Gold nanoparticle probe modified with alkaline phosphatase fluorescent molecule and preparation method

Abstract

The invention relates to a gold nanoparticle probe modified with a fluorescent molecule responsive to alkaline phosphatase and a preparation method thereof, belonging to the field of probe technology. The probe is hCy‑ALP@AuNPs, and the composite structure is mainly composed of gold nanoparticles of about 20 nm, and the surface is modified with a fluorescent molecule responsive to alkaline phosphatase through a bioorthogonal reaction of thiol-polyethylene glycol-diphenylcyclooctyne. The fluorescent molecule consists of a recognition group of alkaline phosphatase and a near-infrared fluorescent dye IR700. hCy‑ALP@AuNPs can achieve the effect of fluorescence onset under the hydrolysis of the tumor marker alkaline phosphatase. Compared with a single fluorescent probe, the grafted gold nanoparticles of the present invention have a high retention (EPR) effect in solid tumors, which can enhance their accumulation effect in the tumor site, increase the imaging window time, and achieve the purpose of long-retention imaging. The present invention has important application value in the fields of drug delivery carriers, bioimaging, medicine, etc.

Description

Technical Field

[0001] The present invention is a diagnostic fluorescent molecule-gold nanoparticle composite probe, which relates to a preparation method of a gold nanoparticle probe modified with a fluorescent molecule that responds to alkaline phosphatase. The ability of the probe to respond to alkaline phosphatase and extend the tumor imaging window is verified through near-infrared fluorescence imaging at the cellular and animal levels. Background Art

[0002] Gold nanoparticles, also known as colloidal gold, are small nanoparticles formed by the gradual aggregation of gold atoms in chloroauric acid (HAuCl4) under the reduction of reducing agents such as white phosphorus, ascorbic acid, sodium citrate, and tannic acid. Gold nanoparticles are typically a colloidal suspension of gold, with particle sizes ranging from 1 to 100 nm and colors ranging from orange-yellow to purple-red. The binding of reducing ligands to gold is often associated with Au-S bonding. Gold forms a stable Au-S bond with sulfhydryl (-SH) groups, creating an extremely stable chemical bond and resulting in a stable colloid. Gold nanoparticles are typically negatively charged in aqueous solutions and alkaline environments, allowing them to form strong bonds with positively charged groups (such as proteins and peptides) without affecting the biological properties of the proteins. In addition to proteins, gold nanoparticles can also bind to many other biomacromolecules, such as SPA, PHA, and ConA. Some physical properties of gold nanoparticles, such as high electron density, particle size, shape and color reaction, combined with the immune and biological properties of the conjugates, make gold nanoparticles widely used in fields such as immunology, histology, pathology and cell biology.

[0003] Alkaline phosphatase, abbreviated as ALP, is a class of phosphohydrolases widely distributed in the liver, kidney, intestine, and bone. This enzyme catalyzes the removal of 5'-phosphate groups from nucleic acid molecules, converting the 5'-P termini of DNA or RNA fragments into 5'-OH termini. It also catalyzes the conversion of organic compounds bearing phosphate groups. Overexpression of ALP is often associated with major diseases such as cirrhosis, hepatocellular carcinoma, bile duct diseases, viral hepatitis, alcoholic hepatitis, drug-induced liver damage, and other malignancies. Therefore, probes designed based on ALP are widely used in medical diagnostics. ALP levels are high in adolescents, reaching as high as 0.75 U / mL. In normal adults, ALP levels range from 0.04 to 0.15 U / mL. Levels significantly higher than this indicate abnormal conditions. Therefore, studying ALP levels can be used to diagnose related diseases, and developing an effective method for measuring ALP levels in vivo holds great medical promise.

