Macrophage-targeting compound and use thereof

Abstract

The present invention relates to the technical field of biology, and specifically relates to a macrophage-targeting compound and a use thereof. The compound uses a saccharide as a raw material, and the problem of functional instability caused by the non-uniform structural composition of the compound is avoided while the high targeting performance of the compound is maintained. By selecting the basic unit of a saccharide as a targeting moiety and then linking to a hydrophobic chain, the aggregation of saccharide units in water is completed, ensuring the high targeting performance of the design material. The saccharide-containing compound and a complex thereof can specifically target macrophages in plaques without affecting other cells, thereby achieving in vivo specific targeting of macrophages.

Description

A compound targeting macrophages and applications thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a compound targeting macrophages and applications thereof. BACKGROUND

[0002] Cardiovascular disease has been the leading cause of death globally (Safiri, S., et al., Burden of ischemic heart disease and its attributable risk factors in 204 countries and territories, 1990-2019. European Journal of Preventive Cardiology, 2022. 29(2): p. 420-431.). According to statistics, 2050 million people died of cardiovascular disease in 2021, accounting for about one-third of the total number of deaths worldwide. One of the main causes of cardiovascular disease is atherosclerosis (Wu, Y., et al., Recent Advances in the Development of Theranostic Nanoparticles for Cardiovascular Diseases. Nanotheranostics, 2021. 5(4): p. 499-514.), which is a disease caused by inflammation of the blood vessel wall, and ischemic heart disease (coronary heart disease) is a heart disease caused by coronary atherosclerosis leading to myocardial ischemia and hypoxia. Global burden of ischemic heart disease (coronary heart disease) reports from 1990 to 2019 show that in 2019, about 914 million people died of coronary heart disease worldwide, and an estimated 1.97 million people worldwide suffered from coronary heart disease. Among them, the number of deaths in China reached 1.87 million, ranking among the countries with the most deaths from coronary heart disease worldwide. Most patients with coronary heart disease have no symptoms at ordinary times, which also leads to the difficulty of early diagnosis and timely intervention for coronary heart disease. Therefore, developing a non-invasive method for diagnosing coronary heart disease is conducive to the timely discovery of coronary heart disease and reduces the risk of disease in patients.

[0003] In the early stage of atherosclerosis formation, monocytes are recruited to accumulate at the arterial vessel wall and then differentiate into macrophages. Macrophages will take up a large amount of low-density lipoprotein-derived cholesterol, and the excessive accumulation of cholesterol will further promote the inflammatory response and induce the formation of vulnerable plaques, triggering atherosclerotic lesions (Chung, E.J. and M. Tirrell, Recent Advances in Targeted, Self-Assembling Nanoparticles to Address Vascular Damage Due to Atherosclerosis. Advanced Healthcare Materials, 2015. 4(16): p. 2408-2422.). Macrophages play a key role in the process of atherosclerotic lesions, so they are considered an important diagnostic and therapeutic target for atherosclerotic cardiovascular disease (Chen, W., et al., Macrophage-targeted nanomedicine for the diagnosis and treatment of atherosclerosis. Nature Reviews Cardiology, 2021. 19(4): p. 228-249.). However, there is a high degree of similarity between macrophages involved in atherosclerosis and host defense, which has led to the difficulty of designing materials targeting atherosclerotic macrophages. During the atherosclerotic lesion process, smooth muscle transdifferentiated macrophages play an important role, which accounts for more than 60% in late-stage plaques (Xu, H., et al., Vascular Macrophages in Atherosclerosis. Journal of Immunology Research, 2019. 2019: p. 1-14.), but there is currently no development and application of targeting agents for smooth muscle transdifferentiated macrophages. Experiments have shown that macrophage scavenger receptor 1 (MSR1) is overexpressed on the surface of smooth muscle transdifferentiated macrophages (Zhai, M., et al., Extracellular traps from activated vascular smooth muscle cells drive the progression of atherosclerosis. Nature Communications, 2022. 13(1.). Therefore, MSR1 receptor is a potential target for smooth muscle transdifferentiated macrophages and can be further used for diagnosis and treatment of atherosclerosis.Fucoidan is a natural polysaccharide that can target the MSR1 receptor on the surface of macrophages, and is also considered to be beneficial for the treatment of cardiovascular disease (Chollet, L., et al., Fucoidans in Nanomedicine. Marine Drugs, 2016. 14(8).). However, due to the complex structure and non-uniform composition of fucoidan, its effect may vary with different extraction batches, and even cause opposite effects (Suprunchuk, V. E., Low-molecular-weight fucoidan: Chemical modification, synthesis of its oligomeric fragments and mimetics. Carbohydrate Research, 2019. 485.). At the same time, due to the complex structure of fucoidan, the complexation of fucoidan with functional groups such as developing agents is relatively complex, and therefore the application of fucoidan is limited.

