Compound-nucleic acid nanoparticles, medicaments, uses, and methods of preparation

Abstract

A compound-nucleic acid nanoparticle includes: a nucleic acid; and a compound of formula I, # imgabs0, in which n is an integer selected from 1 to 14, and R is a positively charged group containing one or more nitrogen atoms. The invention also relates to a medicament comprising the compound-nucleic acid nanoparticle, a use of the compound-nucleic acid nanoparticle, a method for preparing the compound-nucleic acid nanoparticle, and a use of the compound of formula I in the preparation of a product for introducing the nucleic acid into a cell.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid drugs, and particularly to compound-nucleic acid nanoparticles, drugs comprising compound-nucleic acid nanoparticles, uses of compound-nucleic acid nanoparticles, preparation methods of compound-nucleic acid nanoparticles, and uses of compounds in the preparation of products for introducing nucleic acids into cells. Background Art

[0002] Nucleic acid drugs can regulate genes related to protein expression based on the principle of base complementary pairing. Nucleic acid drugs can be designed based on the base sequence of the target gene.

[0003] In recent years, the research and development of nucleic acid drugs has attracted extensive attention. As of 2022, 4 small interfering ribonucleic acid (siRNA) drugs, 9 antisense oligonucleotide (ASO) drugs, and 2 messenger ribonucleic acid (mRNA) vaccines against COVID-19 have been launched on the market, and there are also hundreds of candidate drugs in the clinical research stage.

[0004] Although remarkable progress has been made, nucleic acid drugs still face important challenges in pharmacokinetics, including systemic circulation, cellular uptake, and lysosome / endosome escape. First, nucleic acid drugs are extremely vulnerable to nuclease degradation, so it is difficult for nucleic acid drugs to reach the site of action during circulation in the body. Second, the hydrophilic, negatively charged, and macromolecular properties of nucleic acid drugs make it difficult for them to cross the cell membrane and enter the cytoplasm. Finally, endosome / lysosome capture is also a stumbling block for nucleic acid drugs to exert their effects. Nucleic acid drugs usually enter cells through endocytosis. During endocytosis, the acidic microenvironment of endosomes and lysosomes and the abundance of nucleases cause most nucleic acid drugs to be degraded, and only a very small amount can escape from endosomes / lysosomes and enter the cytoplasm. In summary, developing special drug formulations or drug carriers to protect nucleic acid drugs from degradation and deliver them to the cytoplasm is one of the keys to ensuring the efficacy of nucleic acid drugs.

[0005] Currently, liposome nanoparticles are commonly used clinically to load and deliver nucleic acid drugs. In addition, polypeptides, cationic polymers, nucleic acid nanostructures, etc. have also been actively designed for nucleic acid drug delivery. Although they have many excellent properties, including good biocompatibility and strong cellular uptake, the main bottleneck lies in their endocytosis-dependent pathway for entering cells, resulting in their easy capture and degradation by endosomes / lysosomes. Research shows that liposome nanoparticles can only deliver 0.05%-1% of nucleic acids into the cytoplasm through the endocytic pathway, and the delivery efficiency is extremely low.

[0006] Therefore, it is necessary to provide new technical solutions to solve one or more of the above problems, and / or other problems. Summary of the Invention

[0007] In view of this, an object of the present invention is to provide new technical solutions to solve one or more of the above problems, and / or other problems.

[0008] One aspect of an embodiment of the present invention provides a compound-nucleic acid nanoparticle, which comprises: a nucleic acid; and a compound of formula I, wherein, n is an integer selected from 1 to 14, and R is a positively charged group containing one or more nitrogen atoms.

[0009] In some embodiments, R is a positively charged group that can be ionized when the pH value of its environment is less than its pKa value.

[0010] In some embodiments, R includes a primary amine group secondary amine group tertiary amine group quaternary amine group amidinyl and guanidyl one or more of.

[0011] In some embodiments, the nucleic acid includes one or more of small interfering ribonucleic acid, micro ribonucleic acid, antisense oligonucleotide, messenger ribonucleic acid, long non-coding ribonucleic acid, circular ribonucleic acid, small nuclear ribonucleic acid, modified small interfering ribonucleic acid, modified micro ribonucleic acid, modified antisense oligonucleotide, modified messenger ribonucleic acid, modified long non-coding ribonucleic acid, modified circular ribonucleic acid, and modified small nuclear ribonucleic acid.

[0012] In some embodiments, the sizes of the small interfering ribonucleic acid and the modified small interfering ribonucleic acid are respectively in the range of 15bp - 30bp.

[0013] In some embodiments, the lengths of the micro ribonucleic acid and the modified micro ribonucleic acid are respectively in the range of 15nt - 30nt.

[0014] In some embodiments, the lengths of the antisense oligonucleotide and the modified antisense oligonucleotide are respectively in the range of 15nt - 30nt.

[0015] In some embodiments, the lengths of the messenger ribonucleic acid and the modified messenger ribonucleic acid are respectively independently >500nt, or in the range of 500nt - 3000nt.

[0016] In some embodiments, R includes guanidyl n > 1.

[0017] In some embodiments, R comprises a guanidyl group The nucleic acid comprises one or more of an antisense oligonucleotide, a messenger ribonucleic acid, a modified antisense oligonucleotide, and a modified messenger ribonucleic acid.

[0018] In some embodiments, the compound of Formula I comprises one or more compounds having the structural formula and the nucleic acid comprises an antisense oligonucleotide and / or a modified antisense oligonucleotide.

[0019] In some embodiments, the compound of Formula I comprises a compound having the structural formula and the nucleic acid comprises a small interfering ribonucleic acid, a modified small interfering ribonucleic acid, a messenger ribonucleic acid, a modified messenger ribonucleic acid, an antisense oligonucleotide, and / or a modified antisense oligonucleotide.

[0020] In some embodiments, the compound of Formula I comprises a compound having the structural formula and the nucleic acid comprises one or more of a small interfering ribonucleic acid, a micro ribonucleic acid, a messenger ribonucleic acid, a modified small interfering ribonucleic acid, a modified micro ribonucleic acid, and a modified messenger ribonucleic acid.

[0021] In some embodiments, the compound of Formula I comprises a compound having the structural formula and the nucleic acid comprises one or more of a double-stranded small interfering ribonucleic acid, a single-stranded micro ribonucleic acid, a long-chain messenger ribonucleic acid, a modified double-stranded small interfering ribonucleic acid, a modified single-stranded micro ribonucleic acid, and a modified long-chain messenger ribonucleic acid.

[0022] In some embodiments, the compound of Formula I comprises a compound having the structural formula and the nucleic acid comprises a messenger ribonucleic acid and / or a modified messenger ribonucleic acid.

[0023] In some embodiments, the ratio of the number of moles of the compound of Formula I to the number of moles of phosphate groups in the nucleic acid ranges from 100:1 to 0.5:1.

[0024] In some embodiments, the ratio of the number of moles of the compound of Formula I to the number of moles of phosphate groups in the nucleic acid ranges from 40:1 to 10:1.

[0025] In some embodiments, the ratio of the number of moles of the compound of Formula I to the number of moles of phosphate groups in the nucleic acid is 40:1, 20:1, 15:1, or 10:1.

[0026] In some embodiments, the particle size of the compound-nucleic acid nanoparticle ranges from 30 nanometers to 600 nanometers.

[0027] In some embodiments, the particle size of the compound-nucleic acid nanoparticles ranges from 47.42 nm to 208.44 nm.

[0028] Another aspect of the embodiments of the present invention relates to a drug comprising a pharmaceutical carrier and the compound-nucleic acid nanoparticles as described in the present invention.

[0029] In some embodiments, the drug is one or more of a drug for preventing and treating tumors, a drug for preventing and treating cancers, a drug for preventing and treating neurodegenerative diseases, a protein replacement drug, a vaccine, a gene editing drug, and a drug for preventing and treating pulmonary fibrosis.

[0030] In some embodiments, the drug is one or more of a small interfering ribonucleic acid drug, a micro ribonucleic acid drug, an antisense oligonucleotide drug, a messenger ribonucleic acid drug, a long non-coding ribonucleic acid drug, a circular ribonucleic acid drug, a small nuclear ribonucleic acid drug, a modified small interfering ribonucleic acid drug, a modified micro ribonucleic acid drug, a modified antisense oligonucleotide drug, a modified messenger ribonucleic acid drug, a modified long non-coding ribonucleic acid drug, a modified circular ribonucleic acid drug, and a modified small nuclear ribonucleic acid drug.

[0031] In some embodiments, the drug is a drug for preventing and treating leukemia.

[0032] In some embodiments, the drug is a drug for preventing and treating pancreatic cancer.

[0033] In some embodiments, the drug is a drug for preventing and treating cervical cancer.

[0034] In some embodiments, the drug is a drug for preventing and treating lung cancer.

[0035] Another aspect of the embodiments of the present invention relates to the use of the compound-nucleic acid nanoparticles as described in the present invention in the preparation of a product for introducing nucleic acid into cells.

[0036] In some embodiments, the cells are mammalian cells.

[0037] Another aspect of the embodiments of the present invention relates to the use of the compound-nucleic acid nanoparticles as described in the present invention in the preparation of a product for in vivo delivery of nucleic acid to be administered to a mammal.

[0038] Another aspect of the embodiments of the present invention relates to the use of the compound-nucleic acid nanoparticles as described in the present invention in the preparation of a drug to be administered to a mammal.

[0039] In some embodiments, the drug is one or more of a drug for preventing and treating tumors, a drug for preventing and treating cancers, a drug for preventing and treating neurodegenerative diseases, a protein replacement drug, a vaccine, a gene editing drug, and a drug for preventing and treating pulmonary fibrosis.

[0040] In some embodiments, the drug is one or more of small interfering ribonucleic acid drugs, micro ribonucleic acid drugs, antisense oligonucleotide drugs, messenger ribonucleic acid drugs, long non-coding ribonucleic acid drugs, circular ribonucleic acid drugs, small nuclear ribonucleic acid drugs, modified small interfering ribonucleic acid drugs, modified micro ribonucleic acid drugs, modified antisense oligonucleotide drugs, modified messenger ribonucleic acid drugs, modified long non-coding ribonucleic acid drugs, modified circular ribonucleic acid drugs, and modified small nuclear ribonucleic acid drugs.

[0041] In some embodiments, the drug is a drug for the prevention and treatment of leukemia.

[0042] In some embodiments, the drug is a drug for the prevention and treatment of pancreatic cancer.

[0043] In some embodiments, the drug is a drug for the prevention and treatment of cervical cancer.

[0044] In some embodiments, the drug is a drug for the prevention and treatment of lung cancer.

[0045] Another aspect of the embodiments of the present invention relates to a method for preparing the compound-nucleic acid nanoparticle of the present invention, which includes: dissolving the compound of formula I in a solvent to obtain a solution of the compound of formula I; dissolving the nucleic acid in water to obtain a nucleic acid solution; and mixing the solution of the compound of formula I with the nucleic acid solution to obtain the compound-nucleic acid nanoparticle.

