Tumor cell glycan shielding network based on click chemistry and construction method thereof

Abstract

The invention discloses a construction method of tumor cell glycan shielding based on click chemistry. The method comprises the following steps: modifying glycosyl on the surface of a tumor cell through glycometabolism engineering to enable the glycosyl to have an azide reaction group, and then constructing a staggered polysialic acid network (PSA-N) on a tumor cell membrane by utilizing polysialic acid (PSA) with an aza-benzocyclooctyne (DBCO) group through an orthogonal click reaction between azide and cycloalkyne. The polysialic acid network constructed by the method provided by the invention has good biological safety, can relieve the stimulation induction effect of various cell factors or additional stimulating factors and the like on tumor cells, and finally reduces the distal tissue metastasis of in-situ tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a tumor cell glycan shielding network based on click chemistry and a construction method thereof, which can reduce the distant tissue metastasis of in situ tumors by shielding membrane protein glycosylation sites. Background Art

[0002] Transforming growth factor β (TGFβ) is an important cytokine with both tumor suppressor and oncogenic functions. In the early stages of tumor development, TGFβ is an antiproliferative factor, whereas in advanced cancers, it becomes an oncogenic factor that induces cancer cell proliferation, invasion, and metastasis. TGFβ also plays a key role in the activation of epithelial-mesenchymal transition (EMT), which promotes tumor cells to have stem cell properties, drug resistance, and metastatic spread. Activation of the TGFβ signaling pathway depends on the binding of TGFβ ligands to TGFβ receptors such as type II (TGFβRII) and type I (TGFβRI) receptors, and the subsequent formation of heteromeric complexes to activate downstream signaling pathways in cells. Therefore, blocking the interaction between TGFβ ligands and TGFβ receptors in tumor cells is highly desirable for selectively inhibiting TGFβ activation or signaling.

[0003] At present, the commonly used methods to block the interaction between TGFβ and TGFβ receptor focus on protein antibodies or small molecule inhibitors. Protein antibodies are used to specifically bind to TGFβ or TGFβ receptor to reduce the binding ability of TGFβ to TGFβ receptor. Commonly used small molecule inhibitors inhibit the binding of TGFβ to TGFβ receptor by reducing the activity of TGFβ receptor. Various cell signaling pathways are interconnected, and changes in TGFβ and TGFβ receptor signaling pathways may cause abnormalities in other signaling pathways.

[0004] Glycosylation is one of the most abundant post-translational modifications of membrane proteins, which affects protein biosynthesis, folding, stability and biological activity by manipulating the structure of proteins and their interactions with other molecules. It has a positive guiding significance to use endogenous glycan molecules in the form of an interlaced network to shield and protect the glycosylation sites of cell proteins to block the binding of stimulatory factors in the tumor microenvironment with corresponding cell surface receptors. Summary of the invention

[0005] The present invention proposes for the first time the technical concept of using polysaccharides to shield and protect the glycosylation sites of cell proteins to block the signal transduction mediated by the ligand and receptor recognition of tumor cells, and successfully anchors polysialic acid to the glycosylation sites of tumor cells with covalent bonds through sugar metabolism engineering and orthogonal click chemistry to shield / protect the glycosylation sites of cell proteins. The experimental results confirm that the polysaccharide network constructed by the method of the present invention can effectively inhibit the invasion and spread of tumor cells and ultimately reduce the in vivo distal metastasis of tumors.

[0006] The technical solution of the present invention is as follows:

[0007] A method for constructing a tumor cell glycan shielding network based on click chemistry, wherein the glycosides and glycans on the surface of tumor cells are functionalized respectively, so that the glycosides and glycans on the surface of tumor cells can be covalently linked based on a click chemistry reaction, and the polysialic acid grafted on the surface of tumor cells is staggered to form a glycan shielding network.

[0008] Click chemistry, also known as linking chemistry and dynamic combinatorial chemistry, is a combinatorial chemistry method that uses readily available raw materials to achieve carbon-heteroatom connections (C—X—C) through modular, reliable, efficient and highly selective chemical transformations, thereby rapidly synthesizing various new compounds at low cost.

[0009] Based on click chemistry technology, the groups carried by the sugar groups and polysaccharides on the surface of the tumor cells are the following reactive group pairs: alkynyl-azide group, amino-carboxyl group, hydroxyl-carboxyl group, thiol-double bond, thiol-maleimide, halogen-amino group, halogen-hydroxyl group or halogen-carboxyl group.

