Novel H2O2-driven nano motor H-CeO-D-PM and preparation method and application thereof

Abstract

The invention discloses a novel H2O2-driven nano motor H-CeO-D (at) PM as well as a preparation method and application thereof, and relates to the technical field of biological medicines, and the key points of the technical scheme are as follows: a lung injury targeted antioxidant nano system-H-CeO-D (at) PM with pathogenesis adaptive DDS and ROS catalytic movement is constructed; the compound is used for sequential regulation of ROS accumulation and alveolar epithelial cell regeneration in ARDS treatment of dynamic inflammation. The H-CeO-D (at) PM nano system is established on hollow mesoporous cerium oxide which is coated with a Caspase-3 response peptide and an inflammation-targeted platelet membrane, wherein the Caspase-3 response peptide and the inflammation-targeted platelet membrane are conjugated with 7, 8-dihydroxyflavone. The H-CeO-D-coated PM constructed by the invention has broad-spectrum and efficient ROS (reactive oxygen species) removal capability, has a self-diffusion driving behavior under physiological H2O2 concentration, and can be responsively released; the drug can target lung tissue, sequentially remove ROS, promote alveolar cell regeneration and repair alveolar capillary barrier, so as to effectively treat ARDS; good biological safety is realized in vitro and in vivo.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically, to a novel H2O2-driven nanomotor H-CeO-D@PM and a preparation method and application thereof. Background Art

[0002] Acute respiratory distress syndrome (ARDS) is a very common clinical destructive disease that seriously endangers the life and health of patients. Its clinical features include acute hypoxemia, respiratory failure and bilateral pulmonary infiltration. Although advanced technologies have been developed in lung protective ventilation and fluid management, no drug has been proven to be a specific drug for ARDS. The pathogenesis of ARDS is very complex, but lung damage caused by inflammatory storm is one of the main pathogenesis. A large amount of reactive oxygen species (ROS) produced locally at the site of inflammation causes severe oxidative stress damage to biological macromolecules such as proteins, lipids, glycoproteins and DNA, leading to mitochondrial dysfunction, cell death and recruitment of inflammatory cells, destroying the endothelial cell barrier and alveolar epithelial cell barrier, and then leading to pulmonary edema, hyaline membrane formation and pulmonary fibrosis. It is well known that the inflammatory cascade of ARDS has spatiotemporal specificity. In the initial stage of the disease, immune cells secrete a large amount of ROS and proinflammatory cytokines to resist pathogen infection. In this process, vascular permeability increases, and then plasma enters the interstitium, and inflammation spreads and intensifies. Afterwards, the uncontrolled inflammatory cascade causes a sharp increase in epithelial cell apoptosis and destructive collapse of the alveolar capillary barrier, accompanied by the release of a large amount of cysteine-containing aspartate protease (caspase), which eventually leads to pulmonary edema, respiratory failure, and even death. How to remove a large amount of ROS as quickly as possible and relieve the inflammatory storm, as well as the subsequent repair of the alveolar-capillary barrier, is a key factor in the patient's survival time and wins the opportunity for comprehensive treatment in the late stage. Although single therapy at specific stages of the disease has shown some effects, individual differences and the sequential nature of the pathogenesis of ARDS still largely affect the recovery of the disease.

[0003] NPs antioxidants and nano-engineered drug delivery systems (DDSs) have been shown to be effective in treating ARDS. However, there are at least four bottlenecks in ARDS that limit their therapeutic efficacy. First, few studies have focused on sequential treatments in the initial and advanced stages, which are critical for ARDS treatment. Co-delivery of antioxidants and proliferative drugs leads to poor efficacy because they should act at different times. Second, the pathophysiological changes after ARDS vary from patient to patient, which requires precise repair of the alveolar-capillary barrier according to on-demand strategies after regulating the inflammatory storm. Recent stimuli-responsive DDS, including ROS- and pH-triggered systems, are still unsatisfactory. This is because increased oxidative stress and low pH are pathological features of the initial and late stages of ARDS, respectively. Third, the damaged but edema-fluid-filled alveolar-capillary barrier prevents traditional passive permeation methods. Finally, there is a lack of nanomaterials that can integrate the above functions into a single system. Summary of the invention

[0004] The purpose of the present invention is to solve the above problems and provide a new type of H2O2-driven nanomotor H-CeO-D@PM and its preparation method and application. The motor has the characteristics of removing active oxygen, targeting ability, hollow structure, and loading DHF.

[0005] The above technical objectives of the present invention are achieved through the following technical solutions: a new type of H2O2-driven nanomotor H-CeO-D@PMH-CeO-D@PM, the new type of H2O2-driven nanomotor H-CeO-D@PM has a hollow mesoporous structure, presents uniformly distributed and regularly shaped spherical particles, and the fluid dynamics diameter is about 224nm.

[0006] The present invention further discloses a method for preparing the novel H2O2-driven nanomotor H-CeO-D@PM, comprising the following steps:

[0007] S1: Preparation of silica NPs by the classic Stober method;

[0008] S2: preparing H-CeO NPs by mixing the prepared silica NPs with cerium (III) acetate hydrate;

[0009] S3: Pep-D is dissolved in a mixed solution of DMSO and water, and after the reaction, H-CeO loaded with Pep-D, i.e., H-CeO-D, is collected, and unreacted Pep-D is removed.

[0010] S4: Extract PM by repeated freeze-thaw method.

[0011] The present invention is further configured as follows: S1 specifically comprises the following steps:

[0012] S11: A mixed solution containing 8.4 mL of 28-30 wt % ammonium hydroxide, 280 mL of anhydrous ethanol and 56 mL of deionized water was stirred at room temperature for 0.5 h;

[0013] S12: Then, 8 ml of LTEOS was quickly added to the mixture under vigorous stirring and reacted for 24 h;

[0014] S13: The product was collected by centrifugation at 8000 rpm for 15 min and washed 3 times with ethanol and water for further use.

[0015] The present invention is further configured as follows: S2 specifically comprises the following steps:

[0016] S21: 3 mg / mL silica NPs aqueous solution and 5 mg / mL cerium (III) acetate hydrate aqueous solution were mixed together in a 100 mL beaker and stirred for 0.5 h to form a homogeneous solution;

[0017] S22: The mixture was transferred into a 100 mL polytetrafluoroethylene-lined stainless steel autoclave and heated at 160 °C for 16 h. The product was collected by centrifugation at 5000 rpm for 10 min and washed three times with anhydrous ethanol and water. Subsequently, the excess silica was etched by incubation with 5 mol / L sodium hydroxide for 2 days and washed three times with deionized water to obtain H-CeO.

[0018] The present invention is further configured as follows: S3 specifically comprises the following steps:

[0019] S31: dissolving Pep-D in a mixed solution of DMSO and water, wherein the ratio of DMSO to water is 1:4, to prepare Pep-D solutions of different concentrations ranging from 0 to 1 mg / mL;

[0020] S32: H-CeO was mixed with it and stirred for reaction at room temperature; after 24 hours, H-CeO loaded with Pep-D, i.e., H-CeO-D, was collected by repeated centrifugation with deionized water for 3 times, each time at 5000 rpm for 10 min, and unreacted Pep-D was removed.

[0021] The present invention is further configured as follows: S4 comprises the following steps:

[0022] S41: Dissolve the mixed protease inhibitor tablets in PBS to prepare protease inhibitor buffer;

[0023] S42: Fresh SD rat platelets were dispensed into 1 mL / tube, centrifuged at 800 × g for 20 min, the supernatant was discarded, and the precipitate was resuspended in the mixed protease inhibitor buffer prepared in S41;

[0024] S43: The suspension obtained by resuspending S42 was frozen at -80°C for 30 min, thawed at room temperature, centrifuged at 5000×g for 5 min, the supernatant was discarded, and the precipitate was resuspended in mixed protease inhibitor buffer;

[0025] S44: Repeat S42-S41 three times, collect the platelet membrane vesicles purified by centrifugation and mix them with H-CeO-D, and use an Avanti micro extruder to extrude the platelet membrane vesicles and H-CeO-D mixture through a 200 nm polycarbonate membrane to produce a final product.

[0026] The present invention further discloses the use of a novel H2O2-driven nanomotor H-CeO-D@PM in the preparation of drugs for treating acute respiratory distress syndrome. The H-CeO-D@PM nanomotor performs self-driving motion in a physiological H2O2 solution.

[0027] The present invention further discloses the use of a novel H2O2-driven nanomotor H-CeO-D@PM in the preparation of drugs for treating acute respiratory distress syndrome, wherein the H-CeO-D@PM achieves the therapeutic purpose by responsively releasing drugs.

[0028] The present invention further discloses the use of a novel H2O2-driven nanomotor H-CeO-D@PM in the preparation of drugs for treating acute respiratory distress syndrome. The H-CeO-D@PM achieves the therapeutic purpose by efficiently removing ROS, promoting alveolar cell regeneration and repairing the alveolar capillary barrier.

[0029] The present invention further discloses the use of a novel H2O2-driven nanomotor H-CeO-D@PM in the preparation of a drug for treating acute respiratory distress syndrome. The H-CeO-D@PM targets lung tissue, sequentially removes ROS, promotes alveolar cell regeneration, and repairs the alveolar capillary barrier, thereby effectively treating ARDS.

[0030] The present invention further discloses the use of a novel H2O2-driven nanomotor H-CeO-D@PM in the preparation of a drug for treating acute respiratory distress syndrome. The H-CeO-D@PM reduces inflammation of lung tissue by inhibiting the TNF signaling pathway.

[0031] The present invention further discloses the use of a novel H2O2-driven nanomotor H-CeO-D@PM in the preparation of a drug for treating acute respiratory distress syndrome. The H-CeO-D@PM activates the RAS signaling pathway, promotes the proliferation and regeneration of alveolar cells, repairs the alveolar capillary barrier, and thereby effectively treats ARDS.

[0032] The present invention further discloses the use of a novel H2O2-driven nanomotor H-CeO-D@PM in the preparation of a drug for treating acute respiratory distress syndrome. The drug administration routes include intravenous systemic administration, inhalation administration and oral administration.

[0033] In summary, the present invention has the following beneficial effects:

[0034] (1) The present invention successfully prepared H-CeO-D@PM based on the construction principle and characterized it. H-CeO-D@PM showed a broad-spectrum and efficient ROS scavenging ability, self-diffusion-driven behavior under physiological H2O2 concentrations, and responsive release.

[0035] (2) In vitro cell experiments showed that H-CeO-D@PM could effectively remove ROS, promote alveolar cell regeneration and repair of the alveolar capillary barrier simulated in vitro.

[0036] (3) In vivo animal experiments showed that H-CeO-D@PM could target lung tissue, sequentially remove ROS, promote alveolar cell regeneration, and repair the alveolar capillary barrier, thereby effectively treating ARDS.

