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

Abstract

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

Description

Technical Field

[0001] The present 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, and a preparation method and application thereof. Background Art

[0002] Inflammation is ubiquitous in human daily life. Common pulmonary asthma, inflammatory bowel inflammation, arthritis and allergies are common inflammatory reactions that plague thousands of people. (See Baechle, JJ; Chen, N.; Makhijani, P.; Winer, S.; Furman, D.; Winer, D. Molecular Metabolism, 2023, 74). Most inflammatory reactions are caused by pathogens. When the host is infected by pathogens, the immune cells in the body will first detect pathogen-related molecular patterns through pattern recognition receptors, thereby being activated. The activated immune cells will directly phagocytize or secrete proteases to form cell traps to achieve pathogen clearance (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 signal transduction pathways in immune cells. For example, the activation of the κ-light chain enhanced signaling pathway of nuclear factor-activated B cells initiates an immune cascade reaction, produces inflammatory cytokines, and leads to a cytokine storm. In the case of immune dysregulation, other inflammatory mediators such as free nucleic acids, reactive oxygen species, and nitric oxide are also produced. Ultimately, long-term, excessive inflammatory responses may cause organ damage. 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] At present, the clinical diagnosis of inflammation mainly includes etiological tests, routine blood tests and imaging tests, which may have the disadvantages of being time-consuming, having poor specificity and radiation exposure. As a new type of biological imaging tool, fluorescence imaging technology has the advantages of high sensitivity, non-invasiveness, and real-time monitoring. It does not require a professional medical background and can effectively analyze the imaging results only through changes in fluorescence intensity. However, it also has problems such as high background signals 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, it is urgent 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 proposed. Summary of the invention

[0007] The purpose of the present invention is to provide an activatable hydrophilic near-infrared fluorescent probe, a liposome fluorescent probe, a cell membrane fluorescent probe, and a preparation method and application thereof. The fluorescent probe of the present invention can actively target the inflammation site, respond to the highly expressed neutrophil elastase in the inflammatory microenvironment, and then turn on the fluorescence to achieve in situ detection of inflammation.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly 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 and a hydrophilic polymer;

[0010] Wherein, 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 fluorescent shielding group.

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

[0012]

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

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

[0015]

[0016]

[0017] Preferably, the hydrophilic polymer comprises any one of polyvinyl pyrrolidone, distearoyl phosphatidylethanolamine-methoxy polyethylene glycol or polyethylene glycol, or a combination of at least two thereof, preferably distearoyl phosphatidylethanolamine-methoxy polyethylene glycol and / or polyethylene glycol.

[0018] Preferably, the weight average molecular weight of the aqueous polymer is 1000-5000, preferably 2000.

[0019] Preferably, the hydrophilic polymer is a hydrophilic polymer having an alkynyl group at its terminal group.

[0020] Preferably, the activatable fluorescent probe and the hydrophilic polymer are grafted by forming a 1,2,3-triazole ring group.

[0021] 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:

[0022] The activatable fluorescent probe and the hydrophilic polymer undergo a click reaction to obtain the activatable hydrophilic near-infrared fluorescent probe.

[0023] Preferably, the temperature of the click reaction is 25 to 40° C., and the time of the click reaction is 6 to 18 hours.

[0024] Preferably, the click reaction is carried out in the presence of a catalyst; wherein the catalyst is sodium ascorbate.

[0025] Preferably, the click reaction is carried out in the presence of a promoter; wherein the promoter is copper sulfate.

[0026] Preferably, the click reaction is carried out in the presence of a solvent; wherein the solvent comprises dimethyl sulfoxide and water.

[0027] Preferably, the volume ratio of dimethyl sulfoxide to water is 1:(1-2).

[0028] Preferably, the molar ratio of the activatable fluorescent probe, the hydrophilic polymer, the catalyst and the accelerator is 1:(1-1.5):(0.1-0.2):(0.1-0.3).

[0029] Preferably, after the click reaction is completed, the following post-treatment step is further included: dialyzing and freeze-drying the reaction solution obtained by the click reaction to obtain the activatable hydrophilic near-infrared fluorescent probe.

[0030] Preferably, the molecular weight of the dialyzed solution is 2000-3000 Dalton.

[0031] In a third aspect, the present invention provides a liposome fluorescent probe, wherein the liposome fluorescent probe is obtained by self-assembly of lipids and the activatable hydrophilic near-infrared fluorescent probe as described in the first aspect.

[0032] Preferably, the lipid comprises any one of distearoylphosphatidylcholine, didecyldimethylammonium bromide or cholesterol or a combination of at least two thereof, preferably a combination of distearoylphosphatidylcholine, didecyldimethylammonium bromide and cholesterol.

[0033] Preferably, the mass ratio of the distearoylphosphatidylcholine, didecyldimethylammonium bromide, cholesterol and the activatable hydrophilic near-infrared fluorescent probe is (1-12):(1-7.2):1:(1-5), preferably 12:3.6:1:2.5.

[0034] In a fourth aspect, the present invention provides a method for preparing a liposome fluorescent probe as described in the third aspect, wherein the method for preparing a liposome fluorescent probe comprises the following steps:

[0035] 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 ultrasonic treatment is performed to obtain the liposome fluorescent probe.

[0036] Preferably, the solvent is chloroform.

[0037] 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 50-200W.

[0038] In a fifth aspect, the present invention provides a cell membrane fluorescent probe, which is obtained by self-assembly of a macrophage membrane and the 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 the liposome fluorescent probe as described in the third aspect.

