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Difunctional modified ferritin heavy chain subunit nanoparticles as well as preparation method and application thereof

A ferritin heavy chain and nanoparticle technology, applied in the field of biomedical engineering, can solve the problems of single function, inconvenient observation and analysis, and insufficient traceability.

Inactive Publication Date: 2014-09-03
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to provide a new ferritin heavy-duty protein for the defects of the existing nanoparticles in the process of targeting tumor cells, such as insufficient traceability, inconvenient observation and analysis, and single function. Chain subunit nanoparticles, preparation method and use thereof

Method used

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  • Difunctional modified ferritin heavy chain subunit nanoparticles as well as preparation method and application thereof
  • Difunctional modified ferritin heavy chain subunit nanoparticles as well as preparation method and application thereof
  • Difunctional modified ferritin heavy chain subunit nanoparticles as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Expression of EGFP-G4S-FTH1 and EGF-FTH1 fusion protein

[0047] 1. Use overlapping PCR technology to amplify the hEGF::FTH1 gene, insert the fragments into pET28a(+) vector, and transform into DH5αt competent cells, then transform the expression vector with the correct sequence into Ecoli.BL21(DE3) ) Expression strains to obtain target engineering bacteria. The amino acid sequence of the EGF-FTH1 target protein is shown in SEQ ID NO.2.

[0048] For the preparation and washing of EGF-FTH1 inclusion bodies, please refer to the 2011 Li Xu graduation thesis of the School of Bioengineering, East China University of Science and Technology, "Preparation of Functionalized Heavy Chain Subunit Ferritin Nanoparticles and Research on Breast Cancer Cell Targeting".

[0049] 2. Using overlapping PCR technology to amplify the gene of EGFP-G4S-FTH1, insert the fragment into pET28a(+) vector, and transform it into DH5α competent cells, then transform the expression vector with the ...

Embodiment 2

[0051] Example 2 Preparation of hybrid nanoparticles EGF-FTH1 / EGFP-G4S-FTH1

[0052] 1. Degeneration of inclusion bodies

[0053] Add 25ml of Buffer C to the relatively pure inclusion bodies, ultrasonically resuspend until no particles are visible to the naked eye, and denature overnight at a constant temperature oscillator at 28°C. After filtering with a 0.22μm filter, SDS-PAGE was performed to detect the purity of the target protein after denaturation. Two denatured proteins were obtained: EGFP-G4S-FTH1 and EGF-FTH1.

[0054] 2. Refolding: Refolding of inclusion bodies by gradient dialysis. First handle the dialysis bag: take the dialysis bag, boil it in Buffer D for 10 minutes, rinse it with double distilled water, boil it in Buffer E for 10 minutes, cool it, and keep it at 4°C for use. Mix the denatured EGFP-G4S-FTH1 and EGF-FTH1 at a molar ratio of 4:6, the volume is 50ml, and the total protein mass is 5mg. After mixing, put them into the pretreated dialysis bag, and seal the ...

Embodiment 3

[0062] Example 3 Study on cell targeting of FTH1 / EGFP-G4S-FTH1 hybrid protein nanoparticles

[0063] (1) Trypsinize MDA-MB-468 cells to 2×10 5 The concentration of each cell in each well is plated in a 24-well cell culture plate that has been added to the cell slide. After the cells are mixed, 37℃, 5% CO 2 Incubate overnight in an incubator to allow the cells to adhere.

[0064] (2) After cells adhere to the wall, wash 3 times with 1×PBS buffer, add 0μM, 0.02μM, 0.1μM EGF-FTH1 / EGFP-G4S-FTH1 nanoparticles, and add 10μM EGF to a cell sample and incubate for 3 hours .

[0065] (3) After the incubation is completed, wash with 1×PBS buffer for 3 times to wash away the nanoparticles that have not been internalized into the cells, and then add 4% paraformaldehyde to fix the cells at room temperature for 5 minutes.

[0066] (4) Wash the cells 3 times with 1×PBS buffer to remove residual paraformaldehyde solution, 200ng / mL DAPI, 200μL per well, DAPI solution is prepared with 1×PBS solution. I...

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Abstract

The invention discloses difunctional modified ferritin heavy chain subunit nanoparticles as well as a preparation method and an application thereof. The N end or the C end of FTH1 of the difunctional modified ferritin heavy chain subunit nanoparticles is modified with enhanced green fluorescent protein. The ferritin heavy chain subunit nanoparticles are modified with the enhanced green fluorescent protein (EGFP), are subjected to denaturation and renaturation together with nanoparticles modified with EGF at the same time and are self-assembled in vitro; and formed hybrid protein nanoparticles can specifically target to tumor cells and keep fluorescence activity. According to the preparation method of the nanoparticles, novel method and reference are provided for realizing functionalized modification of biomacromolecular nanocarriers, a very good model is provided for tumor cell targeted therapy of subsequently selected drugs, and the application prospect is very good.

Description

Technical field [0001] The invention belongs to the technical field of biomedical engineering, and specifically relates to a bifunctional modified ferritin heavy chain subunit nanoparticle, and a preparation method and application thereof. Background technique [0002] The characteristics of nanomaterials make it have many advantages in tumor diagnosis and analysis. The recent development of nanotechnology enables nano-scale carriers to be connected to various functional molecules at the same time, including tumor-specific ligands, antibodies, anti-cancer drugs and imaging Probes etc. The biocage protein itself is a natural biological macromolecule and has good biological compatibility. They are usually composed of multiple polypeptide subunits combined to form a hollow structure. The outer and hollow diameters are both at the nanometer scale, making it easy for nanomaterials to be modified on their surface, inside and between the interfaces of protein subunits to assemble into ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12P21/02C07K1/113A61K47/42A61P35/00B82Y30/00C12R1/19
Inventor 曹旭妮蒋晓丹王焦清邱李辉
Owner EAST CHINA UNIV OF SCI & TECH
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