TA polypeptide, TA polypeptide-modified drug delivery system, preparation method of TA polypeptide-modified drug delivery system and application of TA polypeptide and TA polypeptide-modified drug delivery system

A peptide modification and delivery system technology, applied in the field of medicine, can solve problems such as easy inactivation, inconvenience to patients, unstable physical and chemical properties, etc., and achieve strong target binding ability and the effect of inhibiting proliferation

Active Publication Date: 2020-05-08
山东德升生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Endostatin has the following disadvantages: its purification process is complicated and its cost is high; its physical and chemical properties are unstable and easy to inactivate; it needs frequent administration, which causes great inconvenience to patients; because it is a protein macromolecular drug, it is difficult to pass through the blood-brain barrier
[0004] Based on the above deficiencies, the inventors found that if ES genes are delivered to glioma vascular endothelial cells and tumor cells by using a gene-targeted delivery system and highly expressed, angiogenesis and tumor growth can be inhibited in an autocrine or paracrine manner. It will greatly improve its curative effect and clinical application value; however, the Endostatin gene targeted delivery system for the treatment of glioma needs to overcome the following two problems: blood-brain barrier (Blood–Brain Barrier, BBB), blood-brain barrier - The blocking effect of the tumor barrier (Blood–Brain-Tumor Barrier, BBTB) on the drug, and the non-selectivity of the gene drug on the tumor tissue

Method used

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  • TA polypeptide, TA polypeptide-modified drug delivery system, preparation method of TA polypeptide-modified drug delivery system and application of TA polypeptide and TA polypeptide-modified drug delivery system
  • TA polypeptide, TA polypeptide-modified drug delivery system, preparation method of TA polypeptide-modified drug delivery system and application of TA polypeptide and TA polypeptide-modified drug delivery system
  • TA polypeptide, TA polypeptide-modified drug delivery system, preparation method of TA polypeptide-modified drug delivery system and application of TA polypeptide and TA polypeptide-modified drug delivery system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Example 1 Synthesis and characterization of TA, Fluorescein-TA.

[0102] (1) Put a certain amount of resin into the reactor, add dichloromethane (DCM) to swell for half an hour, then remove the DCM, add the first amino acid in the sequence and diisopropylethylamine (DIEA), an appropriate amount DMF, nitrogen bubbling reaction for 60min. Then add methanol, react for half an hour, remove the reaction solution, and wash with DMF and MEOH;

[0103] (2) Add the second amino acid in the sequence, 1-hydroxyl, benzo, trichlorazole tetramethylhexafluorophosphate (HBTU) and DIEA to the reactor, react with nitrogen bubbles for half an hour, wash off the liquid, indene Triketone detection followed by capping with pyridine and acetic anhydride. Finally, wash, add an appropriate amount of decapping solution to remove the 9-fluorenylmethoxycarbonyl (Fmoc) protecting group, wash, and detect ninhydrin; add different amino acids in the sequence according to the above method until th...

Embodiment 2

[0109] Example 2 Synthesis and characterization of PEI-PEG-TA.

[0110] Weigh 13.50 mg of TA and 9.0 mg of NHS-PEG-MAL and dissolve them in 500 μL DMSO, react for 12 h under nitrogen protection, transfer the reaction product to a Sephadex S30 gel column, and buffer it with phosphate buffer at a flow rate of 1 ml / min Liquid flow relative to the product was separated and purified, and the product NHS-PEG-TA was lyophilized. Weigh 15mgPEI and dissolve in 0.1mM pH

[0111] In the phosphate buffer solution of 7.2, weigh 6.40mg NHS-PEG-TA and dissolve it in 500μL DMSO, add it dropwise to the PEI solution under the condition of stirring, and react overnight under the protection of nitrogen, with a molecular weight cut-off of 8000-14000Da The dialysis bag was dialyzed for 48h, and the product was obtained by freeze-drying. 1 H NMR characterizes the final product as image 3 shown.

Embodiment 3

[0112] Example 3 The binding ability of TA to VEGFR-2 and NRP-1 receptor protein was determined.

[0113] (1) Using surface plasmon resonance (SPR) technology, ① coupled protein. The best coupling solutions for VEGFR-2 and NRP-1 proteins were selected by pH scouting test: sodium acetate solution with pH 4.0. First use EDC / EHS to activate the carboxyl group of the CM5 chip, dilute the VEGFR-2 and NRP-1 proteins to 50 μg / mL with sodium acetate solution at pH 4.0, and form a covalent bond by reacting the amino group of the protein with the carboxyl group of the CM5 chip Coupled to channels 2 and 4 in the same way, and then blocked with ethanolamine; channels 1 and 3 were only activated by EDC / NHS and blocked with ethanolamine as reference channels. ②Binding force analysis. Through the surface test, the analyte TA mother solution was diluted to 0.25 μmoL as the highest concentration of injection, and six concentrations were serially diluted, injected at a flow rate of 30 μL / m...

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Abstract

The invention provides a TA polypeptide, a TA polypeptide-modified drug delivery system, a preparation method of the TA polypeptide-modified drug delivery system and application of the TA polypeptideand TA polypeptide-modified drug delivery system. The polypeptide contains an amino acid sequence RKKRRQRRR and an amino acid sequence ATWLPPR, and has the ability of penetration of biological barriers, and the proliferation, migration and other activity of new vessels can be inhibited through target binding to VEGFR-2 and NRP-1 receptors. The polypeptide can be applied to targeted therapy and diagnosis of tumor angiogenesis, after the gene drug delivery system is modified by the TA polypeptide, therapeutic genes can be carried, and penetrate a blood-brain barrier BBB and a blood-tumor barrierBBTB, and uptake and expression of gene drugs at sites of tumors and angiogenesis, so that targeted therapy for in-situ glioma is achieved effectively.

Description

technical field [0001] The present invention relates to the field of medicine, in particular to a TA polypeptide and its modified drug delivery system as well as its preparation method and application. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily to be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Glioma is the most common intracranial tumor with a high incidence rate and mostly malignant manifestations. It is difficult to completely remove it by surgery and has a high recurrence rate. The current treatment for glioma is mainly based on surgical resection, supplemented by radiotherapy and chemotherapy. However, because glioma infiltrates and grows in normal brain tissue before surgery, and the boundary is blurr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K47/42A61K49/00A61P9/00A61P35/00C08G65/334C08G73/04
CPCC07K7/06A61K47/42A61K49/0056A61P35/00A61P9/00C08G65/3348C08G73/0206C07K2319/10
Inventor 张新科路璐王珑坤王凤山程艳娜
Owner 山东德升生物工程有限公司
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