WO2024136254A1 - Lipid nanoparticles comprising low-concentration ionizable lipids, and preparation method therefor - Google Patents

Abstract

The present invention relates to lipid nanoparticles comprising low-concentration ionizable lipids, and a preparation method therefor, and has a low amount of ionizable lipids in the lipid nanoparticles such that cytotoxicity can be reduced. In addition, according to the preparation method for lipid nanoparticles, of the present invention, the lipid nanoparticles have a uniform spherical shape and an excellent nucleic acid encapsulation rate, and, when lipid nanoparticles are prepared, amounts of non-ionized lipids and fusogenic lipids are increased such that a plurality of lipid nanoparticles can be prepared using an amount of nucleic acids which are the same as those used for conventional lipid nanoparticles, and, when the same nucleic acids are injected, the number of lipid nanoparticles to be injected into the body is increased such that the protein expression level of the nucleic acids can be increased.

Description

Lipid nanoparticles containing low concentration ionized lipids and method for producing the same

The present invention relates to lipid nanoparticles containing low concentration ionized lipids and a method for producing the same.

Nucleic acid-based medicines, which started about 40 years ago by injecting plasmid DNA into the human body to help produce deficient proteins, have since been reported in various types such as antigene, decoy, antisense, siRNA, and miRNA that inhibit gene transcription and translation.

Nucleic acid-based medicines target DNA or RNA rather than proteins and have received attention as personalized treatments through complementary binding to DNA or RNA of a specific sequence. Nucleic acid-based medicines are used not only as therapeutic agents but also as preventive agents that protect against diseases by injecting genes that can express antigens for specific diseases.

Gene-based vaccines are divided into DNA vaccines, RNA vaccines, and viral vector vaccines. Among these, RNA vaccines are viral vector-based vaccines that inject mRNA encoding an antigen into the human body to express the antigen within the body and induce the formation of antibodies against it. It has been spotlighted as an effective response to COVID-19, which broke out in 2019, as it has no concerns about infection with vaccines or the potential risk of genetic mutation with DNA vaccines, and has the advantage of being able to be developed quickly.

However, nucleic acid-based medicines are easily degraded by nucleic acid hydrolyzing enzymes in the human body and are negatively charged macromolecules, so they have the disadvantage of not being easily delivered into cells, so a method of delivering them to the desired location stably and efficiently is needed.

As delivery systems for nucleic acids, delivery techniques based on various materials such as lipids, polymers, dendrimer, and inorganic metal materials have been reported, including patisiran, the first siRNA new drug approved by the FDA in 2018, as well as COVID-19 approved for emergency use in 2020. The mRNA vaccine for Korea also used lipid nanoparticles.

Current lipid nanoparticles are generally used in the form of a mixture of four components: ionized lipid, phospholipid (helper lipid), cholesterol (structure-maintaining lipid), and PEG-lipid in a certain ratio.

The lipid nanoparticle contains mRNA inside, and the lipid nanoparticle protects the mRNA from being decomposed by enzymes in vivo and neutralizes the negative charge of the mRNA to increase cell membrane penetration efficiency. Additionally, lipid nanoparticles that enter the cell are separated from the mRNA and decomposed, and the mRNA participates in the protein expression process in the cytoplasm.

These lipid nanoparticle-based carriers necessarily contain positively charged ionized lipids in order to contain negatively charged mRNA, but the toxicity of the ionized lipids may be a problem.

Therefore, the development of new lipid nanoparticles is necessary.

[Prior art literature]

[Patent Document]

KR 10-2569192 B1

The object of the present invention relates to lipid nanoparticles containing low concentration ionized lipids and a method for producing the same.

Another object of the present invention is to provide lipid nanoparticles that can reduce cytotoxicity due to the low content of ionized lipids in the lipid nanoparticles.

Another object of the present invention is that it has a uniform spherical shape, has an excellent encapsulation rate of nucleic acids, and increases the content of non-ionized lipids and fusible lipids when producing lipid nanoparticles, thereby increasing the content of the same nucleic acid compared to conventional lipid nanoparticles. It provides a method for producing lipid nanoparticles that can be manufactured into a large number of lipid nanoparticles using , and when the same nucleic acid is injected, the number of lipid nanoparticles injected into the body increases, thereby increasing the protein expression level of the nucleic acid.

In order to achieve the above object, the present invention relates to lipid nanoparticles containing low concentration ionized lipids, including nucleic acids, ionizable lipids, non-ionizable lipids, neutral lipids, and fusion lipids. , the ionized lipid may be included in an amount of 25 mol% or less based on the total weight of lipid in the lipid nanoparticle.

Additionally, the ionized lipid and the non-ionized lipid may be included in a molar ratio of 1:0.3 to 1:3.

Additionally, the ionized lipid and the fusible lipid may be included in a molar ratio of 1:1 to 1:10.

Additionally, the lipid nanoparticles may have a uniform spherical shape and have a polydispersity index of 0.05 to 0.1.

Additionally, the average diameter of the lipid nanoparticles may be 70 nm to 100 nm.

In addition, the nucleic acids include RNA, DNA, short interfering RNA (siRNA), messenger RNA (mRNA), aptamer, antisense oligodeoxynucleotide (ODN), antisense RNA, ribozyme, and DNA enzyme. (DNAzyme) and mixtures thereof.

