Activatable hydrophilic near-infrared fluorescent probes, liposome fluorescent probes, cell membrane fluorescent probes, preparation method and application thereof

Abstract

The application provides an activatable hydrophilic near-infrared fluorescent probe, a liposome fluorescent probe, a cell membrane fluorescent probe, a preparation method and application thereof, and relates to the field of biomedical new materials.The activatable hydrophilic near-infrared fluorescent probe is obtained after grafting of an activatable fluorescent probe and a hydrophilic polymer; the activatable fluorescent probe is a fluorescent probe that specifically responds to neutrophil elastase; and the activatable fluorescent probe comprises a hemicyanine fluorescent group and a fluorescent shielding group.The activatable hydrophilic near-infrared fluorescent probe, the liposome fluorescent probe and the cell membrane fluorescent probe provided by the application can be activated by neutrophil elastase that is highly expressed in an inflammatory environment, have the advantages of wide application range, various detection modes, accurate detection effect, good biocompatibility and the like.The application has the ability of active targeting of an inflammatory site, and can realize real-time detection of early inflammation under the guidance of fluorescence imaging.

Description

Technical Field

[0001] This invention relates to the field of new biomedical materials, and in particular to an activatable hydrophilic near-infrared fluorescent probe, a liposome fluorescent probe, a cell membrane fluorescent probe, its preparation method, and its application. Background Technology

[0002] Inflammation is ubiquitous in human daily life. Common inflammatory responses such as asthma, inflammatory bowel disease, arthritis, and allergies affect millions of people. (See Baechle, JJ; Chen, N.; Makhijani, P.; Winer, S.; Furman, D.; Winer, D. Molecular Metabolism, 2023, 74). Most inflammatory responses are caused by pathogens. When a host is infected by a pathogen, the body's immune cells are activated by detecting pathogen-related molecular patterns through pattern recognition receptors. Activated immune cells either engulf pathogens directly or secrete proteases to form cellular traps to clear the pathogens. (See Kolaczkowska, E.; Kubes, P. Nature Reviews Immunology, 2013, 13, 159; Yamamoto, H.; Ichikawa, Y.; Hirano, SI; Sato, B.; Satoh, F. International Journal of Molecular Sciences, 2021, 22).

[0003] The presence of pathogens also activates intracellular signal transduction pathways in immune cells. For example, activation of the κ-light chain enhancement signaling pathway in B cells activated by nuclear factor initiates an immune cascade, producing inflammatory cytokines and leading to a cytokine storm. In cases of immune dysregulation, other inflammatory mediators such as free nucleic acids, reactive oxygen species, and nitric oxide are also produced. Ultimately, a prolonged and excessive inflammatory response may cause organ dysfunction. Therefore, early diagnosis of inflammation is crucial. (See Kudiabor H. Nature, 2024, 631, 717; Poole S.; Clark T. The Journal of Infection, 2020, 80, 1; Cilloniz C.; Liapikou A.; Torres A. Current opinion in pulmonary medicine, 2020, 26).

[0004] Currently, the clinical diagnosis of inflammation mainly includes etiological testing, routine blood tests, and imaging examinations, which may have drawbacks such as long processing time, poor specificity, and radiation exposure. As a novel bioimaging tool, fluorescence imaging technology has advantages such as high sensitivity, non-invasiveness, and real-time monitoring. It does not require a professional medical background and can effectively analyze imaging results simply by observing changes in fluorescence intensity. However, it also has problems such as high background signal and poor specificity. (See Baechle, JJ; Chen, N.; Makhijani, P.; Winer, S.; Furman, D.; Winer, D. Molecular Metabolism, 2023, 74; Ouyang, J.; Sun, L.; Zeng, F.; Wu, S. Coordination Chemistry Reviews, 2022, 458, 214438).

[0005] Therefore, there is an urgent need to develop an activatable fluorescent probe with low background signal, accurate detection, and inflammation specificity to achieve real-time detection of early inflammation and provide accurate guidance for the clinical diagnosis and treatment of inflammation.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The purpose of this invention is to provide an activatable hydrophilic near-infrared fluorescent probe, a liposome fluorescent probe, a cell membrane fluorescent probe, its preparation method, and its application. The fluorescent probe of this invention can actively target inflammatory sites, responding to the highly expressed neutrophil elastase in the inflammatory microenvironment, thereby activating fluorescence and achieving in situ detection of inflammation.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0009] In a first aspect, the present invention provides an activatable hydrophilic near-infrared fluorescent probe, wherein the activatable hydrophilic near-infrared fluorescent probe is obtained by grafting an activatable fluorescent probe with a hydrophilic polymer.

[0010] The activatable fluorescent probe is a fluorescent probe that specifically responds to neutrophil elastase; and the activatable fluorescent probe includes a hemicyanine fluorescent group and a fluorescence shielding group.

[0011] Preferably, the structural formula of the hemicyanine fluorescent group is shown in Formula I below;

[0012]

[0013] Preferably, the fluorescent shielding group comprises an acetyl-alanine-alanine-proline-valine polypeptide.

[0014] Preferably, the structure of the activatable fluorescent probe is shown in Formula II below:

[0015]

[0016] Preferably, the hydrophilic polymer includes any one or a combination of at least two of polyvinylpyrrolidone, distearylphosphatidylethanolamine-methoxy polyethylene glycol, or polyethylene glycol, and more preferably distearylphosphatidylethanolamine-methoxy polyethylene glycol and / or polyethylene glycol.

[0017] Preferably, the weight-average molecular weight of the aqueous polymer is 1000-5000, and more preferably 2000.

[0018] Preferably, the hydrophilic polymer is a hydrophilic polymer with alkynyl groups at the end.

[0019] Preferably, the activatable fluorescent probe and the hydrophilic polymer are grafted together by forming a 1,2,3-triazole ring group.

[0020] In a second aspect, the present invention provides a method for preparing an activatable hydrophilic near-infrared fluorescent probe as described in the first aspect, the preparation method comprising the following steps:

[0021] The activatable fluorescent probe and the hydrophilic polymer are reacted via a click reaction to obtain the activatable hydrophilic near-infrared fluorescent probe.

[0022] Preferably, the temperature of the click reaction is 25–40°C, and the time of the click reaction is 6–18 hours.

[0023] Preferably, the click reaction is carried out in the presence of a catalyst; wherein the catalyst is sodium ascorbate.

