Degradable liposomes and their nanocomplexes for delivery of active molecules

Abstract

The present invention relates to a liposome for forming a nano-drug carrier, the liposome can form a nanocomplex that selectively and efficiently delivers a medicament active molecule, and can reduce or minimize toxicity associated with the treatment. The present invention further relates to a nanocomplex comprising the liposome and a medicament active molecule, and a pharmaceutical composition comprising the nanocomplex.

Description

[Technical field]

[0001] The present invention relates to a degradable liposomal compound for delivery of active molecules, preferably a liposomal compound for delivery of biologically active molecules, which can efficiently deliver active molecules (including, but not limited to, nucleic acids, proteins, small molecule drugs, etc.) and reduce or minimize toxicity associated with treatment. The present invention relates to a method for delivering nucleic acids using the degradable liposomes, or the use of the degradable liposomes for nucleic acid delivery. The present invention further relates to a nanocomplex comprising the liposomal compound and an active molecule, and a pharmaceutical composition comprising the nanocomplex. [Background technology]

[0002] Targeted drug delivery can improve the therapeutic selectivity of drugs to diseased cells, and can minimize the toxicity of delivery carriers and bioactive molecules to normal cells, as well as the toxic side effects of drugs. Biopolymer drugs such as nucleic acids and proteins are expected to be widely applied to the treatment and / or prevention of serious and intractable diseases. However, biopolymer drugs are easily degraded and cannot enter mammalian cells independently, and new drug delivery techniques are extremely important for their biological treatment. It is extremely important to develop new drug delivery carriers to efficiently and safely introduce biopolymer drugs into target cells and enrich them in diseased tissues, thereby improving the therapeutic effect of biological drugs. For example, messenger RNA (mRNA) needs to enter the cytoplasm of target cells to be translated to produce functional proteins. However, mRNA is easily degraded, has a large molecular weight, is highly hydrophilic, and cannot enter cells independently. Therefore, the application of mRNA drug biological therapy depends on a safe, effective and stable delivery system to protect nucleic acids from degradation, promote cellular uptake of mRNA, and control the efficient and selective release of mRNA from the carrier in cells. Viral carriers have been widely used and applied in gene drug delivery and gene therapy, and are one of the delivery carriers for biopolymer drugs. However, when a viral carrier delivers a gene drug, there are problems such as a limited dose of the gene drug that can be encapsulated, strong immunogenicity, and cytotoxicity. Nanomaterials such as non-viral carriers are widely used in the delivery of biopolymer drugs due to their low toxicity and high delivery efficiency, and liposomes, inorganic nanomaterials, metal-organic framework materials, polymeric polymers, proteins and peptides are all used in the delivery of nucleic acid and protein drugs. Liposomes are widely used in the delivery of biopolymers such as DNA and RNA due to their excellent delivery efficiency and good biocompatibility. To date, several liposomal formulations have been approved for clinical application for small interfering RNA delivery, among which mRNA vaccines encapsulating lipid nanoparticles have been used to prevent COVID-19 infection. Liposomes encapsulating nucleic acids usually enter cells via endocytosis and need to further escape from lysosomes to release the nucleic acids into the cytoplasm for the expression of the corresponding proteins.Currently, there are two main mechanisms by which liposome nanoparticles escape from the lysosome. One is that the liposome interacts with the lysosomal membrane to destabilize the lysosomal membrane. The other is that the liposome acts as a proton sponge, allowing a large amount of negative ions and water to flow into the interior for charge and concentration balance, causing the lysosome to swell and burst.

[0003] Efficiently adjusting and controlling the release of biopolymer drugs in target cells is extremely important for improving the drug delivery and biological therapy efficiency. Therefore, by developing a delivery carrier suitable for new bioactive molecules, especially nucleic acids, the bioactive molecules can be more efficiently delivered to targets such as target cells or target organs, and not only can the damage to normal tissues or cells caused by drug administration be avoided, but the drug can also be efficiently released in diseased cells or lesion tissues, further improving the delivery efficiency of biopolymer drugs, developing targeted therapy, and reducing or minimizing the risk of toxicity associated with treatment. Summary of the Invention

[0004] The present invention provides a nano drug delivery carrier, which is a degradable liposome based on reactive oxygen species (ROS) responsiveness, which can deliver pharmacoactive molecules to target diseased cells more efficiently and selectively, and not only can improve the release efficiency of the pharmacoactive molecules in the diseased cells, but also can further reduce drug toxicity to normal cells by targeting the diseased tissue.

[0005] In a first aspect, the present invention provides a liposome of formula (I): JPEG2025507982000002.jpg24161 [wherein, R 1a , R 2a , R 3a and R 4a are each independently a hydrogen atom, a monovalent aliphatic hydrocarbon group, a monovalent heteroaliphatic hydrocarbon group, a monovalent aromatic hydrocarbon group, a monovalent heteroaromatic hydrocarbon group, or Ht. t and s each independently represent 0 or 1, and when t or s is zero, the moiety directly represents a single bond. A1, A2, and A3 each independently represent a single bond, a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group, a divalent heteroaromatic hydrocarbon group, or a combination of two of the above. Ht for each occurrence is independently -R1-X-R2-Y-R3-Z-R4; however, Each occurrence of R1 is independently a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group, or a divalent heteroaromatic hydrocarbon group. Each occurrence of X is JPEG2025507982000003.jpg51161 (wherein m, n, p, q and r each independently represent an integer of 1 to 6. W is O, S or NR c It is. L1, L3, L5, L7 and L9 are directly linked to R1 or R2, and each independently represents a single bond, O, S or NR d Represents. L2, L4, L6, L8 and L 10 each independently represents a single bond, O, S, or NR e It is. V is an aliphatic hydrocarbon group, OR f , S.R. g or NR h R i It is. However, R b , R c , R d , R e , R f , R g , R h and R i are each independently a hydrogen atom, a hydroxy group, an aliphatic hydrocarbon oxy group, a monovalent aliphatic hydrocarbon group, a monovalent heteroaliphatic hydrocarbon group, a monovalent aromatic hydrocarbon group, or a monovalent heteroaromatic hydrocarbon group. Y and Z are each independently S or O at each occurrence. Each occurrence of R2 is independently a single bond, a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group, or a divalent heteroaromatic hydrocarbon group. Each occurrence of R3 is independently a single bond, a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group, or a divalent heteroaromatic hydrocarbon group. R4, at each occurrence, is independently a hydrophobic group. The N atom in the framework of formula (I) is R 1a and R 4a is hydrogen; and The compound may be cationized if it is not one of the compounds described below. JPEG2025507982000004.jpg63161〕

[0006] In some embodiments, the liposome of formula (I) is in an ionizable form, i.e., one or more N atoms in the backbone structure of formula (I) may be ionized. In further embodiments, the liposome of formula (I) is in an ionizable form, thereby forming the following structure and containing the corresponding counterion: JPEG2025507982000005.jpg205161 [In the formula, R 1b , R 2b , R 3b and R 4b are each independently a hydrogen, a monovalent aliphatic hydrocarbon group, a monovalent heteroaliphatic hydrocarbon group, a monovalent aromatic hydrocarbon group, a monovalent heteroaromatic hydrocarbon group, or Ht, and the remaining variables are as defined in formula (I).

[0007] In some embodiments, the counterion forms the anion of the salt with the positively charged N atom.Suitable anions include, but are not limited to, chloride, bromide, iodide, sulfate, nitrate, sulfate, citrate, methanesulfonate, trifluoroacetate, acetate, malate, toluenesulfonate, tartrate, fumarate, glutamate, glucuronate, lactate, glutarate, and maleate.

[0008] In some embodiments, R 1a or R 4a is hydrogen, or R 1a and R 4a are hydrogen.

[0009] In some embodiments, t and s are both 0; alternatively, t is 0 and s is 1; or alternatively, t is 1 and s is 0.

[0010] In some embodiments, t and s are both 0, and R 1a is Ht and R 4a is hydrogen, or t and s are both 0, and R 1a is hydrogen and R 4a is Ht.

[0011] In some embodiments, R 2a and R 3a each independently, if present, is hydrogen, a monovalent aliphatic hydrocarbon group, or a monovalent heteroaliphatic hydrocarbon group, preferably hydrogen, a monovalent C 1-6 Alkyl group, monovalent C 1-6 Heteroalkyl groups, such as amino C 1-6 Alkyl group, di(C 1-6 Alkyl)aminoalkyl group, mono(C 1-6 Alkyl)aminoalkyl group, C 1-6 Alkoxy C 1-6 Alkyl group, hydroxy C 1-6Alkyl group, C 1-6 Alkylthio C 1-6 Alkyl group or mercapto group C 1-6 It is an alkyl group.

[0012] In some embodiments, the fragment JPEG2025507982000006.jpg1971 is A1-A3 [wherein A1 and A3 are each independently a C1-C6 divalent aliphatic hydrocarbon group (e.g., a C1-C4 divalent aliphatic hydrocarbon group, e.g., a C1-C6 divalent alkyl group, preferably a C1-C4 divalent alkyl group)], or ●A1-(NR 2a )-A3 [wherein A1 and A3 are each independently a C1-C6 divalent aliphatic hydrocarbon group (e.g., a C1-C4 divalent aliphatic hydrocarbon group, e.g., a C1-C6 divalent alkyl group, preferably a C1-C4 divalent alkyl group), and R 2a is hydrogen, a monovalent aliphatic hydrocarbon group, or a monovalent heteroaliphatic hydrocarbon group, preferably hydrogen, a monovalent C 1-6 Alkyl group, monovalent C 1-6 Heteroalkyl groups, such as amino C 1-6 Alkyl group, di(C 1-6 Alkyl)aminoalkyl group, mono(C 1-6 Alkyl)aminoalkyl group, C 1-6 Alkoxy C 1-6 Alkyl group, hydroxy C 1-6 Alkyl group, C 1-6 Alkylthio C 1-6 Alkyl group or mercapto group C 1-6 is an alkyl group; ●A1-(NR 2a )-A2-(NR 2b )-A3 [wherein A1, A2, and A3 are each independently a C1-C6 divalent aliphatic hydrocarbon group (e.g., a C1-C4 divalent aliphatic hydrocarbon group, e.g., a C1-C6 divalent alkyl group, preferably a C1-C4 divalent alkyl group), and R 2a and R 2bare each independently hydrogen, a monovalent aliphatic hydrocarbon group, or a monovalent heteroaliphatic hydrocarbon group, and are preferably hydrogen, a monovalent C 1-6 Alkyl group, monovalent C 1-6 Heteroalkyl groups, such as amino C 1-6 Alkyl group, di(C 1-6 Alkyl)aminoalkyl group, mono(C 1-6 Alkyl)aminoalkyl group, C 1-6 Alkoxy C 1-6 Alkyl group, hydroxy C 1-6 Alkyl group, C 1-6 Alkylthio C 1-6 Alkyl group or mercapto group C 1-6 is an alkyl group; A1-A2-A3 [wherein A1 and A3 are each independently a C1-C6 divalent aliphatic hydrocarbon group (e.g., a C1-C4 divalent aliphatic hydrocarbon group, e.g., a C1-C6 divalent alkyl group, preferably a C1-C4 divalent alkyl group), and A2 is a divalent cyclic heteroaliphatic hydrocarbon group, e.g., a 3- to 7-membered monocyclic cyclic heteroaliphatic hydrocarbon group containing 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur, preferably a divalent piperazinyl group.] and Here, the asterisk * indicates that the group is bonded to the terminal N atom in formula (I) or (I-1) to (I-7).

[0013] In some embodiments, the fragment JPEG2025507982000007.jpg1971 is ●-(CH2) m1 [wherein m1 is an integer of 2 to 12, preferably 2 to 10 or 3 to 8, or an integer of 3], or ●-(CH2) m2 -(NR 2a )-(CH2) m3 - [wherein m2 and m3 are each independently an integer of 1 to 6, preferably an integer of 2 to 6, or 2 to 4, or 2 or 3; R 2a is hydrogen, monovalent C 1-6Alkyl group, amino C 1-6 Alkyl group, di(C 1-6 Alkyl)aminoalkyl group, mono(C 1-6 Alkyl)aminoalkyl group, C 1-6 Alkoxy C 1-6 Alkyl group, hydroxy C 1-6 Alkyl group, C 1-6 Alkylthio C 1-6 Alkyl group or mercapto group C 1-6 is an alkyl group; or ●-(CH2) m2 -(NR 2a )-(CH2) m4 -(NR 2b )-(CH2) m3 [wherein m2, m3, and m4 each independently represent an integer of 1 to 6, preferably an integer of 2 to 6, or 2 to 4, or 2 or 3; R 2a and R 2b are each independently hydrogen, monovalent C 1-6 Alkyl group, amino C 1-6 Alkyl group, di(C 1-6 Alkyl)aminoalkyl group, mono(C 1-6 Alkyl)aminoalkyl group, C 1-6 Alkoxy C 1-6 Alkyl group, hydroxy C 1-6 Alkyl group, C 1-6 Alkylthio C 1-6 Alkyl group or mercapto group C 1-6 is an alkyl group; ●-(CH2) m2 -A2-(CH2) m3 [wherein m2 and m3 are each independently an integer of 1 to 6, preferably an integer of 2 to 6 or 2 to 4 or 2 or 3, and A2 is a divalent cyclic heteroaliphatic hydrocarbon group, for example a 3- to 7-membered monocyclic cyclic heteroaliphatic hydrocarbon group containing 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur, and is preferably a divalent piperazinyl group.] Here, the asterisk * is bonded to the terminal N atom in formula (I) or (I-1) to (I-7).