[0004] Small molecule fluorescent probes are a class of molecular devices that rely on excitation light to provide energy, transition from a ground state to an excited state, and release energy to emit fluorescence. They are fluorescent molecules whose physical and chemical properties (excitation and emission wavelengths, UV spectra, lifetime, etc.) can sensitively change with the properties of their environment (polarity, refractive index, viscosity, etc.). Near-infrared fluorescent probes are the most widely used in bioimaging. Commonly used near-infrared fluorophores include rhodamines, cyanines, BODIPYs, and porphyrins. These fluorophores fluoresce in the near-infrared (NIR) region (700-1700 nm). Near-infrared light is well-suited to penetrating deep tissues, resulting in minimal light loss and background interference in biological samples. This is a highly advantageous characteristic of near-infrared fluorescent probes for in vivo imaging.

[0005] In order to solve the problem of how to connect two modules (fluorophore and reaction site, drug molecules, drug molecules and DNA), scientist Sharpless discovered a bioorthogonal Click reaction. Its essence is to modify one end of each of the two molecules with a terminal that can react and connect. The use of an appropriate catalyst can connect the terminals to achieve the effect of conjugation between the two molecules. The reaction is fast, effective, and has high utilization rate. Its classification basically includes azide-alkyne reaction, ene-thiol reaction, and azide-amino reaction. The biggest advantage of this reaction is modularity, high yield, environmental pollution-free, simple reaction conditions, and it is suitable for conjugation reactions of various biological proteins, drugs, DNA, etc. The emergence of the Click reaction has made the connection between the two modules simple and easy, and also made the application of bioorthogonal reactions in life sciences show broad prospects.

[0006] The present invention relates to a preparation method for a small molecule synthesized by a biological click reaction between a hemicyanine dye fluorescent molecule IR700 and thiol-polyethylene glycol-diphenylcyclooctyne (DBCO-PEG-SH), and then modified onto gold nanoparticles via an Au-S bond. First, auric acid is reduced to conjugate citrate to the surface of Au atoms, generating citrate-protected gold nanoparticles. The hemicyanine dye IR700 is then linked to an ALP enzyme recognition site, enabling the fluorescent molecule to recognize the overexpressed ALP enzyme in tumors and accumulate at the tumor site. Next, the fluorescent molecule is stably linked to the DBCO-PEG-SH via a biological click reaction to synthesize a surface ligand for the probe. Finally, a ligand exchange method is used to replace the citrate binding to the gold particles, leveraging the strong affinity of the thiol groups on the fluorescent molecule, and the modification is completed. Due to the high retention (EPR) effect of the grafted gold nanoparticles in solid tumors, their accumulation at the tumor site can be enhanced, increasing the imaging window time and achieving the purpose of long-retention imaging. The present invention has important application value in the fields of drug delivery carriers, biological imaging, medicine, etc. Summary of the Invention

[0007] The gold nanoparticle probe modified with alkaline phosphatase fluorescent molecules of the present invention is hCy-ALP@AuNPs, and the structure of hCy-ALP@AuNPs is as follows:

[0008]

[0009] The preparation method of hCy-ALP@AuNPs specifically includes the following steps:

[0010] (1) Preparation of compound 5, comprising the following steps: (a) dissolving 2,3,3-trimethylindole (compound 1), potassium iodide and 1-azido-4-chloro-butane in anhydrous acetonitrile under nitrogen protection, and heating the reaction solution to 90°C and reflux for 9 hours, and recrystallizing from petroleum ether to obtain purple compound 2; (b) dissolving compound 2, 2-chloro-3-(hydroxymethylene)cyclohex-1-enecarboxaldehyde and sodium methoxide in anhydrous acetic anhydride, and heating the reaction solution to 130°C and reflux for 3 hours, and recrystallizing from petroleum ether to obtain green compound 3; (c) ) Compound 3, m-diphenol, and triethylamine were dissolved in anhydrous acetonitrile, and the reaction solution was heated to 50° C. for 6 hours, and purified by column chromatography (methanol: dichloromethane = 1:50-1:10) to obtain a blue-green compound 4; (d) Compound 4 and phosphorus oxychloride were dissolved in a certain volume of anhydrous pyridine, and the reaction solution was reacted at room temperature, such as for 12 hours, and then the same volume of ice water was added and reacted at room temperature, such as for 12 hours. After the reaction, the mixture was extracted with ethyl acetate and water, the organic phase was removed, and the aqueous phase was retained. After lyophilization, the mixture was separated by high performance liquid chromatography to obtain a blue compound 5.