[0039] Preferably, the mass ratio of the macrophage membrane to the activatable hydrophilic near-infrared fluorescent probe is 1:(0.1-10), preferably 1:5.

[0040] In a sixth aspect, the present invention provides a method for preparing a cell membrane fluorescent probe as described in the fifth aspect, wherein the method for preparing a cell membrane fluorescent probe comprises the following steps:

[0041] The macrophage membrane and the activatable hydrophilic near-infrared fluorescent probe are mixed in a buffer solution and incubated, and then ultrasonically treated to obtain the cell membrane fluorescent probe.

[0042] Preferably, the buffer comprises phosphate buffer.

[0043] Preferably, the mixing temperature is 2-6°C.

[0044] Preferably, the incubation time is 2 to 8 hours, preferably 4 hours.

[0045] 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 50-200W.

[0046] In a seventh aspect, the present invention provides a use of the activatable hydrophilic near-infrared fluorescent probe according to the first aspect, 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.

[0047] Furthermore, the application includes the use of the activatable hydrophilic near-infrared fluorescent probe, the liposome fluorescent probe, or the cell membrane fluorescent probe as an active targeted detection agent for inflammatory sites.

[0048] Furthermore, the application includes real-time detection of inflammation in situ and in urine using the activatable hydrophilic near-infrared fluorescent probe, or the liposome fluorescent probe, or the cell membrane fluorescent probe under the guidance of fluorescence imaging.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] (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 the inflammatory site and can be activated by neutrophil elastase highly expressed in the inflammatory microenvironment, thereby achieving real-time imaging of inflammation and non-invasive urine analysis.

[0051] (2) The present invention utilizes activatable hydrophilic near-infrared fluorescent probes, liposome fluorescent probes, and cell membrane fluorescent probes to not only detect inflammation in real time at an early stage, but also assist in the screening and dosage selection of inflammatory therapeutic drugs, thus providing a new imaging tool for inflammation detection and drug efficacy evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0053] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the activatable fluorescent probe provided in Preparation Example 1.

[0054] Figure 2 This is the mass spectrum of the activatable fluorescent probe provided in Preparation Example 1.

[0055] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of the activatable hydrophilic near-infrared fluorescent probe provided in Example 2.

[0056] Figure 4 This is the mass spectrum of the activatable hydrophilic near-infrared fluorescent probe provided in Example 2.

[0057] Figure 5 This is a transmission electron microscopy image of the activatable hydrophilic near-infrared fluorescent probe provided in Example 2.

[0058] Figure 6 This is a schematic diagram of the structure of the preparation process of the liposome fluorescent probe provided in Examples 3 to 5.

[0059] Figure 7 This is a transmission electron microscopy image of the liposome fluorescent probe provided in Example 4.

[0060] Figure 8 This is a schematic diagram of the structure of the preparation process of the cell membrane fluorescent probe provided in Example 6.

[0061] Fig. 9 This is a transmission electron microscopy image of the cell membrane fluorescent probe provided in Example 6.

[0062] Fig. 10A This is a UV-visible absorption spectrum of the sample of the activatable hydrophilic near-infrared fluorescent probe provided in Example 2 after incubation with elastase.

[0063] Fig. 10B This is a UV-visible absorption spectrum of the liposome fluorescent probe sample provided in Example 4 after incubation with elastase.

[0064] Fig. 10C This is a UV-visible absorption spectrum of the sample of the cell membrane fluorescent probe provided in Example 6 after incubation with elastase.

[0065] Fig.11AThis is a fluorescence spectrum diagram of the sample of the activatable hydrophilic near-infrared fluorescent probe provided in Example 2 after incubation with elastase.

[0066] Fig. 11B This is a fluorescence spectrum diagram of the liposome fluorescent probe sample provided in Example 4 after incubation with elastase.

[0067] Fig. 11C This is a fluorescence spectrum diagram of the sample of the cell membrane fluorescent probe provided in Example 6 after incubation with elastase.

[0068] Fig.12 This is a diagram for verifying the neutrophil responsiveness of the activatable hydrophilic near-infrared fluorescent probe provided in Example 2.

[0069] Fig.13 This is an in vivo fluorescence imaging image of the probe sample provided in Test Example 4 injected into a pneumonia mouse.

[0070] Fig.14A A diagram showing real-time detection of pneumonia by the activatable hydrophilic near-infrared fluorescent probe provided in Example 2.

[0071] Fig. 14B This is a diagram of the real-time detection of pneumonia by the liposome fluorescent probe provided in Example 4.

[0072] Fig. 14C This is a diagram of the real-time detection of pneumonia by the cell membrane fluorescent probe provided in Example 6.

[0073] Fig.15A This is a urine detection diagram of the activatable hydrophilic near-infrared fluorescent probe provided in Example 2.

[0074] Fig. 15B This is a urine detection diagram of the liposome fluorescent probe provided in Example 4.

[0075] Fig. 15C This is a urine detection diagram of the cell membrane fluorescent probe provided in Example 6.

[0076] Fig.16 This is a biosafety verification diagram provided for Example 2. DETAILED DESCRIPTION

[0077] Unless otherwise defined herein, scientific and technological terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0078] It should be noted that specific details are described in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in a variety of other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0079] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0080] 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 and a hydrophilic polymer;

[0081] Wherein, 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 fluorescent shielding group.