[0010] In a preferred embodiment of the present invention, the glycosyl groups on the surface of tumor cells are functionalized to carry azide groups, and the polysaccharides are functionalized to carry alkynyl groups.

[0011] A preferred embodiment of the present invention is to azidate the glycosylation functions on the surface of tumor cells based on sugar metabolism engineering, and the azidation reagent is selected from one or more of 9-azidosialic acid, tetraacetyl-6-azido-6-deoxy-aD-pyranose glucose, and tetraacetyl-N-azidoacetylmannosamine.

[0012] In a specific example of the present invention, the azidation reagent is selected from the sugar metabolism precursor tetraacetyl-N-azidoacetylmannosamine (Ac4ManNAz), and the sugar group on the cell surface is provided with an azide group through sugar metabolism engineering. The tetraacetyl-N-azidoacetylmannosamine has the following structure:

[0013]

[0014] The alkynyl group of the polysaccharide used in the present invention after functionalization is a terminal alkynyl or cyclooctyne. In a specific example of the present invention, the alkynyl group is azadibenzocyclooctyneamino.

[0015] The polysaccharide described in the present invention can be an endogenous polysaccharide in an organism, such as hyaluronic acid, chondroitin sulfate or polysialic acid. In a specific example of the present invention, the polysaccharide is polysialic acid with a molecular weight of 150 kDa.

[0016] The polysialic acid modified with azadibenzocyclooctyne group has the following structure:

[0017]

[0018] The method of the present invention specifically comprises the following steps:

[0019] (1) dissolving polysialic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in an organic solvent, adding azadibenzocyclooctynamine to react, and removing the organic solvent and impurities after the reaction is completed;

[0020] (2) injecting / applying an azidating agent to the tumor / tumor cells in situ;

[0021] (3) In situ injection / application of the azabenzocyclooctyne-polysialic acid prepared in step (1) into the tumor / tumor cells of step (2) to graft polysialic acid onto the glycosides on the surface of the tumor cells to form a polysaccharide shielding network.

[0022] Another object of the present invention is to provide a tumor cell glycan shielding network prepared by the above-mentioned method.

[0023] Another object of the present invention is to provide a drug combination for preventing tumor metastasis, characterized in that it includes reagent 1: azabenzocyclooctyne-polysialic acid and reagent 2: tetraacetyl-N-azidoacetylmannosamine.

[0024] Advantages of the present invention:

[0025] The method of the present invention anchors polysialic acid on the surface of tumor cells in the form of covalent bonds, forming a staggered network structure on the cell surface that can shield the glycosylation sites of cell membrane proteins, and further can alleviate the epithelial-mesenchymal transition process induced by TGFβ and reduce tumor metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Benzocyclooctyne-polysialic acid (DPSA) 1 H NMR spectrum, D2O, 400 MHz.

[0027] Figure 2 Fluorescence imaging and semi-quantitative graph of 4T1 cell sugar metabolism engineering markers.

[0028] Figure 3 Schematic diagram of the shielding effect of cell membrane proteins.

[0029] Figure 4 Laser confocal imaging and semi-quantitative data of the adhesion and shielding effects of the polysialic acid network on the 4T1 cell membrane.

[0030] Figure 5Laser confocal images of the effect of polysialic acid network on E-cadherin in 4T1 cells induced by TGFβ.

[0031] Figure 6 Laser confocal images of the effect of polyethylene glycol loading on E-cadherin in TGFβ-induced 4T1 cells.

[0032] Figure 7 This is an IVIS in vivo imaging image of the polysialic acid network inhibiting 4T1-luc tumor metastasis. DETAILED DESCRIPTION

[0033] The content of the present invention is further explained below in conjunction with embodiments, but these embodiments do not limit the protection scope of the present invention.

[0034] Example 1: Preparation of benzocyclooctyne-polysialic acid (DPSA)

[0035] Polysialic acid (PSA) (100 mg, 0.3 mM COOH), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (128 mg, 0.67 mM) and N-hydroxysuccinimide (NHS) (38.3 mg, 0.33 mM) were dissolved in 7 mL of anhydrous formamide solvent and stirred for 4 hours under ice-water bath conditions. DBCO-NH2 (26 mg, 0.1 mM) dissolved in anhydrous dichloromethane was then added dropwise and stirred at room temperature for 24 hours. After the reaction was completed, dichloromethane was removed by vacuum rotary evaporation, and the remaining reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzed in ultrapure water for 96 hours, and finally freeze-dried for 48 hours.

