KR102618727B1 - Lipid nanoparticle for delivering nucleic acid - Google Patents

Abstract

The present invention relates to lipid nanoparticles for nucleic acid delivery within the body, and more specifically, to lipid nanoparticles for nucleic acid delivery containing cationic sterol derivatives. According to the present invention, it was confirmed that lipid derivatives developed for different purposes were used together to deliver nucleic acids in vivo more effectively than existing delivery vehicles.

Description

Lipid nanoparticle for delivering nucleic acid}

The present invention relates to lipid nanoparticles for in vivo nucleic acid delivery, and more specifically, to lipid nanoparticles for nucleic acid delivery containing cationic sterol derivatives.

Gene therapy refers to a method of treating or preventing cancer, infectious diseases, genetic diseases, etc. by introducing therapeutic genes into the patient's body to correct defective genes or add new functions to cells. Recently, gene therapy, a method of treating diseases using nucleic acids (DNA, RNA), is rapidly emerging as a new clinical treatment for various diseases.

In order to effectively perform gene therapy, it is necessary to develop gene transfer technology that can deliver therapeutic genes to desired target cells and achieve high expression efficiency. In other words, the development of genetically engineered nucleic acids and delivery vehicles for delivering them, such as lipid nanoparticles, are essential.

The introduction of purified nucleic acid molecules into eukaryotic cells using lipid nanoparticles is called transfaction. It is one of the basic research methods applied in various bio fields such as advanced therapeutic drug development, cell line production, protein production, and virus production. .

In developing a new drug, there are methods such as deriving the structure of a new compound, cocktail therapy that mixes various drugs, and drug reinvention that uses an existing drug for a different purpose.

A similar strategy to the previously mentioned drug development process can be applied to the development of new gene delivery systems. In other words, there is an approach to start anew by deriving a novel lipid structure by synthesizing a new derivative based on an existing lipid or changing the binding site of the derivative. In addition, research methods to derive an optimized ratio by combining existing lipids with other lipids or phospholipids are also being actively conducted.

Cationic polymers used as gene carriers under recent research combine with genes to form cationic polymer-gene complexes, thereby inducing transfection of eukaryotic cells. Specific examples of such cationic polymers include cationic homopolymers such as polyethyleneimine (PEI), polyamidoamine, polyL-lysine, polyaminoester, polypropyleneimine, and polyethylene glycol (PEG)-block-poly. Cationic block nanocomposites such as L-lysine and PEG-PEI can be mentioned.

However, many cationic polymers have the disadvantage that genes do not enter cells smoothly in vivo. Therefore, the development of a gene delivery system containing novel cationic lipids that can solve these shortcomings is required.

Republic of Korea Patent No. 10-1769666 (registered on August 11, 2017)

The purpose of the present invention is to develop a gene transfer technology that can achieve high expression efficiency by transferring genes.

In order to solve the above problem, the present invention includes (i) one or more nucleic acid molecules, (ii) cationic cholesterol derivative A represented by Formula 1, (iii) cationic sitosterol derivative B represented by Formula 2, and (iv) phospholipid. Provided is a lipid nanoparticle comprising:

According to one embodiment of the present invention, the nucleic acid molecule is pDNA or siRNA.

In addition, the molar mixing ratio of derivative A: derivative B: phospholipid in the lipid nanoparticle is 2-3: 2-3: 0.5-1.

Additionally, the phospholipid is characterized as 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE).

The present invention also provides a pharmaceutical composition comprising the lipid nanoparticles and a pharmaceutically acceptable carrier.

Additionally, the present invention provides a method of delivering nucleic acids to cells comprising contacting the cells with the lipid nanoparticles.

Additionally, the present invention provides a pharmaceutical composition that exhibits inhibitory effect on the proliferation of ovarian cancer and breast cancer cells using lipid nanoparticles.

In the present invention, it was confirmed that by using a mixture of cationic cholesterol derivatives and cationic sitosterol derivatives as a gene delivery vehicle, it is more effective in RNA delivery than when used alone.

