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A PEGylated Fe based on functional peptide R9 modification 3+ /pei gene carrier and its preparation method and application

A gene carrier and functional peptide technology, applied in the medical field, can solve the problems of poor targeting of polyethyleneimine, prone to dissociation, strong cytotoxicity, etc., to improve nuclear delivery capacity, improve transfection efficiency, and reduce cytotoxicity. Effect

Active Publication Date: 2019-08-27
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are two outstanding problems in the use of polyethyleneimine as a gene carrier: first, there is a contradiction between transfection efficiency and cytotoxicity
Although small molecule PEI has low cytotoxicity, it is easy to dissociate with DNA at physiological ion concentration, resulting in poor transfection effect; although PEI with a molecular weight above 20kd has a relatively ideal transfection efficiency, but because the surface of PEI is rich in positive High molecular weight PEI exhibits strong cytotoxicity due to its charge and non-degradability in vivo
Second, polyethyleneimine has poor targeting: it uses its own positive charge to combine with negatively charged receptors on the cell surface through electrostatic interaction, so the specificity of cell selection is poor, and solving the targeting problem has become a Top concerns in non-viral vectors

Method used

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  • A PEGylated Fe based on functional peptide R9 modification <sup>3+</sup> /pei gene carrier and its preparation method and application
  • A PEGylated Fe based on functional peptide R9 modification <sup>3+</sup> /pei gene carrier and its preparation method and application
  • A PEGylated Fe based on functional peptide R9 modification <sup>3+</sup> /pei gene carrier and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Polypeptide R9 modified PEG-Fe 3+ / PEI gene vector preparation and function verification (1)

[0044] 1. PEG-Fe 3+ / PEI synthesis

[0045] Separately add 20 μL FeCl 3 ·6H 2 O (250mg / mL), 30μL bPEI (250mg / mL), 10μL vinyl sulfone (10mg / mL), 15μL PEG-NHS (Mw=5000, 10mg / mL) were added 1mL Tween 80 in cyclohexane solution (250mg / mL), at room temperature, fully stirred for 12h. After the reaction, add excess ethanol to the emulsion, centrifuge at 10000r / min for 1 hour, carefully remove the supernatant, and disperse the precipitate in ethanol again by sonic degradation. Repeat three times. After centrifugation, use 1mL ultra Pure water dissolves the precipitate and the result is PEG-Fe 3+ / PEI polymer solution.

[0046] 2. Synthesis of functional peptide R9

[0047] The sequence of functional peptide R9 is Arg-Gly-Asp-Cys-Lys-Lys-Lys-Arg-Lys, synthesized by Shanghai Gill Biochemical Co., Ltd. by solid phase method.

[0048] 3. PEG-Fe modified with polypeptide R9 3+ / PEI pr...

Embodiment 2

[0054] Example 2 Polypeptide R9 modified PEG-Fe 3+ / PEI gene vector preparation and function verification (2)

[0055] PEG-Fe 3+ / PEI-R9 preparation, cytotoxicity test, and in vitro transfection experiment are the same as in Example 1, except that: the PEG-Fe prepared in this example 3+ / PEI-R9, where PEI and Fe 3+ The molar ratio of PEG is 1:6, the molecular weight of PEI is 5KDa, and the molar ratio of PEGylated FE3+ / PEI to polypeptide R9 is 1:5.

[0056] Cytotoxicity test results show: PEG-Fe 3+ / PEI-R9 is almost non-toxic, the PEG-Fe of this example 3+ / PEI-R9 has low cytotoxicity and ensures PEG-Fe 3+ The feasibility of / PEI-R9 as a gene carrier. The results of the in vitro transfection experiment are shown in Table 1.

Embodiment 3

[0057] Example 3 Polypeptide R9 modified PEG-Fe 3+ / PEI gene vector preparation and function verification (3)

[0058] PEG-Fe 3+ / PEI-R9 preparation, cytotoxicity test, and in vitro transfection experiment are the same as in Example 1, except that: the PEG-Fe prepared in this example 3+ / PEI-R9, where PEI and Fe 3+ The molar ratio of PEG is 1:10, the molecular weight of PEI is 25KDa, and the molar ratio of PEGylated FE3+ / PEI to polypeptide R9 is 1:10.

[0059] Cytotoxicity test results show: PEG-Fe 3+ / PEI-R9 is almost non-toxic, the PEG-Fe of this example 3+ / PEI-R9 has low cytotoxicity and ensures PEG-Fe 3+ The feasibility of / PEI-R9 as a gene carrier. The results of the in vitro transfection experiment are shown in Table 1.

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Abstract

The invention relates to a PEGylated Fe3+ / PEI genetic vector based on functional peptide R9 modification and a preparation method and application of the PEGylated Fe3+ / PEI genetic vector. The genetic vector contains polyethylene glycol modified Fe3+ / polyethyleneimine and a conjugate formed by functional peptides R9. The PEGylated Fe3+ / PEI genetic vector based on functional peptide R9 modification has the advantages that metal ions Fe3 react with PEI at first, PEG modification is carried out to form PEGylated Fe3+ / PEI, then the functional peptide R9 which has ability of improving nuclear delivery and is targeted at alpha v beta 3 is synthesized, R9 is coupled with a PEG-Fe3+ / PEI by a crosslinking technology, and therefore, a novel virogene-free vector system PEG-Fe3 + / PEI is established. On the basis of reducing PEI cytotoxicity, in-vivo targeting property and nuclear delivery ability are improved, then the transfection efficiency of the vector in vivo is improved, and an effective way is searched for final application of gene therapy on clinical treatment.

Description

Technical field [0001] The present invention relates to the field of medical technology, in particular to a PEGylated Fe modified based on functional peptide R9 3+ / PEI gene vector and its preparation method and application. Background technique [0002] Gene therapy is a new type of treatment method based on genetic engineering technology and molecular genetics in recent years. Because the biological basis of tumor occurrence and development is gene mutation, gene therapy has now become the most promising field for conquering tumors. There are three important links in gene therapy, namely the target gene, the transgenic vector and the target cell. The gene delivery system is the core technology of gene therapy. The biggest problem at this stage is that the ideal gene carrier has not yet been found. The currently used vectors include viral vectors and non-viral vectors. Viral vectors have high transfection efficiency but have problems such as low carrying capacity and potential...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/64C07K7/06A61K47/42A61K48/00A61K31/713A61P35/00
Inventor 刘克海张敏邬军文余倩张庭瑞吴文惠
Owner SHANGHAI OCEAN UNIV
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