, the lung tissue is collected for subsequent analysis ( Figure 5a In CPPC-treated mice, the lung wet / dry weight ratio was calculated by removing the right upper lobe of the lung, removing the connective tissue, washing with saline and blotting with filter paper, weighing the wet weight, and drying it in a 70°C constant temperature drying oven for more than 72 hours. The lung tissue dry weight was weighed and recorded, and the mouse lung W / D value was calculated. 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°C. The precipitated cells were resuspended in 250 μL of saline, mixed, and the total cells were counted using an automatic cell counter. The results showed that ( Figure 5c ), CPPC treatment resulted in a significant decrease in the total cell count in BALF. The protein concentration in BALF was measured by the BCA method: Reagent A and reagent B in the BCA kit were fully mixed in a ratio of A:B=50:1 to prepare the BCA working solution; protein standards were added to the 96-well plate in sequence. The sample to be tested was added to the 96-well plate, 20 μL was added to each well; 200 μL of BCA working solution was added to each well and incubated in a 37°C incubator for 30 minutes; the absorbance value was detected at 562 nm with an enzyme reader, a protein standard curve was drawn, and the protein concentration was calculated. The results showed that ( Figure 5d ), CPPC treatment resulted in a significant decrease in protein levels in BALF.

[0085] Mouse BLAF, lung tissue homogenate supernatant and serum samples were obtained according to the instructions of the ELISA kit of Dakoway Biotechnology Co., Ltd. to detect the content of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in mouse lung tissue homogenate and BLAF. Specific operation steps: before use, the kit was equilibrated at room temperature for 30 minutes; blank wells: no sample was added, only colorimetric reagents A, B and stop solution were added for zero adjustment; standard wells: 100 μL of diluted standard was added to each well, and then 50 μL of biotin antigen working solution was added; zero wells: 100 μL of standard / sample diluent was added. Then add 50 μL of biotin antigen working solution; Sample well: add 100 μL of sample, then add 50 μL of biotin antigen working solution; shake gently, cover with sealing film, and incubate in a 37°C incubator for 90 minutes; dilute the 50-fold concentrated washing solution with distilled water 50 times for later use; First wash: carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 60 seconds and then discard, repeat this 4 times, and pat dry; add 100 μL TMB to zero well, standard well, and standard well. Sample and sample wells, shake gently, cover with sealing film, and incubate in a 37°C incubator for 30 minutes; second wash: carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 60 seconds and then discard, repeat 4 times, and pat dry; color development: add 100μL of color developer to each well, shake gently to mix, and color at 37°C for 10 minutes; stop: add 100μL of stop solution to each well to stop the reaction; determination: adjust the blank well to zero, and measure the absorbance of each well at a wavelength of 450nm. 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 an efficacy superior to that of the PBS treatment group ( Figure 5e , Figure 5f , Figure 5g These cytokines are known to be key contributors to the high mortality rate of cytokine storm and sepsis-induced acute lung injury. Overall, these findings highlight the potential of CPPC to suppress cytokine storm and mitigate lung injury in vivo.