[0082] In the present invention, the hydrophilic polymer and the activatable fluorescent probe are grafted to form an activatable hydrophilic near-infrared fluorescent probe. Due to the introduction of the hydrophilic polymer, the activated probe can be quickly excreted outside the body through the kidneys, thereby realizing urine detection of inflammation. In particular, the design of the activatable hydrophilic near-infrared fluorescent probe also effectively reduces the accumulation of fluorescent probes in non-target sites, reduces the background signal of fluorescent imaging, increases the specificity of inflammation detection, improves the sensitivity of imaging, and realizes in situ and urine detection of early inflammation. It should be noted that the activatable fluorescent probe of the present invention can specifically respond to neutrophil elastase highly expressed in an inflammatory environment, and a significant enhancement of fluorescence occurs.

[0083] As an optional embodiment, 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 through a condensation reaction, and then using phosphorus tribromide to introduce a hemicyanine fluorescent group to obtain the activatable fluorescent probe.

[0084] As an optional embodiment, the activatable fluorescent probe is prepared by the following steps:

[0085] (a) synthesizing acetyl-alanine-alanine-proline-valine polypeptide (AAPV) using standard solid phase synthesis method;

[0086] (b) mixing acetyl-alanine-alanine-proline-valine polypeptide (AAPV), 4-aminobenzyl alcohol (PABA), and diethyldithiocarbamate diethylamine in dichloromethane to react to obtain an intermediate polypeptide (AAPV-PABA);

[0087] (c) mixing the intermediate product polypeptide (AAPV-PABA) and a bromination reagent in tetrahydrofuran to react to obtain a brominated intermediate product polypeptide (AAPV-PABA-Br);

[0088] (d) mixing the brominated intermediate polypeptide (AAPV-PABA-Br), the hemicyanine fluorescent group and a base in acetonitrile to react and obtain the activatable fluorescent probe.

[0089] As an optional embodiment, in step (b), the temperature of the mixed 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 mixed reaction is 3-5h, for example, it can be 3h, 3.2h, 3.4h, 3.5h, 3.6h, 3.8h, 4h, 4.2h, 4.4h, 4.5h, 4.6h, 4.8h, 5h, etc., preferably 4h.

[0090] As an optional embodiment, in step (b), the molar ratio of AAPV, PABA and diethyldithiocarbamate diethylamine is 1:(1.5-2):(1.5-2);

[0091] Wherein, PABA: "1.5-2" can be, for example, 1.5, 1.6, 1.7, 1.8, 1.9, 2, etc.;

[0092] Among them, diethylamine diethyldithiocarbamate: "1.5 to 2" can be, for example, 1.5, 1.6, 1.7, 1.8, 1.9, 2, etc.

[0093] As an optional embodiment, in step (c), the temperature of the mixed 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., and is preferably 0°C; the time of the mixed reaction is 1 to 3h, for example, it can be 1h, 1.2h, 1.4h, 1.5h, 1.6h, 1.8h, 2h, 2.2h, 2.4h, 2.5h, 2.6h, 2.8h, 3h, etc., and is preferably 1h.

[0094] As an optional embodiment, in step (c), the bromination reagent is phosphorus tribromide.

[0095] As an optional embodiment, in step (c), the molar ratio of the AAPV-PABA to the bromination reagent is 1:(2.5-3.5), 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.

[0096] As an optional embodiment, in step (d), the temperature of the mixed reaction is 50-60°C, for example, it can be 50°C, 51°C, 52°C, 53°C, 54°C, 56°C, 57°C, 58°C, 59°C, 60°C, etc., and is preferably 55°C; the time of the mixed reaction is 18-30h, for example, it can be 18h, 19h, 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h, etc., and is preferably 24h.

[0097] As an optional embodiment, in step (d), the base is N,N-diisopropylethylamine.

[0098] As an optional embodiment, in step (d), the molar ratio of the AAPV-PABA-Br, the hemicyanine fluorescent group and the base is 1:(0.02-0.06):(0.06-0.18);

[0099] Here, “0.02-0.06” may be, for example, 0.02, 0.03, 0.04, 0.05, 0.06, etc.;

[0100] Here, “0.06-0.18” can be, for example, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18, etc.

[0101] As an optional embodiment, the structural formula of the hemicyanine fluorescent group is shown in Formula I below:

[0102]

[0103] As an optional embodiment, the fluorescent shielding group includes acetyl-alanine-alanine-proline-valine polypeptide (AAPV).

[0104] As an optional embodiment, the structural formula of the activatable fluorescent probe is shown in Formula II below:

[0105]

[0106] As an optional embodiment, the hydrophilic polymer includes any one of polyvinyl pyrrolidone, distearoyl phosphatidylethanolamine-methoxy polyethylene glycol or polyethylene glycol, or a combination of at least two thereof, preferably distearoyl phosphatidylethanolamine-methoxy polyethylene glycol and / or polyethylene glycol.

[0107] As an optional embodiment, the weight average molecular weight of the aqueous polymer is 1000-5000, for example, it can be 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, etc., preferably 2000.

[0108] As an optional embodiment, the hydrophilic polymer is a hydrophilic polymer having an alkynyl group at its terminal group.

[0109] As an optional embodiment, the activatable fluorescent probe and the hydrophilic polymer are grafted by forming a 1,2,3-triazole ring group.

[0110] As an optional embodiment, the structural formula of the activatable hydrophilic near-infrared fluorescent probe is shown in the following formula III:

[0111]

[0112] Among them, R1-L1-R2 can activate the fluorescent probe group, R1 represents the hemicyanine fluorescent group, L1 represents the connecting group, R2 represents the fluorescent shielding group, and R3 represents the hydrophilic polymer group.