[0036] The structural characterization data of the product are as follows Figure 1 As shown, the red letter a represents the characteristic peak of the DBCO group, with a chemical shift between 7.0-7.8ppm; the blue letter b represents the characteristic peak of polysialic acid, with the chemical shift of the methyl group at the C5 position of the monomer sialic acid being around 2.0ppm. The molecular weight of polysialic acid is 150KDa, and by calculating the peak area integrals of 2.0ppm and 7.0-7.8ppm, it can be seen that there are an average of 6 DBCO groups attached to each polysialic acid chain.

[0037] Its structure is as follows:

[0038]

[0039] Example 2: Glycolytic Engineering to Express Azide on the Surface of Tumor Cells

[0040] After 4T1 cells were digested, 1×10 4The density of 1000 μg / dish was inoculated in a small dish with a diameter of 35 mm and a glass slide. The cells were cultured overnight in 2 mL RPMI-1640 medium, and then 25 μM Ac4ManNAz was added to continue culturing for 48 hours. The cells were washed three times with PBS, 2 mL fresh RPMI-1640 medium was added, and then 5 μM DBCO-Cy5 was added and cultured in an incubator for 1 hour. After the culture, the cells were washed three times with PBS, fixed with 4% paraformaldehyde at room temperature in the dark for 10 minutes, washed three times with PBS, and stained with DAPI for 10 minutes at room temperature in the dark, and washed three times with PBS again. The cells were observed with a laser confocal microscope under a 63x oil lens.

[0041] The results are as follows Figure 2 As shown, sugar metabolism engineering can successfully express azide groups on the surface of 4T1 tumor cells.

[0042] Example 3: Construction of polysialic acid cross-linked network to shield tumor cells

[0043] After 4T1 cells were digested, 1×10 4 / dish were inoculated in a small dish with a diameter of 35mm with a glass slide. 25μM Ac4ManNAz was added to co-culture with the cells for 48 hours, rinsed three times with PBS, and DPSA-Cy5 (300μg / mL PSA) was added to the small dish and cultured at 37℃ in the dark for 1 hour. Cells that were not co-cultured with M-NPs and directly added with DPSA-Cy5 were used as controls. After washing, fixing, and nuclear staining, the cells were observed under a laser confocal microscope.

[0044] Schematic diagram of DPSA's shielding effect on tumor cells through sugar metabolism engineering and in vivo orthogonal click reaction strategy. Figure 3 DPSA produces a shielding effect on the cell surface through cross-linking adhesion. Figure 4 As shown. Compared with the blank group, the cells co-cultured with DPSA-Cy5 showed enhanced fluorescence, indicating that DPSA can adhere to the cell surface to a certain extent but the amount of adhesion is very small. Compared with the cells co-cultured with DPSA-Cy5 alone, the cells pretreated with 25μM Ac4ManNAz and then co-cultured with DPSA-Cy5 showed a nearly 3-fold increase in fluorescence intensity, and the fluorescence was concentrated on the cell membrane surface. The results show that the present invention successfully constructs an interlaced polysialic acid network on the cell surface through sugar metabolism engineering and in vivo orthogonal click reaction strategy.

[0045] Example 4: Effect of polysialic acid network on TGFβ-induced epithelial-mesenchymal transition

[0046] After 4T1 cells were digested, 1×10 4The cells were seeded at a density of 100 μg / dish in a small dish with a diameter of 35 mm and a glass slide. 25 μM Ac4ManNAz was added to co-culture with the cells for 48 hours, and PBS was rinsed three times. DPSA (300 μg / mL PSA) was added to the small dish and cultured at 37°C in the dark for 1 hour. PBS was rinsed three times, TGFβ (5 ng / mL) was added to the small dish and cultured for 24 hours. Cells that were directly added with DPSA-Cy5 without co-culture with M-NP and cells that were co-cultured only with 25 μM Ac4ManNAz were used as controls. After washing, fixing, and nuclear staining, the cells were observed under a laser confocal microscope.

[0047] The results are as follows Figure 5 As shown, the polysialic acid network can effectively alleviate the epithelial-mesenchymal transition of 4T1 cells induced by exogenous TGFβ.

