pH-RESPONSIVE LIPID DERIVATIVE

Abstract

The present invention provides a pH-responsive lipid derivative represented by formula (1): R1-L1-R2 (in the formula, R1 represents a lipid moiety, L1 represents NH or O, and R2 represents a polymer moiety having a repeating unit represented by formula (2) and a repeating unit represented by formula (3)), wherein the amount of a repeating unit (B) based on the total amount of a repeating unit (A) and the repeating unit (B) is 6 to 50 mol% (the symbols in formulae (2) and (3) are as defined in the description).

Description

pH-responsive lipid derivatives

[0001] This invention relates to pH-responsive lipid derivatives. The invention also relates to drug delivery carriers that exhibit high tumor tissue accumulation by detecting minute pH changes around tumor tissue and undergoing physical property changes.

[0002] Nucleic acid drugs can control the expression of disease-causing proteins at the gene level, which could not be targeted by conventional small molecule or antibody drugs, and are expected to lead to drug discovery in many disease areas. Drug delivery systems (DDS) are being investigated to deliver nucleic acid drugs to target cells more efficiently. In addition, research is being conducted to deliver conventional small molecule drugs more efficiently to target cells and reduce side effects.

[0003] Some drug delivery systems utilize lipid nanoparticles (LNPs), which are primarily composed of phospholipids, sterols, PEG lipids, or other lipids. Among the components of LNPs, PEG lipids play an important role in inhibiting aggregation between carriers and improving blood retention in the body.

[0004] In carriers using PEG lipids (e.g., LNPs), the zeta potential representing the charge on their surface is known to be -5 mV to 0 mV at a blood pH of 7.4, indicating that the surface is anionic to neutral.

[0005] The improved blood retention of carriers by PEG lipids is based on the fact that the carrier surface is anionic, and the hydrated layer formed by PEG covers the carrier surface, suppressing opsonization such as serum protein adsorption. As a result, it can avoid phagocytosis by macrophages and uptake by reticuloendothelial tissue (hereinafter, this property will be referred to as "stealth"). On the other hand, it suppresses not only interactions with blood components and normal tissues, but also interactions with tumor tissue, leading to a problem of reduced accumulation of carriers in tumor tissue and decreased efficiency of intracellular uptake (hereinafter, this problem will be referred to as the "PEG dilemma").

[0006] To overcome the dilemma of PEG, a drug delivery system is known that involves a complex of an antibody or antibody fragment and a carrier, with the aim of improving accumulation in target cells and efficiency of uptake into cells (for example, Patent Document 1). While this complex can be delivered to specific cells by being given target-directivity by the antibody, it is necessary to select a suitable antibody, which limits its versatility in target cells. Furthermore, since antibodies are expensive to produce, the cost of the complex is also high.

[0007] As one method to solve the above problems, Patent Document 2 reports a pH-responsive polymer consisting of a biocompatible polymer to which a group that is electrically neutral in a pH environment above 7 and changes to cationic at pH 7 or below is bonded. This polymer is neutral at pH 7.4 in normal tissue, so it exhibits stealth properties similar to PEG, and changes to cationic at pH 6.0 to 6.6 around tumor tissue, thus improving accumulation and uptake efficiency in tumor tissue. However, Patent Document 2 does not show the function of LNPs or liposomes containing this polymer.

[0008] Patent Document 3 describes LNPs containing pH-responsive lipid derivatives, and it has been shown that they exhibit superior antitumor effects compared to LNPs containing commonly used PEG lipids. LNPs containing PEG lipids have a zeta potential of approximately -5 mV to 0 mV (anionic to neutral) in a neutral environment (pH 7.4) surrounding normal tissue. On the other hand, Patent Document 3 shows that the zeta potential of the LNPs containing the pH-responsive lipid derivatives is 4 mV to 9 mV (weakly cationic) at pH 7.4.

[0009] International Publication No. 2010 / 090222, International Publication No. 2018 / 110366, International Publication No. 2023 / 282296

[0010] It is believed that adding a pH-responsive lipid derivative to LNPs such that the zeta potential of the LNPs at pH 7.4 is 0 mV or less (weakly anionic) will improve the blood retention of the LNPs and result in a higher antitumor effect. However, the structural optimization of pH-responsive lipid derivatives such that the zeta potential of LNPs becomes anionic has not been investigated in Patent Document 3.

[0011] An object of the present invention is to provide a pH-responsive lipid derivative capable of forming a carrier such that the zeta potential of the carrier is -5 mV or less (weakly anionic) in a neutral environment (around pH 7.4) around normal tissues, and the zeta potential of the carrier is 10 mV or more (cationic) in a weakly acidic environment (around pH 6.5) around tumor tissues.

[0012] The present invention that achieves the above object is as follows. [1] Formula (1): R １ -L １ -R ２ (1) (wherein R １ is a lipid moiety, L １ is NH or O, and R ２ is a polymer moiety having a repeating unit A represented by formula (2):

[0013]

[0014] (wherein * is a binding site) and a repeating unit B represented by formula (3):

[0015]

[0016] (wherein a1 and c1 are each independently 1 or 2, b1 is 0 or 1, X １ is an ester bond, an amide bond, or a thioester bond, Y １ is a carboxy group or a sulfo group, and * is a binding site), which is a pH-responsive lipid derivative represented by: wherein the amount of the repeating unit B relative to the total of the repeating unit A and the repeating unit B is 6 to 50 mol%.

[0017] [2] The pH-responsive lipid derivative according to [1], which has a number average molecular weight of 3,000 to 50,000. [3] The pH-responsive lipid derivative according to [1] or [2], wherein in formula (3), a1 and c1 are both 2, b1 is 1, X １ is an amide bond, and Y １ is a carboxy group. [4] R １ is represented by formula (4):

[0018]

[0019] (In the formula, R ３ and R ４ Each of these is independently an aliphatic hydrocarbon group having 8 to 24 carbon atoms, an acyl group having 8 to 24 carbon atoms, or a sterol residue, and M is a trivalent hydrocarbon group having 3 to 7 carbon atoms or *-N(CH ２ CH ２ -*)-* represents a trivalent nitrogen-containing hydrocarbon group, R ５ A is a hydrogen atom, an alkali metal atom, or ammonium, and a2, a3, b2, c2, and d1 are each independently 0 or 1, and * is a binding position.) A pH-responsive lipid derivative according to any one of [1] to [3] above, which is a lipid moiety represented by ).

[0020] [5] A drug delivery carrier comprising a pH-responsive lipid derivative as described in any one of [1] to [4] above. [6] The drug delivery carrier according to [5], which is a solid lipid nanoparticle. [7] The drug delivery carrier according to [5] or [6], which has an average particle size of 10 to 250 nm.

[0021] [8] The drug delivery carrier according to [5], further comprising at least one selected from the group consisting of phospholipids, ionic lipids, and sterols. [9] The drug delivery carrier according to [8], wherein the content of the pH-responsive lipid derivative is 0.1 to 30 mol%, the content of the phospholipid is 5 to 60 mol%, the content of the ionic lipid is 5 to 60 mol%, and the content of the sterol is 5 to 60 mol%, relative to the total of the pH-responsive lipid derivative and lipids optionally included in the drug delivery carrier.

[10] The drug delivery carrier according to any one of [5] to [9], wherein the drug transported by the drug delivery carrier is at least one selected from the group consisting of nucleic acids, nucleic acid derivatives, peptides, small molecule drugs, and anticancer drugs.

[0022]

[11] Formula (5): R １ -L １ -R６ (5) (wherein, R １ This is a lipid region, L １ is NH or O, and R ６ Equation (6):

[0023]

[0024] (In the formula, a1 is 1 or 2, P １ is a protecting group for the carboxyl group, and * is the bond position. ) is a polymer moiety containing repeating units represented by ). ) and formula (7): X ２ - (CH ２ ) ｃ１ -Y ２ (7) (wherein c1 is 1 or 2, X ２ Y is a hydroxyl group, an amino group, or a sulfanyl group. ２ *-CO-OP ２ , sulfo group, or *-SO ２ -OP ３ P ２ P is a protecting group for the carboxyl group. ３ A is a protecting group for the sulfo group, and * indicates the bond position.) A compound represented by formula (8): H ２ N-CH ２ CH ２ -NH-CH ２ CH ２ -NH-CH ２ CH ２ - CO-OP ４ (8) (wherein, P ４ A method for producing a pH-responsive lipid derivative, comprising: step (A) reacting with any of the compounds represented by ) to obtain intermediate 1; step (B) reacting intermediate 1 obtained in step (A) with the remainder of the compound represented by formula (7) or formula (8) that was not used in step (A) to obtain intermediate 2 having a protected carboxyl group; and step (C) deprotecting intermediate 2 obtained in step (B) to obtain the pH-responsive lipid derivative described in [1] above, in which b1 is 1.

[0025] The pH-responsive lipid derivative of the present invention has the property of becoming electrically anionic under neutral conditions (pH 7.4) and changing to cationic under weakly acidic conditions (pH 6.5) around tumor tissue. A drug delivery carrier (particularly a solid LNP) containing the pH-responsive lipid derivative of the present invention becomes anionic on its surface in the neutral environment of blood components or normal tissues in vivo, and its cationicity increases in response to minute pH changes around tumor tissue.

[0026] <pH-responsive lipid derivatives> In this specification, "lipid derivative" means a derivative having a lipid moiety. In this specification, "pH-responsive lipid derivative" means a lipid derivative whose electrical properties (anion or cation) change depending on the surrounding pH. In this specification, numerical ranges defined using "~" include the values ​​at both ends (upper and lower limits) of "~". For example, "6 to 50 mol%" means "6 mol% or more and 50 mol% or less". In this specification, each description can be combined with others unless it is clearly stated that they cannot be combined.

[0027] The pH-responsive lipid derivative of the present invention (hereinafter sometimes abbreviated as "the derivative of the present invention") is of formula (1): R １ -L １ -R ２ (1) is expressed as R １ This is the lipid portion. １ (That is, R １ and R ２ The linker (which bonds with L) is NH or O. １ From the viewpoint of ease of synthesis of the derivatives of the present invention, NH is preferred. ２ This is the polymer part. ２ It has a repeating unit A represented by the following formula (2) and a repeating unit B represented by the following formula (3).