[0011]

[0012] (2) The preparation method of compound 6 comprises the following steps: dissolving compound 5 in dimethyl sulfoxide, adding the mixture together with mercapto-polyethylene glycol-diphenylcyclooctyne to at least 9 volumes of water, and performing a click reaction in water for 1-3 hours to obtain compound 6.

[0013]

[0014] (3) A method for preparing gold nanoparticles, comprising the following steps: according to the preparation method in the literature, chloroauric acid is added to a round-bottom flask that has been soaked in aqua regia in advance, heated to 95°C, and a large amount of sodium citrate is added dropwise while maintaining high-speed stirring. The reaction is carried out for 35 minutes, and after the gold colloid is formed, gold particles protected by sodium citrate are obtained, which are purple-red in color and have a particle size of about 20 nm as observed under a transmission electron microscope.

[0015]

[0016] (4) The preparation method of hCy-ALP@AuNPs comprises the following steps: ligand exchange of gold particles protected by sodium citrate with compound 6, slowly dripping the gold particles into the solution of step (2), stirring at room temperature for 12 hours, and then dialysis and purification for 12 hours to obtain the fluorescent molecule-modified gold nanoparticles hCy-ALP@AuNPs solution.

[0017]

[0018] In step (1), the molar concentration ratio of 2,3,3-trimethylindole, potassium iodide and 1-azido-4-chloro-butane in (a) is 1:2:1, the reaction temperature is 90° C., and the reaction time is 9 hours.

[0019] The molar concentration ratio of the compound 2 described in (b) in step (1) to 2-chloro-3-(hydroxymethylene)cyclohex-1-enecarbaldehyde is 1:0.5, the reaction temperature is 130° C., and the reaction time is 3 hours.

[0020] In step (1), the molar concentration ratio of compound 3, m-diphenol, and triethylamine is 1:3:2.5, the reaction temperature is 50°C, and the reaction time is 6 hours. In column chromatography purification, the volume ratio of the solvent is methanol:dichloromethane = 1:50-1:10.

[0021] In step (1), the molar concentration ratio of compound 4 described in (d) to phosphorus oxychloride is 1:1, the reaction temperature is room temperature, and the reaction time is 12 hours.

[0022] The molar concentration ratio of compound 5 and mercapto-polyethylene glycol-diphenylcyclooctyne described in step (2) is 1:1, the reaction temperature is room temperature, and the reaction time is 1-3 hours.

[0023] The molar concentration ratio of compound 6 and gold nanoparticles in step (4) is 1:1, the reaction temperature is room temperature, the reaction time is 12 hours, and the dialysis purification time is 12 hours.

[0024] The gold nanoparticles hCy-ALP@AuNPs obtained by the present invention are used for near-infrared fluorescence imaging of tumor sites.

[0025] Advantages of the present invention: The gold nanoparticle fluorescent probe of the present invention can effectively prolong the retention time of the probe in tumor cells through the EPR effect of the gold nanoparticles.

[0026] The fluorescent probe of the present invention can quickly and sensitively identify and respond to ALP, and exhibits good imaging effects at both the cellular level and the in vivo level. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1This is the mass spectrum of compound 5.

[0028] Figure 2 is the H NMR spectrum of compound 5.

[0029] Figure 3 is the C NMR spectrum of compound 5.

[0030] Figure 4 The state and particle size of the probe hCy-ALP@AuNPs under TEM.

[0031] Figure 5 (a) is the UV absorption spectrum of the probe hCy-ALP@AuNPs incubated with ALP, (b) is the fluorescence spectrum of the probe hCy-ALP@AuNPs incubated with different concentrations of ALP, (c) is the linear relationship between the fluorescence intensity of the probe hCy-ALP@AuNPs and the incubation with different concentrations of ALP, and (d) is the linear relationship between the fluorescence intensity of the probe hCy-ALP@AuNPs and the change with time after ALP response.

