A vascular endothelial cell targeted drug-loaded micelle and its preparation method and application

Abstract

The present invention discloses a vascular endothelial cell targeted drug-loaded micelle and a preparation method and application thereof. The vascular endothelial cell targeted drug-loaded micelle comprises a vascular endothelial cell targeted micelle and an antioxidant encapsulated in the vascular endothelial cell targeted micelle; the vascular endothelial cell targeted micelle is self-assembled by LSRQ-PEG-PLGA graft and PEG-PLGA; the LSRQ-PEG-PLGA graft is obtained by condensation reaction of an amino group in an LSRQ peptide and a carboxyl group in PEG-PLGA; the amino acid sequence of the LSRQ peptide is LSRQPVGGHPKC. The vascular endothelial cell targeted drug-loaded micelle prepared by the present invention can increase the distribution of drugs in target cells damaged by inflammation, has strong specificity, has a good lung targeting effect, and has good safety, and can achieve safe and efficient targeted treatment of acute lung injury.

Description

Technical Field

[0001] The present invention relates to the technical field of novel drug delivery systems, and in particular to a vascular endothelial cell targeted drug-loaded micelle, and a preparation method and application thereof. Background Art

[0002] Acute Respiratory Distress Syndrome (ARDS) is a severe manifestation of systemic inflammatory response in the lungs. In the early stage, it manifests as acute lung injury (ALI), which then develops into severe lung injury ARDS. In the late stage, it often develops into or is combined with multiple organ dysfunction or even multiple organ failure. At present, the incidence of ARDS is still high, and there are no specific drugs and means for clinical treatment, which leads to high mortality. It is one of the common acute and critical diseases in clinical practice. Inflammation plays a key and decisive role in its pathogenesis. Its inflammatory mechanism includes the migration and aggregation of inflammatory cells in the lungs, the release of inflammatory mediators, and the damage and increased permeability of alveolar capillaries. Once a large amount of inflammatory mediators are released, an inflammatory factor waterfall occurs, and the patient's condition will take a sharp turn for the worse, which is extremely difficult to treat, and is usually accompanied by secondary systemic diseases such as sepsis and shock, thus endangering life. Therefore, how to explore a new drug or new treatment method based on the pathogenesis of ALI has become more and more concerned by everyone.

[0003] Recent studies have found that pulmonary vascular endothelial cells are the main target cells for the occurrence and development of ALI caused by various pathogenic factors. Pulmonary vascular endothelial cells are located on the inner side of blood vessels. They not only act as a selective permeable barrier, but also have important endocrine functions. They are important in regulating vasomotor state and permeability, resisting platelet aggregation, and maintaining vascular wall integrity. Pulmonary vascular endothelial cells are important target cells and effector cells that mediate ALI. In the early stages of ALI, the barrier function and endocrine function of endothelial cells have changed, which is manifested as increased vascular permeability and excessive release of various inflammatory mediators. Therefore, it is crucial to improve endothelial barrier function by inhibiting endothelial cell activation for the treatment of ALI.

[0004] Given the key role of vascular endothelial cells in the pathophysiological mechanism of ALI, pulmonary vascular endothelial cells may become a new target for ALI drug treatment. However, although pulmonary vascular endothelial cells play an important role in the pathogenesis of ALI, current therapeutic drugs have no specific selection for them. After entering the body, they cannot concentrate on damaged target cells, that is, the local drug concentration is too low to reach an effective therapeutic concentration, and often also triggers many different degrees of extrapulmonary effects. Therefore, how to distribute as many drugs as possible to lung tissue and achieve active targeted delivery of pulmonary vascular endothelial cells is the key to achieving safe and efficient treatment of ALI. Fortunately, by using receptors specifically expressed by vascular endothelial cells when ALI occurs, such as intercellular adhesion molecule-1 (CD54 / ICAM-1), vascular cell adhesion molecule-1 (CD106 / VCAM-1), selectin family (L-selectin, E-selectin, P-selectin), etc., combined with nano-targeted drug delivery technology, drugs can be delivered to pulmonary vascular endothelial cells, making targeted drug delivery of vascular endothelial cells when ALI occurs a reality. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the technical defects of the background technology and provide a vascular endothelial cell targeted drug-loaded micelle and its preparation method and application. The present invention uses a targeting peptide LSRQ peptide ( Figure 1 , amino acid sequence LSRQPVGGHPKC) is the vascular endothelial cell targeting group, and the targeting peptide is chemically grafted onto the biomaterial polyethylene glycol-polylactic acid-glycolic acid copolymer (PEG-PLGA) with high biosafety to synthesize LSRQ-PEG-PLGA graft. Then, equal proportions of LSRQ-PEG-PLGA graft and PEG-PLGA are used as carrier materials, and the dialysis method is used to prepare melatonin-loaded micelles. The targeting and therapeutic effects are clarified at the cellular and animal levels, and based on the pathogenesis of acute lung injury, a new safe and efficient targeted treatment approach for acute lung injury is obtained.