In other words, it was found that the gene delivery ability was greatly improved by mixing cationic lipid derivatives with structural differences.

Figure 1 is a graph showing the size and distribution results of the lipid nanoparticles of the present invention.
Figure 2 is a graph showing the results of plasmid DNA expression of lipid nanoparticles using various ratios.
Figure 3 is a graph showing the results of siRNA delivery using BES 2 particles.
Figure 4 shows the results of transmission electron microscopy of lipid nanoparticles before and after mixing.
Figure 5 is a graph showing the results of flow cytometry analysis of lipid nanoparticles before and after mixing.

The present invention will be described in detail below, focusing on examples.

1.1. Cells and Chemicals

All cell lines used were purchased from the Korea Cell Line Bank (KCLB), and the recommended medium containing 10% FBS was used and cultured at 37°C with 5% CO ₂ .

The delivered pDNA contained eGFP and a CMV vector, and siRNA was ordered and used from Macrogen.

Compounds represented by Formula 1 and Formula 2 were each synthesized directly according to the literature.

[Formula 1]

This structure is a structure in which putrescine and cholesterol are linked through a carbamate bond, and is a structure that exhibits the form of a cation at pH 7 or lower using trifluoroacetate salt.

[Formula 2]

This structure is a structure in which the primary amine structure is connected to sitosterol through an ester bond, and it is a structure that shows the form of a cation at pH 7 or lower using a trifluoroacetate salt.

In the present invention, lipid nanoparticles using a cationic cholesterol derivative (hereinafter referred to as "ED") synthesized to increase binding efficiency with nucleic acid and lipid nanoparticles using a cationic sitosterol derivative used for radiation treatment and enhancement of its effectiveness (hereinafter referred to as "ED") SD") was used in combination to form a new carrier, BES 2.

1.2. Lipid nanoparticle manufacturing

Dissolve the lipid mixture in chloroform and methanol so that the molar ratio of the cationic derivative of Formula 1 and the cationic derivative of Formula 2 and the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) is 5:1. Each was prepared. The lipid mixture was formed into a thin lipid film using a rotary evaporator, and the film was hydrated by exposure to distilled water for one day. The hydrated film was formed into lipid nanoparticles through an ultrasonic generator and stored at 4°C for the experiment.

Particles with a molar ratio of the derivative of Formula 1 and DOPE of 5:1 were denoted as ED, particles with a molar ratio of the derivative of Formula 2 and DOPE of 5:1 were denoted as SD, and the derivative of Formula 1, the derivative of Formula 2, and DOPE were denoted as ED. Lipid nanoparticles mixed at a molar ratio of 2.5:2.5:1 were designated as BES 2.

1.3. Physicochemical analysis of lipid nanoparticles

Before analysis, each lipid nanoparticle was diluted 20 to 50 times with distilled water and analyzed using a Nano-ZS zeta potential analyzer (Malvern Inc., UK). The particle size and distribution were expressed as average and standard deviation values by performing 15 repeated measurements three times.

1.4. Transmission electron microscopy observation

Lipid nanoparticles were fixed through a 4% formaldehyde solution, and samples were produced using grids (200 meshes, TED PELLA, USA) coated with nickel-based formava-carbon. The produced grid was observed through an electron microscope (H-7000B, Hitachi, Japan).

1.5. Transfection experiment using lipid nanoparticles

In order to compare the efficiency according to the mixing ratio of ED and SD that make up lipid nanoparticles, five lipid nanoparticles were formed by changing the mixing ratio of the derivatives of Formula 1 and Formula 2, and plasmid DNA carrying eGFP in three types of cells. Expression was expressed as a relative value and analyzed. The plasmid DNA expression experiment was performed in a 96-well plate, and 10,000 A549 cells and 10,000 CHO cells or 12,000 CHO cells were used per well, and 100ng of plasmid DNA was used.