[0032] Figure 6 The probe hCy-ALP@AuNPs specifically detects ALP.

[0033] Figure 7 Detection and imaging of endogenous ALP in HeLa cells using the probe hCy-ALP@AuNPs. Figure 8 Imaging of ALP detection in HeLa tumor-bearing mice using the probe hCy-ALP@AuNPs. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below with reference to specific examples, but the present invention is not limited to the following examples.

[0035] Example 1

[0036] Synthesis of gold nanoparticle probes modified with alkaline phosphatase fluorescent molecules Synthesis of compound 2

[0037] Under nitrogen protection, 2,3,3-trimethylindole, potassium iodide and 1-azido-4-chloro-butane were dissolved in anhydrous acetonitrile, and the reaction solution was heated to 90°C and refluxed for 9 hours, and recrystallized from petroleum ether to obtain purple compound 2.

[0038] Synthesis of compound 3

[0039] Compound 2 and 2-chloro-3-(hydroxymethylene)cyclohex-1-enecarboxaldehyde were dissolved in anhydrous acetic anhydride, and the reaction solution was heated to 130° C. and refluxed for 3 hours, and recrystallized from petroleum ether to obtain green compound 3.

[0040] Synthesis of compound 4

[0041] Compound 3, m-diphenol and triethylamine were dissolved in anhydrous acetonitrile, and the reaction solution was heated to 50° C. for 6 hours. The mixture was purified by column chromatography (methanol:dichloromethane=1:50-1:10) to obtain a blue-green compound 4.

[0042] Synthesis of compound 5

[0043] Compound 4 and phosphorus oxychloride were dissolved in a certain volume of anhydrous pyridine, and the reaction solution was reacted at room temperature for 12 hours. The same volume of ice water was then added and reacted at room temperature for 12 hours. After the reaction, ethyl acetate and water were extracted, the organic phase was removed, and the aqueous phase was retained. After freeze-drying, it was separated by high performance liquid chromatography to obtain blue compound 5.

[0044] Synthesis of compound 6

[0045] Compound 5 was dissolved in dimethyl sulfoxide, and added together with mercapto-polyethylene glycol-diphenylcyclooctyne into 9 times volume of water, mixed, and subjected to click reaction in water for 1-3 hours to obtain compound 6.

[0046] Synthesis of gold nanoparticles

[0047] According to the preparation method in the literature, add an appropriate concentration of chloroauric acid to a round-bottom flask soaked in aqua regia in advance, heat to 95°C, add a large amount of sodium citrate dropwise, and keep stirring at a high speed. React for 35 minutes until the gold colloid is formed and obtain gold particles protected by sodium citrate. They are purple-red in color and have a particle size of 20 nm under transmission electron microscopy.

[0048] Synthesis of hCy-ALP@AuNPs composite

[0049] The sodium citrate-protected gold particles were ligand-exchanged with compound 6, and the gold particles were slowly dripped into the reaction solution of compound 6 in step 6. The reaction was stirred at room temperature for 12 hours, and then dialyzed and purified for 12 hours to obtain the fluorescent molecule-modified gold nanoparticle solution.

[0050] Example 2

[0051] TEM and particle size statistics verify the particle size of probe hCy-ALP@AuNPs

[0052] The molecular size of the probe hCy-ALP@AuNPs was characterized by transmission electron microscopy (TEM) and particle size statistics. It can be clearly observed that the particle size of the probe is about 20 nm (see Figure 2). Figure 1 ).

[0053] Example 3

[0054] Measurement of absorption and fluorescence spectra of probe hCy-ALP@AuNPs

[0055] All spectra were measured in Tris-HCl buffer at 37°C. The probe hCy-ALP@AuNPs was diluted to 0.01 mg / mL with Tris-HCl buffer for in vitro measurement. The fluorescence spectrum was measured using an Edinburgh FS-5 fluorescence spectrophotometer, and the UV spectrum was measured using a Shimadzu UV-2600 UV-visible spectrophotometer. The UV spectrum scanning range was 400-800nm, the fluorescence spectrum scanning excitation was 670nm, and the emission range was 695-800nm. The UV absorption spectrum test showed an increase in absorption at 650nm in the UV absorption spectrum and the generation of a new peak at 700nm, proving that the probe structure changed through the reaction. At the same time, it can be found that after adding a gradient of 0-5U / mL of ALP enzyme, the fluorescence spectrum has an enhancement of nearly 10 times, which proves that the fluorescence light is turned on (see Appendix). Figure 2 ).