[0006] The technical solution adopted by the present invention to solve the above technical problems is as follows:

[0007] The first aspect of the present invention provides a vascular endothelial cell targeted drug-loaded micelle, which comprises a vascular endothelial cell targeted drug-loaded micelle and an antioxidant encapsulated in the vascular endothelial cell targeted micelle; the vascular endothelial cell targeted micelle is self-assembled by an LSRQ-PEG-PLGA graft and PEG-PLGA; the LSRQ-PEG-PLGA graft is obtained by a condensation reaction between an amino group in an LSRQ peptide and a carboxyl group in PEG-PLGA; the LSRQ peptide is composed of 12 amino acid sequences, and its amino acid sequence is LSRQPVGGHPKC, and its molecular weight is 1292Da, which binds to the CD106 receptor highly expressed by vascular endothelial cells when ALI occurs, thereby increasing the distribution of the drug-loaded micelle at the target site.

[0008] Preferably, the LSRQ-PEG-PLGA graft is obtained by condensing the amino group in the LSRQ peptide and the carboxyl group in PEG-PLGA and connecting them with an amide bond; the LSRQ-PEG-PLGA graft and PEG-PLGA have good biosafety and contain hydrophilic and hydrophobic parts, so they can self-assemble to form micelles in water; the micelles can be used as drug carriers to encapsulate antioxidants to achieve active targeted treatment of acute lung injury.

[0009] Preferably, the mass ratio of the LSRQ-PEG-PLGA graft to PEG-PLGA is 1:1.

[0010] Preferably, the molar ratio of LSRQ peptide to PEG-PLGA in the LSRQ-PEG-PLGA graft is 1:1.

[0011] Preferably, the antioxidant is melatonin, which has a molecular weight of 232 Da. The micelles formed by melatonin, LSRQ-PEG-PLGA graft and PEG-PLGA have pharmacological effects and achieve targeting of vascular endothelial cells and efficient distribution in lung tissues.

[0012] Preferably, the mass ratio of the vascular endothelial cell targeting micelles to melatonin is 1:5.

[0013] Preferably, the particle size of the vascular endothelial cell targeted drug-loaded micelles is about 360 nm.

[0014] The second aspect of the present invention provides a method for preparing the vascular endothelial cell targeted drug-loaded micelles as described above, comprising the following steps:

[0015] (1) Synthesis of LSRQ-PEG-PLGA graft: dissolving the amphiphilic polymer PEG-PLGA in a solvent, adding a dehydrating agent and a catalyst, and then adding the LSRQ peptide. After the reaction is continued, the reaction product is placed in a dialysis bag, dialyzed, and freeze-dried to obtain the LSRQ-PEG-PLGA graft;

[0016] (2) Preparing micelles encapsulating the antioxidant by dialysis: mixing the LSRQ-PEG-PLGA graft prepared in step (1) with PEG-PLGA, dissolving them in a solvent, and adding the antioxidant to obtain a mixed solution; adding the mixed solution into water, stirring, dialyzing, and drying to obtain the vascular endothelial cell targeted drug-loaded micelles.

[0017] Preferably, the step (1) comprises the following preparation steps: dissolving 50 mg of the amphiphilic polymer PEG-PLGA in 5 mL of anhydrous dimethyl sulfoxide, adding 10.3 mg of N,N′-dicyclohexylcarbodiimide and 2 mg of 4-dimethylaminopyridine, and then adding 21.5 mg of the LSRQ peptide. After continuing the reaction for 48 hours, the reaction product is placed in a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzed in deionized water for 48 hours, and freeze-dried to obtain the LSRQ-PEG-PLGA graft.

[0018] Preferably, in the step (2), the LSRQ-PEG-PLGA graft and PEG-PLGA are dissolved in a solvent at a mass ratio of 1:1.