Each lipid nanoparticle was combined with nucleic acid for 15 minutes, and then a transfection experiment was performed for 24 hours in the presence of serum. The expressed eGFP protein was measured through a fluorometer by lysing the cells in each well. The fluorescence intensity was recorded. The intensity of fluorescence was expressed as a relative value based on the highest BES 2 fluorescence intensity.

The siRNA expression experiment was performed in a 12-well plate, 80,000 KB cells and 80,000 MCF 7 cells were used per well, and siRNA was treated at a concentration of 25nM. Each lipid nanoparticle was bound for 15 minutes, then a transfection experiment was performed for 24 hours in the presence of serum, and RNA was extracted using Trizol. cDNA was extracted using a reverse transcription kit (thermofisher scientific Inc., USA) and amplified using transcription and reverse transcription primers for Lamin A/C, Bcl-2, and GAPDH.

The resulting value of the mRNA level corresponding to each sequence was expressed relative to the GAPDH mRNA level of the negative control. The sequences of primers used for each siRNA are as follows. Lamin A/C primer (forward) 5'-TGGAAGATGATCCCTTGCTGA-3', (reverse) 5'-GCATGGCCACTTCTTCCCA-3'; Bcl-2 primers (forward) 5'- CTGCACCTGACGCCCTTCAC-3'' and (reverse) 5''- CACATGACCCC-ACCGAACTCAAAGA-3'',; GAPDH primer (forward) 5' AACGGGAAGCTTGTCATCAATGGAAA-3', (reverse) 5'-GCATCAGCAGAGGGGGCAGA-3'.

1.6. Endocytosis and gene expression experiments incorporating flow cytometry

To analyze intracellular gene transfer and expression patterns, fluorescence distribution was observed 24 hours after transfection. 50,000 CHO cells were cultured per well of a 24-well plate, and 500ng nucleic acid and lipid nanoparticles were combined for 15 minutes before transfection. The transfected cells were separated using Trpsin-EDTA, washed twice with PBS, and then suspended in 1% formaldehyde solution for analysis.

Ten thousand cells were analyzed using a flow cytometer (CytoFlex, Beckman, Germany), and the results were displayed as a histogram using a program provided by the manufacturer.

To observe intracellular transfection, fluorescent lipid nanoparticles were prepared by mixing 1% rhodamine-B, and fluorescence distribution was observed 1 hour after transfection.

1.7. Statistical analysis processing

All graphs and statistical processing were performed using one way-ANOVA using Graph pad prism 7, and were expressed as the average value ± standard deviation of three repeated experiments. As a result of statistical analysis, statistical significance was indicated by the number of * according to the P value.

<Example 1>

Cholesterol is a basic component of biological membranes, has excellent biocompatibility, and is an essential component of the LNP structure for mRNA delivery, which has recently been in the spotlight. As the proportion of cholesterol in the nanoparticle composition increases, the hardness of the particle increases. Accordingly, the efficiency of internal substance release from lipid nanoparticles decreases, but the stability of the particle increases.

In other words, the appropriate cholesterol ratio is very important in composing nanoparticles, and this affects not only the physical and chemical properties of the particles but also their biological activity. In addition to these advantages, recent research has shown that increasing intracellular cholesterol improves gene expression efficiency.

Meanwhile, there are reports of an increase in mRNA delivery efficiency when cholesterol is replaced with sitosterol, a plant sterol. According to the literature, lipid nanoparticles formed from sitosterol are observed to have a polygonal shape instead of the existing spherical shape, and are said to improve gene transfer by increasing the contact area with cells.

In the initial development stage, the derivative of Chemical Formula 1 was produced using cholesterol and showed excellence in gene transfer alone without mixing with phospholipids. The derivative of Formula 2 improved the efficiency of drug delivery by directly conjugating sitosterol to an anticancer drug and using it to increase retention time.

However, the gene delivery vehicle using only the derivative of Formula 1 showed limitations in delivery ability. To solve this, five types of lipid nanoparticles were newly formed by fusing the derivatives of Formula 2.