In addition, the non-ionized lipids include distearoylphosphatidylcholine (DSPC), dioleolphosphatidyl ethanolamine (DOPE), bis(diphenylphosphino)ethane (DPPE), diacyl phosphatidylcholine, diacylphosphatidylethanolamine, and diacylphosphatidylethanolamine. sphatidylserine ) and mixtures thereof.

In addition, the neutral lipid is a group consisting of polyethylene glycol 2000 distearoylphosphatidylethanolamine (PEG (2000) DSPE), DMG-PEG, PEG-DMPE, DPPE-PEG, DPG-PEG, PEG-DOPE, and mixtures thereof. can be selected from

Additionally, the fusible lipid may be selected from the group consisting of phospholipids, cholesterol, tocopherol, and mixtures thereof.

A method for producing lipid nanoparticles containing a low concentration of ionized lipid according to another embodiment of the present invention includes ionizable lipid in an organic solvent; non-ionizable lipid; neutral lipid; and dissolving the fusible lipid to prepare an oily solution; Preparing an aqueous solution containing nucleic acids; and manufacturing lipid nanoparticles by injecting the oil phase solution and the aqueous solution into a chip for producing lipid nanoparticles, wherein the chip for producing lipid nanoparticles includes an oil phase solution supply unit, an aqueous solution supply unit, a mixer unit, and a discharge unit; , the oil phase solution is injected into the oil phase solution supply section, the aqueous phase solution is injected into the aqueous phase solution supply section, the oil phase solution is injected into the mixer section through the oil phase solution supply section, and the aqueous phase solution is injected into the mixer section through the aqueous phase solution supply section. , the oil phase solution and the aqueous phase solution are mixed in the mixer unit to form lipid nanoparticles, and the ionized lipid may be included in an amount of 25 mol% or less based on the total weight of lipids in the lipid nanoparticles.

The present invention has a low content of ionized lipids in lipid nanoparticles, which can reduce cytotoxicity.

In addition, according to the method for producing lipid nanoparticles of the present invention, the spherical shape is uniform and the encapsulation rate of nucleic acids is excellent, and when producing lipid nanoparticles, the content of non-ionized lipids and fusible lipids is increased to replace conventional lipid nanoparticles. Compared to nanoparticles, lipid nanoparticles can be manufactured into multiple lipid nanoparticles using the same nucleic acid content, and when the same nucleic acid is injected, the number of lipid nanoparticles injected into the body increases, thereby increasing the protein expression level of nucleic acids. It relates to a manufacturing method.

Figure 1 is a schematic diagram for comparison between the lipid nanoparticles of the present invention and conventional lipid nanoparticles.

Figure 2 is an example of a chip for producing lipid nanoparticles that can produce lipid nanoparticles of the present invention.

Figure 3 is an example of a chip for producing lipid nanoparticles that can produce lipid nanoparticles of the present invention.

Figure 4 is an example of a chip for producing lipid nanoparticles that can produce lipid nanoparticles of the present invention.

Figure 5 shows the average diameter and PDI measurement results of lipid nanoparticles according to an embodiment of the present invention.

Figure 6 shows the results of the encapsulation rate of lipid nanoparticles according to an embodiment of the present invention.

Figure 7 shows the results of a test on the shape change of cells treated with lipid nanoparticles according to an embodiment of the present invention.

Figure 8 shows the results of a cytotoxicity test of lipid nanoparticles according to an embodiment of the present invention.

Figure 9 shows the results of a cytotoxicity test on conventional lipid nanoparticles and lipid nanoparticles containing low concentration ionized lipid according to an embodiment of the present invention.

Figure 10 shows the results of a test on the level of protein expression for conventional lipid nanoparticles and lipid nanoparticles containing low concentration ionized lipid according to an embodiment of the present invention.

Figure 11 is a test result of the number of lipid nanoparticles formed depending on the concentration of ionized lipid according to an embodiment of the present invention.

Figure 12 is a test result of the number of lipid nanoparticles formed depending on the concentration of ionized lipid according to an embodiment of the present invention.

The present invention relates to nucleic acids; Ionizable lipid; non-ionizable lipid; neutral lipid; and a fusible lipid, wherein the ionized lipid is contained in an amount of 25 mol% or less based on the total weight of the lipid in the lipid nanoparticle.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement it. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein.

mRNA is short for messenger ribonucleic acid, and is an intermediate that connects DNA and proteins in the process where DNA containing genetic information becomes mRNA and proteins are synthesized using this.

Interest in and development of mRNA vaccines is focused due to COVID-19. mRNA vaccines have several advantages over other types of vaccines.　The biggest advantage of mRNA vaccines is that lipid nanoparticles (LNPs) containing mRNA are a platform technology, enabling rapid technology development against viruses that frequently mutate, such as COVID-19.　

Specifically, mRNA can be produced by identifying a protective protein antigen and sequencing the gene for the antigen.　When new mRNA is manufactured using this method and the formulation design and manufacturing process of conventional mRNA vaccines are used, rapid production of mRNA vaccines is possible. This means that since mRNA encoding different antigens is chemically and physically very similar, the formulation design and manufacturing process of a new mRNA vaccine can proceed in the same steps as the formulation and manufacturing process of a conventional mRNA vaccine.