[0019] Preferably, the step (2) includes the following preparation steps: mixing the LSRQ-PEG-PLGA graft prepared in step (1) with PEG-PLGA in a mass ratio of 1:1, dissolving the mixture in DMSO, and adding the antioxidant in a carrier to drug mass ratio of 1:5 to obtain a mixed solution; adding the mixed solution dropwise into 50 mL of deionized water, stirring at room temperature for 2 hours, placing the stirred mixed solution in a dialysis bag with a molecular weight cutoff of 3500Da, dialyzing in deionized water for 48 hours, and freeze-drying to obtain the vascular endothelial cell targeted drug-loaded micelles.

[0020] The third aspect of the present invention provides a use of the vascular endothelial cell targeted drug-loaded micelle as described above in the preparation of a drug for treating acute lung injury.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The vascular endothelial cell targeted drug-loaded micelles prepared by the present invention can increase the distribution of drugs in target cells damaged by inflammation, have strong specificity, have good lung targeting effect, and have good safety, and can achieve safe and efficient targeted treatment of acute lung injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the chemical structural formula of the LSRQ peptide described in Example 1 of the present invention;

[0024] Figure 2 This is the synthetic route of the LSRQ-PEG-PLGA graft described in Example 1 of the present invention;

[0025] Figure 3 This is the H-NMR spectrum of the LSRQ-PEG-PLGA graft described in Example 1 of the present invention;

[0026] Figure 4 The particle size distribution of the LSRQ / MLT drug-loaded micelles prepared in Example 1 of the present invention;

[0027] Figure 5 This is a transmission electron microscope photograph of the LSRQ / MLT drug-loaded micelles prepared in Example 1 of the present invention;

[0028] Figure 6 The cumulative release rate of melatonin from the LSRQ / MLT drug-loaded micelles prepared in Example 1 of the present invention in the release medium;

[0029] Figure 7 The uptake of LSRQ / cou drug-loaded micelles of the present invention in inflammatory vascular endothelial cells;

[0030] Figure 8 The distribution of LSRQ / cou drug-loaded micelles of the present invention in multiple organs of acute lung injury model animals;

[0031] Fig. 9 The LSRQ / MLT drug-loaded micelles prepared in Example 1 of the present invention reduce the generation of reactive oxygen species in inflammatory vascular endothelial cells;

[0032] Fig.10 The LSRQ / MLT drug-loaded micelles prepared in Example 1 of the present invention improve the pulmonary edema in the acute lung injury model animal;

[0033] Fig.11 The LSRQ / MLT drug-loaded micelles prepared in Example 1 of the present invention improve oxidative stress injury in the lungs of animals with acute lung injury model;

[0034] Fig.12 The LSRQ / MLT drug-loaded micelles prepared in Example 1 of the present invention improve the pathological progression of lung tissue in animals with acute lung injury model;

[0035] Fig.13 This is a safety evaluation of the nanoformulation prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0036] In order to better understand the content of the present invention, further description is given below in conjunction with specific embodiments and accompanying drawings. It should be understood that these embodiments are only used to further illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content described in the present invention, those skilled in the art may make some non-essential changes or adjustments to the present invention, which still belong to the protection scope of the present invention.

[0037] Example 1

[0038] A method for preparing vascular endothelial cell-targeted drug-loaded micelles comprises the following steps:

[0039] (1) Synthesis of LSRQ-PEG-PLGA graft: 50 mg of the amphiphilic polymer PEG-PLGA was dissolved in 5 mL of anhydrous dimethyl sulfoxide, and 10.3 mg of N, N′-dicyclohexylcarbodiimide and 2 mg of 4-dimethylaminopyridine were added as dehydrating agents. 21.5 mg of LSRQ peptide (amino acid sequence: LSRQPVGGHPKC, molecular weight: 1292 Da) was then added. After the reaction was continued for 48 h, the reaction product was placed in a dialysis bag (molecular weight cutoff: 3500 Da), dialyzed in deionized water for 48 h, and freeze-dried to obtain LSRQ-PEG-PLGA graft ( Figure 2 );

[0040] (2) Preparing micelles encapsulating melatonin by dialysis, labeled as LSRQ / MLT drug-loaded micelles: The LSRQ-PEG-PLGA graft prepared in step (1) was mixed with PEG-PLGA at a mass ratio of 1:1, dissolved in DMSO, and melatonin (molecular weight of 232 Da) was added at a drug loading ratio of 1:5 (mass ratio of carrier to drug) to obtain a mixed solution; the mixed solution was dropwise added into 50 mL of deionized water, stirred at room temperature for 2 h, and the stirred mixed solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da. After dialysis in deionized water for 48 h, the LSRQ / MLT drug-loaded micelles were obtained after freeze-drying.

