Nitrogen-doped carbon quantum dot coupled ICAM-1 monoclonal antibody nano-composite and transformation application method of nitrogen-doped carbon quantum dot coupled ICAM-1 monoclonal antibody nano-composite in case of influence of manganese on progress of sepsis

Abstract

The invention provides a nano-composite of a nitrogen-doped carbon quantum dot coupled ICAM-1 monoclonal antibody and a transformation application method of the nano-composite in the situation that manganese affects the progress of sepsis, and relates to the technical field of biological medicine. The nano complex is a fluorescent carbon nano particle coupled monoclonal antibody. According to the nano complex of the nitrogen-doped carbon quantum dot coupled ICAM-1 monoclonal antibody, a nano material nitrogen-doped carbon quantum dot is taken as a carrier, ICAM-1 is taken as a targeting molecule, a targeting nano carrier ICAM-1 (at) N-CDs is formed through covalent coupling, the nitrogen-doped carbon quantum dot is delivered into an inflammation part, excessive ROS in and out of endothelial cells and macrophages is rapidly and efficiently eliminated, and the inflammation part of the endothelial cells and the macrophages is inhibited. The inflammatory microenvironment of a focus part is improved, and the treatment effect of ALI / ARDS and septicopyemia is improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a nanocomplex of nitrogen-doped carbon quantum dots coupled with ICAM-1 monoclonal antibodies and a method for transforming and applying the nanocomplex in the process of manganese-induced sepsis progression. Background Art

[0002] Sepsis is a life-threatening organ dysfunction syndrome caused by a dysregulated host response to infection. It is characterized by a systemic inflammatory response syndrome that can lead to organ damage, organ failure, septic shock, and even death. Almost all pathogens can cause sepsis. The most common site of infection leading to sepsis is the lungs (64%), followed by the abdomen (20%), bloodstream (15%), and kidneys and urogenital tract (14%). The pathophysiological mechanism of sepsis is complex. When pathogens invade the host, they induce an immune response that cannot be restored to the physiological balance in the body, resulting in excessive inflammatory response and immunosuppression. Organ dysfunction caused by sepsis involves multiple mechanisms, including endothelial and microvascular dysfunction, immune and autonomic nervous system regulation disorders, and cell metabolic reprogramming. Endothelial and microvascular dysfunction: One of the important characteristics of sepsis is microvascular dysfunction. Sepsis can affect various aspects of endothelial cell function, which is a key factor in the progression of sepsis to organ failure. During sepsis, endothelial cells shift to a pro-apoptotic, pro-inflammatory, pro-adhesive, and pro-coagulant phenotype. In addition, changes in endothelial cells include increased leukocyte adhesion, vasodilation, and loss of barrier function, leading to widespread tissue edema. In sepsis, endothelial dysfunction is induced by damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs), oxidative stress, and altered nitric oxide production. Endothelial cells are both targets and sources of inflammatory responses, and they also serve as a bridge between local and systemic immune responses. In response to cytokines produced by immune cells, endothelial cells express adhesion molecules and produce vasoactive compounds, inflammatory cytokines, and chemokines, thereby shifting from an anticoagulant to a procoagulant state. In addition, microcirculatory disturbances lead to a decrease in the proportion of perfused vessels, an increase in the proportion of vessels with poor blood flow, a decrease in total vascular density, and an increase in the heterogeneity of blood flow distribution in patients with sepsis.

[0003] Oxidative stress plays an important role in the development of ALI / ARDS. When the body is stimulated by ALI / ARDS risk factors, excessive reactive oxygen species (ROS) including free radicals are produced. Normally, cells express various proteins such as superoxide dismutase (SOD), glutamate-cysteine ​​ligase catalytic subunit (GCLC), NAD(P)H, quinone-1 (NQO1), catalase (CAT), glutathione peroxidase (GSH-Px), and heme oxygenase 1 (HO-1) that scavenge ROS to protect cells from oxidant-induced damage. However, excess ROS that exceeds the scavenging capacity of antioxidants can lead to unsaturation of fatty acids in cell membranes, reduce membrane fluidity and increase membrane permeability, leading to pulmonary edema and lung expansion. Excessive ROS produced by damaged endothelium / epithelium and recruited leukocytes play a major role in ARDS progression and lung injury. ROS can upregulate the expression of proinflammatory cytokines and adhesion molecules, amplifying tissue damage and pulmonary edema. ROS have several different effects on the endothelial barrier. Initially, in the transcellular pathway, caveolin-1 is affected by ROS, resulting in increased vascular permeability. In addition, increasing evidence supports that the extravasation of polymorphonuclear leukocytes (PMNs) induced by inflammatory stimuli is regulated by oxidative stress. First, the upregulated level of ROS produced by damaged tissues can act as a chemoattractant for immune cells. Large amounts of ROS production can lead to an enhanced ability of immune cells to invade damaged tissues. ROS also regulate the expression of cell adhesion molecules (CAMs) directly and through transcription factors (NF-κB, AP-1) that have a significant impact on PMN migration. There are a variety of enzyme systems in the body that can neutralize free radicals and ROS through catalytic reactions, and these form endogenous defense mechanisms in the body to prevent oxidant-induced cell damage, such as superoxide dismutase (SOD), catalase, glutathione and related enzymes. Proper oxidant-antioxidant balance is essential for vascular homeostasis, so systems that lead to excessive ROS production can become targets for ARDS treatment. Oxidative stress is caused by excessive ROS production or insufficient antioxidant defense capacity, both of which can occur in sepsis. Oxidative stress is closely related to the pathological process of sepsis and is a key factor affecting the severity of sepsis. Studies have shown that patients with sepsis who died had higher levels of oxidative stress than those who survived. In addition, the levels of the main antioxidants GSH and catalase decreased in patients with sepsis who died, and the GSH level of patients who died showed a downward trend over time, while the GSH level of patients who survived remained unchanged. In sepsis, some oxidized products of ROS have a protective effect in acute inflammatory states. Neutrophils can exert antibacterial effects through oxidants, proteases, and antimicrobial peptides. The oxidants ROS and RNS produced by neutrophils can not only directly kill microbial pathogens, but also act as signal molecules to participate in the regulation of multiple physiological signaling pathways, further regulating the expression of key cytokines and chemokines of inflammatory responses.In the inflammatory response, ROS and RNS can regulate phagocytosis, secretion, gene expression, and apoptosis. In addition, in sepsis, oxidative stress can promote adaptive responses to hypoxia and repair of the endothelium after injury. However, excessive oxidants are produced in sepsis, and the oxidative burst in plasma occurs in the early stage of sepsis and is regulated by neutrophils, macrophages, and endothelial cells. These cells are the first line of defense against microbial invasion, and the ROS and RNS they produce are important for host defense, as demonstrated in studies of mice with insufficient O2- production, which is associated with decreased bacterial clearance. However, persistent and excessive production of ROS and RNS is associated with increased morbidity and mortality in sepsis. ROS is an important mediator of cell damage, which can cause oxidative damage to all cellular components and participate in the development of sepsis. The proinflammatory properties of ROS include endothelial cell injury, chemokine formation, neutrophil recruitment, lipid peroxidation and oxidation, DNA damage, release of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) 1β, and formation of hyperoxic nitrite. In sepsis, there is an imbalance between ROS and RNS and the antioxidant system. In sepsis, immune activation initiates a redox cascade that includes sustained production of ROS and RNS, activation of DNA transcription, and impairment of mitochondrial function, ultimately leading to multi-organ dysfunction and death. NO and ROS together directly inhibit the electron transport chain within mitochondria, resulting in impaired energy production and accumulation of mitochondrial superoxide. Mitochondrial dysfunction is at the core of the deleterious proinflammatory and prooxidative pathways in sepsis.