[0028]

[0029] The following explains the definitions of the symbols in formulas (2) and (3). In formulas (2) and (3), * represents a bond position, not a carbon atom. The same applies to * in other formulas. Therefore, in this specification, both "*-" and "-*" represent a single bond.

[0030] In equation (3), a1 and c1 are independently 1 or 2, and b1 is 0 or 1. Here, "b1 is 0" means that (X) in equation (3) １ ) ｂ１ This means that it does not exist. From the viewpoint of the ease of obtaining raw materials, a1 and c1 are preferably both 2. Similarly, from the viewpoint of the ease of obtaining raw materials, b1 is preferably 1.

[0031] In formula (3), X １ These are ester bonds (*-CO-O-*), amide bonds (*-CO-NH-*), or thioester bonds (*-CO-S-*). １ Preferably on the carbonyl group side (CH ２ ) ａ１ It combines with it. See X for details. １ In any of the ester bonds (*-CO-O-*), amide bonds (*-CO-NH-*), or thioester bonds (*-CO-S-*), the carbonyl group (CO) side is (CH ２ ) ａ１ It bonds with X. From the viewpoint of ease of synthesis of the derivative of the present invention, １ In formula (3), Y １ This is a carboxyl group or a sulfo group, preferably a carboxyl group.

[0032] In one embodiment of the present invention, in formula (3), a1 and c1 are both 2, b1 is 1, and X １ However, it is an ester bond or an amide bond, and Y １ However, it is preferable that it be a carboxyl group or a sulfo group, a1 and c1 are both 2, b1 is 1, X １ However, it is an ester bond or an amide bond, and Y １ However, a carboxyl group is more preferable.

[0033] In another embodiment of the present invention, in formula (3), a1 and c1 are both 2, b1 is 1, and X １ However, it is an amide bond, and Y １ However, it is preferable that it be a carboxyl group or a sulfo group, a1 and c1 are both 2, b1 is 1, X １ However, it is an amide bond, and Y １ However, a carboxyl group is more preferable.

[0034] Repeating unit B is preferably a repeating unit represented by the following formula (3a), a repeating unit represented by the following formula (3b), or a repeating unit represented by the following formula (3c). In this specification, the "repeating unit represented by formula (3a)" may be abbreviated as "unit (3a)". Repeating units, groups, polymers, compounds, and intermediates represented by other formulas may also be abbreviated in the same way as the "repeating unit represented by formula (3a)". Repeating unit B is more preferably unit (3a) or unit (3b), and even more preferably unit (3a).

[0035]

[0036] R ２ The carbonyl group (CO) at the end of either repeating unit A or repeating unit B inside is L １ It is preferable that it is bonded with L. １ R that is not coupled with ２ It is preferable that the imino group (NH) at the end of either repeating unit A or repeating unit B is bonded to a hydrogen atom (H).

[0037] If there are two or more repeating units B in the polymer moiety, the two or more repeating units B may be the same or different from each other. In other words, if there are two or more repeating units B in the polymer moiety, the two or more a1 may be the same or different from each other, the two or more b1 may be the same or different from each other, the two or more c1 may be the same or different from each other, and the two or more X １ These can be the same or different, and there are two or more Y１ These repeating units B may be the same or different. When two or more repeating units B are present in the polymer moiety, from the viewpoint of ease of derivative synthesis, the two or more repeating units B are preferably the same. In other words, from the viewpoint of ease of derivative synthesis, the polymer moiety preferably has one type of repeating unit B.

[0038] The amount of repeating unit B relative to the total of repeating unit A and repeating unit B is 6 to 50 mol% from the viewpoint of blood retention and tumor accumulation of the drug delivery carrier containing the derivative of the present invention (hereinafter sometimes abbreviated as "carrier of the present invention"). If the amount is less than 6 mol%, the zeta potential of the carrier of the present invention at pH 7.4 will not be -5 mV or less, and if the amount exceeds 50 mol%, the zeta potential of the carrier of the present invention at pH 6.5 will not be 10 mV or more, making it impossible to achieve both blood retention and tumor accumulation of the carrier of the present invention. From the viewpoint of blood retention and tumor accumulation of the carrier of the present invention, the amount is preferably 6 to 40 mol%, and more preferably 6 to 35 mol%.

[0039] The number-average molecular weight of the derivative of the present invention can be appropriately adjusted by adjusting polymerization conditions, etc., so that the required performance can be achieved. The number-average molecular weight is preferably 3,000 to 50,000, more preferably 3,000 to 30,000, and even more preferably 3,000 to 25,000. When the number-average molecular weight is 3,000 or more, the hydrophilicity of the derivative of the present invention increases the blood retention rate of the carrier of the present invention, and when the average molecular weight is 50,000 or less, the carrier of the present invention is more easily taken up by cells. The number-average molecular weight of the derivative of the present invention is, for example, １ The number of repeating units can be calculated by 1H NMR measurement, and the structure can be determined from the number of repeating units obtained.

[0040] R ２ The total number of repeating units A and B within the carrier is preferably 10 to 200, more preferably 10 to 140, and even more preferably 10 to 120, from the viewpoint of blood retention and tumor accumulation of the carrier of the present invention.

[0041] R ２ The polymer portion may have repeating units different from repeating unit A or repeating unit B (hereinafter abbreviated as "other repeating units"), to the extent that it does not impair the effects of the present invention. The amount of other repeating units is preferably 5 mol% or less, and more preferably 2 mol% or less, relative to the total number of repeating units (i.e., the sum of repeating unit A, repeating unit B, and other repeating units). ２ It is even more preferable that it does not have other repeating units.

[0042] R ２ The polymer portion may be in the form of a gradient copolymer, a block copolymer, or a random copolymer. ２ Preferably, it is in the form of a random copolymer.

[0043] R １ Preferably, the lipid moiety is represented by the following formula (4).

[0044]

[0045] The following explains the definition of the symbols in equation (4). In equation (4), R ３ and R ４ Each of these is independently an aliphatic hydrocarbon group having 8 to 24 carbon atoms, an acyl group having 8 to 24 carbon atoms, or a sterol residue.

[0046] In this specification, the aliphatic hydrocarbon group may be linear, branched, or cyclic, and may be saturated or unsaturated. The aliphatic hydrocarbon group having 8 to 24 carbon atoms is preferably an alkyl group having 8 to 24 carbon atoms.

[0047] In this specification, alkyl groups may be linear or branched. Examples of alkyl groups include octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, henicosyl, docosyl, tricosyl, and tetracosyl groups.

[0048] In this specification, the acyl group may be either an aromatic acyl group or an aliphatic acyl group. In this specification, "aromatic acyl group (Ar-CO-*)" means an acyl group in which the organic group (Ar) bonded to the carbonyl group (CO) is an aromatic group, and "aliphatic acyl group (R-CO-*)" means an acyl group in which the organic group (R) bonded to the carbonyl group (CO) is an aliphatic group. The acyl group is preferably an aliphatic acyl group.

[0049] The aliphatic acyl group may be linear, branched, or cyclic. The aliphatic acyl group may also contain unsaturated bonds. Examples of aliphatic acyl groups include octanoyl, nonanoyl, decanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl, nonadecanoyl, eicosanoyl, henicosanoyl, docosanoyl, octaenoyl, nonaenoyl, decaenoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl, nonadecanoyl, eicosanoyl, henicosanoyl Examples include yl groups, docosaenoyl groups, octadienoyl groups, nonadienol groups, decadienol groups, undecadienol groups, dodecadienol groups, tridecadienol groups, tetradecadienol groups, pentadecadienol groups, hexadecadienol groups, heptadecadienol groups, octadecadienol groups, nonadecadienol groups, eicosadienoyl groups, henicosadienoyl groups, docosadienol groups, octadecatrienoyl groups, eicosatrienoyl groups, eicosatetraenoyl groups, eicosapentaenoyl groups, docosahexaenoyl groups, isostearoyl groups, tetramethylhexadecanoyl groups (phytanoyl groups), retinoyl groups, etc.

[0050] As used herein, the term "sterol residue" refers to a monovalent group having a structure obtained by removing a hydroxy group (OH) from a sterol. The "cholesterol residue" and the like described below have the same meaning as the "sterol residue". Examples of the sterol residue include a cholesterol residue, a cholestanol residue, a stigmasterol residue, a β-sitosterol residue, a lanosterol residue, and an ergosterol residue.

[0051] R ３ and R ４ are each independently preferably an alkyl group having 8 to 24 carbon atoms, an aliphatic acyl group having 8 to 24 carbon atoms, or a sterol residue, more preferably an alkyl group having 8 to 24 carbon atoms or an aliphatic acyl group having 8 to 24 carbon atoms, still more preferably an octadecyl group (C １８ H ３７ -*), a tetradecanoyl group (C １３ H ２７ -CO-*), or an octadecanoyl group (C １７ H ３５ -CO-*). When b2 is 1, R ３ and R ４ are preferably the same as each other.

[0052] In formula (4), M is a trivalent hydrocarbon group having 3 to 7 carbon atoms or a trivalent nitrogen-containing hydrocarbon group represented by *-N(CH ２ CH ２ -*)-*. Here, "*-N(CH ２ CH ２ -*)-*" is specifically represented by the following formula:

[0053]

[0054] which means the trivalent nitrogen-containing hydrocarbon group represented. M is preferably a trivalent hydrocarbon group having 3 to 7 carbon atoms, more preferably a propane-1,2,3-triyl group.

[0055] In formula (4), R ５ is a hydrogen atom, an alkali metal atom, or ammonium. R ５ is preferably a hydrogen atom.

[0056] In equation (4), a2, a3, b2, c2, and d1 are each independently either 0 or 1. Here, "a2 is 0" means that (O) in equation (4) ａ２ This means that it does not exist, and "a3 is 0" means that (O) in equation (4) ａ３ This means that it does not exist, and "b2 is 0" means that in equation (4) [M - (O) ａ３ -R ４ ] ｂ２ This means that it does not exist, and "d1 is 0" means that in equation (4) [P(=O)(OR ５ ) - OCH ２ CH ２ ] ｄ１ This means that it does not exist.