[0046] In some embodiments, the temperature for mixing the solution of the compound of formula I with the nucleic acid solution ranges from 25°C to -80°C.

[0047] In some embodiments, the time for mixing the solution of the compound of formula I with the nucleic acid solution ranges from 15 minutes to 48 hours.

[0048] Another aspect of the embodiments of the present invention relates to the use of the compound of formula I in the preparation of a product for introducing nucleic acid into cells, wherein n is an integer selected from 1 to 14, and R is a positively charged group containing one or more nitrogen atoms.

[0049] In some embodiments, R is a positively charged group that can be ionized when the pH value of its environment is less than its pKa value.

[0050] In some embodiments, R includes a primary amine group a secondary amine group a tertiary amine group a quaternary amine group an imidazolyl group and a guanidyl group and one or more of them.

[0051] In some embodiments, the nucleic acid includes one or more of small interfering ribonucleic acid, micro ribonucleic acid, antisense oligonucleotide, messenger ribonucleic acid, long non-coding ribonucleic acid, circular ribonucleic acid, small nuclear ribonucleic acid, modified small interfering ribonucleic acid, modified micro ribonucleic acid, modified antisense oligonucleotide, modified messenger ribonucleic acid, modified long non-coding ribonucleic acid, modified circular ribonucleic acid, and modified small nuclear ribonucleic acid.

[0052] In some embodiments, the sizes of the small interfering ribonucleic acid and the modified small interfering ribonucleic acid are respectively in the range of 15 bp - 30 bp.

[0053] In some embodiments, the lengths of the micro ribonucleic acid and the modified micro ribonucleic acid are respectively in the range of 15 nt - 30 nt.

[0054] In some embodiments, the lengths of the antisense oligonucleotide and the modified antisense oligonucleotide are respectively in the range of 15 nt - 30 nt.

[0055] In some embodiments, the lengths of the messenger ribonucleic acid and the modified messenger ribonucleic acid are respectively independently > 500 nt or in the range of 500 nt - 3000 nt.

[0056] In some embodiments, R includes a guanidyl group n > 1.

[0057] In some embodiments, R includes a guanidyl group The nucleic acid includes one or more of antisense oligonucleotide, messenger ribonucleic acid, modified antisense oligonucleotide, and modified messenger ribonucleic acid.

[0058] In some embodiments, the compound of Formula I includes one or more of the compounds having the structural formula and the nucleic acid includes antisense oligonucleotide and / or modified antisense oligonucleotide.

[0059] In some embodiments, the compound of Formula I includes the compound having the structural formula and the nucleic acid includes small interfering ribonucleic acid, modified small interfering ribonucleic acid, messenger ribonucleic acid, modified messenger ribonucleic acid, antisense oligonucleotide, and / or modified antisense oligonucleotide.

[0060] In some embodiments, the compound of Formula I includes the compound having the structural formula and the nucleic acid includes one or more of small interfering ribonucleic acid, micro ribonucleic acid, messenger ribonucleic acid, modified small interfering ribonucleic acid, modified micro ribonucleic acid, and modified messenger ribonucleic acid.

[0061] In some embodiments, the compound of Formula I includes a compound having the structural formula and the nucleic acid includes one or more of double-stranded small interfering ribonucleic acid, single-stranded micro ribonucleic acid, long-chain messenger ribonucleic acid, modified double-stranded small interfering ribonucleic acid, modified single-stranded micro ribonucleic acid, and modified long-chain messenger ribonucleic acid.

[0062] In some embodiments, the compound of Formula I includes a compound having the structural formula and the nucleic acid includes messenger ribonucleic acid and / or modified messenger ribonucleic acid.

[0063] The technical features of each embodiment in the present invention can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in each embodiment are described. However, as long as these combinations of technical features can be implemented by those skilled in the art, they should be considered as falling within the scope described in this specification.

[0064] In order to make the objectives, technical solutions, features and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to the scope of the described embodiments. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention. Description of the Drawings

[0065] Figure 1 It is the mass spectrometry diagrams of Compounds 1, 2, and 3 in Example 1.

[0066] Figure 2 It is the mass spectrometry diagram of Compound 4 in Example 2.

[0067] Figure 3 It is the mass spectrometry diagrams of Compounds 5, 6, 7, and 8 in Example 3.

[0068] Figure 4 It is the particle size number distribution data diagram of the compound-nucleic acid nanoparticles formed by Compounds 1 to 5 containing different positively charged groups and antisense oligonucleotide (ASO) in Example 4.

[0069] Figure 5 It is the particle size number distribution data diagram of the compound-nucleic acid nanoparticles formed by Compounds 6 to 8 with different chain lengths and ASO in Example 5.

[0070] Figure 6 It is the particle size number distribution data diagram of the compound-nucleic acid nanoparticles formed by the assembly of Compound 2 and ASO with different sequences in Example 6.

[0071] Figure 7 Particle size number distribution data graphs of compound-nucleic acid nanoparticles formed by Compound 3 in Example 7 with small interfering ribonucleic acid (siRNA), micro ribonucleic acid (miRNA), and messenger ribonucleic acid (mRNA), respectively.

[0072] Figure 8 It is a particle size number distribution data graph of compound-nucleic acid nanoparticles formed by assembling Compound 2 with different chemically modified ASOs in Example 8.

[0073] Figure 9 Flow cytometry results graphs of the cellular uptake of Compound 1-ASO nanoparticles, Compound 2-ASO nanoparticles, Compound 3-ASO nanoparticles, Compound 4-ASO nanoparticles, and Compound 5-ASO nanoparticles in Example 9.

[0074] Figure 10 It is a flow cytometry results graph of the study on the cellular uptake mechanism of Compound 1-ASO nanoparticles, Compound 2-ASO nanoparticles, Compound 3-ASO nanoparticles, Compound 4-ASO nanoparticles, and Compound 5-ASO nanoparticles in Example 10.

[0075] Figure 11 Flow cytometry results graphs of the cellular gene therapy of Compound 2-siRNA nanoparticles in Example 11.

[0076] Figure 12 It is a results graph of the in vitro inhibition of the proliferation of lung cancer cells by Compound 2-ASO nanoparticles in Example 12.

[0077] Figure 13 Results graphs of the protein expression in cells of Compound 5-mRNA nanoparticles and Compound 1-mRNA nanoparticles in Example 13.

[0078] Figure 14 It is a results graph of the treatment of pulmonary fibrosis in mice with Compound 2-siRNA nanoparticles in Example 14. Detailed implementation manners

[0079] One aspect of the embodiments of the present invention provides a compound-nucleic acid nanoparticle, which comprises: a nucleic acid; and a Compound of Formula I, wherein, n is an integer selected from 1 to 14, and R is a positively charged group containing one or more nitrogen atoms.

[0080] The compound-nucleic acid nanoparticles can contribute to the efficient delivery of nucleic acid drugs to the cytoplasm, solving important problems faced in the fields of chemistry and biomedicine. The compound-nucleic acid nanoparticles can utilize dynamic covalent thiol-exchange chemistry. The compound of formula I can be referred to as a thiol compound. The disulfide unit of the compound-nucleic acid nanoparticles can undergo a dynamic thiol-exchange reaction with the thiols on the outer surface of the cell membrane, thereby covalently binding to the cell surface and being further rapidly transported into the cytoplasm. Under the action of endogenous glutathione in the cytoplasm, the carrier is cleaved to release the nucleic acid drug. Compared with the traditional endocytic delivery pathway, the compound-nucleic acid nanoparticles can effectively avoid the capture and degradation by endosomes and lysosomes through thiol-exchange-mediated cell delivery, greatly improving the nucleic acid delivery efficiency.

[0081] In the present invention, unless otherwise specifically stated and / or the context clearly indicates, a numerical range may include any sub-range therein. For example, the selection range of n may include 1-14, 2-14, 3-14, 4-14, 5-14, 6-14, 7-14, 8-14, 9-14, 10-14, 11-14, 12-14, 13-14, 1-13, 2-13, 3-13, 4-13... 1-7... and so on.

[0082] In the examples of the present invention, the compounds of formula I with different n values (carbon chain lengths) can all form nanoparticles with smaller and uniform particle sizes with nucleic acids. As the n value increases, the nanoparticle size increases slightly. The formed compound-nucleic acid nanoparticles can all exhibit good delivery effects, and the nucleic acids delivered via the compound-nucleic acid nanoparticles can exert corresponding therapeutic effects.

[0083] Specifically, n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some embodiments, n is 1, 3, 5, or 7.

[0084] In the examples of the present invention, the strength of R has no significant effect on the formation of the compound-nucleic acid nanoparticles, and the formed compound-nucleic acid nanoparticles can all exhibit good delivery effects.

[0085] R can include one nitrogen (N) atom, two nitrogen (N) atoms, three nitrogen (N) atoms, or more nitrogen (N) atoms. R can include other atoms, such as hydrogen (H) atoms and carbon (C) atoms. R can include substituents, such as alkyl groups like methyl (-CH3).

[0086] R can be a positively charged group that helps the compound of formula I bind to the negatively charged nucleic acid. R can be a positively charged group that is ionizable under certain conditions. The acid dissociation constant (pKa) value of R is a specific equilibrium constant that represents its ability to dissociate hydrogen ions. The pKa value of R can be obtained by prediction through computer theoretical simulation.

[0087] In some embodiments, R is a positively charged group that is ionizable when the pH value of its environment is less than its pKa value.

[0088] When the pH value of the environment (such as a solution) where the compound of formula I is located is less than the pKa value of R, R can be ionized and form a stable salt bridge with the negatively charged phosphate group in the nucleic acid, thereby promoting the assembly of the compound of formula I and the nucleic acid to form nanoparticles.

[0089] In some embodiments, R includes a primary amine group a secondary amine group a tertiary amine group a quaternary amine group an amidino group and a guanidino group or one or more of them.

[0090] For example, the pKa values of the primary amine group the secondary amine group the tertiary amine group the quaternary amine group the amidino group the guanidino group obtained by computer simulation prediction are 12.08, 10.70, 11.57, 8.91, 13.39, and 12.74 respectively.

[0091] The compound-nucleic acid nanoparticles can help achieve thiol-mediated cytoplasmic delivery of various types of nucleic acids. In the embodiments of the present invention, perhaps because the mode of action is that the positively charged group of the compound of formula I forms a non-covalent interaction with the negatively charged phosphate group of the nucleic acid, the type of nucleic acid, the sequence of the nucleic acid, whether the nucleic acid is single-stranded or double-stranded, whether the nucleic acid is chemically modified, the type of nucleic acid chemical modification, and the amount of nucleic acid chemical modification have no obvious effect on the formation of the compound-nucleic acid nanoparticles. The compound of formula I can form nanoparticles with various nucleic acids, and the compound-nucleic acid nanoparticles can exert a good delivery effect on various nucleic acids. The nucleic acid delivered via the compound-nucleic acid nanoparticles can exert the corresponding therapeutic effect. Therefore, the compound of formula I and the compound-nucleic acid nanoparticles have broad application prospects in nucleic acid drug delivery.