Because of the negative charge of the phosphate group, mRNA is a polyanionic macromolecule in the pH range commonly used for parenteral use. As described above, lipid nanoparticles can be manufactured using positively charged ionisable lipids (or cationic lipids), using the electrical properties of negatively charged mRNA. Specifically, ionized lipids are positively charged lipids that bind strongly to negatively charged mRNA through electrical attraction. In addition to the ionized lipids, lipid nanoparticles are formed by additionally including non-ionizable lipids, neutral lipids, and fusion lipids.

The nucleic acid-lipid particle of US 9364435 B2 comprises (a) nucleic acid, (b) cationic lipid, (c) non-cationic lipid and (d) fusible lipid, wherein, based on the total content of lipids in the particle, the above It is disclosed as comprising 50 mol% to 85 mol% of cationic lipid, 13 mol% to 49.5 mol% of non-cationic lipid, and 0.5 mol% to 2 mol% of fusible lipid. .

In addition, the nucleic acid-lipid particle of EP 2279254 B1 contains 50 mol% to 65 mol% of the cationic lipid and 49.5 mol% or less of the non-cationic lipid, based on the total content of lipids in the particle. , cholesterol or its derivatives are included at 30 mol% to 40 mol%, and fusion lipids are disclosed at 0.5 mol% to 2 mol%.

As described above, lipid nanoparticles containing mRNA have been confirmed to contain a large amount of ionized lipids (cationic lipids) within the particles.

However, some concerns still remain regarding the safety of lipid nanoparticles (LNPs) containing mRNA as described above due to their toxicity in vitro and in vivo. This toxicity occurs primarily based on non-specific charge interactions. In other words, the toxicity problem of positively charged ionized lipids is becoming an issue, and research to supplement this is continuing.

It is most intuitive to attempt to reduce the amount of cationic ionized lipids used, but when low concentrations of ionized lipids below the threshold are used, the produced lipid nanoparticles (LNPs) do not release mRNA well after entering the cell, leading to a decrease in protein expression rate. there is. On the other hand, in the lipid nanoparticles containing low concentration ionized lipids of the present invention, the number of nucleic acids and ionized lipids contained in one lipid nanoparticle is reduced in order to reduce the toxicity of ionized lipids, but the lipid nanoparticles are made using the same amount of nucleic acids. When producing particles, compared to the conventional method of producing lipid nanoparticles, the overall number of lipid nanoparticles produced is increased, thereby increasing the number of lipid nanoparticles entering cells. As the number of lipid nanoparticles injected into the cell increases, the amount of ionized lipid generated when the lipid nanoparticles escape the endosome within the cell is reduced, resulting in less cytotoxicity.

Accordingly, the present invention relates to lipid nanoparticles containing a low concentration of ionized lipids. Although the concentration of ionized lipids in the lipid nanoparticles is low, the lipid nanoparticles are of uniform size and have a low content of ionized lipids, which can reduce cytotoxicity. It is characterized by being able to increase the protein expression level of nucleic acids contained in lipid nanoparticles.

Specifically, lipid nanoparticles containing low concentration ionized lipids according to an embodiment of the present invention include nucleic acids; Ionizable lipid; non-ionizable lipid; neutral lipid; and fusible lipids, wherein the ionized lipids may be included in an amount of 25 mol% or less based on the total weight of lipids in the lipid nanoparticles.

As described above, conventional lipid nanoparticles containing nucleic acids are known to contain about 50 mol% of ionized lipids, which means that during the formation process of lipid nanoparticles, the negative and positive ionized lipids of nucleic acids are self-assembled. To form lipid nanoparticles, a large amount of positively charged ionized lipids must be included in order to produce lipid nanoparticles through electrostatic attraction with nucleic acids.

However, as mentioned above, there is a risk of toxicity in lipid nanoparticles, and it is presumed that this toxicity problem is caused by ionized lipids. Therefore, the lipid nanoparticles of the present invention contain ionized lipids based on the total weight of lipids in the lipid nanoparticles. In comparison, it may be included at 25 mol% or less, 8 mol% to 22 mol%, and 8.1 mol% to 20 mol%. When ionized lipids are included in lipid nanoparticles within the above range, the protein expression level of nucleic acids can be increased.

Even if the content of ionized lipids in the lipid nanoparticles is low as described above, unlike the prior art, it is possible to produce uniform lipid nanoparticles because the ionized lipids and non-ionized lipids are 1:0.3 to 1:3. and the ionized lipid and fusible lipid are included in a molar ratio of 1:1 to 1:10.

Figure 1 is a conceptual diagram for comparison between the lipid nanoparticles (200) of the present invention and the conventional lipid nanoparticles (100).

Figure 1 shows the lipid nanoparticles (100, 200) formed when producing lipid nanoparticles (100, 200) using 5 mRNAs (1) and 25 ionized lipids (2) among nucleic acids, which are conventional lipid nanoparticles (100, 200). It can be confirmed that the nanoparticle 100 contains all 5 mRNAs (1) and 25 ionized lipids (2) in one particle. In other words, the conventional lipid nanoparticle 100 contains a plurality of mRNAs (1) in one particle, and as it contains a plurality of mRNAs (1), the negatively charged mRNA (1) has a positive charge around it. Ionized lipid (2) is located adjacent to each other by electrostatic attraction and is contained within the lipid nanoparticle (100).