[0048] Example 5: Effect of tumor cells loaded with polyethylene glycol on epithelial-mesenchymal transition induced by TGFβ

[0049] After 4T1 cells were digested, 1×10 4 / dish were inoculated in a small dish with a diameter of 35 mm and a glass slide. 25 μM Ac4ManNAz was added to co-culture with the cells for 48 hours, rinsed three times with PBS, DPEG (300 μg / mL PEG) was added to the small dish and cultured at 37°C in the dark for 1 hour. PBS was rinsed three times, TGFβ (5 ng / mL) was added to the small dish and cultured for 24 hours. After washing, fixing, and nuclear staining, the cells were observed under a laser confocal microscope.

[0050] The results are as follows Figure 6 As shown, polyethylene glycol loaded on the surface of tumor cells could not alleviate the epithelial-mesenchymal transition of 4T1 cells induced by exogenous TGFβ.

[0051] Example 6: Effect of polysialic acid network on 4T1 tumor metastasis

[0052] will contain 5×10 6 150 μL of saline containing 4T1-luc cells was subcutaneously injected into the mammary pad of the left lower limb of seven-week-old female nude mice. Three days later, a highly metastatic breast cancer model was established in mice. The 4T1-luc tumor-bearing mice were divided into four groups (three mice in each group). The first group was injected with PBS (0.01 mM, 100 μL) as the control group, and the second group was injected with Ac4ManNAz (20 mg kg -1 Ac4ManNAz, 100 μL), and the third group was injected with DPSA (15 mg / kg on days 9, 11, 16, 18, 23, and 25. -1DPSA, 100 μL), and the fourth group was injected with Ac4ManNAz (20 mg kg -1 Ac4ManNAz, 100 μL), and DPSA (15 mg kg -1 DPSA, 100 μL). All reagents were injected peritumorally with 50 μL and intratumorally with 50 μL. Healthy female nude mice were used as negative controls and named NC (three mice). The mice in the above five groups were anesthetized with isoflurane on days 7, 15, 23, and 31, and then imaged using the IVIS in vivo imaging system.

[0053] IVIS in vivo imaging results Figure 7 As shown, the results indicate that shielding tumor cell membrane proteins through sugar metabolism engineering and in vivo orthogonal click chemistry-mediated cross-linking strategy can alleviate the metastasis of 4T1-luc tumor cells and reduce the metastatic sites in organs.

Claims

1. A method for constructing a tumor cell glycan shielding network based on click chemistry, characterized in that The glycosides and glycans on the surface of tumor cells are functionalized separately so that they can be covalently linked based on click chemistry reactions, and the polysialic acids grafted on the surface of tumor cells are staggered to form a glycan shielding network.

2. The method according to claim 1, characterized in that The groups carried by the sugars and polysaccharides on the surface of tumor cells are the following reactive group pairs: alkynyl-azide group, amino-carboxyl group, hydroxyl-carboxyl group, sulfhydryl-double bond, sulfhydryl-maleimide, halogen-amino group, halogen-hydroxyl group or halogen-carboxyl group.

3. The method according to claim 2, characterized in that The functionalized glycosides on the surface of tumor cells carry azide groups, and the functionalized polysaccharides carry alkyne groups.

4. The method according to claim 3, characterized in that Based on sugar metabolism engineering, the glycosides on the surface of tumor cells are azidated, and the azidation reagent is selected from one or more of 9-azidosialic acid, tetraacetyl-6-azido-6-deoxy-aD-pyranose glucose, and tetraacetyl-N-azidoacetylmannosamine.

5. The method according to claim 3, characterized in that The alkyne group carried by the polysaccharide after functionalization is a terminal alkyne or a cyclooctyne.

6. The method according to claim 5, characterized in that The alkynyl group is azadibenzocyclooctynylamino group.

7. The method according to any one of claims 1 to 6, characterized in that The polysaccharide is selected from hyaluronic acid or chondroitin sulfate or polysialic acid.

8. The method according to claim 7, characterized in that The polysaccharide is polysialic acid with a molecular weight of 150 kDa.

9. The method according to claim 1, characterized in that The steps include: (1) dissolving polysialic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in an organic solvent, adding azadibenzocyclooctynamine to react, and removing the organic solvent and impurities after the reaction is completed; (2) injecting / applying an azidating agent to the tumor / tumor cells in situ; (3) In situ injection / application of the azabenzocyclooctyne-polysialic acid prepared in step (1) into the tumor / tumor cells of step (2) to graft polysialic acid onto the glycosides on the surface of the tumor cells to form a polysaccharide shielding network.

10. A tumor cell glycan shielding network, characterized in that The method is prepared by any one of claims 1 to 8.

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