[0092] In some embodiments, the nucleic acid includes one or more of small interfering ribonucleic acid (siRNA), micro ribonucleic acid (miRNA), antisense oligonucleotide (ASO), messenger ribonucleic acid (mRNA), long non-coding ribonucleic acid (lncRNA), circular ribonucleic acid (circRNA), small nuclear ribonucleic acid (snRNA), modified siRNA, modified miRNA, modified ASO, modified mRNA, modified lncRNA, modified circRNA, and modified snRNA.

[0093] The compound-nucleic acid nanoparticles can be used for the delivery of nucleic acids of any sequence. The compound-nucleic acid nanoparticles can facilitate the thiol-mediated cytoplasmic delivery of nucleic acids of various sizes. In the embodiments of the present invention, the formation of the compound-nucleic acid nanoparticles is independent of the sequence of the nucleic acid, and nucleic acids of any sequence can form nanoparticles with the compound of formula I.

[0094] In some embodiments, the sizes of the small interfering ribonucleic acid and the modified small interfering ribonucleic acid are respectively in the range of 15 bp - 30 bp. The compound-nucleic acid nanoparticles can target growth factor receptors, signal transduction molecules, cyclins, apoptosis regulators, proteolytic enzymes, vascular endothelial growth factor, etc., and regulate their gene expression levels for the treatment of tumors, neurodegenerative diseases, etc.

[0095] In some embodiments, the lengths of the micro ribonucleic acid and the modified micro ribonucleic acid are respectively in the range of 15 nt - 30 nt. The compound-nucleic acid nanoparticles can target growth factor receptors, signal transduction molecules, cyclins, apoptosis regulators, proteolytic enzymes, vascular endothelial growth factor, etc., and regulate their gene expression levels for the treatment of tumors, neurodegenerative diseases, etc.

[0096] In some embodiments, the lengths of the antisense oligonucleotide and the modified antisense oligonucleotide are respectively in the range of 15 nt - 30 nt. The compound-nucleic acid nanoparticles can target growth factor receptors, signal transduction molecules, cyclins, apoptosis regulators, proteases, vascular endothelial growth factor, etc., and regulate their gene expression levels for the treatment of tumors, neurodegenerative diseases, etc.

[0097] In some embodiments, the lengths of the messenger ribonucleic acid and the modified messenger ribonucleic acid are respectively independently > 500 nt or in the range of 500 nt - 3000 nt. The compound-nucleic acid nanoparticles can be used for protein replacement therapy to encode relevant proteins for treating diseases or for mRNA vaccines to encode relevant proteins that can cause an immune response in the human body.

[0098] The compound-nucleic acid nanoparticles may include the compound of formula I having one or more structures.

[0099] In some embodiments, R includes a guanidyl group n > 1.

[0100] In some embodiments, R includes a guanidyl group The nucleic acid includes one or more of antisense oligonucleotides, messenger ribonucleic acid, modified antisense oligonucleotides, and modified messenger ribonucleic acid.

[0101] The molecular formula of the compound of formula I may be C9H 16 NOS2(CH2) n R. The molecular formula of the compound of formula I may be C 10 H 20 N2OS2, C 11 H 22 N2OS2, C 12 H 24 N2OS2, C 13 H 26 N2OS2, C 11 H 22 N4OS2, C 13 H 27 N4OS2, C 15 H 31 N4OS2, or C 17 H 35 N4OS2.

[0102] In some embodiments, the compound of formula I includes one or more of the compounds having the structural formula and the nucleic acid includes antisense oligonucleotides and / or modified antisense oligonucleotides.

[0103] In some embodiments, the compound of Formula I includes a compound having the structural formula and the nucleic acid includes small interfering ribonucleic acid, modified small interfering ribonucleic acid, messenger ribonucleic acid, modified messenger ribonucleic acid, antisense oligonucleotide, and / or modified antisense oligonucleotide.

[0104] In some embodiments, the compound of Formula I includes a compound having the structural formula and the nucleic acid includes one or more of small interfering ribonucleic acid, micro ribonucleic acid, messenger ribonucleic acid, modified small interfering ribonucleic acid, modified micro ribonucleic acid, and modified messenger ribonucleic acid.

[0105] In some embodiments, the compound of Formula I includes a compound having the structural formula and the nucleic acid includes one or more of double-stranded small interfering ribonucleic acid, single-stranded micro ribonucleic acid, long-chain messenger ribonucleic acid, modified double-stranded small interfering ribonucleic acid, modified single-stranded micro ribonucleic acid, and modified long-chain messenger ribonucleic acid.

[0106] In some embodiments, the compound of Formula I includes a compound having the structural formula and the nucleic acid includes messenger ribonucleic acid and / or modified messenger ribonucleic acid.

[0107] In the embodiments of the present invention, the nitrogen-to-phosphorus ratio of the compound-nucleic acid nanoparticle can be beneficial for the compound-nucleic acid nanoparticle to have a smaller and uniform particle size. The nitrogen-to-phosphorus ratio refers to the ratio of the number of moles of the compound of Formula I to the number of moles of phosphate groups in the nucleic acid, and can be represented by N / P.

[0108] In some embodiments, the ratio of the number of moles of the compound of Formula I to the number of moles of phosphate groups in the nucleic acid is in the range of 100:1 to 0.5:1.

[0109] Unless otherwise specifically stated or clearly indicated by the context, the numerical values in the present invention may include errors such as measurement errors, precision errors, and measurement errors, for example, errors within ±5%. For example, 100 may include values within the range of 100×(1±5%), that is, values within the range of 95 to 105, such as 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, and so on.

[0110] In some embodiments, the ratio of the number of moles of the compound of Formula I to the number of moles of phosphate groups in the nucleic acid is in the range of 40:1 to 10:1.

[0111] In some embodiments, the molar ratio of the compound of Formula I to the phosphate groups in the nucleic acid is 40:1, 20:1, 15:1, or 10:1.

[0112] The particle size of the compound-nucleic acid nanoparticles can be measured using, for example, a Malvern particle size analyzer. The particle size of the compound-nucleic acid nanoparticles can be measured in nanometers. The particle size of the compound-nucleic acid nanoparticles can be the average particle size. The particle size of the compound-nucleic acid nanoparticles can be the average diameter. The particle size of the compound-nucleic acid nanoparticles can facilitate the efficient entry of the compound-nucleic acid nanoparticles into the cytoplasm through thiol-mediated cellular uptake.

[0113] In some embodiments, the particle size of the compound-nucleic acid nanoparticles ranges from 30 nanometers to 600 nanometers. In some embodiments, the particle size of the compound-nucleic acid nanoparticles ranges from 47.42 nanometers to 208.44 nanometers. The particle size of the compound-nucleic acid nanoparticles can be, for example, 101.2 ± 20.87 nanometers, 82.52 ± 23.92 nanometers, 122.8 ± 29.6 nanometers, 112.3 ± 37.14 nanometers, 103.9 ± 25.97 nanometers, 77.15 ± 26.82 nanometers, 72.18 ± 24.76 nanometers, 133 ± 43.82 nanometers, 152.5 ± 55.94 nanometers, 108.4 ± 43.13 nanometers, or 86.8 ± 26.87 nanometers, and so on.

[0114] The polydispersity index (PDI) of the compound-nucleic acid nanoparticles can be, for example, 0.042, 0.088, 0.041, 0.129, 0.030, 0.174, 0.210, 0.264, 0.108, 0.148, or 0.134, and so on.

[0115] The compound-nucleic acid nanoparticles not only retain the ability to undergo dynamic covalent thiol exchange reactions with cell membranes but also improve the interaction between the compound of Formula I and the nucleic acid, facilitating the release of the nucleic acid in the cytoplasm.

[0116] The compound-nucleic acid nanoparticles can be applied to the treatment of various diseases. In some embodiments, the compound-nucleic acid nanoparticles have achieved good therapeutic effects in the treatment of pulmonary fibrosis by aerosol inhalation.

[0117] The compound-nucleic acid nanoparticles can be administered alone or in combination with a pharmaceutical carrier. The compound-nucleic acid nanoparticles can be administered directly at the target site or at an administration site away from the target site.

[0118] Another aspect of the embodiments of the present invention relates to a drug, which comprises a pharmaceutical carrier and the compound-nucleic acid nanoparticles as described in the present invention.

[0119] The pharmaceutical carrier may include substances and / or compositions that do not cause allergic reactions or similar adverse reactions when administered to mammals such as humans.

[0120] The pharmaceutical carrier can be selected according to the route of administration and standard pharmaceutical practice. Examples of the pharmaceutical carrier may include water, physiological saline, 0.4% saline, 135-150 mM saline, phosphate buffer, 0.3% glycine, etc. Other examples of the pharmaceutical carrier may include glycoproteins for increasing stability, such as albumin, lipoproteins, globulins, etc.

[0121] The pharmaceutical carrier can be a solvent, dispersion medium, vehicle, coating, diluent, antibacterial agent, antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, etc.

[0122] The pharmaceutical carrier can be added after the formation of the compound-nucleic acid nanoparticles. In some embodiments, after the formation of the compound-nucleic acid nanoparticles, the compound-nucleic acid nanoparticles can be diluted into the pharmaceutical carrier.

[0123] The concentration of the compound-nucleic acid nanoparticles in the drug can vary widely, that is, from less than about 0.05% by weight, equal to or at least about 2-5% by weight, to at most about 10-90% by weight, and is mainly selected according to the specific administration mode chosen through fluid volume, viscosity, etc. For example, the concentration can be increased to reduce the fluid load associated with treatment.

[0124] The drug can be sterilized by sterilization techniques.

[0125] The drug can contain pharmaceutical adjuvant substances as needed to approximate physiological conditions, such as pH regulators, pH buffers, tonicity regulators, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride, etc.

[0126] The drug can contain: excipients, binders, gelatin, lubricants, lipid carriers and / or flavoring agents.

[0127] The drug can be tablets, capsules, pills, lozenges, elixirs, mouthwashes, suspensions, oral sprays, syrups, cachets, etc.

[0128] For in vivo administration, the administration can be carried out in any manner known in the art, for example, by injection, infusion, oral administration, inhalation (e.g., intranasal or intratracheal), transdermal administration, or rectal administration. The administration can be achieved by single or multiple doses. The drug can be administered parenterally, i.e., intra-articularly, intravenously, intraperitoneally, intravesically, subcutaneously, intrathecally, or intramuscularly. In some embodiments, the drug can be administered intravenously or intraperitoneally by bolus injection. In some embodiments, the drug can be administered by intramuscular injection, tail vein injection, and / or pulmonary inhalation.