[0113] 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, wherein the method for preparing an activatable hydrophilic near-infrared fluorescent probe comprises the following steps:

[0114] The activatable fluorescent probe and the hydrophilic polymer undergo a click reaction to obtain the activatable hydrophilic near-infrared fluorescent probe; the reaction formula of the click reaction is as follows:

[0115]

[0116] As an optional embodiment, the temperature of the click reaction is 25-40°C, for example, it can be 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 6-18h, for example, it can be 6h, 6.5h, 7h, 7.5h, 8h, 8.5h, 9h, 9.5h, 10h, 10.5h, 11h, 11.5h, 12h, 12.5h, 13h, 13.5h, 14h, 14.5h, 15h, 15.5h, 16h, 16.5h, 17h, 17.5h, 18h, etc., preferably 12h.

[0117] As an optional embodiment, the click reaction is performed in the presence of a catalyst.

[0118] As an optional embodiment, the catalyst for the click reaction is sodium ascorbate.

[0119] As an optional embodiment, the click reaction is performed in the presence of a promoter.

[0120] As an optional embodiment, the accelerator of the click reaction is copper sulfate, preferably anhydrous copper sulfate.

[0121] As an optional embodiment, the click reaction is performed in the presence of a solvent.

[0122] As an optional embodiment, the solvent for the click reaction includes dimethyl sulfoxide and water.

[0123] As an optional embodiment, 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.

[0124] As an optional embodiment, the molar ratio of the activatable fluorescent probe, the hydrophilic polymer, the catalyst and the accelerator is 1:(1-1.5):(0.1-0.2):(0.1-0.3);

[0125] Here, “1-1.5” can be, for example, 1, 1.1, 1.2, 1.3, 1.4, 1.5, etc.;

[0126] Here, “0.1-0.2” can be, for example, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, etc.;

[0127] Here, “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.

[0128] As an optional embodiment, after the click reaction is completed, the following post-treatment step is further included: dialyzing and freeze-drying the reaction solution obtained by the click reaction to obtain the activatable hydrophilic near-infrared fluorescent probe.

[0129] As an optional embodiment, the molecular weight of the dialysis 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.

[0130] In a third aspect, the present invention provides a liposome fluorescent probe, wherein the liposome fluorescent probe is obtained by self-assembly of lipids and the activatable hydrophilic near-infrared fluorescent probe as described in the first aspect.

[0131] As an optional embodiment, the lipid includes any one of distearoylphosphatidylcholine, didecyldimethylammonium bromide or cholesterol, or a combination of at least two thereof.

[0132] As a preferred embodiment, the lipid is a combination of distearoylphosphatidylcholine, didecyldimethylammonium bromide and cholesterol.

[0133] As an optional embodiment, the mass ratio of the distearoylphosphatidylcholine, didecyldimethylammonium bromide, cholesterol and the activatable hydrophilic near-infrared fluorescent probe is (1-12):(1-7.2):1:(1-5);

[0134] Here, “1-12” can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.;

[0135] Here, “1-7.2” can be, for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 7.2, etc.;

[0136] Among them, “1~5” can be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc.

[0137] As a preferred embodiment, the mass ratio of the distearoylphosphatidylcholine, didecyldimethylammonium bromide, cholesterol and the activatable hydrophilic near-infrared fluorescent probe is 12:3.6:1:2.5.

[0138] In a fourth aspect, the present invention provides a method for preparing a liposome fluorescent probe as described in the third aspect, wherein the method for preparing a liposome fluorescent probe comprises the following steps:

[0139] 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 ultrasonic treatment is performed to obtain the liposome fluorescent probe.

[0140] As an optional embodiment, the solvent is chloroform.

[0141] As an optional embodiment, the temperature of the ultrasonic treatment is 25-30°C, for example, it can be 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, etc., the time of the ultrasonic treatment is 1-10min, for example, it can be 1min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min, 10min, etc., and the power of the ultrasonic treatment is 50-200W, for example, it can be 50W, 60W, 80W, 100W, 120W, 140W, 150W, 160W, 180W, 200W, etc.

[0142] In a fifth aspect, the present invention provides a cell membrane fluorescent probe, which is obtained by self-assembly of a macrophage membrane and the 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 the liposome fluorescent probe as described in the third aspect.

[0143] As an optional embodiment, 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.

[0144] In a sixth aspect, the present invention provides a method for preparing a cell membrane fluorescent probe as described in the fifth aspect, wherein the method for preparing a cell membrane fluorescent probe comprises the following steps:

[0145] The macrophage membrane and the activatable hydrophilic near-infrared fluorescent probe are mixed in a buffer solution and incubated, and then ultrasonically treated to obtain the cell membrane fluorescent probe.

[0146] As an optional embodiment, the buffer comprises phosphate buffer.

[0147] As an optional embodiment, the mixing temperature is 2-6°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.

[0148] As an optional embodiment, the temperature of the ultrasonic treatment is 25-40°C, for example, it can be 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, etc., the time of the ultrasonic treatment is 1-10min, for example, it can be 1min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min, 10min, etc., and the power of the ultrasonic treatment is 50-200W, for example, it can be 50W, 60W, 80W, 100W, 120W, 140W, 150W, 160W, 180W, 200W, etc.

[0149] In a seventh aspect, the present invention provides a use of the activatable hydrophilic near-infrared fluorescent probe according to the first aspect, 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.

[0150] As an optional embodiment, 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 targeted detection agent for inflammatory sites.