[0086] Lung tissue damage was observed by hematoxylin-eosin (H&E) staining. 24 hours after modeling, 30 mg kg sodium pentobarbital was injected intraperitoneally. -1Mice were anesthetized, and the right middle lobe tissue was taken and placed in precooled physiological saline to drain the residual blood in the lungs. The tissues were transferred to 10% formalin and fixed for 24 hours. The tissues were rinsed with running water, dehydrated with gradient ethanol (concentrations of 30%, 50%, 80%, 95%, 95%, 100%, 100%), and then treated with xylene for two times for wax embedding to prepare paraffin sections with a thickness of 5 μm. The tissues were then dewaxed with xylene, hydrated with descending gradient alcohol, stained with hematoxylin and eosin, dehydrated with ascending gradient alcohol, transparentized with xylene, and sealed with neutral gum to prepare pathological sections. The morphological changes of lung tissue were observed under an optical microscope (200×). The results showed that the lung tissues of mice treated with LPS showed obvious structural damage, including varying degrees of pulmonary interstitial edema, hemorrhage, necrosis, alveolar inflammatory cell infiltration, and alveolar wall thickening. In contrast, the lung tissue structure of the CPPC-treated group was clearer, the alveolar wall was only slightly edematous and thickened, the immune cell infiltration was reduced, and the lung damage was significantly alleviated compared with the other groups ( Figure 5h ). In order to more objectively evaluate the degree of lung injury, a standardized lung injury scoring system was used to quantify the degree of injury. The scoring criteria include lung tissue hemorrhage, edema, inflammatory cell infiltration, alveolar damage or hyaline membrane formation, etc. Three visual fields were randomly selected for each slice to evaluate the score, with 0-5 points representing different degrees of injury: 0 points for no injury, 1 point for mild injury, 2 points for moderate injury, 3 points for moderate to severe injury, 4 points for severe injury, and 5 points for extremely severe injury. Consistent with the histological results, the CPPC treatment group had the lowest lung injury score of 1.2±0.45 ( Figure 5i ).

[0087] Myeloperoxidase (MPO) immunohistochemical staining was used to observe the leukocyte infiltration of lung tissue. The paraffin sections were subjected to immunohistochemical staining according to the following steps: ① Antigen repair: Pour citric acid antigen repair buffer (pH 6.0) into the repair box, heat it in a microwave oven until it boils, and then put the sections into the repair box for antigen repair. Repair on high heat for 10 minutes. During this process, excessive evaporation of the buffer should be prevented, and be careful not to dry the sections. After natural cooling, transfer to PBS solution and wash on a decolorizing shaker for 5 minutes, repeat 3 times; ② Block endogenous peroxidase: transfer the slices to 3% hydrogen peroxide solution, incubate at room temperature in the dark for 25 minutes, place in PBS solution and wash on a decolorizing shaker for 5 minutes, repeat 3 times; ③ BSA blocking: gently shake the slices dry, draw a circle around the tissue with a tissue 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, add primary antibody to the slices, add a small amount of water to the wet box, place the slices horizontally in the wet box, and incubate overnight in a refrigerator at 4℃; ⑤ Secondary antibody incubation: transfer the slices to PBS solution, wash on a decolorizing shaker for 5 minutes, repeat 3 times. After the slices are gently shaken dry, add the secondary antibody of the corresponding species to the primary antibody in the circle to evenly cover the tissue, and incubate at room temperature for 50 minutes; ⑥ DAB color development: place the slides in PBS solution and wash on a decolorizing shaker for 5 minutes, repeat 3 times. The slices were gently shaken dry, and the newly prepared AB coloring solution was dropped into the circle. The coloring time was observed under a microscope. The positive color was brown-yellow. The slices were rinsed with tap water to stop the coloring. ⑦ Re-staining of cell nuclei: Re-staining with hematoxylin for about 3 minutes, washing with tap water, differentiation with 1% hydrochloric acid alcohol for 3 seconds, rinsing with tap water, blueing with ammonia water, and washing with running water; ⑧ Dehydration and sealing: The slices were successively transferred 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 make them transparent, then dried and sealed with neutral gum. The depth and area of ​​staining were observed under a microscope and photographed. MPO immunohistochemical staining images ( Figure 5j )CPPC treatment significantly reduced the expression of MPO in the lung. Quantitative analysis of immunohistochemical images was performed using Image J software ( Figure 5k ) showed that neutrophil infiltration was significantly reduced. This may be related to CPPC reducing neutrophil inflammation in lung tissue and releasing Cor.

