Nucleic acid delivery composition, nucleic acid vaccine, anticancer agent, and use of fine particles as nucleic acid delivery composition

Abstract

To provide a composition for delivering a nucleic acid capable of delivering the nucleic acid into a cell and obtaining the function of the nucleic acid.SOLUTION: The composition for delivering a nucleic acid comprises a fine particle containing a polyamino acid and a nucleic acid held by the fine particle.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a composition for nucleic acid delivery, a nucleic acid vaccine, an anticancer agent, and the use of fine particles as a composition for nucleic acid delivery. [Background technology]

[0002] Nucleic acid vaccines, in which antigen-producing mRNA or DNA is encapsulated in nanoscale liposomes, have been used against the novel coronavirus (SARS-CoV-2) that causes SARS (Severe Acute Respiratory Syndrome).In addition to nucleic acid vaccines, development is also underway on drug delivery systems that deliver nucleic acids with anti-cancer properties to cancer cells.

[0003] The minimum functions required for nucleic acid delivery vehicles are to take up nucleic acids and fuse with the cell membrane. Generally, liposomes containing phospholipids, which have components similar to those of the cell membrane and form a lipid bilayer that easily fuses with the cell membrane, are used. Most liposomes form an electrostatic complex between the negative charge of DNA and the positive charge of cationic phospholipids.

[0004] Electrostatic complexes such as liposomes can be prepared relatively easily, but their sphere size is difficult to control and their ability to release nucleic acids after passing through a cell membrane is poor. Therefore, nucleic acid delivery vehicles containing polypeptides whose structure can be easily controlled by amino acid sequence have also been developed. For example, Patent Document 1 discloses a polyion complex containing a block copolymer in which polyethylene glycol and a polypeptide having a cationic group in its side chain are linked via a disulfide group, and nucleic acid.

[0005] Preparation of nucleic acid delivery vehicles containing polypeptides with controlled structures is complicated. Therefore, as disclosed in Non-Patent Document 1, proteinoid microspheres have been investigated as spherical structures. Proteinoid microspheres are produced by dissolving a proteinoid in hot water and then cooling. By producing proteinoid microspheres in the presence of nucleic acids, nucleic acids can be retained in the proteinoid microspheres. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2007 / 099660 [Non-patent literature]

[0007] [Non-Patent Document 1] Kiyoka Takahashi and Hajime Mita, Proceedings of the 103rd Annual Meeting of the Chemical Society of Japan (2023), K303-1vn-09, "Nucleic Acid Adsorption to Proteinoid Microspheres," Chemical Society of Japan, March 8, 2023 Summary of the Invention [Problem to be solved by the invention]

[0008] To deliver nucleic acids to cells and exert their functions, the nucleic acids must be taken up by the cells and released within the cells. However, it is unclear whether nucleic acids held in proteinoid microspheres are released within the cells.

[0009] The present invention has been made in consideration of the above circumstances, and aims to provide a nucleic acid delivery composition that can deliver nucleic acids into cells and obtain the functions of the nucleic acids, a nucleic acid vaccine, an anticancer agent, and the use of microparticles as a nucleic acid delivery composition. [Means for solving the problem]

[0010] The nucleic acid delivery composition according to the first aspect of the present invention comprises: microparticles containing polyamino acids; Nucleic acid retained on the microparticles; Includes.

[0011] The polyamino acid is containing anhydropolyaspartic acid, This may also be the case.

[0012] The nucleic acid vaccine according to the second aspect of the present invention comprises: The nucleic acid delivery composition according to the first aspect of the present invention is included, The nucleic acid is It encodes an antigenic polypeptide.

[0013] The anticancer agent according to the third aspect of the present invention comprises: The nucleic acid delivery composition according to the first aspect of the present invention is included, The nucleic acid is It is an anti-cancer nucleic acid.