[0004] Computed tomography (CT) is a non-destructive 3D imaging technique. In clinical practice, due to its high spatial resolution, short acquisition time and other advantages, CT has become a powerful tool for monitoring coronary atherosclerotic macrophages (Hyafil, F., et al., Noninvasive detection of macrophages using a nanoparticulate contrast agent for computed tomography. Nature Medicine, 2007. 13(5): p. 636-641.). Therefore, the development of a CT imaging material that can target atherosclerotic macrophages will help non-invasive diagnosis and treatment of coronary heart disease and monitoring of disease-related macrophages. Currently, the materials used for atherosclerotic CT imaging mainly include small molecule contrast agents, iodinated nanoparticles and gold nanoparticles. Among them, gold nanoparticles have attracted widespread attention as contrast agents in recent years due to their stability, low toxicity and high X-ray attenuation coefficient (Wu, Y., et al., Recent Advances in the Development of Theranostic Nanoparticles for Cardiovascular Diseases. Nanotheranostics, 2021. 5(4): p. 499-514.). In addition, gold nanoparticles are easier to modify, and can be modified with targeting groups on their surface, which can greatly reduce the amount of contrast agent used and facilitate its clinical translation. Targeted modification of imaging gold nanoparticles mostly chooses antibody protein modification or synthetic molecule modification. Although antibody modification has high selectivity, its high cost makes it difficult to translate. Synthetic molecule modification has relatively low cost, but due to the low targeting of synthetic molecules and the difficulty of cell-specific targeting, it is very difficult but also has great application prospect to develop a contrast agent for targeting macrophages modified by synthetic molecules. SUMMARY

[0005] Based on the deficiencies of the prior art, the present application provides a compound which uses sugar as a raw material, retains its high targeting while avoiding the problem of functional instability caused by uneven sugar structure composition; by selecting the basic unit of sugar as the targeting part, and combining with the hydrophobic chain to complete the aggregation of the sugar unit in water, the high targeting performance of the designed material is ensured, that is, the compound can specifically target the smooth muscle transdifferentiation macrophages and myeloid-derived macrophages in the plaque, without affecting other cells, achieving specific targeting of macrophages in vivo. Based on this, the present application is completed.

[0006] In a first aspect, the present application provides a compound that can target macrophages, the compound being a sugar-containing amphiphilic small molecule of Formula (I) or a sugar-containing polymer of Formula (II),

[0007] wherein the oxygen-containing ring represents a sugar, R is a compound containing a lipid chain or a peptide chain, and Linker is a linker connecting the oxygen-containing ring and the compound chain, preferably an amide bond or a triazole ring;

[0008] wherein 1 black main chain line represents a polymer backbone, and 2 oxygen-containing ring represents a sugar.

[0009] Further, the sugar of Formula (I) is selected from one or more of mannose, glucose, galactose, fucose, and their corresponding sulfated sugars.

[0010] Further, the sugar of Formula (II) is selected from one or more of mannose, mannodiose, galactose, and / or fucose.

[0011] Preferably, the sugar is sulfated fucose.

[0012] Further, the type of sulfated fucose is selected from tri-substitution, di-substitution, and / or mono-substitution.

[0013] Further, the lipid chain is selected from one or more of single-chain, double-chain, and / or different length lipid chains.

[0014] Further, the peptide chain is selected from one or more of 2F (phenylalanine-phenylalanine), 3F (phenylalanine-phenylalanine-phenylalanine), KYF (lysine-tyrosine-phenylalanine), KYY (lysine-tyrosine-tyrosine), KFF (lysine-phenylalanine-phenylalanine), and / or KYW (lysine-tyrosine-tryptophan).

[0015] Further, the polymer backbone of Formula (II) is selected from one or more of a polyester, a polyacrylate, or a polyamino acid.

[0016] Further, the polymer structure includes, but is not limited to, one or more of homopolymerization, random copolymerization, or block copolymerization.

[0017] Further, the way the polymer backbone is connected to the sugar includes an amide bond or a triazole ring connection.

[0018] In an embodiment of the present application, when R is a compound containing a lipid chain, the compound of Formula (I) is a glycolipid molecule, and the structure is as follows:

[0019] In one embodiment of the present application, when R is a compound containing a peptide chain, the compound of formula (I) is a glycopeptide molecule, and the structure is selected from any one of the following:

[0020] Further, the macrophages include smooth muscle transdifferentiation macrophages and myeloid-derived macrophages.