[0057] The combinations of a2, a3, b2, c2, and d1 are preferably such that a2, a3, and b2 are all 1 and c2 and d1 are both 0, or a2, a3, b2, c2, and d1 are all 1, or a2, a3, b2, c2, and d1 are all 0, more preferably a2, a3, and b2 are all 1 and c2 and d1 are both 0, or a2, a3, b2, c2, and d1 are all 1, and even more preferably a2, a3, and b2 are all 1 and c2 and d1 are both 0.

[0058] The following are some preferred combinations of symbols in formula (4): (i) R ３ and R ４ However, each is independently an alkyl group having 8 to 24 carbon atoms or an aliphatic acyl group having 8 to 24 carbon atoms, M is a trivalent hydrocarbon group having 3 to 7 carbon atoms, a2, a3, and b2 are all 1, and c2 and d1 are all 0; (ii) R ３ and R ４ However, each is independently an alkyl group having 8 to 24 carbon atoms or an aliphatic acyl group having 8 to 24 carbon atoms, M is a trivalent hydrocarbon group having 3 to 7 carbon atoms, and R ５ is a hydrogen atom and a2, a3, b2, c2, and d1 are all 1; or (iii)R ３The residue is a sterol residue, and a2, a3, b2, c2, and d1 are all 0.

[0059] A more preferred combination of symbols in formula (4) is as follows: (i) R ３ and R ４ However, each is independently an alkyl group having 8 to 24 carbon atoms or an aliphatic acyl group having 8 to 24 carbon atoms, M is a trivalent hydrocarbon group having 3 to 7 carbon atoms, a2, a3, and b2 are all 1, and c2 and d1 are all 0; or (ii)R ３ and R ４ However, each is independently an alkyl group having 8 to 24 carbon atoms or an aliphatic acyl group having 8 to 24 carbon atoms, M is a trivalent hydrocarbon group having 3 to 7 carbon atoms, and R ５ is a hydrogen atom, and a2, a3, b2, c2, and d1 are all 1.

[0060] A more preferred combination of symbols in formula (4) is as follows: (i) R ３ and R ４ However, each independently, the octadecyl group (C １８ H ３７ -*), tetradecanoyl group (C １３ H ２７ -CO-*), or octadecanoyl group (C １７ H ３５ (-CO-*) where M is a propane-1,2,3-triyl group, a2, a3, and b2 are all 1, and c2 and d1 are all 0; or (ii)R ３ and R ４ However, each independently, the octadecyl group (C １８ H ３７ -*), tetradecanoyl group (C １３ H ２７ -CO-*), or octadecanoyl group (C １７ H ３５ -CO-*) where M is a propane-1,2,3-triyl group, R ５ is a hydrogen atom, and a2, a3, b2, c2, and d1 are all 1.

[0061] The following are some particularly preferred combinations of symbols in formula (4): R ３ and R ４ However, each independently, the octadecyl group (C １８ H ３７ -*), tetradecanoyl group (C １３ H ２７ -CO-*), or octadecanoyl group (C １７ H ３５ The compound is -CO-*), where M is a propane-1,2,3-triyl group, a2, a3, and b2 are all 1, and c2 and d1 are both 0.

[0062] In one embodiment of the present invention, R １ Preferably, the group is represented by the following formula (4a), the group represented by the following formula (4b), or the group represented by the following formula (4c):

[0063]

[0064] (In the formula, C １８ H ３７ It is an octadecyl group, C １３ H ２７ is a tridecyl group, and C １７ H ３５ R is a heptadecyl group.) In one embodiment of the present invention, R １ The more preferable group is (4a).

[0065] R １ , L １ , and R ２ The following are some preferred combinations: R １ However, it is group (4a), group (4b), or group (4c), L １ NH is and R ２ However, the polymer moiety has unit (2) as repeating unit A, and unit (3a), unit (3b), or unit (3c) as repeating unit B. The explanations for the above combinations, such as the amount of repeating unit B relative to the total of repeating unit A and repeating unit B, the number-average molecular weight of the derivative of the present invention, and the amount of other units relative to the total repeating units, are as described above.

[0066] R １ , L １ , and R ２ A more preferable combination is the following: R １ However, it is group (4a), group (4b), or group (4c), L １ NH is and R ２ However, the polymer moiety has unit (2) as repeating unit A and unit (3a) or unit (3b) as repeating unit B. The explanations for the above combination, such as the amount of repeating unit B relative to the total of repeating units A and B, the number-average molecular weight of the derivative of the present invention, and the amount of other units relative to the total repeating units, are as described above.

[0067] R １ , L １ , and R ２ Even more preferred combinations include: R １ However, it is group (4a), group (4b), or group (4c), L １ NH is and R ２ However, the polymer portion has unit (2) as repeating unit A and unit (3a) as repeating unit B. The explanations for the above combination, such as the amount of repeating unit B relative to the total of repeating units A and B, the number-average molecular weight of the derivative of the present invention, and the amount of other units relative to the total repeating units, are as described above.

[0068] R １ , L １ , and R ２ Particularly preferred combinations include the following: R １ However, it is the base (4a), L １ NH is and R ２ However, the polymer portion has unit (2) as repeating unit A and unit (3a) as repeating unit B. The explanations for the above combination, such as the amount of repeating unit B relative to the total of repeating units A and B, the number-average molecular weight of the derivative of the present invention, and the amount of other units relative to the total repeating units, are as described above.

[0069] <Method for producing the derivative of the present invention> pH-responsive lipid derivatives can be produced, for example, by synthesizing a polymer having a peptide structure by "ring-opening polymerization" according to the synthesis method described in International Publication No. 2023 / 282297 and International Publication No. 2023 / 282296, followed by an "addition reaction" in which an amine compound or the like is added to the polymer side chain, and a "deprotection reaction" in which a protecting group is removed.

[0070] The derivative of the present invention in which b1 is 1 is given by formula (5): R １ -L １ -R ６ (5) (wherein, R １ This is a lipid region, L １ is NH or O, and R ６ Equation (6):

[0071]

[0072] (In the formula, a1 is 1 or 2, P １ is a protecting group for the carboxyl group, and * is the bond position. ) is a polymer moiety containing repeating units represented by ). ) and formula (7): X ２ - (CH ２ ) ｃ１ -Y ２ (7) (wherein c1 is 1 or 2, X ２ Y is a hydroxyl group, an amino group, or a sulfanyl group. ２ *-CO-OP ２ , sulfo group, or *-SO ２ -OP ３ P ２ P is a protecting group for the carboxyl group. ３ A is a protecting group for the sulfo group, and * indicates the bond position.) A compound represented by formula (8): H ２ N-CH ２ CH ２ -NH-CH ２ CH ２ -NH-CH ２ CH ２ - CO-OP ４ (8) (wherein, P ４The present invention can be produced by a manufacturing method comprising: step (A) reacting with any of the compounds represented by ) to obtain intermediate 1; step (B) reacting intermediate 1 obtained in step (A) with the remainder of the compound represented by formula (7) or formula (8) that was not used in step (A) to obtain intermediate 2 with a protected carboxyl group; and step (C) deprotecting intermediate 2 obtained in step (B) to obtain a derivative of the present invention in which b1 is 1. Steps (A) to (C) will be described in order below.

[0073] [Step (A)] Step (A) is a step in which a polymer (5) having unit (6) is reacted with either compound (7) or compound (8) to obtain intermediate 1.

[0074] In formula (5), R １ This is a lipid region, L １ is NH or O, and R ６ This is a polymer moiety having unit (6). １ and L １ The explanation is as stated above.

[0075] In formula (6), a1 is 1 or 2, and P １ is a protecting group for the carboxyl group. a1 is preferably 2. P １ The protecting group is preferably a carboxyl group having 1 to 8 carbon atoms. Examples of carboxyl group protecting groups include the methyl group, ethyl group, tert-butyl group, allyl group, and benzyl group. １ It is more preferably a benzyl group.

[0076] The number of units (6) in the polymer (5) is preferably 10 to 300, more preferably 10 to 200, and even more preferably 10 to 150, from the viewpoint of ease of synthesis.

[0077] R ６ The carbonyl group (CO) at the end of the unit (6) inside is L １ It is preferable that it is bonded with L. １ R that is not coupled with ６It is preferable that the terminal imino group (NH) of the unit (6) inside is bonded to a hydrogen atom (H).

[0078] In equation (7), c1 is 1 or 2, and X ２ c1 is preferably 2 from the viewpoint of ease of obtaining raw materials. ２ From the viewpoint of ease of obtaining raw materials, it is preferably a hydroxyl group or an amino group, and from the viewpoint of reactivity, it is more preferably an amino group.

[0079] In formula (7), Y ２ *-CO-OP ２ , sulfo group, or *-SO ２ -OP ３ P ２ is a protecting group for the carboxyl group, and P ３ P is a protecting group for sulfo groups. The explanation of protecting groups for carboxyl groups is as described above. ２ The group is preferably a tert-butyl group.

[0080] Examples of protecting groups for sulfo groups include methyl groups, ethyl groups, and 2,2,2-trifluoromethyl groups. ３ Preferably, this is a 2,2,2-trifluoromethyl group.

[0081] Y ２ Preferably *-CO-OP ２ (In the formula, P ２ (is a protecting group for the carboxyl group) or a sulfo group, more preferably *-CO-OP ２ (In the formula, P ２ (is a protecting group for the carboxyl group), and more preferably a tert-butoxycarbonyl group (i.e., P ２ *-CO-OP is a tert-butyl group ２ )

[0082] In formula (8), P ４ P is a protecting group for the carboxyl group. The explanation of the protecting group for the carboxyl group is as described above. ４ The group is preferably a tert-butyl group.

