A mesoporous silicon probe for visually misleading intracellular sialylation and preparation method thereof

Abstract

The present invention relates to a mesoporous silicon probe for visually misleading intracellular sialylation. It uses mesoporous silica nanoparticles as carriers, and the surface is simultaneously modified with galactose, folic acid and functional double-stranded DNA molecules modified with carboxyfluorescein (FAM) quenched by black hole quencher 1 (BHQ1), and the interior is loaded with functional single-stranded DNA molecules with phenylboronic acid and cyanine 5 fluorescein (Cy5) at both ends. When the mesoporous silicon probe enters the cell through the cell endocytosis mediated by folic acid on its surface, the sialyltransferase in the cell will connect sialic acid with the galactose on the surface of the mesoporous silicon probe as the substrate, and the fluorescence of Cy5 at one end of the functional single-stranded DNA molecule released from the mesoporous silicon is used for tracing, and the phenylboronic acid at the other end will specifically bind to the connected sialic acid, and induce the adjacent chain substitution to replace the single chain modified with BHQ1 in the functional double-stranded DNA molecule, restore the fluorescence of FAM, and realize the visual misleading of intracellular sialylation.

Description

1. Technical Field

[0001] The invention relates to a mesoporous silicon probe for visualizing sialylation in misleading cells and a preparation method thereof. 2. Background Technology

[0002] Sialic acid is an important biological molecule. It is usually located at the end of glycoproteins or glycolipid molecules on the cell surface. It mediates a large number of physiological and pathological processes and plays an important role in the immune regulation of tumor cells. Sialic acid is generally produced by the sialylation process mediated by intracellular sialyltransferases. Misdirecting the sialylation process in tumor cells can directly reduce the expression of sialic acid on the cell surface, further inhibit the immune escape of tumors, and promote the immunotherapy effect on tumor cells.

[0003] In order to achieve the regulation of cell surface sialic acid for tumor suppression, this patent prepared a mesoporous silica probe and established an analytical method for misleading and visually monitoring the intracellular sialylation process. III. Summary of the invention

[0004] The purpose of the present invention is to aminosilane mesoporous silicon nanoparticles, covalently connect three different functional polyethylene glycol molecules on the surface, connect a functional double-stranded DNA molecule to the end of one of the functional polyethylene glycol molecules, and finally load the functional single-stranded DNA molecule inside the mesoporous silicon nanoparticles to prepare a mesoporous silicon probe ( Figure 1 The detection scheme directly incubates the mesoporous silica probe with cells and utilizes the folic acid groups modified on the surface of the mesoporous silica probe to mediate cell endocytosis ( Figure 2 ); the intracellular sialyltransferase will use the galactose group modified on the surface of the mesoporous silica probe as a substrate and connect sialic acid molecules to it to achieve the misdirection of intracellular sialylation; the functional single-stranded DNA molecules inside the mesoporous silica probe will be released from the pores of the mesoporous silica probe and traced using cyanine 5 fluorescein (FAM) at one end, and the phenylboronic acid molecules at the other end will specifically bind to the sialic acid molecules; the functional single-stranded DNA molecules bound to the surface of the mesoporous silica probe will replace the single strand containing black hole quencher 1 (BHQ1) in the functional double-stranded DNA molecules bound to the surface of the mesoporous silica probe through a DNA chain substitution reaction induced by the ortho effect (ortho-chain substitution), restore the fluorescence of the single strand containing carboxyfluorescein, and achieve visualization of the intracellular sialylation misdirection process.

[0005] The present invention is achieved through the following technical solutions:

[0006] Hydroxylated mesoporous silica nanoparticles were used as carriers, and three different functional polyethylene glycol molecules were covalently modified at the same time after amino silanization, with one end of each of them being a succinimide group, and the other ends being a galactose group, a succinimide group, and a folic acid group; then, the polyethylene glycol molecule with a succinimide group at the other end was further connected to two functional double-stranded DNA molecules modified with FAM and BHQ1 at the same end; a functional single-stranded DNA molecule with one end modified with a phenylboronic acid molecule and the other end modified with cyanine 5 fluorescein (Cy5) was loaded inside the mesoporous silica to obtain a mesoporous silica probe ( Figure 1 ).

[0007] Working principle of the present invention:

[0008] The preparation process of the mesoporous silicon probe for visualizing intracellular sialylation in the present invention is as follows: Figure 1 As shown. Hydroxylated mesoporous silica nanoparticles were used as carriers, and three different functional polyethylene glycol molecules were covalently modified at the same time after aminosilanization, with one end of each being a succinimide group, and the other ends being a galactose group, a succinimide group, and a folic acid group; then, the polyethylene glycol molecule with a succinimide group at the other end was further connected to two functional double-stranded DNA molecules modified with FAM and BHQ1 at the same end; a functional single-stranded DNA molecule modified with a phenylboronic acid molecule at one end and Cy5 at the other end was loaded inside the mesoporous silica to obtain a mesoporous silica probe.

[0009] The working principle of the present invention is as follows Figure 2 As shown. The mesoporous silicon probe is directly incubated with cells, and the folic acid group modified on the surface of the mesoporous silicon probe is used to mediate the cell's endocytosis; the intracellular sialyltransferase uses the galactose group modified on the surface of the mesoporous silicon probe as a substrate to connect sialic acid molecules on it; the functional single-stranded DNA molecules inside the mesoporous silicon probe are released from the mesoporous silicon probe pores, and are traced using Cy5 at one end, while the phenylboronic acid molecules at the other end specifically bind to the sialic acid molecules; the functional single-stranded DNA molecules bound to the mesoporous silicon surface replace the single-stranded DNA molecules containing BHQ1 in the functional double-stranded DNA molecules bound to the mesoporous silicon surface through adjacent chain substitution, restoring the fluorescence of the single-stranded DNA containing FAM, and realizing the visual misleading of intracellular sialylation.

