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Nano probe used for nucleolin cross-linking to induce tumor cell apoptosis, and preparation method and application of nano probe

A technology of tumor cell apoptosis and nano-probe, which is applied in the field of nucleolin crosslinking-induced tumor cell apoptosis nano-probe and its preparation, which can solve the problem of inability to selectively induce tumor cell apoptosis, probe degradation or Endocytosis, poor stability and other issues

Active Publication Date: 2020-10-20
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are two main problems in the research on the induction of tumor cell apoptosis based on the cross-linking of cell membrane receptors: (1) The types of cell membrane receptors studied are relatively single, and they are not specifically expressed on the surface of tumor cells, so selective induction cannot be achieved. Tumor cell apoptosis; (2) Due to cell membrane turnover, internalization and enzymatic degradation, etc., the probe may be degraded or endocytized, etc., and the stability is poor

Method used

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  • Nano probe used for nucleolin cross-linking to induce tumor cell apoptosis, and preparation method and application of nano probe
  • Nano probe used for nucleolin cross-linking to induce tumor cell apoptosis, and preparation method and application of nano probe
  • Nano probe used for nucleolin cross-linking to induce tumor cell apoptosis, and preparation method and application of nano probe

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Experimental program
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Embodiment 1

[0036] Preparation of nanoprobes for nucleolin cross-linking to induce tumor cell apoptosis

[0037](1) Glycol chitosan (GC) and succinimidyl ester-polyethylene glycol-cholesterol (chol, purchased from NANOCS, USA) were dissolved in PBS buffer with a concentration of 0.0067M and a pH of 7.4 In the method, the ethylene glycol chitosan solution with a concentration of 1.34 mg / mL and the succinimide ester-polyethylene glycol-cholesterol solution with a concentration of 5.00 mg / mL were obtained, and the two solutions were mixed in equal volumes and stirred at room temperature After reacting for 4 hours, the final reaction solution was transferred to a dialysis bag with a molecular weight cut-off of 10KD, dialyzed in deionized water for 3 days, and freeze-dried to obtain an ethylene glycol chitosan-cholesterol compound. The mass fraction of cholesterol in the compound was 30 %;

[0038] (2) The ethylene glycol chitosan-cholesterol compound and the A chain (apt, 5'-COOH-ttttttttttt...

Embodiment 2

[0043] Seed HEp-2 cells (human laryngeal carcinoma epithelial cells) in imaging dishes (1×10 4 per well), at 37°C and 5% CO 2 The culture medium was cultured for 24 hours under the condition of 24 hours, then the culture medium was aspirated, washed 3 times with PBS buffer, and then the GC-chol-apt-cDNA solution in Example 1 with a concentration of 60 μg / mL was added, and incubated at 37°C for 2 hours. After the incubation, wash off the unbound GC-chol-apt-cDNA with PBS, then add DiO cell membrane dye (5 μM), and incubate at 37°C for 25 min. After the incubation, wash off the unbound DiO cell membrane dye with PBS, and Fluorescence signals were collected using a confocal fluorescence microscope (Olympus IX-81). The result is as Figure 5 As shown, after GC-chol-apt-cDNA interacted with HEp-2 cells, Cy3 red fluorescence appeared, and it had a high co-localization phenomenon with Dio cell membrane dye, indicating that GC-chol-apt-cDNA could specifically interact with Nucleoli...

Embodiment 3

[0045] Validation of the ability of GC-chol-apt-cDNA to regulate nucleolin cross-linking and anchoring to the cell membrane

[0046] Seed HEp-2 cells (human laryngeal carcinoma epithelial cells) in imaging dishes (1×10 4 per well), at 37°C and 5% CO 2 Cultured under the condition of 24h, then aspirated the medium, washed 3 times with PBS buffer solution, then added the apt solution with a concentration of 60nM and the GC-chol-apt-cDNA solution in Example 1 with a concentration of 60μg / mL respectively, at 37°C Incubate for 0, 0.5, 2, 4, 6 hours. After the incubation, unbound apt and GC-chol-apt-cDNA were washed away with PBS, and fluorescent signals were collected using a confocal fluorescence microscope (Olympus IX-81). The result is as Figure 6 As shown, GC-chol-apt-cDNA can stay on the membrane for up to 6h, while the single Cy3-labeled A chain (apt) enters the cell after 0.5h.

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Abstract

The invention relates to a nano probe used for nucleolin cross-linking to induce the tumor cell apoptosis, and a preparation method and application of the nano probe, and belongs to the technical field of biology. The nano probe uses ethylene glycol chitosan as a polymer backbone, the polymer backbone is grafted with cholesterol and an A chain, the 5'end of the A chain is modified with a carboxylgroup, the 3'end of the A chain is modified with a dye Cy3, the A chain contains a nucleic acid adapter AS1411 gene sequence, and the A chain partially hybridizes with a B chain modified with BHQ2 atthe 5'end. The nano probe can selectively kill tumor cells with the overexpression of nucleolin, but has relatively little effects on normal cells, is expected to become a method for the treatment ofmalignant tumors, and provides a new alternative receptor for the idea of receptor cross-linking to induce apoptosis. The nano probe is simple in synthesis, separation and purification, and is suitable for expanded production.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a nanoprobe for nucleolin cross-linking to induce tumor cell apoptosis, a preparation method and application thereof. Background technique [0002] Malignant tumor (cancer) has become one of the major public health problems that seriously threaten human health. Although modern medical technology and diagnostic methods have been developed to a certain level, the morbidity and mortality of tumors are still high due to their fast growth and easy metastasis, which seriously threaten the health and even lives of patients. Developing new cancer treatments that effectively kill tumor cells without harming normal tissue has long been a goal of researchers. [0003] Cell signal transduction is a very important process in biological systems. It can exchange information through various signaling molecular network structures to realize the control of intracellular and extracellular a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/61A61K47/54A61K31/7088A61P35/00
CPCA61K47/61A61K47/54A61K31/7088A61P35/00
Inventor 李春梅程凤
Owner SOUTHWEST UNIV
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