[0037] (4) H-CeO-D@PM has good biosafety both in vivo and in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is a structural diagram of Pep-D in Example 1 of the present invention;

[0039] Figure 2 This is a schematic diagram of the construction of H-CeO-D@PM in Example 1 of the present invention;

[0040] Figure 3 is a representative transmission electron microscope image of H-CeO in Example 1 of the present invention (scale bar 200 nm);

[0041] Figure 4 is the hydrodynamic diameter distribution of different NPs in Example 1 of the present invention;

[0042] Figure 5 is the X-ray diffraction spectrum of H-CeO in Example 1 of the present invention;

[0043] Figure 6 is a high-resolution transmission electron microscope image of H-CeO in Example 1 of the present invention (scale bar 5 nm, 10 nm);

[0044] Figure 7 is the H-CeOX ray photoelectron spectrum in Example 1 of the present invention;

[0045] Figure 8is the N2 adsorption-desorption isotherm and BJH pore size distribution curve in Example 1 of the present invention;

[0046] Fig. 9 is a representative transmission electron microscope image of H-CeO-D in Example 1 of the present invention (scale bar 200 nm);

[0047] Fig.10 is a diagram of the enzyme-responsive release structure of H-CeO-D in Example 1 of the present invention;

[0048] Fig.11 is the infrared spectra of different samples in Example 1 of the present invention;

[0049] Fig.12 is the Zeta potential diagram of different NPs in Example 1 of the present invention;

[0050] Fig.13 is the element distribution diagram of H-CeO-D@PM in Example 1 of the present invention (scale bar 100 nm);

[0051] Fig.14 This is a representative transmission electron microscopy image of H-CeO-D@PM in Example 1 of the present invention. Scale bar 200 nm;

[0052] Fig.15 (a) Western blot analysis and (b) quantification of CD42b, CD61 and CD62P in Plt, PM and H-CeO-D@PM in Example 1 of the present invention;

[0053] Fig.16 The protein composition of H-CeO-D@PM in Example 1 of the present invention, wherein (a) Venn diagram shows the protein overlap between Plt, PM and H-CeO-D@PM; (b) Volcano plot shows the differential proteins between PM and H-CeO-D@PM; (c) Classification of H-CeO-D@PM proteins by biological process; (d) Classification of H-CeO-D@PM proteins by molecular function; (e) Expression abundance of proteins related to platelet adhesion and immune escape properties in PM and H-CeO-D@PM;

[0054] Fig.17 is a schematic diagram of H-CeO-D@PM removing ROS in Example 1 of the present invention;

[0055] Fig.18 is the scavenging rate of ROS by H-CeO-D@PM at different concentrations in Example 1 of the present invention, wherein (a) the scavenging rate of H2O2 by H-CeO-D@PM; (b) the scavenging rate of O2· by H-CeO-D@PM, (c) the scavenging rate of ·OH by H-CeO-D@PM;

[0056] Fig.19 It is the X-ray photoelectron spectrometer spectrum of small CeONPs in Example 1 of the present invention;

[0057] Fig. 20 is the electron spin resonance spectrum of ·OH in the presence of different concentrations of H-CeO-D@PM in Example 1 of the present invention;

[0058] Fig.21 It is the generation of O2 in the catalytic reaction in the presence of H-CeO-D@PM in Example 1 of the present invention;

[0059] Fig. 22 is a Raman spectrum of H2O2 repeatedly removed by H-CeO-D@PM in Example 1 of the present invention;

[0060] Fig.23 It is the motion trajectory of H-CeO-D@PM in different concentrations of H2O2 in Example 1 of the present invention;

[0061] Fig.24 is the average MSD curve of H-CeO-D@PM before and after adding different concentrations of H2O2 in Example 1 of the present invention;

[0062] Fig.25 It is the motion trajectory of the H-CeO-D@PM nanomotor in Example 1 of the present invention in different concentrations of H2O2;

[0063] Fig.26 is the loading capacity of H-CeO at different Pep-D concentrations in Example 1 of the present invention;

[0064] Fig. 27 It is the release behavior triggered by Casp3 in Example 1 of the present invention, wherein (a) the cumulative drug release profile of H-CeO-D@PM under different conditions within 48 h; (b) the drug release profile of H-CeO-D@PM in the presence of different concentrations of Casp3;

[0065] Fig.28 It is a schematic diagram of the in vitro removal of ROS by H-CeO-D@PM in Example 2 of the present invention;

[0066] Fig.29 The fluorescent dye DCFH-DA in Example 2 of the present invention is used to measure the ROS level, wherein (a) the ROS level in HUVEC cells is measured by laser confocal microscopy; (b) the ROS level is measured by laser confocal microscopy quantitative statistical graph; (c) the ROS level is measured by microplate reader. Scale bar 50 μm;

[0067] Fig.30The CCK-8 analysis and evaluation of cell viability in Example 2 of the present invention, wherein (a) the flow cytometry method is used to detect the apoptosis and necrosis ratio of HUVEC; (b) the quantitative statistical graph of the apoptosis and necrosis ratio of HUVEC detected by flow cytometry; (c) the CCK-8 method is used to detect the cell viability of HUVEC;

[0068] Fig.31 This is the EdU detection of cell proliferation in Example 2 of the present invention, wherein (a) EdU detection of MLE-12 cell proliferation; (b) EdU detection of MLE-12 cell proliferation quantitative statistical graph; (c) Ki67 detection of MLE-12 cell proliferation; (d) Ki67 detection of MLE-12 cell proliferation quantitative statistical graph. Scale bar 50 μm;

[0069] Fig.32 The voltmeter in Example 2 of the present invention measures the transendothelial resistance to monitor the integrity of the double-layer cells, wherein (a) is a schematic diagram of the alveolar capillary barrier damage cell model; (b) the TEER value is measured to evaluate the integrity of the alveolar capillary barrier; Fig.33 It is a schematic diagram of chest micro-CT and tissue ICP-MS examination after the ARDS model is constructed in Example 3 of the present invention;

[0070] Fig.34 It is a schematic diagram of body weight, survival rate recording, lung tissue and BALF detection after the ARDS model is constructed in Example 3 of the present invention;

[0071] Fig.35 The chest CT in Example 3 of the present invention evaluates the targeting ability of H-CeO-D@PM in ARDS mice, wherein (a) CT attenuation rate of H-CeO-D@PM at different concentrations; (b) representative images of mouse Micro-CT chest scans;

[0072] Fig.36 The chest CT in Example 3 of the present invention is used to image the lungs, wherein (a) representative Micro-CT images of different treatment groups; (b) quantitative CT values ​​of different treatment groups;

[0073] Fig.37 The ICP-MS test ce content in Example 3 of the present invention, wherein (a) the ce content of lung tissues in different treatment groups; (b) the distribution of ce in different tissues and at different time periods;

[0074] Fig.38The weight change, survival curve, lung wet-to-dry weight ratio and protein concentration in BALF of ARDS mice treated with H-CeO-D@PM in Example 3 of the present invention, wherein (a) weight change of ARDS mice treated with different treatments; (b) survival curve of ARDS mice treated with different treatments; (c) protein concentration in BALF of ARDS mice treated with different treatments; (d) lung wet-to-dry weight ratio of ARDS mice treated with different treatments;

[0075] Fig.39 (a) nucleated cell count in BALF of ARDS mice treated with different methods in Example 3 of the present invention; (b) neutrophil ratio in flow cytometry; (c) quantitative analysis of neutrophil ratio;

[0076] Fig.40 (a) HE staining of lung tissue of ARDS mice treated with different treatments in Example 3 of the present invention; (b) lung injury scores of ARDS mice treated with different treatments. Scale bar 100 μm, 50 μm. ;

[0077] Fig.41 (a) DHE staining of lung tissue of ARDS mice treated with different treatment methods in Example 3 of the present invention; (b) quantitative analysis of DHE staining; (c) SOD activity. Scale bar 100 μm;

[0078] Fig.42 (a) F4 / 80 staining in lung tissue of ARDS mice treated with different treatments; (b) quantitative analysis of F4 / 80 staining in lung tissue of ARDS mice treated with different treatments; (c) CD11b staining in lung tissue of ARDS mice treated with different treatments; (d) quantitative analysis of CD11b staining in lung tissue of ARDS mice treated with different treatments in Example 3 of the present invention. Scale bar 100 μm;

[0079] Fig.43 are the concentrations of TNF-α, IL-6 and IL-1β in (a) BALF and (b) lung tissue of ARDS mice treated with different methods in Example 3 of the present invention;

[0080] Fig.44 (a) PDPN staining of lung tissue of ARDS mice treated with different treatments; (b) quantitative analysis of PDPN staining of lung tissue of ARDS mice treated with different treatments in Example 3 of the present invention. Scale bar: 100 μm.

[0081] Fig.45 (a) VE-Cadherin staining of lung tissue of ARDS mice treated with different treatments in Example 3 of the present invention; (b) quantitative analysis of VE-Cadherin staining of lung tissue of ARDS mice treated with different treatments. Scale bar: 100 μm;

[0082] Fig.46 is the cell survival rate of H-CeO-D@PM in Example 4 of the present invention after co-culture with (a) HUVEC cells and (b) MLE-12 cells for 24 h;

[0083] Fig.47 The results of in vitro hemolysis experiments of H-CeO-D@PM with different concentrations in Example 4 of the present invention are shown;

[0084] Fig.48 The blood routine and biochemical parameters of mice 48 hours after administration of H-CeO-D@PM in Example 4 of the present invention;

[0085] Fig.49 It is the main organ of mice 48 hours after administration of H-CeO-D@PM in Example 4 of the present invention. DETAILED DESCRIPTION

[0086] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0087] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0088] Embodiment 1:

[0089] Preparation and characterization of a reactive oxygen species-catalyzed exercise-induced lung injury-targeted antioxidant nanosystem (H-CeO-D@PM).

[0090] 1.1 Materials

[0091] 1.1.1 Reagents and consumables

[0092] Cerium acetate trihydrate (Aladdin, China), tetraethyl orthosilicate (Jingzewang, China), concentrated ammonia (Aladdin, China), anhydrous ethanol (McLean, China), terminal thiol peptide (Baonuo, China), cerium nitrate hexahydrate (Aladdin, China), 7,8-DHF (Aladdin, China), protease inhibitor tablets (Roche, Germany), PBS (Solarbio, China), 30% H2O 2(Kelon, China), dialysis bag (Solaibao, China), carbon support membrane copper mesh (Zhongjing Technology, China), BCA kit (Thermo Fisher, USA), RIPA lysis buffer (Biyuntian, China), protease phosphatase inhibitor (Biyuntian, China), 5× loading buffer (Biyuntian, China), one-step PAGE gel rapid preparation kit (Yazyme, China), PVDF membrane (Millipore, USA), TBST solution (Biyuntian, China), primary antibody, secondary antibody diluent (Biyuntian, China), HRP-labeled goat anti-rabbit IgG antibody (Biyuntian, China), rabbit anti-CD42b (abcam, USA), rabbit anti-CD62p (abcam, USA), rabbit anti-Integrinbeta3 (Affinity, China), rabbit extracellular anti-CD47 (CST, USA), rabbit intracellular anti-CD47 (Affinity, China), 1.1.2 Instruments and equipment

[0093] Milli-Q water purification system (Millipore, USA), vortex machine (IKA, Germany), electronic balance scale (Sartorius, Germany), glass container (Hinwil, China), vacuum dryer (Shanzhi, China), high-speed centrifuge (Thermo Fisher, USA), pipettes of different ranges (Eppendorf, Germany), gel imager (Invitrogen, USA), transmission electron microscope (Shimadzu, Japan), transmission electron microscope energy dispersive spectrometer (Bruker, USA), scanning electron microscope (Zeiss, Germany).

[0094] 1.1.3 Experimental animals

[0095] Male SD rats weighing 300–350 g and aged 8–10 weeks were purchased from the Experimental Animal Center of the Army Medical University. All animal experiments were approved by the Animal Ethics Committee of the Army Medical University in accordance with the Guide for the Care and Use of Laboratory Animals of the Army Medical University, No. AMUWEC20224137.

[0096] 1.2 Methods

[0097] 1.2.1 Synthesis of H-CeO-D@PM

[0098] (1) Synthesis of H-CeO:

[0099] First, H-CeO nanoparticles (NPs) with broad-spectrum ROS scavenging ability were prepared, and then Casp3-responsive drugs 7,8-DHF, which promotes alveolar capillary barrier repair, were loaded on the surface and internal hollow of NPs. In order to target the site of lung damage, PM was coated on the surface of drug-loaded NPs to construct a new nanozyme system that improves lung tissue penetration, achieves ROS scavenging and precise controlled drug release, and promotes alveolar epithelial cell repair and regeneration. The details are as follows:

[0100] Through the classic Silica NPs were prepared by a method. Typically, a mixed solution containing ammonium hydroxide (8.4 mL, 28-30 wt%), anhydrous ethanol (280 mL) and deionized water (56 mL) was stirred at room temperature for 0.5 h. Then, TEOS (8 mL) was quickly added to the mixture under vigorous stirring for 24 h. Finally, the product was collected by centrifugation at 8000 rpm for 15 min and washed 3 times with ethanol and water for further use. Then, a 3 mg / mL silica NPs aqueous solution and a 5 mg / mL cerium (III) acetate hydrate aqueous solution were mixed together in a 100 mL beaker and stirred for 0.5 h to form a homogeneous solution. Then, the mixture was transferred to a 100 mL polytetrafluoroethylene-lined stainless steel autoclave and heated at 160 ° C for 16 h. The product was collected by centrifugation at 5000 rpm for 10 min and washed 3 times with anhydrous ethanol and water. Subsequently, the excess silicon dioxide was etched by incubating with 5 mol / L sodium hydroxide for 2 days and washed with deionized water three times to obtain H-CeO.

