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A preparation method of chitosan derivative nanoparticles delivering siRNA

A chitosan derivative and nanoparticle technology, applied in the field of gene biology, to achieve the effect of promoting the release of siRNA, inhibiting proliferation and migration, and the method is simple

Active Publication Date: 2022-02-11
HEFEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no nanoparticles formed by encapsulating siRNA with carboxymethyl chitosan and chitosan hydrochloride, which can promote the uptake of siRNA by cells, protect siRNA from degradation in gastric juice, and control the release of siRNA in the intestinal tract

Method used

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  • A preparation method of chitosan derivative nanoparticles delivering siRNA
  • A preparation method of chitosan derivative nanoparticles delivering siRNA
  • A preparation method of chitosan derivative nanoparticles delivering siRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The concrete operating steps of the chitosan hydrochloride powder of preparation FITC mark are as follows:

[0026] 10 mg chitosan hydrochloride was completely dissolved in 10 mg deionized water to obtain a chitosan hydrochloride solution with a concentration of 1 mg / mL. Add 10 mL of fluorescein isothiocyanate (FITC) solution (5.8 mg / L, anhydrous methanol) with a concentration of 5.8 mg / L to the chitosan hydrochloride solution with a concentration of 1 mg / mL, and stir under magnetic stirring (500 rpm ) for 4 h. Then add 1 M concentration of hydroxide (NaOH) to adjust the pH value to 10, and precipitate FITC-labeled chitosan hydrochloride. The free FITC was washed with ultrapure water and centrifuged until no fluorescence was detected in the supernatant. Finally, FITC-labeled chitosan hydrochloride powder was obtained by freeze-drying.

[0027] Prepare siRNA-CDNPs according to siRNA in Table 1, different volumes of CHC and different volumes of CMC (600 μL, 500 μL, 400...

Embodiment 2

[0033] Application of siRNA-CDNPs in the controlled release of siRNA in a simulated environment of colon cancer cells:

[0034]The siRNA-CDNPs prepared in Example 1 were incubated in a gastric environment (pH value = 2.2, 120 rpm) at 37°C for different times, up to 120 min. The above acidic solution was then mixed with NaOH (1 M) to adjust the pH to a weak acid (pH = 5.5). Then sonicate for 8 min, incubate at 37°C, and shake well (120 rpm) for 112 min. Samples were taken at predetermined times (0, 30, 60, 90, 120, 128, 150, 180, 210 and 240 min). Samples were analyzed on 5% (w / v) polyacrylamide gel, electrophoresis was performed at 120 mV in 1× TBE buffer, the gel was stained with ethidium bromide, and observed with a gel imager FLA 7000, the results were as follows figure 2 shown.

[0035] The release of siRNA from siRNA-CDNPs was detected by polyacrylamide gel electrophoresis by analyzing the siRNA content at pH 2.2 and pH 5.5. figure 2 The red and white arrows in ind...

Embodiment 3

[0037] Application of siRNA in siRNA-CDNPs efficiently internalized by HT-29 cells:

[0038] Incubate colon cancer cells (HT-29) with siRNA-CDNPs prepared in Example 1, and analyze the transfection effect of siRNA. The specific steps are as follows: Add 100 μL siRNA-CDNPs (containing 0.55 nmol siRNA) to HT-29 cells (1×10 5 / well) for 16 h. The added siRNA-CDNPs were then gently absorbed, and the cells were washed three times with PBS. Cells were fixed with 4% paraformaldehyde in PBS for 15 min, washed twice with PBS, and nuclear stained with DAPI (4′,6-diamino-2-phenylindole) for 10 min. Cells were washed again with PBS, rinsed with ultrapure water, and placed on microscope slides. Fluorescence images were taken with a fluorescence microscope, and the results were as follows image 3 shown.

[0039] The siRNA-CDNPs prepared in Example 1 were added to colon cancer cells (HT-29), and the transfection effect of siRNA was analyzed. The results were as follows: image 3 shown...

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Abstract

The invention relates to a preparation method of chitosan derivative nanoparticles for delivering siRNA, belonging to the technical field of gene biology. The present invention mixes positively charged chitosan hydrochloride solution with siRNA solution that can inhibit colon cancer cell proliferation and migration-related protein expression, and then adds negatively charged carboxymethyl chitosan solution, through intermolecular electrostatic interaction function and self-assemble into chitosan-derived nanoparticles (siRNA‑CDNPs) for delivery of siRNA. The chitosan derivative nanoparticles for delivering siRNA can effectively protect siRNA under the condition of pH value 1.2, and realize the controllable release of siRNA by responding to external stimuli (ultrasound) under the condition of pH value 5.5 in the environment of colon cancer cells ; siRNA‑CDNPs can effectively inhibit the expression of β‑catenin protein in colon cancer cells, and is expected to inhibit the proliferation and migration of cancer cells. The preparation method of the invention is simple to operate, environmentally friendly and low in cost.

Description

technical field [0001] The invention belongs to the technical field of gene biology, and in particular relates to the preparation and application of a pH-sensitive siRNA / chitosan derivative nano system. Background technique [0002] Gene-based therapeutic approaches hold great potential to modulate the silencing of large numbers of target mRNA molecules, ultimately reducing target protein levels. However, the application of this gene silencing technology still needs to overcome challenges, including the rapid degradation of siRNA under physiological conditions and the difficulty of crossing the negatively charged plasma membrane. A key challenge in achieving siRNA therapeutic efficacy is the development and design of novel degradable vectors with safe and sufficient gene delivery efficacy. Currently, the 5-year survival rate for patients with colon cancer liver metastases treated with chemotherapy or resection is less than 50%. It is generally believed that compared with r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61K31/713A61K9/51A61K47/69A61P35/00A61P35/04
CPCA61K31/713A61K9/5161A61K47/6939A61P35/00A61P35/04
Inventor 郑磊颜玲刘长虹高胜杰刘帅
Owner HEFEI UNIV OF TECH
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