An hbi derivative and a method for preparing the same, a self-assembled body of the hbi derivative and applications thereof

Abstract

This invention discloses an HBI derivative and its preparation method, a self-assembled form of the HBI derivative, and its applications. The HBI derivative is obtained by derivatization using the green fluorescent protein chromophore (4-(4-hydroxybenzyl)-5-imidazolinone) as the backbone structure. The preparation steps are simple, the preparation conditions are easy to control, and it can be prepared on a large scale. The HBI derivative of this invention can rapidly self-assemble in a mixture of organic solvent and water to form hollow sheet-like, hollow columnar, or hollow rod-like self-assembled forms. The assembled self-assembled forms exhibit green fluorescence and have great application prospects in cell bioimaging and drug delivery systems.

Description

Technical Field

[0001] This invention relates to the field of fluorescent small molecule self-assembly technology, specifically to an HBI derivative and its preparation method, a self-assembled HBI derivative and its application. Background Technology

[0002] Self-assembly is a fundamental process in nature, with significant implications in materials science, biology, and chemistry. Research on self-assembly has been ongoing for over a century, leading to the emergence of numerous specific research areas, such as the combination of self-assembly with the emerging aggregation-induced emission (AIE). Due to their fascinating photophysical properties, AIE-luminescent materials have shown great potential in this field. AIE processes are often accompanied by molecular aggregation (i.e., AIEgens), and aggregation typically refers to self-assembly; therefore, AIEgens are natural self-assembly building blocks. Furthermore, the photophysical properties of AIEgens can be easily modulated by supramolecular aggregates. Due to their aggregation-induced emission properties, they show great potential for application in visualizing dynamic self-assembly; however, to date, related research is limited due to technical complexity. Therefore, monitoring self-assembly processes using AIEgens will receive increasing attention.

[0003] The concept of AIE was first proposed by Tang Benzhong's research group. In 2001, they discovered a series of organic molecules with distorted conformations. For example, hexaphenylsilane (HPS) and tetraphenylethylene (TPE) do not emit light in their molecularly dissolved state, but emit strong fluorescence in their aggregated state (e.g., ...). Figure 1 As shown in the image, this phenomenon, contrary to the traditional properties of fluorophores, is called aggregation-induced emission (AIE). Mechanistic studies have shown that the restriction of intramolecular motion (RIM), including rotation and oscillation, is the main cause of aggregation-induced emission (AIE). Currently, aggregation-induced luminescent molecules are recognized as an ideal class of compounds for constructing supramolecular materials.

[0004] Literature review revealed that, in addition to TPE-based AIE luminescent materials, the green fluorescent protein chromophore (4-hydroxybenzylidene-imidazolinone, HBI) also exhibits AIE properties, such as... Figure 2As shown, free-state HBI undergoes rapid internal conversion due to free torsional motion around the C-C bonds, quickly returning to the ground state via nonradiative transition. When intramolecular motion is restricted, the energy of the excited-state molecule is released in the form of fluorescence, causing the chromophore to exhibit the property of restricted molecular motion luminescence. Since HBI originates from the chromophore of green fluorescent protein, its derivative self-assemblies exhibit better biocompatibility and lower cytotoxicity compared to TPE-like molecules, making them more suitable for applications in the biomedical field. Summary of the Invention

[0005] Based on the above-mentioned prior art, the present invention provides an HBI derivative and its preparation method, a self-assembled HBI derivative and its application. The HBI derivative is obtained by derivatization using green fluorescent protein chromophore as the backbone structure. It can rapidly self-assemble in a mixed system to form fluorescent materials with specific morphologies, and has great application prospects in the fields of cell bioimaging and drug carriers.

[0006] The technical solution adopted to achieve the above-mentioned objectives of this invention is as follows:

[0007] An HBI derivative has the following general structural formula:

[0008]

[0009] Wherein, R1 is dimethylamino and R2 is n-hexyl.

[0010] A method for preparing an HBI derivative includes the following steps:

[0011] 1. Under nitrogen protection and at room temperature, compound (1) undergoes a condensation reaction with n-hexylamine to produce compound (2), the reaction formula of which is as follows:

[0012]

[0013] Wherein, R1 is dimethylamino and R2 is n-hexyl;

[0014] 2. Under alkaline conditions and at room temperature, glycine methyl ester hydrochloride undergoes a substitution reaction with ethyl acetylimine hydrochloride to produce compound (3), the reaction formula of which is as follows:

[0015]

[0016] 3. Compounds of formula (2) and (3) undergo a cycloaddition reaction at room temperature to generate HBI derivatives, as shown in the following reaction formula:

[0017]

[0018] Furthermore, the alkali mentioned is potassium carbonate.