[0014] In some embodiments, R is C 1-6 (For example, C 1-4 ) a divalent aliphatic hydrocarbon group or C 1-6 (For example, C 1-4 ), preferably C 1-4 or C 1-4 is a divalent heteroalkyl group.

[0015] In some embodiments, X is JPEG2025507982000008.jpg51161, wherein each variable is as defined in formula (I). In further embodiments, L1, L3, L5, L7 and L9 are linked to R1 and each independently are a single bond, O, S or NH. In further embodiments, X is JPEG2025507982000009.jpg109161 (in the formula, R d and R e is as defined in formula (I). In some embodiments, R d and R e are each independently H or C 1-4 is a monovalent aliphatic hydrocarbon group, preferably H or C 1-4 is a monovalent alkyl group.

[0016] In some embodiments, Y and Z are both S; alternatively, Y is S and Z is O; alternatively, Y is O and Z is S; or alternatively, Y and Z are both O.

[0017] In some embodiments, each occurrence of R2 is independently a bond or C 1-6 Divalent aliphatic hydrocarbon groups (e.g., C 1-4 is a divalent aliphatic hydrocarbon group, preferably 1-4 More preferably, the divalent alkyl group is C 1-2 It is a divalent alkyl group.

[0018] In some embodiments, each occurrence of R3 is independently a single bond or C 1-6 Divalent aliphatic hydrocarbon groups (e.g., C 1-4 is a divalent aliphatic hydrocarbon group, preferably 1-4 In a further embodiment, each occurrence of R3 is independently a single bond or JPEG2025507982000010.jpg2122 or methylene group ( JPEG2025507982000011.jpg2121), preferably The image is JPEG2025507982000012.jpg2122.

[0019] In some embodiments, each occurrence of R4 is independently a hydrophobic group, the hydrophobic group being selected from the group consisting of long chain alkyl groups, alkenyl groups, aryl groups, alkylaryl groups, arylalkyl groups, arylalkenyl groups, cyclic groups, alicyclic groups, and polycyclic groups, and the hydrophobic group may have at least one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur. In further embodiments, each occurrence of R4 is independently a C8-C 30 Alkyl groups, C8-C 30 Alkenyl group or C8-C 30 In a further embodiment, R4 is The image is JPEG2025507982000013.jpg121161.

[0020] In further embodiments, each occurrence of R is independently -(CHCHO). m -C8-C 30 Alkyl groups, -(CH2CH2O) y -C8-C 30 Alkenyl group or -(CH2CH2O) y -C8-C 30 alkynyl group, where y is 0 or 1 or 2, e.g., R4 is -CH2CH2O-C8H 17 , -CH2CH2O-C 10 H 21, -CH2CH2O-C 12 H 25 , -CH2CH2O-C 14 H 29 or -CH2CH2O-C 15 H 31 It is.

[0021] In some embodiments, the pKa value of the amine head is greater than 4, preferably greater than 6, and more preferably greater than 8.

[0022] In some embodiments, A1 and A3 are each independently a single bond or a divalent aliphatic hydrocarbon group, and A2 is a divalent aliphatic hydrocarbon group or a divalent heteroaliphatic hydrocarbon group. In further embodiments, A1 and A3 are each independently a C1-C6 divalent aliphatic hydrocarbon group (e.g., a C1-C4 divalent aliphatic hydrocarbon group, preferably a C1-C4 divalent alkyl group), and A2 is a divalent cyclic heteroaliphatic hydrocarbon group, such as a 3- to 7-membered monocyclic cyclic heteroaliphatic hydrocarbon group containing 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. In further embodiments, A1 and A3 are each independently an ethylene group, and A2 is a divalent piperazinyl group.

[0023] In some embodiments, the liposome of formula (I) is as follows: JPEG2025507982000014.jpg231161 [wherein A1, A2, A3 and R 4a is as defined in formula (I), -(CHCHO)-R 44 is R4 as defined in formula (I), Y and Z are both S, or Y is S and Z is O, or Y is O and Z is S, or Y and Z are both O, and R3 at each occurrence is independently a single bond or JPEG2025507982000015.jpg2122 or methylene group ( JPEG2025507982000016.jpg2121), and preferably, A1 and A3 are each independently -(CH2) m -;A2 is -(CH2) m In a further embodiment, R is a - or divalent cyclic heteroaliphatic hydrocarbon group, for example a 3- to 7-membered monocyclic cyclic heteroaliphatic hydrocarbon group containing 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur, preferably a divalent piperazinyl group, with the proviso that m in each occurrence is independently an integer of 1 to 6, preferably an integer of 2 to 6, or 2 to 4, or 2 or 3. 44 each occurrence independently represents an alkyl group C u H 2u+1 or alkenyl C u H 2u-1 (wherein u is 8 to 30, preferably 8, 12, 14 or 16). In a further embodiment, A2 is a divalent cyclic heteroaliphatic hydrocarbon group, preferably a divalent piperazinyl group, and A1 and A3 are each independently C 1-4 and divalent alkyl groups such as methylene, ethylene, and propylene.

[0024] In some embodiments, the liposome of formula (I) is as follows: JPEG2025507982000017.jpg230161 (However, A2 and R 4a is as defined in formula (I), -(CHCHO)-R 44 is R4 as defined in formula (I), Y and Z are both S, or Y is S and Z is O, or Y is O and Z is S, or Y and Z are both O, and R3 at each occurrence is independently a single bond or JPEG2025507982000018.jpg2122 or methylene group ( JPEG2025507982000019.jpg2121), and preferably, A2 is -(CH2) mIn a further embodiment, R is a - or divalent cyclic heteroaliphatic hydrocarbon group, for example a 3- to 7-membered monocyclic cyclic heteroaliphatic hydrocarbon group containing 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur, preferably a divalent piperazinyl group, and m is independently an integer of 1 to 6 in each occurrence, preferably an integer of 2 to 6, or 2 to 4, or 2 or 3. 44 each occurrence independently represents an alkyl group C u H 2u+1 or alkenyl C u H 2u-1 (wherein u is 8 to 30, preferably 8, 12, 14 or 16.) In a further embodiment, A2 is a divalent cyclic heteroaliphatic hydrocarbon group, preferably a divalent piperazinyl group.]

[0025] In some embodiments, the liposome of formula (I) is as follows: JPEG2025507982000020.jpg125161 (wherein v is 6 to 28, and preferably v is 10. A2 and R 4a is as defined in formula (I), preferably A2 is -(CH2) m - or a divalent cyclic heteroaliphatic hydrocarbon group, for example a 3- to 7-membered monocyclic cyclic heteroaliphatic hydrocarbon group containing 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur, preferably a divalent piperazinyl group, and m, each occurrence, is independently an integer of 1 to 6, preferably an integer of 2 to 6, or 2 to 4, or 2 or 3. In a further embodiment, A2 is a divalent cyclic heteroaliphatic hydrocarbon group, preferably a divalent piperazinyl group.]

[0026] In some embodiments, the liposome of formula (I) is as follows: JPEG2025507982000021.jpg171161 [However, R d and R e are as defined in formula (I), and preferably each independently represents hydrogen or a monovalent aliphatic hydrocarbon group, e.g., C1-4 is an alkyl group.

[0027] In some embodiments, an exemplary liposome is as follows: JPEG2025507982000022.jpg247161 JPEG2025507982000023.jpg252161 JPEG2025507982000024.jpg242161 JPEG2025507982000025.jpg252161 JPEG2025507982000026.jpg76161

[0028] The liposomes of formula (I) of the present invention contain ketal-responsive units (particularly units corresponding to thioketals when both Y and Z are S) in the Ht group, and are therefore degradable. Such liposomes can efficiently link with mRNA to form assembled nanostructures, and after entering cells, selectively degrade in response to the oxidative environment in the cells, and then efficiently release the mRNA and express the responsive protein. Furthermore, the inventors of the present invention have found that by reserving at least one hydrogen atom (i.e., not replacing with Ht or other groups) for the N atom on the backbone structure of the compound of formula (I), the delivery efficiency can be further improved, drugs can be delivered to the diseased tissue in a targeted manner, and the toxicity associated with drug administration, such as drug toxicity to normal cells and liver toxicity, can be reduced.

[0029] In a second aspect, the present invention provides a method of a liposome of formula (I) in which R1 is an ethylene group, the method comprising: A process for reacting an acrylic compound of formula (II), CH2=CH-X-R2-Y-R3-Z-R4 (II), (wherein each variable is as defined in formula (I)) with a hydrophilic amine in a molar ratio, wherein the hydrophilic amine contains at least one primary amine group (-NH2) or at least two secondary amine groups (-NH-), and the molar ratio of the acrylate hydrate of formula (II) to the hydrophilic amine is smaller than the number of moles of free hydrogen atoms on amino groups in the hydrophilic amine, but is greater than 1, e.g., the molar ratio of the acrylate hydrate of formula (II) to the hydrophilic amine ... a molar ratio of the acrylate hydrate of formula (II) to a hydrophilic amine containing 3 free hydrogen atoms on an amino group is less than 3, for example, 1.1 to 2.7, for example, 1.5 to 2.4, or further for example, 2.4; a molar ratio of the acrylate hydrate of formula (II) to a hydrophilic amine containing 4 free hydrogen atoms on an amino group is less than 4, for example, 1.1 to 3.5, for example, 1.1 to 1.8 or 2.1 to 3.3, or further for example, 3.3; and then purifying the product by chromatography to obtain the desired liposome of formula (I), The variables R2, X, Y, R3, Z and R4 are as defined in formula (I). In some embodiments, X is JPEG2025507982000027.jpg51161, wherein each variable is as defined in formula (I). In further embodiments, L1, L3, L5, L7 and L9 are linked to R1 and each independently are a single bond, O, S or NH. In further embodiments, X is JPEG2025507982000028.jpg109161 (However, R d and R e As defined in formula (I). In some embodiments, R d and R e are each independently H or C 1-4is a monovalent aliphatic hydrocarbon group, preferably H or C 1-4 is a monovalent alkyl group.

[0030] Specifically, the present invention provides a method of a liposome of formula (I), wherein R1 is an ethylene group and X is -C(O)-O-, the method comprising: A process for reacting a ketal-containing acrylate, CH2=CH-C(O)O-R2-Y-R3-Z-R4 (wherein each variable is as defined in formula (I)) with a hydrophilic amine in a molar ratio, wherein the hydrophilic amine contains at least one primary amine group (-NH2) or at least two secondary amine groups (-NH-), the molar ratio of the ketal-containing acrylate to the hydrophilic amine is smaller than the number of moles of free hydrogen atoms on the amino groups in the hydrophilic amine, but the molar ratio of the ketal-containing acrylate to the hydrophilic amine is greater than 1, e.g., the number of moles of the ketal-containing acrylate to the hydrophilic amine is greater than 2 (or 2 moles). a molar ratio of the ketal-containing acrylate to the hydrophilic amine containing 3 free hydrogen atoms on the amino group is less than 3, for example, 1.1 to 2.7, for example, 1.5 to 2.4, or further for example, 2.4; a molar ratio of the ketal-containing acrylate to the hydrophilic amine containing 4 free hydrogen atoms on the amino group is less than 4, for example, 1.1 to 3.5, for example, 1.1 to 1.8, or further for example, 2.1 to 3.3, and then purifying the product, optionally by chromatography, to obtain the desired liposome of formula (I), The variables R2, Y, R3, Z and R4 are as defined in formula (I).

[0031] In some embodiments, the method includes a molar ratio of the ketal-containing acrylate to the hydrophilic amine that is 1 or 2 less than the number of moles of free hydrogen atoms on the amino groups in the hydrophilic amine, but the molar ratio of the ketal-containing acrylate to the hydrophilic amine is greater than 1. In some embodiments, the hydrophilic amine contains two primary amine groups (-NH2), i.e., four free hydrogen atoms on the amino groups, and the molar ratio of the ketal-containing acrylate to the hydrophilic amine is greater than 1 and not greater than 3.3 or not greater than 2.4. Alternatively, the method includes a hydrophilic amine that contains one primary amine group (-NH2) and one secondary amine group (-NH-), and the molar ratio of the ketal-containing acrylate to the hydrophilic amine is greater than 1 and not greater than 2.4. Alternatively, in the method, the hydrophilic amine contains two secondary amine groups (-NH-), and the molar ratio of the ketal-containing acrylate to the hydrophilic amine is greater than 1 and less than or equal to 1.5.

[0032] In some embodiments, in method (a), the product is purified by chromatography to obtain the desired liposomes of Formula (I), and methods of chromatographic purification are well known in the art.

[0033] In some embodiments, the amine head has a pKa value greater than 4, preferably greater than 6, and more preferably greater than 8. The hydrophilic amine in the method is JPEG2025507982000029.jpg231161.

