Cationic liposome-protamine-mRNA tumor vaccine and preparation method and application method thereof

A cationic liposome and protamine technology, applied in the field of nanomaterials and nucleic acid vaccines, can solve the problems of weak immunogenicity, poor patient compliance, single vaccination route, etc., and achieve high safety, simple cost, synthesis and purification fast effect

Pending Publication Date: 2020-03-17
NINGXIA MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to provide a cationic liposome-protamine-mRNA tumor vaccine, thereby overcoming the defects of weak immunogenicity, single inocul

Method used

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  • Cationic liposome-protamine-mRNA tumor vaccine and preparation method and application method thereof
  • Cationic liposome-protamine-mRNA tumor vaccine and preparation method and application method thereof
  • Cationic liposome-protamine-mRNA tumor vaccine and preparation method and application method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1: Preparation of DDA / TDB-protamine-(CK19) mRNA vaccine by film dispersion method

[0037] Put DDA and TDB in a 50 ml round bottom flask with a molar ratio of 6-10:1, add 1-2 ml of chloroform and methanol to dissolve (chloroform:methanol 9:1), and rotate under reduced pressure in a constant temperature water bath at 37°C Remove the organic solvent by evaporation to form a uniform lipid film, and then pass through 2 minN 2 , remove the residual solvent. Then add 5ml of Tris-HCl buffer (pH 6.8-9) with a concentration of 10 mM, and hydrate at 60°C for 30 minutes to obtain blank liposomes with a particle size of about 50-800 nm. Then according to liposome: protamine: mRNA mass ratio of 5:1:1 ~ 15:2:1, the protamine condensed mRNA (protamine is added to mRNA to condense and stand still). Add it into the above-mentioned blank liposome, make it fully fused, and let it rest for 20-40 minutes to obtain a cationic liposome-protamine-mRNA vaccine, which is ready for use....

Embodiment 2

[0038] Example 2: Preparation of DOTAP / Chol / DSPE-PEG200-protamine-(CK19) mRNA vaccine by film dispersion method

[0039] Put DOTAP, Chol and DSPE-PEG2000 in a molar ratio of 1:1:0.1 to 1:1:1 in a 50 ml round bottom flask, add 1 to 2 ml of chloroform and methanol to dissolve (chloroform:methanol 9:1), Remove the organic solvent by rotary evaporation under reduced pressure under heating in a water bath to form a uniform lipid film, and then pass it through for 2minN 2 , remove the residual solvent. Then add 5 ml of Tris-HCl buffer solution (pH 5-9) with a concentration of 10 mM, and hydrate at 60°C for 30 min to obtain blank liposomes with a particle size of about 50-800 nm. Then according to liposome:protamine:mRNA mass ratio of 5:1:1~15:2:1, protamine condensed mRNA (add protamine to mRNA to condense, stand still) add to In the above-mentioned blank liposome, make it fully fused, and stand still for 20 to 40 minutes to obtain a cationic liposome-protamine-mRNA vaccine, which...

Embodiment 3

[0041] Example 3: Preparation of DOTAP / Chol / DSPE-PEG200-protamine-(CK19) mRNA vaccine by freeze-drying method

[0042] Put DOTAP, Chol and DSPE-PEG2000 in a molar ratio of 1:1:0.1 to 1:1:1 in a 50 ml round bottom flask, add 1 to 3 ml of chloroform and methanol to dissolve (chloroform:methanol 9:1), Remove the organic solvent by rotary evaporation under reduced pressure under heating in a water bath to form a uniform lipid film, and then pass it through for 2 to 4 minutes under N 2 Remove residual solvent. Then add 2 ml of Tris-HCl buffer (pH 5-9) with a concentration of 10 mM, and hydrate at 50-70°C for 30 min-1 h to obtain blank liposomes with a particle size of about 50-800 nm. Then press liposome:protamine:mRNA mass ratio is 5:1:1~15:2:1, and add concentration and be 2% mannitol, sucrose, lactose, or trehalose as freeze-drying protection agent, make it Fully fused, then put it into the refrigerator to pre-freeze for 4, 8, and 12 hours, take it out, and freeze-dry it in a ...

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Abstract

The invention relates to a cationic liposome-protamine-mRNA tumor vaccine and a preparation method and an application method thereof. According to the tumor vaccine, a cationic liposome-protamine compound is used as a delivery carrier and adjuvant of mRNA, wherein the mass ratio of the cationic liposome to protamine to mRNA is 5:1: 1 to 15: 2:1, and the optimal mass ratio is preferably 10:1:1; theparticle size of the constructed cationic liposome-protamine-mRNA tumor vaccine is 50 to 800nm; the Zeta potential is 10 to 60mv; and the encapsulation efficiency is 70% to 95%. The cationic liposome-protamine-mRNA tumor vaccine prepared by the invention can effectively promote antigen uptake of APCs, induce DC stimulation and maturation, promotes secretion of cytokines and cause anti-tumor immune response. The tumor vaccine is used for immunotherapy through intramuscular and subcutaneous injection or nasal mucosa administration, wherein nasal mucosa administration can show a more excellent tumor treatment effect. The tumor vaccine has a wide application prospect in the aspect of tumor treatment.

Description

technical field [0001] The invention belongs to the technical field of nanomaterials and the field of nucleic acid vaccines, in particular to a cationic liposome-protamine-mRNA tumor vaccine and its preparation method and application in tumor treatment. Background technique [0002] In the face of life-threatening diseases such as cancer, vaccination is currently the most effective method for human prevention and treatment. With the in-depth study of tumor immunotherapy, therapeutic vaccines have great potential in the treatment of cancer and infectious diseases. Proteins, peptides and DNA plasmids are traditionally used as antigens for vaccine development. Protein and peptide vaccines are relatively easy to prepare and can be produced on a large scale, but they have weak immunogenicity, short duration of immune response, and limitations in the selection of antigenic peptides. Plasmid DNA vaccines have low immune effects and risks of uncontrolled genome mutation and immune t...

Claims

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Application Information

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IPC IPC(8): A61K39/00A61K39/39A61K9/127A61K47/28A61P35/00
CPCA61K39/001186A61K39/0011A61K39/001156A61K39/001106A61K39/001182A61K39/39A61K9/127A61K47/28A61P35/00A61K2039/53A61K2039/55516A61K2039/543A61K2039/876A61K2039/86
Inventor 杨建宏买亚萍郭珏铄苟国敬侯延辉李莉
Owner NINGXIA MEDICAL UNIV
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