[0019] A self-assembled HBI derivative was prepared by the following method:

[0020] The HBI derivative was added to an organic solvent and dissolved completely. Then it was added to ultrapure water and mixed evenly to obtain a mixture. The mixture was then allowed to stand to obtain the self-assembled HBI derivative.

[0021] Furthermore, the organic solvent is one of methanol, ethanol, acetonitrile, and tetrahydrofuran, or any combination thereof.

[0022] Furthermore, the concentration of the HBI derivative in the mixture is 0.2-10 mg / mL.

[0023] Furthermore, the volume ratio of the organic solvent to ultrapure water is 0.05-0.95:1.

[0024] Furthermore, the settling time is 0.1-72 hours.

[0025] Application of a self-assembled HBI derivative in the preparation of cell imaging agents.

[0026] Compared with the prior art, the beneficial effects and advantages of the present invention are as follows:

[0027] 1. The HBI derivative of the present invention is obtained by derivatization using the green fluorescent protein chromophore (4-(4-hydroxybenzyl)-5-imidazolinone) as the backbone structure. The preparation steps are simple, the preparation conditions are simple and easy to control, and it can be prepared on a large scale.

[0028] 2. The HBI derivative of the present invention can rapidly self-assemble in a mixture of organic solvent and water to form sheet-like, hollow columnar, hollow rod-like, or hollow sheet-like self-assembled bodies. It can be assembled at room temperature, and the assembly process is minimally affected by temperature. The assembled self-assembled bodies exhibit green fluorescence and have great application prospects in the fields of cell bioimaging and drug delivery. Attached Figure Description

[0029] Figure 1 The image shows the fluorescence emission of the self-assembled hexaphenylsilane in a mixture of tetrahydrofuran and water.

[0030] Figure 2 This is a diagram illustrating the luminescence mechanism of the green fluorescent protein chromophore HBI.

[0031] Figure 3 Morphological image of the self-assembled XSC prepared in Example 4: Figure 3 (a) is a morphological image of the self-assembled XSC under a fluorescence microscope; Figure 3(b) shows the overall morphology of the self-assembled XSC under a high-resolution cold field scanning electron microscope; Figure 3 (c) is a cross-sectional morphology of the self-assembled XSC under an ultra-high resolution cold field scanning electron microscope.

[0032] Figure 4 Morphological images of XSC at different assembly time points: Figure 4 (a) is the morphology of XSC under a fluorescence microscope after standing for 0 min; Figure 4 (b) is the morphology of XSC under a fluorescence microscope after standing for 30 min; Figure 4(c) is the morphology of XSC under a fluorescence microscope after standing for 2 h. Figure 4 (d) shows the morphology of XSC under a fluorescence microscope after standing for 6 hours; Figure 4 (e) is the morphology of XSC under a high-resolution cold field scanning electron microscope after standing for 0 min; Figure 4 (f) shows the morphology of XSC under a high-resolution cold field scanning electron microscope after standing for 30 min; Figure 4 (g) is the morphology of XSC under a high-resolution cold field scanning electron microscope after standing for 6 hours.

[0033] Figure 5 Morphological image of the self-assembled FSC prepared in Example 5: Figure 5 (a) is a morphological image of the self-assembled FSC under a fluorescence microscope; Figure 5 (b) shows the overall morphology of the self-assembled FSC under an ultra-high resolution cold field scanning electron microscope; Figure 5 (c) is a cross-sectional morphology of the self-assembled FSC under an ultra-high resolution cold field scanning electron microscope.

[0034] Figure 6 Topographic images of FSC at different assembly time points: Figure 6 (a) is the morphology of FSC under a fluorescence microscope after standing for 0 min; Figure 6 (b) shows the morphology of FSC under a fluorescence microscope after standing for 30 min; Figure 6 (c) shows the morphology of FSC under a fluorescence microscope after standing for 2 hours; Figure 6 (d) shows the morphology of FSC under a fluorescence microscope after standing for 6 hours; Figure 6 (e) is the morphology of FSC under a high-resolution cold field scanning electron microscope after standing for 0 min; Figure 6 (f) shows the morphology of FSC under an ultra-high resolution cold field scanning electron microscope after standing for 30 min; Figure 6 (g) is the morphology of FSC under an ultra-high resolution cold field scanning electron microscope after standing for 6 hours.

[0035] Figure 7 Morphological image of the self-assembled ZSC prepared in Example 5: Figure 7 (a) is a morphological image of the self-assembled ZSC under a fluorescence microscope; Figure 7 (b) shows the overall morphology of the ZSC self-assembled assembly under an ultra-high resolution cold field scanning electron microscope; Figure 7 (c) is a cross-sectional morphology of the ZSC self-assembled part under an ultra-high resolution cold field scanning electron microscope.