[0014] The use according to the fourth aspect of the present invention comprises: The present invention relates to the use of microparticles containing polyamino acids as a composition for nucleic acid delivery. [Effects of the Invention]

[0015] According to the present invention, nucleic acids can be delivered into cells to obtain the functions of the nucleic acids. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 is a view showing a scanning electron microscope image of a proteinoid microsphere according to an embodiment of the present invention. [Figure 2] FIG. 1 shows the results of elemental analysis of proteinoid microspheres according to an embodiment of the present invention by an electron probe microanalyzer. [Figure 3] FIG. 1 shows a fluorescent microscope image of Escherichia coli cultured in a medium containing proteinoid microspheres according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0017] Embodiments of the present invention will be described with reference to the drawings. Note that the present invention is not limited to the following embodiments and drawings. Note that in the following embodiments, the expressions "have," "include," or "contain" also include the meaning of "consisting of" or "consisting of."

[0018] (Embodiment 1: Nucleic acid delivery composition) The nucleic acid delivery composition according to the present embodiment comprises microparticles containing polyamino acids and nucleic acids held by the microparticles. Polyamino acids are amino acid chains formed by dehydration of hydrates, free of peptide and polypeptide bonds. The microparticles are also called proteinoid microspheres, which are particles primarily composed of polyamino acids. Preferably, the polyamino acids contain anhydropolyaspartic acid. Proteinoid microspheres containing anhydropolyaspartic acid are hydrophobic substances, having abundant five-membered ring imide structures and almost no carboxyl or amino groups. Therefore, although they are poorly water-soluble and only slightly soluble in hot water, they form tiny spherical substances upon cooling.

[0019] Proteinoids are produced by heating and melting amino acids or amino acid precursors. For example, proteinoids can be obtained by heating monoammonium malate at 140 to 180°C for 6 to 24 hours. In addition to monoammonium malate, proteinoids can also be obtained by heating glutamic acid, aspartic acid, lysine, etc. Considering the ease of forming proteinoid microspheres, it is preferable to use proteinoids obtained by heating hydrophobic amino acids such as valine, leucine, isoleucine, phenylalanine, methionine, tryptophan, proline, and alanine rather than hydrophilic amino acids.

[0020] As described above, proteinoid microspheres are produced by cooling proteinoids dissolved in hot water. The particle size of proteinoid microspheres varies depending on the cooling rate. When microparticles are used as nucleic acid delivery vehicles, a small spherical diameter is preferable. The particle size of the microparticles is, for example, on the order of micrometers, preferably on the order of nanometers. For example, the particle size of the microparticles is 10 to 1,000 nm, 30 to 1,000 nm, 40 to 800 nm, or 50 to 600 nm. The particle size can be measured by sieving, sedimentation, microscopy, dynamic light scattering, laser diffraction / scattering, electrical resistance testing, observation with a transmission electron microscope, observation with a scanning electron microscope, or the like. The particle size may also be measured using a known particle size distribution analyzer. The particle size can be expressed as a Stokes equivalent diameter, a circle equivalent diameter, a sphere equivalent diameter, or the like, depending on the measurement method. The particle size may also be expressed as an average particle size, volume average particle size, area average particle size, or the like, expressed as an average of multiple particles.

[0021] The nucleic acid encapsulated in the microparticles is not particularly limited and may be DNA or RNA. The nucleic acid may be a chemically modified nucleic acid. The base length of the nucleic acid is not particularly limited as long as the nucleic acid is retained in the microparticles. The base sequence of the nucleic acid can be flexibly designed depending on the function of the nucleic acid, etc. When the nucleic acid is delivered for the purpose of gene expression, the nucleic acid is one or more nucleic acids encoding one or more proteins, peptides, or polypeptides. When the nucleic acid is delivered as a nucleic acid vaccine, the nucleic acid encodes an antigenic polypeptide. In this case, the nucleic acid may preferably be RNA, particularly mRNA, or may be RNA having an open reading frame. When the nucleic acid is delivered for the purpose of gene expression inhibition (gene silencing), the nucleic acid is a gene-inhibitory nucleic acid or a regulatory nucleic acid. Examples of gene-inhibitory nucleic acids and regulatory nucleic acids include antisense nucleic acids (DNA or RNA), siRNA, shRNA, miRNA, CRISPRi, etc.