[0041] Effect Example

[0042] 1. Determination of drug loading and encapsulation efficiency of melatonin in LSRQ / MLT drug-loaded micelles

[0043] 10 mg of the LSRQ / MLT drug-loaded micelles prepared in Example 1 were accurately weighed, dissolved in dimethyl sulfoxide, filtered to remove impurities, and then the drug loading amount and encapsulation efficiency of melatonin in the LSRQ / MLT drug-loaded micelles were determined by high performance liquid chromatography.

[0044] It was determined that the drug loading and encapsulation efficiency of the LSRQ / MLT drug-loaded micelles prepared in Example 1 were 8.5±0.7% and 90.8±1.2%, respectively.

[0045] 2. 1H-NMR determination of the molecular structure of LSRQ-PEG-PLGA grafts

[0046] The chemical shift of the protons on the LSRQ peptide is mainly concentrated around δ=7.5-9, while the chemical shift peak of the protons on the terminal carboxyl group of PEG-PLGA is at δ=12.6ppm. In the 1H-NMR spectrum of the LSRQ-PEG-PLGA graft prepared in Example 1, the chemical shift of the LSRQ peptide around δ=7.5-9 can be clearly seen, and the chemical shift peak of the protons on the terminal carboxyl group of PEG-PLGA at δ=12.6ppm disappears ( Figure 3 ), which is due to the amide reaction between the carboxyl group and the amino group on the LSRQ peptide. Therefore, it can be concluded that the LSRQ peptide is successfully connected to PEG-PLGA.

[0047] 3. Particle size and Zeta potential determination of LSRQ / MLT drug-loaded micelles

[0048] The LSRQ / MLT drug-loaded micelles prepared in Example 1 were diluted with deionized water to a 0.1 mg / mL dispersion, and the particle size distribution and Zeta potential thereof were measured using a particle size potential meter.

[0049] The particle size distribution of LSRQ / MLT drug-loaded micelles was measured by Malvern particle size analyzer. The results showed that the particle size of LSRQ / MLT drug-loaded micelles was around 360nm ( Figure 4 ), the polydispersity coefficient PDI is 0.263, the Zeta potential is -8.46, and the surface of the nanomicelles is negatively charged.

[0050] 4. Observation of Nanoparticle Morphology by Transmission Microscopy

[0051] The LSRQ / MLT drug-loaded micelles prepared in Example 1 were diluted with deionized water to a 0.1 mg / mL dispersion, 10 μL was dropped on a copper mesh, and after natural evaporation, negative staining was performed with 1% phosphotungstic acid solution for 1 minute. The surface liquid was absorbed by filter paper, and the appearance of the LSRQ / MLT drug-loaded micelles was observed under a transmission electron microscope.

[0052] like Figure 5 It can be seen that the LSRQ / MLT drug-loaded micelles have a round appearance, a smooth surface, and a relatively uniform particle size distribution.

[0053] 5. Investigation of in vitro release of LSRQ / MLT drug-loaded micelles

[0054] The LSRQ / MLT drug-loaded micelles prepared in Example 1 were dispersed with pH 7.4 phosphate buffer, placed in a dialysis bag (molecular weight cutoff 3500Da), and then immersed in 50mL pH 7.4 phosphate buffer, and oscillated for 48h at 37°C and 200rpm. At a predetermined time point, 0.2mL of release solution was drawn and the same volume of fresh phosphate buffer was added. After the release solution was diluted, the melatonin concentration was determined by high performance liquid chromatography, and the cumulative release of LSRQ / MLT drug-loaded micelles at different time points was calculated.

[0055] The in vitro cumulative release results showed that ( Figure 6 ), compared with simple free drugs, LSRQ / MLT drug-loaded micelles have a sustained release effect on drugs; and whether the LSRQ peptide is modified or not does not affect the release behavior of the encapsulated drugs.

