A high molecular probe for evaluating sialylation level in vivo and a preparation method thereof

Abstract

This invention relates to a polymeric probe for assessing sialylation levels in vivo. It uses a dendritic polymer with alkyne-terminated branches as a carrier, and its surface is simultaneously modified with galactose, cyanidin 5 fluorescent dye, and folic acid. The detection protocol involves subcutaneously injecting the prepared polymeric probe into the tumor region of tumor-bearing mice. Folic acid-mediated targeted endocytosis allows the polymeric probe to enter the tumor cells. In vivo sialylates bind sialic acid to the galactose terminal of the polymeric probe. Sodium periodate is used to oxidize the sialic acid terminal in mouse tumor tissue sections to form an aldehyde group, which is then further coupled with an acylhydrazide fluorescein molecule. Fluorescence signals from cyanidin 5 and fluorescein are simultaneously collected using fluorescence microscopy, and the fluorescence intensity of fluorescein in the superimposed region of the two fluorescence signals is obtained using software, which can then be used to assess the sialylation level of the tumor tissue.

Description

I. Technical Field

[0001] This invention belongs to the field of biological detection technology and relates to a polymeric probe for assessing the level of saliva acidification in vivo and its preparation method. II. Background Technology

[0002] Sialization is an important glycosylation process that modifies sialic acid to the outermost ends of glycan chains on proteins, lipids, or nucleic acids. As the terminal group of glycans, sialic acid is a major executor of glycan function, mediating various physiological and pathological processes, particularly tumor cell migration and immune escape. Therefore, assessing in vivo sialylation levels can more precisely reveal sialic acid-related biological processes and provide new theoretical guidance and technical support for the clinical diagnosis and treatment of tumors. Although many methods have enabled the direct detection of intracellular sialic acid, methods for in situ assessment of sialylation at the in vivo level remain scarce.

[0003] To achieve in-situ assessment of salivation levels in vivo, this patent developed a polymeric probe and established an analytical method for assessing in-situ salivation levels in vivo. III. Summary of the Invention

[0004] The purpose of this invention is to prepare a polymer probe by using a dendritic polymer with alkynyl groups at the branch ends as a carrier, and connecting polyethylene glycol molecules with an azide group at one end and galactose, amino group, and folic acid at the other end via a click chemical reaction between the alkynyl group and azide group. Then, a coupling reaction between the amino group and N-hydroxysuccinimide is used to connect a cyanocyanate 5 fluorescent dye containing N-hydroxysuccinimide. Figure 1 The detection protocol involves subcutaneously injecting the prepared polymeric probe into the tumor region of tumor-bearing mice. Figure 2 Under folic acid-mediated targeted endocytosis, polymeric probes can rapidly enter tumor cells. In vivo, sialyl transferases bind galactose to the terminal portion of the probe, forming sialic acid. After a period of time, mouse tumor tissue is collected and prepared into sections, which are then incubated with sodium periodate solution to specifically oxidize the terminal portion of sialic acid to form an aldehyde group. Subsequently, fluorescein hydrazide molecules are added, and the sialic acid molecules are labeled with fluorescein using the coupling reaction between the hydrazide and the aldehyde group. The prepared sections are then imaged using fluorescence microscopy, simultaneously collecting fluorescence signals from cyanidin 5 and fluorescein. Image processing software is used to obtain the fluorescein fluorescence region overlapping with the cyanidin 5 fluorescence region; the fluorescence intensity of this dual-color fluorescence overlap region can be used to assess the sialylation level of tumor tissue.

[0005] This invention is achieved through the following technical solution:

[0006] Dendritic polymers with alkynyl groups at the branch ends were reacted with azido-polyethylene glycol galactose, azido-polyethylene glycol amino, azido-polyethylene glycol folic acid, copper sulfate solution, 2-(4-((bis((1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)acetic acid, and sodium ascorbate, and stirred overnight at room temperature. After the reaction, the dendritic polymers were purified by ultrafiltration, and then N-hydroxysuccinimide cyanogen 5 was added and reacted overnight at room temperature. After the reaction, the polymer probe was purified again by ultrafiltration. Figure 1 ).

[0007] Working principle of the invention:

[0008] The preparation process of the polymeric probe for assessing in vivo sialylation levels proposed in this invention is as follows: Figure 1 As shown, using dendritic polymers with alkynyl groups at the branch ends as carriers, three different functional polyethylene glycol molecules are covalently modified simultaneously via click chemistry of alkynyl groups and azido groups. Each molecule has an azido group at one end and galactose, amino, and folic acid polyethylene glycol groups at the other end, respectively. Then, a coupling reaction between amino groups and N-hydroxysuccinimide is used to connect the polymer probe to a cyanocyanate-5 fluorescent dye containing N-hydroxysuccinimide.

[0009] The working principle of this invention is as follows: Figure 2 As shown, the prepared polymeric probe was subcutaneously injected into the tumor region of tumor-bearing mice. Folic acid-mediated targeted endocytosis facilitated the entry of the polymeric probe into the tumor cells. In vivo sialyltransferase linked sialic acid to the galactose terminal of the polymeric probe. Mouse tumor tissue sections were incubated with sodium periodate solution to oxidize the sialic acid terminal to an aldehyde group. The aldehyde group and hydrazide were coupled to a fluorescein hydrazide molecule. Fluorescence signals from cyanogen 5 and fluorescein were simultaneously collected using fluorescence microscopy. Image processing software was used to obtain the superposition region of cyanogen 5 and fluorescein fluorescence; the fluorescein fluorescence intensity in this region can be used to assess the sialylation level of the tumor tissue.