On the other hand, the lipid nanoparticles (200) of the present invention are formed when producing lipid nanoparticles (200) using 5 identical mRNAs (1) and 25 ionized lipids (2). It can be seen that the number of mRNAs (1) contained in the lipid nanoparticles (200) is small, unlike the conventional lipid nanoparticles (100), and as a result, the number of ionized lipids (2) contained in the lipid nanoparticles (200) is 5. You can see the difference in the number of others.

To prepare the lipid nanoparticles of the present invention, ionized lipids and non-ionized lipids are included in a molar ratio of 1:0.3 to 1:3, 1:0.6 to 1:2.8, and 1:0.7 to 1. :2.5, and may be included in a molar ratio of 1:0.8 to 1:2.4.

In addition, the ionized lipid and the fusible lipid are included in a molar ratio of 1:1 to 1:10, included in a molar ratio of 1:2 to 1:9.5, included in a molar ratio of 1:2.5 to 1:9, and 1 It may be included at a molar ratio of :3 to 1:8.9.

In other words, when lipid nanoparticles are formed by including a high concentration of non-ionized lipids and fusible lipids included to produce lipid nanoparticles as described above, a high concentration of non-ionized lipids and fusible lipids are included, making them relatively It enabled the formation of uniform lipid nanoparticles even when small nucleic acids and ionized lipids were included.

The lipid nanoparticles have a uniform spherical shape, have a polydispersity index of 0.05 to 0.1, and the average diameter of the lipid nanoparticles may be 70 nm to 100 nm.

Specifically, the polydispersity index of the lipid nanoparticles is 0.05 to 0.1, 0.05 to 0.09, and is formed within a uniform diameter range, and the average diameter of the lipid nanoparticles is 70 nm to 100 nm, and may be 70 nm to 95 nm. As described above, the lipid nanoparticles of the present invention may be nanoparticles with a small diameter and a uniform spherical shape.

The lipid nanoparticles of the present invention may have a nucleic acid encapsulation rate of 90% or more, 92% or more, 93% to 100%, or 93% to 99%. The lipid nanoparticles not only have a uniform spherical shape, but can also exhibit an excellent encapsulation rate for nucleic acids used to produce lipid nanoparticles.

The nucleic acids include RNA, DNA, short interfering RNA (siRNA), messenger RNA (mRNA) aptamer, antisense oligodeoxynucleotide (ODN), antisense RNA, ribozyme, and DNAzyme. and mixtures thereof, preferably mRNA, but is not limited to the above examples.

The nucleic acid is used to prevent or treat disease, and as an example, it synthesizes a spike protein to combat the COVID-19 virus, such as the COVID-19 vaccine. Without being limited to the above examples, any nucleic acid for preventing or treating disease can be used.

The ionized lipid may be ALC-0315 (Genevant), ALC-0159 (Genevant), DLinDAP, Dlin-MC3-DMA, or SM102 (Arbutus). The ionized lipid is not limited to examples, and any ionized lipid that can be used in the production of lipid nanoparticles can be used without limitation.

The non-ionized lipids include distearoylphosphatidylcholine (DSPC), dioleolphosphatidyl ethanolamine (DOPE), bis(diphenylphosphino)ethane (DPPE), diacyl phosphatidylcholine, diacylphosphatidylethanolamine, and diacylphosphatidylserine. tidylserine) and It is selected from the group consisting of mixtures thereof, preferably DSPC, but is not limited to the above examples.

The fusion lipid may be selected from the group consisting of cholesterol, tocopherol, and mixtures thereof, preferably cholesterol, but is not limited to the above examples.

The neutral lipids can act as a steric barrier to prevent aggregation during storage, and reduce the protein adsorption of lipid nanoparticles in-vivo, thereby reducing immunogenic reactions and reducing the attack of lipid nanoparticles by macrophages. It plays a role in giving, specifically polyethylene glycol 2000 distearoylphosphatidylethanolamine (PEG (2000) DSPE), DMG-PEG, PEG-DMPE, DPPE-PEG, DPG-PEG, PEG-DOPE and mixtures thereof. It may be selected from the group consisting of DMG-PEG, but is not limited to the above examples.

A method for producing lipid nanoparticles containing a low concentration of ionized lipid according to another embodiment of the present invention includes ionizable lipid in an organic solvent; non-ionizable lipid; neutral lipid; and dissolving the fusible lipid to prepare an oily solution; Preparing an aqueous solution containing nucleic acids; and manufacturing lipid nanoparticles by injecting the oil phase solution and the aqueous solution into a chip for producing lipid nanoparticles, wherein the chip for producing lipid nanoparticles includes an oil phase solution supply unit, an aqueous solution supply unit, a mixer unit, and a discharge unit; , the oil phase solution is injected into the oil phase solution supply section, the aqueous phase solution is injected into the aqueous phase solution supply section, the oil phase solution is injected into the mixer section through the oil phase solution supply section, and the aqueous phase solution is injected into the mixer section through the aqueous phase solution supply section. , the oil phase solution and the aqueous phase solution are mixed in the mixer unit to form lipid nanoparticles, and the ionized lipid may be included in an amount of 25 mol% or less based on the total weight of lipids in the lipid nanoparticles.

Specifically, in the step of preparing the oil phase solution, ionized lipids, non-ionizable lipids, neutral lipids, and fusible lipids are dissolved in an organic solution to prepare an oil phase solution.

