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Ferritin nanoparticles of C-terminal modified tumor penetrating peptide RGERPPR and preparation method and application of ferritin nanoparticles

A technology of tumor penetrating peptides and nanoparticles, which is applied in the field of ferritin nanoparticles, can solve the problems of insufficient targeting ability and efficiency of nanoparticles to tumor cells, and achieve strong targeting ability, strong cytotoxicity, and strong cell The effect of penetration

Active Publication Date: 2021-03-02
埃特尼特(上海)生命科学有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the defects that the existing nanoparticles have insufficient targeting ability and efficiency to tumor cells, the present invention provides a ferritin nanoparticle with a C-terminally modified tumor-penetrating peptide RGERPPR, its preparation method and application, which is expected to realize While targeting tumor cells, it is effectively delivered to tumor neovascularization and penetrates blood vessels to achieve dual targeting effects on tumor cell transferrin receptor 1 (TfR1) and neuropilin receptor 1 (NRP-1), It provides a very good model for the subsequent selection of drug-targeted tumor cell therapy, and has a good application prospect

Method used

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  • Ferritin nanoparticles of C-terminal modified tumor penetrating peptide RGERPPR and preparation method and application of ferritin nanoparticles
  • Ferritin nanoparticles of C-terminal modified tumor penetrating peptide RGERPPR and preparation method and application of ferritin nanoparticles
  • Ferritin nanoparticles of C-terminal modified tumor penetrating peptide RGERPPR and preparation method and application of ferritin nanoparticles

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Construction of HFtn-MMP2-RGE and HFtn-mMMP2-RGE recombinant ferritin:

[0044] Based on the HFtn coding gene, the tumor penetrating peptide RGERPPR (Shanghai Jierui Bioengineering Co., Ltd.) was connected to the C-terminus of human heavy chain ferritin (Nanjing Sipujin Biology) through a linking sequence at its 3' end. The coding gene for adding MMP-2 restriction site is: GGTGGTGGTGGTAGCGGTCCGCTGGGTCTGGCAGGTGGTGGTGGTGGTAGCGGTGGTGGTGGTAGCGGTGGTGGTGGTAGCCGTGGTGAACGTCCGCCGCGT, and the gene sequence was subcloned into the pET-20b(+) plasmid vector to obtain the HFtn-MMP2-RGE plasmid;

[0045] Based on the HFtn coding gene, the tumor-penetrating peptide RGERPPR was connected to the C-terminus of human heavy chain ferritin through a connecting sequence at its 3' end, and the coding gene that added a mutant MMP-2 restriction site in the connecting sequence was GGTGGTGGTGGTAGCGGTGCACTGGGTGCAGCAGGTGGTGGTGGTGGTAGCGGTGGTGGTGGTAGCGGTGGTGGTGGTAGCCGTGGTGAACGTCCGCCGCGT , subcloning t...

Embodiment 2

[0048] Expression of HFtn-MMP2-RGE and HFtn-mMMP2-RGE recombinant ferritin:

[0049] Inoculate positive recombinant bacteria of HFtn-MMP2-RGE and HFtn-mMMP2-RGE at 1% into LB medium containing ampicillin (final concentration 100 μg / ml), shake at 210 r / min at 37°C Activate the strain overnight for 12 hours; add the activated bacterial solution to 400 mL of LB medium containing ampicillin (final concentration: 100 μg / ml) at 1% inoculum size, cultivate at 37°C and 210 r / min until the bacterial solution OD 600 reached 0.7, added IPTG with a final concentration of 0.5mM to the culture medium, induced culture at 30°C for 9h, centrifuged at 4°C at 8000×g for 5min to collect the bacteria, discarded the supernatant, and added 5mL of Lysis buffer (50mM NaH 2 PO 4 , 300mM NaCl, 10mM Imidazole pH 8) to resuspend, centrifuge again under the same conditions after resuspension, discard the supernatant to collect the bacteria, resuspend in 5mL Lysis buffer again, and store at 4°C.

Embodiment 3

[0051] Separation and purification of HFtn-MMP2-RGE and HFtn-mMMP2-RGE recombinant ferritin:

[0052] Sonicate the resuspended bacterial solution of HFtn-MMP2-RGE and HFtn-mMMP2-RGE. The sonication conditions are as follows: sonicate for 5 s, intermittently for 5 s, and sonicate for 30 min in total, then centrifuge at 8000×g for 10 min to collect the supernatant, and place the supernatant in a 60°C water bath 10min, centrifuged again at 8000×g for 30min to collect the supernatant, and purified the target recombinant ferritin several times by size exclusion chromatography (SEC) to obtain the target recombinant ferritin HFtn-MMP2-RGE and HFtn-mMMP2-RGE, Characterization results such as figure 1 shown.

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Abstract

The invention discloses ferritin nanoparticles of C-terminal modified tumor penetrating peptide RGERPPR and a preparation method and application of the ferritin nanoparticles. The C terminal of the ferritin nanoparticle is modified with a tumor penetrating peptide RGERPPR, the tumor penetrating peptide RGERPPR is connected with the C terminal of ferritin through a connecting sequence, and the tumor penetrating peptide RGERPPR is exposed to the outside of ferritin. The C-terminal modified tumor penetrating peptide RGERPPR ferritin nanoparticles have strong targeting ability and cell penetratingpower, and can be strongly combined with tumor cells, and the prepared anti-tumor drug has strong cytotoxicity, provides a very good carrier model for subsequent drug targeting tumor cell treatment,and has a very good application prospect.

Description

technical field [0001] The invention relates to a C-terminally modified tumor-penetrating peptide RGERPPR ferritin nanoparticle, its preparation method and application, and belongs to the field of anti-tumor technology. Background technique [0002] Protein cages have good stability, biocompatibility, and biodegradability, and have been widely used in drug delivery and vaccine development. Among them, ferritin is self-assembled by 24 protein subunits to form a hollow nano-cage structure. The nano-cage has good water solubility, strong biocompatibility, good stability in vivo, and uniform size, and the ferritin itself has targeting properties. , can specifically bind to transferrin receptor 1 (TfR1) overexpressed in tumor cells. Although the inherent tumor-targeting ability of ferritin makes it a simple drug delivery vehicle, it is not limited to targeting only the natural receptor TfR1, and additional targeting motifs can be modified at any time for a variety of use. [0...

Claims

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Application Information

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IPC IPC(8): A61K47/42A61K31/337A61P35/00B82Y5/00B82Y40/00C07K19/00C12N15/70
CPCA61K31/337A61K47/42A61P35/00B82Y5/00B82Y40/00C07K14/47C07K2319/10C12N15/70
Inventor 张瑜马原蒙王飞李迅董亦馨
Owner 埃特尼特(上海)生命科学有限公司
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