[0021] In a second aspect, the present application provides a composite nanoparticle, which is composed of the compound of the first aspect and an encapsulated particle, wherein the surface of the encapsulated particle is modified by the compound of the first aspect; the composite nanoparticle can be enriched at the site of atherosclerotic lesions, and early plaques and late plaques can be distinguished by imaging techniques.

[0022] Further, the encapsulated particle includes, but is not limited to, a gold nanoparticle or an iron particle.

[0023] Further, the gold nanoparticle has a particle size of 1-50 nm.

[0024] Further, the gold nanoparticle has a particle size of 5-40 nm.

[0025] Preferably, the gold nanoparticle has a particle size of 10-30 nm.

[0026] Further, the iron particle includes ferroferric oxide and / or iron oxide.

[0027] Further, the imaging technique includes, but is not limited to, optical coherence tomography (OCT), magnetic resonance imaging (MRI), and micro-computed tomography (micro-CT).

[0028] In a third aspect, the present application provides a targeted imaging agent, which comprises the compound of the first aspect and a fluorescent molecule, and the targeted imaging agent can be targeted and enriched at the site of atherosclerotic lesions of blood vessels, reflecting the progression of plaque lesions.

[0029] Further, the fluorescent molecule includes, but is not limited to, one or more of FITC, Cy5, Cy3, and / or ICG.

[0030] Further, the targeted imaging agent is a contrast agent.

[0031] In a fourth aspect, the present application provides a targeted pharmaceutical composition, which comprises the compound of the first aspect and a drug molecule.

[0032] Further, the drug molecule includes, but is not limited to, one or more of carmustine, paclitaxel, docetaxel, vinblastine, navelbine, and / or statins.

[0033] In a fifth aspect, the present application provides use of the compound of the first aspect or the composite nanoparticle of the second aspect in the preparation of a targeted imaging reagent for macrophages, which can be targeted to accumulate in the vascular site of an atherosclerotic lesion, reflecting the progression of plaque lesions.

[0034] Further, the macrophages include smooth muscle transdifferentiation macrophages and myeloid-derived macrophages.

[0035] Further, the targeted imaging reagent is a contrast agent.

[0036] In a sixth aspect, the present application provides use of the targeted pharmaceutical composition of the fourth aspect in the preparation of a medicament for treating an atherosclerosis-related disease, which exerts an effect by transporting a drug to the macrophages in the atherosclerotic plaque.

[0037] Further, the atherosclerosis-related disease includes, but is not limited to, one or more of coronary heart disease, stroke, abdominal aortic aneurysm, peripheral arterial disease, hypertension, diabetes, hyperlipidemia and / or hypercholesterolemia.

[0038] Further, the macrophages include smooth muscle transdifferentiation macrophages and myeloid-derived macrophages. Advantages

[0039] 1. The compound material prepared by the present application is derived from sugar, which avoids the problem of functional instability caused by the uneven structure of sugar while retaining its high targeting property; by selecting a sugar basic unit as the targeting part, and then combining a hydrophobic lipid chain or a peptide chain to complete the aggregation of the sugar unit in water, the high targeting performance of the designed material is ensured.

[0040] 2. The compound prepared by the present application can also be very simple to complex drug molecules or fluorescent molecules and other functional groups due to the introduction of lipid chains or peptide chains, so as to realize the targeted imaging and drug delivery of smooth muscle transdifferentiation macrophages.

[0041] 3. The compound prepared by the present application can also be complexed with a packaging particle to distinguish the early and late stage plaques by the change of CT development area, which is expected to be used for non-invasive diagnosis and treatment of atherosclerosis-related diseases.

[0042] 4. The compound and its complex prepared by the present application can specifically target smooth muscle transdifferentiation macrophages and myeloid-derived macrophages in the plaque, without affecting other cells, so as to realize the in vivo specific targeting of macrophages and facilitate the tracking research of such cells.

[0043] 5. The compound and its complex of the present application have good biocompatibility. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 is a nuclear magnetic hydrogen spectrum of product 3.

[0045] Figure 2 is a nuclear magnetic hydrogen spectrum of product 4.

[0046] Figure 3 is a nuclear magnetic hydrogen spectrum of product 5.

[0047] Figure 4 is a nuclear magnetic hydrogen spectrum of product 6.

[0048] Figure 5 is a nuclear magnetic hydrogen spectrum of SFGL.

[0049] Figure 6 is a nuclear magnetic carbon spectrum of SFGL.

[0050] Figure 7 is a mass spectrum of SFGL.

[0051] Figures 8-12 are nuclear magnetic hydrogen spectra of glycopeptide molecules.