[0101] (2) Synthesis of H-CeO-D:

[0102] Pep-D was ordered from Beijing Baonuo Technology Co., Ltd. The structure diagram is as follows Figure 1 As shown. Pep-D was dissolved in a mixed solution of DMSO and water, where the ratio of DMSO to water was 1:4, to prepare Pep-D solutions of different concentrations (0-1 mg / mL). H-CeO was then mixed with it and stirred for reaction at room temperature. After 24 hours, H-CeO loaded with Pep-D (H-CeO-D) was collected by centrifugation with deionized water (5000 rpm, 10 min) for 3 times, and unreacted Pep-D was removed.

[0103] (3) Synthesis of H-CeO-D@PM

[0104] Extraction of PM: Fresh SD rat platelets (PLT) were aliquoted. PM was obtained through repeated freezing and thawing [9]. In brief, aliquots of platelet suspension were first frozen at -80°C for 30 min, thawed at room temperature, and centrifuged at 5000×g for 5 min. The supernatant was discarded and resuspended in PBS solution containing protease inhibitors. After repeating the above steps three times, the precipitated PM was resuspended in deionized water.

[0105] A 1 mg / mL H-CeO-D aqueous solution was mixed with 1 mL of about 1×10 9 The platelet PM solution was mixed under ultrasound conditions (75 W, 50 Hz) for 5 min. The mixture was extruded through a polycarbonate membrane (200 nm) using an Avanti microextruder to produce the final product

[30] .

[0106] (4) Detection of important functional protein markers of PM using Western blot technology

[0107] Extraction of PM protein: Plt precipitate was added with RIPA lysis buffer and 50× protease and phosphatase inhibitors and lysed on ice for 30 min.

[0108] Protein sample concentration determination, sample preparation and denaturation: Preparation of BCA solution: Prepare a mixed solution in a ratio of A:B solution equal to 50:1, and finally obtain solution C after thorough mixing; dilute the protein sample 5 times: add 5μL of protein sample solution to 20μL of RIPA lysis solution, and mix by pipetting; prepare standard protein samples of different concentrations in turn; add 25μL of diluted protein sample solution to 200μL of solution C, mix by pipetting, and incubate at 37℃ for 30min; take 200μL of liquid from each tube of sample and add it to a 96-well plate. Use an enzyme reader to detect the absorbance value of the sample at 562nm. Draw a standard curve based on the results. Substitute the absorbance value of the sample to be tested into the standard curve formula to obtain the protein concentration of the sample to be tested.

[0109] The protein samples were diluted to a concentration of 5 μg / μL using 5× loading buffer and RIPA lysis buffer; after boiling for 5 min, the samples were naturally cooled to room temperature and stored in aliquots.

[0110] Immunoblotting detection: Use 7.5% one-step method for PAGE gel preparation; align and fix two 1.5mm thick clean and dry glass plates. Take 4.0mL of lower gel and 4.0mL of lower gel buffer, blow and mix well for later use. Then, take 1.0mL of upper gel and 1.0mL of colored upper gel buffer, blow and mix well for later use. Next, slowly add 80μL of improved coagulant to the mixed lower gel, blow and mix well, and use a 1mL pipette to carefully inject the solution into the glass plate for gel making. Add 20μL of improved coagulant to the mixed upper gel, blow and mix well, and immediately inject the mixed solution carefully into the glass plate for gel making, and insert the comb teeth. After the gel solidifies for about 15 minutes, remove the comb teeth and use it for electrophoresis. Add 10μL 1×5 loading buffer at the beginning and end of the lane, then add maker, add 5μL at the beginning and 2μL at the end, and add 10μL of protein sample with a concentration of 5μg / μL in the middle lane; start electrophoresis after connecting the power supply, 150V, 80min, until the bromophenol blue dye is electrophoresed to the bottom of the lower gel. Cut a 6.5×5.5cm PVDF membrane, soak it in methanol for 30s to activate it, and then transfer it to the electrotransfer solution. Soak the PVDF membrane, gel, and sponge pad in 1× electrotransfer solution. After aligning the black and white plates, close the lid, be careful to avoid the membrane from being entangled, install it in the electrotransfer tank, pay attention to the positive and negative poles, put a plastic ice bag in the tank, and then place the electrophoresis tank in a basin filled with crushed ice. Set the program: 100V, 70min, start electrotransfer. After the transfer is completed, turn off the power and take out the PVDF membrane. Place in blocking solution and place on a shaker, block at room temperature for 15 minutes; remove the PVDF membrane and cut the membrane according to the marker position and the molecular weight of the target protein. Use primary antibody diluent to dilute the primary antibody: rabbit anti-CD42b (1:1000), rabbit anti-CD61 (1:1000) and mouse anti-CD62p (1:2000), and the antibody needs to completely cover the membrane. Incubate overnight at 4°C. The next day, HRP-labeled goat anti-rabbit IgG antibody (1:1000) and HRP-labeled goat anti-mouse IgG antibody (1:1000) diluted with secondary antibody diluent are ready for use. Wash the membrane with 1×TBST for 5 minutes and wash it repeatedly for 5 times, then add the secondary antibody and incubate at room temperature for 1 hour. Wash the membrane with 1×TBST for 5 minutes and wash it repeatedly for 5 times, then pour ECL color developing solution on it and expose it in a gel imager.

[0111] (5) PM proteomics analysis using non-protein marker quantitative techniques:

[0112] In order to determine the composition and retention of platelet membrane protein functions in H-CeO-D@PM, Shanghai Meiji Biopharmaceutical Technology Co., Ltd. was commissioned to perform proteomics sample processing and sequencing. Quantitative proteomics analysis of PLT, purified PM and H-CeO-D@PM was performed using non-protein labeling technology. In brief, the protein was cleaved with protease lysis buffer and the protein content was determined by the BCA method. Further proteolysis, peptide desalting and quantification were performed, followed by non-protein labeling quantitative technology mass spectrometry detection, non-protein labeling quantitative technology data analysis and bioinformatics analysis.

[0113] 1.2.2 Characterization of ROS scavenging performance of H-CeO-D@PM

[0114] (1) H2O2 scavenging performance of H-CeO-D@PM: The H2O2 scavenging activity of H-CeO-D@PMNPs was evaluated using a hydrogen peroxide assay kit. H-CeO-D@PM with various ce concentrations (25-400 μg / mL) was mixed with 1 mM H2O2 at 37 °C for 1 h. Then, the inhibition rate of H2O2 by H-CeO-D@PM was evaluated by measuring the absorbance value at 571 nm. At the same time, the H2O2 inhibition rate of small-particle CeO NPs as a control group was also detected according to the above method. The scavenging of H2O2 by H-CeO-D@PM NPs is accompanied by the generation of oxygen. 1 mL of H-CeO-D@PM NPs (100 μg / mL) was transferred to a 5 mL glass reaction bottle, and H2O2 with different reaction concentrations (0, 0.5 and 1 mM) was added and stirred continuously for 3 min. The oxygen concentration in the above reaction solution was measured using a dissolved oxygen tester.

[0115] (2) ·OH scavenging performance of H-CeO-D@PM: The ·OH scavenging activity of H-CeO-D@PM NPs was determined using a hydroxyl radical antioxidant capacity assay kit. H-CeO-D@PM NPs (25-400 μg / mL) with various ce concentrations were incubated with a fluorescent solution containing ·OH at 37 °C for 0.5 h. The scavenging rate of ·OH by H-CeO-D@PM was measured by fluorescence values ​​(Ex: 490 nm, Em: 530 nm). The ·OH scavenging rate of small-particle CeO NPs as a control group was also detected according to the above method. The ·OH scavenging activity of H-CeO-D@PM NPs was also detected by electron spin resonance spectroscopy. ·OH was produced by the Fenton reaction between FeSO4 (70 μL, 0.735 mM) and H2O2 (25 μL, 0.315 mM). The ·OH elimination efficiency of H-CeO-D@PM NPs (100 μL, 0 and 200 μg / mL) was evaluated by using 5,5′-dimethylpyrroline-1-oxide (DMPO) as a spin trapping agent. The change of the 1:2:2:1 intensity multipeak in the electron spin resonance spectrum of the DMPO-·OH adduct indicated the ·OH elimination efficiency of H-CeO-D@PM NPs.

[0116] (3) O2·-scavenging activity of H-CeO-D@PM NPs: The O2·-scavenging activity of H-CeO-D@PM was detected using a SOD assay kit. H-CeO-D@PM with ce concentrations of 25, 50, 100, 200 and 400 μg / mL were incubated with WST-1 working solution at 37°C for 20 min. Finally, the absorbance value (450 nm) of the above mixture was measured by using an ELISA instrument. The O2·-scavenging rate of small-particle CeONPs as a control group was also detected according to the above method.

[0117] (4) Repeated antioxidant capacity of H-CeO-D@PM: The repeated scavenging capacity of H-CeO-D@PM for H2O2 was detected by Raman spectroscopic microscopy. Specifically, 0.1 mL of an aqueous solution containing H-CeO-D@PM (100 μg / mL) and H2O2 (1 mM) was dropped onto a glass slide and scanned using a 488 nm laser at 0, 15, 30, and 60 min, respectively. Immediately after the first cycle of 60 min, 0.1 mL of H2O2 (1 mM) was dropped onto the previous glass slide again and scanned at the same time point.

[0118] 1.2.3 Determination of the motion behavior of H-CeO-D@PM nanomotors

[0119] The movement behavior of NPs in different solutions was recorded at room temperature by using a nanoparticle tracking analyzer equipped with a CMOS camera. Specifically, 10 μL of NPs (100 μg / mL) was added to 0.99 mL of PBS (pH 7.4) and fresh H2O2 solutions of different concentrations (0.5 and 1.0 mM), and then diluted to a concentration suitable for detection by the nanoparticle tracking analyzer (110 particles / frame). NPs were injected into a 1 mL nanomicroscope sample chamber and recorded at a rate of 25 frames / second in a field of view of 100×80×10 μm. The obtained videos were analyzed by image J and its plug-in (MtrackJ) to track the x and y trajectories of NPs, and then the mean square displacement (MSD), diffusion coefficient and movement speed of NPs in different solutions were calculated based on the trajectories.

[0120] 1.2.4 Drug release behavior of H-CeO-D@PM

[0121] (1) Drug loading and encapsulation efficiency of Pep-D: The drug loading and encapsulation efficiency of Pep-D in H-CeO-D@PM were evaluated by the absorbance at 207 nm using a micro-UV spectrophotometer. After reacting different concentrations of Pep-D (31.25, 62.5, 125, 250, 500, 1000 μg / mL) with H-CeO, the supernatant was taken to detect the absorbance intensity at 207 nm, and the linear regression equation of Pep-D was fitted with the absorbance intensity (n=3). The amount of Pep-D in the supernatant was calculated using the linear regression equation of Pep-D (n=3). The drug loading and encapsulation efficiency of Pep-D were calculated according to the following formula:

[0122] Drug loading % = weight of Pep-D in H-CeO-D@PM / weight of H-CeO-D@PM×100%.

[0123] Encapsulation efficiency % = weight of Pep-D in H-CeO-D@PM / weight of added Pep-D × 100%.

[0124] (2) Drug release study of H-CeO-D@PM:

[0125] The Casp3 cleavage behavior of Casp3 responsive peptide (Pep-D) was studied by incubating Pep-D (50 μM) and 10 μg / mL Casp3 in Casp3 buffer at 37°C for 0.5, 1, 2 and 8 h, respectively. The cleavage products including DHF were analyzed by high-resolution mass spectrometry and high-performance liquid chromatography. The release profiles of DHF under different conditions in vitro were studied by micro-UV spectrophotometry. In detail, 1 mg / mL of H-CeO-D@PM was incubated with PBS (pH 7.4), fresh H2O2 (1 mM) and different concentrations of Casp3 (0, 0.1 and 1 μg / mL) in Casp3 buffer (10 mL) at 37°C in the dark. At predetermined time points (0.5, 1, 2, 4, 8, 24, 36 and 48 h), the supernatant was obtained by centrifugation (5000 rpm, 10 min), and then 100 μL of the supernatant was taken and the same volume of Casp3 buffer was added to resuspend the NPs. Finally, the DHF release amount of H-CeO-D@PM in vitro was calculated according to the linear regression equation of the absorbance of DHF at 207 nm (n = 3).