[0024] Preferably, the click reaction is carried out in the presence of a promoter, wherein the promoter is copper sulfate.

[0025] Preferably, the click reaction is carried out in the presence of a solvent; wherein the solvent includes dimethyl sulfoxide and water.

[0026] Preferably, the volume ratio of dimethyl sulfoxide to water is 1:(1-2).

[0027] Preferably, the molar ratio of the activatable fluorescent probe, the hydrophilic polymer, the catalyst, and the promoter is 1:(1-1.5):(0.1-0.2):(0.1-0.3).

[0028] Preferably, the following post-processing steps are further included after the click reaction is completed: dialyzing and lyophilizing the reaction solution obtained from the click reaction to obtain the activatable hydrophilic near-infrared fluorescent probe.

[0029] Preferably, the molecular weight of the dialysis solution is 2000-3000 Dalton.

[0030] Thirdly, the present invention provides a liposomal fluorescent probe, which is obtained by self-assembly of lipids and an activatable hydrophilic near-infrared fluorescent probe as described in the first aspect.

[0031] Preferably, the lipid comprises any one or a combination of at least two of distearylphosphatidylcholine, didecyldimethylammonium bromide, or cholesterol, and more preferably a combination of distearylphosphatidylcholine, didecyldimethylammonium bromide, and cholesterol.

[0032] Preferably, the mass ratio of distearate phosphatidylcholine, didecyl dimethyl ammonium bromide, cholesterol, and the activatable hydrophilic near-infrared fluorescent probe is (1-12):(1-7.2):1:(1-5), and more preferably 12:3.6:1:2.5.

[0033] Fourthly, the present invention provides a method for preparing a liposome fluorescent probe as described in the third aspect, the method comprising the following steps:

[0034] The lipid and the activatable hydrophilic near-infrared fluorescent probe are dispersed in a solvent, the solvent is removed by evaporation, water is added, and the mixture is sonicated to obtain the liposome fluorescent probe.

[0035] Preferably, the solvent is trichloromethane.

[0036] Preferably, the temperature of the ultrasonic treatment is 25-30°C, the time of the ultrasonic treatment is 1-10 min, and the power of the ultrasonic treatment is 50-200 W.

[0037] Fifthly, the present invention provides a cell membrane fluorescent probe, wherein the cell membrane fluorescent probe is obtained by self-assembly of a macrophage membrane and an activatable hydrophilic near-infrared fluorescent probe as described in the first aspect; or the cell membrane fluorescent probe is obtained by self-assembly of a macrophage membrane and a liposome fluorescent probe as described in the third aspect.

[0038] Preferably, the mass ratio of the macrophage membrane to the activatable hydrophilic near-infrared fluorescent probe is 1:(0.1-10), and more preferably 1:5.

[0039] Sixthly, the present invention provides a method for preparing a cell membrane fluorescent probe as described in the fifth aspect, the method comprising the following steps:

[0040] The macrophage cell membrane and the activatable hydrophilic near-infrared fluorescent probe were mixed in a buffer solution and incubated. After sonication, the cell membrane fluorescent probe was obtained.

[0041] Preferably, the buffer solution comprises a phosphate buffer solution.

[0042] Preferably, the mixing temperature is 2–6°C.

[0043] Preferably, the incubation time is 2 to 8 hours, and more preferably 4 hours.

[0044] Preferably, the temperature of the ultrasonic treatment is 25-40°C, the time of the ultrasonic treatment is 1-10 min, and the power of the ultrasonic treatment is 50-200 W.

[0045] In a seventh aspect, the present invention provides the application of the activatable hydrophilic near-infrared fluorescent probe according to the first aspect, or the liposome fluorescent probe according to the third aspect, or the cell membrane fluorescent probe according to the fifth aspect, as an inflammation detection reagent.

[0046] Furthermore, the application includes the use of the activatable hydrophilic near-infrared fluorescent probe, or the liposome fluorescent probe, or the cell membrane fluorescent probe as an active targeting detection reagent for inflammatory sites.

[0047] Furthermore, the application includes the real-time detection of inflammation in situ and in urine under the guidance of fluorescence imaging using the activatable hydrophilic near-infrared fluorescent probe, or the liposome fluorescent probe, or the cell membrane fluorescent probe.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] (1) The activatable hydrophilic near-infrared fluorescent probe, liposome fluorescent probe and cell membrane fluorescent probe provided by the present invention have the ability to actively target inflammatory sites and can be activated by neutrophil elastase highly expressed in the inflammatory microenvironment, thereby realizing real-time imaging of inflammation and non-invasive urine analysis.

[0050] (2) This invention utilizes activatable hydrophilic near-infrared fluorescent probes, liposome fluorescent probes, and cell membrane fluorescent probes to not only detect inflammation in the early stage in real time, but also to assist in the screening and dosage selection of anti-inflammatory drugs, providing a novel imaging tool for inflammation detection and drug efficacy evaluation. Attached Figure Description

[0051] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0052] Figure 1 The proton NMR spectrum of the activatable fluorescent probe provided in Example 1.

[0053] Figure 2 The mass spectrum of the activatable fluorescent probe provided in Example 1.

[0054] Figure 3 The 1H NMR spectrum of the activatable hydrophilic near-infrared fluorescent probe provided in Example 2.

[0055] Figure 4 The mass spectrum of the activatable hydrophilic near-infrared fluorescent probe provided in Example 2.

[0056] Figure 5 Transmission electron microscopy image of the activatable hydrophilic near-infrared fluorescent probe provided in Example 2.

[0057] Figure 6 The diagram shows the structural schematics of the preparation process of the liposome fluorescent probes provided in Examples 3-5.

[0058] Figure 7 Transmission electron microscopy image of the liposome fluorescent probe provided in Example 4.

[0059] Figure 8 This is a schematic diagram of the preparation process of the cell membrane fluorescent probe provided in Example 6.

[0060] Figure 9 Transmission electron microscopy image of the cell membrane fluorescent probe provided in Example 6.

[0061] Figure 10A The UV-Vis absorption spectrum of the sample containing the activatable hydrophilic near-infrared fluorescent probe provided in Example 2 after incubation with elastase.

[0062] Figure 10B The UV-Vis absorption spectrum of the liposome fluorescent probe provided in Example 4 after incubation with elastase.

