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Immune nano-carrier for conveying siRNA (small interfering Ribonucleic Acid) and preparation method and application thereof

A nano-carrier and carrier technology, applied in the field of biomedicine, can solve the problems of large molecular weight of antibodies and limited clinical application, and achieve the effects of reducing cytotoxicity, flexible structure, and small molecular weight

Inactive Publication Date: 2012-02-01
黄开红 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the clinical application of antibodies prepared by traditional methods is limited due to their large molecular weight and the ability to stimulate the human immune response, and it is difficult to pass through the gap between vascular endothelial cells.

Method used

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  • Immune nano-carrier for conveying siRNA (small interfering Ribonucleic Acid) and preparation method and application thereof
  • Immune nano-carrier for conveying siRNA (small interfering Ribonucleic Acid) and preparation method and application thereof
  • Immune nano-carrier for conveying siRNA (small interfering Ribonucleic Acid) and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1, scFv CD44v6 Expression purification

[0059] Follow the steps below to implement scFv CD44v6 Expression purification

[0060] (1) Obtain the CD44v6 single-chain antibody gene through T7Select phage display library screening, and carry out PCR amplification of this gene fragment.

[0061] The CD44v6 single-chain antibody gene was obtained through the screening of the T7Select phage display library, and contained T7Select primer sequences in the upstream and downstream of the cloning region. Among them, the human-derived CD44v6 single-chain antibody gene has the nuclide sequence as SEQ ID NO. 1, and the human-derived CD44v6 single-chain antibody has the amino acid sequence as SEQ ID NO.2.

[0062] Among them, the upstream and downstream of the cloning region respectively contain T7Select primer sequences:

[0063] T7Select Up primer: 5′-GGAGCTGTCGTATTCCAGTC-3′

[0064] T7Select Down primer: 5'-AACCCCTCAAGACCCGTTTA-3'

[0065] (2) Digest and ligate the amplified product ...

Embodiment 2

[0078] Example 2. Preparation of PPS nanoparticles

[0079] 1. Synthesis of polyethylene glycol grafted polyethyleneimine (mPEG-g-PEI).

[0080] Weigh 4.0 g of monomethyl ether polyethylene glycol (mPEG-OH, 2kD) in a dry two-neck flask at 60° C. and vacuum dry for 8 hours. After cooling, add 30 mL of dried tetrahydrofuran (THF) under Ar protection to fully dissolve. Weigh 3.2 g of N,N'-carboxydiimidazole (CDI) and place it in another dry two-necked flask. Under the protection of Ar, slowly add the mPEG-OH dissolved THF solution to the two-necked flask, and continue to stir at room temperature for 12h. . 200μL of water was added to inactivate the excess CDI, the reaction was carried out for 0.5h, precipitated with a large amount of anhydrous ether twice, filtered and dried to obtain a white powdery solid.

[0081] Weigh 1.1 g of branched polyethyleneimine (hy-PEI, 25kD) and 0.8 g of the above white powder and dissolve them in 20 mL CHCl 3 , Continue to stir the reaction at room temp...

Embodiment 3

[0085] Example 3. Preparation of PPS / siRNA and s-PPS / siRNA complex and determination of its physical and chemical properties

[0086] 1. Preparation of PPS / siRNA complex and determination of its physical and chemical properties

[0087] (1) PPS is dissolved in sterile deionized water. According to different N / P ratios, a certain amount of siRNA solution and PPS solution are gently mixed in deionized water or PBS, and incubated at room temperature for 10-15 minutes , That is, PPS / siRNA complexes with different N / P values ​​are obtained.

[0088] Among them, the formula for calculating the ratio of PPS / siRNA N / P

[0089]

[0090] (2) Measurement of particle size and potential

[0091] According to the N / P ratio of 5, 10, 15, 20 to form PPS / siRNA complexes, the amount of siRNA contained in each complex is set to 10μg, diluted with deionized water to 500μl, and the Zeta-Plus particle size analyzer is used to determine the complex Particle size and potential. Each sample is measured 5 tim...

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Abstract

The invention discloses an immune nano-carrier for conveying siRNA (small interfering Ribonucleic Acid). An active ingredient of the immune nano-carrier is a coupling product of a single-chain antibody and a siRNA conveying carrier; and the siRNA conveying carrier is polyethylene glycol-polyethylene imine-superparamagnetic ferric oxide nanoparticles formed by coupling superparamagnetic ferric oxide nanoparticles with polyethylene glycol-polyethylene imine. Moreover, the invention further discloses a preparation method of the immune nano-carrier. In the immune nano-carrier, the single-chain antibody is coupled with the siRNA conveying carrier, so that the immune nano-carrier has the physicochemical properties of small particle size and appropriate surface potential, high siRNA compounding capability, low cell toxicity, high transfection efficiency, high targeted conveying characteristic, and good application prospects on siRNA conveying and RNA interference-based disease treatment.

Description

Technical field [0001] The invention relates to the field of biomedicine, in particular to an immune nano carrier for delivering siRNA, and a preparation method and application thereof. Background technique [0002] RNA interference (RNA interference, RNAi) is an ancient biological phenomenon that has been discovered in recent years, and is a product of biological evolution. Specifically refers to the double strand RNA (double strand RNA, dsRNA) guidance, with the participation of specific enzymes, exogenous or endogenous mRNA as the target of degradation, the specific target gene silencing at the post-transcriptional level, that is, no expression. Current research shows that RNAi is widely present in most eukaryotes ranging from fungi to higher plants, from invertebrates to mammals, and prokaryotes have not yet been discovered. RNAi not only participates in the regulation of normal cell growth, development, senescence and apoptosis, but also prevents genes from being expressed...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/87C08G65/00A61K48/00A61P35/00
Inventor 黄开红陈茵婷
Owner 黄开红
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