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

[0011] The mesoporous silicon probe prepared by the invention can realize the misleading and visual monitoring of intracellular sialylation.

[0012] Compared with the existing intracellular glycosylation process monitoring methods, the present invention has the following advantages:

[0013] 1. The mesoporous silicon probe described in the present invention is a signal-responsive type, has a low fluorescence background, and can realize real-time monitoring of the intracellular sialylation misdirection process.

[0014] 2. The mesoporous silica probe described in the present invention can achieve the misdirection of sialylation in tumor cells, inhibit the expression of sialic acid on the cell surface, and promote tumor immunotherapy. IV. Description of the drawings

[0015] Figure 1 .Schematic diagram of mesoporous silica probe preparation

[0016] Figure 2 Visualizing the process of misdirected intracellular sialylation using mesoporous silica probes V. Specific implementation methods

[0017] Example 1: Combination Figure 1 Preparation and synthesis of mesoporous silica probes for visualization of misguided intracellular sialylation

[0018] 0.5 g of mesoporous silica nanoparticles (particle size 80 nm) were first stirred overnight at room temperature with 10 ml of piranha acid (containing 7 ml of concentrated sulfuric acid and 3 ml of 30% hydrogen peroxide). After washing and drying, the obtained hydroxylated mesoporous silica nanoparticles were added with 10 ml of anhydrous ethanol containing 1% aminosilane reagent and stirred overnight at 36 degrees Celsius. After washing three times with ethanol and water respectively, the obtained amino-modified mesoporous silica nanoparticles were dispersed in triethylenediaminetetraacetic acid buffer (pH 8.0). The amino-modified mesoporous silica nanoparticles (10 nM final concentration) were stirred overnight at room temperature with three different functional polyethylene glycol molecules (50 μM final concentration) with succinimide groups at one end and galactose groups, succinimide groups and folic acid groups at the other ends in 2 ml of Tris-EDTA buffer (pH 8.0). After washing three times by centrifugation with triethylenediaminetetraacetic acid buffer, the obtained polyethylene glycol-modified mesoporous silica nanoparticles (10 nM final concentration) and functional double-stranded DNA molecules (5 μM final concentration) with two single-stranded functional double-stranded DNA molecules modified with FAM and BHQ1 at one end were stirred overnight at room temperature in 2 ml of triethylenediaminetetraacetic acid buffer (pH 8.0). After washing three times by centrifugation with phosphate buffer (pH 7.4), the obtained double-stranded DNA and polyethylene glycol-modified mesoporous silica nanoparticles (10 nM final concentration) and functional single-stranded DNA molecules (100 μM final concentration) modified with phenylboronic acid molecules at one end and Cy5 at the other end were stirred overnight at room temperature in 0.5 ml of phosphate buffer (pH 7.4) to obtain a mesoporous silica probe.

[0019] Example 2: Combination Figure 2 Visualizing misdirected intracellular sialylation using mesoporous silica probes

[0020] About 10,000 MCF-7 cells were seeded in a 10 mm single-well confocal dish and incubated overnight, and washed three times with PBS buffer. 100 μL of mesoporous silica probe (5 nM) dispersed in phosphate buffer (pH 7.4) was added and incubated in a cell culture incubator at 37 degrees Celsius for 4 hours. Fluorescence imaging was then performed directly using a confocal microscope to detect the fluorescence of FAM and Cy5 in the cells. The fluorescence of Cy5 was used to trace the mesoporous silica probe, and the fluorescence of FAM was used to visualize the misdirected process of sialylation in the cell.

Claims

1. A mesoporous silicon probe for visually misleading intracellular sialylation, characterized in that: The mesoporous silicon probe is prepared by using mesoporous silicon nanoparticles as carriers, first modifying three different functional polyethylene glycol molecules on the surface, then connecting a functional double-stranded DNA molecule to the end of one of the functional polyethylene glycol molecules, and finally loading a functional single-stranded DNA molecule inside the mesoporous silicon nanoparticles; the modification of the three different functional polyethylene glycol molecules is to firstly amino-silane the surface of the mesoporous silicon dioxide nanoparticles, then covalently modifying one end with a succinimide group, and the other ends with a galactose group, a succinimide group and a folic acid group. Three polyethylene glycol molecules; the two single-stranded ends of one end of the connecting functional double-stranded DNA molecule are respectively modified with carboxyfluorescein and black hole quencher 1, and the other end is covalently connected to the polyethylene glycol molecule with succinimide groups on both ends of the mesoporous silicon surface through the modified amino group; the functional single-stranded DNA molecule is modified with a phenylboronic acid molecule at one end and with cyanine 5 fluorescein at the other end, and is loaded into the mesoporous silicon after incubation with the mesoporous silicon modified with the three functional polyethylene glycol molecules and the connecting functional double-stranded DNA molecule.

2. The mesoporous silicon probe according to claim 1, characterized in that: The folic acid group mediates the endocytosis of the mesoporous silicon probe into the cell; the galactose group serves as a substrate for the intracellular sialyltransferase to connect to the sialic acid group; the cyanine 5 fluorescein at one end of the functional single-stranded DNA molecule is used for tracing, and the phenylboronic acid at the other end specifically binds to the sialic acid group on the surface of the mesoporous silicon probe, triggering the adjacent chain substitution to replace the single chain modified with the black hole quencher 1 in the functional double-stranded DNA molecule, and restoring the fluorescence of the cyanine 5 fluorescein in the functional double-stranded DNA molecule, thereby realizing the visualization misleading of the intracellular sialic acid process.

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