[0129] In certain embodiments, the drug can be delivered by intranasal spray, inhalation, and / or other aerosol delivery vehicles. The drug can be formulated into an aerosol preparation (i.e., they can be "atomized") alone or in combination with other suitable components, so as to be administered by inhalation (e.g., intranasal or intratracheal). The aerosol preparation can be placed in a propellant that allows pressurization, such as dichlorodifluoromethane, propane, nitrogen, etc.

[0130] The drug can play a role in the prevention and treatment of various diseases through administration methods such as aerosol inhalation, intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection, etc.

[0131] In some embodiments, the drug is one or more of a drug for tumor prevention and treatment, a drug for cancer prevention and treatment, a drug for neurodegenerative disease prevention and treatment, a protein replacement drug, a vaccine, a gene editing drug, a drug for pulmonary fibrosis prevention and treatment.

[0132] In some embodiments, the drug is one or more of a small interfering ribonucleic acid drug, a micro ribonucleic acid drug, an antisense oligonucleotide drug, a messenger ribonucleic acid drug, a long non-coding ribonucleic acid drug, a circular ribonucleic acid drug, a small nuclear ribonucleic acid drug, a modified small interfering ribonucleic acid drug, a modified micro ribonucleic acid drug, a modified antisense oligonucleotide drug, a modified messenger ribonucleic acid drug, a modified long non-coding ribonucleic acid drug, a modified circular ribonucleic acid drug, and a modified small nuclear ribonucleic acid drug.

[0133] In some embodiments, the drug is a drug for leukemia prevention and treatment. In some embodiments, the drug is a drug for pancreatic cancer prevention and treatment. In some embodiments, the drug is a drug for cervical cancer prevention and treatment. In some embodiments, the drug is a drug for lung cancer prevention and treatment.

[0134] Another aspect of the embodiments of the present invention relates to the use of the compound-nucleic acid nanoparticles of the present invention in the preparation of a product for introducing nucleic acid into cells.

[0135] In some embodiments, the product may be a drug comprising the compound-nucleic acid nanoparticle of the present invention. For specific details of the product, reference may be made to the relevant description of the drug comprising the compound-nucleic acid nanoparticle of the present invention above.

[0136] In some embodiments, when nucleic acid needs to be introduced into cells, the product comprising the compound-nucleic acid nanoparticle of the present invention may be contacted with the cells.

[0137] In some embodiments, the cells are mammalian cells.

[0138] Unless otherwise specifically stated, mammals involved in the present invention may include, for example, primates (e.g., humans, chimpanzees, and other non-human primates), canines, felines, equines, bovines, sheep, goats, rodents (e.g., hamsters, guinea pigs, rats, and mice), lagomorphs, and livestock (pigs), etc.

[0139] Unless otherwise specifically stated, cells involved in the present invention may include, for example, hematopoietic progenitor (stem) cells, fibroblasts, keratinocytes, hepatocytes, endothelial cells, skeletal and smooth muscle cells, osteoblasts, neurons, quiescent lymphocytes, terminally differentiated cells, slow or non-cycling primary cells, parenchymal cells, lymphoid cells, epithelial cells, human embryonic kidney cells (HEK293T cells), macrophages, and osteocytes, etc.

[0140] Unless otherwise specifically stated, cells involved in the present invention may include, for example, cancer cells, such as lung cancer cells, colon cancer cells, rectal cancer cells, anal cancer cells, bile duct cancer cells, small intestine cancer cells, gastric cancer cells, esophageal cancer cells, gallbladder cancer cells, liver cancer cells, pancreatic cancer cells, appendiceal cancer cells, breast cancer cells, ovarian cancer cells, cervical cancer cells, prostate cancer cells, kidney cancer cells, central nervous system cancer cells, glioblastoma tumor cells, skin cancer cells, lymphoma cells, choriocarcinoma tumor cells, head and neck cancer cells, osteogenic sarcoma tumor cells, and blood cancer cells, etc.

[0141] Another aspect of the embodiments of the present invention relates to the use of the compound-nucleic acid nanoparticle of the present invention in the preparation of a product for in vivo delivery of nucleic acid to be administered to a mammal.

[0142] In some embodiments, the product may be a drug comprising the compound-nucleic acid nanoparticle of the present invention. For specific details of the product, reference may be made to the relevant description of the drug comprising the compound-nucleic acid nanoparticle of the present invention above.

[0143] Unless otherwise specifically stated, the administration involved in the present invention may include, but is not limited to, oral, intranasal, intravenous, intraperitoneal, intramuscular, intra-articular, intralesional, intratracheal, subcutaneous, and intradermal.

[0144] Unless otherwise specifically stated, the delivery involved in the present invention can be carried out in any manner known in the art, including, for example, intravenous, subcutaneous, and intraperitoneal.

[0145] The term "delivery", unless otherwise specifically stated, may refer to systemic delivery that results in widespread biodistribution of the nucleic acid in a living organism, which can expose an effective amount, such as a therapeutic amount, of the nucleic acid to most parts of the body and prevent the nucleic acid from being rapidly degraded or cleared before reaching the disease site far from the administration site, such as not being degraded or cleared by first-pass organs (liver, lung, etc.) or non-specific cells. Systemic delivery for in vivo treatment can, for example, deliver a therapeutic nucleic acid to target cells through a body system such as the circulatory system.

[0146] The term "delivery", unless otherwise specifically stated, may also refer to local delivery that directly delivers the nucleic acid to a target site in a living organism. For example, the compound-nucleic acid nanoparticles can be locally delivered by direct injection into a disease site such as a tumor, other target sites such as an inflammatory site, or a target organ such as the liver, heart, pancreas, kidney, etc.

[0147] Another aspect of the embodiments of the present invention relates to the use of the compound-nucleic acid nanoparticles described in the present invention in the preparation of a medicament for administration to a mammal.

[0148] The preparation, composition, etc. of the medicament for administration to a mammal can refer to the relevant descriptions of the preparation, composition, etc. of the above-mentioned medicament.

[0149] In some embodiments, the medicament is one or more of an anti-tumor prevention and treatment medicament, an anti-cancer prevention and treatment medicament, a neuro-degenerative disease prevention and treatment medicament, a protein replacement medicament, a vaccine, a gene editing medicament, and a pulmonary fibrosis prevention and treatment medicament.

[0150] In some embodiments, the medicament is one or more of a small interfering ribonucleic acid medicament, a micro ribonucleic acid medicament, an antisense oligonucleotide medicament, a messenger ribonucleic acid medicament, a long non-coding ribonucleic acid medicament, a circular ribonucleic acid medicament, a small nuclear ribonucleic acid medicament, a modified small interfering ribonucleic acid medicament, a modified micro ribonucleic acid medicament, a modified antisense oligonucleotide medicament, a modified messenger ribonucleic acid medicament, a modified long non-coding ribonucleic acid medicament, a modified circular ribonucleic acid medicament, and a modified small nuclear ribonucleic acid medicament.

[0151] In some embodiments, the drug is a drug for preventing and treating leukemia. In some embodiments, the drug is a drug for preventing and treating pancreatic cancer. In some embodiments, the drug is a drug for preventing and treating cervical cancer. In some embodiments, the drug is a drug for preventing and treating lung cancer.

[0152] Embodiments of the present invention can utilize the non-covalent interaction force between the compound of Formula I and the nucleic acid, such as electrostatic interaction force, to form the compound-nucleic acid nanoparticles by complexation, providing a delivery carrier suitable for nucleic acid drug delivery, screening delivery carriers that meet various requirements such as higher delivery efficiency and different nucleic acid types, providing more possibilities for the clinical application of the compound in nucleic acid drugs, and providing a solid theoretical basis and technical guidance for the clinical trials of nucleic acid drugs.

[0153] Another aspect of the embodiments of the present invention relates to the compound-nucleic acid nanoparticles prepared by the following preparation method, which includes: dissolving the compound of Formula I in a solvent to obtain a solution of the compound of Formula I; dissolving the nucleic acid in water to obtain a nucleic acid solution; and mixing the solution of the compound of Formula I with the nucleic acid solution to obtain the compound-nucleic acid nanoparticles.

[0154] Another aspect of the embodiments of the present invention relates to the preparation method of the compound-nucleic acid nanoparticles of the present invention, which includes: dissolving the compound of Formula I in a solvent to obtain a solution of the compound of Formula I; dissolving the nucleic acid in water to obtain a nucleic acid solution; and mixing the solution of the compound of Formula I with the nucleic acid solution to obtain the compound-nucleic acid nanoparticles.

[0155] The solvent can be methanol, dimethyl sulfoxide (DMSO), etc. After the compound of Formula I is dissolved in the solvent, it can be diluted in a buffer solution or diethylpyrocarbonate (DEPC) water, etc. according to the required nitrogen-phosphorus ratio to obtain the solution of the compound of Formula I.

[0156] The water can be diethylpyrocarbonate (DEPC) water. After the nucleic acid is dissolved in diethylpyrocarbonate (DEPC) water, it can be diluted with diethylpyrocarbonate (DEPC) water, etc. to obtain the nucleic acid solution.

[0157] The step of mixing the solution of the compound of formula I with the nucleic acid solution may include that after the solution of the compound of formula I and the nucleic acid solution are mixed evenly, they are allowed to stand or react with shaking at a certain temperature for a certain period of time. The temperature and time of the standing or shaking reaction may be respectively included in the temperature and time of mixing the solution of the compound of formula I with the nucleic acid solution.

[0158] In some embodiments, the temperature for mixing the solution of the compound of formula I with the nucleic acid solution ranges from 25°C to -80°C. The temperature for mixing the solution of the compound of formula I with the nucleic acid solution may be, for example, 25°C, 4°C, -20°C, or -80°C.

[0159] In some embodiments, the time for mixing the solution of the compound of formula I with the nucleic acid solution ranges from 15 minutes to 48 hours. The time for mixing the solution of the compound of formula I with the nucleic acid solution may be, for example, 15 minutes, 30 minutes, 1 hour, 4 hours, 8 hours, 24 hours, or 48 hours.

[0160] The formed compound-nucleic acid nanoparticles can be purified by means such as high-speed centrifugation, dialysis or tangential flow separation to remove unreacted nucleic acid and the compound of formula I, and the solvent can be removed by freeze-drying for long-term preservation. When the compound-nucleic acid nanoparticles are needed for in vitro or in vivo use, they can be dispersed with a buffer solution or the like.

[0161] Another aspect of the embodiments of the present invention relates to the use of the compound of formula I in the preparation of a product for introducing nucleic acid into cells, wherein n is an integer selected from 1 to 14, and R is a positively charged group containing one or more nitrogen atoms.

[0162] The embodiments of the present invention relate to the application of the compound of formula I with various structures having different positively charged groups R and various carbon chain lengths n in nucleic acid delivery, which can help fill the gap in this aspect.

[0163] In some embodiments, R is a positively charged group that can be ionized when the pH value of its environment is less than its pKa value.