[0036] Figure 8 Morphological images of ZSC at different assembly time points: Figure 8 (a) is the morphology of ZSC under a fluorescence microscope after standing for 0 min; Figure 8 (b) shows the morphology of ZSC under a fluorescence microscope after standing for 30 min; Figure 8 (c) is the morphology of ZSC under a fluorescence microscope after standing for 2 hours; Figure 8 (d) shows the morphology of ZSC under a fluorescence microscope after standing for 6 hours; Figure 8 (e) is the morphology of ZSC under a high-resolution cold field scanning electron microscope after standing for 0 min; Figure 8 (f) shows the morphology of ZSC under an ultra-high resolution cold field scanning electron microscope after standing for 30 min; Figure 8 (g) is the morphology of ZSC under an ultra-high resolution cold field scanning electron microscope after standing for 6 hours.

[0037] Figure 9 The images show the morphology of self-assembled XSC structures at different temperatures under a fluorescence microscope. Figure 9 (a) is a morphological diagram of the self-assembled body formed by XSC at 40℃; Figure 9 (b) is a morphological diagram of the self-assembled body formed by XSC at 4℃.

[0038] Figure 10 The images show the morphology of the self-assembled structures formed by FSC at different temperatures under a fluorescence microscope. Figure 10 (a) is a morphological diagram of the self-assembled body formed by FSC at 40℃; Figure 10 (b) is a morphological diagram of the self-assembled body formed by FSC at 4℃.

[0039] Figure 11 The images show the morphology of the self-assembled ZSCs at different temperatures under a fluorescence microscope. Figure 11 (a) is a morphological diagram of the self-assembled body formed by ZSC at 40℃; Figure 11 (b) is a morphological diagram of the self-assembled body formed by ZSC at 4℃.

[0040] Figure 12 Fluorescence spectra of XSC, FSC, and ZSC before and after assembly: Figure 12 (a) Fluorescence spectra of XSC before and after assembly; Figure 12 (b) Fluorescence spectra of the FSC before and after assembly; Figure 12 (c) shows the fluorescence spectra of ZSC before and after assembly.

[0041] Figure 13 Fluorescence microscopy images of MCF-7 cells stained with self-assemblies in XSC, FSC, and ZSC: Figure 13 (a) A fluorescence microscope image of cells stained with XSC self-assemblies under white light; Figure 13 (b) A fluorescence microscope image of cells stained with XSC self-assemblies under fluorescence conditions; Figure 13 (c) A fluorescence microscope image of cells stained with FSC self-assemblies under white light; Figure 13 (d) is a fluorescence microscope image of cells stained with FSC self-assemblies under fluorescence conditions; Figure 13 (e) is a fluorescence microscope image of cells stained with ZSC self-assemblies under white light conditions;

[0042] Figure 13 (f) is a fluorescence microscope image of cells stained with ZSC self-assemblies under fluorescence conditions.

[0043] Figure 14 This is a comparison of the toxicity of self-assembled XSC, FSC, and ZSC cells to U87 (human glioma cell line) cells. Detailed Implementation

[0044] The present invention will now be described with reference to specific embodiments, but these are not intended to limit the scope of the invention.

[0045] Example 1

[0046] 1. Take a 500mL round-bottom flask, add 200mL of diethyl ether, 32mL of deionized water and 10g of glycine methyl ester hydrochloride, then add 11g of anhydrous potassium carbonate, stir for 10min to mix well, then add 9.8g of ethyl acetylimine hydrochloride, stir for 10min to react, separate the resulting mixture through a separatory funnel, add 200mL of diethyl ether to the aqueous phase again, continue to react for 10min, combine the organic phases, add anhydrous magnesium sulfate to the organic phase to dry, filter, and evaporate the filtrate to dryness to obtain a pale yellow liquid;

[0047] 2. Take a 50 mL dry eggplant-shaped flask, add 4-dimethylaminobenzaldehyde (1.49 g, 10 mmol) and n-hexylamine (1.45 mL, 11 mol), add 20 mL of anhydrous ethanol as solvent, stir for 12 h under nitrogen atmosphere and room temperature, and evaporate the resulting mixed product to dryness to obtain a solid product.

[0048] 3. Add a pale yellow liquid (1.25 g, 11 mmol) to the obtained solid product, add 20 mL of anhydrous ethanol as solvent, stir the reaction at room temperature for 12 h, after the reaction is completed, evaporate the solvent, and purify the obtained residual solid by chromatography column to obtain a brownish-yellow powder (yield 65%). Characterization shows that the obtained brownish-yellow powder is (Z)-5-(4-dimethylaminophenylmethylene)-2-n-hexyl-3-methylimidazolinone, labeled as XSC.