[0052] Figure 13 is a glycolipid characterization. (A) Negative staining transmission electron microscopy image (the red circle in the figure is a small ball formed by glycolipid molecules); (B) Cryo-EM image (the red circle in the figure is a small ball formed by glycolipid molecules); (C) Zeta potential graph.

[0053] Figure 14 is a fluorescence emission spectrum. The black line represents the glycolipid solution, and the red line represents the aqueous solution.

[0054] Figure 15 is a glycolipid modified gold nanoparticle characterization. (A) UV-Vis absorption spectrum (the black line represents unmodified gold nanoparticles, and the red line represents glycolipid modified gold nanoparticles); (B) Dynamic light scattering spectrum; (C) Transmission electron microscopy image of unmodified gold nanoparticles (the scale bar in the figure represents 50 nm); (D) Transmission electron microscopy image of glycolipid modified gold nanoparticles (the scale bar in the figure represents 50 nm); (E) Process diagram of glycolipid modified gold nanoparticles.

[0055] Figure 16 is an in vivo near-infrared fluorescence imaging experiment to verify the performance of targeting diseased blood vessels. (A) In vivo atherosclerotic aorta fluorescence imaging image and related oil red o staining results of atherosclerotic aorta; (B) Representative fluorescence images of different organs at different time points after glycolipid injection; Liver: liver; Kidney: kidney; Lung: lung; Heart: heart; Spleen: spleen; Aorta: aorta; (C) Quantification of the mean fluorescence index of different organs at different time points after glycolipid injection.

[0056] Figure 17 is the experimental results of different concentrations of glycolipid molecules incubated with RAW264.7 cells. (A) Representative immunofluorescence of SFGL binding positive RAW264.7 macrophages incubated with different concentrations of glycolipid solution; (B) Quantitative analysis of positive SFGL+ cells / total cells in Figure A, ****p<0.0001; (C) Flow cytometry analysis chart of RAW264.7 incubated with different concentrations of glycolipid solution; (D) Quantitative results of SFGL + cells / total cells in flow cytometry of Figure C.

[0057] Figure 18 is the flow cytometry analysis chart of endothelial cells (A) and smooth muscle cells (C) after co-incubation with different concentrations of glycolipid solution; Figures B and D are the quantitative results of SFGL+ cell ratio of Figures A and C, respectively.

[0058] Figure 19 is the targeting of glycolipid in the myocardial infarction model. (A) Glycolipid targeting chart at the aortic vascular plaque of the myocardial infarction model. (B) Glycolipid targeting chart at the spleen of the myocardial infarction model. (C) Quantitative analysis chart of Figure A, showing the glycolipid targeting at the aortic vascular plaque of the myocardial infarction model mice and healthy mice.

[0059] Figure 20 is the specific targeting of smooth muscle transdifferentiation macrophages by glycolipid. (A) The upper three charts represent smooth muscle cell targeting flow cytometry analysis chart, and the lower two charts represent smooth muscle transdifferentiation macrophage targeting flow cytometry analysis chart. The abscissa represents the fluorescence intensity, reflecting the binding ability of glycolipid molecules; the ordinate represents the side scatter light intensity, reflecting the cell type; (B) Quantitative statistical result chart of Figure A.

[0060] Figure 21 is the targeting of fluorescently labeled glycolipid to plaques at different stages. (A) Arteriosclerotic plaque section images of early and late stage regions. Among them, DAPI represents the location of cell nucleus, zsGreen represents cells unrelated to smooth muscle cells, tdTomato represents smooth muscle cells, Cd68 represents macrophages, and the overlapping area of tdTomato and Cd68 represents smooth muscle transdifferentiation macrophages. Scale bar = 100 μm; (B) Representative image of Masson staining of plaques in Figure A. Scale bar = 100 μm; (C) The proportion of CD68+Td+ cells to total Td+ cells in Figure B; (D) The average fluorescence intensity of SFGL in the atherosclerotic plaques of Figure B; (E) Quantification of the ratio of necrotic area to plaque area (%) in Figure B.

[0061] Figure 22 is a sugar lipid gold nanoparticle complex targeting different stages of plaque and its CT imaging results. (A) CT imaging of sugar lipid gold nanoparticle complex targeting atherosclerotic plaque; (B) Oil red O staining results of the aorta used in Figure A; (C) CT images of SFGL complex gold nanoparticles in the mouse aorta of atherosclerotic plaque at different stages; (D) Quantification of plaque volume in Figure C; (E) Masson staining image of plaque in Figure C; (F) Quantification of the ratio of necrotic area / plaque area and fibrosis area / plaque area in Figure E.