[0056] Example 4

[0057] Specific detection of ALP by probe hCy-ALP@AuNPs

[0058] All spectra were measured in Tris-HCl buffer at 37°C. The probe hCy-ALP@AuNPs was diluted to 0.01 mg / mL with Tris-HCl buffer for specific detection. The analytes included BSA, NaClO, FA, Gly, NaCl, MgCl2, CaCl2, H2O2, NaHS, Cys, GSH, vitamin C (1 mM each), alkaline phosphatase (ALP), β-galactosidase (β-Gal), leucine aminopeptidase (LAP) (5 U / mL each in distilled water) and Escherichia coli nitroreductase (NTR) (5 μg / mL in distilled water). The excitation wavelength was 670 nm and the emission wavelength was 710 nm. The experiments showed that the probe was only turned on in the presence of ALP, demonstrating good ALP selectivity (attached Figure 3 ).

[0059] Example 5

[0060] Detection of endogenous ALP in HeLa cells using probe hCy-ALP@AuNPs

[0061] HeLa cells were cultured in a glass-bottom confocal culture dish for 24 hours before the experiment. Fluorescence imaging of hCy-ALP@AuNPs (0.02 mg / mL) in HeLa cells was divided into three groups: blank group, probe group and probe + inhibitor (100 μM sodium orthovanadate) group. The cells in the probe + inhibitor group were incubated in culture medium containing 100 μM sodium orthovanadate for 4 hours in advance, and the other two groups did not require prior treatment. The cells were then incubated with hCy-ALP@AuNPs (0.02 mg / mL) probe under normal conditions (37°C, 5% CO2 and 21% O2) for 2 hours, and the fluorescence images of the cells were captured by a Nikon Ti-e microscope (λex = 635 nm, λem = 663-738 nm). Confocal experiments found that the probe group cells emitted red fluorescence, while the blank group and the probe + inhibitor did not emit red fluorescence. This proves that the probe has the ability to recognize endogenous ALP enzyme in cells (see Appendix). Figure 4 ).

[0062] Example 6

[0063] Detection of endogenous ALP in HeLa tumor-bearing mice using probe hCy-ALP@AuNPs All animal procedures were performed in accordance with animal ethics procedures. Female BALB / c mice (weighing approximately 18-20 g) were housed under standard conditions. A HeLa xenograft model was obtained by subcutaneously injecting HeLa cells into the lower limbs of mice for 2 weeks. When the tumors grew to an appropriate size, the mice were used for in vivo fluorescence imaging. During the in vivo fluorescence imaging experiment in mice, the mice were anesthetized with 2% isoflurane delivered via a nose cone. After the mice were intratumorally injected with probe hCy-ALP@AuNPs (0.05 mg / mL), they were placed in an IVIS system and imaged at different time points (0, 15, 30, 60, 90, 120, 180, and 240 min). The excitation wavelength of the IVIS system was 640 nm, while the emission wavelength was 680-700 nm. Imaging experiments have shown that the probe hCy-ALP@AuNPs is continuously turned on in tumors, and has a strong fluorescence imaging effect at 240 minutes, which proves the imaging ability of the probe in tumor tissues and has the effect of continuously accumulating and extending the imaging window (Appendix Figure 5 ).