Overall, the ratio of total cationic lipids and phospholipids (DOPE) was set to a molar ratio of 5:1, and to analyze the role of each cationic lipid, the molar mixing ratio of derivative of Formula 1: derivative of Formula 2: phospholipid was used, respectively. It was classified in detail as 5:0:1 (ED), 4:1:1, 2.5:2.5:1 (BES 2), 1:4:1, and 0:5:1 (SD).

When analyzing the physicochemical properties of the five types of particles using DLS equipment, the particles were measured to be approximately 30 nm larger than ED and SD, and the PDI values were all below 0.2, indicating that they were produced relatively uniformly. (see Figure 1)

Next, to evaluate the gene delivery ability of the lipid nanoparticles, eGFP plasmid DNA was transfected into CHO, HeLa, and A549 cells, and after 24 hours, the cells in each well were lysed to measure the fluorescence value. Most lipid nanoparticles formed of the derivatives of Formula 1 and the derivative of Formula 2 at various molar ratios showed similar efficiency in gene expression, but in particular, the BES 2 group showed a synergistic effect. (see Figure 2)

The above synergistic effect appears to be due to differences in the structures of each derivative. In general, ISO-type molecules with a branched structure have lower physical and chemical properties, such as boiling point, than regular molecules, but this is due to three-dimensional structural disorder.

The lipids used in the present invention, cholesterol derivatives and sitosterol derivatives, have structural differences. In particular, the branched structure of sitosterol is believed to impede the formation of lipid nanoparticles and increase the fluidity of the lipid membrane.

After this, the experiment was conducted focusing on the three types of ED, SD, and BES 2.

<Example 2> Evaluation of siRNA gene delivery ability of lipid nanoparticles

To confirm the effect of siRNA sequence delivery and expression of BES 2, siRNA that interferes with the expression of the Lamin A/C gene was treated with ovarian cancer cell line KB cell line and breast cancer cell line MCF 7 cell line.

Lamin A/C, like GAPDH, is a housekeeping gene often used as a positive control and is an essential gene required to maintain the nuclear membrane within cells. The prepared cationic lipid nanoparticles showed different efficiencies depending on the ratio of the corresponding components, and BES 2, which contains the derivative of Formula 1 and the derivative of Formula 2 in equal proportions, showed the best efficiency. (See Figure 3) In particular, BES 2 showed superior results than competing reagents currently available on the market, and this trend was similar to that of plasmid DNA transfection results.

Additionally, when transfecting siRNA that suppresses Bcl-2, an essential gene for cancer cell survival, BES 2 showed the best results. When cancer cells with Bcl-2 suppressed were observed during the experiment, it was confirmed that the negative and positive control groups were normal, but the cell shape of the experimental group changed and they were no longer able to survive and entered the death stage.

This confirmed that the gene carrier of the present invention can exhibit cancer treatment effects by suppressing genes involved in cancer cell survival or continuously delivering genes necessary for cancer cell death, and that it can be used as a carrier candidate for gene therapy.

<Example 3> Observation of lipid nanoparticles and confirmation of intracellular transfection

After the derivative of Formula 1 and the derivative of Formula 2 were fused, they were observed using an electron microscope to check whether the shape of the carrier changed. The sample produced using only ED appeared to be in the form of a double-layered membrane, while the sample produced using only SD showed a multicystic form with additional small spheres inside spherical particles.

BES 2 showed a similar shape to the ED used sample and confirmed the shape of the liposome forming a phospholipid bilayer. (See Figure 4) The shape of the nanoparticles is similar to the previously used derivative of Chemical Formula 1, but it showed a significant improvement in delivery efficiency, so additional experiments were performed to prove the results.

To investigate the endocytosis of nanoparticles, fluorescent lipids were added to each particle and intracellular fluorescence was analyzed after treatment. ED and SD showed different aspects of intracellular transfection. ED, which used only the derivative of Formula 1, had many lipid nanoparticles transfected into cells, but the number of cells showing strong fluorescence was small. In other words, although delivery into cells was excellent, there was a limit to the number of lipid nanoparticles transfected per cell.