[0083] In step (A), the polymer (5) and compound (7) are preferably reacted. The ratio of compound (7) or compound (8) (preferably compound (7)) used in step (A) is determined by the *-CO-OP of the polymer (5). １ The amount is preferably 2 to 10 mol, more preferably 2 to 8 mol, and even more preferably 3 to 6 mol per 1 mol. If the amount ratio is less than 2 mol, the reaction rate will be slow, the reaction time will be long, or the reaction may stop midway. If the amount ratio is more than 10 mol, the reaction rate will be fast, making it difficult to control, and excess compound will have to be discarded.

[0084] A catalyst may be used in the reaction of step (A). Examples of catalysts include 2-hydroxypyridine, pyridine, and triethylamine. One catalyst may be used alone, or two or more catalysts may be used in combination.

[0085] When a catalyst is used in process (A), the amount ratio of the catalyst is such that the polymer (5) has *-CO-OP １ Preferably, the amount is 2 to 10 mol, more preferably 2 to 8 mol, and even more preferably 3 to 6 mol per 1 mol.

[0086] The reaction in step (A) can be carried out in a solvent. The solvent is not particularly limited as long as it does not react with the reactants (i.e., polymer (5), and compound (7) or compound (8)) and the product (i.e., intermediate 1), and examples include tetrahydrofuran, toluene, acetonitrile, chloroform, N-methyl-2-pyrrolidone, N,N-dimethylformamide, etc. Only one solvent may be used, or two or more may be used in combination. From the viewpoint of solubility of the reactants and product, the solvent is preferably at least one selected from the group consisting of N-methyl-2-pyrrolidone and tetrahydrofuran.

[0087] The ratio of solvent used in the reaction of step (A) is preferably 1.5 to 70 mL, more preferably 1.8 to 50 mL, and even more preferably 2 to 30 mL per 1 g of polymer (5).

[0088] The reaction temperature in step (A) varies depending on the reactants, solvent, and catalyst used, but is preferably 0 to 100°C. The reaction time in step (A) varies depending on the reaction temperature, etc., but is preferably 3 to 72 hours.

[0089] Intermediate 1 obtained in step (A) may be used in the next step (B) without purification, but it is preferable to purify it by treatment such as reprecipitation, gel filtration chromatography, membrane purification, or dialysis before using it in the next step (B).

[0090] When compound (7) is used in step (A), intermediate 1 obtained in step (A) contains unit (6) in addition to formula (p3):

[0091]

[0092] (The definitions of the symbols in the formula are as described above.) It has a repeating unit represented by Y. ２ *-CO-OP ２ If that is the case, then P ２ By removing it, from the unit (p3), Y １ A unit (3) is formed in which the carboxyl group is Y ２ If it is a sulfo group, the unit (p3) is Y １ This corresponds to the unit (3) which is a sulfo group. ２ *-SO ２ -OP ３ And under the conditions of process (A), P ３ If not removed, P ３ By removing it, from the unit (p3), Y １ A unit (3) is formed, which is a sulfo group.

[0093] When compound (8) is used in step (A), intermediate 1 obtained in step (A) contains unit (6) in addition to formula (p2):

[0094]

[0095] (The definitions of the symbols in the formula are as described above.) It has a repeating unit represented by . In step (C), P ４ By removing it, unit (2) is formed from unit (p2).

[0096] [Step (B)] Step (B) is a step in which intermediate 1 obtained in step (A) is reacted with the remainder of compound (7) or compound (8) that was not used in step (A) (i.e., compound (8) if compound (7) was used in step (A), or compound (7) if compound (8) was used in step (A)) to obtain intermediate 2 having a protected carboxyl group.

[0097] In step (B), intermediate 1 is preferably reacted with compound (8). The ratio of compound (7) or compound (8) (preferably compound (8)) used in step (B) is such that intermediate 1 has *-CO-OP １ 1 mol (i.e., *-CO-OP that did not react in process (A)) １ The amount is preferably 2 to 10 mol, more preferably 2 to 8 mol, and even more preferably 3 to 6 mol per 1 mol. If the amount ratio is less than 2 mol, the reaction rate will be slow, the reaction time will be long, or the reaction may stop midway. If the amount ratio is more than 10 mol, the reaction rate will be fast, making it difficult to control, and excess compound will have to be discarded.

[0098] In step (B), a catalyst can be used in the same manner as in step (A). The description of the catalyst used in step (B) is the same as the description for step (A). When a catalyst is used in step (B), the amount ratio of the catalyst is preferably 2 to 10 mol, more preferably 2 to 8 mol, and even more preferably 3 to 6 mol.

[0099] In step (B), the same solvent can be used as in step (A). The description of the solvent used in step (B) is the same as the description for step (A). The ratio of the amount of solvent used in the reaction of step (B) is 1.5 to 70 mL, preferably 1.8 to 50 mL, and most preferably 2 to 30 mL per 1 g of intermediate. The description of the reaction temperature and reaction time in step (B) is the same as the description for step (A).

[0100] The intermediate 2 obtained in step (B) may be used in the next step (C) without purification, but it is preferable to purify it by treatment such as reprecipitation, gel filtration chromatography, membrane purification, or dialysis before using it in the next step (C). In step (B), intermediate 2 having the above-mentioned units (p2) and (p3) is obtained.

[0101] [Step (C)] The intermediate 2 obtained in step (B) has a protected carboxyl group in the unit (p2) (i.e., *-CO-OP ４ ) has. Y ２ The protected carboxyl group (i.e., *-CO-OP ２ If this is the case, then intermediate 2 has *-CO-OP in its unit (p3). ２ It has Y. ２ The protected sulfo group (i.e., *-SO ２ -OP ３ ) and if the sulfo group protected under the conditions of step (A) or step (B) is not deprotected, intermediate 2 has *-SO in unit (p3). ２ -OP ３ It has. Step (C) is to deprotect intermediate 2 (for details, see P ４ Remove or P ２ Or P ３ If P exists, ４ And, P ２ Or P ３ This is a step to obtain the derivative of the present invention in which b1 is 1, by removing (and).

[0102] Deprotection is well known to those skilled in the art, and deprotection can be carried out under known conditions depending on the protecting group. For example, P ２ or P ４ However, if it is a tert-butyl group, hydrolysis under acidic conditions will result in P ２ or P ４ The P group is removed, and a carboxyl group is formed. ３ However, if it is a 2,2,2-trifluoromethyl group, hydrolysis under basic conditions will result in P ３ The sulfo group is removed and a sulfo group is formed.

[0103] [Method for producing polymer (5) used in step (A)] The polymer (5) used in step (A) is, for example, formula (9): R １ -L ２ (9) (wherein, R １ is a lipid site, and L ２ The following compounds are used as initiators, where (10) is an amino group or a hydroxyl group:

[0104]

[0105] (wherein a1 is 1 or 2, and P １ (It is a protecting group for the carboxyl group.) It can be produced by ring-opening polymerization using the compound as a monomer.

[0106] R in equation (9) １ The explanation of the (lipid portion) is as described above. L in formula (9) ２ a1 and P in formula (10) are preferably amino groups. １ The explanation of the (carboxyl group protecting group) is as described above. From the viewpoint of use in pharmaceutical applications, compound (10) is preferably an acid anhydride derived from a naturally occurring L-amino acid.

[0107] Examples of solvents used in ring-opening polymerization include tetrahydrofuran, acetonitrile, N-methyl-2-pyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dichloromethane, and chloroform. Only one solvent may be used, or two or more may be used in combination. Preferably, the solvent is at least one selected from the group consisting of N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, dichloromethane, and chloroform.

[0108] The ratio of solvent used in ring-opening polymerization is preferably 3 to 100 mL, more preferably 5 to 50 mL, and even more preferably 7 to 25 mL per 1 g of compound (10). When using a mixed solvent, the ratio of each solvent is not particularly limited.

[0109] The ring-opening polymerization temperature is preferably 20 to 60°C, and more preferably 25 to 40°C. The ring-opening polymerization time varies depending on the polymerization temperature and other factors, but is preferably 3 to 24 hours.

[0110] The obtained polymer (5) may be used in step (A) without purification, but it is preferable to purify it by treatment such as reprecipitation, gel filtration chromatography, membrane purification, or dialysis before using it in step (A).

[0111] <Drug Delivery Carrier> The present invention provides a drug delivery carrier containing the derivative of the present invention.

[0112] The form of the carrier of the present invention or the form of the dispersion containing the carrier of the present invention is not particularly limited, but examples include single-layer liposomes, multilayer liposomes, O / W emulsions, W / O / W emulsions, spherical micelles, solid lipid nanoparticles, amorphous layered structures, etc. The carrier of the present invention is preferably solid lipid nanoparticles.

[0113] The carrier of the present invention (particularly solid LNP) has a structure in which the hydrophilic portions of the derivative of the present invention are arranged toward the aqueous phase side of the interface. Among the derivatives of the present invention, R １ The (lipid portion) exhibits hydrophobicity, R ２ Because the (polymer portion) exhibits hydrophilicity, R is formed on the surface of the carrier of the present invention. ２ This is exposed. The carrier of the present invention, like the derivative of the present invention, is pH responsive (i.e., under neutral conditions (pH 7.4), its zeta potential becomes -5 mV or less (weakly anionic), and under weakly acidic conditions around tumor tissue (pH 6.5), its zeta potential becomes 10 mV or more (cationic)).

[0114] The carrier of the present invention (particularly the solid LNP) may contain general lipids in addition to the derivative of the present invention. Examples of such lipids include ionic lipids, sterols, phospholipids, neutral lipids, saturated or unsaturated fatty acids, etc. The carrier of the present invention (particularly the solid LNP) preferably contains at least one selected from the group consisting of phospholipids, ionic lipids, and sterols in addition to the derivative of the present invention.

[0115] Examples of phospholipids include phosphatidylcholine, glycerophospholipids, sphingophospholipids, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. More specifically, examples include dioleylphosphatidylethanolamine (abbreviated as DOPE) and dioleylphosphatidic acid (abbreviated as DOPA). Among these, DOPE is preferred.