[0056] VI. Uptake of LSRQ / cou drug-loaded micelles in inflammatory vascular endothelial cells

[0057] Human umbilical vein endothelial cells were cultured at 5×10 4 The cells were inoculated into a 12-well plate at a density of 10 cells / well and cultured overnight at 37°C. The fluorescent molecule coumarin (cou) was used to replace the melatonin in Example 1 to prepare LSRQ / cou drug-loaded micelles. The cou drug-loaded micelles without LSRQ modification were used as controls. After incubation with inflammatory cells for a period of time, the transport of drug-loaded micelles in cells was observed under a fluorescence microscope.

[0058] Fluorescence imaging was used to investigate the transport of LSRQ peptide-modified micelles in inflammatory vascular endothelial cells. Figure 7 As shown, after the micelles were modified with LSRQ peptides, their transport in inflammatory vascular endothelial cells was significantly improved (fluorescence signal enhancement), and the additional free LSRQ peptide would compete with the LSRQ / cou micelles for binding to the CD106 receptor, thereby affecting the transport of LSRQ / cou micelles in inflammatory vascular endothelial cells, indicating that the drug-loaded micelles modified with LSRQ peptides can increase their uptake by inflammatory vascular endothelial cells.

[0059] VII. Distribution of LSRQ / cou drug-loaded micelles in multiple organs of acute lung injury model animals

[0060] An acute lung injury mouse model was established by exposed tracheal instillation of lipopolysaccharide. The mice were anesthetized with 5% chloral hydrate (450 mg / kg) and lipopolysaccharide (2 μg / g, body weight) dissolved in 50 μL of normal saline was slowly instilled through the exposed tracheal instillation method.

[0061] Coumarin (cou) was used instead of melatonin in Example 1 to encapsulate the micelles to prepare LSRQ / cou drug-loaded micelles. The cou drug-loaded micelles without LSRQ modification were used as the control. The acute lung injury model mice were injected with LSRQ / cou drug-loaded micelles or cou drug-loaded micelles through the tail vein. The mice were killed 6 hours after the administration, and the heart, liver, spleen, lung and kidney were obtained to make frozen sections. The distribution of the two drug-loaded micelles in various organs was observed under an inverted fluorescence microscope.

[0062] The distribution of LSRQ peptide-modified micelles in various organs and tissues of acute lung injury model animals was investigated by fluorescence imaging technology. Figure 8 As shown, the distribution of LSRQ / cou micelles in lung tissue is higher than that of micelles not modified with LSRQ peptide, indicating that LSRQ peptide can increase the distribution of micelles in lung tissue and provide a basis for the lung-targeted delivery of melatonin.

[0063] 8. LSRQ / MLT drug-loaded micelles reduce the production of reactive oxygen species in inflammatory vascular endothelial cells

[0064] Human umbilical vein endothelial cells were cultured at 5×10 4 The cells were inoculated into a 12-well plate at a density of 10 cells / well and cultured overnight at 37°C. After 12 hours of lipopolysaccharide stimulation, MLT, MLT drug-loaded micelles or LSRQ / MLT drug-loaded micelles prepared in Example 1 were added and incubated for 12 hours. The fluorescent probe DCFH-DA was used to detect the level of reactive oxygen in vascular endothelial cells. DCFH-DA itself has no fluorescence and can freely pass through the cell membrane. After entering the cell, it can be hydrolyzed by the esterase in the cell to generate DCFH. However, DCFH cannot penetrate the cell membrane, making it easy for the probe to be loaded into the cell. The reactive oxygen in the cell can oxidize the non-fluorescent DCFH to generate fluorescent DCF. The level of intracellular reactive oxygen is evaluated by detecting the fluorescent signal of intracellular DCF, and the intracellular DCF fluorescent signal is observed by a fluorescent inverted microscope.

[0065] The fluorescent probe DCFH-DA was used to detect the level of reactive oxygen species in endothelial cells. Fig. 9 As shown, after endothelial cells were stimulated by lipopolysaccharide, the level of reactive oxygen species increased significantly (fluorescence signal enhanced), while after intervention with MLT, MLT drug-loaded micelles or LSRQ / MLT drug-loaded micelles, the level of reactive oxygen species was reduced to varying degrees, and the effect of LSRQ / MLT drug-loaded micelles was the most significant.

