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Lipid nanoparticle preparation and application thereof

A technology of lipid nanoparticles and preparations, which can be used in medical preparations without active ingredients, medical preparations containing active ingredients, DNA/RNA vaccination, etc., can solve problems such as limited application space, and achieve good in vitro transfection The effect of efficiency

Pending Publication Date: 2021-03-16
南京澄实生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, DNA vaccines have limited their application space due to the risk of high recombination with the host genome and can cause severe immune rejection (McNamara, Nair et al.2015)

Method used

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  • Lipid nanoparticle preparation and application thereof
  • Lipid nanoparticle preparation and application thereof
  • Lipid nanoparticle preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Embodiment 1 Lipid nanoparticle multi-component production procedure

[0081] First, DOTAP, DOPE and cholesterol were prepared into 10 mg / ml solutions in chloroform respectively. According to the molar ratio of DOTAP:DOPE:cholesterol=40:10:48, the above three solutions were mixed uniformly in a round bottom flask. Rotary evaporation under vacuum at 37°C for 1 h formed a film-like solid. Then add DPEC water to prepare a suspension with a concentration of 1 mg / ml. Sonicate for 1.5h, then overnight at 4°C, and sonicate again for 1h the next day to ensure full dissolution. First filter three times with a 0.45um PES filter, then filter three times with a 0.22μm PES filter, and finally filter three times with a 0.1um PES pinhole filter, so as to obtain unloaded nano lipids whose particle size meets the experimental requirements.

[0082] The concentration of the prepared unloaded nano-lipid is 1 mg / ml, and the unloaded nano-lipid and mRNA are prepared according to the char...

Embodiment 2

[0084] Example 2 explores the optimal ratio of nano-lipid and mRNA combination to obtain the best transfection effect

[0085] The charge carried by the cationic lipid in the nano-lipid, that is, the positive charge, and the charge carried by the phosphate group in the mRNA, that is, the negative charge, are converted so that the molar ratio of the positive charge to the negative charge (i.e. the quantity ratio) is 3:1, 2: 1, 1:1, 1.7:2, 1.5:2, 1:2 and 1:3. The specific experiment is to use HEK293T and DC2.4 cells as the research objects respectively. First, a certain number of HEK293T and DC2.4 cells are transferred to a 24-well culture plate for culture. Transfection was started when the cell number reached 50-60% by observation under an inverted phase-contrast microscope. Transfect according to the amount of transfection 1ug EGFP mRNA per well, at this time, the molar ratio of positive charge to negative charge (that is, the number ratio) is 3:1, 2:1, 1:1, 1.7:2, 1.5:2,1 ...

Embodiment 3

[0088] Example 3 Cell experiments assess the impact of clinically used injections on the transfection efficiency of preparations

[0089] The purpose of this experiment is to select the most suitable clinical commonly used injection, ensure that the preparation can exist stably in the injection and can effectively transfect cells, and at the same time ensure that the injection can be used in mammals without causing adverse reactions. Currently known injections commonly used clinically include physiological saline, sodium bicarbonate injection, sodium lactate Ringer injection and sodium potassium magnesium calcium glucose injection. In the experiment, normal saline, PBS (phosphate buffer saline), sodium bicarbonate injection, Opti-MEM, lactated Ringer injection and sodium potassium magnesium calcium glucose injection were used as transfection reagents to prepare EGFP mRNA-LNP3. The specific operation is to first transfer a certain number of HEK293T cells to a 24-well plate for ...

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Abstract

The invention discloses a lipid nanoparticle preparation and application thereof. The lipid nanoparticle preparation comprises lipid nanoparticles and a buffer system; the lipid nanoparticles are obtained by encapsulating at least one RNA molecule for encoding an antigen with non-load nano lipid; and the non-loaded nanolipid comprises cationic lipid and neutral lipid at least selected from the combination of DOPE and cholesterol. The three-component combination preparation adopted by the invention is obviously superior to a two-component combination preparation when mRNA transfection is carried out in vitro. According to the invention, compared with a nano-lipid formed by a cationic lipid and a neutral lipid, the three-component lipid nano-particle formed by combining two neutral lipids and the cationic lipid in a specific ratio shows higher transfection efficiency and mRNA expression efficiency in the mRNA coating and in-vitro cell transfection processes.

Description

technical field [0001] The invention belongs to the field of pharmaceutical preparations, and relates to a lipid nanoparticle preparation and an application thereof. Background technique [0002] Vaccines usually consist of antigens that are recognized by the immune system and are often given with adjuvants that boost immunity. Vaccines are generally divided into two main categories, therapeutic vaccines and prophylactic vaccines. Preventive vaccines are mainly used to prevent diseases by producing antibodies and immune cells against pathogens. Therapeutic vaccines are used for treatment after the onset of disease, and can produce immune cells (mainly CD4+ and CD8+ T cells) and neutralizing antibodies that kill pathogens in a short period of time. Therapeutic vaccines have great application value in the treatment of life-threatening diseases including tumors and infectious diseases. [0003] In previous vaccine development, protein peptides and DNA plasmids were usually u...

Claims

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Application Information

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IPC IPC(8): A61K9/08A61K9/19A61K9/51A61K39/00A61K47/26A61K47/28A61K47/24A61K47/18
CPCA61K9/0019A61K9/08A61K9/19A61K9/5123A61K39/00A61K47/26A61K2039/5154A61K2039/5156A61K2039/53
Inventor 韩悌云徐实
Owner 南京澄实生物科技有限公司
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