[0063] Figure 10C The UV-Vis absorption spectrum of the cell membrane fluorescent probe provided in Example 6 after incubation with elastase.

[0064] Figure 11A The fluorescence spectrum of the sample containing the activatable hydrophilic near-infrared fluorescent probe provided in Example 2 after incubation with elastase.

[0065] Figure 11B The fluorescence spectrum of the liposome fluorescent probe provided in Example 4 after incubation with elastase.

[0066] Figure 11CThe fluorescence spectrum of the cell membrane fluorescent probe provided in Example 6 after incubation with elastase.

[0067] Figure 12 This is a verification diagram of the neutrophil responsiveness of the activatable hydrophilic near-infrared fluorescent probe provided in Example 2.

[0068] Figure 13 The in vivo fluorescence imaging of the probe sample provided for test example 4 injected into a mouse with pneumonia.

[0069] Figure 14A This is a diagram illustrating the real-time detection of pneumonia using the activatable hydrophilic near-infrared fluorescent probe provided in Example 2.

[0070] Figure 14B This is a diagram showing the real-time detection of pneumonia using the liposome fluorescent probe provided in Example 4.

[0071] Figure 14C This is a diagram illustrating the real-time detection of pneumonia using the cell membrane fluorescent probe provided in Example 6.

[0072] Figure 15A This is a urine detection image of the activatable hydrophilic near-infrared fluorescent probe provided in Example 2.

[0073] Figure 15B This is a urine detection image of the liposome fluorescent probe provided in Example 4.

[0074] Figure 15C This is a urine detection image of the cell membrane fluorescent probe provided in Example 6.

[0075] Figure 16 This is a biosafety verification diagram provided for Example 2. Detailed Implementation

[0076] Unless otherwise defined herein, scientific and process terms used in conjunction with this invention should have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms should be clear; however, in any case of potential ambiguity, the definitions provided herein take precedence over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0077] It should be noted that specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many ways other than those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0078] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0079] In a first aspect, the present invention provides an activatable hydrophilic near-infrared fluorescent probe, wherein the activatable hydrophilic near-infrared fluorescent probe is obtained by grafting an activatable fluorescent probe with a hydrophilic polymer.

[0080] The activatable fluorescent probe is a fluorescent probe that specifically responds to neutrophil elastase; and the activatable fluorescent probe includes a hemicyanine fluorescent group and a fluorescence shielding group.

[0081] In this invention, an activatable hydrophilic near-infrared fluorescent probe is formed by grafting a hydrophilic polymer with an activatable fluorescent probe. Due to the introduction of the hydrophilic polymer, the activated probe can be rapidly excreted from the body via the kidneys, enabling urine detection of inflammation. Specifically, the design of the activatable hydrophilic near-infrared fluorescent probe effectively reduces the accumulation of fluorescent probes at non-target sites, reduces background signals in fluorescence imaging, increases the specificity of inflammation detection, improves imaging sensitivity, and enables in situ and urine detection of early inflammation. It is noteworthy that the activatable fluorescent probe of this invention specifically responds to the highly expressed neutrophil elastase in the inflammatory environment, exhibiting a significant enhancement of fluorescence.

[0082] As an optional implementation, the activatable fluorescent probe is prepared by the following steps: introducing 4-aminobenzyl alcohol into an acetyl-alanine-alanine-proline-valine polypeptide synthesized by solid-phase synthesis via a condensation reaction, followed by introducing a hemicyanine fluorescent group using phosphorus tribromide bromination to obtain the activatable fluorescent probe.

[0083] As an optional implementation, the activatable fluorescent probe is prepared by the following steps:

[0084] (a) Synthesizing acetyl-alanine-alanine-proline-valine polypeptide (AAPV) using a standard solid-phase synthesis method;

[0085] (b) Acetyl-alanine-alanine-proline-valine polypeptide (AAPV), 4-aminobenzyl alcohol (PABA), and diethyldithiocarbamate diethylamine were mixed and reacted in dichloromethane to obtain the intermediate product polypeptide (AAPV-PABA).

[0086] (c) The intermediate polypeptide (AAPV-PABA) and the brominating reagent were mixed and reacted in tetrahydrofuran to obtain the brominated intermediate polypeptide (AAPV-PABA-Br).

[0087] (d) The brominated intermediate polypeptide (AAPV-PABA-Br), hemicyanine fluorescent group, and base are mixed and reacted in acetonitrile to obtain the activated fluorescent probe.

[0088] As an optional implementation, in step (b), the temperature of the mixing reaction is 10-40°C, for example, it can be 10°C, 12°C, 14°C, 15°C, 16°C, 18°C, 20°C, 22°C, 24°C, 25°C, 26°C, 28°C, 30°C, 32°C, 34°C, 35°C, 36°C, 38°C, 40°C, etc.; the time of the mixing reaction is 3-5 hours, for example, it can be 3 hours, 3.2 hours, 3.4 hours, 3.5 hours, 3.6 hours, 3.8 hours, 4 hours, 4.2 hours, 4.4 hours, 4.5 hours, 4.6 hours, 4.8 hours, 5 hours, etc., preferably 4 hours.

[0089] As an optional implementation, in step (b), the molar ratio of AAPV, PABA and diethyldithiocarbamate diethylamine is 1:(1.5-2):(1.5-2).

[0090] In PABA: "1.5~2" can be, for example, 1.5, 1.6, 1.7, 1.8, 1.9, 2, etc.

[0091] Diethyldithiocarbamate diethylamine: "1.5~2" can be, for example, 1.5, 1.6, 1.7, 1.8, 1.9, 2, etc.

[0092] As an optional implementation, in step (c), the temperature of the mixing reaction is -5 to 5°C, for example, it can be -5°C, -4°C, -3°C, -2°C, -1°C, 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, etc., preferably 0°C; the time of the mixing reaction is 1 to 3 hours, for example, it can be 1 hour, 1.2 hours, 1.4 hours, 1.5 hours, 1.6 hours, 1.8 hours, 2 hours, 2.2 hours, 2.4 hours, 2.5 hours, 2.6 hours, 2.8 hours, 3 hours, etc., preferably 1 hour.

[0093] As an optional implementation, in step (c), the brominating agent is phosphorus tribromide.

[0094] As an optional implementation, in step (c), the molar ratio of AAPV-PABA to the brominated reagent is 1:(2.5 to 3.5), for example, it can be 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, etc.