[0049] The XSC characterization of the brownish-yellow powder is as follows:

[0050] HRMS: calculated for [M+H]:314.22, measured:314.2224;

[0051] 1 H NMR (400MHz, DMSO, 298K): δ (ppm): 8.05 (d, J = 9.0Hz, 2H), 6.85 (s, 1H), 6.74 (d, J = 9.1Hz, 2H), 3.53 (t,J=7.3Hz,2H),3.01(s,6H),2.33(s,3H),1.68–1.43(m,2H),1.26(s,6H),0.86(t,J=6.8Hz,3H);

[0052] 13 CNMR(101MHz,DMSO,298K):δ(ppm):170.13,160.43,151.73,134.90,134.26 ,126.94,122.05,112.10,40.08,31.30,29.14,26.33,22.48,15.73,14.37.

[0053] Example 2

[0054] 1. Take a 500mL round-bottom flask, add 200mL of diethyl ether, 32mL of deionized water and 10g of glycine methyl ester hydrochloride, then add 11g of anhydrous potassium carbonate, stir for 10min to mix well, then add 9.8g of ethyl acetylimine hydrochloride, stir for 10min to react, separate the resulting mixture through a separatory funnel, add 200mL of diethyl ether to the aqueous phase again, continue to react for 10min, combine the organic phases, add anhydrous magnesium sulfate to the organic phase to dry, filter, and evaporate the filtrate to dryness to obtain a pale yellow liquid;

[0055] 2. Take a 50 mL dry eggplant-shaped flask, add 3-dimethylaminobenzaldehyde (1.49 g, 10 mmol) and n-hexylamine (1.45 mL, 11 mol), add 20 mL of anhydrous ethanol as solvent, stir for 12 h under nitrogen atmosphere and room temperature, and evaporate the resulting mixed product to obtain a solid product.

[0056] 3. Add a pale yellow liquid (1.25 g, 11 mmol) to the obtained solid product, add 20 mL of anhydrous ethanol as solvent, stir the reaction at room temperature for 12 h, after the reaction is completed, evaporate the solvent, and purify the obtained residual solid by chromatography column to obtain a pale yellow powder (yield 69%). Characterization shows that the pale yellow powder is (Z)-5-(3-dimethylaminophenylmethylene)-2-n-hexyl-3-methylimidazolinone, labeled as FSC.

[0057] The pale yellow powder FSC is characterized as follows:

[0058] HRMS: calculated for[M+H]:314.22, measured:314.2226;

[0059] 1 H NMR (400MHz, DMSO, 298K): δ (ppm): 7.58 (t, J = 5.1Hz, 2H), 7.24 (t, J = 7.9Hz, 1H), 6.91 (s, 1H), 6.78 (dd, J = 8.2, 2.1Hz, 1H),3.54(t,J=7.3Hz,2H),2.92(s,6H),2.37(s,3H),1.62–1.44(m,2H),1.27(d,J=3.8Hz,6H),0.86(t,J=6.8Hz,3H);

[0060] 13CNMR(101MHz,DMSO,298K):δ(ppm):170.46,163.91,150.94,138.76,134.94,129.53,12 6.60,120.61,116.35,114.74,40.53,40.31,31.28,29.01,26.31,22.47,15.98,14.36.

[0061] Example 3

[0062] 1. Take a 500mL round-bottom flask, add 200mL of diethyl ether, 32mL of deionized water and 10g of glycine methyl ester hydrochloride, then add 11g of anhydrous potassium carbonate, stir for 10min to mix well, then add 9.8g of ethyl acetylimine hydrochloride, stir for 10min to react, separate the resulting mixture through a separatory funnel, add 200mL of diethyl ether to the aqueous phase again, continue to react for 10min, combine the organic phases, add anhydrous magnesium sulfate to the organic phase to dry, filter, and evaporate the filtrate to dryness to obtain a pale yellow liquid;

[0063] 2. Take a 50 mL dry eggplant-shaped flask, add 2-dimethylaminobenzaldehyde (1.49 g, 10 mmol) and n-hexylamine (1.45 mL, 11 mol), add 20 mL of anhydrous ethanol as solvent, stir for 12 h under nitrogen atmosphere and room temperature, and evaporate the resulting mixed product to dryness to obtain a solid product.