[0066] IX. LSRQ / MLT drug-loaded micelles improve pulmonary edema in acute lung injury model animals

[0067] An acute lung injury mouse model was constructed by exposed tracheal instillation of lipopolysaccharide, and the drug was administered through the tail vein to investigate the therapeutic effect of the LSRQ / MLT drug-loaded micelles prepared in Example 1 on acute lung injury. The experimental animals were divided into the following groups: Group A, normal group; Group B, acute lung injury + saline group; Group C, acute lung injury + MLT group; Group D: acute lung injury + MLT drug-loaded micelle group; Group E: acute lung injury + LSRQ / MLT drug-loaded micelle group. The mice were killed 24 hours after administration, and the right lower lung lobe was taken. The surface blood and water were absorbed with filter paper, and the wet weight was weighed. It was placed in a 70°C oven for 48 hours, and the dry weight was weighed to calculate the wet weight / dry weight (W / D) value.

[0068] An acute lung injury mouse model was established by instilling lipopolysaccharide in exposed trachea, and the drug was administered through the tail vein to investigate the therapeutic effect of the LSRQ / MLT drug-loaded micelles prepared in Example 1 on acute lung injury. Fig.10 As shown in the figure, the wet / dry ratio of lung tissue in mice with acute lung injury increased significantly, while after intervention with MLT, MLT drug-loaded micelles or LSRQ / MLT drug-loaded micelles, the wet / dry ratio of lung tissue was reduced to varying degrees, and the effect of LSRQ / MLT drug-loaded micelles was the most significant.

[0069] 10. LSRQ / MLT drug-loaded micelles improve oxidative stress injury in the lungs of animals with acute lung injury model

[0070] The acute lung injury mouse model was established by instilling lipopolysaccharide in exposed trachea, and the drug was administered through the tail vein to investigate the therapeutic effect of the LSRQ / MLT drug-loaded micelles prepared in Example 1 on acute lung injury. The experimental animals were divided into the following groups: Group A, normal group; Group B, acute lung injury + saline group; Group C, acute lung injury + MLT group; Group D: acute lung injury + MLT drug-loaded micelle group; Group E: acute lung injury + LSRQ / MLT drug-loaded micelle group. Mice were killed 24 hours after administration, and alveolar lavage fluid was collected to measure the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in the lavage fluid.

[0071] The levels of superoxide dismutase (SOD) and malondialdehyde (MDA), indicators of oxidative stress in the lung tissue of mice with acute lung injury, were measured. Fig.11 As shown in the figure, the SOD level in the lung tissue of mice with acute lung injury was significantly reduced, while after intervention with MLT, MLT-loaded micelles or LSRQ / MLT-loaded micelles, the SOD level was increased to varying degrees, and the effect of LSRQ / MLT-loaded micelles was the most significant ( Fig.11 A). The MDA level in the lung tissue of mice with acute lung injury was significantly reduced. After intervention with MLT, MLT-loaded micelles or LSRQ / MLT-loaded micelles, the MDA level was reduced to varying degrees, and the effect of LSRQ / MLT-loaded micelles was the most significant ( Fig.11 B).

[0072] XI. LSRQ / MLT drug-loaded micelles improve the pathological changes of lung tissue in animals with acute lung injury model

[0073] An acute lung injury mouse model was constructed by exposed tracheal instillation of lipopolysaccharide, and the drug was administered through the tail vein to investigate the therapeutic effect of the LSRQ / MLT drug-loaded micelles prepared in Example 1 on acute lung injury. The experimental animals were divided into the following groups: Group A, normal group; Group B, acute lung injury + saline group; Group C, acute lung injury + MLT group; Group D: acute lung injury + MLT drug-loaded micelle group; Group E: acute lung injury + LSRQ / MLT drug-loaded micelle group. Mice were killed 24 hours after administration, and the left lung lobe was taken and fixed in formalin for 24 hours. After fixation, it was dehydrated, transparent, paraffin-embedded, and sectioned, and hematoxylin-eosin (H&E) staining was performed. Pulmonary edema and inflammatory cell infiltration were observed under a microscope, and pathological changes in lung tissue were observed.

[0074] like Fig.12 As shown, after the acute lung injury mice were treated with MLT, MLT drug-loaded micelles or LSRQ / MLT drug-loaded micelles prepared in Example 1, the pathological conditions of the lung tissue sections were improved to a certain extent compared with the model group. The pathological section photos of the mice in the treatment group were compared with the model group: the number of inflammatory cells infiltrating in the alveoli was significantly reduced, and the alveolar structure was visible, among which the improvement in the LSRQ / MLT drug-loaded micelle experimental group was the most significant. The analysis results show that LSRQ / MLT drug-loaded micelles can play a protective role against acute lung injury symptoms in the model animals of the present invention.