[0095] As an optional implementation, in step (d), the temperature of the mixing reaction is 50-60°C, for example, 50°C, 51°C, 52°C, 53°C, 54°C, 56°C, 57°C, 58°C, 59°C, 60°C, etc., preferably 55°C; the time of the mixing reaction is 18-30h, for example, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h, etc., preferably 24h.

[0096] As an optional implementation, in step (d), the base is N,N-diisopropylethylamine.

[0097] As an optional implementation, in step (d), the molar ratio of AAPV-PABA-Br, hemicyanine fluorescent group and base is 1:(0.02~0.06):(0.06~0.18);

[0098] Among them, "0.02~0.06" can be, for example, 0.02, 0.03, 0.04, 0.05, 0.06, etc.;

[0099] Among them, "0.06~0.18" can be, for example, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18, etc.

[0100] As an optional embodiment, the structural formula of the hemicyanine fluorescent group is shown in Formula I below:

[0101]

[0102] As an optional implementation, the fluorescent shielding group includes an acetyl-alanine-alanine-proline-valine polypeptide (AAPV).

[0103] As an optional implementation, the structure of the activatable fluorescent probe is shown in Formula II below:

[0104]

[0105] As an optional embodiment, the hydrophilic polymer includes any one or a combination of at least two of the following: polyvinylpyrrolidone, distearylphosphatidylethanolamine-methoxy polyethylene glycol, or polyethylene glycol, preferably distearylphosphatidylethanolamine-methoxy polyethylene glycol and / or polyethylene glycol.

[0106] As an optional embodiment, the weight-average molecular weight of the aqueous polymer is 1000 to 5000, for example, it can be 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, etc., preferably 2000.

[0107] As an optional embodiment, the hydrophilic polymer is a hydrophilic polymer with alkynyl groups at the end.

[0108] As an optional implementation, the activatable fluorescent probe and the hydrophilic polymer are grafted together by forming a 1,2,3-triazole ring group.

[0109] As an optional implementation, the structure of the activatable hydrophilic near-infrared fluorescent probe is shown in Formula III below:

[0110]

[0111] Among them, R1-L1-R2 can activate fluorescent probe groups, R1 represents hemicyanine fluorescent group, L1 represents linking group, R2 represents fluorescent shielding group, and R3 represents hydrophilic polymer group.

[0112] In a second aspect, the present invention provides a method for preparing an activatable hydrophilic near-infrared fluorescent probe as described in the first aspect, the method comprising the following steps:

[0113] An activatable fluorescent probe and a hydrophilic polymer are reacted via a click reaction to obtain the activatable hydrophilic near-infrared fluorescent probe; the reaction formula for the click reaction is shown below:

[0114]

[0115] As an optional implementation, the temperature of the click reaction is 25-40°C, for example, 25°C, 26°C, 28°C, 30°C, 32°C, 34°C, 35°C, 36°C, 38°C, 40°C, etc.; the time of the click reaction is 6-18 hours, for example, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours, 11.5 hours, 12 hours, 12.5 hours, 13 hours, 13.5 hours, 14 hours, 14.5 hours, 15 hours, 15.5 hours, 16 hours, 16.5 hours, 17 hours, 17.5 hours, 18 hours, etc., preferably 12 hours.

[0116] As an optional implementation, the click reaction is carried out in the presence of a catalyst.

[0117] As an optional implementation, the catalyst for the click reaction is sodium ascorbate.

[0118] As an optional implementation, the click reaction is carried out in the presence of a promoter.

[0119] As an optional implementation, the promoter of the click reaction is copper sulfate, preferably anhydrous copper sulfate.

[0120] As an optional implementation, the click reaction is carried out in the presence of a solvent.

[0121] As an optional implementation, the solvent for the click reaction includes dimethyl sulfoxide and water.

[0122] As an optional implementation, the volume ratio of dimethyl sulfoxide to water is 1:(1-2), for example, it can be 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, etc.

[0123] As an optional implementation, the molar ratio of the activatable fluorescent probe, hydrophilic polymer, catalyst, and accelerator is 1:(1-1.5):(0.1-0.2):(0.1-0.3);

[0124] Among them, "1 to 1.5" can be, for example, 1, 1.1, 1.2, 1.3, 1.4, 1.5, etc.;

[0125] Among them, "0.1~0.2" can be, for example, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, etc.;

[0126] Among them, "0.1~0.3" can be, for example, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.22, 0.24, 0.26, 0.28, 0.3, etc.

[0127] As an optional implementation, the following post-processing steps are further included after the click reaction is completed: dialyzing and lyophilizing the reaction solution obtained from the click reaction to obtain the activatable hydrophilic near-infrared fluorescent probe.

[0128] As an optional implementation, the molecular weight of the dialysis sample is 2000-3000 Dalton, for example, it can be 2000 Dalton, 2100 Dalton, 2200 Dalton, 2300 Dalton, 2400 Dalton, 2500 Dalton, 2600 Dalton, 2700 Dalton, 2800 Dalton, 2900 Dalton, 3000 Dalton, etc.

[0129] Thirdly, the present invention provides a liposomal fluorescent probe, which is obtained by self-assembly of lipids and an activatable hydrophilic near-infrared fluorescent probe as described in the first aspect.

[0130] As an optional implementation, the lipid includes any one or a combination of at least two of distearylphosphatidylcholine, didecyldimethylammonium bromide, or cholesterol.

[0131] In a preferred embodiment, the lipid is a combination of distearate phosphatidylcholine, didecyl dimethyl ammonium bromide, and cholesterol.

[0132] As an optional implementation, the mass ratio of the distearate phosphatidylcholine, bisdecyldimethylammonium bromide, cholesterol, and the activatable hydrophilic near-infrared fluorescent probe is (1-12):(1-7.2):1:(1-5);

[0133] Among them, "1 to 12" can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0134] Among them, "1 to 7.2" can be, for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 7.2, etc.;

[0135] Among them, "1 to 5" can be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc.

[0136] In a preferred embodiment, the mass ratio of distearate phosphatidylcholine, bisdecyldimethylammonium bromide, cholesterol, and the activatable hydrophilic near-infrared fluorescent probe is 12:3.6:1:2.5.