[0062] Figure 23 is the CCK-8 detection results after the sugar lipid and cells are co-incubated. DETAILED DESCRIPTION

[0063] The specific embodiments of the present application are described below. It should be noted that the description of these embodiments is used to help understand the present application, but does not constitute a limitation on the present application. In addition, the technical features involved in the following described embodiments can be combined with each other as long as they do not conflict with each other.

[0064] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all commercially available unless otherwise specified.

[0065] MSR1: Macrophage scavenger receptor 1 (Macrophage Scavenger Receptor 1) or CD204, belongs to a class of pattern recognition receptors mainly expressed in macrophages. Scavenger receptor A is a member of the scavenger receptor family, and MSR1 protein exists in three types: type I, type II and type III; they are derived from the selective splicing of the same gene. Type I and type II isoforms are functional receptors that can mediate the endocytosis of low-density lipoprotein, and type III isoform does not embed into the membrane and has no uptake function.

[0066] Attenuation coefficient: describes the probability of interaction between rays and matter, which is affected by factors such as the energy of the rays, the atomic number and density of the material.

[0067] Example 1 Molecular synthesis circuit

[0068] 1.1 Preparation of the basic structure of sugar lipid

[0069] Synthesis route of sugar lipid molecule (SFGL)

[0070] 1.1.1 Synthesis of compounds 1-3

[0071] In a round bottom flask, add L-fucose, acetic anhydride and pyridine, stir overnight at room temperature, extract and dry to get compound 1. Then, dissolve compound 1 in tetrahydrofuran, add benzylamine dropwise, stir at room temperature for 12 h, and pass through a column to get compound 2. In a round bottom flask, add compound 2, potassium bicarbonate, tert-butyl bromoacetate and DMF in sequence, stir for two days. After the reaction is completed, filter out the salt, and purify by column to get the product, which is compound 3.

[0072] 1 H NMR (400 MHz, Chloroform-d) δ 5.41 (dd, J = 10.5, 3.4 Hz, 1H), 5.31 (dd, J = 3.4, 1.3 Hz, 1H), 5.19 - 5.12 (m, 2H), 4.28 (dtd, J = 6.6, 6.0, 1.2 Hz, 1H), 4.16 - 4.00 (m, 2H), 2.16 (s, 3H), 2.13 (s, 3H), 1.98 (s, 3H), 1.45 (s, 9H), 1.13 (d, J = 6.5 Hz, 3H). See Figure 1 for the NMR spectrum.

[0073] 1.1.2 Synthesis of compound 4

[0074] Dissolve compound 3 in a mixed solvent of dichloromethane and trifluoroacetic acid, stir at room temperature for 3 h. After the reaction is completed, co-evaporate with toluene twice, and purify by column to get the product, which is compound 4.

[0075] 1 H NMR (400 MHz, Chloroform-d) δ 5.40 (ddt, J = 10.3, 3.4, 1.6 Hz, 1H), 5.32 (dd, J = 3.3, 1.3 Hz, 1H), 5.20 - 5.11 (m, 2H), 4.36 - 4.20 (m, 3H), 2.17 (s, 3H), 2.11 (s, 3H), 2.00 (s, 3H), 1.15 (d, J = 6.5 Hz, 3H). See Figure 2 for the NMR spectrum.

[0076] 1.1.3 Synthesis of compound 5:

[0077] In a reaction bottle, add compound 4, pentadecylamine, 1-ethyl-(3- dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole and tetrahydrofuran in sequence, stir overnight at room temperature. After the reaction is completed, purify by column to get the product, which is compound 5 in the form of light yellow oil.

[0078] 1H NMR (400 MHz, Chloroform-d) δ 6.50 (t, J = 6.0 Hz, 1H), 5.58 - 5.15 (m, 3H), 5.01 (d, J = 3.5 Hz, 1H), 4.23 - 3.89 (m, 3H), 3.29 (dt, J = 9.4, 6.0 Hz, 2H), 2.17 (d, J = 2.5 Hz, 3H), 2.08 (d, J = 2.6 Hz, 3H), 2.00 (d, J = 2.6 Hz, 3H), 1.59 - 1.46 (m, 3H), 1.44 - 1.18 (m, 23H), 1.15 (dd, J = 6.6, 2.5 Hz, 3H), 0.87 (td, J = 6.9, 2.5 Hz, 3H). See Figure 3 for NMR spectra.

[0079] 1.1.4 Synthesis of compound 6:

[0080] Compound 6 was dissolved in methanol, and sodium methoxide solution was added dropwise. The reaction was stirred at room temperature for 3 h. After the reaction was completed, Dowex hydrogen ion exchange resin was added to adjust the pH of the solution to neutral. The solution was filtered and concentrated to give white solid product, which was compound 6.