[0034] In some embodiments, the ketal-containing acrylate CH2=CH-C(O)O-R2-Y-R3-Z-R4 is JPEG2025507982000030.jpg20117, and the remainder of the variables are as defined in formula (I).

[0035] In some embodiments, the hydrophilic amine is JPEG2025507982000031.jpg1149, and the ketal-containing acrylate is JPEG2025507982000032.jpg60161 (C u H 2u+1 represents an alkyl group, C u H 2u-1 represents an alkenyl group, and u is 8 to 30, and preferably 8, 12, 14, or 16. In one specific embodiment, the ketal-containing acrylate is 2-((2-((2-(dodecyloxy)ethyl)thio)propan-2-yl)thio)ethyl acrylate.

[0036] In some embodiments, the reaction is an addition reaction, preferably a Michael addition reaction.

[0037] In some embodiments, the reaction is carried out at a temperature of 60 to 85°C.

[0038] The ketal-containing acrylate according to the present invention may be synthesized according to a method known in the art, for example, the method disclosed in CN110101665A. The (thio)ketal-containing acrylate is an ester compound obtained by esterification of acrylic acid with an alcohol compound containing a (thio)ketal.

[0039] Other liposomes of the present invention may be prepared using other suitable starting materials by the above synthetic routes and other routes known in the art. The above methods may include one or more additional steps to add or remove appropriate protecting groups to ultimately allow synthesis of the liposome. It is noted that each synthetic step may be performed in an alternative order or sequence to obtain the desired material. Synthetic chemical transformations and protecting group methods (protection and deprotection) available for the synthesis of applicable liposomes are known in the art.

[0040] In a third aspect, the present invention provides a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and a nanocomplex comprising the liposome of formula (I) of the present invention and a pharma- ceutical active molecule.

[0041] In some embodiments, the pharmacoactive molecule is a small molecule drug, a protein, a peptide, a nucleic acid, a sugar or a combination thereof, preferably a nucleic acid.In further embodiments, the pharmacoactive molecule is an mRNA.In further embodiments, the mRNA can be an RFP mRNA expressing red fluorescent protein, an GFP mRNA expressing green fluorescent protein, or an DUF5 mRNA expressing RAS protease DUF5, and in further embodiments, the pharmacoactive molecule can be a plasmid DNA, such as a plasmid DNA expressing GFP.

[0042] In some embodiments, the nanocomposite has a particle size of 50-500 nm (eg, 50-300 nm and 50-180 nm).

[0043] In some embodiments, the liposomes of Formula (I) are ionizable.

[0044] In some embodiments, the liposome and the pharmacoactive molecule are associated by non-covalent interactions, covalent bonds, or a combination of both.

[0045] In some embodiments, the nanocomplex further comprises another lipid selected from the group consisting of cholesterol, dioleoylphosphatidylethanolamine (DOPE, CAS: 4004-05-1) and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000, CAS: 147867-65-0) or a combination thereof. In further embodiments, the nanocomplex further comprises cholesterol, dioleoylphosphatidylethanolamine and distearoylphosphatidylethanolamine-polyethylene glycol 2000. In one embodiment, the mass ratio of liposome:cholesterol:DOPE:DSPE-PEG2000 in the present example is 4:1:1:1 or 4:1:1:8.

[0046] In some embodiments, the nanocomposite of the present invention is prepared as follows: the liposome of the present invention, cholesterol, and DOPE are dissolved in trichloromethane to prepare a mixed solution, which is then evaporated to form a film, and the solution is dissolved in absolute ethanol by ultrasonication, and then dropped into a sodium acetate buffer solution containing DSPE-PEG2000 and stirred at high speed to obtain liposome nanoparticles.

[0047] In some embodiments, the pharmaceutical composition comprising the nanocomplex of the present invention is prepared as follows: A pharmacologic active molecule is mixed with the nanocomplex in a mass of 5 to 20 times (N / P ratio range of liposome to nucleic acid is 3:1 to 20:1) and incubated. In further embodiments, the pharmacologic active molecule is mRNA.

[0048] In the fourth aspect, the present invention provides a method for delivering nucleic acid, the method being to deliver nucleic acid to a target cell using the liposome of formula (I) of the present invention to release intracellular nucleic acid medicine in the target cell; alternatively, the present invention provides the use of the liposome of formula (I) as a drug delivery carrier, the drug being a nucleic acid. Alternatively, the present invention provides the use of the liposome of formula (I) as a drug delivery carrier to prepare a nucleic acid medicine, the drug delivery carrier delivering the nucleic acid medicine to a target cell to release intracellular nucleic acid medicine in the target cell.

[0049] In some embodiments, the liposome of formula (I) is as follows: JPEG2025507982000033.jpg24161 [wherein, R 1a , R 2a , R 3a and R 4a are each independently a hydrogen atom, a monovalent aliphatic hydrocarbon group, a monovalent heteroaliphatic hydrocarbon group, a monovalent aromatic hydrocarbon group, a monovalent heteroaromatic hydrocarbon group, or Ht. t and s each independently represent 0 or 1, and when t or s is zero, the moiety directly represents a single bond. A1, A2, and A3 each independently represent a single bond, a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group, or a divalent heteroaromatic hydrocarbon group, or a combination of two of the above. Each occurrence of Ht is independently -R1-X-R2-Y-R3-Z-R4. however, Each occurrence of R1 is independently a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group, or a divalent heteroaromatic hydrocarbon group. Each occurrence of X is JPEG2025507982000034.jpg51161 (wherein m, n, p, q and r each independently represent an integer of 1 to 6. W is O, S or NR c It is. L1, L3, L5, L7, and L9 are directly linked to R1 or R2 and each independently represents a single bond, O, S, or NR d It is. L2, L4, L6, L8 and L 10 each independently represents a single bond, O, S, or NR e It is. V is an aliphatic hydrocarbon group, OR f , S.R. g or NR h R i It is. However, R b , R c , R d , R e , R f , R g , R h and R i are each independently a hydrogen atom, a hydroxy group, an aliphatic hydrocarbon oxy group, a monovalent aliphatic hydrocarbon group, a monovalent heteroaliphatic hydrocarbon group, a monovalent aromatic hydrocarbon group, or a monovalent heteroaromatic hydrocarbon group. Y and Z are each independently S or O at each occurrence. Each occurrence of R2 is independently a single bond, a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group, or a divalent heteroaromatic hydrocarbon group. Each occurrence of R3 is independently a single bond, a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group, or a divalent heteroaromatic hydrocarbon group. R4 at each occurrence is independently a hydrophobic group. The N atom in the framework of formula (I) is R 1a and R 4a When at least one of the groups is hydrogen, the compound may be cationized.

[0050] In some embodiments, the liposome of formula (I) is in ionizable form, i.e., one or more N atoms in the backbone structure of formula (I) are ionizable. In further embodiments, the liposome of formula (I) is in ionizable form, thereby forming the following structure and containing the corresponding counterion: JPEG2025507982000035.jpg205161 [wherein, R 1b , R 2b , R 3b and R 4b are each independently a hydrogen, a monovalent aliphatic hydrocarbon group, a monovalent heteroaliphatic hydrocarbon group, a monovalent aromatic hydrocarbon group, a monovalent heteroaromatic hydrocarbon group, or Ht, and the remaining variables are as defined in formula (I).

[0051] In some embodiments, the counterion forms the anion of the salt with the positively charged N atom.Suitable anions include, but are not limited to, chloride, bromide, iodide, sulfate, nitrate, sulfate, citrate, methanesulfonate, trifluoroacetate, acetate, malate, toluenesulfonate, tartrate, fumarate, glutamate, glucuronate, lactate, glutarate, and maleate.

[0052] In some embodiments, R 1a or R 4a is hydrogen, or R 1a and R 4a are hydrogen.

[0053] In some embodiments, t and s are both 0; alternatively, t is 0 and s is 1; or alternatively, t is 1 and s is 0.

[0054] In some embodiments, t and s are both 0, and R 1a is Ht and R 4ais hydrogen, or t and s are both 0, and R 1a is hydrogen and R 4a is Ht.

[0055] In some embodiments, R 2a and R 3a each independently, if present, is hydrogen, a monovalent aliphatic hydrocarbon group, or a monovalent heteroaliphatic hydrocarbon group, preferably hydrogen, a monovalent C 1-6 Alkyl group, monovalent C 1-6 Heteroalkyl groups, such as amino C 1-6 Alkyl group, di(C 1-6 Alkyl)aminoalkyl group, mono(C 1-6 Alkyl)aminoalkyl group, C 1-6 Alkoxy C 1-6 Alkyl group, hydroxy C 1-6 Alkyl group, C 1-6 Alkylthio C 1-6 Alkyl group or mercapto group C 1-6 It is an alkyl group.

[0056] In some embodiments, the fragment JPEG2025507982000036.jpg1971 is A1-A3 [wherein A1 and A3 are each independently a C1-C6 divalent aliphatic hydrocarbon group (e.g., a C1-C4 divalent aliphatic hydrocarbon group, e.g., a C1-C6 divalent alkyl group, preferably a C1-C4 divalent alkyl group)], or ●A1-(NR 2a )-A3 [wherein A1 and A3 are each independently a C1-C6 divalent aliphatic hydrocarbon group (e.g., a C1-C4 divalent aliphatic hydrocarbon group, e.g., a C1-C6 divalent alkyl group, preferably a C1-C4 divalent alkyl group), and R 2a is hydrogen, a monovalent aliphatic hydrocarbon group, or a monovalent heteroaliphatic hydrocarbon group, preferably hydrogen, a monovalent C 1-6 Alkyl group, monovalent C 1-6 Heteroalkyl groups, such as amino C 1-6 Alkyl group, di(C1-6 Alkyl)aminoalkyl group, mono(C 1-6 Alkyl)aminoalkyl group, C 1-6 Alkoxy C 1-6 Alkyl group, hydroxy C 1-6 Alkyl group, C 1-6 Alkylthio C 1-6 Alkyl group or mercapto group C 1-6 is an alkyl group; ●A1-(NR 2a )-A2-(NR 2b )-A3 [wherein A1, A2, and A3 are each independently a C1-C6 divalent aliphatic hydrocarbon group (e.g., a C1-C4 divalent aliphatic hydrocarbon group, e.g., a C1-C6 divalent alkyl group, preferably a C1-C4 divalent alkyl group), and R 2a and R 2b are each independently hydrogen, a monovalent aliphatic hydrocarbon group, or a monovalent heteroaliphatic hydrocarbon group, and are preferably hydrogen, a monovalent C 1-6 Alkyl group, monovalent C 1-6 Heteroalkyl groups, such as amino C 1-6 Alkyl group, di(C 1-6 Alkyl)aminoalkyl group, mono(C 1-6 Alkyl)aminoalkyl group, C 1-6 Alkoxy C 1-6 Alkyl group, hydroxy C 1-6 Alkyl group, C 1-6 Alkylthio C 1-6 Alkyl group or mercapto group C 1-6 is an alkyl group; A1-A2-A3 [wherein A1 and A3 are each independently a C1-C6 divalent aliphatic hydrocarbon group (e.g., a C1-C4 divalent aliphatic hydrocarbon group, e.g., a C1-C6 divalent alkyl group, preferably a C1-C4 divalent alkyl group), and A2 is a divalent cyclic heteroaliphatic hydrocarbon group, e.g., a 3- to 7-membered monocyclic cyclic heteroaliphatic hydrocarbon group containing 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur, preferably a divalent piperazinyl group]; Here, the asterisk * indicates that the group is bonded to the terminal N atom in formula (I) or (I-1) to (I-7).

[0057] In some embodiments, the fragment JPEG2025507982000037.jpg1971 is ●-(CH2) m1 [wherein m1 is an integer of 2 to 12, preferably 2 to 10 or 3 to 8, or an integer of 3], or ●-(CH2) m2 -(NR 2a )-(CH2) m3 - [wherein m2 and m3 are each independently an integer of 1 to 6, preferably an integer of 2 to 6, or 2 to 4, or 2 or 3; R 2a is hydrogen, monovalent C 1-6 Alkyl group, amino C 1-6 Alkyl group, di(C 1-6 Alkyl)aminoalkyl group, mono(C 1-6 Alkyl)aminoalkyl group, C 1-6 Alkoxy C 1-6 Alkyl group, hydroxy C 1-6 Alkyl group, C 1-6 Alkylthio C 1-6 Alkyl group or mercapto group C 1-6 is an alkyl group; or ●-(CH2) m2 -(NR 2a )-(CH2) m4 -(NR 2b )-(CH2) m3 [wherein m2, m3, and m4 each independently represent an integer of 1 to 6, preferably an integer of 2 to 6, or 2 to 4, or 2 or 3; R 2a and R 2b are each independently hydrogen, monovalent C 1-6 Alkyl group, amino C 1-6 Alkyl group, di(C 1-6 Alkyl)aminoalkyl group, mono(C 1-6 Alkyl)aminoalkyl group, C 1-6 Alkoxy C 1-6 Alkyl group, hydroxy C1-6 Alkyl group, C 1-6 Alkylthio C 1-6 Alkyl group or mercapto group C 1-6 is an alkyl group; ●-(CH2) m2 -A2-(CH2) m3 [wherein m2 and m3 are each independently an integer of 1 to 6, preferably an integer of 2 to 6 or 2 to 4 or 2 or 3, and A2 is a divalent cyclic heteroaliphatic hydrocarbon group, for example a 3- to 7-membered monocyclic cyclic heteroaliphatic hydrocarbon group containing 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur, and is preferably a divalent piperazinyl group.] Here, the asterisk * is bonded to the terminal N atom in formula (I) or (I-1) to (I-7).