[0164] In some embodiments, R includes a primary amine group a secondary amine group a tertiary amine group a quaternary amine group an imidazolyl group and a guanidyl group or one or more of them.

[0165] In some embodiments, the nucleic acid includes one or more of small interfering ribonucleic acid, micro ribonucleic acid, antisense oligonucleotide, messenger ribonucleic acid, long non-coding ribonucleic acid, circular ribonucleic acid, small nuclear ribonucleic acid, modified small interfering ribonucleic acid, modified micro ribonucleic acid, modified antisense oligonucleotide, modified messenger ribonucleic acid, modified long non-coding ribonucleic acid, modified circular ribonucleic acid, and modified small nuclear ribonucleic acid.

[0166] In some embodiments, the sizes of the small interfering ribonucleic acid and the modified small interfering ribonucleic acid are respectively in the range of 15 bp - 30 bp. The compound of Formula I can be used to target growth factor receptors, signal transduction molecules, cyclins, apoptosis regulators, proteases, vascular endothelial growth factor, etc., and regulate their gene expression levels for the treatment of tumors, neurodegenerative diseases, etc.

[0167] In some embodiments, the lengths of the micro ribonucleic acid and the modified micro ribonucleic acid are respectively in the range of 15 nt - 30 nt. The compound of Formula I can be used to target growth factor receptors, signal transduction molecules, cyclins, apoptosis regulators, proteases, vascular endothelial growth factor, etc., and regulate their gene expression levels for the treatment of tumors, neurodegenerative diseases, etc.

[0168] In some embodiments, the lengths of the antisense oligonucleotide and the modified antisense oligonucleotide are respectively in the range of 15 nt - 30 nt. The compound of Formula I can be used to target growth factor receptors, signal transduction molecules, cyclins, apoptosis regulators, proteases, vascular endothelial growth factor, etc., and regulate their gene expression levels for the treatment of tumors, neurodegenerative diseases, etc.

[0169] In some embodiments, the lengths of the messenger ribonucleic acid and the modified messenger ribonucleic acid are respectively independently > 500 nt or in the range of 500 nt - 3000 nt. The compound of Formula I can be used for protein replacement therapy of related proteins that can treat diseases or for mRNA vaccines encoding related proteins that can cause an immune response in the human body.

[0170] In some embodiments, R includes a guanidyl group n > 1.

[0171] In some embodiments, R includes a guanidyl group The nucleic acid includes one or more of antisense oligonucleotide, messenger ribonucleic acid, modified antisense oligonucleotide, and modified messenger ribonucleic acid.

[0172] In some embodiments, the compound of Formula I includes a structural formula as one or more of the compounds, wherein the nucleic acid comprises an antisense oligonucleotide and / or a modified antisense oligonucleotide.

[0173] In some embodiments, the compound of Formula I comprises a compound having the structural formula and the nucleic acid comprises small interfering ribonucleic acid, modified small interfering ribonucleic acid, messenger ribonucleic acid, modified messenger ribonucleic acid, antisense oligonucleotide, and / or modified antisense oligonucleotide.

[0174] In some embodiments, the compound of Formula I comprises a compound having the structural formula and the nucleic acid comprises one or more of small interfering ribonucleic acid, micro ribonucleic acid, messenger ribonucleic acid, modified small interfering ribonucleic acid, modified micro ribonucleic acid, and modified messenger ribonucleic acid.

[0175] In some embodiments, the compound of Formula I comprises a compound having the structural formula and the nucleic acid comprises one or more of double-stranded small interfering ribonucleic acid, single-stranded micro ribonucleic acid, long-chain messenger ribonucleic acid, modified double-stranded small interfering ribonucleic acid, modified single-stranded micro ribonucleic acid, and modified long-chain messenger ribonucleic acid.

[0176] In some embodiments, the compound of Formula I comprises a compound having the structural formula and the nucleic acid comprises messenger ribonucleic acid and / or modified messenger ribonucleic acid.

[0177] Another aspect of the embodiments of the present invention relates to a method for preventing and treating a disease, which comprises administering the drug according to the present invention to a mammal.

[0178] Unless otherwise specifically stated, the administration involved in the present invention may include, but is not limited to, oral, intranasal, intravenous, intraperitoneal, intramuscular, intra-articular, intralesional, intratracheal, subcutaneous, and intradermal.

[0179] Another aspect of the embodiments of the present invention relates to a method for antiviral and / or cell infection, which comprises administering the drug according to the present invention to a mammal.

[0180] Another aspect of the embodiments of the present invention relates to a method for introducing nucleic acid into a cell, which comprises: contacting the cell with the compound-nucleic acid nanoparticle according to the present invention.

[0181] The method for introducing the nucleic acid into the cell can be carried out in vitro or in vivo by the following steps: first forming the compound-nucleic acid nanoparticle as described in the present invention, and then contacting the compound-nucleic acid nanoparticle with the cell for a period of time sufficient to effect the delivery of the nucleic acid to the cell.

[0182] For in vitro applications, the nucleic acid can be delivered to any cell growing in culture, regardless of plant or animal origin, vertebrate or invertebrate, and any tissue or type. In a preferred embodiment, the cell can be an animal cell, such as a mammalian cell, e.g., a human cell.

[0183] Contact between the cell and the compound-nucleic acid nanoparticle, when carried out in vitro, can occur in a biocompatible medium.

[0184] Yet another aspect of the embodiments of the present invention relates to a method for introducing nucleic acid into a cell, which comprises: contacting the cell with the compound-nucleic acid nanoparticle of the present invention in vitro.

[0185] Yet another aspect of the embodiments of the present invention relates to a method for in vivo delivery of nucleic acid, which comprises administering the compound-nucleic acid nanoparticle of the present invention to a mammal.

[0186] Unless otherwise specifically stated, administration as referred to in the present invention can include, but is not limited to, oral, intranasal, intravenous, intraperitoneal, intramuscular, intra-articular, intralesional, intratracheal, subcutaneous, and intradermal.

[0187] Yet another aspect of the embodiments of the present invention relates to a method for using the compound-nucleic acid nanoparticle of the present invention for in vivo delivery of nucleic acid, which comprises administering the compound-nucleic acid nanoparticle to a mammal.

[0188] Yet another aspect of the embodiments of the present invention relates to a method for treating a disease or disorder in a mammal, which comprises administering an effective therapeutic dose of the compound-nucleic acid nanoparticle of the present invention to the mammal.

[0189] The effective therapeutic dose can be an amount sufficient to produce the desired therapeutic effect.

[0190] Yet another aspect of the embodiments of the present invention relates to a method for using the compound-nucleic acid nanoparticle of the present invention for treating a disease or disorder in a mammal, which comprises administering an effective therapeutic dose of the compound-nucleic acid nanoparticle to the mammal.

[0191] In some embodiments, the disease or disorder is selected from neurodegenerative diseases, tumors, cancers, and pulmonary fibrosis.

[0192] Yet another aspect of the embodiments of the present invention relates to the use of the compound-nucleic acid nanoparticle of the present invention in gene therapy.

[0193] In some embodiments, the gene therapy includes one or more of small interfering ribonucleic acid therapy, microRNA therapy, antisense oligonucleotide therapy, messenger ribonucleic acid therapy, long non-coding ribonucleic acid therapy, circular ribonucleic acid therapy, and small nuclear ribonucleic acid therapy.

[0194] The present invention will be further illustrated below in conjunction with experimental examples. The experimental examples are only for illustrative purposes and are not intended to limit the scope of protection of the present invention in any way.

[0195] Example

[0196] Unless otherwise specified, the process methods involved in the present invention are all conventional process methods. Unless otherwise specified, the materials, reagents, etc. involved in the present invention can be obtained from commercial sources. The reagents used in the chemical synthesis processes of Compounds 1-8 in Examples 1-3 are from Sigma-Aldrich or Shanghai Macklin Biochemical Co., Ltd.

[0197] The sequence list of the nucleic acids used in the examples is shown in Table 1 below.

[0198] Table 1

[0199]

[0200]

[0201] Example 1: Synthesis and Mass Spectral Characterization of Compounds 1-3

[0202] The synthesis processes of Compounds 1-3 are shown below.

[0203]

[0204] Specifically, three portions of purchased α-lipoic acid (784 mg, 3.8 mmol) and three portions of N,N'-carbonyldiimidazole (812 mg, 5.0 mmol) were respectively dissolved in three portions of 25 ml of anhydrous dichloromethane to obtain three identical solutions. One portion of the solution was added dropwise to 7 ml of anhydrous dichloromethane containing 30 mmol of amine solution (ethylenediamine, N-methylethylenediamine, or N,N-dimethylethylenediamine) and maintained at 0 °C to obtain three reaction mixtures. Each reaction mixture was stirred at 0 °C for 40 minutes, stirred at room temperature for 30 minutes, and then washed with brine (3 × 20 ml) to obtain the corresponding organic layer. Each organic layer was dried with anhydrous Na2SO4 and concentrated under reduced pressure to obtain yellow oily products 1-3. Products 1-3 were respectively dissolved in methanol and subjected to mass spectrometry tests, and the results are shown in Figure 1 .

[0205] As Figure 1As shown, for the product 1 synthesized by mass spectrometry, its main mass-to-charge ratio is located at 249.1102, which is consistent with the molecular weight of compound 1 (C 10 H 20 N2OS2), indicating the successful synthesis of compound 1. The main mass-to-charge ratio of product 2 is located at 263.1246, which is consistent with the molecular weight of compound 2 (C 11 H 22 N2OS2), indicating the successful synthesis of compound 2. The main mass-to-charge ratio of product 3 is located at 277.1389, which is consistent with the molecular weight of compound 3 (C 12 H 24 N2OS2), indicating the successful synthesis of compound 3.

[0206] Example 2: Synthesis and Mass Spectral Characterization of Compound 4

[0207] The synthesis process of compound 4 is as follows.

[0208]

[0209] Specifically, 1.5 mmol of lipoic acid NHS-ester was dissolved in 9 ml of 1,4-dioxane to obtain a lipoic acid NHS-ester solution. 2.25 mmol of (2-aminoethyl) trimethylammonium chloride hydrochloride was dissolved in 9 ml of 0.25 N sodium carbonate buffer solution, and the pH was adjusted to 7.6 by adding an aqueous NaOH solution to obtain a (2-aminoethyl) trimethylammonium chloride hydrochloride solution. The lipoic acid NHS-ester solution and the (2-aminoethyl) trimethylammonium chloride hydrochloride solution were slowly mixed together at -5°C and stirred at room temperature for 24 hours. The by-products were removed by vacuum filtration. The solvent was evaporated using a rotary evaporator to obtain a crude product. The crude product was dissolved in the minimum volume of aqueous NaOH solution and extracted into the isopropanol / chloroform / ethyl acetate (EA) layer. It was dried with sodium sulfate, the solvent was evaporated to dryness, and an intermediate product was obtained. The intermediate product was redissolved in water. The undissolved by-products were removed by filtration to obtain a crude product solution. The crude product solution was lyophilized to obtain the product, and mass spectrometry was performed. The results are shown in Figure 2 .