[0004] Intercellular cell adhesion molecule-1 (ICAM-1), also known as CD54, is a type of transmembrane glycoprotein that is mainly expressed on the luminal surface of endothelial cells. ICAM-1 is expressed at a low level on resting endothelial cells, and its expression is significantly increased when the endothelium is in an inflammatory pathological state. Studies have reported that the level of ICAM-1 measured in plasma is also positively correlated with the development of ALI symptoms. ICAM-1 can mediate the adhesion of fibrinogen to leukocytes and vascular endothelium, the adhesion and transmembrane migration of neutrophils, and the migration of lymphocytes. By affecting inflammatory mediators, leukocytes, etc., it recruits neutrophils circulating in the blood to migrate to tissues and organs, thereby accelerating the progression of ALI / ARDS. By using the specific binding of ICAM-1 monoclonal antibody to ICAM-1 protein on the surface of vascular endothelial cells, a nano drug delivery system with targeting to the pulmonary vascular endothelium overexpressing ICAM-1 under the pathological state of ALI is constructed, which is expected to promote the efficient delivery of drugs in inflamed lungs and improve the long-term efficacy of drugs.

[0005] In order to promote the aggregation of nitrogen-doped carbon quantum dots at the inflammation site and improve the efficiency of removing reactive oxygen species (ROS), the nitrogen-doped carbon quantum dots are delivered to the inflammation site by coupling with ICAM-1 monoclonal antibody for targeted positioning. In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a nanocomposite of nitrogen-doped carbon quantum dots coupled with ICAM-1 monoclonal antibody and its transformation and application method in the process of manganese affecting the progression of sepsis. The nanocomposite of the present invention uses nanomaterial nitrogen-doped carbon quantum dots as carriers and ICAM-1 as targeting molecules, and the targeted nanocarrier N-CDs-ICAM-1 is formed by covalent coupling, and its particle size is 8.08±0.18nm. The nanocomposite of the present invention can target the inflammatory site of the lung through the ICAM-1 antibody, and quickly and efficiently eliminate the excessive ROS inside and outside the endothelial cells and macrophages to improve the inflammatory microenvironment of the lesion site and improve the therapeutic effect of ALI / ARDS.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A nanocomposite of nitrogen-doped carbon quantum dots coupled with ICAM-1 monoclonal antibody. The nitrogen-doped carbon quantum dots coupled with ICAM-1 monoclonal antibody uses nanomaterial nitrogen-doped carbon quantum dots as carriers and ICAM-1 as a targeting molecule to form a targeting nanocarrier ICAM-1@N-CDs through covalent coupling.

[0009] The nitrogen-doped carbon quantum dots are prepared by the following method: 4.2 g of citric acid and 0.2 g of arginine are dissolved in 20 ml of ultrapure water, stirred and mixed evenly, and nitrogen is introduced into the solution to remove dissolved oxygen in the solution; the obtained solution is transferred to a reactor for thermal decomposition to obtain a crude N-CDs product, and an N-CDs solution is obtained after purification.

[0010] The specific coupling process is as follows: first, the amino functional group on the ICAM-1 monoclonal antibody is deactivated with N-hydroxysuccinimide ester (NHS) and 1-methyl-3-(3-dimethylaminopropyl)-carbonamide (EDC); the activated antibody solution is added to the N-CDs solution, magnetically stirred overnight at 4°C, and the obtained solution is centrifuged to obtain the coupling product.

[0011] The pyrolysis conditions are: pyrolysis at 180° C. for 8 hours.

[0012] The purification is divided into two steps. The first step is to centrifuge the crude N-CDs product. The second step is to dialyze the crude N-CDs product after centrifugation to obtain a purified product.

[0013] In the first step, the crude N-CDs product is centrifuged at 10,000 rpm for 30 minutes.

[0014] The dialysis conditions are 25° C., a stirring speed of 60 rpm and a magnetic stirring condition for 48 hours, and the molecular weight cutoff of the dialysis bag is 1000 Da.

[0015] During coupling, the mass ratio of nitrogen-doped carbon quantum dots to ICAM-1 monoclonal antibody was controlled at 5:1-1:5.

[0016] The method for preparing a nanocomposite of nitrogen-doped carbon quantum dots coupled with ICAM-1 monoclonal antibodies comprises the following steps:

[0017] (1) Preparation of nitrogen-doped carbon quantum dots

[0018] N-CDs were prepared by a hydrothermal method using citric acid as a carbon source and arginine as a nitrogen doping agent. The preparation process was as follows: 4.2 g of citric acid and 0.2 g of arginine were dissolved in 20 mL of ultrapure water, stirred and mixed evenly, and nitrogen was introduced into the solution for 30 min to remove dissolved oxygen in the solution; the obtained solution was transferred to a reactor and pyrolyzed at 180° C. for 8 hours, and the reactor was naturally cooled to room temperature to obtain a crude N-CDs product; the crude N-CDs product was centrifuged to remove large-volume impurities, and then the supernatant after centrifugation was dialyzed for 48 hours under magnetic stirring conditions, and the molecular weight cutoff of the dialysis bag was 1000 Da to remove unreacted reactants to obtain a purified N-CDs solution; the purified N-CDs solution was vacuum freeze-dried;

[0019] (2) Nanocomplex of nitrogen-doped carbon quantum dots coupled with ICAM-1 monoclonal antibody

[0020] First, the amino functional groups on the ICAM-1 monoclonal antibody were deactivated with N-hydroxysuccinimide ester (NHS) and 1-methyl-3-(3-dimethylaminopropyl)-carboxamide (EDC); the activated antibody solution was added to the nitrogen-doped carbon quantum dot solution and magnetically stirred overnight at 4°C; the coupling product was extracted from the reaction mixture by centrifugation at 10,000 rpm for 30 minutes; finally, the purified final product was freeze-dried and stored at 4°C.

[0021] The use of the nanocomposite of nitrogen-doped carbon quantum dots coupled with ICAM-1 monoclonal antibodies or the preparation method of the nanocomposite of nitrogen-doped carbon quantum dots coupled with ICAM-1 monoclonal antibodies as described in claim 2 in the preparation of drugs for preventing or treating septic acute lung injury, sepsis, acute respiratory distress syndrome, oxidative stress injury-related diseases and inflammatory diseases.