[0058] In some embodiments, R is C 1-6 (For example, C 1-4 ) a divalent aliphatic hydrocarbon group or C 1-6 (For example, C 1-4 ), preferably C 1-4 or C 1-4 is a divalent heteroalkyl group.

[0059] In some embodiments, X is JPEG2025507982000038.jpg51161, wherein each variable is as defined in formula (I). In further embodiments, L1, L3, L5, L7 and L9 are linked to R1 and each independently are a single bond, O, S or NH. In further embodiments, X is JPEG2025507982000039.jpg109161 (However, R d and R e As defined in formula (I). In some embodiments, R d and R e are each independently H or C 1-4 is a monovalent aliphatic hydrocarbon group, preferably H or C1-4 is a monovalent alkyl group.

[0060] In some embodiments, Y and Z are both S; alternatively, Y is S and Z is O; alternatively, Y is O and Z is S; or alternatively, Y and Z are both O.

[0061] In some embodiments, each occurrence of R2 is independently a bond or C 1-6 Divalent aliphatic hydrocarbon groups (e.g., C 1-4 is a divalent aliphatic hydrocarbon group, preferably 1-4 More preferably, the divalent alkyl group is C 1-2 It is a divalent alkyl group.

[0062] In some embodiments, each occurrence of R3 is independently a single bond or C 1-6 Divalent aliphatic hydrocarbon groups (e.g., C 1-4 is a divalent aliphatic hydrocarbon group, preferably 1-4 In a further embodiment, each occurrence of R3 is independently a single bond or JPEG2025507982000040.jpg2121 or methylene group ( JPEG2025507982000041.jpg2121), preferably The image is JPEG2025507982000042.jpg2121.

[0063] In some embodiments, each occurrence of R4 is independently a hydrophobic group, the hydrophobic group being selected from the group consisting of long chain alkyl groups, alkenyl groups, aryl groups, alkylaryl groups, arylalkyl groups, arylalkenyl groups, cyclic groups, alicyclic groups, and polycyclic groups, and the hydrophobic group may have at least one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur. In further embodiments, each occurrence of R4 is independently a C8-C 30 Alkyl groups, C8-C 30Alkenyl group or C8-C 30 In a further embodiment, R4 is The image is JPEG2025507982000043.jpg121161.

[0064] In further embodiments, each occurrence of R is independently -(CHCHO). m -C8-C 30 Alkyl groups, -(CH2CH2O) y -C8-C 30 Alkenyl group or -(CH2CH2O) y -C8-C 30 An alkynyl group (wherein y is 0, 1, or 2), for example, R4 is -CH2CH2O-C8H 17 , -CH2CH2O-C 10 H 21 , -CH2CH2O-C 12 H 25 , -CH2CH2O-C 14 H 29 or -CH2CH2O-C 15 H 31 It is.

[0065] In some embodiments, the pKa value of the amine head is 4, preferably greater than 6, and more preferably greater than 8.

[0066] In some embodiments, A1 and A3 are each independently a single bond or a divalent aliphatic hydrocarbon group, and A2 is a divalent aliphatic hydrocarbon group or a divalent heteroaliphatic hydrocarbon group. In further embodiments, A1 and A3 are each independently a C1-C6 divalent aliphatic hydrocarbon group (e.g., a C1-C4 divalent aliphatic hydrocarbon group, preferably a C1-C4 divalent alkyl group), and A2 is a divalent cyclic heteroaliphatic hydrocarbon group, such as a 3- to 7-membered monocyclic cyclic heteroaliphatic hydrocarbon group containing 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. In further embodiments, A1 and A3 are each independently an ethylene group, and A2 is a divalent piperazinyl group.

[0067] In some embodiments, the liposome of formula (I) is as follows: JPEG2025507982000044.jpg231161 [However, A1, A2, A3 and R 4a is as defined in formula (I), -(CHCHO)-R 44 is R4 as defined in formula (I), Y and Z are both S, or Y is S and Z is O, or Y is O and Z is S, or Y and Z are both O, and R3 at each occurrence is independently a single bond or JPEG2025507982000045.jpg2121 or methylene group ( JPEG2025507982000046.jpg2121). In a further embodiment, R 44 each occurrence independently represents an alkyl group C u H 2u+1 or alkenyl C u H 2u-1 (wherein u is 8 to 30, preferably 8, 12, 14 or 16). In a further embodiment, A2 is a divalent cyclic heteroaliphatic hydrocarbon group, preferably a divalent piperazinyl group, and A1 and A3 are each independently C 1-4 and divalent alkyl groups such as methylene, ethylene, and propylene.

[0068] In some embodiments, the liposome of formula (I) is the following compound: JPEG2025507982000047.jpg230161 (However, A2 and R 4a is as defined in formula (I), -(CHCHO)-R 44 is R4 as defined in formula (I), Y and Z are both S, or Y is S and Z is O, or Y is O and Z is S, or Y and Z are both O, and R3 at each occurrence is independently a single bond or JPEG2025507982000048.jpg2121 or methylene group ( In a further embodiment, R 44 each occurrence independently represents an alkyl group C u H 2u+1 or alkenyl C u H 2u-1 (wherein u is 8 to 30, preferably 8, 12, 14 or 16.) In a further embodiment, A2 is a divalent cyclic heteroaliphatic hydrocarbon group, preferably a divalent piperazinyl group.]

[0069] In some embodiments, the liposome of formula (I) is the following compound: JPEG2025507982000050.jpg125161 [wherein v is 6 to 28, and preferably v is 10. A2 and R 4a is as defined in formula (I). In a further embodiment, A2 is a divalent cyclic heteroaliphatic hydrocarbon group, preferably a divalent piperazinyl group.

[0070] In some embodiments, the liposome of formula (I) is as follows: JPEG2025507982000051.jpg178161 [wherein, R d and R e are as defined in formula (I), and preferably each independently represents hydrogen or a monovalent aliphatic hydrocarbon group, e.g., C 1-4 is an alkyl group.

[0071] In some embodiments, an exemplary liposome is as follows:

[0072] JPEG2025507982000052.jpg242161 JPEG2025507982000053.jpg252161 JPEG2025507982000054.jpg242161 JPEG2025507982000055.jpg91161

[0073] In some embodiments, the target cell is a cancer cell, a cell infected by a pathogen, or a cell mediating a disease, e.g., selected from the group consisting of an endothelial cell, an epithelial cell, a muscle cell, a brain cell, a neuronal cell, a skin cell, a hair cell, a progenitor cell, and a pericyte.

[0074] In some embodiments, the nucleic acid is selected from the group consisting of an oligonucleotide, an aptamer, a single stranded DNA, a double stranded DNA, a plasmid DNA, a short isomer, an antisense molecule, a small interfering RNA (siRNA), an asymmetric interfering RNA (aiRNA), a microRNA (miRNA), a double-stranded RNA (dsRNA), a small / short hairpin RNA (shRNA), a transfer RNA (tRNA), a messenger RNA (mRNA), a small active RNA, and a circRNA, and other forms of RNA molecules known in the art.

[0075] In some embodiments, the nucleic acid is in the form of a nanocomplex, hi some embodiments, the nanocomplex further comprises other lipids.

[0076] In the present invention, A1, A2 and A3 and the N linked thereto together constitute the amine head of the liposome of the present invention, which can be reacted with the tail of hydrophobic lipid to obtain a series of liposomes, and since the amine head can be protonated, such liposome nanoparticles can be positively charged as a whole, and can electrostatically interact with negatively charged nucleic acids, such as mRNA and cell membranes and lysosomal membranes, so that such liposomes can effectively encapsulate and deliver nucleic acids. The inventors of the present invention have found that liposomes with incompletely substituted nitrogen in the amine head have stronger proton-sponge action than those with complete substitution, improving the lysosomal escape ability and further improving the delivery ability of nucleic acid medicines, and also enhancing the release of drugs through a specific response to ROS substances in the lesion cells, thereby reducing the toxicity associated with drug administration. The inventors of the present invention have designed and synthesized ROS-responsive liposomes, selected liposomes that efficiently deliver plasmids such as the BAmP-TK-12 of the present invention, and realized efficient and selective delivery of RAS protease DUF5 mRNA to various KRAS mutant cancer cells and living animals, thereby solving the problem of the difficulty of universally regulating and controlling tumor RAS mutants. The inventors of the present invention have used a self-assembled complex (BAmP-TK-12 / DUF5 mRNA) of liposomes and DUF5 mRNA to inhibit abnormal RAS / RAF / MAPK / ERK and RAS / PI3K / AKT signaling pathways and tumor growth in vitro and in vivo, thereby achieving the goal of targeting various mutant KRAS in cancer cells. The present invention explores the potential of the BAmP-TK-12 / DUF5 mRNA complex for cancer therapy, and provides a new approach to cancer therapy that targets and inhibits RAS. Definition

[0077] The term "aliphatic" refers to a saturated or unsaturated, straight or branched chain, acyclic, cyclic or polycyclic hydrocarbon moiety, including, but not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl and cycloalkynyl moieties.

[0078] The term "alkyl group" refers to a hydrocarbon group selected from the group consisting of linear and branched saturated hydrocarbon groups having 1 to 30 carbon atoms (e.g., 1 to 24, 1 to 18, e.g., 1 to 12, further e.g., 1 to 10, and further e.g., 1 to 8 or 1 to 6 or 1 to 4). Examples of monovalent alkyl groups or alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pendadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, and triacontyl. Examples of divalent alkyl groups, i.e., alkylene groups, include, but are not limited to, methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, dodecylene, tridecylene, tetradecylene, pentadecylene, hexadecylene, heptadecylene, octadecylene, nonadecylene, icosylene, and triacotylene.

[0079] A monovalent radical is a radical obtained by removing one hydrogen atom from the corresponding hydrocarbon moiety, and a divalent radical is a radical obtained by removing two hydrogen atoms from the corresponding hydrocarbon moiety.

[0080] The term "alkenyl group" includes a hydrocarbon group having at least one C=C double bond and selected from the group consisting of straight and branched chain hydrocarbon groups having 2 to 30 carbon atoms (e.g., 2 to 24, 2 to 18, such as 2 to 8, further such as 2 to 6). Examples of alkenyl groups, for example C2-6 alkenyl groups, include, but are not limited to, vinyl groups (ethenyl / vinyl), prop-1-enyl groups, prop-2-enyl groups, 2-methylprop-1-enyl groups, but-1-enyl groups, but-2-enyl groups, but-3-enyl groups, but-1,3-dienyl groups, 2-methylbut-1,3-dienyl groups, hex-1-enyl groups, hex-2-enyl groups, hex-3-enyl groups, hex-4-enyl groups, hex-1,3-dienyl groups, dodecenyl groups, tetradecenyl groups, hexadecenyl groups, and tenoctoalkenyl groups.

[0081] The term "alkynyl group" includes at least one C≡C triple bond and a hydrocarbon group selected from the group consisting of linear and branched chain hydrocarbon groups having 2 to 30 carbon atoms (e.g., 2 to 24, 2 to 18, e.g., 2 to 8, further e.g., 2 to 6). Alkynyl groups, e.g., C 2-6 Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl (propargyl), 1-butynyl, 2-butynyl, and 3-butynyl groups.

[0082] The term "cycloalkyl group" includes hydrocarbon groups selected from the group consisting of saturated cyclic hydrocarbon groups, including monocyclic and polycyclic (e.g., bicyclic and tricyclic) groups, including fused, bridged or spiroalkyl groups. The cycloalkyl group may contain 3 to 30, 3 to 12 (e.g., 3 to 10, further e.g., 3 to 8, further e.g., 3 to 6, 3 to 5 or 3 to 4) carbon atoms. Further, for example, the cycloalkyl group may be selected from the group consisting of monocyclic groups containing 3 to 12 (e.g., 3 to 10, further e.g., 3 to 8, 3 to 6) carbon atoms. Examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecanyl, cycloundecyl, and cyclododecyl groups. The term "cycloalkenyl group" refers to a non-aromatic cyclic hydrocarbon moiety containing at least one double bond, such as cyclohexenyl and cyclohexenylene groups. The term "cycloalkynyl" refers to a non-aromatic cyclic hydrocarbon moiety containing at least one triple bond, such as cyclooctynyl and cyclooctynylidene. Similarly, cycloalkylene, cycloalkenylene, and cycloalkynylene are the corresponding divalent radicals.