[0088] Select tissue sections with good quality and uniform thickness and dissolve them in paraffin. After dewaxing, quickly put the baked sections into xylene twice, 5 minutes each time; then undergo gradient hydration: place in 100%, 90%, 80%, 70% ethanol for 5 minutes each, and place in distilled water for 5 minutes; endogenous peroxidase blocking: add 3% hydrogen peroxide, place in a wet box at room temperature for 10 minutes, and then place in distilled water for 1 minute; antigen repair: place in 1x In CB, microwave at 80% power for 4 minutes, 40% power for 8 minutes, cool to room temperature; permeabilize with 0.3% Triton at room temperature for 15 minutes; block: dry the liquid around the tissue, add 10% NGS, place in a wet box, and incubate at room temperature for 2 hours; primary antibody: dilute the antibody according to the ratio recommended in the antibody manual; dry the liquid around the tissue, add the diluted primary antibody at 4°C overnight; place in 1xPBS and wash 3 times, 5 minutes each time; secondary antibody: dry the liquid around the tissue, add the diluted fluorescent secondary antibody, incubate at room temperature in the dark for 1 hour, and then wash three times with PBS solution, 5 minutes each time; nuclear staining: add Hoechst fluorescent dye to mark the cell nucleus, incubate at room temperature for 15 minutes, and then wash twice with PBS solution, 5 minutes each time; sealing: add a drop of anti-fluorescence quenching sealing agent on the tissue, cover the slide with a coverslip, and observe and take pictures under a microscope. Immunofluorescence staining ( Figure 5l , Figure 5m )The results showed that CPPC significantly reduced the levels of IL-6 and TNF-α proinflammatory factors in lung tissue.

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

[0090] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Claims

1. A method for preparing drug-loaded nanoparticles Cor@PDA-PEG@CD11b, characterized in that: The following steps are involved: S1, mixing the cordycepin solution with the PDA aqueous solution, so that the cordycepin and PDA form a complex Cor@PDA through π-π stacking; S2, performing PEG modification on the obtained complex Cor@PDA to obtain a complex Cor@PDA@PEG; S3. Disperse the obtained Cor@PDA@PEG in buffer, add anti-CD11b antibody, react for 24 hours, centrifuge, wash, collect the precipitate, and dry to obtain Cor@PDA-PEG@CD11b.

2. The method for preparing the drug-loaded nanoparticle Cor@PDA-PEG@CD11b according to claim 1, characterized in that: In step S1, the concentration of the PDA aqueous solution is 2 mg mL -1 , the concentration of cordycepin solution is 2 mg mL -1 , the mixing volume ratio of cordycepin solution and PDA aqueous solution is 1:

1.

3. The method for preparing the drug-loaded nanoparticle Cor@PDA-PEG@CD11b according to claim 2, characterized in that: In step S1, equal volumes of cordycepin solution and PDA aqueous solution are mixed, stirred at room temperature for 24 hours, centrifuged, washed, and the precipitate is collected to obtain the complex Cor@PDA.

4. The method for preparing the drug-loaded nanoparticle Cor@PDA-PEG@CD11b according to claim 1, characterized in that: In step S2, the complex Cor@PDA is dispersed in double distilled water, SH-PEG-streptavidin and NH4OH are added, and the reaction is stirred at room temperature for 16-24 hours, centrifuged, and the precipitate is collected to obtain the complex Cor@PDA@PEG.

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

5.

6. The method for preparing the drug-loaded nanoparticle Cor@PDA-PEG@CD11b according to claim 1, characterized in that: 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.

7. The method for preparing the drug-loaded nanoparticle Cor@PDA-PEG@CD11b according to claim 1, characterized in that: In step S3, the mass ratio of Cor@PDA@PEG to anti-CD11b antibody was 5:

1.

8. A drug-loaded nanoparticle Cor@PDA-PEG@CD11b, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 7.

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

10. The use according to claim 9, characterized in that: The lung injury is sepsis-induced acute lung injury.

11. The use according to claim 9, characterized in that: The drug is an intravenous preparation.

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