[0137] Fourthly, the present invention provides a method for preparing a liposome fluorescent probe as described in the third aspect, the method comprising the following steps:

[0138] The lipid and the activatable hydrophilic near-infrared fluorescent probe are dispersed in a solvent, the solvent is removed by evaporation, water is added, and the mixture is sonicated to obtain the liposome fluorescent probe.

[0139] As an optional implementation, the solvent is trichloromethane.

[0140] As an optional implementation, the temperature of the ultrasonic treatment is 25-30°C, for example, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, etc., the time of the ultrasonic treatment is 1-10 minutes, for example, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, etc., and the power of the ultrasonic treatment is 50-200W, for example, 50W, 60W, 80W, 100W, 120W, 140W, 150W, 160W, 180W, 200W, etc.

[0141] Fifthly, the present invention provides a cell membrane fluorescent probe, wherein the cell membrane fluorescent probe is obtained by self-assembly of a macrophage membrane and an activatable hydrophilic near-infrared fluorescent probe as described in the first aspect; or the cell membrane fluorescent probe is obtained by self-assembly of a macrophage membrane and a liposome fluorescent probe as described in the third aspect.

[0142] As an optional implementation, the mass ratio of the macrophage membrane to the activatable hydrophilic near-infrared fluorescent probe is 1:(0.1-10), for example, it can be 1:0.1, 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, etc., preferably 1:5.

[0143] Sixthly, the present invention provides a method for preparing a cell membrane fluorescent probe as described in the fifth aspect, the method comprising the following steps:

[0144] The macrophage cell membrane and the activatable hydrophilic near-infrared fluorescent probe were mixed in a buffer solution and incubated. After sonication, the cell membrane fluorescent probe was obtained.

[0145] As an optional implementation, the buffer solution includes a phosphate buffer.

[0146] As an optional implementation, the mixing temperature is 2 to 6°C, for example, it can be 2°C, 2.5°C, 3°C, 3.5°C, 4°C, 4.5°C, 5°C, 5.5°C, 6°C, etc., preferably 4°C.

[0147] As an optional implementation, the temperature of the ultrasonic treatment is 25-40℃, for example, 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 32℃, 34℃, 36℃, 38℃, 40℃, etc., the time of the ultrasonic treatment is 1-10min, for example, 1min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min, 10min, etc., and the power of the ultrasonic treatment is 50-200W, for example, 50W, 60W, 80W, 100W, 120W, 140W, 150W, 160W, 180W, 200W, etc.

[0148] In a seventh aspect, the present invention provides the application of the activatable hydrophilic near-infrared fluorescent probe according to the first aspect, or the liposome fluorescent probe according to the third aspect, or the cell membrane fluorescent probe according to the fifth aspect, as an inflammation detection reagent.

[0149] As an optional implementation, the application includes the use of the activatable hydrophilic near-infrared fluorescent probe, or the liposome fluorescent probe, or the cell membrane fluorescent probe as an active targeting detection reagent for inflammatory sites.

[0150] As an optional implementation, the application includes real-time detection of inflammation in situ and in urine under the guidance of fluorescence imaging, using the activated hydrophilic near-infrared fluorescent probe, the liposome fluorescent probe, or the cell membrane fluorescent probe.

[0151] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0152] Preparation Example 1

[0153] This preparation example provides an activatable fluorescent probe, the structural formula of which is shown in Formula II below:

[0154]

[0155] The activatable fluorescent probe shown in Formula II is prepared by the following steps:

[0156] (a) Synthesizing acetyl-alanine-alanine-proline-valine polypeptide (AAPV) using a standard solid-phase synthesis method;

[0157] (b) AAPV, PABA and diethyldithiocarbamate diethylamine in a molar ratio of 1:1.5:1.5 were mixed in dichloromethane and reacted at room temperature for 4 h to obtain the intermediate product polypeptide AAPV-PABA.

[0158] (c) AAPV-PABA and phosphorus tribromide in a molar ratio of 1:3 were mixed in tetrahydrofuran and reacted at 0°C for 2 h to obtain the brominated intermediate polypeptide AAPV-PABA-Br.

[0159] (d) AAPV-PABA-Br, the hemicyanine fluorescent group shown in Formula I, and N,N-diisopropylethylamine were mixed in acetonitrile at a molar ratio of 1:0.02:0.04 and reacted at 55°C for 24 h to obtain the activated fluorescent probe.

[0160] in, Figure 1 The proton NMR spectrum of the activatable fluorescent probe provided in Example 1 is shown in the attached diagram. For detailed proton NMR structural analysis, please refer to [link to example 1]. Figure 1 . Figure 2 The mass spectrum of the activatable fluorescent probe provided in Example 1. Figure 2 As shown, a peak with a molecular weight of 953 appeared in the proton NMR spectrum. This proves that the activatable fluorescent probe provided in Preparation Example 1 was successfully synthesized.

[0161] Example 1

[0162] This embodiment provides an activatable hydrophilic near-infrared fluorescent probe, which is obtained by grafting an activatable fluorescent probe of Formula II with distearate phosphatidylethanolamine-methoxy polyethylene glycol; the structural formula of the activatable hydrophilic near-infrared fluorescent probe is shown in Formula III-1 below:

[0163]

[0164] The activatable hydrophilic near-infrared fluorescent probe is prepared by the following steps:

[0165] The activatable fluorescent probe of Formula II, in a molar ratio of 1:1:1:0.4, distearylphosphatidylethanolamine-methoxy polyethylene glycol-alkynyl (Mw = 2000), anhydrous copper sulfate, and sodium ascorbate were mixed in a mixed solution of dimethyl sulfoxide and water (V). 二甲基亚砜 :V 水 In a ratio of 2:1, the mixture was reacted at room temperature for 12 hours, dialyzed (the molecular weight of the dialyzed sample was 2000 Dalton), and lyophilized to obtain the activated hydrophilic near-infrared fluorescent probe.

[0166] Example 2

[0167] This embodiment provides an activatable hydrophilic near-infrared fluorescent probe, which is obtained by grafting an activatable fluorescent probe of Formula II with polyethylene glycol; the structural formula of the activatable hydrophilic near-infrared fluorescent probe is shown in Formula III-2 below:

[0168]

[0169] The activatable hydrophilic near-infrared fluorescent probe is prepared by the following steps:

[0170] The activatable fluorescent probe of Formula II, polyethylene glycol-alkynyl (Mw = 2000), anhydrous copper sulfate, and sodium ascorbate were mixed in a molar ratio of 1:1:1:0.4 in a mixed solution of dimethyl sulfoxide and water (V). 二甲基亚砜 :V 水 =2.5:) was reacted at room temperature for 12 h, dialyzed (the dialyzed molecular weight was 2000 Dalton), and lyophilized to obtain the activated hydrophilic near-infrared fluorescent probe.