[0083] The term "heteroaliphatic" refers to an aliphatic moiety having at least one heteroatom selected from the group consisting of N, O, P, B, S, Si, Sb, Al, Sn, As, Se, and Ge. Heteroaliphatic hydrocarbon groups of the present invention include alkyl, alkenyl, or alkynyl groups having at least one heteroatom selected from the group consisting of N, O, P, B, S, Si, Sb, Al, Sn, As, Se, and Ge, and cycloalkyl, cycloalkenyl, or cycloalkynyl groups having at least one heteroatom selected from the group consisting of N, O, P, B, S, Si, Sb, Al, Sn, As, Se, and Ge. Examples of the cyclic heteroaliphatic hydrocarbon group include a 3- to 7-membered monocyclic heteroaliphatic hydrocarbon group and a 7- to 12-membered bicyclic heteroaliphatic hydrocarbon group, and the cyclic heteroaliphatic hydrocarbon group contains 1 to 3 or more (e.g., 1 to 3) heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. In some embodiments, the cyclic heteroaliphatic hydrocarbon group is a 3- to 7-membered monocyclic heteroaliphatic hydrocarbon group containing 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur.Examples of the cyclic heteroaliphatic hydrocarbon group include a pyrrolidinyl group, an imidazolidinyl group, a piperidinyl group, a piperazinyl group, a pyranyl group, a morpholinyl group, an oxiranyl group, an aziridinyl group, a thiiranyl group, an azetidinyl group, an oxetanyl group, a thietanyl group, a dithietanyl group, a dihydropyridinyl group, a tetrahydropyridinyl group, a thiomorpholinyl group, a homopiperazinyl group, a homopiperidinyl group, an azepanyl group, an oxepanyl group, a thiepanyl group, an oxathiyl group, a dioxepanyl group, an oxathiepanyl group, an oxazepanyl group, a dithiepanyl group, a thiazepanyl group, a diazepanyl group, an azathianyl group, an oxazepi ... Representative heteroaliphatic hydrocarbon groups include, but are not limited to, heteroaryl, diazepinyl, thiazepinyl, dihydrothienyl, dihydropyranyl, dihydrofuranyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl, pyrrolinyl, indolinyl, dioxanyl, dioxolanyl, pyrazolinyl, pyrazolidinyl, dithianyl, dithiolanyl, pyrimidinonyl, dioxo-thiomorpholinyl, azabicyclo[3.1.0]hexanyl, azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, and the like. Representative heteroaliphatic hydrocarbon groups are heteroalkyl groups, i.e., alkyl groups containing at least one heteroatom selected from the group consisting of N, O, or S, such as C containing one N atom. 1-6 Alkyl groups (e.g., aminoalkyl groups, alkylaminoalkyl groups, etc., and also, for example, amino C 1-6 Alkyl group, di(C 1-6 Alkyl)aminoalkyl group, mono(C 1-6 alkyl)aminoalkyl) C containing 1 O atom 1-6 Alkyl groups (e.g., alkoxyalkyl groups, hydroxyalkyl groups, etc.) and also, for example, C 1-6 Alkoxy C 1-6 Alkyl group, hydroxy C 1-6 alkyl group), or C containing one S atom 1-6 Alkyl groups (e.g., alkylthioalkyl groups, mercaptoalkyl groups, etc.) and also, for example, C 1-6 Alkylthio C 1-6Alkyl group, mercapto C 1-6 alkyl group) or C containing one N atom 1-4 Heteroalkyl groups, C containing one O atom 1-4 Heteroalkyl groups or C containing one S atom 1-4 It is a heteroalkyl group.

[0084] The term "oxyaliphatic" refers to an -O-aliphatic hydrocarbon group. Examples of oxyaliphatic hydrocarbon groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, s-butoxy, and t-butoxy groups.

[0085] The term "aryl group (aromatic hydrocarbon group, aryl)" refers to a C6 monocyclic, C 10 Two rings, C 14 The three rings, C 20 Four rings or C 24 Examples of aryl groups include phenyl, phenylene, naphthyl, naphthylene, anthryl, anthrylene, pyrenyl, and picenylene groups.

[0086] The term "heteroaryl group (heteroaromatic hydrocarbon group, heteroaryl)" refers to aromatic 5- to 8-membered monocyclic, 8- to 12-membered bicyclic, 11- to 14-membered tricyclic, and 15- to 20-membered tetracyclic ring systems containing one or more heteroatoms (e.g., O, N, S, or Se). Examples of heteroaryl groups include furanyl, furanylene, fluorenyl, fluorenylene, pyrrolyl, pyrrolylene, thienyl, thiophenylene, oxazolyl, oxazolylene, imidazolyl, imidazolylene, benzimidazolyl, benzimidazolylene, thiazolyl, thiazolylene, pyridyl, pyridinylene, pyrimidyl, pyrimidinylene, quinazolinyl, quinazolinylene, quinolyl, quinolinylene, isoquinolyl, isoquinolinylene, indolyl, and indolylene groups.

[0087] Unless otherwise specified, the aliphatic hydrocarbon groups, heteroaliphatic hydrocarbon groups, aliphatic hydrocarbonoxy groups, alkyl groups, alkylene groups, alkenyl groups, alkenylene groups, alkynyl groups, alkynylene groups, cycloalkyl groups, cycloalkylene groups, cycloalkenyl groups, cycloalkenylene groups, cycloalkynyl groups, cycloalkynylene groups, heterocycloalkyl groups, heterocycloalkylene groups, heterocycloalkenyl groups, heterocycloalkenylene groups, aryl groups, and heteroaryl groups described herein include both substituted and unsubstituted moieties. Possible substituents on the cycloalkyl groups, cycloalkylene groups, cycloalkenyl groups, cycloalkenylene groups, cycloalkynyl groups, cycloalkynylene groups, heterocycloalkyl groups, heterocycloalkylene groups, heterocycloalkenyl groups, heterocycloalkenylene groups, aryl groups, and heteroaryl groups include C1-C 10 Alkyl groups, C2-C 10 Alkenyl groups, C2-C 10 Alkynyl groups, C3-C 20 Cycloalkyl groups, C3-C 20 Cycloalkenyl groups, C2-C 20 Heterocycloalkyl groups, C3-C 20 Heterocycloalkenyl groups, C1-C 10 Alkoxy group, aryl group, aryloxy group, heteroaryl group, heteroaryloxy group, amino group, C1-C 10 Alkylamino groups, C2-C 20 Dialkylamino group, arylamino group, diarylamino group, C1-C 10 Alkylsulfonylamino group, arylsulfonylamino group, C1-C 10 Alkylimino group, arylimino group, C1-C 10 Alkyl sulfonylimino group, aryl sulfonylimino group, hydroxy group, halogeno, thio, C1-C 10Examples of the substituents include, but are not limited to, alkylthio, arylthio, C1-C10 alkylsulfonyl, arylsulfonyl, acylamino, aminoacyl, aminothioacyl, amido, amidino, guanidino, urea, thiourea, cyano, nitro, nitroso, azide, acyl, thioacyl, acyloxy, carboxy, and carboxylic acid ester groups. On the other hand, possible substituents on the aliphatic hydrocarbon group, heteroaliphatic hydrocarbon group, aliphatic hydrocarbonoxy group, alkyl group, alkylene group, alkenyl group, alkenylene group, alkynyl group, and alkynylene group include all the substituents listed above, but are not limited to C1-C10 alkylsulfonyl, arylsulfonyl, acylamino, aminoacyl, aminothioacyl, amido, amidino, guanidino, urea, thiourea, cyano, nitro, nitroso, azido, acyl, thioacyl, acyloxy, carboxy, and carboxylic acid ester groups. 10 Except for alkyl groups, cycloalkyl groups, cycloalkylene groups, cycloalkenyl groups, cycloalkenylene groups, heterocycloalkyl groups, heterocycloalkylene groups, heterocycloalkenyl groups, heterocycloalkenylene groups, aryl groups and heteroaryl groups may further be condensed with each other.

[0088] The term "pharmaceutical active molecule" refers to any chemical compound used for the medical diagnosis, cure, treatment or prevention of disease, and includes small and large molecule drugs.

[0089] The term "small molecule" refers to organic compounds having a molecular weight of less than 800 daltons (eg, less than 500 daltons), including oligopeptides, oligosaccharides, and oligonucleotides.

[0090] The term "polymeric drug" includes "peptides" or "proteins" and refers to polymers of natural or unnatural amino acids linked through amide bonds and having a molecular weight of 800 daltons or greater.

[0091] The term "nucleic acid" refers to a polymer of nucleotides linked via phosphodiester bonds. Any of these polymers may be chemically modified. Specifically, the nucleic acid includes any form of nucleic acid molecule, including but not limited to oligonucleotides, aptamers, single-stranded DNA, double-stranded DNA, plasmid DNA, short isomers, antisense molecules, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dsRNA (Dicer-substrate RNA), shRNA (small / short hairpin RNA), transfer RNA (tRNA), messenger RNA (mRNA), small active RNA and circRNA, and other forms of RNA molecules known in the art. The term "nucleic acid drug" refers to a nucleic acid molecule having the above-mentioned disease treatment function. The nucleic acid molecule may further include any other pharma- ceutically acceptable carrier or excipient as a nucleic acid drug.

[0092] The term "drug delivery system" refers to a composition for administering a drug (particularly a nucleic acid) to a subject, such as a human or an animal, that needs it. In some embodiments, the drug delivery system comprises the liposome of the present invention and / or other liposomes as a drug delivery carrier, and can deliver the liposome encapsulating a drug (nucleic acid) to a site of interest in a human or an animal, preferably selectively in a target cell, such as a cancer cell, rather than to other sites in the body or organ, and more preferably, to a target cell by endocytosis.

[0093] The term "drug delivery carrier" refers to a substance that releases a drug in vivo, for example to a target cell, during the drug delivery process, enhancing the selectivity, functional efficacy, safety and / or pharmacokinetics of the drug.

[0094] The term "target cell" refers to a cell to which a drug is delivered by a drug delivery system, typically a cancer cell, a cell infected by a pathogen or a cell mediating a disease, such as an endothelial cell, an epithelial cell, a muscle cell, a brain cell, a neuronal cell, a skin cell, a hair cell, a progenitor cell and a pericyte.

[0095] The term "non-covalent interactions" refers to any non-covalent bond, including ionic interactions, hydrogen bonds, van der Waals interactions, and hydrophobic interactions. [Brief description of the drawings]

[0096] FIG. 1 shows RFP mRNA delivery by liposomes. Figure 2 shows the structure-activity relationship study of BAmP-TK-12 liposomes, in which a) is the chemical structure of BAmP-TK-12 and BAmP-NTK, b) is the encapsulation efficiency of RFP mRNA by BAmP-TK-12 and BAmP-NTK, c) is the mRNA release under ROS stimulation of BAmP-TK-12 and BAmP-NTK, d) is the Cy3-mRNA delivery efficiency of BAmP-TK-12 and BAmP-NTK in HeLa cells, and e) is the RFP mRNA delivery efficiency of BAmP-TK-12 and BAmP-NTK in HeLa cells. FIG. 3 shows the effect of the number of hydrophobic lipid tails in 21TK (ie, BAmP) amine-headed liposomes on delivery. FIG. 4 shows the delivery efficiency of the liposomes of the present invention for different amounts of GFP mRNA in HeLa cells. FIG. 5 shows that BAmP-TK-12 delivers DUF5 mRNA to inhibit tumor cell proliferation, including a) BAmP-TK-12 delivers DUF5 mRNA to inhibit KRAS G13D a) inhibits the proliferation of colorectal cancer cell line HCT-116, which contains a KRAS mutation; b) shows that BAmP-TK-12 delivers DUF5 mRNA to inhibit the proliferation of KRAS G13C c) inhibits the proliferation of colorectal cancer cell line H358 containing the KRAS mutation; and c) demonstrates that BAmP-TK-12 delivers DUF5 mRNA to inhibit the proliferation of KRAS G12V c) inhibits the proliferation of colorectal cancer cell line SW480 containing the KRAS mutation; and c) demonstrates that BAmP-TK-12 delivers DUF5 mRNA to inhibit the proliferation of KRAS G12Sd) shows that DUF5 mRNA delivered by BAmP-TK-12 inhibits the phosphorylation of AKT and ERK proteins by cleaving RAS protein in HCT-116, H358, SW480, and A549 cell lines, thereby inhibiting the proliferation of tumor cells. Figure 6 shows the biodelivery and tumor inhibition of DUF5 mRNA by BAmP-TK-12 liposomes. a) Biodistribution of BAmP-TK-12 / DUF5 mRNA. b) Relative tumor volume growth curve of HCT-116 tumor-bearing mice. c) Mouse tumor tissue at the end of the study. d) Western blot study of tumor tissue. FIG. 7 shows the NMR spectrum of compound TK-21 (BAmP-TK-12). FIG. 8 shows the NMR spectrum of compound TK-21-4 (BAmP containing 4 TK-12). FIG. 9 shows a test of pDHFR-GFP plasmid (5185 bp) delivery by BAmP-TK-12. FIG. 10 shows a pDNMT3A-GFP (9746 bp) plasmid delivery test by BAmP-TK-12. FIG. 11 shows a test of DUF5 mRNA and GFP mRNA delivery by the liposome of the present invention. FIG. 12 shows a test of P53 mRNA delivery by BAmP-TK-12. FIG. 13 shows a test of GFP protein and GFP mRNA delivery by BAmP-TK-12. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0097] The present invention will be described in more detail below with reference to specific embodiments, and the examples described are only for illustrating the present invention, and are not intended to limit the scope of the present invention. The examples provided below are not intended to limit the present invention in any manner, but serve as a guide for those skilled in the art to make further improvements.

