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ph selective anti-tumor polypeptide and its application

An anti-tumor and selective technology, applied in the field of anti-tumor drug development and application, can solve problems such as toxic and side effects, and achieve the effect of enhancing selectivity

Inactive Publication Date: 2016-04-06
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Secondly, peptide drugs generally do not accumulate in the liver and kidney like small molecule drugs, causing toxic side effects to them

Method used

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  • ph selective anti-tumor polypeptide and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1. Preparation of polypeptide

[0069] (1) The ribosome display vector (pRDV, Genbank No. AY327136) was used for screening, the empty vector and the library were cut with restriction endonucleases BamHI and HindIII respectively, and the library was inserted into the restriction sites of the vector BamHI and HindIII (Such as figure 1 ). DNA libraries containing 19, 22 and 25 random polypeptide sequences consist of a GNN codon and 18, or 21 or 24 NNS codons, N is adenine (A), guanine (G), A mixture of cytosine (C) and thymine (T), S is a mixture of C and G. NNS can encode all 20 essential amino acids, but only one of the three terminators. The library can encode polypeptides of 19 amino acids, 22 amino acids and 25 amino acids in length. Then, PCR amplifies the above vector inserted into the library, and introduces the T7 RNA polymerase promoter sequence, ribosome binding position (sequence) and 5' stem-loop structure of stable structure before the seque...

Embodiment 2

[0082] Example 2. Anticancer Activity Test of Polypeptides

[0083] The anticancer activity of the polypeptide was detected in human lung cancer cell line A549, human breast cancer cell line MCF-7, human lung fibroblast cell line CCD-13Lu and human breast epithelial cell line MCF-10A. Press 5×10 3 The cells were seeded into a 96-well plate at a density of 1 cell / well and incubated at 37°C for 24 hours. Dilute the polypeptide to different concentrations with culture solution (pH to be tested) to replace the original culture solution. Incubate at 37°C for 2 hours. Then, according to the standard experimental method of MTT, the activity of the polypeptide at different pH values ​​was measured.

[0084] Table 2. Activity of polypeptides at pH 7.5 and 5.5.

[0085]

[0086] *:IC 20 (μM): Peptide concentration that inhibits 20% cell viability.

[0087] pH selectivity = (IC at pH 7.5 50 ) / (IC at pH5.5 50 )

[0088] The results showed that all the peptides studied above ...

Embodiment 3

[0089] Example 3. In vivo experiments

[0090] Feeding and maintenance of Nu / Nu nude mice. Mice were housed in pathogen-free cages on a 12-hour light / 12-hour dark rhythm. Subcutaneously inject 100mL containing 1.7′10 6 A549 cell suspension. Every other day, the tumor size was measured with a vernier caliper, and the volume was calculated according to the following formula: volume=0.5'W'L'H, where W, L and H represent width, length and height, respectively. When the tumor grows to about 100mm 3 start intravenous injection. Mice were divided into four groups: (1) saline (control); (2) ZTU0 in saline (pH 7.5); (3) ZTU17 in saline (pH 7.5); (4) ZTU17 in saline In brine (pH5.5). Each group consisted of 6 to 7 mice. Peptide injection (20nmol polypeptide / mouse, dissolved in 100ml saline) was injected every three days. Tumor volumes were measured every four days after the start of polypeptide injection. 24 days after the start of injections, all mice were sacrificed and ...

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Abstract

The invention relates to a high-efficiency pH-selective anti-tumor polypeptide and its application, and belongs to the technical field of research and development and application of anti-tumor drugs. The present invention uses ribosome display technology to construct a series of histidine-rich polypeptides with membrane cleavage and anti-tumor activity, and uses the special acidity coefficient of histidine imidazole side chain to construct a series of polypeptides that are effective for normal physiological conditions and tumors. Selective anti-tumor polypeptides in characteristic acidic conditions. They have killing effects on both lung cancer cells and breast cancer cells, and can selectively kill tumor cells under acidic conditions; inhibit the growth of tumors in mouse xenograft models, especially highly selectively inhibit tumor cells under acidic conditions Tumor growth. Through microscope observation, lactate dehydrogenase experiment, fluorescence quenching and polypeptide circular dichroism, it is proved that these polypeptides increase their permeability by inserting and cleaving the cell membrane, resulting in decreased cell activity and death.

Description

technical field [0001] The invention relates to a high-efficiency pH-selective anti-tumor polypeptide and its application, and belongs to the technical field of research and development and application of anti-tumor drugs. Background technique [0002] Multidrug resistance of tumors has always been a major obstacle to cancer treatment. High-efficiency anti-cancer peptides, as a candidate drug that can directly and powerfully destroy tumor cells, are receiving more and more attention due to their special mechanism of action, which is not prone to tumor drug resistance. The design of highly efficient and specific anti-cancer peptides is a recognized hotspot in the academic circle. The development of anti-tumor polypeptides with medicinal value has broad market prospects. However, anti-cancer peptides also have their own limitations, mainly poor selectivity. The present invention designs a series of anticancer polypeptides with pH value selectivity, which significantly incre...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C07K14/00A61K38/10A61K38/16A61P35/00
Inventor 屠志刚
Owner JIANGSU UNIV
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