[0151] As an optional embodiment, the application includes real-time detection of inflammation in situ and in urine under the guidance of fluorescence imaging using the activatable hydrophilic near-infrared fluorescent probe, the liposome fluorescent probe, or the cell membrane fluorescent probe.

[0152] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0153] Preparation Example 1

[0154] This preparation example provides an activatable fluorescent probe, the structural formula of the activatable fluorescent probe is shown in the following formula II:

[0155]

[0156] The activatable fluorescent probe shown in Formula II is prepared by the following steps:

[0157] (a) synthesizing acetyl-alanine-alanine-proline-valine polypeptide (AAPV) using standard solid phase synthesis method;

[0158] (b) mixing AAPV, PABA and diethyldithiocarbamate in a molar ratio of 1:1.5:1.5 in dichloromethane, and reacting at room temperature for 4 hours to obtain an intermediate product, polypeptide AAPV-PABA;

[0159] (c) mixing AAPV-PABA and phosphorus tribromide in a molar ratio of 1:3 in tetrahydrofuran, and reacting at 0° C. for 2 h to obtain a brominated intermediate polypeptide AAPV-PABA-Br;

[0160] (d) Mixing AAPV-PABA-Br, the hemicyanine fluorescent group shown in Formula I, and N,N-diisopropylethylamine in a molar ratio of 1:0.02:0.04 in acetonitrile, and reacting at 55° C. for 24 hours to obtain the activatable fluorescent probe.

[0161] in, Figure 1 This is the hydrogen NMR spectrum of the activatable fluorescent probe provided in Preparation Example 1. The hydrogen spectrum structure analysis is detailed in Figure 1 . Figure 2 The mass spectrum of the activatable fluorescent probe provided in Preparation Example 1. Figure 2 As shown, a peak with a molecular weight of 953 appeared in the hydrogen spectrum analysis. This proves that the activatable fluorescent probe provided in Preparation Example 1 was successfully synthesized.

[0162] Example 1

[0163] This embodiment provides an activatable hydrophilic near-infrared fluorescent probe, which is obtained by grafting the activatable fluorescent probe shown in Formula II with distearoylphosphatidylethanolamine-methoxypolyethylene glycol; the structural formula of the activatable hydrophilic near-infrared fluorescent probe is shown in the following Formula III-1:

[0164]

[0165] The activatable hydrophilic near-infrared fluorescent probe is prepared by the following steps:

[0166] The activatable fluorescent probe represented by formula II, distearoyl phosphatidylethanolamine-methoxy polyethylene glycol-alkynyl (Mw=2000), anhydrous copper sulfate and sodium ascorbate in a mixed solution of dimethyl sulfoxide and water (V 二甲基亚砜 :V 水 =2:1), react at room temperature for 12 h, dialyze (dialysis molecular weight is 2000 Dalton), and freeze-dry to obtain the activatable hydrophilic near-infrared fluorescent probe.

[0167] Example 2

[0168] This embodiment provides an activatable hydrophilic near-infrared fluorescent probe, which is obtained by grafting the activatable fluorescent probe shown in Formula II with polyethylene glycol; the structural formula of the activatable hydrophilic near-infrared fluorescent probe is shown in Formula III-2 below:

[0169]

[0170] The activatable hydrophilic near-infrared fluorescent probe is prepared by the following steps:

[0171] The activatable fluorescent probe represented by Formula II, polyethylene glycol-alkynyl (Mw=2000), anhydrous copper sulfate and sodium ascorbate in a molar ratio of 1:1:1:0.4 were mixed in a mixed solution of dimethyl sulfoxide and water (V 二甲基亚砜 :V 水 =2.5:), react at room temperature for 12 h, dialyze (dialysis molecular weight is 2000 Dalton), and freeze-dry to obtain the activatable hydrophilic near-infrared fluorescent probe.

[0172] in, Figure 3 This is the hydrogen nuclear magnetic resonance 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 about 2900 appeared, confirming the successful grafting of PEG. This proves that the activatable hydrophilic near-infrared fluorescent probe provided in Example 2 was successfully synthesized.

[0173] in, Figure 5 This is a transmission electron microscopy 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.

[0174] Example 3

[0175] This embodiment provides a liposome fluorescent probe, which is prepared by the following steps:

[0176] Distearoylphosphatidylcholine, didecyldimethylammonium bromide, cholesterol and the activatable hydrophilic near-infrared fluorescent probe prepared in Example 1 in a mass ratio of 12:1.8:1:2.5 were dispersed in chloroform, the solvent was removed by rotary evaporation, water was added, and ultrasonic treatment was performed at 25°C and 100W for 2 minutes to obtain the liposome fluorescent probe.

[0177] Example 4

[0178] This embodiment provides a liposome fluorescent probe, which is prepared by the following steps:

[0179] Distearoylphosphatidylcholine, didecyldimethylammonium bromide, cholesterol and the activatable hydrophilic near-infrared fluorescent probe prepared in Example 1 in a mass ratio of 12:3.6:1:2.5 were dispersed in chloroform, the solvent was removed by rotary evaporation, water was added, and ultrasonic treatment was performed at 25°C and 100W for 2 minutes to obtain the liposome fluorescent probe.

[0180] Example 5

[0181] This embodiment provides a liposome fluorescent probe, which is prepared by the following steps:

[0182] Distearoylphosphatidylcholine, didecyldimethylammonium bromide, cholesterol and the activatable hydrophilic near-infrared fluorescent probe prepared in Example 1 in a mass ratio of 12:7.2:1:2.5 were dispersed in chloroform, the solvent was removed by rotary evaporation, water was added, and ultrasonic treatment was performed at 25°C and 100W for 2 minutes to obtain the liposome fluorescent probe.