[0098] For the quantitative tests in the following examples, each test was repeated three times and the average value of the results was calculated.

[0099] Unless otherwise specified, the test methods in the following examples are all common methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available products.

[0100] In the following examples, cholesterol is a product of Miclin Co., Ltd. (C10006595, CAS: 57-88-5).

[0101] In the following examples, DOPE (dioleoylphosphatidylethanolamine) is a product of Ponsure Biological Corporation (CAS: 4004-05-1).

[0102] In the following examples, DSPE-PEG2000 is a product of Ponsure Biological Corporation (E05275, CAS: 147867-65-0).

[0103] In the following examples, the plasmids pDHFR-GFP and pDNMT3A-GFP are products of Wuhan Miaoling Biology Science and Technology Limited Company.

[0104] In the following examples, GFP protein is a product of Beijing Soleibao Technology Co., Ltd.

[0105] In the following examples, HeLa cells, HCT-116 cells, SW480 cells, and A549 cells are products of Beijing Union Cell Center.

[0106] In the following examples, the Nu / Nu mice are products of Beijing Weitong Lihua Testing Animal Technology Co., Ltd. Working Example

[0107] Example 1: Liposome synthesis and complex preparation Example 1a: Synthesis of Liposomes The liposomes are synthesized by Michael addition reaction of a hydrophilic amine compound and a hydrophobic tail, where the hydrophobic tail is an acrylate containing a ketal, specifically an acrylate containing a thioketal. Among them, the acrylate containing a thioketal, i.e., 2-((2-((2-(dodecyloxy)ethyl)thio)propan-2-yl)thio)ethyl acrylate, is prepared according to the method of Examples 1 and 2 of CN110101665A. The prepared 2-((2-((2-(dodecyloxy)ethyl)thio)propan-2-yl)thio)ethyl acrylate 1 The H NMR is as follows: 1 H NMR(300MHz,DMSO)δ6.18(s,1H),5.97(d,J=10.2Hz,1H),4.26(s,2H),3.51(s,4H),3.34(s ,6H),2.87(s,1H),2.74(s,1H),1.55(s,4H),1.50-1.39(m,1H),1.25(s,6H),0.86(s,1H).

[0108] Then, the prepared 2-((2-((2-(dodecyloxy)ethyl)thio)propan-2-yl)thio)ethyl acrylate was mixed with hydrophilic amine compound 1,4-Bis(3-aminopropyl)piperazine (CAS:7209-38-3, BAmP) in a molar ratio of 3.3:1, heated at 80°C for 24h, and the crude product was purified on a silica gel column using dichloromethane / methanol as eluent to obtain liposome TK-21 (also called BAmP-TK-12).

[0109] In a similar manner, prepare the corresponding liposome compound with an appropriate ratio of the corresponding hydrophilic amine (for hydrophilic amines containing four free hydrogen atoms on the amino group, the molar ratio of 2-((2-((2-(dodecyloxy)ethyl)thio)propan-2-yl)thio)ethyl acrylate to the hydrophilic amine is 3.3:1, and for hydrophilic amines containing three free hydrogen atoms on the amino group, the molar ratio of 2-((2-((2-(dodecyloxy)ethyl)thio)propan-2-yl)thio)ethyl acrylate to the hydrophilic amine is 2.4:1).

[0110] JPEG2025507982000056.jpg213161 JPEG2025507982000057.jpg201161 JPEG2025507982000058.jpg185161 JPEG2025507982000059.jpg198161 JPEG2025507982000060.jpg205161 JPEG2025507982000061.jpg165161 JPEG2025507982000062.jpg185161

[0111] Example 1b: Preparation of liposome complexes In order to construct a delivery carrier with high transfection efficiency and excellent biocompatibility, the inventors prepared a liposome complex, the specific method being as follows: The liposomes prepared in Example 1a, cholesterol, and DOPE were dissolved in trichloromethane to prepare a mixed solution with a concentration of 10 mg / mL, the mixed solution of the three was evaporated at a predetermined ratio to form a film, and then dissolved by ultrasonic waves using absolute ethanol, and then dropped into a sodium acetate buffer solution containing DSPE-PEG2000 at 5 mg / mL (200 mM, pH=5.2), and stirred at 2000 rpm for 5 minutes to prepare a liposome complex. In this example, the liposomes (TK-2, TK-3, TK-5, TK-6, TK-7, TK-8, TK-10, TK-11, TK-13, TK-14, TK-15, TK-16, TK-17, TK-18, TK-20, TK-21) prepared in Example 1a were selected and used, and combined into 16 kinds of liposome complexes having a mass ratio of liposome:cholesterol:DOPE:DSPE-PEG2000 of 16:4:4:1 in sequence according to the method described above.

[0112] According to the same method, the 16 kinds of liposome complexes prepared above are used to deliver RFP mRNA, and the RFP-positive cell rate is detected to select the liposomes. The specific method is as follows.

[0113] 0.33μg / mL of RFP mRNA was added to sodium acetate buffer solution (25mM, pH=5.2), and mixed with 15 times the mass of each of the 16 liposome complexes prepared above. After uniform mixing, the mixture was incubated for 15 minutes to complete liposome / RFP mRNA self-assembly. 50μL of each self-assembly system was added to HeLa cells, and after 8h incubation, the medium was replaced with fresh medium. After 40h, the RFP-positive cell rate was analyzed using a flow cytometer.

[0114] The results are shown in Figure 1. All 16 types of liposomes were able to deliver RFP mRNA, and among them, liposome TK-21 (also called BAmP-TK-12) had the highest RFP-positive cell rate (see Figure 1).

[0115] Example 2: Study on the activity of BAmP-TK-12 liposome structure 1. Effect of thioketal structure on the ability of BAmP-TK-12 to respond to ROS stimulation In the present invention, a thioketal structure capable of being cleaved by ROS is introduced into the tail of the hydrophobic lipid of the liposome, and the liposome is decomposed by the stimulation of intracellular ROS to release the encapsulated mRNA. In order to verify the effect of the thioketal structure on the ROS stimulation response ability of BAmP-TK-12, first, a BAmP-NTK liposome (see FIG. 2A for the structural formula) having the same amine head structure BAmP as that of BAmP-TK-12 but not containing a thioketal in the hydrophobic tail is synthesized. That is, the BAmP amine head and the hydrophobic tail octadecyl acrylate (CAS: 4813-57-4) were mixed in a molar ratio of 1:3.3, heated at 80°C for 24h, and the crude product was purified in a silica gel column using dichloromethane / methanol as an eluent to obtain liposome BAmP-NTK.

[0116] To compare the mRNA encapsulation efficiency and ROS stimulation response ability of BAmP-TK-12 and BAmP-NTK, BAmP-TK-12 and BAmP-TK, respectively, and RFP mRNA with one-fifteenth of their mass were self-assembled in sodium acetate buffer solution (25 mM, pH = 5.2), and after 15 min of incubation, the concentration of unencapsulated mRNA was measured using an iQuant broad range RNA quantitation kit (GeneCopoeia). The measurement results were normalized by dissolving an equal amount of mRNA directly in the buffer solution to the corresponding concentration of 100%, and the results are shown in Figure 2B. Both BAmP-TK-12 and BAmP-TK had mRNA encapsulation efficiencies close to 100%. In order to simulate the process in which ROS in diseased cells stimulates the rupture of liposomal particles to release mRNA, BAmP-TK-12 / RFP mRNA and BAmP-TK / RFP mRNA were added with a final concentration of 25 mM H2O2, incubated at 37℃ for 24h, and the released mRNA concentration was measured. An equal amount of mRNA was dissolved in a buffer solution as is, and the corresponding concentration was set to 100% to normalize the measurement results, which are shown in Figure 2C. As can be seen from the figure, after being treated with H2O2, BAmP-NTK only released a small amount of mRNA, while BAmP-TK-12 released a large amount of mRNA, proving that BAmP-TK-12 has ROS-stimulation responsiveness and the ability to release mRNA due to the thioketal structure.

[0117] To compare the mRNA delivery performance of BAmP-TK-12 and BAmP-NTK, different concentrations of BAmP-TK-12 and BAmP-NTK were used with Cy3-labeled mRNA (Cy3-mRNA) at one-fifteenth of their mass to self-assemble in sodium acetate buffer solution (25 mM, pH = 5.2), incubated for 15 min, and then added to HeLa cells. After 8 h of incubation, the Cy3-positive cell rate was analyzed using a flow cytometer. The test results are shown in Figure 2D. As a result, it was clear that under each concentration condition, BAmP-TK-12 and BAmP-NTK were close to the Cy3-positive cell rate of the mRNA complex, proving that the cell entry efficiency of both was similar. Furthermore, BAmP-TK-12 and BAmP-NTK were applied to the delivery of RFP mRNA as described above, and the results are shown in Figure 2E. By comparing the RFP-positive cell rates, it was found that under each concentration condition, BAmP-TK-12 could burst and release mRNA in response to relatively high ROS in HeLa cells, and therefore its delivery efficiency was higher than that of BAmP-NTK.

[0118] 2. Effect of number of hydrophobic long chains on delivery efficiency In the present invention, a series of liposomes are obtained by the addition reaction of different amine heads with the tails of hydrophobic lipids, and the N atom in the amine head can be protonated as a proton acceptor, and such liposome nanoparticles are positively charged overall, so they can electrostatically interact with negatively charged mRNA, cell membranes, and lysosomal membranes. Therefore, the N structure of non-complete replacement by hydrophobic tails is an important factor for the liposomes to effectively encapsulate and deliver mRNA. In order to study how different amine head structures affect the mRNA delivery ability of liposomes, BAmP-TK-12 containing three thioketal hydrophobic tails and TK-21-4 containing four thioketal hydrophobic tails (i.e., BAmP containing four thioketal hydrophobic tails) were synthesized, and the NMR spectrum of TK-21 (BAmP-TK-12) is shown in Figure 7, and the NMR spectrum of TK-21-4 (BAmP containing four TK-12) is shown in Figure 8, and their mRNA delivery abilities were compared. Different concentrations of BAmP-TK-12 and TK-21-4, and GFP mRNA at one-fifteenth of their mass, were self-assembled in sodium acetate buffer solution (25 mM, pH=5.2), incubated for 15 min, and then added to HeLa cells. After 8 h of incubation, the solution was replaced, and the GFP-positive cell rate was analyzed after 36 h using a flow cytometer. The test results are shown in Figure 3. The results showed that under each concentration condition, BAmP-TK-12 could efficiently deliver GFP mRNA, whereas TK-21-4 could not. The test demonstrated that the non-complete replacement of N by hydrophobic tail in the amine head was an important factor in the liposome's ability to effectively encapsulate and deliver mRNA.

[0119] 3. Delivery efficiency of different liposomes To further prove that the non-completely substituted N at the amine head by the hydrophobic tail is an important factor in the liposome's effective encapsulation and delivery of mRNA, liposomes with non-completely substituted N at the amine head were used to deliver GFP mRNA. Liposomes TK-12, TK-19, TK-21, TK-22, TK-23, TK-24, and TK-25, cholesterol, and DOPE were dissolved in trichloromethane to prepare a mixed solution with a concentration of 10 mg / mL. The mixed solution of the three was evaporated at a predetermined ratio to form a film, and then dissolved by ultrasonication using absolute ethanol. The liposome complex was then dropped into a sodium acetate buffer solution containing DSPE-PEG2000 at 5 mg / mL (200 mM, pH=5.2) and stirred at 2000 rpm for 5 minutes to prepare a liposome complex. One of TK-12, TK-19, TK-21, TK-22, TK-23, TK-24, and TK-25 prepared in this example was combined into seven kinds of liposome complexes with a mass ratio of liposome:cholesterol:DOPE:DSPE-PEG2000 of 16:4:4:1 in sequence according to the above method. 37.5, 75, 150, 300, and 600 ng / mL of GFP mRNA was added to a sodium acetate buffer solution (25 mM, pH = 5.2), and mixed with 15 times the mass of the seven kinds of liposome complexes prepared above, respectively, and after uniform mixing, incubated for 15 minutes to complete the self-organization of liposome / GFP mRNA. 50 μL of each self-organization system was added to HeLa cells, and after 8 h of incubation, fresh medium was replaced, and the GFP-positive cell rate was analyzed by a flow cytometer after 40 h. The results are shown in Figure 4. Studies have demonstrated that liposomes containing non-fully substituted N in the amine head can also efficiently deliver mRNA.

[0120] Example 3: Delivery of DUF5 mRNA into cells by BAmP-TK-12 liposomes and inhibition of cancer cell proliferation 1.Inhibitory effect of BAmP-TK-12 / DUF5 mRNA on colon cancer cells HCT-116 The gene encoding the DUF5 protein, i.e., the DUF5 gene, is a fragment in the V. vulnificus rtxA1 gene (GI: 27366913; VV2_0479) 3596-4079, and its sequence is referred to as PDB ID: 5W6L. The DUF5 gene was recombinantly cloned using pcDNA3.1 as a carrier to obtain the recombinant expression carrier pcDNA3.1-DUF5 plasmid for the DUF5 protein. The pcDNA3.1-DUF5 plasmid was linearized with XhoI endonuclease (NEB), and the linearized plasmid was used as a template to perform the RiboMAX TM The DNA was transcribed using the Large Scale RNA Production System-T7 (Promega) reagent kit, followed by tailing with E. coli Poly(A) Polymerase (NEB) to obtain DUF5 mRNA.

