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Antitumor peptide with pH selectivity and application of antitumor peptide

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: 2014-07-23
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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  • Antitumor peptide with pH selectivity and application of antitumor peptide
  • Antitumor peptide with pH selectivity and application of antitumor peptide
  • Antitumor peptide with pH selectivity and application of antitumor peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1. Preparation of polypeptides

[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 of stable structure before the sequence of the l...

Embodiment 2

[0082] Example 2. Anticancer activity test of polypeptide

[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 peptides 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 pH 5.5 50 )

[0088] The results showed that all the peptide...

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 100 mL 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 100 mm 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 (pH 5.5). Each group consisted of 6 to 7 mice. Peptide injection (20 nmol peptide / mouse, dissolved in 100 ml 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 sacri...

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Abstract

The invention relates to an antitumor peptide with efficient pH selectivity and an application of the antitumor peptide, belonging to the technical field of antitumor drug research and development and application. A series of peptides which are rich in histidine and have membrane lysis and antitumor activities are constructed by adopting the ribosome display technology and a series of antitumor peptides with normal physiological condition and tumor characteristic acidic condition selectivity are constructed by utilizing the special acidity coefficient of the imidazole side chain of histidine. The peptides have killing effects on lung cancer cells and breast cancer cells and can selectively kill the tumor cells in acidic conditions, inhibit tumor growth in mice xenotransplantation models and especially inhibit tumor growth in acidic conditions highly selectively. Microscopic observation, lactic dehydrogenase experiments, fluorescence quenching and circular dichroism spectrums of the peptides prove that the permeability of the peptides is increased by inserting and carrying out lysis on cell membranes, thus leading cell activity reduction and cell 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 Applications(China)
IPC IPC(8): C07K7/08C07K14/00A61K38/10A61K38/16A61P35/00
Inventor 屠志刚
Owner JIANGSU UNIV
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