[0022] The manner in which nucleic acids are held on microparticles is not particularly limited, and nucleic acids may be encapsulated in the microparticles, adsorbed to the surface of the microparticles, or both. Alternatively, a portion of the nucleic acid may be encapsulated in the microparticles, with the remaining portion exposed to the outside of the microparticles. To hold nucleic acids on microparticles, proteinoid microspheres may be mixed with nucleic acids, or proteinoid microspheres may be formed by mixing nucleic acids with hot water in which a proteinoid has been dissolved.

[0023] As shown in the examples below, the microparticles contained in the nucleic acid delivery composition according to this embodiment have the property of incorporating hydrophobic substances and can retain nucleic acids via the hydrophobic portion of the nucleic acid. By exposing the nucleic acid delivery composition to cells, the nucleic acid is delivered into the cells by a mechanism similar to that of fusion between liposomes and cells.

[0024] As shown in the following examples, the nucleic acid delivery composition according to this embodiment can deliver nucleic acids into cells and express proteins encoded by the nucleic acids, thereby providing the functions of the nucleic acids.

[0025] In another embodiment, the use of the microparticles as a composition for nucleic acid delivery is provided. In another embodiment, a nucleic acid drug is provided, comprising the microparticles and a nucleic acid held by the microparticles. The nucleic acid contained in the nucleic acid drug is not particularly limited as long as it has anti-cancer activity, anti-inflammatory activity, antibacterial activity, antiviral activity, or the like. The nucleic acid drug may be a nucleic acid vaccine according to the following embodiment 2 or an anti-cancer drug according to the following embodiment 3.

[0026] The dosage form of the nucleic acid drug is not particularly limited and may be, for example, an injection, oral preparation, rectal suppository, vaginal suppository, nasal absorption agent, transdermal absorption agent, pulmonary absorption agent, or oral absorption agent. The nucleic acid drug may also be a combination drug containing a pharmacologically acceptable carrier. The pharmacologically acceptable carrier is various organic or inorganic carrier substances. The pharmacologically acceptable carrier is incorporated into the nucleic acid drug as, for example, an excipient, lubricant, binder, or disintegrant in a solid preparation, or as a solvent, solubilizer, suspending agent, isotonicity agent, buffer, or soothing agent in a liquid preparation. Furthermore, additives such as preservatives, antioxidants, colorants, and sweeteners can also be used as needed.

[0027] Examples of excipients include lactose, sucrose, D-mannitol, starch, crystalline cellulose, light anhydrous silicic acid, etc. Examples of lubricants include magnesium stearate, calcium stearate, talc, colloidal silica, etc. Examples of binders include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, etc. Examples of disintegrants include starch, carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, carboxymethylstarch sodium, etc.

[0028] Examples of solvents include water for injection, alcohol, propylene glycol, macrogol, etc. Examples of solubilizers include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, etc. Examples of suspending agents include surfactants, hydrophilic polymers, etc., such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glycerin monostearate, polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, etc.

[0029] Examples of isotonic agents include sodium chloride, glycerin, D-mannitol, etc. Examples of buffers include phosphate, acetate, carbonate, citrate buffer solutions, etc. Examples of soothing agents include benzyl alcohol, etc. Examples of preservatives include parahydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, etc. Examples of antioxidants include sulfites, ascorbic acid, etc.

[0030] The nucleic acid drug is produced by a known method and contains the nucleic acid held on the microparticles as an active ingredient. The anticancer drug contains, for example, 0.1 to 99 wt %, 1 to 50 wt %, and preferably 1 to 20 wt % of the nucleic acid as an active ingredient.

[0031] The nucleic acid medicine can be administered to humans and non-human animals, preferably mammals, including primates such as humans and chimpanzees, laboratory animals such as rats, mice, and rabbits, livestock animals such as pigs, cows, horses, sheep, and goats, and pet animals such as dogs and cats.