[0126] 1.3 Results

[0127] 1.3.1 Rational design, synthesis and characterization of H-CeO-D@PM

[0128] Smaller CeO NPs are more likely to accumulate in the kidneys (<6nm), spleen, or liver (20-150nm) rather than in the lungs (150-1000nm). Considering that nanoparticles with a particle size range of 100-200nm are more conducive to prolonging the blood circulation residence time, NPs with a diameter of about 200nm, H-CeO-D@PM, were selected for ARDS treatment to improve their accumulation in the lungs. First, using silica as a template, hydrophilic hollow porous cerium oxide nanoparticles (H-CeO NPs) were synthesized by mixing silica and cerium acetate in water under simple and green reaction conditions by a hydrothermal method without any other reagents. Figure 2 After the silicon dioxide etching treatment, uniform monodispersed H-CeO NPs with a diameter and shell thickness of approximately 183 nm and 22 nm were obtained ( Figure 3 Meanwhile, the hydrodynamic diameter of H-CeO NPs was about 190 nm ( Figure 4 ). X-ray diffraction spectrum ( Figure 5 ) and high-resolution transmission electron microscopy images ( Figure 6 ) further proved that H-CeO is formed by the accumulation of many small particles (4-6nm) of crystalline CeO with good crystallinity. X-ray photoelectron spectroscopy signals such as Figure 7The results show that the split peaks of Ce3d3 / 2 and Ce3d5 / 2 in H-CeO are 885.7, 902.2 eV and 883.4, 889.9, 899.2, 907.6, 909.6 and 917.6 eV, respectively, confirming the mixed valence state. By fitting the peaks of Ce 3d spectrum, the content of cerium (III) oxide in the synthesized H-CeO is about 35.5%, and the content of the remaining cerium (IV) oxide is about 64.5% ( Figure 7 ). It has higher Ce than ultra-small CeONPs (5.68nm). 3+ / Ce 4+ This is beneficial for the removal of ROS. The Brunauer-Emmett-Teller surface area and average pore size of H-CeO are 201.7 m2 / g and 9.5 nm ( Figure 8 ), which paves the way for large-scale drug loading and effective ROS scavenging. The designed Casp3-responsive peptide (Pep) was successfully coupled with 7,8-DHF via esterification reaction (Pep-D)( Fig. 9 Then, through the coordination reaction between the thiol group in Casp3 at the end of Pep-D and the Ce in H-CeO, the enzyme-responsive release of the DHF behavior of H-CeO-D and Casp3 was obtained ( Fig.10 Compared with the infrared spectrum of H-CeO, the aromatic ring of H-CeO-D showed new skeleton vibration absorption peaks corresponding to Pep-D at 1455, 1508 and 1575 cm-1 ( Fig.11 After connecting Pep-D, the zeta potential changes from -20.07mV of H-CeO to -23.33mV of H-CeO-D ( Fig.12 ). All these results revealed the successful connection between H-CeO and Pep-D. Since PM has a high binding affinity to vascular injury sites, PM was encapsulated on the H-CeO-D surface to improve the delivery efficiency of the nanosystem to the inflammation site. In order to obtain H-CeO-D@PM, the prepared H-CeO-D was uniformly mixed with PM obtained by repeated freeze-thaw process, and then the mixture was treated by ultrasonic and repeated extrusion treatment to reduce material loss. Transmission electron microscopy images showed that the P, S and N elements in PM were uniformly distributed on the surface of H-CeO-D ( Fig.13 ). The stem diameter of NPs increased by about 11.4 nm ( Fig.13 and Fig.14 ), the hydrodynamic diameter changes from about 192 nm for H-CeO-D to about 224 nm for H-CeO-D@PM ( Figure 4), and the increased thickness is consistent with the thickness of monolayer PM reported in other literature, indicating the presence of a monolayer PM coating around the H-CeO-D core. Due to the insertion of negatively charged PMs by lipid membrane fusion, the zeta potential of the NPs increased from -23.33 mV (H-CeO-D) to -27.87 mV (H-CeO-D@PM)( Fig.12 In addition, Western blot analysis and quantification ( Fig.15 -a, b) confirmed the presence of three important platelet protein markers CD42b, CD61 and CD62P from PM extracted after repeated freeze-thaw cycles, and these markers were retained in the subsequent PM-encapsulated H-CeO-D@PM. The protein composition of H-CeO-D@PM was studied using a label-free quantitative technique. Fig.16 As shown in Figure 2, after repeated freezing and thawing, the extracted PM retained 5086 proteins, accounting for 91.08% of the total protein species in the original Plt population. H-CeO-D@PM retained 90.97% of the total protein species in PM. Fig.16 -b, H-CeO-D@PM retains most of the proteins in PM. At the same time, proteins in purified PM and H-CeO-D@PM are expressed according to their biological processes ( Fig.16 -c) and molecular functions ( Fig.16 -d) classification, indicating that Plt proteins were well preserved in H-CeO-D@PM. Next, the abundance of proteins related to adhesion and immune system interaction was analyzed (16-e), mainly including intercellular response proteins and membrane integrin receptors: such as GP1BA, GP1BB, GP5, GP6, GP9, CD62P, CD47 and CD58, all retained in the extracted PM and H-CeO-D@PM to achieve their strong binding affinity with damaged blood vessels. These results confirm that H-CeO-D@PM inherits the ability of native platelets and provides basic support for the targeting function after ARDS injury.

[0129] 1.3.2 Characterization of ROS scavenging performance of H-CeO-D@PM

[0130] The prepared H-CeO-D@PM has high Ce 3+ / Ce 4+ Ratio, where Ce 4+ It can oxidize H2O2 through CAT-like activity, and Ce 3+ Can scavenge superoxide radicals (·O2-) and hydroxyl radicals (·OH) through SOD-like activity and redox reactions ( Fig.17 ). Next, the various ROS scavenging abilities of H-CeO-D@PM were verified by using the corresponding ROS detection kits. Fig.18-a, the removal of H2O2 by H-CeO-D@PM showed a concentration-dependent change, and the removal rate increased with the increase of H-CeO-D@PM concentration, and compared with 5.68nm CeO, the removal efficiency of the two was comparable. When the concentration of ce was 50μg / mL, the removal efficiency of CeO2 and H-CeO-D@PM was 83.90% and 80.08%, respectively. When the concentration of ce increased to 200μg / mL, the removal efficiency of CeO and H-CeO-D@PM was 95.37% and 99.78%, respectively.

[0131] like Fig.18 -b, H-CeO-D@PM to O 2· -'s removal showed a concentration-dependent change, and the removal rate increased with the increase of the concentration of H-CeO-D@PM. Compared with 5.68nm CeO, H-CeO-D@PM had better removal efficiency. When the concentration of Ce was 50μg / mL, the removal efficiencies of CeO and H-CeO-D@PM were 75.60% and 52.52%, respectively. When the concentration of Ce increased to 200μg / mL, the removal efficiencies of CeO and H-CeO-D@PM were 97.01% and 89.28%, respectively. Fig.18 -c shows that the scavenging of ·OH by H-CeO-D@PM shows a concentration-dependent change, and the scavenging rate increases with the increase of the concentration of H-CeO-D@PM, and compared with 5.68nm CeO, the scavenging efficiency of the two is comparable. When the concentration of ce is 50μg / mL, the scavenging efficiencies of CeO and H-CeO-D@PM are 30.45% and 32.17%, respectively. When the concentration of ce is 200μg / mL, the scavenging efficiencies of CeO and H-CeO-D@PM are 61.21% and 64.47%, respectively. Interestingly, H-CeO-D@PM can achieve a ROS elimination rate comparable to that of small CeO NPs. This is because H-CeO-D@PM has a hollow porous structure through the shell formed by interpenetrating cerium oxide nanocrystals (4-6nm), and Ce 3+ / Ce 4+ Compare( Figure 7 ) is slightly higher than that of small-sized CeO NPs ( Fig.19 ). Electron spin resonance spectroscopy such as Fig. 20 As shown, the repeated scavenging effect of H-CeO-D@PMNPs on ·OH was further demonstrated, using 5,5'-dimethylpyrrole-1-oxide as a spin trap, where the 1:2:2:1 multiple peaks in the electron spin resonance spectrum showed the presence of ·OH, and the peaks were steep. After adding H-CeO-D@PMNPs, the signal intensity of ·OH decreased with the increase of H-CeO-D@PM concentration. The generation of O2 was measured using a dissolved oxygen meter. Fig.21As shown, compared with H2O2 alone, the addition of H-CeO-D@PM to the reactants can significantly increase the rate of O2 generation. When 0.5mM H2O2 was added to H-CeO-D@PM, the amount of O2 produced in 1min was 7.38mg / mL, and the amount of O2 produced in 5min was 7.94mg / mL. When 1mM H2O2 was added to H-CeO-D@PM, the amount of O2 produced in 1min was 7.63mg / mL, and the amount of O2 produced in 5min was 8.27mg / mL. The results show that H-CeO-D@PM can catalyze H2O2 to generate O2, and the amount of O2 produced increases with the increase of H2O2 concentration. In addition, H2O2 was selected as a representative of ROS to verify whether H-CeO-D@PM has the reversible ROS elimination ability reported by in situ Raman spectroscopy analysis. After adding H2O2, H-CeO-D@PM was reduced by H2O2, peroxide was adsorbed on the surface, and a new peak was formed at 873cm-1, while the peak at 455cm-1 disappeared, which was attributed to the Ce-O symmetric breathing mode in H-CeO. It is worth noting that the peaks at 455 and 873cm-1 returned to their original state, that is, without H2O2 at about 1h, indicating the ability of H-CeO-D@PM to reversibly release peroxide, thereby restoring its crystal structure to its initial stable state. The intensity change process of the peak at 873cm-1 from appearance to disappearance is defined as the antioxidant cycle. As expected, after adding H2O2 again, the peak change in the second cycle was almost the same as the first cycle, indicating that H-CeO-D@PM has a cyclable antioxidant capacity ( Fig. 22 ).

[0132] 1.3.3 Determination of the motion behavior of H-CeO-D@PM nanomotors

[0133] Currently, most nano antioxidants lack the self-propulsion properties that rely on traditional passive penetration, so one of the difficulties in the treatment of ARDS is the poor permeability of NPs at damaged alveolar capillary barrier sites. Antioxidant nanomaterials with hollow mesoporous structures have been reported to have self-propulsion motion properties. Therefore, we investigated nanomotors in H2O2 at different concentrations by using nanoparticle tracking analysis to verify whether H-CeO-D@PM has self-propulsion properties.

[0134] from Fig.23It can be seen that H-CeO-D@PM only exhibits non-directional Brownian motion in a PBS solution without H2O2. However, after adding H2O2, it was found that the motion trajectory of H-CeO-D@PM was different from that of Brownian motion, and the motion of H-CeO-D@PM was positively correlated with the concentration of H2O2. When the concentration of H2O2 was 0.5mM and 1mM, the motion trajectory of H-CeO-D@PM was significantly extended. These may be attributed to the fact that H-CeO-D@PM catalyzes the conversion of H2O2 into O2 and generates a temperature gradient on the outer shell of H-CeO-D@PM, resulting in violent autothermophoresis. Fig.24 As shown in the figure, the MSD was calculated based on the horizontal and vertical coordinates obtained from the motion trajectory of H-CeO-D@PM. The MSD of H-CeO-D@PM at three H2O2 concentrations (0, 0.5 and 1 mM) followed a parabolic trend with increasing time intervals (Δt), indicating that the motion of H-CeO-D@PM may be caused by the self-propelled diffusion of NPs in the presence of H2O2. Fig.25 As shown, the effective diffusion coefficient was calculated according to the equation Deff = MSD / 4·Δt1. Compared with the Brownian motion (0.52μm 2 / s), the effective diffusion coefficient of H-CeO-D@PM increased significantly to 2.13 and 2.79μm2 / s in the case of 0.5 and 1mM H2O2, respectively, indicating that the diffusion amount per unit area of ​​H-CeO-D@PM per second is positively correlated with the H2O2 concentration. Figure 1-26 -b, we also calculated the corresponding average velocity (v), defined as v = s / t (s represents the movement path over a period of time). The H-CeO-D@PM increased from 0.24μm / s in PBS 7.4 (0mM H2O2) to 1.65μm / s in 0.5mM H2O2 and 2.37μm / s in 1mM H2O2, showing the good movement performance of H-CeO-D@PM in H2O2 environment. In general, the behavior of the self-propelled motor of H-CeO-D@PM in the ROS environment is conducive to driving it through the alveolar capillary barrier and expanding the range of motion to achieve more efficient ROS elimination, which can serve as a multifunctional nano-penetrant for the effective treatment of ARDS.