[0075] 12. Safety evaluation of nanoparticle preparations

[0076] The staining results of pathological sections of important organs can directly reflect the side effects of nanoformulations. After administration (LSRQ / MLT drug-loaded micelles prepared in Example 1), the heart, liver, spleen, lung and kidney tissue sections of each group of mice were stained, and no obvious damage was found in each organ, indicating that the nanocomposite preparation prepared by the present invention has low toxicity ( Fig.13 ).

[0077] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A vascular endothelial cell-targeted drug-loaded micelle, characterized in that: The vascular endothelial cell targeted drug-loaded micelles include vascular endothelial cell targeted micelles and antioxidants encapsulated in the vascular endothelial cell targeted micelles; the vascular endothelial cell targeted micelles are self-assembled by LSRQ-PEG-PLGA grafts and PEG-PLGA; the LSRQ-PEG-PLGA grafts are obtained by condensation reaction of amino groups in LSRQ peptides and carboxyl groups in PEG-PLGA; the amino acid sequence of the LSRQ peptide is LSRQPVGGHPKC.

2. The vascular endothelial cell targeted drug-loaded micelle according to claim 1, characterized in that The mass ratio of the LSRQ-PEG-PLGA graft to PEG-PLGA is 1:

1.

3. The vascular endothelial cell-targeted drug-loaded micelle according to claim 1, characterized in that: The molar ratio of L SRQ peptide to PEG-PLGA in the L SRQ-PEG-PLGA graft is 1:

1.

4. The vascular endothelial cell-targeted drug-loaded micelle according to claim 1, characterized in that: The antioxidant is melatonin.

5. The vascular endothelial cell-targeted drug-loaded micelle according to claim 4, characterized in that: The mass ratio of the vascular endothelial cell targeting micelle to melatonin is 1:

5.

6. The vascular endothelial cell-targeted drug-loaded micelle according to claim 1, characterized in that: The particle size of the vascular endothelial cell targeted drug-loaded micelle is about 360 nm.

7. The method for preparing a vascular endothelial cell-targeted drug-loaded micelle according to any one of claims 1 to 6, characterized in that: The steps include: (1) Synthesis of LSRQ-PEG-PLGA graft: dissolving the amphiphilic polymer PEG-PLGA in a solvent, adding a dehydrating agent and a catalyst, and then adding the LSRQ peptide. After the reaction is continued, the reaction product is placed in a dialysis bag, dialyzed, and freeze-dried to obtain the LSRQ-PEG-PLGA graft; (2) Preparing micelles encapsulating the antioxidant by dialysis: mixing the LSRQ-PEG-PLGA graft prepared in step (1) with PEG-PLGA, dissolving them in a solvent, and adding the antioxidant to obtain a mixed solution; adding the mixed solution into water, stirring, dialyzing, and drying to obtain the vascular endothelial cell targeted drug-loaded micelles.

8. The method for preparing a vascular endothelial cell-targeted drug-loaded micelle according to claim 7, characterized in that: The step (1) comprises the following preparation steps: dissolving 50 mg of the amphiphilic polymer PEG-PLGA in 5 mL of anhydrous dimethyl sulfoxide, adding 10.3 mg of N,N'-dicyclohexylcarbodiimide and 2 mg of 4-dimethylaminopyridine, and then adding 21.5 mg of the LSRQ peptide. After continuing the reaction for 48 hours, the reaction product is placed in a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzed in deionized water for 48 hours, and freeze-dried to obtain the LSRQ-PEG-PLGA graft.

9. The method for preparing a vascular endothelial cell-targeted drug-loaded micelle according to claim 7, characterized in that: The step (2) comprises the following preparation steps: mixing the LSRQ-PEG-PLGA graft prepared in step (1) with PEG-PLGA at a mass ratio of 1:1, dissolving the mixture in DMSO, and adding the antioxidant at a mass ratio of carrier to drug of 1:5 to obtain a mixed solution; adding the mixed solution dropwise into 50 mL of deionized water, stirring at room temperature for 2 hours, placing the stirred mixed solution in a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzing in deionized water for 48 hours, and freeze-drying to obtain the vascular endothelial cell targeted drug-loaded micelles.

10. Use of the vascular endothelial cell targeted drug-loaded micelle according to any one of claims 1 to 6 in the preparation of a drug for treating acute lung injury.

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