[0081] 1 H NMR (400 MHz, Methanol-d4) δ 4.76 (d, J = 2.9 Hz, 1H), 4.16 - 3.91 (m, 3H), 3.84 - 3.74 (m, 2H), 3.68 (dd, J = 2.5, 1.2 Hz, 1H), 3.24 (td, J = 7.0, 3.8 Hz, 2H), 1.54 (p, J = 7.1 Hz, 2H), 1.29 (s, 24H), 1.22 (d, J = 6.6 Hz, 3H), 0.95 - 0.86 (m, 3H). 13 C NMR (151 MHz, MeOD) δ 170.73, 163.48, 100.09, 72.12, 70.05, 68.29, 66.83, 66.51, 56.08, 48.18, 48.03, 47.89, 47.75, 47.61, 47.47, 47.33, 47.18, 38.68, 35.57, 31.68, 30.27, 29.40, 29.38, 29.37, 29.34, 29.28, 29.08, 29.02, 28.95, 26.62, 22.34, 15.77, 15.24, 13.06. See Figure 4 for NMR spectra.

[0082] 1.1.5 Synthesis of SFGL:

[0083] Compound 6 was dissolved in DMF, and sulfur trioxide trimethylamine complex was added. The mixture was stirred at 30-50°C for 5-10 days. After the reaction was completed, the reaction solution was rotary evaporated and then purified by LH-20 column. The eluate was collected and freeze-dried to obtain white solid product, which was SFGL.

[0084] 1.2 Characterization of sugar-containing molecule structure

[0085] The prepared sugar lipid molecules and sugar peptide molecules were characterized and analyzed, and the specific information is shown in the following table:

[0086] Example 2 Preparation of sugar lipid composite material

[0087] 2.1 Preparation of sugar lipid composite fluorescent molecule

[0088] The sugar lipid was dissolved in water, and then the fluorescent molecules Cy5 and FITC were added dropwise into the sugar lipid solution. The mixture was stirred overnight in the dark to obtain the sugar lipid composite fluorescent molecule.

[0089] 2.2 Preparation of sugar lipid modified gold nanoparticles

[0090] Gold nanoparticles modified with sodium citrate were prepared by adding chloroauric acid solution and sodium citrate solution to boiling water.

[0091] Further, the gold nanoparticles were modified with lipid chains. First, the prepared gold nanoparticle aqueous solution was centrifuged and concentrated, and then was added dropwise into a DMF solution containing dodecanethiol. The DMF solution was centrifuged and concentrated, and then was added dropwise into a chloroform solution containing dodecanethiol in the same way. Finally, the chloroform solution containing gold nanoparticles was centrifuged and concentrated. Subsequently, the chloroform solution was mixed with an aqueous solution containing sugar lipid, and the gold nanoparticles were transferred from the organic phase to the aqueous phase, and the surface of the gold nanoparticles was modified with sugar lipid molecules.

[0092] Example 3 Performance test of sugar lipid composite material

[0093] 3.1 Characterization of sugar lipid physical properties

[0094] The sugar lipid was dissolved in water, and its structure information was characterized by negative staining transmission electron microscopy and cryo-electron microscopy. Further, the zeta potential was used to detect the electric potential.

[0095] The results showed that the sugar lipid formed small balls with a diameter of about 6 nm in water, and the surface potential was -86.3 mV, which was consistent with the fact that the surface of the sugar lipid contained three sulfate groups (Figure 13).

[0096] 3.2 Characterization of sugar lipid composite fluorescent molecule physical properties

[0097] The fluorescent molecules were divided into two groups, one group was to drop FITC directly into water, and the fluorescence was quenched; the other group was to dissolve the glycolipid in water first, and then slowly drop the fluorescent molecules into the glycolipid solution. The luminescence intensity of the material was detected by fluorescence spectroscopy, which was used as the basis to judge whether the fluorescent molecules were in the hydrophobic cavity inside the glycolipid molecules.

[0098] As shown in Figure 14, the fluorescent molecule with lipid chain modification was dropped into the glycolipid solution, and the fluorescence was significantly improved, indicating that the FITC with lipid chain modification was in a hydrophobic environment, and the fluorescent molecule and the glycolipid molecule successfully complexed.

[0099] 3.3 Physical property characterization of glycolipid modified gold nanoparticles

[0100] After the glycolipid modified gold nanoparticles, the morphology change before and after the modification of the nanoparticles was detected by negative staining transmission electron microscopy. Then, the ultraviolet-visible-near infrared spectrophotometer and dynamic light scattering instrument were used to further detect the particle size change before and after the modification of the gold nanoparticles.