[0032] The dosage of the nucleic acid drug is determined appropriately based on the subject's gender, age, weight, symptoms, etc. The nucleic acid drug is administered so that the nucleic acid is in an effective amount. When the nucleic acid drug is a nucleic acid vaccine, the effective amount refers to the amount that can induce antibody production in the vaccinated subject. When the nucleic acid drug is an anticancer drug, the effective amount refers to the amount necessary to delay, inhibit, prevent, reverse, or cure the progression of the cancer being treated or treated. The dosage of the nucleic acid drug is typically 0.01 to 1000 mg / kg, preferably 0.1 to 200 mg / kg, and more preferably 0.2 to 20 mg / kg, and can be administered once a day or in divided doses. The nucleic acid drug may be administered at various frequencies, such as daily, every other day, once a week, once every two weeks, or once a month. If necessary, the nucleic acid drug may be used in an amount outside the above range.

[0033] (Embodiment 2: Nucleic Acid Vaccine) The nucleic acid vaccine according to this embodiment will be described mainly focusing on the differences from the above-mentioned embodiment 1. Unless otherwise specified, the explanation of the nucleic acid vaccine can be found in the explanation of the nucleic acid medicine in the above-mentioned embodiment 1.

[0034] The nucleic acid vaccine according to this embodiment includes the nucleic acid delivery composition according to the first embodiment, wherein the nucleic acid encodes an antigenic polypeptide. The antigenic polypeptide is recognized by antibodies in the immune response of the inoculated subject. The antigenic polypeptide includes at least a portion of a protein contained in, for example, a virus or bacterium that causes an infectious disease. Examples of viruses include influenza virus, adenovirus, coronavirus, SARS-CoV-2, herpes virus, human papillomavirus, and hepatitis B virus. Examples of bacteria include Mycobacterium tuberculosis, Clostridium difficile, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, and Pseudomonas aeruginosa.

[0035] The antigenic polypeptide is not particularly limited as long as it is at least a part of a protein that exhibits antigenicity, and examples thereof include hemagglutinin protein, S protein, and the like.

[0036] The nucleic acid vaccine may be administered parenterally or orally. The nucleic acid vaccine is preferably administered by local injection, such as subcutaneous, intradermal, intramuscular, or abdominal cavity. Alternatively, the nucleic acid vaccine may be administered to a subject by inhalation, for example, through the nasal cavity, oral cavity, or lungs. To stimulate subcutaneous immune cells, the nucleic acid vaccine may be administered subcutaneously to a subject. Subcutaneous administration may be by abrasion, or a non-invasive method such as a microneedle.

[0037] The nucleic acid vaccine of this embodiment contains the above-mentioned nucleic acid delivery composition that can deliver nucleic acids to cells, and is therefore particularly useful for RNA vaccines, DNA vaccines, etc., which are effective when nucleic acids encoding antigenic polypeptides are delivered into cells.

[0038] (Embodiment 3: Anticancer agent) The anticancer drug according to this embodiment will be described, focusing mainly on the differences from the above-mentioned embodiment 1. Unless otherwise specified, the explanation of the nucleic acid medicine in the above-mentioned embodiment 1 can be referred to for the anticancer drug.

[0039] The anticancer agent according to this embodiment includes the nucleic acid delivery composition according to the first embodiment, and the nucleic acid is an anticancer nucleic acid. The anticancer nucleic acid may be a gene-suppressing nucleic acid or a regulatory nucleic acid, a DNA aptamer or an RNA aptamer that binds to a cancer-related target, or a nucleic acid that controls post-transcriptional splicing.

[0040] For example, anticancer nucleic acids include CDC shRNA, which suppresses the proliferation and division of cancer cells; P28 shRNA, which suppresses P28, which inhibits the function of tumor suppressor genes; MDM2 shRNA, which suppresses MDM2, which inhibits the function of tumor suppressor genes; and IL6 shRNA, which suppresses IL6, which promotes cancer development and metastasis.