[0135] 1.3.4 Drug release of H-CeO-D@PM

[0136] Casp3 enzyme-responsive DHF (Pep-D) was loaded into H-CeO to achieve sequential treatment of ARDS. Fig.26As shown in the figure, the Pep-D loading capacity of H-CeO as an effective drug carrier at different Pep-D concentrations (31.25, 62.5, 125, 250, 500, 1000 μg / mL) was measured by UV spectrophotometer. The drug loading efficiency of H-CeO was positively correlated with the reaction concentration of Pep-D (31.25-1000 μg / mL). When the concentration of Pep-D was 250 μg / mL, the loading amount of H-CeO reached a plateau and then remained unchanged. According to the maximum drug loading of H-CeO-D@PM and the minimum reaction concentration of Pep-D, 0.25 mg / mL of Pep-D (initial Pep-D concentration was 1 mg / mL) was selected for subsequent research. The calculated drug loading was 36.63% and the encapsulation efficiency was 46.24%. The above results show that H-CeO has a high Pep-D loading efficiency due to its large specific surface area caused by the hollow mesoporous structure. Next, we measured the Casp3-triggered release behavior in vitro using a UV spectrophotometer to further verify whether H-CeO-D@PM has enzyme-responsive drug release behavior. Fig.10 and Fig. 27 -a. Casp3 can trigger drug release when the concentration is as low as 0.1μg / mL, and the amount of drug released is positively correlated with the concentration of Casp3, but a higher Casp3 concentration (1μg / mL) can no longer accelerate its release. This shows that H-CeO-D@PM can be released responsively. Moreover, H-CeO-D@PM shows negligible drug release behavior under H2O2 stimulation, proving the unique Casp3 stimulus responsiveness of H-CeO-D@PM. Fig. 27 -b, 1 μg / ml H-CeO-D@PM and different concentrations of Casp3 (0, 0.01, 0.05, 0.1, 0.5, 1 and 2 μg / ml) were incubated in Casp3 buffer (10 mL) at 37 ° C for 24 h, and the 24-h cumulative release of DHF was calculated by measuring the absorbance value of the supernatant at 207 nm (n = 3). These results indicate that the designed H-CeO-D@PM can provide an on-demand treatment strategy based on the different degrees of apoptosis of lung epithelial cells during the ARDS disease process.

[0137] 1.3.5 Statistical analysis and graphing

[0138] All experiments were repeated three times independently, and the data were expressed as mean ± SEM. Graphpad Prism8.0.2 software was used for statistical analysis and drawing. One-way analysis of variance was used to test the overall significant difference of data from more than two groups. Tukey's multiple comparison analysis method was used to test the significant difference between two groups. P<0.05 was considered to be statistically significant. ns means P>0.05, * means P<0.05, ** means P<0.01, *** means P<0.001

[0139] Embodiment 2:

[0140] Application of H-CeO-D@PM in scavenging reactive oxygen species, promoting alveolar epithelial cell regeneration and repairing alveolar capillary barrier

[0141] 2.1 Cells

[0142] Human umbilical vein endothelial (HUVEC) cell line was purchased from American Type Culture Collection. Murine lung epithelial (MLE) cell line MLE-12 was purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.

[0143] 2.2 Materials

[0144] 2.2.1 Reagents and consumables

[0145] Fetal bovine serum (Gibco, USA), DMEM medium (Gibco, USA), DMEM / F12 medium (Gibco, USA), trypsin (Hyclone, USA), double antibody (Hyclone, USA), cryopreservation solution (Biosharp, China), PBS buffer (Biosharp, China), 30% H2O2 (Biosharp, China), DCFH-DA (Sigma, USA), CCK-8 kit (MCE, USA), AnnexinV-FITC / PI apoptosis detection kit (Lianke Biotechnology, China), EdU cell proliferation detection kit (Biosharp, China), 4% paraformaldehyde (Biosharp, China), 0.3% Triton X-100 (Biyuntian, China), DAPI-containing mounting solution (Biyuntian, China), glutaraldehyde fixative (McLean, China), immunofluorescence primary and secondary antibody diluent (Biyuntian, China), rabbit anti-Ki67 antibody (Abcam, USA), goat anti-rabbit IgG (Abcam, USA), Caspase-3 inhibitor (Biyuntian, China), confocal culture dish (Corning, USA), cell culture flask (Nest, China), centrifuge tube (Nest, China), ELISA plate (Nest, China), cell slide (Biosharp, China), cell culture plate (Corning, USA).

[0146] 2.2.2 Instruments and equipment

[0147] Pipettes of different ranges (Eppendorf, Germany), CO2 cell culture incubator (Thermo Fisher, USA), flow cytometer (Applied Biosystems, USA), multifunctional microplate reader (Thermo Fisher, USA), transmission electron microscope (Shimadzu, Japan), clean bench (Thermo Fisher, USA), centrifuge (Ependorff, Germany), laser scanning confocal microscope (Olympus, Japan).

[0148] 2.3 Methods

[0149] 2.3.1 ROS scavenging ability of H-CeO-D@PM in vitro

[0150] 2.3.1.1 Laser confocal microscopy to detect ROS scavenging ability of H-CeO-D@PM in vitro

[0151] HUVEC were plated at 3 × 10 5The cells were implanted into the confocal culture dish at a density of 100 cells. Until the cells reached 90% fusion. The cells were divided into 5 groups: healthy group, H2O2 group, DHF group, H-CeO@PM group and H-CeO-D@PM group. Subsequently, the DHF group, H-CeO@PM group and H-CeO-D@PM group were treated with fresh culture medium containing 0.075μg / mL DHF, 5μg / mL H-CeO@PM and 5μg / mL H-CeO-D@PM (containing DHF 0.075μg / mL) for 2h. Except for the healthy group, the other groups were added with 200μM H2O2 and cultured at 37℃ for 24h. The next day, DCFH-DA was diluted with serum-free culture medium at a ratio of 1:1000. After incubation with cells for 20min, the cells were washed 3 times with serum-free cell culture medium. The ROS were displayed as green fluorescence using a laser confocal microscope.

[0152] 2.3.1.2 CCK-8 assay to detect the inhibition of H-CeO-D@PM on H2O2-induced cell death. HUVECs were plated at 1.5×10 4 The cells were implanted into a 96-well plate at a density of 100 cells per well until the cells reached 90% fusion. The cell grouping and H2O2 treatment methods were the same as in Example 1. The next day, the old culture medium was discarded, and 100 μL of culture medium containing 10% CCK-8 was added to each well. The 96-well plate was placed in a cell culture incubator and incubated for 2 hours in the dark. The absorbance value of each well at 450 nm was detected by an ELISA reader. The cell viability was calculated using the following formula:

[0153]

[0154] As is the absorbance value of the sample (CCK-8 solution containing cells and H-CeO-D@PM), Ac is the absorbance value of the negative control (CCK-8 solution containing cells and H-CeO-D@PM but not H2O2); Ab is the absorbance value of the blank control (CCK-8 solution without cells and H-CeO-D@PM).

[0155] 2.3.1.3 Flow cytometry to detect the inhibition of H-CeO-D@PM on H2O2-induced apoptosis and necrosis

[0156] HUVECs were plated at 1.5 × 10 5The cells were implanted into a 12-well plate at a density of . Until the cells reached 90% fusion. The cell grouping and H2O2 treatment methods were the same as in Example 1. The next day, after trypsin digestion of the cells, they were centrifuged and washed with pre-cooled PBS to collect all the cells (including the cells in the supernatant). After 5×BindingBuffer was diluted to 1× working solution with double distilled water, 500μl of 1×BindingBuffer was taken to resuspend the cell pellet. 5μlAnnexinV-FITC and 10μlPI were added respectively, and incubated at room temperature away from light for 5 minutes. After the incubation, the cells were detected by flow cytometry through the FITC channel (488nm excitation wavelength, 530nm emission wavelength) and the PI channel (535nm excitation wavelength, 615nm emission wavelength).

[0157] 2.3.2 Ability of H-CeO-D@PM to promote alveolar epithelial cell regeneration in vitro

[0158] 2.3.2.1 Edu method to detect the ability of H-CeO-D@PM to promote alveolar epithelial cell regeneration in vitro

[0159] MLE-12 cells in good condition were collected, and cell slides of appropriate size were placed in a 12-well plate. Then, the MLE-12 cell suspension was plated at 2×10 5The density of cells was gently spread on a 12-well cell crawler until the cells reached 90% confluence. The cells were divided into 6 groups: healthy group, H2O2 group, DHF group, H-CeO@PM group, H-CeO-D@PM+Cas-3inhibitor group and H-CeO-D@PM group. Except for the healthy group, the cells were pretreated with 200μM H2O2 for 2h, and the culture primordium was discarded. Subsequently, fresh culture medium containing 0.075μg / mL DHF, 5μg / mL H-CeO@PM, 5μg / mL H-CeO-D@PM (containing DHF 0.075μg / mL) + 0.35μg / mL Casp3 inhibitor and 5μg / mL H-CeO-D@PM (containing DHF 0.075μg / mL) was added, and fresh culture medium was added to the H2O2 group. Cultured at 37℃ for 24h. The 2× EdU working solution is obtained by diluting EdU in the culture medium at a ratio of 1:500. Add an equal volume of the 2× working solution to a 12-well plate and incubate for 1 hour. Remove the original culture medium, add 500μl 4% paraformaldehyde, and fix for 20 minutes. Wash repeatedly with PBS 3 times. Add 500μl 0.3% TritonX-100 and incubate again at room temperature for 10 minutes. Wash repeatedly with PBS 3 times. Prepare the Click reaction solution. Add 100μl of Click reaction solution to each well and incubate at room temperature for 30 minutes, keeping it away from light. Pour out the reaction solution and wash with PBS for 3 minutes 3 times. Then use the sealing solution containing DAPI stain to co-stain the cell nucleus, observe under a fluorescent inverted microscope, and take pictures to record.

[0160] 2.3.2.2 Ki67 assay to detect the ability of H-CeO-D@PM to promote alveolar epithelial cell regeneration in vitro

[0161] Cell treatment was the same as described above. The fixed and permeabilized samples were blocked with immunostaining blocking solution for 30 minutes. The primary antibody (1:1000) was applied at 4°C overnight and washed with PBS for 5 minutes × 3 times. The secondary antibody (1:500) was incubated at room temperature in the dark for 1 hour and washed with PBS for 5 minutes × 3 times. The slides were sealed with DAPI and nail polish. The slides were stored at 4°C in the dark. The slides were observed and photographed under a fluorescent inverted microscope.

[0162] 2.3.3 Ability of H-CeO-D@PM to repair alveolar capillary barrier model in vitro

[0163] The 12-well transwell was turned upside down and the cell density at the bottom was 2×10 4 After culturing for 12 hours in a cell culture incubator, place the transwell in a matching 12-well plate with the positive orientation and add 2000 μL of fresh DMEM / F12 cell culture medium to the outer groove. The cell density in the inner groove is 2×10 4800μl of MLE-12 per cell / mL. The medium was changed every other day, and the above co-culture system was cultured for no less than 3 days until the stable transendothelial electrical resistance (TEER) of each transwell was measured. The cells were divided into 6 groups: healthy group, H2O2 group, DHF group, H-CeO@PM group, H-CeO-D@PM+Cas-3inhibitor group and H-CeO-D@PM group. Except for the healthy group, the cells were pretreated with 200μM H2O2 for 2h, and the culture primordium was discarded. Subsequently, fresh culture medium containing 5μg / mL of DHFDHF, H-CeO@PM, H-CeO-D@PM+Cas-3inhibitor and H-CeO-D@PM were added respectively, and fresh culture medium was added to the H2O2 group. Cultured at 37℃ for 24h. The TEER test was performed according to the Millicell-ERS instruction manual, with wells without cells inoculated as controls. Each well was measured three times and the average value was taken.

[0164] 2.3.4 Statistical analysis and graphics

[0165] See 1.3.5.