[0022] The beneficial effects obtained by the present invention are:

[0023] The nanocomposite of the present invention uses ICAM-1 as a target, delivers nitrogen-doped carbon quantum dots to systemic inflammatory lesions, and improves the inflammatory microenvironment of the lesions of organs such as the heart, liver, spleen, lung, and kidney by quickly and efficiently eliminating excessive ROS inside and outside endothelial cells and macrophages, thereby improving the therapeutic effect of heavy metal manganese aggravating sepsis-induced acute lung injury, and has the biological effects of removing active oxygen or anti-inflammatory protection. The nanocomposite has fluorescent imaging characteristics. The nanocomposite has a very obvious effect in treating manganese-aggravated sepsis-induced acute lung injury.

[0024] (1) The nanoparticles or targeted nano drug delivery system of the present invention have good storage stability, serum stability and freeze-drying stability. The particle size distribution of the nanoparticles of the present invention is uniform.

[0025] (2) The nanoparticles or targeted nano drug delivery system of the present invention has good targeted drug delivery properties, and can be delivered to the site of lung inflammation through ICAM-1 antibodies, thereby prolonging the circulation time in the body, thereby effectively concentrating the nitrogen-doped carbon quantum dots at the lesion site and increasing the drug concentration.

[0026] (3) The nanoparticles or targeted nano drug delivery system of the present invention can quickly and efficiently eliminate the excessive ROS inside and outside the endothelial cells and macrophages of acute lung injury induced by manganese-aggravated sepsis, thereby improving the inflammatory microenvironment at the lesion site and enhancing the therapeutic effect of ALI / ARDS.

[0027] (4) The present invention provides a new idea and strategy for the treatment of manganese-aggravated sepsis-induced acute lung injury, which has important clinical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a freeze-dried sample picture of the nitrogen-doped carbon quantum dots and the nitrogen-doped carbon quantum dots coupled with ICAM-1 monoclonal antibody nanocomplex of the present invention.

[0029] Figure 2 The fluorescence emission spectra of nanocomplexes of nitrogen-doped carbon quantum dots coupled with ICAM-1 monoclonal antibodies at different concentrations under 685nm excitation.

[0030] Figure 3 The figure is a transmission electron microscope (TEM) image of the nanocomplex of the present invention, which is a nitrogen-doped carbon quantum dot coupled with ICAM-1 monoclonal antibody.

[0031] Figure 4 This is the particle size distribution histogram of ICAM-1@N-CDs particles.

[0032] Figure 5 Zeta potential diagram of N-CDs and ICAM-1@N-CDs.

[0033] Figure 6 The present invention characterizes the physical and chemical properties of the nanocomplex of nitrogen-doped carbon quantum dots coupled with ICAM-1 monoclonal antibody.

[0034] Figure 7 This is the ultraviolet spectrum of the nitrogen-doped carbon quantum dots coupled to the ICAM-1 monoclonal antibody of the present invention.

[0035] Figure 8 This is the infrared spectrum of the nitrogen-doped carbon quantum dots coupled to the ICAM-1 monoclonal antibody of the present invention.

[0036] Fig. 9 This is the ESR spectrum of ICAM-1@N-CDs of the present invention.

[0037] Fig.10 Light microscopic images of RAW264.7 cells in different treatment groups (control, LPS, manganese, LPS+manganese, LPS+manganese+ICAM-1@N-CDs).

[0038] Fig.11 This is a bar graph showing statistical analysis of cell viability of RAW264.7 cells after treatment with different concentrations of Mn2+ for 24 hours using the CCK-8 method.

[0039] Fig.12 This is a bar graph showing the statistical analysis of the cell viability of RAW264.7 cells treated with different concentrations of ICAM-1@N-CDs for 24 hours using the CCK-8 method.

[0040] Fig.13 These are the fluorescence microscopy results of ROS staining using the DCFH-DA fluorescent probe of RAW264.7 cells in different treatment groups (control, LPS, manganese, LPS+manganese, LPS+manganese+ICAM-1@N-CDs(20 mg / kg) and LPS+manganese+ICAM-1@N-CDs(40 mg / kg)).

[0041] Fig.14 This is a graph showing the peak results of ROS staining with DCFH-DA fluorescent probe in RAW264.7 cells in different treatment groups (control, LPS, manganese, LPS+manganese, LPS+manganese+ICAM-1@N-CDs(20mg / kg) and LPS+manganese+ICAM-1@N-CDs(40mg / kg)).

[0042] Fig.15 This is the result of cell apoptosis detection of RAW264.7 cells in different treatment groups (control, LPS, manganese, LPS+manganese, LPS+manganese+ICAM-1@N-CDs(20mg / kg) and LPS+manganese+ICAM-1@N-CDs(40mg / kg)) by flow cytometry.

[0043] Fig.16 The graph shows the localization results of Lyso-Red (targeting lysosomes) and Mito-Deep Red (targeting selective mitochondria) in RAW264.7 cells in different treatment groups (control, LPS, manganese, LPS+manganese, LPS+manganese+ICAM-1@N-CDs).

[0044] Fig.17 The levels of inflammatory factors (TNF-α, IL-6 and IL-1β) in the RAW264.7 cell model of acute lung injury induced by sepsis were changed after different group treatments. The changes in the mRNA expression of inflammatory factors TNF-α, IL-6 and IL-1β in RAW264.7 cells in different treatment groups (control, LPS, manganese, LPS+manganese, LPS+manganese+ICAM-1@N-CDs (20 mg / kg)) indicated that manganese exposure caused an increase in the expression of inflammatory factors in RAW264.7, and carbon dots treatment could reduce the increase in the level of inflammatory factors in RAW264.7 caused by manganese exposure.

[0045] Fig.18 Clustering heat map analysis of differentially expressed genes in RAW264.7 cells after high-throughput mRNA sequencing in different treatment groups (control, LPS, LPS+manganese, LPS+manganese+ICAM-1@N-CDs) for 24 hours.

[0046] Fig.19 Functional enrichment analysis of RAW264.7 cells by high-throughput mRNA sequencing was performed 24 h after treatment with different groups (control, LPS, LPS+manganese, LPS+manganese+ICAM-1@N-CDs).

[0047] Fig. 20 RAW264.7 cells were subjected to PPI protein-protein interaction analysis by high-throughput mRNA sequencing 24 h after different treatment groups (control, LPS, LPS+manganese, LPS+manganese+ICAM-1@N-CDs).

[0048] Fig.21 HE staining detection and analysis of the heart, liver, spleen, lung, kidney and brain of the C57BL / 6 mouse model of sepsis-induced acute lung injury constructed with LPS.

[0049] Fig. 22 TUNEL detection of lung tissue in the LPS-induced sepsis C57BL / 6 mouse model of acute lung injury.

[0050] Fig.23 This is a graph showing the results of the hemolytic experiment of ICAM-1@N-CDs of the present invention.

[0051] Fig.24The diagram shows the heart, liver, spleen, lung, kidney and brain tissues of C57BL / 6 mice treated with ICAM-1@N-CDs (control, 20 mg / kg and 40 mg / kg) of the present invention after a biocompatibility experiment.