[0101] The results showed that the ultraviolet-visible light absorption of the modified gold nanoparticles was slightly red-shifted (Figure 15A), and the dynamic light scattering results showed that the hydration diameter of the modified gold nanoparticles was slightly larger than that before the modification (Figure 15B), which was consistent with the modification of the glycolipid on the surface of the gold nanoparticles. The transmission electron microscopy results showed that there was no obvious change in the diameter of the gold nanoparticles before and after the modification (Figures 15C-D).

[0102] Example 4 Design of material targeting function characterization

[0103] 4.1 In vivo near-infrared fluorescence imaging experiment to verify the targeting of diseased blood vessels

[0104] 200 uL of 1% mass concentration of lipid chain modified FITC complexed glycolipid solution was injected into the tail vein of healthy mice and atherosclerosis model mice, respectively, and the atherosclerotic plaques were observed by oil red o staining. The fluorescence imaging image of the atherosclerotic aorta in the mouse body and the related oil red o staining of the atherosclerotic aorta were observed. Then, the in vivo fluorescence representative images of different organs were observed at 3h, 6h, 12h and 24h after injection, and the average fluorescence index was further detected.

[0105] The near-infrared fluorescence imaging results show that there is no glycolipid enrichment at the blood vessels of healthy mice, and there is a significant fluorescence signal at the blood vessels of atherosclerosis model mice (Figure 16A left), indicating that glycolipids accumulate at the blood vessels. As can be seen from Figure 16A right, the plaque area of the atherosclerosis model mice is significantly larger than that of the healthy mice, indicating that the atherosclerosis model mice are successfully constructed. Time tracking experiments show that the fluorescence signal at the liver is strongest 6 hours after injection, but it decreases rapidly after 12 hours. The signal at the blood vessels reaches a maximum value at 12 hours, and the fluorescence signal is still very strong after 24 hours, indicating that glycolipids are effectively enriched at the blood vessels (Figures 16B-C).

[0106] 4.2 Verification of the targeting function of the material to smooth muscle transdifferentiated macrophages

[0107] We first verified the targeting ability of glycolipids to MSR1 expressing cells. RAW264.7 is a type of cell that expresses MSR1 receptors on the cell membrane surface, while endothelial cells and smooth muscle cells do not express MSR1 receptors. FITC complex glycolipid solutions with concentrations of 10 ug / mL, 50 ug / mL and 100 ug / mL were incubated with RAW264.7, endothelial cells and smooth muscle cells for 12 hours, respectively. The representative immunofluorescence of SFGL binding positive RAW264.7 macrophages incubated with different concentrations of glycolipid solutions and the quantitative results were observed. Further, flow cytometry was used to analyze the quantitative results of RAW164.7 incubated with different concentrations of glycolipid solutions.

[0108] The results show that the targeting of 10 ug / mL glycolipid solution is low, but when the concentration increases to 50 ug / mL and 100 ug / mL, the targeting of glycolipid molecules is greatly improved (Figure 17). After co-incubating glycolipid molecules with endothelial cells and smooth muscle cells, it was found that glycolipid molecules cannot target endothelial cells and smooth muscle cells (Figure 18). This indicates that glycolipid molecules specifically target RAW264.7.

[0109] The myocardial infarction model was used to determine that glycolipids can target MSR1 overexpressing cells in vivo. That is, the myocardial infarction area of the myocardial infarction mice after injection of glycolipid solution was observed by confocal microscopy.

[0110] As shown in Figure 19, a large number of glycolipid molecules can be seen overlapping with the MSR1 receptor in the myocardial infarction group, while there are none in the control group, and the quantitative analysis results further prove this. This indicates that glycolipid molecules can target MSR1 overexpressing cells in vivo.

[0111] After smooth muscle cells transdifferentiate into macrophages, the expression of MSR1 receptors is up-regulated. Therefore, the expression of glycolipid molecules to smooth muscle cells and smooth muscle transdifferentiated macrophages was further detected.

[0112] As shown in Figure 20, the glycolipid molecules do not target smooth muscle cells, but can target smooth muscle transdifferentiated macrophages.

[0113] Example 5 Glycolipid composite for plaque imaging

[0114] 5.1 Verification of the targeting function of the material for different stages of plaques in vivo

[0115] 200 μL of Cy5-labeled glycolipid with a mass concentration of 1% was injected into the tail vein of mice with different stages of plaque models.

[0116] The glycolipid targeting was observed by confocal microscopy, and the collagen content in the atherosclerotic plaque was evaluated by Masson staining technique to determine the correct construction of the mouse atherosclerosis model. The specific staining steps are as follows:

[0117] 1. Paraffin sections were deparaffinized to water.