[0166] 2.4 Results

[0167] 2.4.1 ROS scavenging ability of H-CeO-D@PM in vitro

[0168] The present invention uses HUVEC and H2O2 to simulate the process of H-CeO-D@PM ROS removal in vitro ( Fig.28 ).

[0169] like Fig.29 As shown in the figure, after stimulation with 200 μM H2O2, ROS were overexpressed in HUVEC cells, and DCFH-DA showed bright green fluorescence. Fluorescence quantitative analysis showed that DHF had no effect on scavenging ROS, while H-CeO@PM and H-CeO-D@PM could effectively scavenging ROS. Fig.30 As shown, H-CeO@PM and H-CeO-D@PM can effectively protect cells from H2O2-induced damage.

[0170] 2.4.2 Ability of H-CeO-D@PM to promote alveolar epithelial cell regeneration in vitro

[0171] Fig.31 The results of EdU immunofluorescence staining, quantitative analysis, and Ki67 staining showed that compared with simple DHF, the proliferation effect of the H-CeO-D@PM group, which also has ROS scavenging effect, was superior.

[0172] 2.4.3 Ability of H-CeO-D@PM to repair alveolar capillary barrier model in vitro

[0173] Fig.32 It was shown that compared with simple DHF, the H-CeO-D@PM group had a better ability to repair the alveolar capillary barrier.

[0174] Embodiment 3:

[0175] Application of H-CeO-D@PM in treating ARDS in mice

[0176] 3.1 Materials

[0177] 3.1.1 Reagents and consumables

[0178] LPS (Sigma, USA), PBS (Gibco, USA), Podoplanin primary antibody (Invitrogen, USA), rabbit anti-VE-Cadherin antibody (Abcam, USA), rabbit anti-F4 / 80 antibody (CST, USA), goat anti-rabbit IgG (Alexa Fluor594) (Abcam, USA), rabbit anti-CD11b antibody (CST, USA), rabbit two-step detection kit (Zhongshan Jinqiao, China), immunohistochemistry pen (Zhongshan Jinqiao, China), BCA kit (Thermo Fisher, USA), PerCP / Cyanine5.5 anti-mouse Ly-6G (Biolegend, USA), PE anti-mouse CD11b (Biolegend, USA), sodium pentobarbital dry powder (Solaibao, China), mouse IL-6 ELISA kit (Dakowei, China), mouse IL-1β ELISA kit (Dakowei, China), mouse TNF-α ELISA kit (Dakowei, China), SOD activity detection kit (Sigma, USA), DHE staining solution (Sigma, USA), Anti-fluorescence quenching sealing solution (containing DAPI) (Biyuntian, China), hematoxylin staining solution (Boster, China), eosin staining solution (Boster, China), 4% paraformaldehyde (Boster, China), glutaraldehyde electron microscopy solution (McLean, China), anhydrous ethanol (Chengdu Kelon, China), centrifuge tube (NEST, China), OCT embedding medium (Sakura, USA), vacuum blood collection tube (BD, USA), cover glass (Boster, China), anti-slip slide (Shitai, China), neutral resin (Solabo, China), mouse endotracheal intubation tube (Boruida, China).

[0179] 3.1.2 Instruments and equipment

[0180] Vacuum drying oven (Jinghong, China), cell analyzer (Zhuowei, China), pipettes of different ranges (Eppendorf, Germany), paraffin slicer (Leica, Germany), Micro-CT imaging system (Pingsheng Medical, China), constant temperature drying oven (Yiheng, China), ICP-MS (Agilent, USA), microplate reader (Thermo Fisher, USA), fluorescence inverted microscope (Olympus, Japan), ophthalmic scissors (Biyuntian, China), ophthalmic forceps (Biyuntian, China), cryostat (Leica, Germany), transmission electron microscope (Shimadzu, Japan).

[0181] 3.1.3 Experimental animals

[0182] Male C57BL / 6 mice weighing 18–20 g and aged 8–10 weeks were purchased from the Experimental Animal Center of the Army Medical University. All animal experiments were approved by the Animal Ethics Committee of the Army Medical University in accordance with the Guide for the Care and Use of Laboratory Animals of the Army Medical University, No. AMUWEC20224137.

[0183] 3.2 Methods

[0184] 3.2.1 Construction of mouse ARDS model

[0185] C57BL / 6 mice were taken and anesthetized intraperitoneally with 1% sodium pentobarbital. After successful anesthesia, the upper incisors were hung with rubber bands and fixed in a supine position. The skin in front of the trachea was cut open, and the muscles in front of the trachea were bluntly separated with forceps to expose the trachea, and 50μL of 15mg / kg LPS was slowly pushed in.

[0186] 3.2.2 Targeting ability and distribution of H-CeO-D@PM to the lungs in vivo

[0187] The imaging method of H-CeO-D@PM was operated according to the literature with some improvements. H-CeO-D@PM with a concentration of 0.15-10.0 mg / mL was prepared with deionized water and added to a 96-well plate, 100 μL per well, and scanned using micro-CT and recorded CT values. All images were acquired using a micro-CT scanner, which was acquired using 70 kV and 600 μA on a complementary metal oxide semiconductor detector. The region of interest was manually drawn for standard uptake value calculation. All data were analyzed using Avatar3 software.

[0188] The CT signal enhancement of damaged lung tissue by H-CeO-D@PM was detected by micro-CT scanner. Nine C mice were randomly selected, six were used to establish ARDS model, and the other three were injected with PBS instead of LPS during intratracheal injection as sham operation group. 2h after surgery, the model group was randomly divided into two groups, one group was injected with 100μL of 2mg / kg H-CeO-D through the tail vein; the other group was injected with the same dose of H-CeO-D@PM through the tail vein. The sham operation group was injected with the same dose of H-CeO-D@PM through the tail vein. After isoflurane anesthesia, the mice were continuously recorded with chest CT images at 0h, 0.5h, 1h, 4h, 8h and 24h after the tail vein injection. All images were acquired using a micro-CT scanner, which acquired more than 360 projections using 70kV and 600μA on a complementary metal oxide semiconductor detector, and the images were reconstructed using an improved algorithm. CT images of 1 bed (10min) were acquired and reconstructed using ordered subset expectation maximization. Regions of interest were manually drawn for standard uptake value calculations. All data were analyzed using Avatar3 software.

[0189] In order to further verify the CT results, another 9 C57BL / 6 mice were randomly selected and treated according to the above method. Blood was collected through the eye sockets 4 hours after treatment, and the CE content in the blood of different groups 4 hours after treatment was determined by ICP-MS.

[0190] The time-dependent biodistribution of H-CeO-D@PM was measured using ICP-MS to measure the levels of Ce elements in major organs at different times. Fig.33 As shown. Nine C57BL / 6 mice were randomly selected to establish the ARDS model. 2 hours after surgery, 100 μL of 2 mg / kg H-CeO-D@PM was injected into the tail vein. The heart, liver, spleen, lung, kidney and blood of the mice were collected at 4 hours, 24 hours and 48 hours, and 3 mice were selected in each time period. The level of ce was expressed as the percentage of the injected dose per gram of tissue (%ID g-1).

[0191] 3.2.3 The therapeutic effect of H-CeO-D@PM on ARDS mice

[0192] (1) Body weight changes of ARDS mice after H-CeO-D@PM treatment

[0193] like Fig.3421 mice were randomly divided into 7 groups: sham operation group, model group, DHF group, H-CeO@PM group, H-CeO@PM+DHF group, H-CeO-D@PM+Casp3 inhibitor group and H-CeO-D@PM group. Except for the sham operation group, the ARDS model was induced by the method of 1.2.1 in the rest. Among them, the H-CeO-D@PM+Casp3 inhibitor group was injected with 0.135mg / kg of Casp3 inhibitor at the same time as LPS was injected into the trachea. 2h after the model was successfully established, 0.03mg / kg of DHF, 2mg / kg of H-CeO@PM, 1.97mg / kg of H-CeO@PM+0.03mg / kg of DHF, and 2mg / kg of H-CeO-D@PM (containing 0.03mg / kg of DHF) were injected into the tail vein respectively. The sham operation group and the model group were injected with an equal volume of PBS into the tail vein. All mice were weighed before and 12h, 24h, 36h and 48h after injection, and the changes in body weight were recorded.

[0194] (2) Survival curve of ARDS mice treated with H-CeO-D@PM

[0195] 60 mice with successful ARDS modeling were randomly divided into 6 groups: model group, DHF group, H-CeO@PM group, H-CeO@PM+DHF group, H-CeO-D@PM+Casp3 inhibitor group and H-CeO-D@PM group. The treatment method of each group was the same as (1). The death of mice in each group was observed until the mouse died or 5 days after administration. The survival curve was drawn according to the death of mice.

[0196] (3) Lung wet-to-dry weight ratio of ARDS mice treated with H-CeO-D@PM

[0197] Twenty-one mice were selected and grouped and treated in the same manner as (1). 48 hours after the tail vein injection, the lungs of the mice were collected, and the excess water was absorbed with clean filter paper and then accurately weighed on an electronic balance to obtain the wet weight of the lungs. The lungs were dried in a vacuum oven at 65°C for 24 hours until the tissues were free of water to obtain the dry weight. The wet / dry ratio (W / D) was calculated.

[0198] (4) Lung tissue staining of ARDS mice treated with H-CeO-D@PM

[0199] The same experimental treatment as (1) was applied to another batch of mice, and the left lungs of 7 groups of mice were collected, half of which were cut and fixed in 4% paraformaldehyde; dehydrated in ethanol solutions of different concentrations; immersed in xylene solution to make it transparent; immersed in paraffin wax for embedding; the wax block was placed on the sample stage of the microtome to prepare 5 μm thick slices; the tissue slices were placed in a water tank, scooped up with anti-shedding glass slides, and baked in a 40°C oven overnight; the paraffin slices were baked in a 60°C oven for 1 hour and then dewaxed to water.

[0200] HE staining:

[0201] The paraffin sections were placed in hematoxylin staining solution, then rinsed with running water, differentiated with 75% hydrochloric acid alcohol, rinsed with running water to return to blue, placed in eosin staining solution, and rinsed with running water again; the tissue sections were sequentially immersed in ethanol, anhydrous ethanol, and xylene, and finally sealed with neutral resin; the images were observed and collected under an optical microscope.

[0202] Immunofluorescence staining:

[0203] Antigen retrieval: Heat the water bath in advance during hydration, and use 0.01M sodium citrate (PH=6.0) in the water bath for retrieval for 10 minutes. After the buffer solution is cooled (40 minutes), wash with PBS for 5 minutes; Blocking: Block with blocking solution at room temperature for 30 minutes; Apply primary antibody: Dilute the following primary antibodies with primary antibody diluent: PDPN (1:1000), VE-Cadherin (1:1000) and F4 / 80 (1:200). Draw circles with an immunohistochemistry pen, add 100 μL of diluted primary antibody to each slice, place in a humidified box, and incubate at 4°C overnight; apply secondary antibody: take out the slides the next day, warm them up at room temperature for 30 minutes, pour out the solution, soak them in PBS for 5 minutes, 3 times, dilute the secondary antibody corresponding to the primary antibody with secondary antibody diluent, goat anti-mouse IgG (1:1000), goat anti-rabbit IgG (1:1000), incubate at 37°C in the dark for 30 minutes, wash with PBS for 5 minutes, 3 times; seal the slides: use filter paper to absorb the concentrated matter around the tissue, add 20uL of anti-fluorescence quenching sealing agent (containing DAPI), slowly cover the coverslip from one side, and fix the four corners of the coverslip with nail polish; shoot: collect images with a laser confocal microscope.

[0204] Immunohistochemical staining:

[0205] The antigen repair steps were the same as before; according to the product instructions, IHC staining was performed using the rabbit two-step detection kit. After adding the endogenous peroxidase blocker, incubate for 10 minutes, rinse with PBS three times; block with blocking solution at room temperature for 30 minutes; apply the primary antibody: CD11b (1:200), draw a circle with an immunohistochemistry pen, add 100 μL of the diluted primary antibody, incubate for 60 minutes, and rinse with PBS three times; add 100 μL of reaction enhancement solution, incubate for 20 minutes, and rinse with PBS three times; add 100 μL of enhanced enzyme-labeled goat anti-rabbit IgG polymer, incubate for 20 minutes, and rinse with PBS three times; add DAB color development solution, incubate for 5 minutes; rinse with tap water, incubate with hematoxylin for 20 seconds, rinse with running water, differentiate with 75% hydrochloric acid alcohol, and then rinse with running water to return to blue; immerse the tissue sections in ethanol, anhydrous ethanol, and xylene in turn, and finally seal with neutral resin; observe and collect images under an optical microscope.

