Quaternized chitosan/siRNA (small interfering Ribonucleic Acid) composite particles and preparation method thereof

A technology of quaternized chitosan and composite particles, which can be used in drug combinations, pharmaceutical formulations, preparations for in vivo tests, etc. High density and good solubility

Inactive Publication Date: 2012-09-12
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, although viral siRNA vectors have high delivery efficiency, potential biosafety issues restrict their wide application.

Method used

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  • Quaternized chitosan/siRNA (small interfering Ribonucleic Acid) composite particles and preparation method thereof
  • Quaternized chitosan/siRNA (small interfering Ribonucleic Acid) composite particles and preparation method thereof
  • Quaternized chitosan/siRNA (small interfering Ribonucleic Acid) composite particles and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0028] 37 o C. Add diethyl pyrocarbonate (DEPC) into Milli-Q water under stirring, so that the volume concentration of diethyl pyrocarbonate is 0.1%, at 121 oC , 0.1MPa sterilization for 30min. Prepare quaternized chitosan (quaternary ammonium degree of 32%, molecular weight 5w, degree of deacetylation 85%) solution and 200μg / mL GAPDH-siRNA solution. The GAPDH-siRNA solution is added dropwise in the quaternized chitosan solution, vortexed and mixed evenly to obtain a mixed solution, the vortexed oscillation speed is 3000rpm, and the volume ratio of the GAPDH-siRNA solution to the quaternized chitosan solution is 1: 29. Mix the solution at 37 o C was left to incubate for 1 h, and the ratio of nitrogen / phosphorus (NP) (the molar ratio of N atoms on quaternized chitosan to P atoms on GAPDH-siRNA) was 1, 2.5, 5, 10, 20, 50, respectively. , 100 quaternized chitosan / GAPDH-siRNA composite particles. Thus, composite particles with different sizes, different surface charges, and...

Embodiment 2

[0030] 37 o C. Add diethyl pyrocarbonate (DEPC) into Milli-Q water under stirring, so that the volume concentration of diethyl pyrocarbonate is 0.1%, at 121 o C, 0.1MPa sterilization for 30min. Prepare 17.2μg / mL, 34.5μg / mL, 69μg / mL quaternized chitosan (60% quaternization degree, 150w molecular weight, 88% deacetylation degree) solution and 400μg / mL of Col Ⅰ-siRNA solution. Add the Col Ⅰ-siRNA solution dropwise to the quaternized chitosan solution, vortex and mix evenly to obtain a mixed solution, the vortex oscillation speed is 3000rpm, and the volume ratio of the Col Ⅰ-siRNA solution to the quaternized chitosan solution is 1:58. Mix the solution at 25 o C was left to incubate for 0.5 h to prepare quaternized chitosan / Col Ⅰ-siRNA composite particles with NP ratios of 5, 10, and 20, respectively. Composite particles in DMEM medium with a final concentration of 25% fetal bovine serum37o After incubation in C, after 80 o C water bath for 5 minutes to terminate the serum a...

Embodiment 3

[0032] 37 o C. Add diethyl pyrocarbonate (DEPC) into Milli-Q water under stirring, so that the volume concentration of diethyl pyrocarbonate is 0.1%, and then 121 oC , 0.1MPa sterilization for 30min. Prepare 8.62 μg / mL, 17.2 μg / mL, 34.5 μg / mL, 69 μg / mL, and 138 μg / mL quaternized chitosan (degree of quaternization is 22%, molecular weight 10w, deacetylation degree 82%) solution and 200 μg / mL fluorescently labeled siRNA solution. The siRNA solution was added dropwise into the quaternized chitosan solution, vortexed and mixed evenly to obtain a mixed solution, the vortexed oscillation speed was 3000 rpm, and the volume ratio of the siRNA solution to the quaternized chitosan solution was 1:29. Mix the solution at 25 o C was left to incubate for 1 h to prepare quaternized chitosan / FAM-siRNA composite particles with NP ratios of 2.5, 5, 10, 20, and 50, respectively. After HEK293 cells were cultured for 24 hours, each NP ratio composite particle, naked siRNA and Lipofectamine we...

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Abstract

The invention discloses quaternized chitosan/siRNA (small interfering Ribonucleic Acid) composite particles and a preparation method thereof. Electrostatic interaction between a quaternary ammonium group with positive charges and a siRNA molecule with negative charges on quaternized chitosan is used as driving power; molecule self-assembly is initiated in a water phase through vortex oscillation; and the high-bioactivity quaternized chitosan/siRNA composite particles with controllable size and surface charge are prepared by adjusting and controlling parameters such as molecular charge ratio, incubation temperature and incubation time. According to the quaternized chitosan/siRNA composite particles and the preparation method thereof, the quaternized chitosan is used as a siRNA transfer carrier, and the material has low cytotoxicity, wide source and low cost; a preparation process of the composite particles has mild conditions and is simple and practical; and the bioactivity of the siRNA can be effectively kept. The siRNA used for preparing the composite particles can be effectively intaken by cells and a mum effect of the target gene can be induced aiming at specific design of a target gene; and the composite particles have good application prospect in the fields of cancer treatment, tissue regeneration and repair and the like.

Description

technical field [0001] The invention relates to a siRNA composite particle, in particular to a quaternized chitosan / siRNA composite particle constructed by using quaternized chitosan as an siRNA delivery carrier and a preparation method thereof. Background technique [0002] RNA interference is a specific gene silencing phenomenon that occurs in the post-transcriptional stage, and the RNA interference phenomenon mediated by small interfering RNA (siRNA) homologous to the target gene sequence is the most extensively studied. With the development of chemically synthesized siRNA technology, RNA interference is increasingly used in the fields of diagnosis and therapy. siRNA is a biomacromolecule composed of about twenty nucleotides. In vivo applications, siRNA is easily digested by enzymes, easily cleared by the immune system, and low cellular uptake rate. Therefore, it is of great significance to select a suitable siRNA delivery carrier and prepare safe, stable and efficient c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K47/48A61K49/00A61P35/00
Inventor 马列刘幸高长有
Owner ZHEJIANG UNIV
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