[0118] 2. Chromium treatment or mercuric salt precipitation.

[0119] (1) After deparaffinization of the section, 0.5% iodine alcohol was used;

[0120] (2) Washed with water;

[0121] (3) 5% sodium thiosulfate was used;

[0122] (4) Washed with running water.

[0123] 3. Washed with tap water and distilled water in turn.

[0124] 4. Stained the nucleus with Regaud hematoxylin or Weigert hematoxylin.

[0125] 5. Washed with water thoroughly, and differentiated with hydrochloric alcohol if over-stained.

[0126] 6. Washed with distilled water.

[0127] 7. Used Masson's pink acid fuchsin solution.

[0128] 8. Soaked in 2% aqueous solution of glacial acetic acid for a few moments.

[0129] 9. Differentiated with 1% aqueous solution of phosphomolybdic acid.

[0130] 10. Without water washing, directly stained with aniline blue or light green solution.

[0131] 11. Soaked in 0.2% aqueous solution of glacial acetic acid for a few moments.

[0132] 12. Transparented with 95% alcohol, anhydrous alcohol, xylene, and sealed with neutral gum.

[0133] As shown in FIG. 21, there are almost no smooth muscle transdifferentiation macrophages in the early plaque imaging results, the content of smooth muscle transdifferentiation macrophages in the late plaque results is increased, and the lower glycolipid SFGL color area is increased, and is highly coincident with tdTomato, indicating that SFGL can target smooth muscle transdifferentiation macrophages. Glycolipids will accumulate in large quantities in late plaques and less in early plaques, indicating that the targeting ability of glycolipids for plaques of different periods is different.

[0134] 5.2 Verification of in vivo CT imaging performance of materials for plaques of different periods

[0135] 200 μL of glycolipid composite gold nanoparticle was injected into the mouse body through the tail vein, and the imaging of gold nanoparticles at the blood vessel was observed by Micro-CT 24 h after injection, and the blood vessels of the mouse were stained with oil red o dye to observe the size of the plaque.

[0136] As shown in FIG. 22, glycolipid composite gold nanoparticles can be enriched at the lesion blood vessels. Moreover, as the lesion process of atherosclerosis, the plaque area becomes larger, the number of glycolipid composite gold nanoparticles aggregates correspondingly becomes more, and the imaging area correspondingly becomes larger, and this material can be used for diagnosing the lesion process of plaque.

[0137] Example 6 Biological safety test

[0138] The glycolipid was co-incubated with RAW264.7 cells in vitro for 24 h, and the cell viability was detected by CCK-8 kit.

[0139] As shown in FIG. 23, the cell viability is not affected, indicating that the designed material has good biocompatibility.

Claims

A compound that can target macrophages, the compound being a sugar-containing amphiphilic small molecule of formula (I), wherein The oxygen-containing ring represents a sugar, R is a compound containing a lipid chain or a peptide chain, and Linker is a linker connecting the oxygen-containing ring and the compound chain, preferably an amide bond or a triazole ring. The compound of claim 1, wherein the sugar is selected from one or more of mannose, glucose, galactose, fucose, and their corresponding sulfated sugar forms; preferably, the sugar is sulfated fucose. The compound of claim 1, wherein the macrophages include smooth muscle transdifferentiated macrophages and myeloid-derived macrophages. A composite nanoparticle consisting of the compound of claim 1 and an encapsulated particle, wherein the surface of the encapsulated particle is modified by the compound of claim 1; the composite nanoparticle can be enriched at the site of an atherosclerotic lesion, and early and late plaques can be distinguished by imaging techniques. The composite nanoparticle of claim 4, wherein the encapsulated particle includes, but is not limited to, gold nanoparticles or iron particles. A targeted imaging agent comprising the compound of claim 1 and a fluorescent molecule, which can be targeted to be enriched at the site of a blood vessel of an atherosclerotic lesion, reflecting the progression of plaque lesions. A targeted drug composition comprising the compound of claim 1 and a drug molecule. Use of the compound or the composite nanoparticle of any one of claims 1-5 in the preparation of a targeted imaging agent for macrophages, which can be targeted to be enriched at the site of a blood vessel of an atherosclerotic lesion, reflecting the progression of plaque lesions. Use of the targeted drug composition of claim 7 in the preparation of a medicament for treating an atherosclerosis-related disease, which functions by transporting the drug to macrophages in an atherosclerotic plaque. The use of claim 9, wherein the atherosclerosis-related disease includes, but is not limited to, one or more of coronary heart disease, stroke, abdominal aortic aneurysm, peripheral arterial disease, hypertension, diabetes, hyperlipidemia, and hypercholesterolemia.

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