[0116] Examples of ionic lipids include dioctadecyldimethylammonium chloride (DODAC), N-[2,3-bis(oleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), didodecyldimethylammonium bromide (DDAB), 1,2-dioleoyloxy-3-trimethylammonium propane (DOTAP), and 3β-N-(N',N'-dimethylaminoethane)carbamoyl cholesterol. Cholesterol (abbreviated as DC-Chol), 1,2-dimyristoyloxypropyl-3-dimethylhydroxyethyl ammonium bromide (abbreviated as DMRIE), 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminum trifluoroacetate (abbreviated as DOSPA), 1,2-distearoyl-3-trimethylammonium propane (abbreviated as DOSPA) propane (abbreviation: DSTAP), dioleoyl-3-dimethylammonium-propane (abbreviation: DODAP), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate ((6Z,9Z,28Z,Examples include (31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (abbreviated as D-Lin-MC3-DMA). Among these, DODAP and D-Lin-MC3-DMA are preferred, with D-Lin-MC3-DMA being more preferred.

[0117] Examples of sterols include animal-derived sterols such as cholesterol, cholesterol succinate, lanosterol, dihydrosterol, desmosterol, and dihydrocholesterol; plant-derived sterols (phytosterols) such as stigmasterol, sitosterol, campesterol, and brassicasterol; and microbial-derived sterols such as thymosterol and ergosterol. Among these, cholesterol is preferred.

[0118] The content of the derivative of the present invention in the carrier of the present invention (particularly solid LNP) is preferably 0.1 to 30 mol%, and more preferably 0.1 to 10 mol%, relative to the total amount of the derivative of the present invention and the lipids contained in the carrier of the present invention as needed (hereinafter sometimes abbreviated as "total lipids"). If the content is less than 0.1 mol%, the proportion of the pH-responsive portion on the surface of the carrier of the present invention (particularly solid LNP) becomes small, and it may not be able to acquire sufficient cationic properties for accumulation or uptake by tumor tissue even under weakly acidic conditions. If the content is more than 30 mol%, it may become difficult to manufacture a stable carrier (particularly solid LNP).

[0119] In order to stably produce the carrier of the present invention (particularly solid LNP), the phospholipid content in the carrier of the present invention (particularly solid LNP) is preferably 5 to 60 mol%, more preferably 5 to 30 mol%, the ionic lipid content is preferably 5 to 60 mol%, more preferably 20 to 60 mol%, and the sterol content is preferably 5 to 60 mol%, more preferably 10 to 50 mol%. Note that the above content criteria all refer to the total lipids in the carrier of the present invention.

[0120] The method for producing the carrier of the present invention (especially solid LNPs) is not particularly limited, and the carrier of the present invention can be produced by known methods. For example, when the carrier of the present invention is a solid LNP, the solid LNP can be produced by an alcohol dilution method using a microfluidic channel, a hydration method, etc. Specifically, in the hydration method, the derivative of the present invention and all lipid components are dissolved in an organic solvent such as chloroform, and a dried mixture is formed by drying under reduced pressure using an evaporator or by spray drying using a spray dryer. After forming a lipid film in the dried mixture, an aqueous dispersion medium is added to the dried mixture to form solid LNPs in water. The carrier of the present invention can also be produced by a method that is well known for producing liposomes, such as the reverse-phase atmospheric pressure method. If it is desired to control the size of the carrier of the present invention (especially solid LNPs), extrusion filtration can be performed under high pressure using a membrane filter with uniform pore sizes.

[0121] When the carrier of the present invention (particularly solid LNP) is used in the form of a dispersion, the type of aqueous dispersion medium is not particularly limited and examples include buffers such as phosphate buffer, citrate buffer, and phosphate-buffered saline, physiological saline, and cell culture medium. These aqueous dispersion media can stably disperse the carrier of the present invention (particularly solid LNP), but the dispersion containing the carrier of the present invention may also contain aqueous solutions of sugars such as glucose, lactose, and sucrose, or polyhydric alcohols such as glycerin and propylene glycol, in addition to the aqueous dispersion medium. To stably store the dispersion containing the carrier of the present invention for a long period of time, it is desirable to reduce the electrolyte in the dispersion as much as possible from the viewpoint of physical stability such as aggregation, and it is also desirable to remove dissolved oxygen by nitrogen bubbling. Furthermore, for example, when freezing and storing the dispersion containing the carrier of the present invention, adding an aqueous solution of sugar or an aqueous solution of polyhydric alcohol to the dispersion enables long-term storage. The concentration of the buffering agent in the buffer is preferably 5 to 50 mM, more preferably 10 to 20 mM. The concentration of sugar in the aqueous sugar solution is preferably 2 to 20 w / v%, more preferably 5 to 10 w / v%. The concentration of polyhydric alcohol in the aqueous polyhydric alcohol solution is preferably 1 to 5 w / v%, more preferably 2 to 2.5 w / v%. The amount of the carrier of the present invention in the dispersion is not particularly limited, but the total concentration of the lipid derivative and lipid of the present invention in the dispersion is preferably 0.1 to 500 mM, more preferably 1 to 100 mM.

[0122] Methods for drying a dispersion containing the carrier of the present invention (particularly solid LNP) include, for example, freeze-drying and spray-drying. After producing a dispersion containing the carrier of the present invention (particularly solid LNP), drying it enables long-term storage of the carrier of the present invention. Furthermore, by adding an aqueous drug solution to the dried carrier of the present invention, the drug can be efficiently retained by the carrier of the present invention (particularly solid LNP).

[0123] The average particle diameter of the carrier of the present invention (particularly solid LNP) is preferably 10 to 250 nm, more preferably 20 to 200 nm. Furthermore, the polydispersity index (PDI) of the carrier of the present invention (particularly solid LNP) is preferably 0.5 or less, more preferably 0.45 or less, and even more preferably 0.4 or less. Note that the average particle diameter of the carrier of the present invention (particularly solid LNP) refers to the number-average particle diameter measured by dynamic light scattering (DLS). Measurement by dynamic light scattering can be performed using commercially available DLS equipment.

[0124] The carrier of the present invention (particularly the solid LNP) exhibits weak anionic properties (zeta potential: -5 mV or less) in a neutral environment of pH 7.4, thus exhibiting stealth properties towards blood components or normal tissues. In the vicinity of tumor tissue, it becomes cationic (zeta potential: 10 mV or more) in response to minute pH changes (pH 6.5). Since the cell surface has a negative charge, an increase in cationicity can be expected to improve the accumulation efficiency and uptake efficiency in tumor tissue. As a result, drugs encapsulated in the carrier of the present invention can be efficiently introduced into the vicinity of tumor tissue or into tumor cells.

[0125] The carrier of the present invention (particularly solid LNP) is particularly useful for transporting drugs to tumor tissue. The drugs transported by the carrier of the present invention (particularly solid LNP) are not particularly limited as long as they are transportable. Examples of drugs include nucleic acids, nucleic acid derivatives, peptides, small molecule drugs, anticancer drugs, sugars, metal compounds, and the like.

[0126] Examples of nucleic acids include siRNA, miRNA, shRNA, antisense RNA, messenger RNA (mRNA), single-stranded RNA genomes, double-stranded RNA genomes, RNA replicons, transfer RNA, and ribosomal RNA. Examples of nucleic acid derivatives include nucleic acid aptamers.

[0127] The drug transported by the carrier of the present invention (particularly solid LNP) is preferably at least one selected from the group consisting of nucleic acids or their derivatives, peptides, small molecule drugs, and anticancer agents, more preferably at least one selected from the group consisting of nucleic acids, peptides, small molecule drugs, and anticancer agents, and even more preferably nucleic acids.

[0128] The present invention will be described in detail below based on examples, but the present invention is not limited to the following examples.

[0129] <Example 1 and Comparative Example 1: Synthesis of pH-responsive lipid derivatives> [Example 1-1: Synthesis of pH-responsive lipid derivative A] [Synthesis of intermediate (I-1) (In the following formula (I-1), C １８ H ３７ (This is an octadecyl group, and m1 is 30.)

[0130]

[0131] 2,3-bis(octadecyloxy)-1-propylamine (hereinafter referred to as "DSGE-A") (100.3 mg, 0.168 mmol) was added to a 100 mL round-bottom flask and dissolved with anhydrous dichloromethane (7.3 mL) to obtain a solution of DSGE-A. γ-benzyl-L-glutamic acid NCA (CAS registration number: 26845-84-1, hereinafter referred to as "BLG-NCA") (1.43 g, 5.44 minol) was weighed under nitrogen and dissolved in anhydrous N,N-dimethylformamide (7.3 mL). The solution of DSGE-A was added to the resulting solution and the reaction was carried out at 30°C under a nitrogen flow. Three hours after the start of the reaction... １¹H NMR measurement confirmed a conversion rate of 99.7%, and the reaction was stopped. Dichloromethane was removed by distillation using an evaporator under reduced pressure at 30°C for 1 hour. Methanol (10 mL) and deionized water (20 mL) were added to the reaction solution while stirring to produce a white solid. The white solid was collected using a 0.5 μm polytetrafluoroethylene (hereinafter referred to as "PTFE") filter, and the obtained solid was vacuum-dried for 3 hours. Tetrahydrofuran (7 g, 5 times the weight of the monomer) was added to this solid and dissolved. Ethanol (70 g, 50 times the weight of the monomer) was added to this solution while stirring to precipitate a white solid, and the solution was aged for 30 minutes. After collecting the solid using a 1.0 μm PTFE filter, the obtained solid was vacuum-dried overnight to obtain the intermediate represented by the above formula (I-1) in a yield of 0.97 g. １ H NMR (deuterochloroform, 400MHz): δ 7.00 to 7.40 (m, average degree of polymerization x 5H), 4.80 to 5.30 (m, average degree of polymerization x 2H), 3.80 to 4.40 (m, average degree of polymerization x 1H), 3 .10-3.75 (m, 9H) 1.40-2.90 (m, average degree of polymerization x 4H), 1.15-1.32 (m, 64H), 0.84-0.91 (m, 6H)

[0132] The average degree of polymerization (i.e., m1 in formula (I-1)) of the obtained intermediate (I-1) was calculated using the method described in (Calculation of Average Degree of Polymerization 1) below, and it was found that m1 in formula (I-1) averaged 30. Furthermore, the number-average molecular weight of intermediate (I-1) was calculated to be 7,200 from the above average degree of polymerization. The number-average molecular weight was calculated by rounding to the tens place. The same method was used for the following number-average molecular weights.

