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Drug conjugate of nucleic acid aptamer in targeted combination with CD133 protein and application of drug conjugate

A nucleic acid aptamer and drug conjugate technology, which can be used in drug combinations, medical preparations without active ingredients, and medical preparations containing active ingredients, etc. It can solve the problem of less cytotoxic conjugates and achieve easy Long-term storage, no immune response, strong affinity

Active Publication Date: 2019-03-08
ZHEJIANG CANCER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few related studies on nucleic acid aptamers and their cytotoxic conjugates for thyroid cancer, especially the research on nucleic acid aptamers and cytotoxic conjugates related to ATC has not been reported yet.

Method used

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  • Drug conjugate of nucleic acid aptamer in targeted combination with CD133 protein and application of drug conjugate
  • Drug conjugate of nucleic acid aptamer in targeted combination with CD133 protein and application of drug conjugate
  • Drug conjugate of nucleic acid aptamer in targeted combination with CD133 protein and application of drug conjugate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Expression and localization of CD133 protein in different cell lines

[0031] Such as figure 1 Shown, Caco-2: colon adenocarcinoma cell; FRO: undifferentiated thyroid cancer cell; NTH: normal thyroid cell; 293T: human embryonic kidney cell line.

[0032] Confocal Microscopy was used to observe the expression of CD133 protein in different cell lines ( figure 1 ), red fluorescence (ie figure 1 The membrane-like distribution of bright spots in the four photomicrographs in the lower left corner of the center) represents the expression distribution of the membrane protein CD133 protein, and the blue fluorescence (ie figure 1 Nucleated bright spots) in each micrograph represent DAPI, indicating cell nuclei. The results showed that CD133 protein was expressed on the cell membrane in undifferentiated thyroid cancer FRO cells and colon adenocarcinoma Caco-2 cells as a positive control group, while HEK-293T cells as a negative control group and normal thyroid cells ...

Embodiment 2

[0033] Example 2: Aptamer AP-1-M binds to CD133 positive cells and negative cells.

[0034] Such as figure 2 As shown, by using FITC to label the AP-1-M nucleic acid aptamer, HEK-293T cells overexpressing CD133 protein were regarded as positive cells, and wild-type HEK-293T cells were regarded as negative cells. The 1-M nucleic acid aptamer was incubated with positive cells and negative cells, and then observed under a laser confocal microscope to find that AP-1-M was combined with positive cells and located on the cell membrane ( figure 2 -A), while not binding to negative cells ( figure 2 -B), which also shows that AP-1-M is a targeting nucleic acid aptamer for CD133 protein.

[0035] The results show that AP-1-M is a nucleic acid aptamer that specifically binds to CD133 protein, and the affinity curve of the nucleic acid aptamer is measured by flow cytometry, and the Kd value of AP-1-M is 101.4nM .

Embodiment 3

[0036] Example 3: Binding of aptamer CD133 and undifferentiated thyroid cancer cell FRO.

[0037] Such as image 3 As shown, AP-1-M and FRO cells were incubated at 4°C and 37°C for 30 minutes, respectively, and it was found that at 4°C, because the endocytosis of cells was inhibited, AP-1-M and FRO cells bound to and localized on the cell membrane ( image 3 -A); and at 37°C due to the endocytosis of the cells, AP-1-M was endocytosed into the cells by the FRO cells ( image 3 -B).

[0038] The results indicated that AP-1-M can be combined with FRO cells and located on the cell membrane and can be endocytosed into the cells to achieve the purpose of targeted delivery.

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Abstract

The invention provides a drug conjugate of a nucleic acid aptamer in targeted combination with a CD133 protein and an application of the drug conjugate. The sequence of the nucleic acid aptamer is shown in SEQ ID NO.1, the aptamer in targeted combination with the CD133 protein is screened from an ssDNA library through a cell screening method, and the adapter AP-1-M is chosen. In one scheme of thedrug conjugate, the classic chemotherapy drug adriamycin (DOX) and the AP-1-M are connected finally through a physical embedding mode, a nucleic acid aptamer-cellular poison conjugate (AP-1-M-DOC) intargeted combination with ATC is obtained, the cytotoxic effect of the nucleic acid aptamer-cellular poison conjugate (AP-1-M-DOC) in targeted combination with ATC is explored, a better target is provided for clinically screening and studying a novel targeted drug, and a novel method and idea is provided for clinical treatment of ATC.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to a nucleic acid aptamer-cytotoxic conjugate targeting CD133 protein. Background technique [0002] CD133 protein is a member of the cell membrane protein superfamily. It contains 865 amino acids and a molecular weight of about 120kDa. Its molecular structure includes: extracellular N-terminal, 2 extracellular loops, 5 transmembrane domains, and a Cellular C-terminus of amino acids and 8 N-glycosylation terminals. The predicted size of the CD133 protein is 97 kDa, but the actual glycosylated CD133 has a molecular weight of 120 kDa. In recent years, researchers have discovered that CD133 protein exists on the surface of a variety of stem cell-like tumor cells, including liver cancer, colon cancer and ovarian cancer, which provides a better specific target for clinical screening and research of new drugs, thus providing The development of safer and more effective molec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115A61K47/54A61K31/704A61P35/00
CPCA61K31/704A61K47/549A61P35/00C12N15/115C12N2310/16
Inventor 葛明华朱栩杭那仁满都拉黄萍杨畅张轶雯李清林朱欣张启弘钱杨洋
Owner ZHEJIANG CANCER HOSPITAL
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