[0206] (5) Dihydroethidium (DHE) staining of lung tissue of ARDS mice treated with H-CeO-D@PM

[0207] The right lungs of the 7 groups of mice in (4) were collected and placed on ice; the tissues were quickly frozen in liquid nitrogen; the tissue blocks were then placed in OCT embedding medium to prepare frozen sections with a thickness of 8 μm. After rewarming at room temperature, they were washed with PBS three times; the DHE dye solution was diluted with PBS to a working concentration of 5 μM, added to the sections, incubated at 37°C in the dark for 20 min, and rinsed with PBS three times; the sections were sealed: 20 μL of anti-fluorescence quenching sealing agent (containing DAPI) was added, and a coverslip was slowly applied from one side, and the four corners of the coverslip were fixed with nail polish. Images were collected using a laser confocal microscope.

[0208] (6) Collection and analysis of BALF and lung tissues from ARDS mice treated with H-CeO-D@PM

[0209] To collect BALF from another batch of mice, the grouping and processing were performed as described in (1). Briefly, the mice were anesthetized and suspended by their incisors, fixed, and the trachea was cut open. 500 μL of pre-cooled PBS was slowly injected through the endotracheal cannula each time for a total of 3 lavages, ensuring that the aspiration rate was greater than 60% each time. After BALF lavage, the lungs of the mice were collected, dried with filter paper, and stored at low temperature. 500 μL of BALF was centrifuged at 450 × g for 5 min at 4°C, the supernatant was removed, resuspended in 200 μL of red blood cell lysis buffer, and lysed on ice for 15 min. After resuspending in 500 μL of PBS, 10 μL was aspirated and the number of nucleated cells was determined on a cell analyzer. The remaining BALF was added with antibodies Ly6G (1:200) and CD11b (1:200). Protected from light, incubated on ice for 30 min, and the proportion of neutrophils was analyzed by flow cytometry. The remaining BALF was aliquoted and stored at low temperature. The protein concentration of BALF was determined by BCA kit. The inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in BALF and lung tissue were measured by ELISA kits. The superoxide dismutase (SOD) activity in lung tissue was measured by SOD activity detection kit.

[0210] 3.3 Results

[0211] 3.3.1 Targeting ability and distribution of H-CeO-D@PM to the lungs in vivo

[0212] Pulmonary capillary endothelial damage is a key feature of ARDS. Endothelial damage causes platelets to be mobilized to the site of inflammation and interact with leukocytes by secreting adhesion molecules such as P-selectin and GPIIb / IIIa to close the gaps between endothelial cells and ensure the integrity of inflamed vessels. To verify whether platelet membrane-wrapped NPs can target damaged sites, we evaluated the targeting ability of H-CeO-D@PM in ARDS mice by chest CT. Chest CT is also one of the most intuitive and commonly used methods for evaluating lung conditions clinically. The k-edge of Ce is 40.4keV, slightly higher than iodine (33.2keV), which matches well with the X-ray beam used in medical imaging. Therefore, H-CeO-D@PM can produce strong CT contrast and can be used as a CT contrast agent. The CT attenuation rates of different concentrations of H-CeO-D@PM are shown in Figure 2. Fig.35 -a as shown.

[0213] like Fig.35-b, from left to right, are the CT cross-section, coronal plane, and lung three-dimensional reconstruction images of the model + H-CeO-D@PM 8 hours after intravenous injection. It can be seen that the coronal plane shows the largest vertical range of the lung, so we chose it to measure the CT value of the lung. First, mice were injected intrabronchially with LPS, which is the most commonly used inducer of ARDS in the laboratory. NPs were administered intravenously through the tail vein 2h after modeling. This method is also closer to clinical treatment, that is, treatment is started after the disease occurs. Then, the lungs were imaged by chest CT to visualize the transport of NPs in the lungs ( Fig.36 -a), and quantified by ΔCT value, which was obtained by subtracting the CT value at 0h from the CT value at the corresponding time ( Fig.36 -b).

[0214] Regardless of the NP type, ce gradually accumulated in the lungs of mice within 1 h after intravenous injection. However, the ΔCT value of the model + H-CeO-D@PM group continued to increase and reached a peak at 4 h after injection, indicating that ce accumulated in the damaged lungs in a time-dependent manner. It is worth noting that the peak ΔCT of H-CeO-D@PM in model mice was twice that of H-CeO-D in model mice, and then gradually decreased, indicating that it was the PM coating that significantly promoted the continuous accumulation of H-CeO-D in inflamed lung tissues. In addition, the peak ΔCT of H-CeO-D@PM in model mice was 3.3 times that of H-CeO-D@PM in sham-operated mice, indicating the selective distribution of H-CeO-D@PM and its ability to target damaged blood vessels. Fig.37 As shown in Figure 5A, ICP-MS results showed that 4 hours after intravenous injection of NPs, the ce content of H-CeO-D@PM in model mice was almost twice that of H-CeO-D@PM in model mice and 5 times that of H-CeO-D@PM in sham-operated mice, indicating that H-CeO-D@PM increased alveolar-capillary permeability and retention in damaged lungs.

[0215] To evaluate the distribution of H-CeO-D@PM, mice were euthanized at 4, 24, and 48 h after NPs injection, and the heart, kidney, liver, lung, spleen, and blood were collected to test the ce content by ICP-MS. Fig.37-b, at 4h, Ce was mainly distributed in the lungs (64.52%ID g-1). In addition to the targeted lungs, a large amount of NPs were also accumulated in the spleen (11.55%ID g-1) and liver (11.30%ID g-1), which was attributed to the low blood flow rate and the presence of a large number of phagocytes in these organs, which is consistent with previous studies exploring the organ distribution of CeO nanoparticles. 24h after injection, the Ce content accumulated in the main organs of mice was significantly reduced (lung 12.33%ID g-1, spleen 3.04%ID g-1, liver 3.37%ID g-1). At 48h, the Ce content in the lungs was still 7.26%ID g-1, which may be due to the delayed clearance of NPs after PM coating.

[0216] 3.3.2 Therapeutic effect of H-CeO-D@PM on ARDS mice

[0217] 3.3.2.1 Body weight changes, survival curves, lung wet-to-dry weight ratio, and protein concentration in BALF of ARDS mice treated with H-CeO-D@PM

[0218] To verify the therapeutic effect of H-CeO-D@PM in treating the adaptive mechanism of ARDS mice, a comparative study of sequential regulation was performed. Two hours after LPS modeling, DHF, H-CeO-D@PM, H-CeO-D@PM+Casp3 inhibitor, H-CeO-D@PM+DHF, and H-CeO-D@PM were injected through the tail vein, and then the body weight and mortality were calculated. Fig.38 -a Compared with sham-operated mice, the body weight of ARDS mice decreased by 23.23% below the baseline at 48 h. After treatment, we observed that the mice gained weight, among which the body weight of the H-CeO-D@PM-treated group returned to the level closest to the sham-operated group.

[0219] ARDS is a complex and fatal disease with a mortality rate of 38% to 46%. Fig.38-b, about 60% of ARDS mice died within 5 days. The survival rates of DHF and H-CeO@PM treatment increased to 56.25% and 59.06%, respectively. The survival rate of the H-CeO-D@PM treatment group increased significantly to 100%. It is well known that timely treatment at different stages after injury is the key to improving the therapeutic effect of ARDS. However, individual differences between ARDS patients make it difficult to seize the opportunity to accurately regulate and promote epithelial cell proliferation. Casp3 is a reliable indicator of alveolar-capillary barrier damage and can achieve on-demand drug release. In order to further verify the importance of pathogenesis adaptive sequential treatment, two other different administration methods were used for treatment: H-CeO-D@PM+Casp3 inhibitor and H-CeO@PM+DHF. We found that since Casp3 was inhibited, the Casp3-responsive peptide was not cleaved and the proliferation drug (DHF) could not be released, so the survival rate of H-CeO-D@PM+Casp3 inhibitor was 60%, close to that of the H-CeO@PM group, indicating the importance of combined treatment of ROS scavenging and DHF-proliferation. The survival rate of H-CeO@PM+DHF was 70%, still lower than that of H-CeO-D@PM.

[0220] Lung tissue and BALF were collected at predetermined times according to the experimental design. ARDS is characterized by increased permeability of the capillary endothelial-alveolar epithelial barrier, with protein-rich edema fluid flowing into previously air-only spaces. The lung dry-to-wet ratio is a typical indicator of pulmonary edema. Fig.38 -c showed that the model group had a higher W / D ratio (5.14) compared with the sham group (W / D 4.48), indicating significant pulmonary edema. Compared with the model group, the DHF (4.90) and H-CeO@PM (4.81) groups showed a certain degree of edema inhibition. Importantly, H-CeO-D@PM treatment significantly reduced W / D to 4.54, close to normal levels. The W / D of H-CeO-D@PM+Casp3 inhibitor and H-CeO@PM+DHF were 4.86 and 4.75, respectively, both higher than those of the H-CeO-D@PM group. According to the cause of pulmonary edema, the total protein concentration in BALF was further examined ( Fig.38 -d), also confirmed this trend.

[0221] 3.3.2.2 Proportion of neutrophils in BALF of ARDS mice treated with H-CeO-D@PM

[0222] ARDS is characterized by an influx of protein- and cell-rich edema fluid into the alveoli due to increased permeability of the capillary endothelial alveolar epithelial barrier. Therefore, nucleated cells, mainly neutrophils, macrophages, and monocytes, were collected from BALF to detect lung inflammation and alveolar permeability ( Fig.39-a). CD11b+, Ly6G+ cells in BALF measured by flow cytometry (upper right quadrant), showing the percentage of neutrophils among all cells, and quantified by Flow-J. Fig.39 -b, c. Almost no neutrophils were found in the BALF of the sham group (1.97%). The percentage of neutrophils increased significantly after LPS stimulation to 82.3%, confirming that the LPS-induced ARDS model is neutrophil-dependent. The corresponding percentages of neutrophils decreased slightly after the use of DHF and H-CeO@PM, which were 77.3% and 72.4%, respectively. Compared with the model group, the percentage of neutrophils in the H-CeO-D@PM treatment group was significantly reduced to 48.7%. In addition, the percentage of neutrophils in the H-CeO-D@PM+Casp3 inhibitor group was 65.5%, which was similar to that in the H-CeO@PM group, indicating that the combination treatment was more effective than monotherapy. The percentage of neutrophils in the H-CeO@PM+DHF group was 62.0%, and the treatment effect was lower than that in the H-CeO-D@PM group, indicating that the sequential treatment combination can reduce neutrophil infiltration more effectively than the simple combination. The conclusion was further supported when we examined the concentration of nucleated cells in BALF.

[0223] 3.3.2.3 HE staining of lung tissue and lung injury score of ARDS mice treated with H-CeO-D@PM

[0224] like Fig.40 After different treatments, H-CeO-D@PM showed significant improvement, inhibited interstitial and alveolar edema, and reduced the infiltration of inflammatory cells.

[0225] 3.3.2.4 DHE staining and SOD activity of lung tissues of ARDS mice treated with H-CeO-D@PM

[0226] like Fig.41 After different treatments, the results showed that H-CeO@PM, H-CeO-D@PM+Casp3 inhibitor, H-CeO@PM+DHF, and H-CeO-D@PM had the ability to scavenge ROS derived from H-CeO, thereby reducing DHE and maintaining SOD activity in vivo.

[0227] 3.3.2.5 F4 / 80 and CD11b staining of lung tissues of ARDS mice treated with H-CeO-D@PM

[0228] like Fig.42 As shown, in H-CeO-D@PM, the sequential binding of H-CeO@PM and DHF via Casp3-responsive peptides showed great potential in reducing immune cell infiltration.

[0229] 3.3.2.6 Expression of inflammatory factors in BALF and lung tissues of ARDS mice treated with H-CeO-D@PM

[0230] like Fig.43 , TNF-α, IL-6, and IL-1β in BALF and lung tissues of ARDS mice were higher than those in the sham operation group, and improved after different treatments, among which H-CeO-D@PM had the largest decrease.