[0183] in, Figure 6 This is a schematic diagram of the structure of the preparation process of the liposome fluorescent probe provided in Examples 3 to 5. Figure 7 The transmission electron microscopy image of the liposome fluorescent probe provided in Example 4 is as follows: Figure 7 As shown, the liposome fluorescent probe of the present invention has a round vesicle shape.

[0184] Example 6

[0185] This embodiment provides a cell membrane fluorescent probe, which is prepared by the following steps:

[0186] The macrophage membrane and the activatable hydrophilic near-infrared fluorescent probe prepared in Example 1 in a mass ratio of 1:5 were mixed in a phosphate buffer, incubated at 4° C. for 4 h, and ultrasonically treated at 25° C. and 100 W for 5 min to obtain the cell membrane fluorescent probe.

[0187] in, Figure 8 This is a schematic diagram of the structure of the preparation process of the cell membrane fluorescent probe provided in Example 6. Fig. 9 This is a transmission electron microscope image of the cell membrane fluorescent probe provided in Example 6. Fig. 9 As shown, the liposome fluorescent probe of the present invention has a round vesicle shape.

[0188] Test Example 1

[0189] UV-visible absorption spectrum and fluorescence spectrum test

[0190] Test sample: Example Provided activatable hydrophilic near-infrared fluorescent probe, embodiment The liposome fluorescent probe provided and the cell membrane fluorescent probe provided in Example 6.

[0191] Test method: After incubating elastase and the above test samples in tris(hydroxymethylaminomethane) hydrochloride at 37°C for 2h, the UV-visible absorption spectrum and fluorescence spectrum of the mixed solution were measured to obtain the UV absorption spectrum and fluorescence absorption spectrum of each sample before and after incubation with elastase.

[0192] Test results:

[0193] like Fig. 10A , Fig. 10B , Fig. 10C ,as well as Fig.11A , Fig. 11B Fig. 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 red shift, and the fluorescence intensity was significantly enhanced, indicating 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.

[0194] Test Example 2

[0195] Neutrophil imaging validation

[0196] Test sample: the activatable hydrophilic near-infrared fluorescent probe provided in Example 2.

[0197] Test method: Neutrophils were extracted from mouse bone marrow and 1×10 5 The cells are inoculated in a culture dish at a density of 100 μg / cm2, and after adding the probe or the probe is co-incubated for 2 hours, a confocal microscope is used to observe the activation of the probe or the probe in neutrophils. The cell culture method is not particularly limited and can be based on methods well known to those skilled in the art. In the present invention, the cells are preferably cultured in an incubator containing 10% fetal bovine serum, and the culture conditions are preferably continuous culture in an incubator with a volume fraction of 5% carbon dioxide, and the culture temperature is preferably 37°C.

[0198] Test results:

[0199] like Fig.12 As shown, the activatable hydrophilic near-infrared fluorescent probe provided in Example 2 is activated in neutrophils, and the probe cannot be activated after adding neutrophil elastase inhibitor, indicating that the activatable hydrophilic near-infrared fluorescent probe described in the present invention can be used for imaging of neutrophils.

[0200] Test Example 3

[0201] Renal clearance efficiency test

[0202] Test sample: the activatable hydrophilic near-infrared fluorescent probe provided in Example 2.

[0203] Test method: Inject the above samples into mice via the trachea, collect the urine of mice 3, 6, 9, 12, and 24 hours after tracheal injection using a urine collection device, and measure the content of each sample in the urine to obtain the renal clearance efficiency.

[0204] Test results: The renal clearance efficiency of the activatable hydrophilic near-infrared fluorescent probe provided in Example 2 is 20%, which indicates that the probe can be quickly metabolized by the kidneys and then eliminated through urine, confirming the feasibility of using a hydrophilic probe to detect inflammation through urine.

[0205] Test Example 4

[0206] Targeted testing

[0207] Test sample: liposome fluorescent probe provided in Examples 3 to 5.

[0208] Test method: The above samples were injected into pneumonia mice through the tail vein, and the in vivo fluorescence imaging images were obtained 2, 10, 15, 20, 30, 45, and 60 minutes after the injection of the probe using fluorescence imaging technology.

[0209] Test results:

[0210] like Fig.13 As shown, under the action of lung targeting lipid didecyl dimethyl ammonium bromide and macrophage membrane homologous targeting, the lung fluorescence intensity gradually increases with time, indicating that the liposome fluorescent probes provided in Examples 3 to 5 can actively target the inflammatory site.

[0211] Test Example 5

[0212] 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.

[0213] Test method:

[0214] (1) Construction of pneumonia: Balb / C female mice aged 6-8 weeks were used for model construction. The mice were anesthetized with isoflurane and given lipopolysaccharide (8 mg / kg) through the trachea to complete the construction of the mouse pneumonia model.

[0215] (2) Real-time in situ detection of pneumonia: Pneumonia is detected by administering a probe (2 μm / kg) into the trachea of ​​mice with pneumonia or into the tail vein (5 μm / kg). After administering the probe, a fluorescent imaging device is used to perform whole-body imaging of the mice in vivo to obtain fluorescent imaging images of pneumonia detection. The severity of pneumonia is analyzed using changes in lung fluorescence intensity. The fluorescence imaging test method can be a method familiar to those skilled in the art without any special restrictions.