[0171] in, Figure 3 The 1H NMR spectrum of the activatable hydrophilic near-infrared fluorescent probe provided in Example 2. Figure 4 The mass spectrum of the activatable hydrophilic near-infrared fluorescent probe provided in Example 2. Figure 4 As shown, a polymer peak with a molecular weight of approximately 2900 appeared, confirming the successful grafting of PEG. This demonstrates that the activatable hydrophilic near-infrared fluorescent probe provided in Example 2 was successfully synthesized.

[0172] in, Figure 5 This is a transmission electron microscope (TEM) image of the activatable hydrophilic near-infrared fluorescent probe provided in Example 2. Figure 5 As shown, the morphology of the activatable hydrophilic near-infrared fluorescent probe provided in Example 2 is granular and rod-shaped.

[0173] Example 3

[0174] This embodiment provides a liposome fluorescent probe, which is prepared by the following steps:

[0175] Distearate phosphatidylcholine, didecyl dimethyl ammonium bromide, cholesterol, and the activatable hydrophilic near-infrared fluorescent probe prepared in Example 1 were dispersed in chloroform at a mass ratio of 12:1.8:1:2.5. The solvent was removed by rotary evaporation, water was added, and the mixture was sonicated at 25°C and 100W for 2 min to obtain the liposome fluorescent probe.

[0176] Example 4

[0177] This embodiment provides a liposome fluorescent probe, which is prepared by the following steps:

[0178] Distearate phosphatidylcholine, didecyl dimethyl ammonium bromide, cholesterol, and the activatable hydrophilic near-infrared fluorescent probe prepared in Example 1 were dispersed in chloroform at a mass ratio of 12:3.6:1:2.5. The solvent was removed by rotary evaporation, water was added, and the mixture was sonicated at 25°C and 100W for 2 min to obtain the liposome fluorescent probe.

[0179] Example 5

[0180] This embodiment provides a liposome fluorescent probe, which is prepared by the following steps:

[0181] Distearate phosphatidylcholine, didecyl dimethyl ammonium bromide, cholesterol, and the activatable hydrophilic near-infrared fluorescent probe prepared in Example 1 were dispersed in chloroform at a mass ratio of 12:7.2:1:2.5. The solvent was removed by rotary evaporation, water was added, and the mixture was sonicated at 25°C and 100W for 2 min to obtain the liposome fluorescent probe.

[0182] in, Figure 6 The diagram shows the structural schematics of the preparation process of the liposome fluorescent probes provided in Examples 3-5. Figure 7 Transmission electron microscopy (TEM) image of the liposome fluorescent probe provided in Example 4, as shown below. Figure 7 As shown, the morphology of the liposome fluorescent probe of the present invention is a circular vesicle.

[0183] Example 6

[0184] This embodiment provides a cell membrane fluorescent probe, which is prepared by the following steps:

[0185] Macrophage cell membranes at a mass ratio of 1:5 and the activatable hydrophilic near-infrared fluorescent probe prepared in Example 1 were mixed in phosphate buffer, incubated at 4°C for 4 h, and then sonicated at 25°C and 100 W for 5 min to obtain the cell membrane fluorescent probe.

[0186] in, Figure 8 This is a schematic diagram of the preparation process of the cell membrane fluorescent probe provided in Example 6. Figure 9 This is a transmission electron microscope image of the cell membrane fluorescent probe provided in Example 6. Figure 9 As shown, the morphology of the liposome fluorescent probe of the present invention is a circular vesicle.

[0187] Test Example 1

[0188] UV-Vis absorption spectroscopy and fluorescence spectroscopy tests

[0189] Test Sample: Example Provided activatable hydrophilic near-infrared fluorescent probe, examples The provided liposome fluorescent probe and the cell membrane fluorescent probe provided in Example 6.

[0190] Test method: Elastase and the above test samples were incubated in tris(hydroxymethyl)aminomethane hydrochloride at 37°C for 2 hours. The UV-Vis absorption spectrum and fluorescence spectrum of the mixed solution were then measured to obtain the UV absorption and fluorescence absorption spectra of each sample before and after incubation with elastase.

[0191] Test results:

[0192] like Figure 10A , Figure 10B , Figure 10C ,as well as Figure 11A , Figure 11B Figure 11C As shown, after the addition of elastase, the characteristic peaks of the ultraviolet spectra of the activatable hydrophilic near-infrared fluorescent probe provided in Example 2, the liposome fluorescent probe provided in Example 4, and the cell membrane fluorescent probe provided in Example 6 all showed a red shift, and the fluorescence intensity was significantly enhanced. This indicates that the activatable hydrophilic near-infrared fluorescent probe provided in Example 2, the liposome fluorescent probe provided in Example 4, and the cell membrane fluorescent probe provided in Example 6 can be activated by neutrophil elastase.

[0193] Test Example 2

[0194] Neutrophil imaging verification

[0195] Test sample: The activatable hydrophilic near-infrared fluorescent probe provided in Example 2.

[0196] Test method: Neutrophils were extracted from mouse bone marrow at a concentration of 1×10⁻⁶. 5 Cells are seeded at a density of [insert density here] in culture dishes, and after adding the probe or co-incubating with the probe for 2 hours, the activation of the probe or probe in neutrophils is observed using a confocal microscope. The cell culture method is not particularly limited and can be any method known to those skilled in the art. In this invention, cells are preferably cultured with 10% fetal bovine serum, and the culture conditions are preferably continuous culture in an incubator with 5% carbon dioxide (CO2) at a temperature of 37°C.

[0197] Test results:

[0198] like Figure 12 As shown, the activatable hydrophilic near-infrared fluorescent probe provided in Example 2 was activated in neutrophils. However, the probe could not be activated after the addition of a neutrophil elastase inhibitor, indicating that the activatable hydrophilic near-infrared fluorescent probe of the present invention can be used for neutrophil imaging.