[0210] As Figure 2 shown, for the product synthesized by mass spectrometry, its main mass-to-charge ratio is located at 291.1546, which is consistent with the molecular weight of compound 4 (C 13 H 26 N2OS2), indicating the successful synthesis of compound 4.

[0211] Example 3: Synthesis and Mass Spectral Characterization of Compounds 5 - 8

[0212] The synthesis process of compounds 5 - 8 is as follows.

[0213]

[0214] Specifically, four portions of purchased α-lipoic acid (784 mg, 3.8 mmol) and four portions of N,N'-carbonyldiimidazole (812 mg, 5.0 mmol) were separately dissolved in four portions of 25 ml of anhydrous dichloromethane to obtain four solutions. One portion of the solution was added dropwise to 7 ml of anhydrous dichloromethane containing 30 mmol of an amine solution (ethylenediamine, 1,4-diaminobutane, hexamethylenediamine, or 1,8-diaminooctane), and maintained at 0 °C to obtain the corresponding reaction mixture. Each reaction mixture was stirred at 0 °C for 40 minutes, stirred at room temperature for 30 minutes, and then washed with brine (3 × 20 ml) to obtain the corresponding organic layer. Each organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain yellow oily products 5 - 8. The oily products 5 - 8 were separately dissolved in anhydrous dichloromethane (30 ml), and 1H-pyrazole-1-carboxamidine hydrochloride (3.8 mmol) was separately added to obtain the corresponding suspensions. Each suspension was stirred at room temperature for 4 hours. The solvent was removed under reduced pressure, and each residue was separately dissolved in 2 ml of methanol. Diethyl ether (20 ml) was added to each to induce precipitation. The solids were separately collected and washed with diethyl ether (4 × 10 ml) to obtain light yellow solid products 5 - 8, which were subjected to mass spectrometry tests, and the results are shown in Figure 3 .

[0215] As Figure 3 shown, for the solid product 5 synthesized by mass spectrometry, the main mass-to-charge ratio is located at 291.1285, which is consistent with the molecular weight of compound 5 (C 11 H 22 N4OS2), indicating the successful synthesis of compound 5. The main mass-to-charge ratio of the solid product 6 is located at 319.1637, which is consistent with the molecular weight of compound 6 (C 13 H 27 N4OS2), indicating the successful synthesis of compound 6. The main mass-to-charge ratio of the solid product 7 is located at 347.1932, which is consistent with the molecular weight of compound 7 (C 15 H 31 N4OS2), indicating the successful synthesis of compound 7. The main mass-to-charge ratio of the solid product 8 is located at 375.2283, which is consistent with the molecular weight of compound 8 (C 17 H 35 N4OS2), indicating the successful synthesis of compound 8.

[0216] Example 4: Preparation and Physicochemical Property Investigation of Compound-Nucleic Acid Nanoparticles Composed of Compounds Containing Different Positively Charged Groups and Antisense Oligonucleotides

[0217] Dissolve the antisense oligonucleotide (ASO1) in diethylpyrocarbonate (DEPC) water and dilute it to obtain an ASO1 solution. Dissolve compounds 1, 2, 3, 4, and 5 containing different positively charged groups in methanol respectively, and dilute them according to the nitrogen-to-phosphorus ratios (N / P, where N refers to the molar amount of the thiol compound and P refers to the molar amount of the phosphate group in the nucleic acid) of 15:1, 20:1, 10:1, 40:1, and 15:1 respectively to obtain the corresponding compound solutions 1-5. Add compound solutions 1-5 to the corresponding ASO1 solutions respectively. After mixing evenly, place them at 25 °C and react for 0.5 hours. Centrifuge at 10,000 revolutions per minute for 30 minutes to remove the unreacted ASO1 and compounds, and further disperse them with a buffer solution to prepare compound 1-5-ASO nanoparticles. Measure the particle sizes of the prepared compound 1-5-ASO nanoparticles with a Malvern particle size analyzer respectively, and the obtained number distribution data are shown in Figure 4 .

[0218] As Figure 4 shown, the particle sizes of the obtained compound-ASO nanoparticles are as follows: the particle size of compound 1-ASO nanoparticles is 101.2 ± 20.87 nm, and the PDI is 0.042; the particle size of compound 2-ASO nanoparticles is 82.52 ± 23.92 nm, and the PDI is 0.088; the particle size of compound 3-ASO nanoparticles is 122.8 ± 29.6 nm, and the PDI is 0.041; the particle size of compound 4-ASO nanoparticles is 112.3 ± 37.14 nm, and the PDI is 0.129; the particle size of compound 5-ASO nanoparticles is 103.9 ± 25.97 nm, and the PDI is 0.030.

[0219] The results show that at a certain N / P, compounds 1-5 can all form compound-nucleic acid nanoparticles with smaller and uniform particle sizes with ASO, and the strength of the positively charged group has no significant effect on the formation of compound-nucleic acid nanoparticles.

[0220] Example 5: Preparation and physicochemical property investigation of compound-nucleic acid nanoparticles formed by compounds with different chain lengths and antisense oligonucleotides

[0221] Dissolve antisense oligonucleotide 1 (ASO1) in DEPC water and dilute it to obtain an ASO1 solution. Dissolve guanidine compounds 6, 7, and 8 with different chain lengths in methanol respectively, and dilute them according to the nitrogen-to-phosphorus ratios (N / P) of 15:1, 15:1, and 40:1 respectively to obtain the corresponding compound solutions 6, 7, and 8. Add compound solutions 6 - 8 to the corresponding ASO1 solutions respectively. After mixing evenly, place them at 25 °C and react for 0.5 hours. Centrifuge at 10,000 revolutions per minute for 30 minutes to remove the unreacted ASO1 and compounds, and further disperse them with a buffer solution to prepare compound 6 - 8-ASO nanoparticles. Measure the particle sizes of the prepared compound 6 - 8-ASO nanoparticles with a Malvern particle size analyzer respectively, and the obtained number distribution data are shown in Figure 5 .

[0222] As Figure 5 shown, the particle sizes of the obtained compound 6 - 8-ASO nanoparticles are as follows: the particle size of compound 6-ASO nanoparticles is 77.15 ± 26.82 nm, and the PDI is 0.174; the particle size of compound 7-ASO nanoparticles is 72.18 ± 24.76 nm, and the PDI is 0.210; the particle size of compound 8-ASO nanoparticles is 133 ± 43.82 nm, and the PDI is 0.264.

[0223] The results show that at a certain N / P, compounds with different chain lengths can all form compound-nucleic acid nanoparticles with smaller and more uniform particle sizes with ASO, and the size of the compound-nucleic acid nanoparticles increases slightly with the increase of the carbon chain length.

[0224] Example 6: Influence of nucleic acid sequences on the assembly of compound-nucleic acid nanoparticles

[0225] Dissolve ASO1 and ASO2 with different sequences in DEPC water respectively and dilute them to obtain the corresponding ASO solutions. Dissolve compound 2 in methanol and dilute it according to the nitrogen-to-phosphorus ratio of 20:1 to obtain a compound 2 solution. Add the compound 2 solution to the ASO solutions respectively. After mixing evenly, place them at 25 °C and react for 0.5 hours. Centrifuge at 10,000 revolutions per minute for 30 minutes to remove the unreacted antisense oligonucleotides and compound 2, and further disperse them with a buffer solution to prepare compound 2-ASO(18nt) nanoparticles and compound 2-ASO(22nt) nanoparticles. Measure the particle sizes of the prepared compound 2-ASO(18nt) nanoparticles and compound 2-ASO(22nt) nanoparticles with a Malvern particle size analyzer respectively, and the obtained number distribution data are shown in Figure 6 .

[0226] As Figure 6As shown, ASOs with different sequences can all form compound-nucleic acid nanoparticles with Compound 2, and the particle size is in the range of 30 - 600 nanometers, indicating that the formation of compound-nucleic acid nanoparticles is independent of the nucleic acid sequence, and nucleic acids with any sequence can form compound-nucleic acid nanoparticles with the compound.

[0227] Example 7: Influence of Nucleic Acid Sequence Length and Single- or Double-Strand on the Assembly of Compound-Nucleic Acid Nanoparticles

[0228] Dissolve double-stranded small interfering RNA (siRNA, 21 bp), single-stranded microRNA (miRNA, 22 nt), and long-chain messenger RNA (mRNA, 1000 nt) in DEPC water respectively and dilute them to obtain the corresponding nucleic acid solutions. Dissolve Compound 3 in methanol and dilute it at a nitrogen-to-phosphorus ratio of 10:1 to obtain a Compound 3 solution. Add the Compound 3 solution to each nucleic acid solution respectively, mix well, and place it at 25 °C for reaction for 0.5 hours. Centrifuge at 10,000 revolutions per minute for 30 minutes to remove the unreacted nucleic acid and Compound 3, and further disperse it with a buffer solution to prepare compound-nucleic acid nanoparticles. Measure the particle size of each prepared compound-nucleic acid nanoparticle with a Malvern particle size analyzer, and the obtained number distribution data are shown in Figure 7 .

[0229] As Figure 7 shown, the particle sizes of the obtained compound-nucleic acid nanoparticles are as follows: Compound 3-siRNA nanoparticles are 152.5 ± 55.94 nanometers, and the PDI is 0.108; Compound 3-miRNA nanoparticles are 108.4 ± 43.13 nanometers, and the PDI is 0.148; Compound 3-mRNA nanoparticles are 86.8 ± 26.87 nanometers, and the PDI is 0.134.

[0230] The results show that at a certain N / P, the compound forms compound-nucleic acid nanoparticles with smaller and more uniform particle sizes with various types of nucleic acids. This may be because the mode of action is that the positively charged group of the compound forms a non-covalent interaction with the negatively charged phosphate group of the nucleic acid, and the length of the sequence and the difference between single-stranded or double-stranded have no obvious effect on it. Therefore, the compound has broad application prospects in nucleic acid drug delivery.

[0231] Example 8: Influence of Modified Nucleic Acids on the Assembly of Compound-Nucleic Acid Nanoparticles

[0232] Dissolve chemically modified ASO3, ASO4, and ASO5 in DEPC water respectively and dilute them to obtain the corresponding ASO solutions; dissolve compound 2 in methanol and dilute it according to a nitrogen-to-phosphorus ratio of 20:1 to obtain a compound 2 solution; add the compound 2 solution to the ASO solution, mix evenly, and place it at 25 °C for reaction for 0.5 hour; centrifuge at 10,000 revolutions per minute for 30 minutes to remove unreacted antisense oligonucleotides and compound 2, and further disperse it with a buffer solution to prepare compound 2-nucleic acid nanoparticles. Measure the particle size of each prepared compound 2-nucleic acid nanoparticle with a Malvern particle size analyzer, and the obtained number distribution data are shown in Figure 8 .