The organic solution is alcohol, and may specifically be methanol, ethanol, isopropanol, n-propanol, etc., preferably ethanol, but is not limited to the above examples and may be any organic solvent capable of uniformly dissolving ionized lipids without limitation. Available.

Specific types of ionizable lipids, non-ionizable lipids, neutral lipids, and fusible lipids are as described above.

In the step of preparing the aqueous solution, nucleic acid is mixed with a solvent to prepare an aqueous solution. The solvent is a citric acid solution at pH 3.0, but is not limited to the above example, and any solvent capable of producing lipid nanoparticles by mixing nucleic acids can be used without limitation.

The prepared oil phase solution and aqueous solution can be injected into the oil phase solution supply section and the aqueous phase solution supply section of the lipid nanoparticle production chip to form lipid nanoparticles within the lipid nanoparticle production chip.

An example of the chip for producing lipid nanoparticles is as follows.

A chip for producing lipid nanoparticles, described in KR 10-2361123 B1, including a fluid mixing unit with a bifurcated fluid flow through toric mixing elements as shown in FIG. 2, can be used.

Additionally, a chip for producing lipid nanoparticles according to another embodiment may have the same structure as FIG. 3. The mixer unit within the chip for producing lipid nanoparticles of FIG. 3 may include at least one pair of stabilizing units and mixing units arranged alternately. The mixer unit of the chip for producing lipid nanoparticles in FIG. 3 divides and pulverizes the flow of fluid by transporting particles or substances in the main flow direction of the fluid through advection using laminar flow. The fluid flow channel was designed based on the operating principle of chaotic advection with added chaos effects.

The chaotic advection method overcomes the biggest disadvantage of the existing advection phenomenon, which is that transport is limited to the unidirectional nature of the main fluid flow, and can achieve higher efficiency mixing between solutions.

The chip for producing lipid nanoparticles, as shown in FIG. 3, has a channel design carefully adjusted to increase mixing efficiency and maximize self-assembly at the contact surface of two different solutions so that lipid nanoparticles can be manufactured with high efficiency. . Specifically, in addition to the above-mentioned general characteristics using chaotic advection, symmetric and asymmetric structures are applied to not only efficiently mix solution fluids but also more stably and efficiently manufacture lipid nanoparticles with uniform diameters. It was designed and engineered in a way that can be done.

Additionally, a chip for producing lipid nanoparticles according to another embodiment may have the same structure as FIG. 4. The chip for producing lipid nanoparticles according to FIG. 4 includes an oily solution supply unit; aqueous solution supply unit; and a mixer unit including a spiral structure, wherein the mixer unit is connected to the oil phase solution supply unit and the aqueous solution supply unit, and includes an oil phase solution supplied through the oil phase solution supply unit and an aqueous solution supplied through the aqueous solution supply unit. They can be mixed to form lipid nanoparticles.

As described above, the mixer unit is structurally characterized in that it includes a spiral structure. Specifically, the spiral structure is characterized by including peaks and valleys alternately formed along a spiral trajectory on the circumferential surface.

However, the shape of the chip for producing lipid nanoparticles is not limited to the above examples, and any chip capable of producing lipid nanoparticles can be used.

The chip for producing lipid nanoparticles may be formed on a material selected from the group consisting of a glass substrate, silicon wafer, or polymer film, but examples of the material are not limited to the above examples, and any material capable of forming a channel can be used. .

The polymer film is polyimide, polyethylene, fluorinated ethylene propylene, polypropylene, polyethylene terephthalate, polyethylene naphthalate, polysulfone ( Polysulfone) and mixtures thereof, but is not limited to the above examples.

As an example, aluminum is deposited on a silicon wafer using an e-beam evaporator, and photoresist is patterned on the aluminum using a photolithography technique. Afterwards, aluminum is etched using the photoresist as a mask, and after removing the photoresist, the silicon is etched using DRIE (deep ion reactive etching) using aluminum as a mask. After removing the aluminum, glass is anodized on the wafer to seal it. Thus, the above liposome manufacturing chip can be manufactured.

In another example, the chip for producing lipid nanoparticles of the present invention can be printed and manufactured by a 3D printer.

When manufacturing lipid nanoparticles using the chip for producing lipid nanoparticles of FIG. 3, the prepared oil phase solution is injected into the oil phase solution supply section, the aqueous solution is injected into the aqueous phase solution supply section, and the oil phase solution supply section The oil phase solution is injected into the mixer unit through the aqueous solution supply unit, and the aqueous solution is injected into the mixer unit through the aqueous solution supply unit. The oil phase solution and the aqueous solution are mixed within the mixer unit to form lipid nanoparticles.

The aqueous solution and the oil phase solution injected into the oil phase solution supply unit and the aqueous solution supply unit form a laminar flow at the intersection point and flow through the stirring channel. In addition, as shown in Figure 3, by applying symmetric and asymmetric structures, not only can the solution fluid be mixed efficiently, but lipid nanoparticles with a uniform diameter can be manufactured more stably and efficiently.