[0052] Fig.25 The results of HE staining of heart, liver, spleen, lung, kidney and brain tissue sections of C57BL / 6 mice treated with ICAM-1@N-CDs (control, 20 mg / kg and 40 mg / kg) of the present invention for biocompatibility experiment.

[0053] Fig.26 This is a bright field image of the brain, heart, liver, spleen, lung, kidney, bladder and intestinal tissues of C57BL / 6 mice after treatment with ICAM-1@N-CDs (LPS group, LPS+20 mg / kg) of the present invention.

[0054] Fig. 27 This is an in vitro fluorescence imaging image (excitation wavelength is 685 nm) of the brain, heart, liver, spleen, lung, kidney, bladder and intestinal tissues of C57BL / 6 mice after treatment with ICAM-1@N-CDs (LPS group, LPS+20 mg / kg) of the present invention. DETAILED DESCRIPTION

[0055] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than 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 are within the scope of protection of the present invention.

[0056] Statistical analysis

[0057] The experimental data were expressed as mean ± standard deviation and plotted using Graphpad Prism 8.0. SPSSStatistics ver.19.0 software was used to analyze and process the data. The data were analyzed by Pearson correlation test and showed normal distribution; the two groups of data were analyzed by t test, and multiple groups of data were compared using One-way ANOVA, and the least significant difference test (LSD) method was used for inter-group comparison. Post hoc test was performed using Student-Newman-Keuls posthoc test. Survival curves were analyzed using Log-rank (Mantel-Cox) test. When P < 0.05, it was considered to be statistically significant.

[0058] The nanocomposite of nitrogen-doped carbon quantum dots coupled with ICAM-1 monoclonal antibody with fluorescence imaging characteristics described in the present invention is a targeted nanocarrier N-CDs-ICAM-1 formed by covalent coupling with nanomaterial nitrogen-doped carbon quantum dots as carriers and ICAM-1 as targeting molecules. The particle size is 8.08±0.18nm. The sample is as follows Figure 1 shown.

[0059] Example 1: Preparation of nitrogen-doped carbon quantum dots

[0060] The present invention uses citric acid as a carbon source and arginine as a nitrogen doping agent to prepare N-CDs by a hydrothermal method. The preparation process is as follows: 4.2g of citric acid and 0.2g of arginine are dissolved in 20mL of ultrapure water, stirred and mixed evenly, and nitrogen is introduced into the solution for 30min to remove dissolved oxygen in the solution. The obtained solution is transferred to a 25mL reactor and pyrolyzed at 180°C for 8 hours. The reactor is cooled naturally to room temperature to obtain a crude N-CDs product. The crude N-CDs product is centrifuged at a speed of 10000rpm for 30min to remove large volume impurities, and then the supernatant after centrifugation is dialyzed for 48h under magnetic stirring conditions at 25°C and a stirring speed of 60r / min. The molecular weight cutoff of the dialysis bag is 1000Da to remove the unreacted reactants to obtain a purified N-CDs solution. The purified N-CDs solution is vacuum freeze-dried to obtain a nanomaterial of nitrogen-doped carbon quantum dots.

[0061] Example 2: Nanocomplex of nitrogen-doped carbon quantum dots coupled with ICAM-1 monoclonal antibody

[0062] ICAM-1 was coupled to N-CDs by amidation reaction, and the specific process was as follows: First, the amino functional groups on the ICAM-1 monoclonal antibody were deactivated with N-hydroxysuccinimide ester (NHS) and 1-methyl-3-(3-dimethylaminopropyl)-carbonamide (EDC). The antibody solution (concentration was 1:1000) was added to the carbon quantum dot solution (concentration was 5 mg / mL) and magnetically stirred overnight at 4°C. The above solution was centrifuged at 10,000 rpm for 30 minutes, and the coupling product was extracted from the reaction mixture. Finally, the purified final product was freeze-dried and stored at 4°C.

[0063] Figure 2 The fluorescence emission spectra of the nanocomplex of nitrogen-doped carbon quantum dots coupled with ICAM-1 monoclonal antibody at different concentrations under 685nm excitation. From left to right are pure water control group, 12.5, 25, 50, 100, 200, and 400 mg / mL concentrations. The results show that the carbon quantum dots have excellent fluorescence properties and can be used for in vitro cell imaging and in vivo imaging, respectively.

[0064] Example 3: Characterization of the Nanocomplex of Nitrogen-doped Carbon Quantum Dots Coupled with ICAM-1 Monoclonal Antibody

[0065] (1) Transmission electron microscopy (TEM) characterization of the morphology of nanoparticles ICAM-1@N-CDs

[0066] The test was carried out on a JEM-2100F transmission electron microscope. The sample was dropped on an ultra-thin carbon film and dried at room temperature. The microscopic morphology of the prepared ICAM-1@N-CDs nanomaterial was characterized by transmission electron microscopy (TEM) at an accelerating voltage of 200 kV. The results are shown in Figure 3 shown.

[0067] Figure 4 The particle size distribution histogram of ICAM-1@N-CDs particles; the results show that ICAM-1@N-CDs are uniform spherical nanoparticles, and the average particle size is 8nm according to statistical analysis.

[0068] (2) Potential measurement of nanoparticles ICAM-1@N-CDs

[0069] The Zeta potential of N-CDs and ICAM-1@N-CDs was analyzed using a Malvern Zetasizer Nano ZS potential analyzer. In order to evaluate the successful synthesis of ICAM-1@N-CDs nanomaterials, the average potential of ICAM-1@N-CDs nanomaterials was measured using dynamic light scattering (DLS). Figure 5 The Zeta potential of N-CDs nanoparticles was -15±3.2 mV, and the Zeta potential of ICAM-1@N-CDs nanoparticles prepared after ICAM-1 modification was -10.0±0.5 mV, and the potential was significantly reduced, indicating that the negatively charged polypeptide ICAM-1 was coated on the surface of the nanoparticles, indicating that the nanoparticles ICAM-1@N-CDs were successfully prepared.

[0070] (3) Structural characterization of nanoparticles ICAM-1@N-CDs: XPS

[0071] The test was conducted using a Thermo Scientific ESCALAB 250 photoelectron spectrometer with an aluminum / magnesium target, a high voltage of 14.0 kV, and a power of 250 W. Figure 6 As shown, where: A is the high-resolution full-range X-ray photoelectron spectroscopy (XPS) spectrum of ICAM-1@N-CDs, C 1s, N 1s, O1s; B is the UV-visible absorption spectrum (Abs) of ICAM-1@N-CDs; C is the infrared spectrum of ICAM-1@N-CDs.

[0072] ICAM-1@N-CDs is mainly composed of three elements: C, N, and O, accounting for 57.31%, 4.33%, and 31.67%, respectively. A second-strongest C1s characteristic peak appears at 285.1 eV, a weak N1s characteristic peak appears at 399.1 eV, and a very strong O1s characteristic peak appears at 532.7 eV.