[0064] 3. Add a pale yellow liquid (1.25 g, 11 mmol) to the obtained solid product, add 20 mL of anhydrous ethanol as solvent, stir the reaction at room temperature for 12 h, after the reaction is completed, evaporate the solvent, and purify the obtained residual solid by chromatography column to obtain a yellow powder (yield 62%). Characterization showed that the yellow powder was (Z)-5-(2-dimethylaminophenylmethylene)-2-hexyl-3-methylimidazolinone, labeled ZSC.

[0065] The characterization of yellow ZSC powder is as follows:

[0066] HRMS: calculated for[M+H]:314.22, measured:314.2228;

[0067] 1H NMR (400MHz, DMSO, 298K): δ (ppm): 8.55 (dd, J=7.9, 1.5Hz, 1H), 7.39–7.30 (m, 1H), 7.25 (s, 1H), 7.18–7.01 (m, 2H ),3.63–3.48(m,2H),2.71(s,6H),2.37(s,3H),1.62–1.47(m,2H),1.27(d,J=8.3Hz,6H),0.86(t,J=6.7Hz,3H);

[0068] 13 CNMR(101MHz,DMSO,298K):δ(ppm):170.60,163.78,155.14,138.14,133.12,131.19,12 7.21,122.55,121.99,118.92,45.54,40.36,31.26,29.03,26.33,22.46,15.84,14.35.

[0069] Example 4

[0070] At room temperature, accurately weigh 1 mg of XSC prepared in Example 1 and add it to 200 μL of methanol. Stir to dissolve and, after complete dissolution, add 800 μL of ultrapure water. Mix using a shaker and allow to stand for 6 hours to obtain the self-assembled XSC.

[0071] The obtained XSC self-assembled structures were transferred to a glass plate, and their fluorescence properties and morphology were studied using a fluorescence microscope. The resulting morphology images are shown below. Figure 3 As shown in (a), from Figure 3 (a) It can be seen that XSC can form a self-assembled rod-shaped structure with uniform size in the methanol and water system, and has bright green fluorescence.

[0072] The obtained XSC self-assemblies were freeze-dried, and their morphology was examined using ultra-high resolution cold field scanning electron microscopy. The overall morphology image is shown below. Figure 3 As shown in (b), from Figure 3 (b) It can be seen that the self-assembled XSC is a flat rod-shaped structure with a thickness of approximately 5 μm. The cross-section obtained by cutting the XSC self-assembled with a knife is shown in the figure below. Figure 3 As shown in (c), the flat rod-shaped structure formed by the XSC assembly is a hollow structure, that is, there is a nanoscale hollow in the middle of the flat rod-shaped structure.

[0073] Experiment 1: Study on the Self-Assembly Process of XSC

[0074] Experimental methods:

[0075] At room temperature, accurately weigh 1 mg of XSC prepared in Example 1 and add it to 200 μL of methanol. Stir to dissolve and after complete dissolution, add 800 μL of ultrapure water, mix with a shaker, and let stand. At 0 min, 30 min, 2 h, and 6 h, transfer the solution to a glass plate and study its fluorescence properties and morphology using a fluorescence microscope. At the same time, at 0 min, 30 min, and 6 h, transfer the solution to a silicon wafer, freeze-dry it, and then test its morphology using an ultra-high resolution cold field scanning electron microscope.

[0076] Experimental results:

[0077] Morphological images of self-assembled structures obtained by XSC at different assembly time points under a fluorescence microscope are shown below. Figure 4 (a)- Figure 4 As shown in (d), the morphology of the self-assembled structures obtained by XSC at different assembly time points under a high-resolution cold field scanning electron microscope is as follows. Figure 4 (e)- Figure 4 As shown in (g), by Figure 4 It can be seen that when the droplet has been left to stand for 0 minutes, self-assembly has not yet begun, and only the state of the droplet can be observed. Figure 4 (a) and Figure 4 (e) shows that when XSC is left to stand in a system of methanol and water for 30 minutes, it forms a fine rod-like structure. Figure 4 (b) Display and Figure 4 (f) As shown, with a resting time of 2 hours, the self-assembled structures of XSC gradually aggregate from small rod-shaped structures to form larger rod-shaped structures. Figure 4 (c) shows that when XSC is left to stand in a system of methanol and water for 6 hours, the resulting self-assembled material is a rod-shaped material with bright green fluorescence. Figure 4 (d) and Figure 4 (g)).

[0078] Example 5

[0079] At room temperature, accurately weigh 1 mg of FSC prepared in Example 2 and add it to 200 μL of methanol. Stir to dissolve and after complete dissolution, add 800 μL of ultrapure water, mix with a shaker, and let stand for 6 hours to obtain the self-assembled FSC.