In addition, when producing lipid nanoparticles using the chip for producing lipid nanoparticles of FIG. 4, the prepared oil phase solution is injected into the oil phase solution supply section, the aqueous phase solution is injected into the aqueous phase solution supply section, and the oil phase solution is injected into the oil phase solution supply section. The oil phase solution is injected into the mixer unit through the supply unit, and the aqueous solution is injected into the mixer unit through the aqueous solution supply unit. At this time, the oil phase solution and the aqueous solution move in directions facing each other when injected into the mixer unit, and the oil phase solution and It is injected into the mixer unit at the point where the aqueous solution meets. Accordingly, the oil phase solution and the aqueous phase solution form an interface and flow into the mixer unit, lipid nanoparticles are formed on the interface, and at the same time, the oil phase solution and the aqueous phase solution are mixed to form a mixed solution. The mixer part of the chip for producing lipid nanoparticles in FIG. 4 is characterized in that it has a spiral structure as described above, and a flow of fluid occurs along the circumference of the mixer part, which has a spiral structure, and the flow of fluid includes an oily solution and It can increase the mixing efficiency of the aqueous solution and promote the formation of uniform lipid nanoparticles at the interface.

Using the above-described chip for producing lipid nanoparticles, uniform lipid nanoparticles containing a low concentration of ionized lipid can be formed. As described above, compared to conventional lipid nanoparticles, the lipid nanoparticles can be manufactured into a larger number of lipid nanoparticles when producing lipid nanoparticles using the same nucleic acid, even if they contain a low concentration of ionized lipid. , Lipid nanoparticles can be produced if the above-described molar ratio of the ionized lipid and non-ionized lipid and the molar ratio of the ionized lipid and fusible lipid are met.

The prepared lipid nanoparticles have a low content of ionized lipids in the lipid nanoparticles, which can reduce cytotoxicity.

In addition, the spherical shape is uniform, the encapsulation rate of nucleic acids is excellent, and when manufacturing lipid nanoparticles, the content of non-ionized lipids and fusible lipids is increased to increase the content of nucleic acids compared to conventional lipid nanoparticles. It can be manufactured from lipid nanoparticles, and when the same nucleic acid is injected, the number of lipid nanoparticles injected into the body increases, thereby increasing the protein expression level of the nucleic acid.

Manufacturing example

Manufacturing of lipid nanoparticles

1) For LNP production, ALC0315 (ionized lipid), DSPC (non-ionized lipid), cholesterol (fusible lipid), and PEG (neutral lipid) were each dissolved in absolute ethanol at the following concentrations to prepare an oily solution.

2) An aqueous solution was prepared by mixing mRNA (CleanCap® EGFP mRNA, ~1,000 nucleotides) with citrate solution (pH 3).

3) The aqueous and oily solutions of “2)” were filled into a syringe and then connected to a microfluidic mixer as shown in FIG. 3.

4) Using a syringe pump, each raw material was injected at a flow rate of 51 ml/min for the water phase solution and 17 ml/min for the oil phase solution.

5) Each sample whose mixing was completed in step “4)” was placed in a dialysis cassette (Mw 10k) and dialyzed with 3L of 1x PBS for 2 hours. After the 2-hour dialysis process, it was replaced with fresh 1x PBS, and additional dialysis was performed overnight (approximately 17 hours overnight), and lipid nanoparticles were obtained.

The content and molar ratio of components contained in the aqueous solution and the oil phase solution are shown in Table 1 below:

		Capacity (ug)
		8.1% LNP	10% LNP	15% LNP	20% LNP	25% LNP	50% LNP
mRNA	EGFP (ug)	300	300	300	300	300	300
Lipid	Ionized lipid (ug)	5,001 (8.1)	5,001 (10.0)	5,001 (15.0)	5,001 (20.0)	5,001 (25.0)	5,001 (50.0)
	Non-ionized lipids (ug)	11,970 (18.8)	9,541 (18.5)	6,017 (17.5)	4,229 (16.4)	3,177 (15.4)	1,031 (10.0)
	Fusible lipids (ug)	22,309 (71.6)	17,666 (70.0)	11,105 (66.0)	7,836 (62.1)	5,865 (58.1)	1,943 (38.5)
	Neutral lipid (ug)	3,032 (1.5)	2,456 (1.5)	1,637 (1.5)	1,228 (1.5)	982 (1.5)	491 (1.5)
Weight ratio of ionized and non-ionized lipids		2.39	1.91	1.20	0.85	0.64	0.21
Weight ratio of ionized lipid and neutral lipid		1.86	1.85	1.85	1.85	1.85	1.88

The parentheses relate to the molar ratio of each lipid component to the total lipid content.

Experiment example

Average diameter and PDI analysis (using Malvern Ultra-red DLS equipment)

1ml of the lipid nanoparticles prepared in Table 1 above was loaded into a cuvette for DLS measurement. The cuvette was placed in the DLS equipment and measured.

The experimental results are shown in Figure 5. According to Figure 5, when ionized lipid is included at 8.1 mol%, 10 mol%, 15 mol%, 20 mol%, 25 mol% and 50 mol%, the average diameter is 90nm, 87.5nm, 80.5nm, 75.6nm, 72.2nm. nm and 73.5 nm, and the PDI measurement results were also confirmed to be 0.06, 0.08, 0.07, 0.08, 0.08, and 0.07, confirming that they were manufactured as uniform particles.