[0133] (Calculation of average degree of polymerization 1) Derived from initiator (DSGE-A) １ The peaks (0.84–0.91) of the 1H NMR spectrum are used as the reference (6H), and the hydrogen atom at the α-position relative to the carbonyl group of the amide bond in the polymer backbone (polypeptide) is... １ From the integral values ​​of the peaks (3.80–4.40) of the 1H NMR spectrum, we confirmed that m1 in equation (I-1) averages 30.

[0134] [pH-responsive lipid derivative A (R in formula (1)) １ : Base (4a) (C in formula (4a) １８ H ３７ (The group is an octadecyl group, the same applies below), L in formula (1) １ [Composition of: *-NH-*, Repeating unit A: Unit (2), Number of repeating units A (average): 27, Repeating unit B: Unit (3a), Number of repeating units B (average): 3]

[0135]

[0136] Intermediate (I-1) (300 mg) was dissolved in tetrahydrofuran (1.5 mL). 2-hydroxypyridine (360 mg) and β-alanine tert-butyl ester (hereinafter referred to as "β-alanine tBu") (494 mg) were added and the reaction was carried out at 50°C. １ After confirming by 1H NMR that an average of three benzyloxy groups in intermediate (I-1) were replaced with β-alanine tBu, dialysis was performed using tetrahydrofuran. Three repeating units B are then formed from the three repeating units in which the benzyloxy groups were replaced with β-alanine tBu through a deprotection reaction described later.

[0137] The dialysis solution was concentrated under reduced pressure, and the resulting concentrate was dissolved in tetrahydrofuran (1.5 mL). Then, 2-hydroxypyridine (360 mg) and N-[2-[(2-aminoethyl)amino]ethyl]-β-alanine 1,1-dimethylethyl ester (hereinafter referred to as "DET-CartBu") (1.314 g) were added, and the reaction was carried out at 50°C for 20 hours to replace the remaining 27 benzyloxy groups in intermediate (I-1) with DET-CartBu. From the 27 repeating units in which the benzyloxy groups were replaced with DET-CartBu, 27 repeating units A were formed by a deprotection reaction described later.

[0138] The yellow transparent solution obtained after the above reaction was concentrated under reduced pressure at 50°C for 2 hours. Then, 6M hydrochloric acid was added to the resulting concentrate to adjust the pH to 1.0. This reaction mixture was heated at 50°C for 15 hours to carry out a deprotection reaction, after which a deprotection rate of 99.9% or higher was confirmed. The pH was adjusted to 3.4-3.6 using a 5M sodium hydroxide aqueous solution, and pH-responsive lipid derivative A was obtained in yield of 0.39 g by dialysis using water and freeze-drying. １ H NMR (bihydrochloric acid + heavy water, 400MHz): δ 4.30 to 4.50 (m, average degree of polymerization x 1H), 3.20 to 3.80 (m, average degree of polymerization x 9.2H), 2.85 to 3.00 (t, average degree of polymerization x 1.8H), 2.35 -2.70 (m, average degree of polymerization x 2.2H), 1.85 - 2.25 (m, average degree of polymerization x 2H), 1.10 - 1.30 (m, 64H), 0.84 - 0.91 (m, 6H).

[0139] The average degree of polymerization and number-average molecular weight of the obtained pH-responsive lipid derivative A were calculated using the method described in (Calculation of Average Degree of Polymerization 2) below. The results showed that the average degree of polymerization was 30 and the number-average molecular weight was 9,000.

[0140] (Calculation of average degree of polymerization 2) Derived from the initiator １ The peaks (0.75–0.85) of the 1H NMR spectrum are used as the reference (6H), and the hydrogen atom at the α-position relative to the carbonyl group of the amide bond in the polymer backbone (polypeptide) is... １ The average degree of polymerization was calculated from the integral value of the peak (4.30–4.50) of the 1H NMR spectrum, and it was confirmed that the average degree of polymerization was 30.

[0141] The number of each repeating unit was calculated using the method described below (Calculation of the number of each repeating unit), and the R of pH-responsive lipid derivative A was found to be ２ It was found that the molecule was in the form of a random copolymer having, on average, 27 repeating units A and 3 repeating units B.

[0142] (Calculation of the number of each repeating unit) The hydrogen atom at the α position relative to the carnibonyl group of the amide bond in the polymer backbone (polypeptide) １Using the peaks of the 1H NMR spectrum (2.25–1.80 ppm) as a reference, the hydrogen atom at the α-position relative to the carboxyl group of repeating unit A １ Based on the integral ratio of the peaks (3.00–2.82 ppm) in the 1H NMR spectrum, the number of repeating units A was calculated to be 27. As a result, the number of repeating units B is the remaining 3.

[0143] [Examples 1-2: pH-responsive lipid derivative B (R in formula (1)) １ : Base (4a), L in formula (1) １ [Composition of: *-NH-*, Repeating unit A: Unit (2), Number of repeating units A (average): 24, Repeating unit B: Unit (3a), Number of repeating units B (average): 6]

[0144]

[0145] To the intermediate (I-1) (300 mg) obtained in the same manner as in Example 1-1, tetrahydrofuran (1.5 mL) was added and dissolved. 2-hydroxypyridine (360 mg) and β-alanine tBu (988 mg) were added and the reaction was carried out at 50°C. １ After confirming by 1H NMR that an average of six benzyloxy groups in intermediate (I-1) were replaced with β-alanine tBu, dialysis was performed using tetrahydrofuran. From the six repeating units in which the benzyloxy groups were replaced with β-alanine tBu, six repeating units B were formed by a deprotection reaction described later.

[0146] The dialysis solution was concentrated under reduced pressure, and the resulting concentrate was dissolved in tetrahydrofuran (1.5 mL). Then, 2-hydroxypyridine (360 mg) and DET-CartBu (1.314 g) were added, and the reaction was carried out at 50°C for 20 hours to replace the remaining 24 benzyloxy groups in intermediate (I-1) with DET-CartBu. From the 24 repeating units in which the benzyloxy groups were replaced with DET-CartBu, 24 repeating units A were formed by a deprotection reaction described later.

[0147] The yellow transparent solution obtained after the above reaction was concentrated under reduced pressure at 50°C for 2 hours. Then, 6M hydrochloric acid was added to the resulting concentrate to adjust the pH to 1.0. This reaction mixture was heated at 50°C for 15 hours to carry out a deprotection reaction, after which a deprotection rate of 99.9% or higher was confirmed. The pH was adjusted to 3.4-3.6 using a 5M sodium hydroxide aqueous solution, and pH-responsive lipid derivative B was obtained in yield of 0.40 g by dialysis using water and freeze-drying. １ H NMR (bihydrochloric acid + heavy water, 400MHz): δ 4.30 to 4.50 (m, average degree of polymerization x 1H), 3.20 to 3.80 (m, average degree of polymerization x 8.4H), 2.85 to 3.00 (t, average degree of polymerization x 1.6H), 2.35 to 2.70 (m, average degree of polymerization x 2.4H), 1.85 to 2.25 (m, d, average degree of polymerization x 2H), 1.10 to 1.30 (m, 64H), 0.84 to 0.91 (m, 6H).

[0148] The average degree of polymerization and number-average molecular weight of the obtained pH-responsive lipid derivative B were calculated using the method described in (Calculation of Average Degree of Polymerization 2), and it was found that the average degree of polymerization was 30 and the number-average molecular weight was 8,700. In addition, the number of each repeating unit was calculated using the method described in (Calculation of Number of Each Repeating Unit), and it was found that the R of pH-responsive lipid derivative B ２ It was found that the molecule was in the form of a random copolymer having, on average, 24 repeating units A and 6 repeating units B.

[0149] [Examples 1-3: pH-responsive lipid derivative C (R in formula (1)) １ : Base (4a), L in formula (1) １ [Composition of: *-NH-*, Repeating unit A: Unit (2), Number of repeating units A (average): 24, Repeating unit B: Unit (3b), Number of repeating units B (average): 6]

[0150]

[0151] To the intermediate (I-1) (300 mg) obtained in the same manner as in Example 1-1, tetrahydrofuran (1.5 mL) was added and dissolved. 2-hydroxypyridine (360 mg) and 2-aminoethanesulfonic acid (988 mg) were added and the reaction was carried out at 50°C. １After confirming by 1H NMR that an average of six benzyloxy groups in intermediate (I-1) were replaced with 2-aminoethanesulfonic acid, dialysis was performed using tetrahydrofuran. The six repeating units in which the benzyloxy groups were replaced with 2-aminoethanesulfonic acid correspond to repeating unit B.

[0152] The dialysis solution was concentrated under reduced pressure, and the resulting concentrate was dissolved in tetrahydrofuran (1.5 mL). Then, 2-hydroxypyridine (360 mg) and DET-CartBu (1.314 g) were added, and the reaction was carried out at 50°C for 20 hours to replace the remaining 24 benzyloxy groups in intermediate (I-1) with DET-CartBu. From the 24 repeating units in which the benzyloxy groups were replaced with DET-CartBu, 24 repeating units A were formed by a deprotection reaction described later.

[0153] The yellow transparent solution obtained after the above reaction was concentrated under reduced pressure at 50°C for 2 hours. Then, 6M hydrochloric acid was added to the resulting concentrate to adjust the pH to 1.0. After reacting this reaction mixture at 50°C for 15 hours, a deprotection rate of 99.9% or higher was confirmed. The pH was adjusted to 3.4-3.6 using a 5M sodium hydroxide aqueous solution, and by dialysis using water and freeze-drying, a pH-responsive lipid derivative C was obtained in a yield of 0.41 g. １ H NMR (bihydrochloric acid + heavy water, 400MHz): δ 4.30 to 4.50 (m, average degree of polymerization x 1H), 3.20 to 3.80 (m, average degree of polymerization x 8.8H), 2.85 to 3.00 (t, average degree of polymerization x 1.6H), 2.35 -2.70 (m, average degree of polymerization x 2.0H), 1.85 - 2.25 (m, average degree of polymerization x 2H), 1.10 - 1.30 (m, 64H), 0.84 - 0.91 (m, 6H).

