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Preparation and application of FAPalpha activated polypeptide magnetic nanosphere compound used for diagnosis of tumors

A technology for tumor diagnosis and complexes, applied in peptides, biological tests, material inspection products, etc., can solve the problems of rapid degradation, low biomarkers, and difficulty in dissolving, etc., achieve good application and promotion value, and the preparation method is convenient and easy line effect

Active Publication Date: 2016-04-27
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the very low concentration of biomarkers in the systemic circulation, they are difficult to dissolve in body fluids and are rapidly degraded in vivo and in vitro, the use of naturally occurring biomarkers to diagnose diseases is limited by detection technology and biology.

Method used

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  • Preparation and application of FAPalpha activated polypeptide magnetic nanosphere compound used for diagnosis of tumors
  • Preparation and application of FAPalpha activated polypeptide magnetic nanosphere compound used for diagnosis of tumors
  • Preparation and application of FAPalpha activated polypeptide magnetic nanosphere compound used for diagnosis of tumors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0019] Example 1: Synthesis of FAPa-specific substrates

[0020] 1. Put 2-ChlorotritylChlorideResin resin into the reaction tube, add DCM (15ml / g), shake for 30min;

[0021] 2. Filter out the solvent through the sand core, add 3 times molar excess of Fmoc-L-Cys(Trt)-OH, then add 10 times molar excess of DIEA, and finally add DMF to dissolve, shake for 30 minutes, cap with methanol for 30 minutes;

[0022] 3. Remove the solvent, add 20% piperidine / DMF solution (15ml / g) for 5min, remove and add 20% piperidine / DMF solution (15ml / g) for 15min;

[0023] 4. Take out the piperidine solution, take more than a dozen resins, wash them three times with ethanol, add ninhydrin, KCN, and phenol solution one drop each, heat at 105°C-110°C for 5min, and turn dark blue as a positive reaction;

[0024] 5. DMF (10ml / g) twice, methanol (10ml / g) twice, DMF (10ml / g) twice;

[0025] 6. Protect amino acid Fmoc-L-Asp(OtBu)-OH in three times excess, HBTU in three times excess, dissolve with as little...

Embodiment example 2

[0039] Implementation case 2: Study on the effect of FAM-GP-NWs on tumor diagnosis

[0040] Validation of in vitro tumor diagnosis:

[0041]According to the inhibitory effect of the target polypeptide on the growth of cells expressing different molecular markers, it can directly reflect the in vitro tumor diagnostic activity of FAP-GP-NWs. Therefore, we first selected the appropriate cell lines by detecting the expression of molecular markers in different cell lines, and then detected the tumor diagnostic significance of FAP-GP-NWs by ELISA experiments. The specific operation is as follows:

[0042] Selection of cell lines:

[0043] We selected FAPa+ L929 cell line and FAPa- 4T1 cell line, and detected the expression level of FAPa with Western-bolt.

[0044] Western Blot:

[0045] 1. After the cells are treated according to the experimental requirements, discard the medium, wash with pre-cooled PBS for 3 times, add three decontaminated cell lysates, and place on ice for 20...

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Abstract

The invention discloses a novel FAPalpha activated polypeptide magnetic nanosphere compound FAP-GP-NWs used for diagnosis of tumors. The compound is composed of amino magnetic nanospheres (NWs) and FAPalpha specific enzyme-digested polypeptide substrate with FAM and Biotin labels connected via flexible connexon SM(PEG)<2>; the sequence of a polypeptide substrate is Biotin-<D>Glu-G-<D>Val-<D>Asn-<D>Asp-<D>Asn-<D>Glu-G-<D>Phe-<D>Phe-<D>Ser-<D>Ala-<D>Arg-Lys(5FAM)-GPGPNQC, wherein D represents that the type of amino acid is type D; and SM(PEG)<2> represents NHS-(PEG)<2>-Maleimide connexon and reduce mutual interference between the substrate and the magnetic nanospheres. The invention has the following advantages: the FAPalpha specific substrate is coupled with the magnetic nanospheres for the first time for diagnosis of tumors; polypeptide coupled with the nano-material can circulate for a longer time in the body and is thus conveyed to diseased tissue via blood vessels; activity of fibroblast activator protein, i.e., FAPalpha, abnormally expressed in tumors is utilized to amplify the concentration of biomarkers, so signal molecules are released to urine from blood, which facilitates extraction and identification; and FAP-GP-NWs have no obvious toxicity to mice, so a solid foundation is laid for further clinical research and application.

Description

technical field [0001] The invention belongs to the technical field of biology and medicine, and in particular relates to the preparation and application of a peptide magnetic bead complex FAM-GP-NWs with tumor diagnosis significance. Background technique [0002] The search for biomarkers is aimed at identifying reliable indicators for disease risk assessment, early diagnosis, design of treatment regimens for patients, and effective monitoring of recurrent disease. So far, a wide range of biomolecules such as cell metabolites, peptides, proteins, cell-free nucleic acids, foreign molecules, etc. have been developed into different forms of biomarkers. However, due to the very low concentration of biomarkers in the systemic circulation, they are difficult to dissolve in body fluids and are rapidly degraded in vivo and in vitro, the use of naturally occurring biomarkers to diagnose diseases is limited by detection technology and biology. . Another endogenous biomarker is the ...

Claims

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Application Information

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IPC IPC(8): C07K7/08G01N33/68G01N33/574
Inventor 张革王启帆冯新为刘万里
Owner SUN YAT SEN UNIV
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