[0121] As described above, different amounts of DUF5 mRNA and GFP mRNA were mixed uniformly with 15 times the mass of BAmP-TK-12 liposomes in sodium acetate buffer (25 mM, pH=5.2), incubated for 15 min, and then added to colon cancer cells at final concentrations of 0, 150, 300, and 600 ng / mL. After 8 h of incubation, cell viability was detected. CellTiter-Glo(R) Luminescent Cell Viability Assay Reagent Kit (Promega) was used to detect cell viability. The principle is that luciferase catalyzes the oxidation reaction of fluorescein in the presence of ATP, and at the same time, chemiluminescence is generated. The intensity of chemiluminescence is directly proportional to the ATP content in live cells, so it can indicate cell viability. The intensity of chemiluminescence was measured by ELISA, and the percentage of the chemiluminescence intensity of the test-treated cells relative to the blank control group was calculated to obtain the cell viability. The test results are shown in Figure 5a. Compared with the cells treated with BAmP-TK-12 / GFP mRNA, the cell viability of HCT-116 treated with BAmP-TK-12 / DUF5 mRNA was obviously decreased, and the killing effect gradually increased with the increase of DUF5 mRNA concentration. It was demonstrated that BAmP-TK-12 delivered DUF5 mRNA to colon cancer HCT-116 cells and inhibited their proliferation.

[0122] To investigate the effect of DUF5 mRNA on RAS protein and its downstream signaling pathway in cancer cells, HCT-116 cell lysates incubated with BAmP-TK-12 / DUF5 mRNA or BAmP-TK-12 / GFP mRNA for 8 h or not were collected and Western blot assay was performed. The primary antibodies were anti-RAS (RAS Antibody, CST #3965), anti-Phospho-p44 / 42 MAPK (phosphorylated ERK1 / 2, Thr202 / Tyr204, 197G2, CST #4377), anti-p44 / 42 MAPK (ERK1 / 2, L34F12, CST #4696), anti-Phospho-Akt (Phospho-Akt, Thr308, CST #9275), anti-AKT (anti-pan-AKT, Abcam #ab8805), and anti-β-Actin (anti-β-Actin, 8H10D10, CST #3700). The secondary antibodies were anti-rabbit (anti-rabbit IgG, HRP-linked antibody, CST #7074) and anti-mouse (anti-mouse IgG, HRP-linked antibody, CST #7076). The test results are shown in Figure 5e. Compared with other control groups, after treatment with BAmP-TK-12 / DUF5 mRNA, RAS in HCT-116 cells was obviously cleaved, which caused a decrease in p-ERK (phosphorylated ERK protein) level (ERK protein expression level was not affected), and at the same time, p-AKT (phosphorylated AKT protein) level was slightly decreased (AKT protein expression level was not affected). This demonstrated that after DUF5 mRNA was delivered into HCT-116 cells containing the KRASG13D mutation by BAmP-TK-12, the translated DUF5 protein specifically recognized and cleaved RAS protein and inhibited the phosphorylation activation of ERK and AKT proteins, thereby inhibiting the cell viability and proliferation process of colon cancer cells HCT-116. This demonstrated the regulatory and control function of DUF5 on the RAS / RAF / MEK / ERK and PI3K / AKT signal pathways.

[0123] 2. Inhibitory effect of BAmP-TK-12 / DUF5 mRNA on colon cancer cells SW480 and human non-small cell lung cancer cells A549 The aforementioned study demonstrated that BAmP-TK-12 / DUF5 mRNA inhibits KRAS G13D It has been demonstrated that this system has an inhibitory effect on the cell proliferation of colon cancer HCT-116 containing the KRAS mutation. To further demonstrate that this system has a therapeutic effect on cancer cells caused by various KRAS mutations, G12V Colon cancer cells SW480 and KRAS containing mutations G12S The mutation is used in human non-small cell lung cancer cells A549.

[0124] As mentioned above in "1.," different amounts of DUF5 mRNA or GFP mRNA and 15 times the mass of BAmP-TK-12 liposomes were added to sodium acetate buffer solution (25 mM, pH = 5.2) and mixed uniformly. After incubation for 15 min, they were added to colon cancer cell SW480 or non-small cell lung cancer cell A549, with the final concentrations of mRNA being 0, 150, 300, and 600 ng / mL. After incubation for 8 h, the cell viability was detected. In addition, the cell lysate treated for 8 h was collected for Western blot test. The test results are shown in Figure 5b, c, and d. Compared with other control groups, after treatment with BAmP-TK-12 / DUF5 mRNA, the cell viability of SW480 and A549 cells was obviously reduced, and when the DUF5 mRNA concentration was only 150 ng / mL, the cells were killed quickly for 8 h and the cell viability was reduced to 40%. The underlying mechanism is that RAS is cleaved by DUF5 protein, which causes a decrease in phosphorylated ERK protein levels (ERK protein expression levels were not affected), and at the same time, there is a small decrease in phosphorylated AKT protein levels (AKT protein expression levels were not affected).

[0125] This demonstrated that DUF5 mRNA was delivered into cancer cells by BAmP-TK-12 and translated to produce DUF5 protein, which efficiently cleaved RAS proteins containing G13D, G12V, and G12S mutations, inhibited the phosphorylation activation process of downstream ERK and AKT proteins, and inhibited the proliferation of different types of cancer cells. It was demonstrated that BAmP-TK-12 / DUF5 mRNA has a broad-spectrum therapeutic effect against cancers caused by various RAS mutation types.

[0126] Example 4: Biodelivery of DUF5 mRNA and inhibition of tumors by BAmP-TK-12 liposomes 1. Biodistribution of BAmP-TK-12 / DUF5 mRNA The pcDNA3.1-DUF5 plasmid was linearized with XhoI endonuclease, and the linearized plasmid was used as a template to generate the DNA fragments using HyperScribe. TM Cy5-labeled DUF5 mRNA (Cy5-DUF5 mRNA) was obtained by transcription using the T7 High Yield Cy5 RNA Labeling Kit (Apexbio) reagent kit. The BAmP-TK-12 / Cy5-DUF5 mRNA complex was prepared as described above.

[0127] To obtain a xenograft-bearing mouse model of colon cancer HCT-116 cells, 5.0 × 10 cells were placed in the right armpit of 4-6 week-old female Nu / Nu mice. 6 HCT-116 cells (suspended in PBS solution) were seeded to obtain a tumor volume of 200 mm 3 When the tumor volume reaches 100 mm (the formula for tumor volume is volume(mm 3 )=(l×w 2) / 2, where l is the length of the tumor and w is the width of the tumor), 200 μg of BAmP-TK-12 / Cy5-DUF5 mRNA, 200 μg of Cy5-DUF5 mRNA, or 200 μL of PBS solution was injected via the tail vein. 6 h after tail vein injection, the mice were euthanized and dissected to obtain heart, liver, spleen, lung, kidney, and tumor tissues, which were imaged at an excitation wavelength of 620 nm and an emission wavelength of 660 nm. The test results are shown in Figure 6a. As shown in the figure, Cy5-DUF5 mRNA not complexed with BAmP-TK-12 liposomes was not distributed in any of the major organs and tumor tissues, and its signal intensity was similar to that of the blank control group injected with PBS. In contrast, BAmP-TK-12 / Cy5-DUF5 mRNA was distributed in large amounts in the tumor tissues in addition to accumulating in the liver. Thus, BAmP-TK-12 / Cy5-DUF5 mRNA has the potential to accumulate in and inhibit tumor tissues after circulating in vivo.

[0128] 2. Inhibitory effect of BAmP-TK-12 / DUF5 mRNA on living tumors As shown in “1”, a xenograft mouse model of colon cancer HCT-116 cells was established, and the tumor volume was 100 mm 3 When the tumor reached the end point, BAmP-TK-12 / DUF5 mRNA, BAmP-TK-12 / GFP mRNA, or PBS solution was injected into the tail vein, with the dosage of 200 μg / mouse, and the drug administration frequency was 2 days / time, a total of 6 times. At the same time, the tumor volume and mouse weight were monitored. After reaching the end point of the test, blood was collected, serum was taken for blood biochemistry detection, and the mice were euthanized and dissected to obtain tumor tissue.

[0129] As can be seen from the mouse tumor volume relative growth curve (see FIG. 6b), the mouse tumors treated with tail vein injection of BAmP-TK-12 / GFP mRNA or PBS solution grew rapidly, while the tumor volume of the mice treated with BAmP-TK-12 / DUF5 mRNA only grew slightly compared to the start of the study. As can be seen from the mouse tumor tissue photograph (see FIG. 6c), at the end of the study, the tumor volume of the mice treated with BAmP-TK-12 / DUF5 mRNA was much smaller than that of the control group, proving that BAmP-TK-12 / DUF5 mRNA effectively inhibited the growth of colorectal cancer malignant tumors at the biological level.

[0130] The peripheral portion of the mouse tumor tissue was cut out and ground with a tissue homogenizer to obtain tumor tissue cell lysate, which was then prepared for Western blot testing, with the antibodies used as described above. Western blot testing (as shown in Figure 6d) showed that BAmP-TK-12 / GFP mRNA or PBS had no effect on the expression levels of RAS protein and its regulated and controlled AKT, ERK, and its phosphorylated proteins p-AKT and p-ERK in mouse tumor site cells, whereas BAmP-TK-12 / DUF5 mRNA reduced the expression levels of p-AKT and p-ERK by cleaving RAS protein. This demonstrated that BAmP-TK-12 / DUF5 mRNA achieved antitumor function in vivo by mediating the enzymatic degradation of RAS protein and the inactivation of downstream signaling pathways.

[0131] Example 5: Different plasmid delivery tests using liposomes of the present invention 1. pDHFR-GFP (5185bp) plasmid and pDNMT3A-GFP (9746bp) plasmid delivery test Liposome TK-21, cholesterol, and DOPE were dissolved in trichloromethane to prepare a mixed solution with a concentration of 10 mg / mL, the mixed solution of the three was evaporated at a predetermined ratio to form a film, and then dissolved by ultrasonic waves using absolute ethanol, and then dropped into a sodium acetate buffer solution containing DSPE-PEG2000 at 5 mg / mL (200 mM, pH = 5.2), and stirred at 2000 rpm for 5 minutes to prepare a liposome complex. In the liposome complex, the mass ratio of liposome: cholesterol: DOPE: DSPE-PEG2000 was 16: 4: 4: 1, respectively. 40, 80, 160, 320, 640ng / mL of pDHFR-GFP (5185bp) plasmid and 15 times the mass of the above-prepared TK-21 liposome complex were added to a sodium acetate buffer solution (25mM, pH=5.2), mixed, and incubated for 15 minutes after uniform mixing to complete the self-organization of TK-21 / pDHFR-GFP plasmid. 50μL of each self-organization system was added to HeLa cells, incubated for 8h, replaced with fresh medium, and analyzed the GFP-positive cell rate after 40h using a flow cytometer. The results are shown in Figure 9. Similarly, the above-prepared TK-21 nanoliposome complex pDNMT3A-GFP (9746bp) plasmid was mixed and used for HeLa cell delivery to detect the GFP-positive cell rate. The results are shown in Figure 10, and as is clear from the results, the liposomes of the present invention, such as TK-21, are suitable for effective delivery of plasmids of various lengths.

[0132] Example 6: Different mRNA delivery tests using liposomes of the present invention 1.DUF5 mRNA delivery test: Liposome, cholesterol, and DOPE were dissolved in trichloromethane to prepare a mixed solution with a concentration of 10 mg / mL, the mixed solution of the three was evaporated at a predetermined ratio to form a film, and then dissolved in absolute ethanol by ultrasonication, and then dropped into a sodium acetate buffer solution containing DSPE-PEG2000 at 5 mg / mL (200 mM, pH=5.2), and stirred at 2000 rpm for 5 minutes to prepare a liposome complex. In this example, one of the prepared TK-12, TK-22, TK-23, TK-24, and TK-25 was selected and used, and five types of liposome complexes with a mass ratio of liposome:cholesterol:DOPE:DSPE-PEG2000 of 16:4:4:1 were combined according to the above method. 0.33 μg / mL of DUF5 mRNA or GFP mRNA and 15 times the mass of the five liposome complexes were added to a sodium acetate buffer solution (25 mM, pH = 5.2) and mixed, and after uniform mixing, incubated for 15 minutes to complete the liposome / mRNA self-assembly. 50 μL of each self-assembly system was added to HeLa cells, and after 8 h incubation, the corresponding cell viability was measured, and the cell viability percentage was normalized to the blank hole. The results are shown in Figure 11.

[0133] 2. p53 mRNA delivery test using TK-21: Similarly, TK-21 nanoliposome complexes were prepared and mixed with 40, 80, 160, 320, and 640ng / mL p53 mRNA, which is 1 / 15 the mass of TK-21, to deliver to HeLa cells, and cell viability was detected after 36h incubation. The results are shown in Figure 12, which shows that TK-21 effectively delivers p53 mRNA and has a killing effect on cells, proving that TK-21 can effectively deliver various functional mRNAs.