[0154] The average degree of polymerization and number-average molecular weight of the obtained pH-responsive lipid derivative C were calculated using the method described in (Calculation of Average Degree of Polymerization 2), and it was found that the average degree of polymerization was 30 and the number-average molecular weight was 9,000. In addition, the number of each repeating unit was calculated using the method described in (Calculation of Number of Each Repeating Unit), and the R ２It was found that the molecule was in the form of a random copolymer having, on average, 24 repeating units A and 6 repeating units B.

[0155] [Example 1-4: Synthesis of pH-responsive lipid derivative (D)] [Synthesis of intermediate (I-2) (in formula (I-2) below, C １８ H ３７ (This is an octadecyl group, and m2 is 15.)

[0156]

[0157] Except for changing the amount of BLG-NCA used from 1.43 g to 0.72 g, the procedure was the same as that for the synthesis of intermediate (I-1), and the resulting solid was vacuum-dried overnight to obtain the intermediate represented by the above formula (I-2) in a yield of 0.49 g. １ H NMR (deuterochloroform, 400MHz): δ 7.00 to 7.40 (m, average degree of polymerization x 5H), 4.80 to 5.30 (m, average degree of polymerization x 2H), 3.80 to 4.40 (m, average degree of polymerization x 1H), 3. 10-3.75 (m, 9H) 1.40-2.90 (m, average degree of polymerization x 4H), 1.15-1.32 (m, 64H), 0.84-0.91 (m, 6H).

[0158] The average degree of polymerization (i.e., m² in formula (I-2)) and number-average molecular weight of the obtained intermediate (I-2) were calculated using the method described in (Calculation of Average Degree of Polymerization 1). As a result, it was found that m² in formula (I-2) averaged 15, and the number-average molecular weight of intermediate (I-2) was 3,900.

[0159] [pH-responsive lipid derivative D (R in formula (1))] １ : Base (4a), L in formula (1) １ [Composition of: *-NH-*, Repeating unit A: Unit (2), Number of repeating units A (average): 13, Repeating unit B: Unit (3c), Number of repeating units B (average): 2]

[0160]

[0161] Intermediate (I-2) (300 mg) was dissolved in tetrahydrofuran (1.5 mL). 2-hydroxypyridine (360 mg) and tert-butyl 3-hydroxypropionate (988 mg) were added, and the reaction was carried out at 50°C. １ After confirming by 1H NMR that an average of two benzyloxy groups in intermediate (I-2) were replaced with tert-butyl 3-hydroxypropionate, dialysis was performed using tetrahydrofuran. Two repeating units B are formed from the two repeating units in which the benzyloxy groups were replaced with tert-butyl 3-hydroxypropionate through a deprotection reaction described later.

[0162] The dialysis solution was concentrated under reduced pressure, and the resulting concentrate was dissolved in tetrahydrofuran (1.5 mL). Then, 2-hydroxypyridine (360 mg) and DET-CartBu (1.314 g) were added, and the reaction was carried out at 50°C for 20 hours to replace the remaining 13 benzyloxy groups in intermediate (I-2) with DET-CartBu. From the 13 repeating units in which the benzyloxy groups were replaced with DET-CartBu, 13 repeating units A were formed by a deprotection reaction described later.

[0163] The yellow transparent solution obtained after the above reaction was concentrated under reduced pressure at 50°C for 2 hours. Then, 6M hydrochloric acid was added to the resulting concentrate to adjust the pH to 1.0. This reaction mixture was heated at 50°C for 15 hours to carry out a deprotection reaction, after which a deprotection rate of 99.9% or higher was confirmed. The pH was adjusted to 3.4-3.6 using a 5M sodium hydroxide aqueous solution, and pH-responsive lipid derivative D was obtained in yield of 0.40 g by dialysis using water and freeze-drying. １ H NMR (bihydrochloric acid + heavy water, 400MHz): δ 4.30 to 4.50 (m, average degree of polymerization x 1H), 3.20 to 3.80 (m, average degree of polymerization x 9.0H), 2.85 to 3.00 (t, average degree of polymerization x 1.8H), 2.35 to 2.70 (m, average degree of polymerization x 2.4H), 1.85 to 2.25 (m, d, average degree of polymerization x 2H), 1.10 to 1.30 (m, 64H), 0.84 to 0.91 (m, 6H).

[0164] The average degree of polymerization and number-average molecular weight of the obtained pH-responsive lipid derivative D were calculated using the method described in (Calculation of Average Degree of Polymerization 2), and it was found that the average degree of polymerization was 15 and the number-average molecular weight was 4,700. In addition, the number of each repeating unit was calculated using the method described in (Calculation of Number of Each Repeating Unit), and the R ２ It was found that the molecule was in the form of a random copolymer having, on average, 13 repeating units A and 2 repeating units B.

[0165] [Examples 1-5: Synthesis of pH-responsive lipid derivative E] [Synthesis of intermediate (I-3) (In formula (I-3) below, C １８ H ３７ (This is an octadecyl group, and m3 is 100.)

[0166]

[0167] Except for changing the amount of BLG-NCA used from 1.43 g to 4.2 g, the procedure was the same as that for the synthesis of intermediate (I-1), and the resulting solid was vacuum-dried overnight to obtain the intermediate represented by the above formula (I-3) in a yield of 2.8 g. １ H NMR (deuterochloroform, 400MHz): δ 7.00 to 7.40 (m, average degree of polymerization x 5H), 4.80 to 5.30 (m, average degree of polymerization x 2H), 3.80 to 4.40 (m, average degree of polymerization x 1H), 3 .10-3.75 (m, 9H) 1.40-2.90 (m, average degree of polymerization x 4H), 1.15-1.32 (m, 64H), 0.84-0.91 (m, 6H)

[0168] The average degree of polymerization (i.e., m3 in formula (I-3)) and the number-average molecular weight of the obtained intermediate (I-3) were calculated using the method described in (Calculation of Average Degree of Polymerization 1). As a result, it was found that m3 in formula (I-3) averaged 100, and the number-average molecular weight of the intermediate (I-3) calculated from this was 22,600.

[0169] [pH-responsive lipid derivative E (R in formula (1)) １ : Base (4a), L in formula (1) １[Composition of: *-NH-*, Repeating unit A: Unit (2), Number of repeating units A (average): 90, Repeating unit B: Unit (3a), Number of repeating units B (average): 10]

[0170]

[0171] Intermediate (I-3) (300 mg) was dissolved in tetrahydrofuran (1.5 mL). 2-hydroxypyridine (360 mg) and β-alanine tert-butyl ester (hereinafter referred to as "β-alanine tBu") (494 mg) were added and the reaction was carried out at 50°C. １ After confirming by 1H NMR that an average of 10 benzyloxy groups in intermediate (I-3) were replaced with β-alanine tBu, dialysis was performed using tetrahydrofuran. Furthermore, 10 repeating units B are formed from these 10 repeating units, where the benzyloxy groups were replaced with β-alanine tBu, through a deprotection reaction described later.

[0172] The dialysis solution was concentrated under reduced pressure, and the resulting concentrate was dissolved in tetrahydrofuran (1.5 mL). Then, 2-hydroxypyridine (360 mg) and DET-CartBu (1.314 g) were added, and the reaction was carried out at 50°C for 20 hours to replace the remaining 90 benzyloxy groups in intermediate (I-3) with DET-CartBu. From the 90 repeating units in which the benzyloxy groups were replaced with DET-CartBu, 90 repeating units A were formed by a deprotection reaction described later.

[0173] The yellow transparent solution obtained after the above reaction was concentrated under reduced pressure at 50°C for 2 hours. 6M hydrochloric acid was then added to the resulting concentrate to adjust the pH to 1.0. This reaction mixture was heated at 50°C for 15 hours to carry out a deprotection reaction, after which a deprotection rate of 99.9% or higher was confirmed. The pH was adjusted to 3.4-3.6 using a 5M sodium hydroxide aqueous solution, and pH-responsive lipid derivative E was obtained in yield of 0.39 g by dialysis using water and freeze-drying. １H NMR (bihydrochloric acid + heavy water, 400MHz): δ 4.30 to 4.50 (m, average degree of polymerization x 1H), 3.20 to 3.80 (m, average degree of polymerization x 8.4H), 2.85 to 3.00 (t, average degree of polymerization x 1.6H), 2.35 to 2.70 (m, average degree of polymerization x 2.4H), 1.85 to 2.25 (m, d, average degree of polymerization x 2H), 1.10 to 1.30 (m, 64H), 0.84 to 0.91 (m, 6H).

[0174] The average degree of polymerization and number-average molecular weight of the obtained pH-responsive lipid derivative E were calculated using the method described in (Calculation of Average Degree of Polymerization 2). The results showed that the average degree of polymerization was 100 and the number-average molecular weight was 28,500. Furthermore, the number of each repeating unit was calculated using the method described in (Calculation of Number of Each Repeating Unit). The results showed that the R of pH-responsive lipid derivative E ２ It was found that the molecule was in the form of a random copolymer having, on average, 90 repeating units A and 10 repeating units B.

[0175] [Examples 1-6: Synthesis of pH-responsive lipid derivative F] [Synthesis of intermediate (I-4) (in formula (I-4) below, C １８ H ３７ (This is an octadecyl group, and m4 is 150.)