[0216] (3) Urine detection of pneumonia: The probe (2 μm / kg) was administered intratracheally or via the tail vein of mice with pneumonia, and then the mouse urine was collected. The fluorescence intensity of the mouse urine was analyzed using a fluorescent imaging device, and pneumonia was detected by the change in the fluorescence intensity of the urine.

[0217] (4) Fluorescence imaging technology was then used to obtain in vivo fluorescence imaging images at 2, 10, 15, 20, 30, 45, and 60 minutes after the probe was administered. Urine from healthy and pneumonia mice after the probe was administered was obtained using a urine collection device and fluorescence imaging was performed.

[0218] Test results:

[0219] like Fig.14A , Fig. 14B and Fig. 14C As shown in Figure 2, the fluorescence intensity in the lungs of mice with pneumonia was significantly greater than that in healthy mice. Fig.15A , Fig. 15B and Fig. 15C As shown in the figure, the fluorescence intensity of urine of pneumonia mice is stronger than that of healthy mice. This shows that the activatable hydrophilic near-infrared fluorescent probe, liposome fluorescent probe, and cell membrane fluorescent probe of the present invention can detect inflammation by in situ imaging and urine detection, which improves the specificity of using fluorescent probes to detect inflammation and increases the signal-to-noise ratio of detection.

[0220] Test Example 6

[0221] Evaluation of the efficacy of drugs for the treatment of pneumonia

[0222] Test sample: the activatable hydrophilic near-infrared fluorescent probe provided in Example 2.

[0223] Test method:

[0224] After the pneumonia mouse model was established, the mice were given pneumonia treatment drugs for treatment. Fluorescence intensity analysis of the mouse lungs and urine was performed using a fluorescent imaging device to evaluate the efficacy of pneumonia treatment drugs. Pneumonia treatment drugs include: low molecular weight heparin (300U / kg), Xuebijing (1.25mL / kg), dexamethasone (10mg / kg) or Sivelex (10mg / kg).

[0225] The test results are shown in Table 1 below:

[0226] Table 1

[0227]

[0228] The experimental results in Table 1 show that the fluorescence intensity of the lungs and urine of drug-treated mice is lower than that of pneumonia mice, and there is no significant difference between the fluorescence intensity of the lungs and urine of mice treated with siveleix and healthy mice. This indicates that the activatable hydrophilic near-infrared fluorescent probe, liposome fluorescent probe, and cell membrane fluorescent probe provided by the present invention can be used for the effective evaluation of pneumonia treatment drugs.

[0229] Test Example 7

[0230] Dose screening of drugs for the treatment of pneumonia

[0231] Test sample: the activatable hydrophilic near-infrared fluorescent probe provided in Example 2.

[0232] Test method:

[0233] After the pneumonia mouse model was established, one of the ulinastatin (5, 10, 20 mg / kg) was used for treatment. Samples were given 12 hours after treatment and the fluorescence intensity of the lungs and urine of healthy mice, pneumonia mice and drug-treated mice was analyzed using a fluorescence imaging device.

[0234] The test results are shown in Table 2 below:

[0235] Table 2

[0236]

[0237] The experimental results in Table 2 show that the fluorescence intensity of the lungs and urine of drug-treated mice is lower than that of pneumonia mice, and there is no significant difference between the fluorescence intensity of the lungs and urine of mice treated with ulinastatin (20 mg / kg) and healthy mice. This indicates that the activatable hydrophilic near-infrared fluorescent probe, liposome fluorescent probe, and cell membrane fluorescent probe provided by the present invention can be used for dose screening of pneumonia treatment drugs.

[0238] Test Case 8

[0239] Biosafety Verification

[0240] Test sample: the activatable hydrophilic near-infrared fluorescent probe provided in Example 2.

[0241] Test method: After the probe (2μm / kg) was administered intratracheally or via the tail vein (5μm / Kg) to healthy mice for 3 days, the main organs and serum of the mice were collected, and the biocompatibility of the probe was evaluated by hematoxylin-eosin staining and liver and kidney toxicity assay. The degree of damage to the main organs of the mice was analyzed by hematoxylin-eosin staining, and the liver and kidney function of the mice was evaluated by liver and kidney toxicity.

[0242] Depend on Fig.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 the present invention, no obvious toxic side effects were caused to the main organs (heart, liver, spleen, lung, and kidney) of mice, indicating that the use of the probe in vivo is safe.

[0243] In summary, the present invention provides an activatable near-infrared fluorescent probe for inflammation detection and its preparation and application. According to the present invention, the activatable near-infrared fluorescent probe can be activated by neutrophil elastase, has the ability to actively target inflammation, can respond to neutrophil elastase highly expressed in the inflammatory microenvironment, and then turn on fluorescence to achieve in situ detection of inflammation. Due to the introduction of hydrophilic polymers, the activated probe can be rapidly excreted to the body through the kidneys to achieve urine detection of inflammation. The use of the activatable near-infrared fluorescent probe reduces the accumulation of fluorescent probes in non-target sites, reduces the background signal of fluorescence imaging, increases the specificity of inflammation detection, and improves the sensitivity of imaging. Under the guidance of fluorescence imaging, the use of the activatable near-infrared fluorescent probe can not only achieve real-time in situ detection and non-invasive urine analysis of early inflammation, but also assist in the screening of drugs for the treatment of inflammation and dose selection.

[0244] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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. An activatable hydrophilic near-infrared fluorescent probe, characterized in that: The activatable hydrophilic near-infrared fluorescent probe is obtained by grafting an activatable fluorescent probe with a hydrophilic polymer; Wherein, 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 fluorescent shielding group.

