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siRNA echovirus delivery system with shell-core structure and application of system

A technology of delivery system and core structure, which is applied in the field of siRNA imitation virus delivery system with shell-core structure, can solve the problems of poor escape ability of endosome, slow intracellular release, limited compression ability, etc., and achieve excellent active targeting performance, High gene load, reduced binding effect

Active Publication Date: 2018-04-06
NANTONG UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The present invention aims at the problems of low gene load, limited compression ability, fast elimination in vivo, poor endosome escape ability and slow intracellular release that still exist in the prior art siRNA delivery carrier, and the structure and function design of the simulated virus has a functional protein shell A virus-like capsid structure carrier composed of a gene compression core, the siRNA delivery system has the functions of extracellular stability, targeted uptake, endosome escape and intracellular rapid release of siRNA

Method used

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  • siRNA echovirus delivery system with shell-core structure and application of system
  • siRNA echovirus delivery system with shell-core structure and application of system
  • siRNA echovirus delivery system with shell-core structure and application of system

Examples

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Embodiment 1

[0037] Embodiment 1 The preparation of the siRNA imitation virus delivery system of the shell-core structure of the present invention

[0038] The c(RGDyK) used in the examples of the present invention was purchased from Gill Biochemical (Shanghai) Co., Ltd.

[0039] 1. Preparation and characterization of core and shell materials

[0040] (1) Core material: preparation of chitosan-disulfide bond-nona-arginine (CS-SS-9R)

[0041] 10 mg of chitosan (CS, MW: 10000) was dissolved in 4 mL of deionized water, and the pH value was adjusted to 6.0 with 2% triethylamine. 9.6 mg of N-succinyl-3-2-dithiopyridine-propionate (SPDP) was dissolved in 4mL DMSO, the CS solution was slowly added dropwise to the SPDP solution, and the reaction was performed under magnetic stirring at room temperature for 24h. The reaction solution was dialyzed with deionized water for 48 hours to remove unreacted raw materials and by-products. Subsequently, 1 mL of 9.5 mg / mL nona-arginine (9R-SH) was added to...

Embodiment 2

[0053] Example 2 Investigating the ability of the siRNA imitation virus delivery system of the present invention to carry siRNA

[0054] The mass ratios of CS-SS-9R and siRNA were selected as 5:1, 10:1, and 20:1, and the mass ratios of CS, 9R and siRNA were both 5:1. Dissolve siRNA in DEPC-treated water, add corresponding doses of CS, 9R, and CS-SS-9R solutions dropwise, vortex for 30 seconds, and place at room temperature for 30 minutes. Gel retardation experiments were used to investigate the binding ability of CS, 9R and CS-SS-9R to siRNA, the results are shown in Figure 4 . The bands condensed in the wells in the figure indicate that the binding ability between the carrier and siRNA is strong, and the migrating bands indicate that the binding ability is weak.

[0055] The results showed that the combination of chitosan and nona-arginine increased the binding capacity of siRNA. When the weight ratio of inner core material to siRNA was 5:1, the blocking ability of CS-SS-...

Embodiment 3

[0057] Example 3 Investigating the GSH-responsive release ability of the siRNA imitation virus delivery system of the shell-core structure of different core materials

[0058] Taking the core material CS-9R as a comparative example, the GSH-responsive release ability of the siRNA imitation virus delivery system with shell-core structure of different core materials was investigated.

[0059] 1. Preparation of CS-9R / siRNA / Arg-BSA-c(RGDyK):

[0060] Preparation of CS-9R: Dissolve 10mg chitosan (MW: 10000) in 4mL water, adjust the pH value to 6.0 with 2% triethylamine, dissolve 13.5mg Sulfo-SMCC in 2mL water, add the Sulfo-SMCC aqueous solution dropwise to the CS solution , the reaction was performed under magnetic stirring for 24 h at room temperature, and the by-products were removed by dialysis for 24 h. Subsequently, 9.5 mg of 9R-SH was added to the dialysate, reacted under magnetic stirring at room temperature for 24 hours, dialyzed in deionized water for 72 hours to remove ...

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Abstract

The invention discloses a siRNA echovirus delivery system with a shell-core structure. The core of the structure refers to polyarginine-modified or polyhistidine-modified chitosan coated siRNA, and the shell refers to albumin modified by polypeptide containing an RGD group sequence and by arginine. The delivery system can recognize tumor cells in a targeted mode, after the system enters in an endosome-lysosome acid environment, the protein shell is removed, the exposed core can escape from the endosome-lysosome, and under the intracellular GSH reduction condition, siRNA is released. The delivery system is good in stability, high in gene loading capacity and excellent in active target performance, and can significantly improve the gene silencing effect of siRNA and effectively inhibit the growth and metastasis of tumor cells.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a shell-core structure siRNA imitation virus delivery system and its application. Background technique [0002] RNA interference (RNA interference, RNAi) is a new technology developed in recent years. It can specifically degrade the target gene mRNA through exogenous or endogenous double-stranded RNA, inhibit gene expression and then down-regulate the target protein. This technology has unique advantages in the clinical treatment of genetic diseases and tumors. Small interfering RNA (siRNA) can be artificially designed and synthesized according to the sequence of the target gene mRNA, which has the characteristics of flexible design and strong pertinence. At present, four siRNA drugs have been used in early clinical research on tumors. Naked siRNA cannot be directly used in tumor gene therapy because it is easily degraded by nucleases in vivo, has a short half-l...

Claims

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Application Information

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IPC IPC(8): A61K47/69A61K47/64A61K47/61A61K31/713A61K48/00A61P35/00
CPCA61K31/713A61K48/0025
Inventor 许伯慧许燕朱红艳苏高星
Owner NANTONG UNIVERSITY
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