[0073] (4) Structural characterization of nanoparticles ICAM-1@N-CDs: ESR

[0074] The scavenging test of ·OH by ICAM-1@N-CDs was conducted on a Bruker A300-10 / 12 spectrometer: First, 500 μL of 50 mM DMPO solution was prepared, and different concentrations of ICAM-1@N-CDs (100, 200 μg / ml) were added thereto. Then, 10 μM Fe2+ and 60 μM H2O2 were added. After the mixed solution was placed at room temperature for 60 s, an EPR test was immediately performed and the EPR spectrum was recorded. The results are shown in Figure 2. Fig. 9 shown. Fig. 9 In the figure, A is the OH· scavenging activity graph of ICAM-1@N-CDs at different action time points; B is the O2·- scavenging activity graph of ICAM-1@N-CDs at different action time points; C is the H2O2 scavenging activity graph of ICAM-1@N-CDs at different action time points. The final ESR spectrum of ICAM-1@N-CDs is shown in Fig. 9 shown.

[0075] (5) Structural characterization of nanoparticles ICAM-1@N-CDs: Fourier transform infrared spectroscopy (FT-IR)

[0076] Fourier transform infrared spectroscopy (FT-IR): Using a Nicolet 670 infrared spectrometer, the sample was mixed with KBr powder and pressed into a tablet for testing. The infrared spectrum of the ICAM-1@N-CDs dialysate prepared at 350°C was tested using a Fourier transform infrared spectrometer. 0.160g of potassium bromide powder was weighed using an electronic analytical balance, and 2mL of the charcoal dialysate and potassium bromide powder were placed in an agate mortar and mixed thoroughly. The mixture was placed in an infrared light box for drying, and the mixture was continuously ground into a uniform fine powder. The powder was continuously dried, and the dried fine powder was evenly distributed in the mold. The pressure was set to 20KPa and maintained for two minutes. The pressure was slowly reduced, and the prepared sample was taken out. It must be immediately placed in an infrared spectrometer for measurement to avoid failure to keep it dry and affect the experimental results. The infrared spectrum results of ICAM-1@N-CDs are shown in the figure. Figure 8As shown, characteristic peaks appeared at 3426.33cm, 2922.31cm, 1634.90cm, and 1034.48cm, among which the absorption peaks at 3423-3431cm indicated the possible existence of -OH bonds, the two peaks at 2920cm and 2850cm indicated the stretching vibration peaks of -CH2-, which are the characteristic peaks of methylene, the strong absorption peaks at 1628-1635cm were the stretching vibration peaks of -C=O bonds, the strong and sharp absorption peaks at 1384cm indicated the possible in-plane bending vibration peaks of -OH, and the absorption peaks at 1034cm indicated the possible presence of -COC- bonds. This indicates that there are functional groups such as hydroxyl, carboxyl, and amino groups on the surface of ICAM-1@N-CDs produced by carbonization.

[0077] (6) Structural characterization of nanoparticles ICAM-1@N-CDs: UV-visible spectroscopy (UV-vis)

[0078] The test was conducted in a 1 cm quartz cuvette using a UH5300 UV-visible absorption spectrometer (Hitachi). In order to further observe the structural characterization of the ICAM-1@N-CDs nanomaterial and confirm whether the nanomaterial is successfully synthesized, the present invention also used a UV-visible spectrophotometer to perform UV-visible absorption spectrum detection on the N-CDs and ICAM-1@N-CDs nanomaterials. Preheat the machine for more than 15 minutes, and debug the instrument: set a medium scan speed of 5nm / s, Averaging 0.9nm, and Bandwiths 1.8nm. The test wavelength is set to 200-800nm. Dilute the above-prepared ICAM-1@N-CDs dialysate in multiple ratios to an appropriate concentration (2mg / mL), draw 2mL and place it in a quartz cuvette, then place the cuvette in the sample slot and prepare for testing. Before testing, use deionized water to determine the baseline. The UV spectrum results are as follows: Figure 7 As shown, a weak absorption peak can be observed in ICAM-1@N-CDs at 260-280 nm, which may be caused by the n-π* transition caused by the unsaturated groups containing heteroatoms.

[0079] Example 4: Cell culture and cytotoxicity assay (CCK8)

[0080] RAW264.7 cells were seeded in 96-well cell plates (density of about 1x104 cells / well) and incubated in a 37°C incubator containing 5% carbon dioxide and 95% air humidity with DMEM medium containing 10% fetal bovine serum, 80 μg / mL streptomycin and 80 U / mL penicillin. After 24 hours of incubation, the culture medium was removed, and the cell wells without cell culture and simply added with culture medium were used as apoptosis, and the control group (untreated), the group containing different concentrations of Mn, the group containing ICAM-1@N-CDs, and the group containing LPS+Mn+ICAM-1@N-CDs were injected into the cell plate with fresh culture medium, and then incubated for 24 hours under the same conditions, and 3 sets of wells were made for each concentration. Next, 10 μL of CCK8 solution (0.5 mg / mL) was added to the wells of the cell plate and incubated in the incubator for 4 hours, and the absorbance (OD) of the obtained mixture was measured at 450 nm using an ELISA reader (Thermo Scientific). The cell viability was estimated using the following equation:

[0081] Cell survival rate (%) = Atest / Acontrol × 100%,

[0082] Among them, Atest represents the absorbance of the experimental group after the addition of drugs, and Acontrol represents the absorbance of the control group without drug treatment.

[0083] Fig.11 and Fig.12 Cell survival rate of RAW264.7 cells after incubation for 24 h in culture medium without any drugs (control group) and with different concentrations of Mn and ICAM-1@N-CDs.

[0084] First, the toxicity of the control group (without any drugs), the group containing different concentrations of Mn, and the ICAM-1@N-CDs group to RAW264.7 cells was tested by CCK8 test. RAW264.7 cells were incubated in the culture medium of the above four groups. The results showed that even after incubation for 24 hours in the culture medium with an ICAM-1@N-CDs content of up to 100μg / ml, the cell survival rate remained above 90%, confirming the low toxicity of ICAM-1@N-CDs to living cells.

[0085] Example 5: Cell apoptosis detection

[0086] The apoptosis experiment was performed on cells using the commercial Annexin V-FITC apoptosis detection kit and following the instructions. RAW264.7 cells or THP-1 cells were incubated for 24 hours in a medium containing no drugs (control group), different concentrations of Mn, ICAM-1@N-CDs, and LPS+Mn+ICAM-1@N-CDs. The culture medium was then collected (suspended cells were retained), and the adherent cells were digested and collected with EDTA-free trypsin (THP-1 cells themselves are suspended growth cells). The suspended cells and adherent cells were combined and collected by centrifugation. The obtained cells were dispersed into 195 μL Annexin V-FITC binding buffer, 5 μL Annexin V-FITC and 10 μL propidium iodide (PI) were added, and the cells were stained in the dark for 15 minutes, and the obtained cells were detected by flow cytometry.