[0199] Test Example 3

[0200] Renal clearance efficiency test

[0201] Test sample: The activatable hydrophilic near-infrared fluorescent probe provided in Example 2.

[0202] Test method: The above samples were injected into mice via trachea. Urine was collected from the mice at 3, 6, 9, 12 and 24 hours after tracheal injection using a urine collection device. The content of each sample in the urine was measured to obtain the renal clearance efficiency.

[0203] Test results: The renal clearance efficiency of the activatable hydrophilic near-infrared fluorescent probe provided in Example 2 was 20%, which indicates that the probe can be rapidly metabolized by the kidneys and excreted in urine, confirming the feasibility of using a hydrophilic probe to detect inflammation through urine.

[0204] Test Example 4

[0205] Targeted testing

[0206] Test samples: Liposome fluorescent probes provided in Examples 3-5.

[0207] Test method: The above samples were injected into pneumonia mice via the tail vein, and in vivo fluorescence imaging images were obtained at 2, 10, 15, 20, 30, 45 and 60 minutes after the injection probe.

[0208] Test results:

[0209] like Figure 13 As shown, under the action of lung-targeting lipid bis(decyldimethylammonium bromide) and macrophage membrane homology targeting, the fluorescence intensity of the lung gradually increases over time, indicating that the liposome fluorescent probes provided in Examples 3-5 can actively target the inflammatory site.

[0210] Test Example 5

[0211] Test samples: the activatable hydrophilic near-infrared fluorescent probe provided in Example 2, the liposome fluorescent probe provided in Example 4, and the cell membrane fluorescent probe provided in Example 7.

[0212] Test method:

[0213] (1) Construction of pneumonia: Balb / C female mice aged 6-8 weeks were used to construct the model. After anesthetizing the mice with isoflurane, lipopolysaccharide (8 mg / kg) was administered intratracheally to complete the construction of the mouse pneumonia model.

[0214] (2) Real-time in situ detection of pneumonia: Pneumonia was detected by administering a probe (2 μm / kg) intratracheally or via the tail vein of mice. After probe administration, in vivo whole-body imaging was performed using a fluorescence imaging device to obtain fluorescence images of the pneumonia. The severity of pneumonia was analyzed using changes in lung fluorescence intensity. Any fluorescence imaging method well-known to those skilled in the art can be used, and there are no special limitations.

[0215] (3) Urine detection of pneumonia: Urine was collected from mice with pneumonia after intratracheal administration of a probe (2 μm / kg) or tail vein administration of a probe (5 μm / kg). The fluorescence intensity of the mouse urine was analyzed using a fluorescence imaging device, and the pneumonia was detected by observing changes in the fluorescence intensity of the urine.

[0216] (4) Subsequently, fluorescence imaging technology was used to obtain in vivo fluorescence imaging images at 2, 10, 15, 20, 30, 45, and 60 minutes after probe administration. Urine from healthy and pneumonia mice after probe administration was obtained using a urine collection device and fluorescence imaging was performed.

[0217] Test results:

[0218] like Figure 14A , Figure 14B and Figure 14C As shown, the fluorescence intensity in the lungs of pneumoniad mice was significantly greater than that in healthy mice. Figure 15A , Figure 15B and Figure 15C As shown, the fluorescence intensity in the urine of pneumoniad mice was stronger than that of healthy mice. This indicates that the activatable hydrophilic near-infrared fluorescent probe, liposome fluorescent probe, and cell membrane fluorescent probe described in this invention can detect inflammation through in situ imaging and urine detection, improving the specificity of inflammation detection using fluorescent probes and increasing the signal-to-noise ratio.

[0219] Test Example 6

[0220] Efficacy evaluation of pneumonia treatment drugs

[0221] Test sample: The activatable hydrophilic near-infrared fluorescent probe provided in Example 2.

[0222] Test method:

[0223] After establishing a mouse model of pneumonia, mice were treated with pneumonia-treating drugs. Fluorescence intensity analysis of mouse lungs and urine was performed using fluorescence imaging to evaluate the efficacy of the pneumonia-treating drugs. The pneumonia-treating drugs included: low molecular weight heparin (300 U / kg), Xuebijing (1.25 mL / kg), dexamethasone (10 mg / kg), or Citric acid (10 mg / kg).

[0224] The test results are shown in Table 1 below:

[0225] Table 1

[0226]

[0227] The experimental results in Table 1 show that the fluorescence intensity in the lungs and urine of drug-treated mice was lower than that in pneumonia-treated mice. Specifically, the fluorescence intensity in the lungs and urine of mice treated with cevelex showed no significant difference compared to healthy mice. This indicates that the activatable hydrophilic near-infrared fluorescent probe, liposome fluorescent probe, and cell membrane fluorescent probe provided by this invention can be used for the effective evaluation of pneumonia treatment drugs.

[0228] Test Example 7

[0229] Dosage screening for pneumonia treatment drugs

[0230] Test sample: The activatable hydrophilic near-infrared fluorescent probe provided in Example 2.

[0231] Test method:

[0232] After establishing a mouse model of pneumonia, mice were treated with one of the following: ulinastatin (5, 10, or 20 mg / kg). Twelve hours after treatment, samples were given, and fluorescence intensity analysis was performed on the lungs and urine of healthy mice, mice with pneumonia, and mice treated with the drug using a fluorescence imaging device.

[0233] The test results are shown in Table 2 below:

[0234] Table 2

[0235]

[0236] The experimental results in Table 2 show that the fluorescence intensity in the lungs and urine of mice treated with the drug was lower than that in mice with pneumonia. Specifically, the fluorescence intensity in the lungs and urine of mice treated with ulinastatin (20 mg / kg) showed no significant difference compared to healthy mice. This indicates that the activatable hydrophilic near-infrared fluorescent probe, liposome fluorescent probe, and cell membrane fluorescent probe provided by this invention can be used for dose screening of drugs for pneumonia treatment.

[0237] Test Example 8

[0238] Biosafety verification

[0239] Test sample: The activatable hydrophilic near-infrared fluorescent probe provided in Example 2.

[0240] Test methods: Healthy mice were administered the probe (2 μm / kg) intratracheally or via tail vein (5 μm / kg) for 3 days. Major organs and serum were collected from the mice. The biocompatibility of the probe was assessed using hematoxylin-eosin staining and hepatorenal toxicity assays. The degree of damage to major organs was analyzed using hematoxylin-eosin staining, and hepatorenal toxicity was used to assess liver and kidney function.