[0233] As Figure 8 shown, the compound-nucleic acid nanoparticles formed by three ASOs containing chemically modified bases are named compound 2-F-ASO nanoparticles, compound 2-2’Me-ASO nanoparticles, and compound 2-F-2’Me-ASO nanoparticles respectively. Different modified ASOs can all form compound-nucleic acid nanoparticles with compound 2, and their particle sizes are in the range of 30 - 600 nanometers, indicating that the formation of compound-nucleic acid nanoparticles is not affected by the chemical modification of nucleic acids, and any chemically modified nucleic acid can form compound-nucleic acid nanoparticles with compound 2.

[0234] Example 9: Cellular uptake of compound-nucleic acid nanoparticles

[0235] Use ASO1 labeled with Cy5 fluorescence as a model drug, and use a flow cytometer to investigate the cellular uptake of the compound-nucleic acid nanoparticles prepared in Reference Example 4. Seed macrophage RAW164.7 cells at a cell number of 5×10 4 cells / well in a 48-well plate and incubate them in an incubator at 37 °C and 5% CO2 for 24 hours. Add compound-nucleic acid nanoparticles with a concentration of 100 nM respectively and incubate them in an incubator at 37 °C and 5% CO2 for 2 hours. After incubation, wash twice with phosphate buffer saline (PBS), then digest the cells with 0.1% EthyleneDiamine Tetraacetic Acid (EDTA), centrifuge to remove EDTA, disperse it in PBS, and perform on-machine detection. The obtained flow analysis statistics are shown in Figure 9 .

[0236] From Figure 9It can be seen that the compound-nucleic acid nanoparticles significantly improve the cellular uptake efficiency of ASO. Among them, the compound 2-ASO nanoparticles have the highest uptake efficiency, followed by the compound 5-ASO nanoparticles, the compound 1-ASO nanoparticles, the compound 3-ASO nanoparticles, and the compound 4-ASO nanoparticles. Thus, it can be seen that by optimizing the structure of the compound, compounds with higher delivery efficiency can be screened out.

[0237] Example 10: Cellular Uptake Mechanism of Compound-Nucleic Acid Nanoparticles

[0238] Using ASO6 labeled with Cy5 fluorescence as a model drug, the cellular uptake mechanism of the compound-nucleic acid nanoparticles prepared in Reference Example 4 was investigated using a flow cytometer.

[0239] Seed cervical cancer Hela cells at a cell density of 5×10 4 cells per well in a 48-well plate and incubate in an incubator at 37 °C and 5% CO2 for 24 hours. Add 4.8 mM thiol inhibitor 5,5’-dithioobis-2-nitrobenzoic acid (DTNB) or endocytosis inhibitors, including 10 μg / mL chlorpromazine (CPZ), 50 nM Wortmannin (W), or 50 μM methyl-β-cyclodextrin (M-β-CD), and incubate with the corresponding cells for 0.5 hours respectively. Then, add 100 nM compound-nucleic acid nanoparticles (compound 1-5-ASO nanoparticles) respectively, and continue to incubate in an incubator at 37 °C and 5% CO2 in the presence of the inhibitor for 2 hours. Wash twice with PBS, then digest the cells with 0.1% EDTA, centrifuge to remove EDTA, disperse in PBS, and detect on the machine. The obtained inhibition rate data are shown in Figure 10 .

[0240] It can be seen from Figure 10 that the endocytosis inhibitors have no obvious inhibition on the cellular uptake of the compound-nucleic acid nanoparticles, and the thiol inhibitor can effectively inhibit the cellular uptake of the compound-nucleic acid nanoparticles, indicating that the compound-nucleic acid nanoparticles mainly enter the cells through a thiol exchange-mediated cellular uptake mechanism, and changing the positively charged group of the compound does not change the way of the compound-nucleic acid nanoparticles entering the cells.

[0241] Example 11: In Vitro Gene Regulation Effect of Compound 2-siRNA Nanoparticles

[0242] Using KRAS-targeting G12DThe mutant siRNA was used as a model drug, and its sequence was: sense sequence: 5’-GUUGGAGCUGAUGGCGUAGdTdT-3’, antisense sequence: 5’-CUACGCCAUCAGCUCCAACdTdT-3’. The gene regulation of the compound 2-siRNA nanoparticles prepared in Reference Example 7 was examined by Western blotting, and at the same time, the commercial RNA transfection kit Lipofectamine RNAi MAX (RNAi MAX) was used as a positive control.

[0243] Inoculate pancreatic cancer PANC-1 cells at a cell number of 1×10 5 per well into a 12-well plate and incubate in an incubator at 37 °C and 5% CO2 for 24 hours. Add 100 nM, 200 nM, 400 nM, and 800 nM of the compound 2-siRNA nanoparticles respectively and co-incubate with the cells for 48 hours. Wash twice with PBS, add cell lysis buffer and protease inhibitor to extract the total protein of the cells. The obtained protein extract was quantified using a BCA protein quantification kit. Mix the protein extract with the loading buffer and perform electrophoresis in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transfer it to a polyvinylidene difluoride (PVDF) membrane. Immerse the membrane in a petri dish containing the blocking solution for blocking. Immerse the membrane in a solution of the primary antibody anti-p-ERK against the target protein and incubate overnight at 4 °C. Then immerse the membrane in a solution of the secondary antibody labeled with HRP, incubate at 4 °C, add the HRP detection reagent for chemiluminescent imaging, and the results are shown in Figure 11 .

[0244] It can be seen from Figure 11 that as the concentration of the compound 2-siRNA composite nanoparticles increases, the expression of phosphorylated ERK (pERK) protein is significantly inhibited, and its inhibitory effect is better than that of the commercial RNA transfection kit. It shows that the compound-nucleic acid nanoparticles can deliver siRNA into cells and exert its gene silencing effect, and have great application potential in the treatment of diseases such as tumors and cancers.

[0245] Example 12: In vitro tumor suppression effect of compound 2-ASO nanoparticles

[0246] Use the ASO targeting miRNA-21 as a model drug, and its sequence is: 5’-TCAACATCAGTCTGATAAGCTA-3’. The growth inhibitory effect of the compound 2-ASO nanoparticles prepared in Reference Example 6 on lung cancer cells A549 was determined by the Cell Counting Kit-8 (CCK-8) method.

[0247] Inoculate A549 cells into a 96-well plate at a cell density of 5×10 3 cells / well and incubate in an incubator at 37°C with 5% CO2 for 24 hours. Then add 100 μL of 5A culture medium containing compound 2-ASO nanoparticles at concentrations of 50 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, and 600 nM respectively, and continue to incubate for 48 hours. After incubation, aspirate the culture medium, add fresh culture medium containing 10% CCK-8, incubate for 1 hour, and measure the optical density (OD) value at 450 nm using a microplate reader. The cell viability data are shown in Figure 12 , and the calculation formula is: Cell viability (%) = [A (drug added) - A (blank)] / [A (0 drug added) - A (blank)] × 100. Where, A (drug added): the optical density value of the well with cells, CCK-8 solution, and nanoparticle solution. A (blank): the optical density value of the well with culture medium and CCK-8 solution but without cells. A (0 drug added): the optical density value of the well with cells and CCK-8 solution but without nanoparticle solution.

[0248] It can be seen from Figure 12 that as the concentration of compound 2-ASO nanoparticles increases, the growth of lung cancer A549 cells is significantly inhibited. This indicates that compound-nucleic acid nanoparticles can deliver ASO into cells and play a role in regulating gene expression, showing great application potential in the treatment of diseases such as tumors and cancers.

[0249] Example 13: Protein expression effects of compound 5-mRNA nanoparticles and compound 1-mRNA nanoparticles in cells

[0250] Using mRNA encoding green fluorescent protein (GFP) with the sequence shown in Table 1 as a model drug, the cellular uptake of compound 5-mRNA nanoparticles and compound 1-mRNA nanoparticles prepared in Reference Example 7 was investigated using a flow cytometer.

[0251] Inoculate HEK293T cells into a 96-well plate at a cell density of 1×10 4 cells / well and incubate in an incubator at 37°C with 5% CO2 for 24 hours. Then add compound-nucleic acid nanoparticles at concentrations of 100 ng, 200 ng, 400 ng, and 800 ng respectively and incubate in an incubator at 37°C with 5% CO2 for 24 hours. After incubation, wash twice with PBS, then digest the cells with 0.1% EDTA, centrifuge to remove EDTA, disperse in PBS, and perform on-machine detection. The obtained flow analysis statistics are shown in Figure 13 .

[0252] It can be seen fromFigure 13 It can be seen that as the concentrations of compound 5-mRNA nanoparticles and compound 1-mRNA nanoparticles increase, the fluorescence intensity gradually increases and the expression of GFP protein increases. It is proved that compound-nucleic acid nanoparticles can deliver mRNA into cells and enable it to express the corresponding protein products in the cytoplasm. Compound-nucleic acid nanoparticles are expected to have potential clinical application value in mRNA drugs including protein replacement therapy, vaccines, gene editing, etc.

[0253] Example 14: Inhalation administration of compound 2-siRNA nanoparticles for treating pulmonary fibrosis in mice

[0254] Using siRNA (siSart1) targeting Sart 1 mRNA as a model drug, its sequence is: sense strand CCCAGAAGACACCGUAUAUTT, antisense strand AUAUACGGUGUCUUCUGGGTT.

[0255] Using random siRNA (siScr) that does not target any mRNA sequence as a control group, its sequence is: sense strand UUCUCCGAACGUGUCACGUTT, antisense strand ACGUGACACGUUCGGAGAATT.

[0256] Female C57BL / 6 mice aged 8 - 10 weeks were used. The mice were anesthetized with sodium pentobarbital (50 mg / kg, prepared with 0.9% normal saline, intraperitoneal injection), and a pulmonary fibrosis model was established by tracheal aerosol inhalation of bleomycin (BLM) (1.5 U / kg).

[0257] On the 11th, 15th, and 19th days after BLM administration, PBS, or compound 2-siScr nanoparticles (random strand control) prepared according to Example 7, or compound 2-siSart1 nanoparticles (experimental group) were inhaled through the trachea. The siRNA content in the nanoparticles was 1.5 mg / kg.

[0258] On the 21st day after BLM administration, lung tissues were taken. The lung tissues were first fixed with 4% paraformaldehyde overnight, then dehydrated with 15% and 30% sucrose gradients overnight, and finally embedded with OCT embedding agent for cryosectioning, with each section having a thickness of 10 μm. Staining was performed using hematoxylin-eosin staining method (H&E staining method) and observed with an inverted microscope (20×). The respective microscope images are shown in Figure 14 .