[0087] Fig.15 RAW264.7 cells were treated with the incubation period of 1 h without any drug (control group), with different concentrations of Mn, ICAM-1@N-CDs, LPS+Mn+ICAM-1@N-CDs (groups Fig.15 There is no correspondence between Fig.15 The middle part shows the apoptosis experiment of cells incubated in the culture medium of control, Mn, LPS, LPS+Mn, LPS+Mn+ICAM-1@N-CDs 20mg / kg, LPS+Mn+ICAM-1@N-CDs 40mg / kg). The lower left quadrant (Annexin V- / PI-) represents living cells; the lower right quadrant (Annexin V+ / PI-) represents early apoptotic cells; the upper right quadrant (Annexin V+ / PI+) represents late apoptotic cells and necrotic cells; the upper left quadrant (AnnexinV- / PI+) is considered to be within the detection error within the allowable range. Regions I, II, III, and IV represent normal cells, early apoptotic cells, late apoptotic cells, and necrotic cells, respectively (it is best to mark regions I, II, III, and IV).

[0088] Example 6: ROS detection

[0089] The cells were cultured in 96-well cell culture plates (Corning, USA). After stable transfection of the plasmid for 48 hours, the cells were stained with 2μmol / L BODIPY 581 / 591C11 (MCE, USA) for 30 minutes, observed under a microscope, and the fluorescence intensity was detected by a fluorescence microplate reader (Beckman, USA). The DCFH-DA probe was diluted with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10μmol / L. After stable transfection of the plasmid for 24 hours, the cells were collected and suspended in the diluted DCFH-DA, incubated in a 37°C cell culture incubator for 20 minutes, and the absorbance was detected using a fluorescence microplate reader. The results are shown in Fig.13 shown.

[0090] Example 7: Mitochondrial and lysosomal localization

[0091] Prepare the staining solution according to the instructions in the manual, and add 1μl Mito-TrackerGreen and 0.8μl Lyso-Tracker Red staining solution to 20ml culture medium at the same time. Aspirate the culture medium, cover the bottom with the 37℃ pre-warmed staining solution just prepared, and incubate at 37℃ for 40 minutes. Aspirate the staining solution and replace it with 37℃ pre-warmed culture medium. Then you can see the fluorescent image under the laser confocal microscope, mitochondria show green, lysosomes show red. The result is as follows Fig.16 As shown in the figure, DAPI is the localization of the cell nucleus, Mitochondria is the mitochondria, Lysosome is the lysosome localization, Merge is the merger of the localization of the cell nucleus, mitochondria and lysosome, and the left side is each different grouping, each group has 3 localization maps and a merged map.

[0092] Example 8: mRNA sequencing

[0093] The original data were obtained by sequencing using the Illumina sequencing platform. DEseq2 software was used to analyze the expression of differentially expressed genes between the H group and the PH group. When P < 0.05, it indicated that the differentially expressed genes between the two groups were statistically significant. Gene function (gene ontology, GO) enrichment analysis and Kyoto Encyclopedia of genes and genomes (KEGG) enrichment analysis were performed on them. Then, the CytoNCA plug-in in the Cytoscape software was used to perform protein-protein interaction network (PPI) analysis, and the key genes ranked in the top 20 in the association node score were screened for further GO and KEGG enrichment analysis. The results are shown in Fig.21 , Fig. 22 shown.

[0094] Example 9: Sepsis-induced acute lung injury experimental animals and grouping

[0095] (1) Experimental animals: C57BL / 6 mice, 6-8 weeks old, half male and half female, weighing 18-22 g, were used as experimental subjects.

[0096] (2) Experimental grouping: After all mice were adaptively fed for 1 week, 30 mice were randomly divided into 5 groups, namely: control group, LPS group, Mn group, LPS+Mn group (Mn was given 1 hour after LPS), LPS+Mn+ICAM-1@N-CDs group (different doses of Mn were given 1 hour after modeling, and ICAM-1@N-CDs was given 1 hour after Mn modeling). Five mice in each group were used for pathological staining, fixation and cryopreservation of samples for molecular biology experiments.

[0097] (3) Animal husbandry: All mice were kept in the animal experiment center after purchase until the end of the experiment. The system environment was SPF level, and all mice were kept in individual ventilated cages (IVC). Humidity was 50-60%, temperature was 22-24℃, light and dark were alternating for 12 hours, and they had free access to food and water. The drinking water was sterilized by high pressure, and the mouse feed was sterilized by cobalt 60 irradiation. They were fasted for 12 hours before the experiment, but they were allowed to drink water freely.

[0098] Example 10: Construction of a mouse model of sepsis-induced acute lung injury by LPS treatment

[0099] Thirty healthy male C57BL / 6 mice, 6-8 weeks old, weighing 21-23 g, were randomly divided into six groups: normal control group (n=6), LPS model group (LPS, n=6), manganese group (Mn, n=6), LPS+manganese group (LPS+Mn, n=6), LPS+manganese group+carbon dots 1 (LPS+Mn+ICAM-1@N-CDs(20 mg / kg), n=6), and LPS+manganese group+carbon dots 2 (LPS+Mn+ICAM-1@N-CDs(40 mg / kg), n=6). The normal control group was intraperitoneally injected with an equal amount of saline, LPS was intraperitoneally injected at 15 mg / kg to establish the model, the manganese group was intraperitoneally injected at 5 mg / kg to establish the model, the LPS+manganese group was intraperitoneally injected with 15 mg / kg of LPS 1 hour after the model was established, and the manganese group was intraperitoneally injected with 5 mg / kg to establish the model; the LPS+manganese+carbon dot group was intraperitoneally injected with 15 mg / kg of LPS 1 hour after the model was established, and the manganese group was intraperitoneally injected with 5 mg / kg of carbon dots 1 hour after the model was established, and then the tail vein was injected with 20 mg / kg and 40 mg / kg of carbon dots. The mice were killed 18 hours after the model was established, and blood and heart, liver, spleen, lung, kidney and brain samples were collected.

[0100] Example 12: ELISA detection of cytokines in mouse serum

[0101] ① Dilution and addition of standard: Set up 10 standard wells on the enzyme-labeled coated plate and dilute the standard according to the instructions of the ELISA kit.

[0102] ② Sample addition: Set up blank wells (blank control wells without adding samples and enzyme-labeled reagents) and test sample wells. First add 40uL of sample diluent to the test sample well on the enzyme-labeled plate, and then add 10uL of the test sample (the final sample dilution is 5 times). When adding samples, add the sample to the bottom of the ELISA plate, try not to touch the well wall, and gently shake to mix.

[0103] ③ Incubation: Seal the plate with a sealing film and incubate in a 37°C incubator for 1 hour.

[0104] ④ Liquid preparation and washing: dilute the concentrated washing solution with distilled water for later use. Carefully peel off the sealing film, discard the liquid in the wells, spin dry, fill each well with washing solution, let it stand for 1 minute, then discard the washing solution, repeat this 5 times, and pat dry.

[0105] ⑤ Add enzyme: Add 50 μl of enzyme-labeled reagent to each well, except for the blank well.