[0010] Compared with the prior art, the present invention has the following characteristics:

[0011] The dendritic polymer probe prepared by this invention has good biocompatibility and can realize in-situ visual assessment of sialylation in vivo.

[0012] Compared with existing methods for assessing sialic acidification, this invention has the following advantages:

[0013] 1. The polymer probe preparation method described in this invention is simple, has a small spatial size, and can achieve high spatial specificity of fluorescence signals.

[0014] 2. The in vivo saliva acidification assessment involved in this invention can provide a visual spatial distribution of saliva acidification levels. IV. Description of the attached drawings

[0015] Figure 1 Schematic diagram of polymer probe preparation

[0016] Figure 2 The process of assessing in vivo sialylation levels using polymeric probes. V. Detailed Implementation Methods

[0017] Example 1: Combination Figure 1 To prepare polymeric probes for assessing in vivo sialylation levels.

[0018] A fifth-generation bis(trimethylolpropane) dendritic polymer with alkynyl groups at the branch ends (dissolved in 1 μL, 5 mmol / L DMSO solution), azidopolyethylene glycol galactose (30 μL, 10 mmol / L), azidopolyethylene glycol folic acid (10 μL, 10 mmol / L), azidopolyethylene glycol amino (10 μL, 10 mmol / L), copper sulfate solution (10 μL, 500 mmol / L), 2-(4-((bis((1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)acetic acid (50 μL, 200 mmol / L), and sodium ascorbate (100 μL, 1 mol / L) were mixed and shaken overnight at room temperature. The mixture was ultrafiltered and washed three times at 10,000 rpm for 10 minutes each time, yielding a pale yellow solution. The pale yellow solution was then brought to a final volume of 250 μL with phosphate buffer, followed by the addition of 10 μL of N-hydroxysuccinimide cyanogen 5 (10 mmol / L), and the reaction was allowed to proceed overnight at room temperature. The reaction solution was then ultrafiltered and washed three times at 10,000 rpm for 10 minutes each time, yielding the polymer probe.

[0019] Example 2: Combination Figure 2 Polymer probes were used to assess the level of sialylation in vivo.

[0020] Mouse breast cancer 4T1 cells (1×10⁻⁶) 6 (One xenograft / mouse) was injected into the hind legs of female nude mice (6-8 weeks old) to establish a mouse 4T1 subcutaneous tumor xenograft model. The tumors were cultured until they reached 1 cm in size. 3Subsequently, 100 μL of molecular probe was subcutaneously injected around the mouse tumor. One hour after injection, the mice were euthanized, and the tumor tissue was collected and fixed with 4% paraformaldehyde. The tissue samples were then frozen and sliced. At room temperature, sodium periodate solution (5 mmol / L) was added to the surface of the tumor tissue slices and incubated for 30 minutes. After washing three times with phosphate buffer, a solution containing aniline (10 mmol / L) and fluorescein hydrazide (100 μmol / L) was added to the surface of the tumor tissue slices, and the reaction was incubated for 1 hour. After washing three more times with phosphate buffer, the slices were sealed with coverslips and fluorescence imaging was performed using a fluorescence microscope. The resulting two-color fluorescence image was analyzed using MATLAB software to obtain the superposition region of cyanogen 5 fluorescence and fluorescein fluorescence. The intensity of fluorescein fluorescence in this region can be used to assess the sialylation level of the tumor tissue.

Claims

1. A polymeric probe for assessing the level of sialylation in vivo, characterized in that, The polymer probe is prepared by first modifying the surface of a dendritic polymer with alkynyl groups at the branch ends with three different functional polyethylene glycol molecules, and then modifying it with cyanocyanate 5 fluorescent dye. The three different functional polyethylene glycol molecules are polyethylene glycol molecules with azido groups at one end and galactose groups, amino groups, and folic acid groups at the other end, respectively. The surface modification is achieved by a click chemical reaction between the alkynyl groups at the end of the dendritic polymer and the azido groups at the end of the three different functional polyethylene glycol molecules. The cyanocyanate 5 fluorescent dye is obtained by coupling N-hydroxysuccinimide cyanocyanate 5 molecules with functional polyethylene glycol molecules that have amino groups at the end.

2. The detection method of a polymeric probe for assessing the level of sialic acidification in vivo according to claim 1, characterized in that... The detection method involves subcutaneously injecting a polymeric probe into the tumor region of a tumor-bearing mouse. The probe then enters the tumor cells via endocytosis mediated by a folic acid group. In vivo, sialic acid groups are attached to sialic acid groups using galactose at the end of the polymeric probe as a substrate. The tumor tissue sections from the mouse are then oxidized with sodium periodate to generate aldehyde groups, which are then coupled with fluorescein hydrazide molecules. The sialylation level of the tumor tissue is assessed using the fluorescence intensity of fluorescein in the superimposed region of cyanogen 5 and fluorescein.

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