[0080] The obtained FSC self-assembled structures were transferred to a glass plate, and their fluorescence properties and morphology were studied using a fluorescence microscope. The resulting morphology images are shown below. Figure 5 As shown in (a), from Figure 5(a) It can be seen that FSC can form a uniformly sized rod-shaped self-assembled structure in the methanol and water system, and has bright green fluorescence. Its width is narrower than that of the self-assembled structure of XSC.

[0081] The obtained FSC self-assembled parts were freeze-dried, and their morphology was examined using ultra-high resolution cold field scanning electron microscopy. The overall morphology image is shown below. Figure 5 As shown in (b), from Figure 5 (b) It can be seen that the self-assembled FSC is a rod-shaped structure with a thickness of approximately 5-20 μm. The cross-section of the FSC self-assembled was observed after cutting it with a knife; the resulting cross-sectional diagram is shown below. Figure 5 As shown in (c) Figure 5 (c) indicates that the assembly of FSC is a hollow cuboid tubular structure with a wall thickness of about 2 μm and a cross-section of the hollow cavity that is 14 μm long and 8 μm wide.

[0082] Experiment 2: Study on the self-assembly process of FSC

[0083] Experimental methods:

[0084] At room temperature, accurately weigh 1 mg of FSC prepared in Example 2 and add it to 200 μL of methanol. Stir to dissolve and after complete dissolution, add 800 μL of ultrapure water and mix with a shaker. Transfer the solution to a glass plate after standing for 0 min, 30 min, 2 h, and 6 h and study its fluorescence properties and morphology using a fluorescence microscope. At the same time, transfer the solution to a silicon wafer after standing for 0 min, 30 min, and 6 h, freeze-dry it, and test its morphology using an ultra-high resolution cold field scanning electron microscope.

[0085] Experimental results:

[0086] Morphological images of self-assembled structures obtained by FSC at different assembly time points under a fluorescence microscope are shown below. Figure 6 (a)- Figure 6 As shown in (d), the morphology of the self-assembled structures obtained by FSC at different assembly time points is shown in the ultra-high resolution cold field scanning electron microscope images. Figure 6 (e)- Figure 6 As shown in (g), by Figure 6 It can be seen that the self-assembly process of FSC is similar to that of XSC. When the mixture is left to stand for 0 minutes (when FSC is just mixed), self-assembly has not yet started, and only the droplet state can be observed. Figure 6 (a) and Figure 6 (e) As shown, after standing for 30 minutes, small rod-shaped structures begin to appear in the self-assembly process. Figure 6 (b) Display and Figure 6(f) As shown, with a settling time of 2 hours, the tiny rod-like structures continue to accumulate into larger rod-like structures. Figure 6 (c) shows that when left to stand for 6 hours, a rod-shaped structure with serrated edges and green fluorescence is eventually formed. Figure 6 (d) and Figure 6 (g)).

[0087] Example 6

[0088] At room temperature, accurately weigh 1 mg of ZSC prepared in Example 3 and add it to 200 μL of methanol. Stir to dissolve and after complete dissolution, add 800 μL of ultrapure water, mix with a shaker, and let stand for 6 hours to obtain the self-assembled ZSC.

[0089] The obtained ZSC self-assembled structures were transferred to a glass plate, and their fluorescence properties and morphology were studied using a fluorescence microscope. The resulting morphology images are shown below. Figure 7 As shown in (a), from Figure 7 (a) It can be seen that ZSC can form a self-assembled, uniformly sized sheet-like structure in a methanol and water system, and has green fluorescence. Its width is wider than that of the self-assembled structures of XSC and FSC.

[0090] The obtained FSC self-assembled parts were freeze-dried, and their morphology was examined using ultra-high resolution cold field scanning electron microscopy. The overall morphology image is shown below. Figure 7 As shown in (b), from Figure 7 (b) It can be seen that the self-assembled structure of ZSC is a sheet-like structure with a width of approximately 100 μm. Observing the cross-section, the obtained cross-sectional diagram is as follows. Figure 7 As shown in (c), the self-assembled ZSC is a sheet-like solid structure.

[0091] Experiment 3: Study on the Self-Assembly Process of ZSC

[0092] Experimental methods:

[0093] At room temperature, accurately weigh 1 mg of ZSC prepared in Example 3 and add it to 200 μL of methanol. Stir to dissolve and after complete dissolution, add 800 μL of ultrapure water and mix with a shaker. Transfer the solution to a glass plate after standing for 0 min, 30 min, 2 h, and 6 h and study its fluorescence properties and morphology using a fluorescence microscope. At the same time, transfer the solution to a silicon wafer after standing for 0 min, 30 min, and 6 h, freeze-dry it, and test its morphology using an ultra-high resolution cold field scanning electron microscope.