[0106] ⑥Incubation: Same operation as ③

[0107] ⑦Washing: Same operation as ④

[0108] ⑧ Color development: Add 50μl of color developer A to each well, then add 50μl of color developer B, gently shake to mix, and develop the color at 37℃ in the dark for 15 minutes.

[0109] ⑨Termination: Add 50 μl of stop solution to each well to terminate the reaction (the blue color immediately turns to yellow).

[0110] ⑩ Determination: Starting from the blank well, measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm.

[0111] Example 13: Preparation of pathological sections from heart, liver, spleen, lung, kidney and brain for HE staining

[0112] (1) Sampling: 24 hours after CLP, mice were anesthetized and killed, and the heart, liver, spleen, lung, kidney, and brain were removed. The removed organs were washed in a beaker containing ice-cold saline and then fixed in 4% paraformaldehyde at room temperature for 48 h.

[0113] (2) Dehydration and transparency: The fixed tissue was washed in 70% alcohol for 1 h, then dehydrated in 80%, 95%, and 100% alcohol for 45 min each, and then transparentized in a mixture of 100% alcohol and xylene (1:1) and xylene for 30 min each;

[0114] (3) Waxing and embedding: Immerse the tissue in a mixture of xylene and paraffin (1:1, 50°C) and hard paraffin solution I / II / III (60°C) for 1 h each. Place the melted paraffin and tissue in a square mold and wait for solidification.

[0115] (4) Sectioning and mounting: Take out the embedded wax block and slice it continuously with a microtome to a thickness of 5 μm. Place the complete wax slice on a glass slide and dry the glass slide at 56°C for 30 min or 37°C for 1 h.

[0116] (5) H&E staining: Take out the slide and place it in xylene for 15 minutes for dehydration, then place it in a mixture of alcohol and xylene (mixed in a ratio of 1:1) for 5 minutes, then place it in 100%, 95%, 80%, 70% alcohol, and distilled water for 2 minutes each. After soaking, place it in a working concentration of hematoxylin solution for staining for 15 minutes, and rinse it with tap water for 15 minutes. After rinsing, soak it in 1% hydrochloric acid alcohol for color separation for 20 seconds, and then rinse it with tap water for 10 minutes. Soak it in 1% ammonia water for anti-blueing for about 30 seconds, and rinse it with tap water for 15 minutes. Stain it with 0.1% eosin solution for 5 minutes, rinse it with tap water for 15 minutes, and then place the slide in 70%, 80%, 95%, 100% alcohol, alcohol and xylene mixture (mixed in a ratio of 1:1), and xylene for 3 minutes each;

[0117] (6) Sealing: Wipe off the xylene around the tissue on the slide, add an appropriate amount of neutral gum, cover with a coverslip, and dry in an oven;

[0118] (7) Observe tissue damage and inflammatory cell infiltration under an optical microscope. The staining results were observed under an optical microscope and the degree of lung damage in each group of mice was scored. The main observation indicators were pulmonary edema and inflammatory cell infiltration.

[0119] Example 14: Preparation of pathological sections from heart, liver, spleen, lung, kidney and brain for TUNEL staining

[0120] 1. Fix tissue sections with freshly prepared fixative (4% paraformaldehyde dissolved in PBS, pH 7.4) at 15-25°C for 20 min.

[0121] 2. Rinse the slides with PBS for 30 minutes. Note: If you need to preserve the fixed tissue sections, dehydrate the sections in anhydrous ethanol for 2 minutes and store them at -15 to -25°C.

[0122] 3. Immerse the slides in blocking solution (3% H2O2, dissolved in methanol) and incubate at 15-25°C for 10 min.

[0123] 4. Rinse the slides with PBS.

[0124] 5. Immerse the slide in permeabilization solution (0.1% Triton X-100 dissolved in 0.1% sodium citrate, freshly prepared) and incubate at 2-8°C for 2 min.

[0125] 6. Pipette 100 μl of the labeling solution (tube 2) into the two negative control samples.

[0126] 7. Add 50 μl of enzyme solution (tube 1) to the remaining labeled solution in tube 2 (about 450 μl) to obtain a total volume of 500 μl of TUNEL reaction mixture.

[0127] 8. Mix the two reagents evenly.

[0128] 9. Rinse the slides twice with PBS.

[0129] 10. Absorb the moisture around the sample.

[0130] 11. Add 50μl TUNEL reaction mixture to the sample. Note: For negative control samples, add 50μl of labeling solution without terminal transferase to each sample. To ensure that the TUNEL reaction mixture is evenly dispersed among the monolayer cells and to avoid evaporation of the solution, the cell sample and the mixture should be covered with sealing film or coverslip during the reaction.

[0131] 12. Cover and react at 37°C in a light-proof and humidified environment for 60 min.

[0132] 13. Rinse the slides three times with PBS.

[0133] 14. Add drops of PBS and analyze the sample under a fluorescence microscope. Use an excitation wavelength of 450-500 nm and detect the fluorescence signal in the range of 515-565 nm (green light).

[0134] 15. Absorb the moisture around the sample.

[0135] 16. Add 50 μl of POD conversion solution (tube 3) to the sample. Note: To ensure that the POD conversion solution is evenly dispersed between the monolayer cells and to avoid evaporation of the solution, it is necessary to cover it with sealing film or coverslip during the reaction.

[0136] 17. React in a humidified environment at 37°C for 30 min.

[0137] 18. Rinse the slides 3 times with PBS.

[0138] 19. Add 50-100 μl DAB substrate or other POD substrate.

[0139] 20. Incubate at 15-25°C for 10 min.

[0140] 21. Rinse the slides 3 times with PBS.

[0141] 22. Cover with a coverslip (add PBS / glycerol, etc.) and analyze the results under an optical microscope.

[0142] Example 15: Total RNA extraction and quantification from mouse lung tissue

[0143] Sample preparation

[0144] ① Prepare an ice box in advance, pre-cool a clean 2 ml EP tube and grinding steel beads, take an appropriate amount of mouse lung tissue, put it in a 2 ml EP tube, cut it into pieces with clean ophthalmic scissors, and add pre-cooled steel beads.

[0145] ② Add 1 ml of Trizol solution to each tube, grind the tissue thoroughly for 2 minutes using a cryogenic grinder, remove the grinding beads, and let stand at room temperature for later use.

[0146] Total RNA extraction

[0147] ① Add 200 μl of chloroform to each tube of the Trizol homogenate prepared in step (1), vortex for 30 seconds to mix thoroughly, and let stand on ice for 10 minutes.

[0148] ② Precool the centrifuge in advance, let it stand, and then centrifuge at 4°C for 15 minutes (12000rpm).

[0149] ③After centrifugation, the liquid in the EP tube can be seen to be clearly stratified. Carefully pipette the upper aqueous phase liquid (about 400 μl) into a new 1.5 ml EP tube for later use.

[0150] RNA precipitation

[0151] ① Add an equal volume of isopropanol to each tube of the RNA solution extracted in step (2), turn the EP tube upside down to mix thoroughly, and let it stand for 10 minutes.

[0152] ② After standing, centrifuge at 4°C for 10 min (12000 rpm). A small amount of white precipitate can be seen at the bottom of the tube.