[0231] 3.3.2.7 PDPN staining of lung tissues of ARDS mice treated with H-CeO-D@PM

[0232] like Fig.44 As shown, H-CeO-D@PM promoted the proliferation of AT1 most effectively compared with other groups.

[0233] 3.3.2.8 VE-Cadherin staining of lung tissues of ARDS mice treated with H-CeO-D@PM

[0234] like Fig.45 As shown, the expression of connexin in the H-CeO-D@PM treatment group was the highest compared with other groups.

[0235] Embodiment 4:

[0236] In vivo and in vitro biosafety studies of H-CeO-D@PM.

[0237] 4.1 Materials

[0238] 4.1.1 Reagents and consumables

[0239] CCK8 kit (MCE, USA), hematoxylin staining solution (Boster, China), eosin staining solution (Boster, China), 24-well and 96-well cell culture plates (Corning, USA)

[0240] 4.1.2 Instruments and equipment

[0241] Pipettes of different ranges (Eppendorf, Germany), multifunctional microplate reader (Thermo Fisher, USA), CO2 cell culture incubator (Thermo Fisher, USA), cell culture clean bench (Thermo Fisher, USA), fully automatic blood cell analyzer (Beckman Coulter, USA), and fully automatic biochemical analyzer (Beckman Coulter, USA).

[0242] 4.1.3 Experimental animals See 1.1.3 and 3.1.3.

[0243] 4.2 Methods

[0244] 4.2.1 In vitro toxicity study of H-CeO-D@PM

[0245] (1) CCK-8 method to detect the cytotoxicity of H-CeO-D@PM

[0246] HUVECs were plated at 1.5 × 10 4 The cells were inoculated into a 96-well plate at a density of 100 cells / mL and cultured overnight until the cells reached 90% fusion. A negative control group and H-CeO-D@PM groups with concentrations of 5, 10, 25, 50 and 100 μg / mL were set up, with 3 replicates in each group. MLE-12 was inoculated in the same way. The 96-well plate was placed in a cell culture incubator and cultured for 24 hours. After 24 hours, the old culture medium was aspirated with a pipette, and CCK8 mixed with the culture medium at a concentration of 10:1 was added and continued to be incubated in the dark for 2 hours. The 96-well plate was placed in a multifunctional microplate reader to detect the OD value at 450 nm.

[0247] (2) Study on blood compatibility of H-CeO-D@PM

[0248] Take 2mL of SD rat whole blood and add it to a centrifuge tube. Centrifuge at 1500rpm for 15min, remove the supernatant and keep the red blood cell pellet; wash the red blood cells with saline and centrifuge at 1500rpm for 15min. Repeat the washing for 3 times; dilute 2mL of red blood cell pellet in 8mL of saline; take 500μL of red blood cells and add them to 500μL of H-CeO-D@PM (10, 25, 50 and 100μg / mL) with different concentrations; place the mixed solution of each experimental group, negative control group and positive control group in a 37℃ cell culture incubator for 3h; centrifuge at 12000rpm for 15min, take photos and record; take 100μL of supernatant and add it to a 96-well flat-bottom plate, and use an enzyme marker to detect the OD value at 540nm.

[0249] (3) In vivo toxicity study of H-CeO-D@PM

[0250] Six C57BL / 6 mice were randomly divided into a control group and a H-CeO-D@PM group, with 3 mice in each group; the mice in the H-CeO-D@PM group were injected with 4 mg / kg and 100 μL of PBS into the tail vein, and the mice in the control group were injected with 100 μL of PBS into the tail vein; 48 hours later, the mice were killed, and about 1 mL of whole blood was collected, 20 μL of which was taken out and the blood routine indicators of the mice were detected using a fully automatic hematology analyzer; the remaining blood was centrifuged at 3000 rpm for 5 min, and the serum was taken out, and the levels of BUN, CRE, ALT, and AST were detected using a fully automatic biochemical analyzer; the heart, liver, spleen, lung, and kidney were collected for HE section staining.

[0251] 4.2.2 Statistical analysis and graphing

[0252] See 1.3.5.

[0253] 4.3 Results

[0254] 4.3.1 In vitro toxicity study of H-CeO-D@PM

[0255] 4.3.1.1 Detection of cytotoxicity of H-CeO-D@PM by CCK-8 method

[0256] The in vitro cytotoxicity of H-CeO-D@PM was tested by measuring cell viability using the CCK-8 method. The experimental results of co-culture of H-CeO-D@PM with different test concentrations and HUVEC and MLE-12 are shown in Figure 2. Fig.46 As shown, after 24 h, H-CeO-D@PM had no significant effect on the viability of HUVEC and MLE-12 cells in the concentration range of 5-100 μg / mL.

[0257] 4.3.1.2 Study on blood compatibility of H-CeO-D@PM

[0258] The in vitro blood compatibility results of H-CeO-D@PM with different concentrations are shown in Figure 2. Fig.47 As shown, the hemolysis rate of H-CeO-D@PM in the concentration range of 10-100 μg / mL is less than 5%, which shows that it has good blood compatibility and can be safely administered by intravenous injection.

[0259] 4.3.2 In vivo toxicity study of H-CeO-D@PM

[0260] The results are as follows Fig.48 Results showed that 48 hours after tail vein injection of 4 mg / kg (twice the dose for treating ARDS in vivo), the results of whole blood cell analysis showed that there were no statistical differences in blood routine related parameters between the H-CeO-D@PM treatment group and the control group (P>0.05), indicating that intravenous injection of 4 mg / kg H-CeO@PM had no obvious acute in vivo toxicity. 48 hours after tail vein injection of H-CeO-D@PM, there were no statistical differences in the concentrations of creatinine and urea nitrogen representing renal function, and serum aspartate aminotransferase and alanine aminotransferase representing liver function between the H-CeO-D@PM treatment group and the control group (P>0.05). Fig.49 The results showed that in the visceral HE sections of mice, no pathological changes such as tissue necrosis, edema, congestion and hemorrhage were found in the heart, liver, spleen, lung and kidney, indicating that intravenous injection of 4 mg / kg H-CeO-D@PM for 48 hours had no obvious acute toxic effects on the main internal organs.

[0261] 4.4 Conclusion

[0262] CCK-8 results showed that H-CeO-D@PM had no significant effect on the survival rate of HUVEC and MLE-12 cells in the concentration range of 5-100μg / mL. The in vivo biosafety of H-CeO-D@PM was further evaluated by in vivo tail vein injection. 48h after tail vein injection of 4mg / kg (twice the in vivo dose for ARDS treatment) of H-CeO-D@PM, no pathological damage was found in the main organs of mice, and there were no abnormal changes in blood routine and liver and kidney function indicators. These results collectively indicate that the therapeutic dose of H-CeO-D@PM has good biosafety. H-CeO-D@PM has no obvious in vitro cytotoxicity and good blood compatibility in the concentration range of 100μg / mL. H-CeO-D@PM has no obvious in vivo toxicity in the dose range of 4mg / kg.

[0263] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A new type of H2O2-driven nanomotor H-CeO-D@PM, characterized by: The novel H2O2-driven nanomotor H-CeO-D@PM has a hollow mesoporous structure and presents uniformly distributed and regularly shaped spherical particles with a hydrodynamic diameter of 224 nm.

2. The method for preparing a novel H2O2-driven nanomotor H-CeO-D@PM according to claim 1, characterized in that: The preparation method of the novel H2O2-driven nanomotor H-CeO-D@PM comprises the following steps: S1: Preparation of silica NPs by the classic Stober method; S2: preparing H-CeO NPs by mixing the prepared silica NPs with cerium (III) acetate hydrate; S3: Pep-D is dissolved in a mixed solution of DMSO and water, and after the reaction, H-CeO loaded with Pep-D, i.e., H-CeO-D, is collected, and unreacted Pep-D is removed. S4: Extract PM by repeated freeze-thaw method.

3. The method for preparing a novel H2O2-driven nanomotor H-CeO-D@PM according to claim 2, characterized in that: The S1 specifically includes the following steps: S11: A mixed solution containing 8.4 mL of 28-30 wt % ammonium hydroxide, 280 mL of anhydrous ethanol and 56 mL of deionized water was stirred at room temperature for 0.5 h; S12: Then, 8 ml of LTEOS was quickly added to the mixture under vigorous stirring and reacted for 24 h; S13: The product was collected by centrifugation at 8000 rpm for 15 min and washed 3 times with ethanol and water for further use.

4. The method for preparing a novel H2O2-driven nanomotor H-CeO-D@PM according to claim 2, characterized in that: The S2 specifically includes the following steps: S21: 3 mg / mL silica NPs aqueous solution and 5 mg / mL cerium (III) acetate hydrate aqueous solution were mixed together in a 100 mL beaker and stirred for 0.5 h to form a homogeneous solution; S22: The mixture was transferred into a 100 mL polytetrafluoroethylene-lined stainless steel autoclave and heated at 160 °C for 16 h. The product was collected by centrifugation at 5000 rpm for 10 min and washed three times with anhydrous ethanol and water. Subsequently, the excess silica was etched by incubation with 5 mol / L sodium hydroxide for 2 days and washed three times with deionized water to obtain H-CeO.

5. The method for preparing a novel H2O2-driven nanomotor H-CeO-D@PM according to claim 2, characterized in that: The S3 specifically includes the following steps: S31: dissolving Pep-D in a mixed solution of DMSO and water, wherein the ratio of DMSO to water is 1:4, to prepare Pep-D solutions of different concentrations ranging from 0 to 1 mg / mL; S32: H-CeO was mixed with it and stirred for reaction at room temperature; after 24 hours, H-CeO loaded with Pep-D, i.e., H-CeO-D, was collected by repeated centrifugation with deionized water for 3 times, each time at 5000 rpm for 10 min, and unreacted Pep-D was removed.

6. The method for preparing a novel H2O2-driven nanomotor H-CeO-D@PM according to claim 2, characterized in that: The S4 comprises the following steps: S41: Dissolve the mixed protease inhibitor tablets in PBS to prepare protease inhibitor buffer; S42: Fresh SD rat platelets were dispensed into 1 mL / tube, centrifuged at 800 × g for 20 min, the supernatant was discarded, and the precipitate was resuspended in the mixed protease inhibitor buffer prepared in S41; S43: The suspension obtained by resuspending S42 was frozen at -80°C for 30 min, thawed at room temperature, centrifuged at 5000×g for 5 min, the supernatant was discarded, and the precipitate was resuspended in mixed protease inhibitor buffer; S44: Repeat S42-S41 three times, collect the platelet membrane vesicles purified by centrifugation and mix them with H-CeO-D, and use an Avanti micro extruder to extrude the platelet membrane vesicles and H-CeO-D mixture through a 200 nm polycarbonate membrane to produce a final product.

7. The use of a novel H2O2-driven nanomotor H-CeO-D@PM according to claim 1 in the preparation of a drug for treating acute respiratory distress syndrome, characterized in that: The H-CeO-D@PM nanomotor performs self-propelled motion in a physiological H2O2 solution.

8. The use of a novel H2O2-driven nanomotor H-CeO-D@PM according to claim 1 in the preparation of a drug for treating acute respiratory distress syndrome, characterized in that: The H-CeO-D@PM achieves therapeutic purposes by responsively releasing drugs.

9. The use of a novel H2O2-driven nanomotor H-CeO-D@PM according to claim 1 in the preparation of a drug for treating acute respiratory distress syndrome, characterized in that: The H-CeO-D@PM achieves the therapeutic purpose by efficiently removing ROS, promoting the regeneration of alveolar cells and the repair of the alveolar capillary barrier.

10. The use of a novel H2O2-driven nanomotor H-CeO-D@PM according to claim 1 in the preparation of a drug for treating acute respiratory distress syndrome, characterized in that: The H-CeO-D@PM targets lung tissue, sequentially removes ROS, promotes alveolar cell regeneration, and repairs the alveolar capillary barrier, thereby achieving effective treatment of ARDS.

11. The use of a novel H2O2-driven nanomotor H-CeO-D@PM according to claim 1 in the preparation of a drug for treating acute respiratory distress syndrome, characterized in that: The routes of drug administration include intravenous systemic administration, inhalation and oral administration.

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