Claims

1. A gold nanoparticle probe modified with a fluorescent molecule responsive to alkaline phosphatase, characterized in that: hCy-ALP@AuNPs, the structure of hCy-ALP@AuNPs is as follows:

2. The method for preparing the gold nanoparticle probe modified with a fluorescent molecule responsive to alkaline phosphatase according to claim 1, characterized in that: The following steps are involved: (1) Preparation of compound 5, comprising the following steps: (a) dissolving 2,3,3-trimethylindole (compound 1), potassium iodide and 1-azido-4-chloro-butane in anhydrous acetonitrile under nitrogen protection, and heating the reaction solution to reflux, and recrystallizing from petroleum ether to obtain purple compound 2; (b) dissolving compound 2, 2-chloro-3-(hydroxymethylene)cyclohex-1-enecarboxaldehyde and sodium methoxide in anhydrous acetic anhydride, and heating the reaction solution to reflux, and recrystallizing from petroleum ether to obtain green compound 3; (c) dissolving compound 3, m-diphenol and triethylamine in anhydrous acetonitrile, and heating the reaction solution to react, and purifying by column chromatography to obtain blue-green compound 4; (d) dissolving compound 4 and phosphorus oxychloride in anhydrous pyridine, and reacting the reaction solution at room temperature, then adding the same volume of ice water to react at room temperature, extracting with ethyl acetate and water after the reaction, removing the organic phase and retaining the aqueous phase, freeze-drying and separating by high performance liquid chromatography to obtain blue compound 5; (2) A method for preparing compound 6, comprising the following steps: dissolving compound 5 in dimethyl sulfoxide, adding the mixture together with thiol-polyethylene glycol-diphenylcyclooctyne to at least 9 volumes of water, and performing a click reaction in water to obtain compound 6; (3) A method for preparing gold nanoparticles, comprising the following steps: adding chloroauric acid to a round-bottom flask pre-soaked in aqua regia, heating, and dropwise adding a large amount of sodium citrate while maintaining high-speed stirring, reacting until gold colloid is formed, and obtaining gold particles protected by sodium citrate; (4) A method for preparing hCy-ALP@AuNPs, comprising the following steps: ligand exchange of gold particles protected by sodium citrate with compound 6, slowly dripping the gold particles into the solution of step (2), stirring at room temperature for reaction, and then dialysis purification to obtain the fluorescent molecule-modified gold nanoparticles hCy-ALP@AuNPs solution; 3. The method according to claim 2, characterized in that In step (1), the molar ratio of 2,3,3-trimethylindole, potassium iodide, and 1-azido-4-chloro-butane described in (a) is 1:2:1, the reaction temperature is 90° C., and the reaction time is 9 hours; In step (1), the molar ratio of compound 2 described in (b) to 2-chloro-3-(hydroxymethylene)cyclohex-1-enecarbaldehyde is 1:0.5, the reaction temperature is 130° C., and the reaction time is 3 hours; In step (1), the molar ratio of compound 3, m-diphenol, and triethylamine is 1:3:2.5, the reaction temperature is 50° C., and the reaction time is 6 hours. In the column chromatography purification, the volume ratio of the solvent is methanol: dichloromethane = 1:50-1:

10. In step (1), the molar ratio of compound 4 described in (d) to phosphorus oxychloride is 1:

1. After adding anhydrous pyridine, the reaction solution is reacted at room temperature for 12 hours, and then the same volume of ice water is added and reacted at room temperature for 12 hours.

4. The method according to claim 2, characterized in that The molar ratio of compound 5 and mercapto-polyethylene glycol-diphenylcyclooctyne in step (2) is 1:1, the reaction temperature is room temperature, and the reaction time is 1-3 hours.

5. The method according to claim 2, characterized in that The particle size of the gold particles protected by sodium citrate in step (3) is 20 nm.

6. The method according to claim 2, characterized in that The molar concentration ratio of compound 6 and gold nanoparticles in step (4) is 1:1, the reaction temperature is room temperature, the reaction time is 12 hours, and the dialysis purification time is 12 hours.

7. Use of the gold nanoparticle probe modified with an alkaline phosphatase-responsive fluorescent molecule according to claim 1 for near-infrared fluorescence imaging of tumor sites; the use is for non-disease diagnosis or treatment.

8. Use according to claim 7, characterized in that The tumor is HeLa; and the application is non-disease diagnosis or treatment.

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