[0259] From Figure 14It can be seen that a mouse model of pulmonary fibrosis was established by inhaling bleomycin, and the mice were treated by inhaling the compound 2-siRNA nanoparticles (BLM+siSart1). Compared with the control groups: only modeling without drug administration (BLM+PBS) and modeling with the administration of compound 2-siScr nanoparticles (BLM+siScr), the experimental group treated with compound 2-siSart1 nanoparticles (BLM+siSart1) reduced the severity of BLM-induced pulmonary fibrosis. It is proved that the compound-nucleic acid nanoparticles can show good therapeutic effects at the in vivo level and have potential clinical application value.

[0260] The above embodiments only represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A compound-nucleic acid nanoparticle, characterized in that, Comprising: Nucleic acid; And Compound of formula I, wherein n is an integer selected from 1 to 14, and R is a positively charged group containing one or more nitrogen atoms.

2. The compound-nucleic acid nanoparticle according to claim 1, characterized in that R is a positively charged group that can be ionized when the pH value of its environment is less than its pKa value; and / or R includes a primary amine group secondary amine group tertiary amine group quaternary amine group amidinyl and guanidyl one or more of; and / or The nucleic acid includes one or more of small interfering ribonucleic acid, micro ribonucleic acid, antisense oligonucleotide, messenger ribonucleic acid, long non-coding ribonucleic acid, circular ribonucleic acid, small nuclear ribonucleic acid, modified small interfering ribonucleic acid, modified micro ribonucleic acid, modified antisense oligonucleotide, modified messenger ribonucleic acid, modified long non-coding ribonucleic acid, modified circular ribonucleic acid, and modified small nuclear ribonucleic acid.

3. The compound-nucleic acid nanoparticle according to claim 2, characterized in that The sizes of the small interfering ribonucleic acid and the modified small interfering ribonucleic acid are respectively in the range of 15 bp - 30 bp; and / or The lengths of the micro ribonucleic acid and the modified micro ribonucleic acid are respectively in the range of 15 nt - 30 nt; and / or The lengths of the antisense oligonucleotide and the modified antisense oligonucleotide are respectively in the range of 15 nt - 30 nt; and / or The lengths of the messenger ribonucleic acid and the modified messenger ribonucleic acid are respectively independently > 500 nt, or in the range of 500 nt - 3000 nt.

4. The compound-nucleic acid nanoparticle according to claim 1, characterized in that R includes a guanidyl group n > 1; and / or R includes a guanidyl group The nucleic acid includes one or more of an antisense oligonucleotide, a messenger ribonucleic acid, a modified antisense oligonucleotide, and a modified messenger ribonucleic acid.

5. The compound-nucleic acid nanoparticle according to claim 1, characterized in that The compound of formula I includes one or more of the compounds with the structural formula and the nucleic acid includes an antisense oligonucleotide and / or a modified antisense oligonucleotide; or The compound of formula I includes a compound having a structural formula of The nucleic acid includes small interfering ribonucleic acid, modified small interfering ribonucleic acid, messenger ribonucleic acid, modified messenger ribonucleic acid, antisense oligonucleotide, and / or modified antisense oligonucleotide; or The compound of formula I includes a compound having the structural formula and the nucleic acid includes one or more of small interfering ribonucleic acid, micro ribonucleic acid, messenger ribonucleic acid, modified small interfering ribonucleic acid, modified micro ribonucleic acid, and modified messenger ribonucleic acid; or The compound of formula I includes a compound with the structural formula and the nucleic acid includes one or more of double-stranded small interfering ribonucleic acid, single-stranded micro ribonucleic acid, long-chain messenger ribonucleic acid, modified double-stranded small interfering ribonucleic acid, modified single-stranded micro ribonucleic acid, and modified long-chain messenger ribonucleic acid; or The compound of formula I includes a compound having the structural formula The nucleic acid includes messenger ribonucleic acid and / or modified messenger ribonucleic acid.

6. The compound-nucleic acid nanoparticle according to any one of claims 1 - 5, characterized in that The molar ratio of the compound of formula I to the phosphate groups in the nucleic acid is in the range of 100:1 to 0.5:1; or The molar ratio of the compound of formula I to the phosphate groups in the nucleic acid is in the range of 40:1 to 10:1; or The molar ratio of the compound of formula I to the phosphate groups in the nucleic acid is 40:1, 20:1, 15:1, or 10:

1.

7. The compound-nucleic acid nanoparticle according to any one of claims 1 - 5, characterized in that The particle size of the compound-nucleic acid nanoparticle is in the range of 30 nanometers to 600 nanometers; or The particle size of the compound-nucleic acid nanoparticle is in the range of 47.42 nanometers to 208.44 nanometers.

8. A drug, characterized in that, Containing a pharmaceutical carrier and the compound-nucleic acid nanoparticle according to any one of claims 1 - 7.

9. The drug according to claim 8, characterized in that One or more of the drugs for tumor prevention and treatment, cancer prevention and treatment, neurodegenerative disease prevention and treatment, protein replacement drugs, vaccines, gene editing drugs, and pulmonary fibrosis prevention and treatment drugs; and / or one or more of small interfering ribonucleic acid drugs, micro ribonucleic acid drugs, antisense oligonucleotide drugs, messenger ribonucleic acid drugs, long non-coding ribonucleic acid drugs, circular ribonucleic acid drugs, small nuclear ribonucleic acid drugs, modified small interfering ribonucleic acid drugs, modified micro ribonucleic acid drugs, modified antisense oligonucleotide drugs, modified messenger ribonucleic acid drugs, modified long non-coding ribonucleic acid drugs, modified circular ribonucleic acid drugs, and modified small nuclear ribonucleic acid drugs.

10. The drug according to claim 8, wherein it is a drug for leukemia prevention and treatment; and / or it is a drug for pancreatic cancer prevention and treatment; and / or it is a drug for cervical cancer prevention and treatment; and / or it is a drug for lung cancer prevention and treatment.

11. Use of the compound-nucleic acid nanoparticle according to any one of claims 1-7 in the preparation of a product for introducing nucleic acid into cells.

12. The use according to claim 11, wherein The cell is a mammalian cell.

13. Use of the compound-nucleic acid nanoparticle according to any one of claims 1-7 in the preparation of a product for in vivo delivery of nucleic acid to be administered to a mammal.

14. Use of the compound-nucleic acid nanoparticle according to any one of claims 1-7 in the preparation of a drug to be administered to a mammal.

15. The use according to claim 14, wherein the drug is one or more of the drugs for tumor prevention and treatment, cancer prevention and treatment, neurodegenerative disease prevention and treatment, protein replacement drugs, vaccines, gene editing drugs, and pulmonary fibrosis prevention and treatment drugs; and / or the drug is one or more of small interfering ribonucleic acid drugs, micro ribonucleic acid drugs, antisense oligonucleotide drugs, messenger ribonucleic acid drugs, long non-coding ribonucleic acid drugs, circular ribonucleic acid drugs, small nuclear ribonucleic acid drugs, modified small interfering ribonucleic acid drugs, modified micro ribonucleic acid drugs, modified antisense oligonucleotide drugs, modified messenger ribonucleic acid drugs, modified long non-coding ribonucleic acid drugs, modified circular ribonucleic acid drugs, and modified small nuclear ribonucleic acid drugs.

16. The use according to claim 14, wherein the drug is a drug for leukemia prevention and treatment; and / or the drug is a drug for pancreatic cancer prevention and treatment; and / or the drug is a drug for cervical cancer prevention and treatment; and / or the drug is a drug for lung cancer prevention and treatment.

17. The method for preparing the compound-nucleic acid nanoparticle according to any one of claims 1-7, characterized in that, Comprising: Dissolving the compound of formula I in a solvent to obtain a solution of the compound of formula I; Dissolving the nucleic acid in water to obtain a nucleic acid solution; And Mixing the solution of the compound of formula I with the nucleic acid solution to obtain the compound-nucleic acid nanoparticle.

18. The preparation method according to claim 17, wherein the temperature for mixing the solution of the compound of formula I with the nucleic acid solution ranges from 25°C to -80°C; and / or the time for mixing the solution of the compound of formula I with the nucleic acid solution ranges from 15 minutes to 48 hours. Use of a compound of formula I in the preparation of a product for introducing nucleic acid into cells, Among them, n is an integer selected from 1 to 14, and R is a positively charged group containing one or more nitrogen atoms.

20. The use according to claim 19, wherein, R is a positively charged group that can be ionized when the pH value of its environment is less than its pKa value; and / or R includes a primary amine group secondary amine group tertiary amine group quaternary amine group imidyl and guanidyl one or more of; and / or the nucleic acid includes one or more of small interfering ribonucleic acid, micro ribonucleic acid, antisense oligonucleotide, messenger ribonucleic acid, long non-coding ribonucleic acid, circular ribonucleic acid, small nuclear ribonucleic acid, modified small interfering ribonucleic acid, modified micro ribonucleic acid, modified antisense oligonucleotide, modified messenger ribonucleic acid, modified long non-coding ribonucleic acid, modified circular ribonucleic acid and modified small nuclear ribonucleic acid.

21. The use according to claim 20, wherein, the sizes of the small interfering ribonucleic acid and the modified small interfering ribonucleic acid are respectively in the range of 15 bp - 30 bp; and / or the lengths of the micro ribonucleic acid and the modified micro ribonucleic acid are respectively in the range of 15 nt - 30 nt; and / or the lengths of the antisense oligonucleotide and the modified antisense oligonucleotide are respectively in the range of 15 nt - 30 nt; and / or the lengths of the messenger ribonucleic acid and the modified messenger ribonucleic acid are respectively independently > 500 nt or in the range of 500 nt - 3000 nt.

22. The use according to any one of claims 19 - 21, wherein, R includes a guanidyl group n > 1; and / or R includes a guanidyl group The nucleic acid includes one or more of an antisense oligonucleotide, a messenger ribonucleic acid, a modified antisense oligonucleotide, and a modified messenger ribonucleic acid.

23. The use according to claim 19, wherein, The compound of formula I includes the structural formula as one or more of the compounds, wherein the nucleic acid comprises an antisense oligonucleotide and / or a modified antisense oligonucleotide; or The compound of formula I includes a compound having the structural formula and the nucleic acid includes small interfering ribonucleic acid, modified small interfering ribonucleic acid, messenger ribonucleic acid, modified messenger ribonucleic acid, antisense oligonucleotide, and / or modified antisense oligonucleotide; or The compound of Formula I includes a compound having a structural formula of The nucleic acid includes one or more of small interfering ribonucleic acid, micro ribonucleic acid, messenger ribonucleic acid, modified small interfering ribonucleic acid, modified micro ribonucleic acid, and modified messenger ribonucleic acid; or The compound of formula I includes a compound having the structural formula and the nucleic acid includes one or more of double-stranded small interfering ribonucleic acid, single-stranded micro ribonucleic acid, long-chain messenger ribonucleic acid, modified double-stranded small interfering ribonucleic acid, modified single-stranded micro ribonucleic acid, and modified long-chain messenger ribonucleic acid; or The compound of formula I includes a compound having the structural formula , and the nucleic acid includes messenger ribonucleic acid and / or modified messenger ribonucleic acid.

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