On the other hand, the histogram of SD using the derivative of Formula 2 was shifted to the right compared to the ED-treated sample, indicating that although there were many lipid nanoparticles transfected per cell, the number of cells showing this result was small.

In the case of BES 2, the rate of incorporation of multiple lipid nanoparticles into cells was much higher, and the strong fluorescence shown by each cell showed that there were also multiple lipid nanoparticles per cell. (See Figure 5(a)).

To investigate protein expression patterns following the transfection pattern of lipid nanoparticles, eGFP fluorescent gene was transfected and analyzed by flow cytometry. ED and SD showed similar fluorescence intensity as the control, and the proportion of cells expressing fluorescence was absolutely low.

On the other hand, the experimental group treated with BES 2 showed a fluorescence intensity that was more than 100 times stronger, and the proportion of expressed cells also increased significantly. (See Figure 5(b)) This shows a similar tendency to the results in Figure 2, indicating that the dramatically increased gene expression ability is related to the pattern of intracellular transfection.

In conclusion, BES 2 analyzed a large number of imported lipid nanoparticles in a large number of cells, and it is expected that the increased fluidity of the membrane structure due to the branched structure of Formula 2 helped release internal substances. These results confirmed that it only works at a certain ratio between the two derivatives.

Therefore, BES 2 showed superior plasmid DNA (pDNA) and siRNA delivery capabilities compared to each of the existing derivatives, and its effect was found to occur only at a certain ratio. By using lipid derivatives developed for different purposes together, it was confirmed to be more effective than existing delivery vehicles. In particular, when analyzed using a flow cytometer, intracellular transfection increased in a short period of time only in samples combined at a specific ratio, helping with gene expression. It was confirmed that this was the case.

<Example 4> Administration of lipid nanoparticles

Lipid nanoparticles (e.g., LNPs) of the invention may be administered alone or in mixture with a pharmaceutically acceptable carrier (e.g., physiological saline or phosphate buffer) selected according to the route of administration and standard pharmaceutical practice. . Typically, normal buffered saline (e.g., 135-150 mM NaCl) will be used as a pharmaceutically acceptable carrier.

As another carrier, water, buffered water, 0.4% saline, 0.3% glycine, etc. may be used and may include glycoproteins for stability such as albumin, lipoprotein, globulin, etc. As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption retarding agents, buffers, carrier solutions, suspensions, colloids, etc. The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that do not cause allergic or similar side effects when administered to humans.

The description so far is illustrative rather than restrictive, and a person skilled in the art will be able to clearly understand the invention through the above description. Therefore, the scope of the invention should be determined not with reference to the foregoing description, but rather with reference to the claims, together with the full scope of equivalents to which such claims are entitled.

Claims (7)

Lipid nanoparticles for nucleic acid delivery,
It is prepared by mixing (i) cationic cholesterol derivative A represented by Formula 1, (ii) cationic sitosterol derivative B represented by Formula 2, and (iii) phospholipids in a certain ratio,
The molar mixing ratio of derivative A: derivative B: phospholipid in the lipid nanoparticle is 2-3: 2-3: 0.5-1.
Lipid nanoparticles for nucleic acid delivery.
[Formula 1]

[Formula 2]
According to paragraph 1,
The nucleic acid is pDNA or siRNA.
Lipid nanoparticles for nucleic acid delivery. delete According to paragraph 1,
The phospholipid is 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE).
Lipid nanoparticles for nucleic acid delivery. A pharmaceutical composition comprising the lipid nanoparticle of any one of claims 1, 2, or 4 and a pharmaceutically acceptable carrier. A method of delivering a nucleic acid into a non-human cell, comprising contacting the lipid nanoparticle of any one of claims 1, 2, or 4 with the non-human cell. delete