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CKIP-1 RNAi compound of macrophage targeted and preparation method and application thereof

A technology for targeting macrophages and complexes, applied in the field of CKIP-1 RNAi complex and its preparation, can solve the problems of high fatality rate and disability rate, lack of treatment methods, etc., to reduce brain edema and enhance transfection efficiency , the effect of increasing the uptake rate

Inactive Publication Date: 2019-01-22
AFFILIATED YONGCHUAN HOSPITAL OF CHONGQING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Intracerebral hemorrhage (ICH) is a common cerebrovascular disease in clinical practice, lacks effective treatment methods, and has high mortality and disability rates

Method used

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  • CKIP-1 RNAi compound of macrophage targeted and preparation method and application thereof
  • CKIP-1 RNAi compound of macrophage targeted and preparation method and application thereof
  • CKIP-1 RNAi compound of macrophage targeted and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Preparation of embodiment 1 fusion peptide

[0041] The fusion peptide is formed by directly linking the carboxyl terminal of the V6 peptide, the amino terminal of the T9 peptide and the amino terminal of the V6 peptide in sequence, and the amino acid sequence is SEQ ID NO.3.

[0042] The fusion peptide was synthesized on an AB-431A peptide synthesizer using a standard Fmoc protocol. Using 0.25mmol p-hydroxymethylphenoxymethyl polystyrene (HMP) resin as the starting resin, according to the amino acid sequence of the fusion peptide H9-V9, the peptide chain is extended from the carboxyl terminal to the amino terminal one by one. After the peptide chain is synthesized, , the resin containing the peptide chain was transferred to the cutting solution (composed of 0.25mL tartrate ethylenediamine, 9.5mL trifluoroacetic acid and 0.25mL deionized water), stirred at room temperature to cleave the peptide chain from the resin, and then used Filter through a G6 glass sand funnel, ...

Embodiment 2

[0044] Example 2 Preparation of CKIP-1 RNAi complex

[0045] The nucleotide sequence of CKIP-1 RNAi is: 5'-CCTGAGTGACTATGAGAAG-3' (SEQ ID No.4). Under the condition of vortexing, to the aqueous solution containing CKIP-1 RNAi at a concentration of 200 μg / mL and citric acid-sodium citrate buffer at a concentration of 0.1 mol / L, add an equal volume of fusion peptide at a concentration of 50 μg / mL dropwise After the dropwise addition of the solution, continue to vortex for 60 minutes, and then stand still for 30 minutes to obtain the CKIP-1 RNAi complex targeting macrophages.

[0046] Drop the freshly prepared CKIP-1 RNAi complex on a 200-mesh copper grid, absorb it for 3 minutes, blot it with absorbent paper, dry it in the air for 30 seconds, negatively stain it with 1% uranyl acetate aqueous solution for 30 seconds, and use absorbent paper Blot dry, dry for 30 seconds, and observe with 80kV transmission electron microscope. The results are attached figure 1 As shown, the obt...

Embodiment 3

[0048] Example 3 Preparation of CKIP-1 RNAi complex

[0049] The nucleotide sequence of KIP-1 RNAi is: 5'-CCTGAGTGACTATGAGAAG-3' (SEQ ID No.4). Under the condition of vortexing, to the aqueous solution containing CKIP-1 RNAi at a concentration of 200 μg / mL and citric acid-sodium citrate buffer at a concentration of 0.1 mol / L, add an equal volume of fusion peptide at a concentration of 50 μg / mL dropwise After the dropwise addition of the solution, continue to vortex for 90 minutes, and then stand still for 60 minutes to obtain the CKIP-1 RNAi complex targeting macrophages.

[0050] Drop the freshly prepared CKIP-1 RNAi complex on a 200-mesh copper grid, absorb it for 3 minutes, blot it with absorbent paper, dry it in the air for 30 seconds, negatively stain it with 1% uranyl acetate aqueous solution for 30 seconds, and use absorbent paper Blot dry, dry for 30 seconds, and observe with 80kV transmission electron microscope. The results are attached figure 1 As shown, the obta...

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Abstract

The invention belongs to the field of biological medicine, and particularly relates to CKIP-1 RNAi compound of macrophage targeted and a preparation method and application thereof. The CKIP-1 RNAi compound is formed by fusion peptides and CKIP-1 RNAi which are connected and combined through an electrostatic interaction; the fusion peptides are composed of V6 peptides and T9 peptides; and the nucleotide sequence of the CKIP-1 RNAi is as shown in SEQ ID NO.4. The fusion peptides are compounded, purified and then are connected and combined with the CKIP-1 RNAi to obtain the CKIP-1 RNAi compound.The CKIP-1 RNAi compound specifically targets CD11b receptors on the surfaces of macrophages and meanwhile efficiently transfects the macrophages, and expression of the CKIP-1 and activation of the macrophages are inhibited, so that inflammatory response of cerebral hemorrhage mediated by the macrophages is inhibited, and the CKIP-1 RNAi compound can be used for preparing anti-inflammatory drugs for treating the cerebral hemorrhage mediated by the macrophages, and good development application prospects are achieved in the cerebral hemorrhage field.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a macrophage-targeted CKIP-1 RNAi complex and a preparation method and application thereof. Background technique [0002] Intracerebral hemorrhage (ICH) is a common clinical cerebrovascular disease, lack of effective treatment, high mortality and disability rates. The secondary neurological injury a few days after cerebral hemorrhage is a critical period of exacerbation. The mechanism of secondary nerve injury is complex and may involve the formation of perihematomal cerebral edema, abnormal metabolism around hematoma, and changes in regional cerebral blood flow (rCBF) around hematoma. Related studies have shown that the inflammatory response mediated by macrophages plays an important role in the secondary nerve injury of cerebral hemorrhage. At present, it is believed that the inflammatory response induced by hematoma components plays an important role in secondary nerve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/42A61K31/7088A61P9/10A61P25/00A61P29/00C07K19/00C07K1/16C07K1/04
CPCA61K31/7088A61K47/42A61P9/10A61P25/00A61P29/00C07K14/705C07K2319/00
Inventor 石会
Owner AFFILIATED YONGCHUAN HOSPITAL OF CHONGQING MEDICAL UNIV
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