[0241] Depend on Figure 16 The experimental results show that after administering the activatable hydrophilic near-infrared fluorescent probe, liposome fluorescent probe, and cell membrane fluorescent probe provided by this invention, no significant toxic side effects were observed in the major organs (heart, liver, spleen, lung, and kidney) of mice, indicating that the probes are safe to use in vivo.

[0242] In summary, this invention provides an activatable near-infrared fluorescent probe for inflammation detection, its preparation, and its application. According to this invention, the activatable near-infrared fluorescent probe can be activated by neutrophil elastase, possessing the ability to actively target inflammation. It responds to the high expression of neutrophil elastase in the inflammatory microenvironment, thereby activating fluorescence and achieving in situ detection of inflammation. Due to the introduction of a hydrophilic polymer, the activated probe can be rapidly excreted through the kidneys, enabling urine detection of inflammation. Using this activatable near-infrared fluorescent probe reduces the accumulation of fluorescent probes at non-target sites, reduces background signals in fluorescence imaging, increases the specificity of inflammation detection, and improves imaging sensitivity. Guided by fluorescence imaging, this activatable near-infrared fluorescent probe can not only achieve real-time in situ detection of early inflammation and non-invasive urine analysis, but also assist in the screening and dosage selection of anti-inflammatory drugs.

[0243] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A liposome fluorescent probe, characterized in that, The liposome fluorescent probe is obtained by self-assembly of lipids and an activatable hydrophilic near-infrared fluorescent probe; The lipid is a combination of distearate phosphatidylcholine, didecyl dimethyl ammonium bromide, and cholesterol. The structural formula of the activatable hydrophilic near-infrared fluorescent probe is shown in Formula III-1 below: Formula III-1; The mass ratio of the distearate phosphatidylcholine, didecyl dimethyl ammonium bromide, cholesterol, and the activatable hydrophilic near-infrared fluorescent probe is 12:3.6:1:2.

5.

2. The liposome fluorescent probe according to claim 1, characterized in that, The activatable hydrophilic near-infrared fluorescent probe is prepared by the following steps: The activatable fluorescent probe and the hydrophilic polymer are reacted via a click reaction to obtain the activatable hydrophilic near-infrared fluorescent probe. The structure of the activatable fluorescent probe is shown in Formula II below: Formula II; The hydrophilic polymer is distearate phosphatidylethanolamine-methoxy polyethylene glycol-alkynyl.

3. The liposome fluorescent probe according to claim 2, characterized in that, The click reaction temperature is 25~40℃, and the click reaction time is 6~18 h.

4. The liposome fluorescent probe according to claim 2, characterized in that, The click reaction is carried out in the presence of a catalyst, wherein the catalyst is sodium ascorbate.

5. The liposome fluorescent probe according to claim 2, characterized in that, The click reaction is carried out in the presence of a promoter, wherein the promoter is copper sulfate.

6. The liposome fluorescent probe according to claim 2, characterized in that, The click reaction is carried out in the presence of a solvent, wherein the solvent includes dimethyl sulfoxide and water.

7. The liposome fluorescent probe according to claim 6, characterized in that, The volume ratio of dimethyl sulfoxide to water is 1:(1~2).

8. The liposome fluorescent probe according to claim 2, characterized in that, The click reaction is carried out in the presence of a catalyst and a promoter, and the molar ratio of the activatable fluorescent probe, the hydrophilic polymer, the catalyst, and the promoter is 1:(1~1.5):(0.1~0.2):(0.1~0.3).

9. The liposome fluorescent probe according to claim 2, characterized in that, The following post-processing steps are included after the click reaction is completed: dialyzing and lyophilizing the reaction solution obtained from the click reaction to obtain the activatable hydrophilic near-infrared fluorescent probe.

10. The liposome fluorescent probe according to claim 9, characterized in that, The molecular weight of the dialysis solution is 2000 Dalton.

11. A method for preparing a liposome fluorescent probe according to any one of claims 1 to 10, characterized in that, The preparation method includes the following steps: The lipid and the activatable hydrophilic near-infrared fluorescent probe are dispersed in a solvent, the solvent is removed by evaporation, water is added, and the mixture is sonicated to obtain the liposome fluorescent probe.

12. The method for preparing the liposome fluorescent probe according to claim 11, characterized in that, The solvent is trichloromethane.

13. The method for preparing the liposome fluorescent probe according to claim 11, characterized in that, The ultrasonic treatment temperature is 25~30℃, the ultrasonic treatment time is 1~10 min, and the ultrasonic treatment power is 50~200 W.

14. A cell membrane fluorescent probe, characterized in that, The cell membrane fluorescent probe is obtained by self-assembly of a macrophage membrane and a liposome fluorescent probe as described in any one of claims 1 to 10.

15. The cell membrane fluorescent probe according to claim 14, characterized in that, The mass ratio of the macrophage membrane to the activatable hydrophilic near-infrared fluorescent probe is 1:(0.1~10).

16. The cell membrane fluorescent probe according to claim 15, characterized in that, The mass ratio of the macrophage membrane to the activatable hydrophilic near-infrared fluorescent probe is 1:

5.

17. A method for preparing a cell membrane fluorescent probe according to any one of claims 14-16, characterized in that, The preparation method includes the following steps: The macrophage cell membrane and the activatable hydrophilic near-infrared fluorescent probe were mixed in a buffer solution and incubated. After sonication, the cell membrane fluorescent probe was obtained.

18. The method for preparing the cell membrane fluorescent probe according to claim 17, characterized in that, The buffer solution includes phosphate buffer.

19. The method for preparing the cell membrane fluorescent probe according to claim 17, characterized in that, The mixing temperature is 2~6℃.

20. The method for preparing the cell membrane fluorescent probe according to claim 19, characterized in that, The mixing temperature is 4°C.

21. The method for preparing the cell membrane fluorescent probe according to claim 17, characterized in that, The incubation time is 2 to 8 hours.

22. The method for preparing the cell membrane fluorescent probe according to claim 21, characterized in that, The incubation time is 4 hours.

23. The method for preparing the cell membrane fluorescent probe according to claim 17, characterized in that, The ultrasonic treatment temperature is 25~40℃, the ultrasonic treatment time is 1~10 min, and the ultrasonic treatment power is 50~200 W.

Images