Positive ion lipid nanometer particle/DNA compound and preparation method thereof

Abstract

The invention provides a positive ion lipid nanometer particle / DNA compound and a preparation technology thereof. The preparation technology comprises the following steps that (1) a positive ion lipidmaterial is dissolved in absolute ethyl alcohol through heating; (2) an ethyl alcohol solution prepared in the step (1) is added to an aqueous phase solution dropwise, and positive ion lipid nanometer particles are formed through self-assembling; (3) residue ethyl alcohol in the positive ion lipid nanometer particles of the step (2) is removed; (4) filtering is carried out; (5) a DNA solution isprepared; (6) the prepared positive ion lipid nanometer particles in the step (4) and the DNA solution prepared in the step (5) are mixed according to a certain weight ratio to form the positive ion lipid nanometer particle / DNA compound; and (7) filtering is carried out. According to the preparation technology of the positive ion lipid nanometer particle / DNA compound, the operation of the preparation method is easy and rapid, the particle diameter of the prepared positive ion lipid nanometer particle / DNA compound is 50-150 nm, PDI<0.3, the positive ion lipid nanometer particle / DNA compound isdistributed in a mono-dispersion mode, the structure is stable, the compound is subjected to filtering sterilization at a terminal, and the security of the compound in clinic application of preparation of tumor curing medicine is effectively ensured.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a cationic lipid nanoparticle / DNA complex and a preparation method thereof. Background technique [0002] The key to the success of gene therapy is whether the therapeutic drug can be safely and effectively delivered into the target cells through the carrier in vivo. Gene therapy vectors are divided into viral vectors and non-viral vectors. Although viral vectors are used as efficient delivery systems to achieve target gene transfection and therapeutic purposes, viral vectors contain immunogenic viral proteins, limited target gene loads, and high prices, making lipids as non-viral vectors Nanoparticles (lipidnanoparticles, LNPs) have attracted widespread attention due to their good stability in vitro, degradability in vivo, safety and reliability, and have been widely used in gene therapy research for congenital and acquired genetic defects. LNPs refer to small v...

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Problems solved by technology

[0006] However, there are still many limiting factors in the ethanol injection method commonly used in industry at present, for example, the particle size and uniformity of the formed LNPs are significantly related to the concentration of lipid solution, stirring speed, lipid composition, and the ratio of oil phase to water phase. In addition, the ethanol injection method often uses mechanical force to inject the oil phase into the corresponding volume of the water phase according to a certain volume ratio to form LNPs. Due to the limitation of the volume of the water phase liquid storage tank, continuous production cannot be realized, which increases the batch production cost. difference between times

The above factors all limit the large-scale production of LNPs.

[0007] LNPs or liposomes prepared by ethanol injection often require a homogenization step (such as high-pressure homogenization, sonication, or extrusion) to improve the uniformity of the nanoparticles, but when using the extrusion method that is most suitable for large-scale production There are still some limitations:

[0008] (1) In the vertical extrusion process of the conventional 47mm liposome extruder, the filter membrane is easily blocked, and even affects the quality of the whole batch of liposome or LNPs products, which seriously limits the liposome or LNPs mass production of

[0009] (2) In order to expand the processing capacity of samples, the purpose of mass production is generally achieved by increasing the film area, but increasing the film area requires a larger extrusion device, which not only greatly increases the production cost, but also cannot guarantee the stability of extrusion

This method has the following problems: first, the cationic lipid nanoparticles and nucleic acid are mixed due to stirring, so there is a shear force, so that the mixing time of the two is short, and effective compounding cannot be achieved to form a stable complex; secondly, in order to prevent the complex from having a high local concentration and a large particle size and poor uniformity, the speed of dropping the nucleic acid is slow and the production time is long; thirdly, this method is limited by the size of the mixer volume and its stirring ability, and the average particle size of the prepared cationic lipid nanoparticle / nucleic acid complex is usually > 220nm, and the terminal filter sterilization of the complex cannot be achieved, so this method needs to be carried out in a clean bench or isolator, thus , this method is not suitable for large-scale production to prepare cationic lipid nanoparticles / nucleic acid complexes

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