[0153] RNA washing

[0154] ① Prepare 75% ethanol solution using anhydrous ethanol and DEPC water.

[0155] ② Carefully discard the supernatant in the EP tube after centrifugation in step (3), add 1 ml of 75% ethanol to each tube, turn it upside down to suspend the RNA precipitate and rinse it thoroughly, and then centrifuge it at 4°C for 5 minutes (12000 rpm).

[0156] ③Repeat step ② and rinse 2-3 times.

[0157] RNA solubilization

[0158] ①After the last rinse, carefully remove the ethanol using a pipette or a disposable 1ml syringe.

[0159] ② Place the EP tube in a clean disposable film glove, air-dry at room temperature for 5-10 minutes, and then add DEPC water to dissolve the RNA precipitate.

[0160] RNA concentration and purity detection: Use Nanodrop instrument to detect the extracted RNA concentration and purity. Continue with subsequent operations if both meet the standards.

[0161] Example 16: RNA Reverse Transcription

[0162] (1) Preparation of reaction system

[0163] ① Take 2μg RNA as the template chain and add it to a 200μl EP tube without enzyme. The total reaction system is 20μl. Calculate the corresponding volume of each reagent according to the instructions in the cDNA reverse transcription kit and the volume of the template chain to be added. Finally, fill it up to 20μl with DEPC water. Vortex to mix, and then use a centrifuge to centrifuge the droplets adhering to the tube wall to the bottom of the tube for reverse transcription.

[0164] (2) RNA reverse transcription ① Place the EP tube in the PCR instrument and set the reaction temperature and time required for reverse transcription. After reverse transcription is completed, take out the EP tube and store it at -20℃ for future use. If long-term storage is required, place it at -80℃.

[0165] Example 17: Real-time fluorescence quantitative PCR

[0166] (1) Add 80 μl of DEPC water to the EP tube containing the cDNA obtained by reverse transcription and dilute it 5 times.

[0167] (2) Prepare the real-time quantitative PCR reaction system according to the reagent instructions.

[0168] (3) Add the desired reaction system into the 96-well plate, then seal the 96-well plate with a plastic sealing film and centrifuge.

[0169] (4) Place the centrifuged 96-well plate in a real-time fluorescence quantitative PCR instrument, set up the reaction process and start.

Claims

1. A nanocomposite of nitrogen-doped carbon quantum dots coupled with ICAM-1 monoclonal antibody, characterized in that: Using the nanomaterial nitrogen-doped carbon quantum dots as the carrier and ICAM-1 as the targeting molecule, the targeted nanocarrier ICAM-1@N-CDs was formed by covalently coupling the nitrogen-doped carbon quantum dots and ICAM-1.

2. The nanocomposite of nitrogen-doped carbon quantum dots coupled with ICAM-1 monoclonal antibody according to claim 1, characterized in that: The nitrogen-doped carbon quantum dots are prepared by the following method: 4.2 g of citric acid and 0.2 g of arginine are dissolved in 20 ml of ultrapure water, stirred and mixed evenly, and nitrogen is introduced into the solution to remove dissolved oxygen in the solution; the obtained solution is transferred to a reactor for thermal decomposition to obtain a crude N-CDs product, and an N-CDs solution is obtained after purification.

3. The nanocomposite of nitrogen-doped carbon quantum dots coupled with ICAM-1 monoclonal antibody according to claim 1, characterized in that: The specific coupling process is as follows: first, the amino functional group on the ICAM-1 monoclonal antibody is deactivated with N-hydroxysuccinimide ester (NHS) and 1-methyl-3-(3-dimethylaminopropyl)-carbonamide (EDC); the activated antibody solution is added to the N-CDs solution, magnetically stirred overnight at 4°C, and the obtained solution is centrifuged to obtain the coupling product.

4. The nanocomposite of nitrogen-doped carbon quantum dots coupled with ICAM-1 monoclonal antibody according to claim 2, characterized in that: The pyrolysis conditions are: pyrolysis at 180° C. for 8 hours.

5. The nanocomposite of nitrogen-doped carbon quantum dots coupled with ICAM-1 monoclonal antibody according to claim 2, characterized in that: The purification is divided into two steps. The first step is to centrifuge the crude N-CDs product. The second step is to dialyze the crude N-CDs product after centrifugation to obtain a purified product.

6. The nanocomposite of nitrogen-doped carbon quantum dots coupled with ICAM-1 monoclonal antibody according to claim 5, characterized in that: In the first step, the crude N-CDs product is centrifuged at 10,000 rpm for 30 minutes.

7. The nanocomposite of nitrogen-doped carbon quantum dots coupled with ICAM-1 monoclonal antibody according to claim 5, characterized in that: The dialysis conditions are 25° C., a stirring speed of 60 rpm and a magnetic stirring condition for 48 hours, and the molecular weight cutoff of the dialysis bag is 1000 Da.

8. The method for preparing a nanocomposite of nitrogen-doped carbon quantum dots coupled with ICAM-1 monoclonal antibody according to claim 1, characterized in that: The following steps are involved: (1) Preparation of nitrogen-doped carbon quantum dots N-CDs were prepared by a hydrothermal method using citric acid as a carbon source and arginine as a nitrogen doping agent. The preparation process was as follows: 4.2 g of citric acid and 0.2 g of arginine were dissolved in 20 mL of ultrapure water, stirred and mixed evenly, and nitrogen was introduced into the solution for 30 min to remove dissolved oxygen in the solution; the obtained solution was transferred to a reactor and pyrolyzed at 180° C. for 8 hours, and the reactor was naturally cooled to room temperature to obtain a crude N-CDs product; the crude N-CDs product was centrifuged to remove large-volume impurities, and then the supernatant after centrifugation was dialyzed for 48 hours under magnetic stirring conditions, and the molecular weight cutoff of the dialysis bag was 1000 Da to remove unreacted reactants to obtain a purified N-CDs solution; the purified N-CDs solution was vacuum freeze-dried; (2) Nanocomplex of nitrogen-doped carbon quantum dots coupled with ICAM-1 monoclonal antibody First, the amino functional groups on the ICAM-1 monoclonal antibody were deactivated with N-hydroxysuccinimide ester (NHS) and 1-methyl-3-(3-dimethylaminopropyl)-carboxamide (EDC); the activated antibody solution was added to the nitrogen-doped carbon quantum dot solution and magnetically stirred overnight at 4°C; the coupling product was extracted from the reaction mixture by centrifugation at 10,000 rpm for 30 minutes; finally, the purified final product was freeze-dried and stored at 4°C.

9. Use of the method for preparing a nanocomposite of nitrogen-doped carbon quantum dots coupled with ICAM-1 monoclonal antibody as claimed in claim 1 or a nanocomposite of nitrogen-doped carbon quantum dots coupled with ICAM-1 monoclonal antibody as claimed in claim 2 in the preparation of drugs for preventing or treating septic acute lung injury, sepsis, acute respiratory distress syndrome, oxidative stress injury-related diseases and inflammatory diseases.

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