2. The activatable hydrophilic near-infrared fluorescent probe according to claim 1, characterized in that: The structural formula of the hemicyanine fluorescent group is shown in Formula I below; Preferably, the fluorescent shielding group comprises an acetyl-alanine-alanine-proline-valine polypeptide; Preferably, the structural formula of the activatable fluorescent probe is as shown in Formula II below: Preferably, the hydrophilic polymer comprises any one of polyvinyl pyrrolidone, distearoyl phosphatidylethanolamine-methoxy polyethylene glycol or polyethylene glycol, or a combination of at least two thereof, preferably distearoyl phosphatidylethanolamine-methoxy polyethylene glycol and / or polyethylene glycol; Preferably, the weight average molecular weight of the aqueous polymer is 1000 to 5000, preferably 2000; Preferably, the hydrophilic polymer is a hydrophilic polymer having an alkyne group at the end group; Preferably, the activatable fluorescent probe and the hydrophilic polymer are grafted by forming a 1,2,3-triazole ring group.

3. A method for preparing an activatable hydrophilic near-infrared fluorescent probe according to claim 1 or 2, characterized in that: The preparation method comprises the following steps: The activatable fluorescent probe and the hydrophilic polymer undergo a click reaction to obtain the activatable hydrophilic near-infrared fluorescent probe.

4. The preparation method according to claim 3, characterized in that: The temperature of the click reaction is 25 to 40° C., and the time of the click reaction is 6 to 18 hours; Preferably, the click reaction is carried out in the presence of a catalyst; wherein the catalyst is sodium ascorbate; Preferably, the click reaction is carried out in the presence of a promoter; wherein the promoter is copper sulfate; Preferably, the click reaction is carried out in the presence of a solvent; wherein the solvent comprises dimethyl sulfoxide and water; Preferably, the volume ratio of dimethyl sulfoxide to water is 1:(1-2); Preferably, the molar ratio of the activatable fluorescent probe, the hydrophilic polymer, the catalyst and the accelerator is 1:(1-1.5):(0.1-0.2):(0.1-0.3); Preferably, after the click reaction is completed, the following post-treatment steps are further included: dialyzing and freeze-drying the reaction solution obtained by the click reaction to obtain the activatable hydrophilic near-infrared fluorescent probe; Preferably, the molecular weight of the dialyzed solution is 2000-3000 Dalton.

5. A liposome fluorescent probe, characterized in that: The liposome fluorescent probe is obtained by self-assembly of lipids and the activatable hydrophilic near-infrared fluorescent probe as claimed in claim 1 or 2.

6. The liposome fluorescent probe according to claim 5, characterized in that: The lipid comprises any one of distearoylphosphatidylcholine, didecyldimethylammonium bromide or cholesterol or a combination of at least two thereof, preferably a combination of distearoylphosphatidylcholine, didecyldimethylammonium bromide and cholesterol; Preferably, the mass ratio of the distearoylphosphatidylcholine, didecyldimethylammonium bromide, cholesterol and the activatable hydrophilic near-infrared fluorescent probe is (1-12):(1-7.2):1:(1-5), preferably 12:3.6:1:2.

5.

7. A method for preparing the liposome fluorescent probe according to claim 5 or 6, characterized in that: The preparation method comprises the following steps: Dispersing the lipid and the activatable hydrophilic near-infrared fluorescent probe in a solvent, removing the solvent by evaporation, adding water, and performing ultrasonic treatment to obtain the liposome fluorescent probe; Preferably, the solvent is chloroform; 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 50-200W.

8. A cell membrane fluorescent probe, characterized in that The cell membrane fluorescent probe is obtained by self-assembly of a macrophage membrane and the activatable hydrophilic near-infrared fluorescent probe as claimed in claim 1 or 2; or the cell membrane fluorescent probe is obtained by self-assembly of a macrophage membrane and the liposome fluorescent probe as claimed in claim 5 or 6; Preferably, the mass ratio of the macrophage membrane to the activatable hydrophilic near-infrared fluorescent probe is 1:(0.1-10), preferably 1:

5.

9. A method for preparing a cell membrane fluorescent probe according to claim 8, characterized in that: The preparation method comprises the following steps: The macrophage membrane and the activatable hydrophilic near-infrared fluorescent probe are mixed in a buffer solution and incubated, and then ultrasonically treated to obtain the cell membrane fluorescent probe; Preferably, the buffer comprises phosphate buffer; Preferably, the mixing temperature is 2 to 6°C, preferably 4°C; Preferably, the incubation time is 2 to 8 hours, preferably 4 hours; 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 50-200W.

10. Use of the activatable hydrophilic near-infrared fluorescent probe according to claim 1 or 2, or the liposome fluorescent probe according to claim 5 or 6, or the cell membrane fluorescent probe according to claim 8 as an inflammation detection reagent; And / or, the application includes the use of the activatable hydrophilic near-infrared fluorescent probe as described in claim 1 or 2, or the liposome fluorescent probe as described in claim 5 or 6, or the cell membrane fluorescent probe as described in claim 8 as an active targeted detection agent for inflammatory sites; And / or, the application includes real-time detection of inflammation in situ and in urine under the guidance of fluorescence imaging using the activatable hydrophilic near-infrared fluorescent probe as described in claim 1 or 2, or the liposome fluorescent probe as described in claim 5 or 6, or the cell membrane fluorescent probe as described in claim 8.

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