[0134] Example 7: Nucleic acid and protein delivery test using liposomes of the present invention 1.Comparison of GFP protein delivery and nucleic acid delivery: TK-21, cholesterol and DOPE were dissolved in trichloromethane to prepare a mixed solution with a concentration of 10 mg / mL, the mixed solution of the three was evaporated at a predetermined ratio to form a film, and then dissolved by ultrasonic waves using anhydrous ethanol, and then dropped into a sodium acetate buffer solution containing DSPE-PEG2000 at 5 mg / mL (200 mM, pH = 5.2), and stirred at 2000 rpm for 5 minutes to prepare a liposome complex, in which the mass ratio of TK-21: cholesterol: DOPE: DSPE-PEG2000 was successively set to 16: 4: 4: 1. The TK-21 liposome complex prepared above at 1.2, 2.4, 4.8, 9.6, and 19.2 μg / mL and 0.6 times the mass of GFP protein were added to the DMEM buffer solution and mixed, respectively, and after uniform mixing, incubated for 15 minutes to complete the self-organization of the liposome / GFP protein. 50 μL of each self-assembly system was added to HeLa cells, and after incubation for 6 hours, the GFP-positive cell rate was analyzed using a flow cytometer. The results are shown in Figure 13, and show that when the concentration of the liposome complex was the same, the nucleic acid delivery ability of the liposome of the present invention was stronger than that of the protein delivery ability.

[0135] The above are merely preferred specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto, and any changes or replacements that a person skilled in the art can easily think of within the technical scope disclosed in the present invention are included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be in accordance with the scope of protection claims.

Claims

1. A compound of formula (I) for use in a method for delivering a nucleic acid drug, The method includes delivering the nucleic acid drug to the target cell by intracellularly releasing the nucleic acid drug into the target cell using the compound of formula (I); the target cell is a cancer cell, a cell infected by a pathogen, or a cell mediating a disease; The compound of formula (I), wherein the nucleic acid is selected from the group consisting of an oligonucleotide, an aptamer, a single-stranded DNA, a double-stranded DNA, a plasmid DNA, a short isomer, an antisense molecule, a small interfering RNA (siRNA), an asymmetric interfering RNA (aiRNA), a microRNA (miRNA), a double-stranded RNA (dsRNA), a small / short hairpin RNA (shRNA), a transfer RNA (tRNA), a messenger RNA (mRNA), a small activating RNA (small activating RNA), and a circRNA. [In the formula, R 1a , R 2a , R 3a and R 4a are each independently hydrogen, a monovalent aliphatic hydrocarbon group, a monovalent heteroaliphatic hydrocarbon group, a monovalent aromatic hydrocarbon group, a monovalent heteroaromatic hydrocarbon group, or Ht. t and s each independently represent 0 or 1, and when t or s is 0, the moiety directly represents a single bond. A 1 , A 2 and A 3 each independently represents a single bond, a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group, a divalent heteroaromatic hydrocarbon group, or a combination of the above. Each occurrence of Ht is independently -R 1 -X-R 2 -Y-R 3 -Z-R 4 and however, R 1 each occurrence is independently a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group, or a divalent heteroaromatic hydrocarbon group. Each occurrence of X is (In the formula, m, n, p, q, and r each independently represent an integer of 1 to 6. W is O, S or NR c is. L 1 , L 3 , L 5 , L 7 and L 9 is R 1 or R 2 and each independently represents a single bond, O, S, or NR d Represents. L 2 , L 4 , L 6 , L 8 and L 10 are each independently a single bond, O, S, or NR e is. V is an aliphatic hydrocarbon group, OR f , S.R. g or NR h R i is. However, R b , R c , R d , R e , R f , R g , R h and R i are each independently hydrogen, a hydroxy group, an aliphatic hydrocarbon oxy group (oxyaliphatic radical), a monovalent aliphatic hydrocarbon group, a monovalent heteroaliphatic hydrocarbon group, a monovalent aromatic hydrocarbon group, or a monovalent heteroaromatic hydrocarbon group. Y and Z are both S. R 2 each occurrence is independently a single bond, a divalent aliphatic hydrocarbon group, a divalent heteroaliphatic hydrocarbon group, a divalent aromatic hydrocarbon group, or a divalent heteroaromatic hydrocarbon group. R 3 are independently is. R 4 is independently a hydrophobic group each time it occurs. The N atom in the backbone structure of formula (I) is R 1a and R 4a When at least one of the groups is hydrogen, the compound may be cationized.

2. 10. The compound of claim 1, wherein the compound is in an ionizable form, thereby forming the following structure and containing the corresponding counterion: [In the formula, R 1b , R 2b , R 3b and R 4b are each independently hydrogen, a monovalent aliphatic hydrocarbon group, a monovalent heteroaliphatic hydrocarbon group, a monovalent aromatic hydrocarbon group, a monovalent heteroaromatic hydrocarbon group, or Ht, and the remaining variables are as defined in formula (I).

3. t and s are both 0, or t is 0 and s is 1, or t is 1 and s is 0, preferably t and s are both 0, and R 1a is Ht and R 4a is hydrogen, or t and s are both 0, and R 1a is hydrogen, and R 4a The compound according to claim 1 or 2, wherein is Ht.

4. piece teeth, ●A 1 -A 3 [However, A 1 and A 3 are each independently C 1 -C 6 a divalent aliphatic hydrocarbon group (e.g., C 1 -C 4 is a divalent aliphatic hydrocarbon group of the formula C 1 -C 6 is a divalent alkyl group of the formula C 1 -C 4 a divalent alkyl group of the formula: ●A 1 - (NR 2a )-A 3 [However, A 1 and A 3 are each independently C 1 -C 6 a divalent aliphatic hydrocarbon group (e.g., C 1 -C 4 is a divalent aliphatic hydrocarbon group of the formula C 1 -C 6 is a divalent alkyl group of the formula C 1 -C 4 is a divalent alkyl group of the formula 2a is hydrogen, a monovalent aliphatic hydrocarbon group, or a monovalent heteroaliphatic hydrocarbon group, preferably hydrogen, a monovalent C 1-6 Alkyl group, monovalent C 1-6 Heteroalkyl groups, such as amino C 1-6 Alkyl group, di(C 1-6 alkyl)aminoalkyl group, mono(C 1-6 alkyl)aminoalkyl group, C 1-6 Alkoxy C 1-6 Alkyl group, hydroxy C 1-6 Alkyl group, C 1-6 Alkylthio C 1-6 Alkyl group or mercapto C 1-6 is an alkyl group, or ●A 1 - (NR 2a )-A 2 - (NR 2b )-A 3 [However, A 1 , A 2 and A 3 are each independently C 1 -C 6 a divalent aliphatic hydrocarbon group (e.g., C 1 -C 4 is a divalent aliphatic hydrocarbon group of the formula C 1 -C 6 is a divalent alkyl group of the formula C 1 -C 4 is a divalent alkyl group of the formula 2a and R 2b are each independently hydrogen, a monovalent aliphatic hydrocarbon group, or a monovalent heteroaliphatic hydrocarbon group, and are preferably hydrogen, a monovalent C 1-6 Alkyl group, monovalent C 1-6 Heteroalkyl groups, such as amino C 1-6 Alkyl group, di(C 1-6 alkyl)aminoalkyl group, mono(C 1-6 alkyl)aminoalkyl group, C 1-6 Alkoxy C 1-6 Alkyl group, hydroxy C 1-6 Alkyl group, C 1-6 Alkylthio C 1-6 Alkyl group or mercapto C 1-6 is an alkyl group, or ●A 1 -A 2 -A 3 [However, A 1 and A 3 are each independently C 1 -C 6 a divalent aliphatic hydrocarbon group (e.g., C 1 -C 4 is a divalent aliphatic hydrocarbon group of the formula C 1 -C 6 is a divalent alkyl group of the formula C 1 -C 4 A is a divalent alkyl group of the formula 2 is a divalent cyclic heteroaliphatic hydrocarbon group, for example, a 3- to 7-membered monocyclic cyclic heteroaliphatic hydrocarbon group containing 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur, and is preferably a divalent piperazinyl group; Here, the asterisk * indicates that the group is bonded to the terminal N atom in formula (I) or (I-1) to (I-7). Preferably, the fragment teeth, ●-(CH 2 ) m1 [wherein m1 is an integer from 2 to 12, preferably from 2 to 10 or from 3 to 8, or is an integer of 3], or ●-(CH 2 ) m2 - (NR 2a )-(CH 2 ) m3 - [where m2 and m3 are each independently an integer of 1 to 6, preferably an integer of 2 to 6, 2 to 4, or 2 or 3, and R 2a is hydrogen, monovalent C 1-6 Alkyl group, amino C 1-6 Alkyl group, di(C 1-6 alkyl)aminoalkyl group, mono(C 1-6 alkyl)aminoalkyl group, C 1-6 Alkoxy C 1-6 Alkyl group, hydroxy C 1-6 Alkyl group, C 1-6 Alkylthio C 1-6 Alkyl group or mercapto C 1-6 is an alkyl group; or ●-(CH 2 ) m2 - (NR 2a )-(CH 2 ) m4 - (NR 2b )-(CH 2 ) m3 [wherein m2, m3, and m4 each independently represent an integer of 1 to 6, preferably an integer of 2 to 6, 2 to 4, or 2 or 3; R 2a and R 2b are each independently hydrogen, monovalent C 1-6 Alkyl group, amino C 1-6 Alkyl group, di(C 1-6 alkyl)aminoalkyl group, mono(C 1-6 alkyl)aminoalkyl group, C 1-6 Alkoxy C 1-6 Alkyl group, hydroxy C 1-6 Alkyl group, C 1-6 Alkylthio C 1-6 Alkyl group or mercapto C 1-6 is an alkyl group, or ●-(CH 2 ) m2 -A 2 - (CH 2 ) m3 [wherein m2 and m3 each independently represent an integer of 1 to 6, preferably an integer of 2 to 6, 2 to 4, or 2 or 3; A 2 is a divalent cyclic heteroaliphatic hydrocarbon group, preferably a divalent piperazinyl group; The compound according to claim 1 or 2, wherein the asterisk * indicates that the compound is bonded to the terminal N atom in formula (I) or (I-1) to (I-7).

5. R 1 is C 1-6 or a divalent aliphatic hydrocarbon group of C 1-6 3. The compound according to claim 1, wherein the divalent heteroaliphatic hydrocarbon group is

6. X is wherein each variable is as defined in formula (I), Preferably, X is and R d and R e 3. The compound according to claim 1 or 2, wherein:

7. R 2 Each occurrence of is independently a single bond or C 1-6 or a divalent aliphatic hydrocarbon group of C 1-6 3. The compound according to claim 1, wherein the divalent heteroaliphatic hydrocarbon group is

8. R 4 each occurrence is independently a hydrophobic group, the hydrophobic group being selected from the group consisting of a long-chain alkyl group, an alkenyl group, an aryl group, an alkylaryl group, an arylalkyl group, an arylalkenyl group, a cyclic group, an alicyclic group, and a polycyclic group, and the hydrophobic group optionally has at least one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur; Preferably, R 4 Each occurrence of 8 -C 30 Alkyl group, C 8 -C 30 Alkenyl group or C 8 -C 30 an alkynyl group, or R 4 Each occurrence of is independently -(CH 2 CH 2 O) m -C 8 -C 30 Alkyl group, -(CH 2 CH 2 O) y -C 8 -C 30 Alkenyl group or -(CH 2 CH 2 O) y -C 8 -C 30 an alkynyl group, where y is 0, 1, or 2, such as R 4 is -CH 2 CH 2 O-C 8 H 17 , -CH 2 CH 2 O-C 10 H 21 , -CH 2 CH 2 O-C 12 H 25 , -CH 2 CH 2 O-C 14 H 29 or -CH 2 CH 2 O-C 15 H 31 3. The compound according to claim 1 or 2, wherein

9. A 1 and A 3 are each independently a single bond or a divalent aliphatic hydrocarbon group, A 2 The compound according to claim 1 or 2, wherein R is a divalent aliphatic hydrocarbon group or a divalent heteroaliphatic hydrocarbon group.

10. The compound of formula (I) is [wherein A 2 is a divalent cyclic heteroaliphatic hydrocarbon group, A 1 and A 3 are each independently a C 1-4 divalent alkyl group, and R 4a is as defined in formula (I), and —(CH 2 CH 2 O)-R 44 is R as defined in formula (I). 4 Y and Z are both S, and R 3 are independently It is. The compound of claim 1, wherein

11. The compound of formula (I) is [However, A 2 and R 4a is as defined in formula (I), and —(CH 2 CH 2 O)-R 44 is R as defined in formula (I). 4 Y and Z are both S, and R 3 are independently It is. The compound of claim 1, wherein

12. The compound is [wherein v is 6 to 28, and A 2 and R 4a is as defined in formula (I). or, [However, R d and R e is as defined in formula (I). The compound of claim 1, wherein

13. The compound is The compound of claim 1, wherein

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