[0041] The cancer targeted by the anticancer agent according to this embodiment is not particularly limited, and may be a blood cancer or a solid cancer. Examples of cancer types include leukemia, lymphoma, hepatocellular carcinoma, gastric cancer, pancreatic cancer, lung cancer, colon cancer, breast cancer, liver cancer (hepatic cancer), kidney cancer (renal cancer), tongue cancer, thyroid cancer, uterine cancer, ovarian cancer, prostate cancer, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, and leiomyoma.

[0042] The anticancer agent according to this embodiment contains the nucleic acid delivery composition capable of delivering nucleic acids to cells, and therefore can efficiently deliver anticancer nucleic acids to cancer cells.

[0043] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples. [Example]

[0044] [Preparation of proteinoids] Monoammonium malate was placed in a test tube and heated at 180°C for 24 hours to obtain a proteinoid.

[0045] [Formation of proteinoid microspheres] A sample prepared by suspending 0.3 g of the resulting proteinoid in 30 mL of purified water was heated in a water bath at 95°C for 2 hours. Plasmid DNA (pGLO, Bio-Rad) carrying an ampicillin resistance gene and a green fluorescent protein (GFP) gene was then added to the sample. After suction filtration, the sample was rapidly cooled by immersion in ice water (4°C). The sample was centrifuged twice (4000 rpm, 15 minutes), and the supernatant was washed away.

[0046] [Observation of proteinoid microspheres and confirmation of nucleic acid uptake] The obtained samples were observed under a scanning electron microscope (SEM, S3000N, Hitachi High-Technologies Corporation). Nucleic acid uptake was assessed by detecting a signal representing P derived from the phosphate group of DNA in the microspheres using elemental analysis using an electron probe microanalyzer (EPMA, JXA-8230, JEOL Ltd.).

[0047] [Intracellular expression of nucleic acids incorporated into proteinoid microspheres] After culturing E. coli in LB medium (5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, 100 mg / L sodium ampicillin, and 6 g / L arabinose) for 8 hours, 10 μL of the proteinoid microsphere suspension containing the plasmid DNA was added to 250 μL of LB medium. 100 mg / L sodium ampicillin and 6 g / L arabinose were added, and the cells were further cultured. Expression of the incorporated DNA within the cells was confirmed using a fluorescence microscope (BZ-X810, Keyence Corporation), as the cells grew in the ampicillin-containing medium and emitted green GFP fluorescence.

[0048] (result) In the SEM image of the proteinoid microspheres shown in Figure 1, spherical particles with a diameter of approximately 100 nm were observed. As shown in Figure 2, elemental analysis revealed a phosphorus (P) signal derived from DNA that overlapped with spherical carbon (C) and nitrogen (N) signals derived from the proteinoid microspheres. This confirmed that DNA had been incorporated into the proteinoid microspheres.

[0049] In the fluorescence microscope image of E. coli shown in Figure 3, green fluorescence was observed due to the GFP protein produced by transcription and translation of the GFP gene within the E. coli. This demonstrated that DNA incorporated into the proteinoid microspheres was successfully delivered into the cells.

[0050] The above-described embodiments are intended to explain the present invention and are not intended to limit the scope of the present invention. That is, the scope of the present invention is defined by the claims, not by the embodiments. Various modifications made within the scope of the claims and within the scope of the meaning of the invention equivalent thereto are considered to be within the scope of the present invention. [Industrial Applicability]

[0051] The present invention is useful for a drug delivery system for nucleic acid drugs.

Claims

1. microparticles containing polyamino acids; Nucleic acid retained on the microparticles; A composition for nucleic acid delivery comprising:

2. The polyamino acid is containing anhydropolyaspartic acid, The nucleic acid delivery composition according to claim 1 .

3. The nucleic acid delivery composition according to claim 1 or 2, The nucleic acid is Encoding an antigenic polypeptide, Nucleic acid vaccines.

4. The nucleic acid delivery composition according to claim 1 or 2, The nucleic acid is It is an anti-cancer nucleic acid, Anticancer drugs.

5. Use of microparticles containing polyamino acids as a composition for nucleic acid delivery.

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