[0094] Experimental results:

[0095] Morphological images of self-assembled ZSCs obtained at different assembly time points under a fluorescence microscope are shown below. Figure 8 (a)- Figure 8 As shown in (d), the morphology of the self-assembled structures obtained by ZSC at different assembly time points under a high-resolution cold field scanning electron microscope is as follows. Figure 8 (e)- Figure 8 As shown in (g), by Figure 8 It can be seen that the self-assembly process of ZSC is similar to that of XSC. When the mixture is left to stand for 0 minutes (when ZSC is just mixed), self-assembly has not yet started, and only the droplet state can be observed. Figure 8 (a) and Figure 8 (e) shows that after standing for 30 minutes, fine sheet-like structures begin to appear during self-assembly. Figure 8 (b) Display and Figure 8 (f) As shown, after 2 hours of settling, the tiny sheet-like structures continue to accumulate into coarser and wider sheet-like structures. Figure 8 (c) As shown), when left to stand for 6 hours, it eventually forms lamellae with green fluorescence. Figure 8 (d) and Figure 8 (g)).

[0096] Experiment 4: The Effect of High Temperature on the Self-Assembly of XSC, FSC, and ZSC

[0097] Experimental methods:

[0098] Accurately weigh 1 mg of XSC, FSC, and ZSC and add them to 200 μL of methanol. Stir to dissolve. After complete dissolution, add 800 μL of ultrapure water at 40 °C, mix with a shaker, and let stand in a water bath at 40 °C for 6 hours to obtain self-assembled XSC, FSC, and ZSC. Transfer the obtained self-assembled XSC, FSC, and ZSC to a glass plate and study their fluorescence properties and morphology using a fluorescence microscope.

[0099] Experimental results:

[0100] The obtained morphological image is as follows Figure 9 As shown in (a), 10(a), and 11(a), from Figure 9 As can be seen from (a), 10(a) and 11(a), at 40℃, XSC, FSC and ZSC can all form rod-shaped structures with green fluorescence through self-assembly in a methanol-water mixture, indicating that slightly higher temperatures have little effect on the self-assembly of XSC, FSC and ZSC.

[0101] Experiment 5: Effect of Low Temperature on Self-Assembly of XSC, FSC, and ZSC

[0102] Experimental methods:

[0103] Accurately weigh 1 mg of XSC, FSC, and ZSC and add them to 200 μL of methanol. Stir to dissolve. After complete dissolution, add 800 μL of ultrapure water at 4 °C, mix with a shaker, and let stand in a refrigerator at 4 °C for 6 hours to obtain self-assembled XSC, FSC, and ZSC. Transfer the obtained self-assembled XSC, FSC, and ZSC to a glass plate and study their fluorescence properties and morphology using a fluorescence microscope.

[0104] Experimental results:

[0105] The obtained morphological image is as follows Figure 9 As shown in (b), 10(b), and 11(b), from Figure 9 As shown in (b), XSC forms an irregular sheet-like structure at 4℃ and cannot assemble into a uniform rod-like structure. From 10(b), it can be seen that FSC can also form a uniform rod-like structure at 4℃, indicating that temperature has little effect on its self-assembly. Figure 11 As can be seen in (b), the structure formed by ZSC at low temperature is irregular, indicating that low temperature has a certain impact on its assembly.

[0106] The above comparison reveals that FSC can self-assemble to form a hollow rod-like structure, and temperature has little effect on it, indicating that FSC has superior performance.

[0107] Experiment 6: Fluorescence properties of self-assembled XSC, FSC, and ZSC

[0108] Experimental methods:

[0109] Accurately weigh 1 mg of XSC, FSC, and ZSC and add them to 200 μL of methanol. Stir to dissolve. After complete dissolution, add 800 μL of ultrapure water at 4℃ and mix with a shaker. After thorough mixing, take samples to test their fluorescence emission spectra. Let the remaining mixture stand for 6 hours to allow them to self-assemble and then test their fluorescence emission spectra. The excitation wavelength of the self-assembled XSC was 455 nm and the slit width was 2.5-2.5. The excitation wavelength of the self-assembled FSC was 473 nm and the slit width was 2.5-2.5. The excitation wavelength of the self-assembled ZSC was 430 nm and the slit width was 5-2.5.

