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Cationic cholesterol derivative, nano-composite and preparation method and application of nano-composite

A technology of cationic cholesterol and nanocomposite, which is applied to other methods of inserting foreign genetic materials, steroids, pharmaceutical formulations, etc., can solve the problems of Linker chain length, inability to detach from auxiliary lipids, and failure to provide preparation methods, etc. Achieve the effect of convenient preparation, significant transfection effect, and easy low-cost large-scale preparation

Pending Publication Date: 2022-04-15
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it still has the following defects: (1) its application in gene transfection preparations cannot break away from the help of auxiliary lipids
(2) fail to provide a kind of stable and controllable preparation method equally
The linker chain length of the lipid-like functional molecule in this prior patent is relatively short, and the stability of binding to siRNA may be lacking, so an auxiliary lipid is needed

Method used

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  • Cationic cholesterol derivative, nano-composite and preparation method and application of nano-composite
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  • Cationic cholesterol derivative, nano-composite and preparation method and application of nano-composite

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051]

[0052] Preparation of Cationic Cholesterol Derivative Chol-6C-Lys

[0053] Step 1: Dissolve 1,6-hexanediol (23.6g, 0.2mol) in 80mL of fully dehydrated and dried dichloromethane, and slowly add dropwise to cholesterol chloride dissolved in 200mL of dry dichloromethane containing 0.4mL pyridine Formic acid ester (22.4g, 0.05mol), stirred at 45°C for 24h, distilled off dichloromethane, and the intermediate cholesterol monosubstituted hexanediol carbonate intermediate was obtained after column chromatography. The synthesis yield was 74%.

[0054] The second step: dissolve the L-lysine (7.3g, 0.05mol) protected by BOC (di-tert-butyl carbonate) in 100mL of dehydrated and dried methylene chloride, and slowly add it dropwise to the dichloromethane dissolved in dichloromethane under the catalysis of pyridine. In the first step of the preparation of methyl chloride, the obtained intermediate cholesterol monosubstituted hexanediol was added with dicyclohexylcarbodiimide (DCC...

Embodiment 2

[0057] Preparation of siRNA / Chol-6C-Lys nanocomplex by microfluidic method.

[0058] 1. Reagent preparation

[0059] Enzyme-free aqueous phase: 5 mL of deionized water was treated with DEPC to make it RNase-free.

[0060] 2. Preparation of nanocomposites

[0061] Weigh the cationic cholesterol derivative Chol-6C-Lys synthesized in Example 1 containing the natural cholesterol skeleton and lysine head group in a centrifuge tube without enzyme treatment, and dissolve it into a derivative solution with the water phase without enzyme treatment, The concentration of Chol-6C-Lys in the derivative solution is 2.5 mg / mL; the siRNA powder is dissolved in an enzyme-free aqueous phase to prepare an siRNA solution with an siRNA concentration of 8 μmol / L. The above-mentioned one derivative solution and one nucleic acid drug solution are respectively loaded into the two injection tubes of the microfluidic pump. The total flow rate of the microfluidic syringe pump was 200 μL / min, the flow ...

Embodiment 3

[0066] Preparation process of microRNA / Chol-6C-Lys nanocomplex.

[0067] 1. Reagent preparation

[0068] Enzyme-free aqueous phase: Treat 5 ml of 10mmol / L phosphate buffer with DEPC to make it free of RNase.

[0069] 2. Preparation of nanocomposites

[0070] Weigh the cationic cholesterol derivative Chol-6C-Lys synthesized in Example 1 containing the natural cholesterol skeleton and lysine head group in a centrifuge tube without enzyme treatment, and dissolve it into a derivative solution with the water phase without enzyme treatment, The concentration of Chol-6C-Lys in the derivative solution is 5 mg / mL; the microRNA powder is dissolved in an enzyme-free aqueous phase to prepare a microRNA solution with a concentration of 8 μmol / L. Take 500 μL cationic cholesterol derivative solution in a centrifuge tube, place it on a vortex mixer, then slowly add 500 μL microRNA solution dropwise, the vortex mixer rotates at 2000 rpm, vortex lasts for 20 seconds, and stands still for 30 ...

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Abstract

The invention provides a cationic cholesterol derivative, a nano-composite as well as a preparation method and application of the nano-composite, and particularly relates to a cationic lipid gene transfection reagent. The invention relates to a preparation method of a cationic cholesterol derivative containing a natural cholesterol skeleton and a lysine head group, in particular to a synthesis method of a cationic cholesterol derivative containing a natural cholesterol skeleton and a lysine head group, a preparation method of a nano-composite and an application of the nano-composite serving as an efficient gene vector to a small interfering RNA (siRNA) and microRNA transfection reagent. According to the cationic cholesterol derivative containing the natural cholesterol skeleton and the lysine head group provided by the invention, the Linker chain length most suitable for siRNA combination is selected, and the nano-composite provided by the invention preferably adopts a micro-fluidic technology to systematically optimize various parameters (including total flow velocity, flow velocity ratio, buffer system, chip structure and the like), so that the stability of the nano-composite is improved, and the stability of the nano-composite is improved. A stable nano compound is formed, and efficient gene delivery capability can be realized without auxiliary lipid.

Description

technical field [0001] The present invention relates to a cationic lipid gene transfection reagent, in particular to a cationic cholesterol derivative, a nanocomposite and its preparation method and application, in particular to a cationic cholesterol derivative containing a natural cholesterol skeleton and a lysine head group , and nanocomposite and its preparation method and its application as high-efficiency gene carrier for small molecule interfering RNA (siRNA) and microRNA transfection reagent. Background technique [0002] Constructing a gene delivery system with good biocompatibility, gene stability and high transfection efficiency is one of the current research hotspots. With the successful application of gene therapy and gene medicine, non-viral vectors may drive further innovations in gene delivery systems. In recent years, nonviral vectors for gene delivery have developed rapidly, among which various cationic lipids and cationic polymers have been reported as pr...

Claims

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Application Information

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IPC IPC(8): C07J9/00C12N15/87A61K47/54A61K31/713A61K31/7088
Inventor 陈剑朱照远张莉
Owner SHANGHAI JIAO TONG UNIV
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