Measurement of encapsulation rate (using Ribogreen assay kit)

The 20x TE buffer contained in the kit was diluted into 1x TE buffer using RNase free buffer. Using 1x TE buffer, 100x Triton was diluted into 1x Triton. For quantitative analysis of mRNA encapsulated in LNP, a sample to create a standard curve of EGFP-mRNA was prepared using 1x Triton. The measurement sample was diluted 50 times using 1x TE buffer and 1x Triton, respectively. The sample was loaded at 100 ul each into a 96 well black plate. The 96-well black plate loaded with all samples was incubated at 37°C for 10 minutes. Ribogreen included in the kit was diluted 200 times with 1x TE buffer. 100ul of diluted Ribogreen was added to each 96-well black plate after the incubation process was completed. Incubation was performed for 10 minutes at room temperature (approximately 25°C). Afterwards, fluorescence was measured at a wavelength of 490 to 520 nm using microplate reader equipment. The measured fluorescence value was substituted into the EGFP-mRNA standard curve to quantify the amount of mRNA inside and outside the LNP (calculate the encapsulation rate and amount of encapsulated RNA using Equation 1 and Equation 2 below.)

[Formula 1]

[Correction 15.02.2024 pursuant to Rule 91]

[Formula 2]

[Correction 15.02.2024 pursuant to Rule 91]

The experimental results are shown in Figure 6. When ionized lipids were included at 8.1 mol%, 10 mol%, 15 mol%, 20 mol%, 25 mol%, and 50 mol%, the encapsulation ratios were 97%, 98%, 99%, 98%, 97%, and 92%. As confirmed, it can be seen that the encapsulation rate of lipid nanoparticles containing 50 mol% of ionized lipid, like conventional lipid nanoparticles, is relatively low.

Cytotoxicity evaluation

Cell morphology confirmation test

Stocked HeLa cells were thawed in a 37°C water bath for 1 minute. 4ml of fresh media was added to the thawed HeLa cells and centrifuged to collect the cells downward. Centrifugation conditions were 1500 rpm, 3 minutes, and 4°C.

After the centrifugation was completed, the supernatant was removed and the cells were resuspended in 10ml of fresh media. The resuspended cells were transferred to a 100 ф culture dish and cultured for 2 days. After 2 days, the cells in culture were subcultured in a 100 ф culture dish (cell stabilization process). When the confluency of subcultured cells reached 90%, cells were seeded in a 24-well culture plate in the following manner.

- Culture media of cells with a density exceeding 90% were removed. 10ml of DPBS was added and the remaining media was washed twice. 5ml of trypsin was added and incubated at 37°C for 3 minutes. Afterwards, the dish was lightly hit to dislodge the cells attached to the bottom of the dish. Trypsin was neutralized by adding 5ml of media, and then the media was replaced with fresh media using centrifugation. At this time, centrifugation conditions were 1500 rpm, 3 minutes, and 4°C. The cells resuspended through the above process were counted under a microscope, and the cells were seeded at a concentration of 7 x 10^(4) cells/well in a 24-well culture plate.

Seeded cells were cultured in a CO ₂ -incubator at 37°C for 24 hours. Afterwards, each lipid nanoparticle was treated according to the experimental concentration (test concentration: 500ng). After treatment with lipid nanoparticles, the cells were cultured in a CO ₂ -incubator at 37°C for 48 hours, and the morphology of the cells was observed using a microscope.

The results of the experiment are shown in Figure 7. Compared to the untreated group (NC), it was confirmed that the cell morphology was not significantly changed in the group treated with 10 mol%, 15 mol%, and 20 mol% of ionized lipid, and in the group treated with 25 mol% and 50 mol% of ionized lipid, It can be seen that there is a significant change in cell shape in the group.

Cell proliferation assay

Lipid nanoparticles were treated in the same manner as the cell shape confirmation test described above, and cultured in a CO ₂ -incubator at 37°C for 72 hours. The reagent of the CCK-8 assay kit was prepared by diluting it 10 times with media. Afterwards, the media of the cells that had been cultured for 72 hours was removed, 0.5ml of the above-prepared reagent was added, and the cells were incubated in a CO ₂ -incubator at 37°C for 3 hours.

Afterwards, the absorbance at 450 nm was measured using a microplate reader device. Cell proliferation was calculated using Equation 3 below.

[Formula 3]

Figure 8 shows the results of the evaluation of cytotoxicity, confirming that the group treated with lipid nanoparticles containing 10 mol% of ionized lipid had lower toxicity compared to the group treated with lipid nanoparticles containing 50 mol% of ionized lipid. .

In addition, the results of confirming the change in cell shape are shown in Figure 9. No change in cell shape was observed in cells treated with lipid nanoparticles containing 10 mol% of ionized lipid, but in cells treated with lipid nanoparticles containing 50 mol% of ionized lipid, a change in shape was observed as indicated. It was confirmed that the concentration of ionized lipid affects cytotoxicity.

Efficiency evaluation test for mRNA protein expression

Cells were seeded in the same manner as the cell morphology confirmation test described above, and after culturing for 24 hours, the lipid nanoparticles were diluted in media so that the RNA concentration was 500ng/ml, and then 1ml was added to each well. Processed. Afterwards, the cells were cultured at 37°C in a CO ₂ -incubator for 48 hours. Afterwards, the GFP expression rate was confirmed using a microscope.

The experimental results are shown in Figure 10. It can be seen that the GFP expression rate was higher in the group treated with lipid nanoparticles containing 10 mol% of ionized lipid compared to the group treated with lipid nanoparticles containing 50 mol% of ionized lipid. This confirmed that the mRNA contained in the lipid nanoparticles of the present invention had a significant difference in protein expression level compared to the lipid nanoparticles containing 50 mol% of conventional ionized lipid.