[0110] Experimental results:

[0111] The fluorescence spectra of XSC, FSC, and ZSC before and after assembly are shown in the figure. Figure 12 As shown, from Figure 12As shown in (a), compared with before XSC assembly, the fluorescence intensity of XSC after assembly is increased by about 1000 times, and the optimal emission wavelength of fluorescence changes from 505 nm before assembly to 496 nm; Figure 12 As can be seen in (b), compared with before FSC assembly, the fluorescence intensity of FSC after assembly is increased by about 36 times, and the optimal emission wavelength of fluorescence changes from 584nm before assembly to 515nm; Figure 12 As shown in (c), compared with ZSC before assembly, the fluorescence intensity of ZSC after assembly is increased by about 2.3 times, and the optimal emission wavelength of fluorescence changes from 550 nm before assembly to 508 nm. This indicates that, compared with before assembly, the optimal emission wavelength of the self-assembled XSC, FSC, and ZSC undergoes a blue shift, and the fluorescence intensity is enhanced in all cases.

[0112] Experiment 7: Cell imaging experiments of self-assembled XSC, FSC, and ZSC cells

[0113] Experimental methods:

[0114] MCF-7 cells (human breast cancer cells) were injected at a rate of 2.5 × 10⁻⁶. 4 Cells were passaged per well in 24-well plates using DMEM culture medium: 10% fetal bovine serum (FBS), 1% penicillin and streptomycin, and 5% carbon dioxide. The plates were incubated at 37°C for 24 hours. Then, 20 μL of self-assembled XSC, FSC, and ZSC cells were added, with a final concentration of 0.2 mM for each cell. Cells were incubated for 4 hours, and cell imaging was observed using a fluorescence microscope. Results:

[0115] Fluorescence microscopy images of MCF-7 cells stained with self-assemblies from XSC, FSC, and ZSC are shown below. Figure 13 As shown, from Figure 13 It can be seen that the self-assembled cells of XSC, FSC, and ZSC all have the ability to stain cells, and the main staining color is green.

[0116] Experiment 8: Cytotoxicity assay of self-assembled XSC, FSC, and ZSC cells

[0117] Experimental methods:

[0118] U87 (human glioma cell line) cells were passaged at 30,000 cells / well in 24-well plates using DMEM culture medium: 10% fetal bovine serum (FBS), 0.1% penicillin and streptomycin, and 5% carbon dioxide. The cells were cultured at 37°C for 24 hours. Then, dimethyl sulfoxide solutions of XSC, FSC, and ZSC were added, with the following concentrations: 1 μM, 10 μM, and 100 μM (final concentrations). For each concentration group of each self-assembly, the following treatment was performed: after adding the self-assembly and incubating for 24 hours, 50 μL of a CCK-8 kit was added to the self-assembly-treated U87 cells, and the cells were incubated at 37°C for another 2 hours. The absorbance at 450 nm was measured using a multi-label microplate reader.

[0119] Laboratory results:

[0120] The comparison of the toxicity of XSC, FSC, and ZSC to U87 cells is shown in the figure below. Figure 14 As shown, by Figure 14 It can be seen that XSC, FSC, and ZSC have very low cytotoxicity to U87 cells; however, as their concentration increases, the self-assembly process produces a weaker cytotoxicity to U87 cells.

Claims

1. An HBI derivative, characterized in that... Its general structural formula is as follows: ； Wherein, R1 is dimethylamino and R2 is n-hexyl.

2. A method for preparing the HBI derivative according to claim 1, characterized in that... Includes the following steps: (2.1) Under nitrogen protection, compound (1) undergoes a condensation reaction with n-hexylamine to produce compound (2), the reaction formula of which is as follows: ， Wherein, R1 is dimethylamino and R2 is n-hexyl; (2.2) Under alkaline conditions, glycine methyl ester hydrochloride reacts with ethyl acetylimine hydrochloride in a substitution reaction to produce compound (3), the reaction formula of which is as follows: ； (2.3) Compounds of formula (2) and (3) undergo a cycloaddition reaction at room temperature to generate HBI derivatives, and the reaction formulas are as follows: 。 3. The method for preparing the HBI derivative according to claim 2, characterized in that: The alkali mentioned is potassium carbonate.

4. A self-assembled HBI derivative, characterized in that... Prepared by the following method: The HBI derivative of claim 1 is added to methanol and dissolved completely. Then, it is added to ultrapure water and mixed evenly to obtain a mixture. The mixture is allowed to stand to obtain a self-assembled HBI derivative, which is in the form of regular sheets.

5. The self-assembled HBI derivative according to claim 4, characterized in that: In the mixture, the concentration of the HBI derivative is 0.2-10 mg / mL.

6. The self-assembled HBI derivative according to claim 4, characterized in that: The volume ratio of methanol to ultrapure water is 0.05-0.95:

1.

7. The self-assembled HBI derivative according to claim 4, characterized in that: The settling time is 0.1-72 hours.

8. The application of a self-assembled HBI derivative of claim 4 in the preparation of a cell imaging agent.

Images