Quantitative analysis test on lipid nanoparticle concentration

1ml of lipid nanoparticles was loaded into the cuvette for DLS measurement. Afterwards, the cuvette was placed in the DLS equipment, the particle concentration function was selected in the software, and measurements were made by pressing the start button.

The test results are shown in Figure 11. According to Figure 11, it can be seen that as the concentration of ionized lipid decreases, the number of lipid nanoparticles formed increases, especially lipid nanoparticles containing 50 mol% of ionized lipid and lipid nanoparticles containing 10 mol% of ionized lipid. In the case of , it can be seen that the number of lipid nanoparticles formed containing 10 mol% of ionized lipid differs by 2.5 times.

This is an experimental result that supports the fact that as the concentration of ionized lipids decreases, as described above, the concentration of non-ionized lipids and fusible lipids increases, and the number of lipid nanoparticles formed increases.

Qualitative analysis test on lipid nanoparticle concentration

Lipid nanoparticles were concentrated at 5000 rpm, 1 hour, and 15°C. Afterwards, encapsulated mRNA was quantified in the same manner as the encapsulation rate measurement method described above, and all of the lipid nanoparticles were diluted so that the encapsulated mRNA concentration was 25ug/ml. Afterwards, using a 200kV JEOL 2100P cryogenic electron microscope, lipid nanoparticles of the same concentration were imaged to confirm the number of particles.

The experimental results are shown in FIG. 12, and it can be seen from the image that the number of lipid nanoparticles containing 10 mol% of ionized lipids is greater than that of lipid nanoparticles containing 50 mol% of ionized lipids.

Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims are also possible. falls within the scope of rights.

The present invention relates to lipid nanoparticles containing low concentration ionized lipids and a method for producing the same.

Claims (10)

1. nucleic acid;

   Ionizable lipid;

   non-ionizable lipid;

   neutral lipid; and

   Contains fusible lipids,

   The ionized lipid contains 25 mol% or less compared to the total weight of lipid in the lipid nanoparticle.

   Lipid nanoparticles containing low concentration of ionized lipids.
2. According to paragraph 1,

   The ionized lipid and the non-ionized lipid are contained in a molar ratio of 1:0.3 to 1:3.

   Lipid nanoparticles containing low concentration of ionized lipids.
3. According to paragraph 1,

   The ionized lipid and fusible lipid are contained in a molar ratio of 1:1 to 1:10.

   Lipid nanoparticles containing low concentration of ionized lipids.
4. According to paragraph 1,

   The lipid nanoparticles have a uniform spherical shape and have a polydispersity index of 0.05 to 0.1.

   Lipid nanoparticles containing low concentration of ionized lipids.
5. According to paragraph 1,

   The average diameter of the lipid nanoparticles is 70nm to 100nm.

   Lipid nanoparticles containing low concentration of ionized lipids.
6. According to paragraph 1,

   The nucleic acids include RNA, DNA, short interfering RNA (siRNA), messenger RNA (mRNA), aptamer, antisense oligodeoxynucleotide (ODN), antisense RNA, ribozyme, and DNAzyme. ) and mixtures thereof.

   Lipid nanoparticles containing low concentration of ionized lipids.
7. According to paragraph 1,

   The non-ionized lipids include distearoylphosphatidylcholine (DSPC), dioleolphosphatidyl ethanolamine (DOPE), bis(diphenylphosphino)ethane (DPPE), diacyl phosphatidylcholine, diacylphosphatidylethanolamine, and diacylphosphatidylserine. tidylserine) and selected from the group consisting of mixtures thereof

   Lipid nanoparticles containing low concentration of ionized lipids.
8. According to paragraph 1,

   The neutral lipid is selected from the group consisting of polyethylene glycol 2000 distearoylphosphatidylethanolamine (PEG (2000) DSPE), DMG-PEG, PEG-DMPE, DPPE-PEG, DPG-PEG, PEG-DOPE, and mixtures thereof. felled

   Lipid nanoparticles containing low concentration of ionized lipids
9. According to paragraph 1,

   The fusion lipid is selected from the group consisting of phospholipids, cholesterol, tocopherol, and mixtures thereof.

   Lipid nanoparticles containing low concentration of ionized lipids.
10. Ionizable lipids in organic solvents; non-ionizable lipid; neutral lipid; and dissolving the fusible lipid to prepare an oily solution;

    Preparing an aqueous solution containing nucleic acids; and

    Including the step of producing lipid nanoparticles by injecting the oil phase solution and the aqueous phase solution into a chip for producing lipid nanoparticles,

    The chip for producing lipid nanoparticles includes an oil phase solution supply section, an aqueous phase solution supply section, a mixer section, and a discharge section,

    The oily solution is injected into the oily solution supply part,

    The aqueous solution is injected into the aqueous solution supply section,

    The oil phase solution is injected into the mixer section through the oil phase solution supply section, and the aqueous phase solution is injected into the mixer section through the aqueous phase solution supply section,

    In the mixer unit, the oil phase solution and the aqueous phase solution are mixed to form lipid nanoparticles,

    The ionized lipid is contained in an amount of 25 mol% or less compared to the total weight of lipids in the lipid nanoparticles.

    Method for producing lipid nanoparticles containing low concentration of ionized lipid.

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