[0176]

[0177] Except for changing the amount of BLG-NCA used from 1.43 g to 6.3 g, the procedure was the same as that for the synthesis of intermediate (I-1), and the resulting solid was vacuum-dried overnight to obtain the intermediate represented by the above formula (I-4) in a yield of 4.2 g. １ H NMR (deuterochloroform, 400MHz): δ 7.00 to 7.40 (m, average degree of polymerization x 5H), 4.80 to 5.30 (m, average degree of polymerization x 2H), 3.80 to 4.40 (m, average degree of polymerization x 1H), 3 .10-3.75 (m, 9H) 1.40-2.90 (m, average degree of polymerization x 4H), 1.15-1.32 (m, 64H), 0.84-0.91 (m, 6H)

[0178] The average degree of polymerization (i.e., m4 in formula (I-4)) and number-average molecular weight of the obtained intermediate (I-4) were calculated using the method described in (Calculation of Average Degree of Polymerization 1). As a result, it was found that m4 in formula (I-4) averaged 150, and the number-average molecular weight of intermediate (I-4) was 33,600.

[0179] [pH-responsive lipid derivative F (R in formula (1))] １ : Base (4a), L in formula (1) １ [Composition of: *-NH-*, Repeating unit A: Unit (2), Number of repeating units A (average): 120, Repeating unit B: Unit (3a), Number of repeating units B (average): 30]

[0180]

[0181] To the intermediate (I-4) (300 mg) obtained in the same manner as in Example 1-1, tetrahydrofuran (1.5 mL) was added and dissolved. 2-hydroxypyridine (360 mg) and β-alanine tBu (988 mg) were added and the reaction was carried out at 50°C. １ After confirming by 1H NMR that an average of 30 benzyloxy groups in intermediate (I-4) were replaced with β-alanine tBu, dialysis was performed using tetrahydrofuran. Furthermore, 30 repeating units B are formed from these 30 repeating units, where the benzyloxy groups were replaced with β-alanine tBu, through a deprotection reaction described later.

[0182] The dialysis solution was concentrated under reduced pressure, and the resulting concentrate was dissolved in tetrahydrofuran (1.5 mL). Then, 2-hydroxypyridine (360 mg) and DET-CartBu (1.314 g) were added, and the reaction was carried out at 50°C for 20 hours to replace the remaining 120 benzyloxy groups in intermediate (I-4) with DET-CartBu. From these 120 repeating units in which the benzyloxy groups were replaced with DET-CartBu, 120 repeating units A were formed by a deprotection reaction described later.

[0183] The yellow transparent solution obtained after the above reaction was concentrated under reduced pressure at 50°C for 2 hours. Then, 6M hydrochloric acid was added to the resulting concentrate to adjust the pH to 1.0. This reaction mixture was heated at 50°C for 15 hours to carry out a deprotection reaction, after which a deprotection rate of 99.9% or higher was confirmed. The pH was adjusted to 3.4-3.6 using a 5M sodium hydroxide aqueous solution, and by dialysis using water and freeze-drying, a pH-responsive lipid derivative F was obtained in a yield of 0.40 g. １ H NMR (bihydrochloric acid + heavy water, 400MHz): δ 4.30 to 4.50 (m, average degree of polymerization x 1H), 3.20 to 3.80 (m, average degree of polymerization x 8.4H), 2.85 to 3.00 (t, average degree of polymerization x 1.6H), 2.35 to 2.70 (m, average degree of polymerization x 2.4H), 1.85 to 2.25 (m, d, average degree of polymerization x 2H), 1.10 to 1.30 (m, 64H), 0.84 to 0.91 (m, 6H).

[0184] The average degree of polymerization and number-average molecular weight of the obtained pH-responsive lipid derivative F were calculated using the method described in (Calculation of Average Degree of Polymerization 2). The results showed that the average degree of polymerization was 150 and the number-average molecular weight was 41,100. Furthermore, the number of each repeating unit was calculated using the method described in (Calculation of Number of Each Repeating Unit). The results showed that the R ２ It was found that, on average, it was in the form of a random copolymer having 120 repeating units A and 30 repeating units B.

[0185] [Examples 1-7: Synthesis of pH-responsive lipid derivative G] [Synthesis of intermediate (I-5) (In formula (I-5) below, C １３ H ２７ (This is a tridecyl group, and m5 is 30.)

[0186] The synthesis of intermediate (I-1) was carried out in the same manner as for intermediate (I-1), except that tetradecanoic acid, 1-(aminomethyl)-1,2-ethanediyl ester (CAS registration number: 229645-69-6) (86 mg, 0.168 mmol) was used instead of DSGE-A (100.3 mg, 0.168 mmol). The resulting solid was vacuum-dried overnight to obtain the intermediate represented by the above formula (I-5) in a yield of 0.97 g. １ H NMR (deuterochloroform, 400MHz): δ 7.00 to 7.40 (m, average degree of polymerization x 5H), 4.80 to 5.30 (m, average degree of polymerization x 2H), 3.80 to 4.40 (m, average degree of polymerization x 1H), 3. 10-3.78 (m, 9H), 1.40-2.90 (m, average degree of polymerization x 4H), 1.15-1.32 (m, 44H), 0.84-0.91 (m, 6H).

[0187] The average degree of polymerization (i.e., m5 in formula (I-5)) and the number-average molecular weight of the obtained intermediate (I-5) were calculated using the method described in (Calculation of Average Degree of Polymerization 1). As a result, it was found that m5 in formula (I-5) is 30, and the number-average molecular weight of intermediate (I-5) is 7,100.

[0188] [pH-responsive lipid derivative G (R in formula (1))] １ : Base (4b) (C in formula (4b) １３ H ２７ ( is a tridecyl group), L in formula (1) １ [Composition of: *-NH-*, Repeating unit A: Unit (2), Number of repeating units A (average): 27, Repeating unit B: Unit (3a), Number of repeating units B (average): 3]

[0189]

[0190] To the intermediate (I-5) (300 mg) obtained in the same manner as in Example 1-1, tetrahydrofuran (1.5 mL) was added and dissolved. 2-hydroxypyridine (360 mg) and β-alanine tBu (988 mg) were added and the reaction was carried out at 50°C. １After confirming by 1H NMR that an average of three benzyloxy groups in the intermediate (I-5) were replaced with β-alanine tBu, dialysis was performed using tetrahydrofuran. Three repeating units B are formed from the three repeating units in which the benzyloxy groups were replaced with β-alanine tBu through a deprotection reaction described later.

[0191] The dialysis solution was concentrated under reduced pressure, and the resulting concentrate was dissolved in tetrahydrofuran (1.5 mL). Then, 2-hydroxypyridine (360 mg) and DET-CartBu (1.314 g) were added, and the reaction was carried out at 50°C for 20 hours to replace the remaining 27 benzyloxy groups in intermediate (I-5) with DET-CartBu. From the 27 repeating units in which the benzyloxy groups were replaced with DET-CartBu, 27 repeating units A were formed by a deprotection reaction described later.

[0192] The yellow transparent solution obtained after the above reaction was concentrated under reduced pressure at 50°C for 2 hours. Then, 6M hydrochloric acid was added to the resulting concentrate to adjust the pH to 1.0. This reaction mixture was heated at 50°C for 15 hours to carry out a deprotection reaction, after which a deprotection rate of 99.9% or higher was confirmed. The pH was adjusted to 3.4-3.6 using a 5M sodium hydroxide aqueous solution, and by dialysis using water and freeze-drying, a pH-responsive lipid derivative G was obtained in a yield of 0.40 g. １ H NMR (bihydrochloric acid + heavy water, 400MHz): δ 4.30 to 4.50 (m, average degree of polymerization x 1H), 3.20 to 3.80 (m, average degree of polymerization x 9.2H), 2.85 to 3.00 (t, average degree of polymerization x 1.8H), 2.35 -2.70 (m, average degree of polymerization x 2.2H), 1.85 - 2.25 (m, average degree of polymerization x 2H), 1.10 - 1.30 (m, 44H), 0.84 - 0.91 (m, 6H).

[0193] The average degree of polymerization and number-average molecular weight of the obtained pH-responsive lipid derivative G were calculated using the method described in (Calculation of Average Degree of Polymerization 2). The results showed that the average degree of polymerization was 30 and the number-average molecular weight was 8,900. Furthermore, the number of each repeating unit was calculated using the method described in (Calculation of Number of Each Repeating Unit). The results showed that the R of pH-responsive lipid derivative G２ It was found that the molecule was in the form of a random copolymer having, on average, 27 repeating units A and 3 repeating units B.

[0194] [Example 1-8: Synthesis of pH-responsive lipid derivative H] [Synthesis of intermediate (I-6) (In formula (I-6) below, C １７ H ３５ (This is a heptadecyl group, and m6 is 30.)

[0195]

[0196] The same procedure as in the synthesis of intermediate (I-1) was followed, except that octadecanoic acid, 1,1'-[1-[[[(2-aminoethoxy)hydroxyphosphinyl]oxy]methyl]-1,2-ethanediyl] ester (CAS registration number: 4537-76-2) (152 mg, 0.168 mmol) was used instead of DSGE-A (100.3 mg, 0.168 mmol). The resulting solid was vacuum-dried overnight to obtain the intermediate represented by the above formula (I-6) in a yield of 0.97 g. １ H NMR (deuterochloroform, 400MHz): δ 7.00 to 7.40 (m, average degree of polymerization x 5H), 4.80 to 5.30 (m, average degree of polymerization x 2H), 3.80 to 4.40 (m, average degree of polymerization x 1H), 3. 10-3.75 (m, 13H) 1.40-2.90 (m, average degree of polymerization x 4H), 1.15-1.32 (m, 60H), 0.84-0.91 (m, 6H).

[0197] The average degree of polymerization (i.e., m6 in formula (I-6)) and number-average molecular weight of the obtained intermediate (I-6) were calculated using the method described in (Calculation of Average Degree of Polymerization 1). As a result, it was found that m6 in formula (I-6) averaged 30, and the number-average molecular weight of intermediate (I-6) was 7,400.

[0198] [pH-responsive lipid derivative H (R in formula (1)) １ : Base (4c), L in formula (1) １[Composition of: *-NH-*, Repeating unit A: Unit (2), Number of repeating units A (average): 27, Repeating unit B: Unit (3a), Number of repeating units B (average): 3]

[0199]

[0200] To the intermediate (I-6) (300 mg) obtained in the same manner as in Example 1-1, tetrahydrofuran (1.5 mL) was added and dissolved. 2-hydroxypyridine (360 mg) and β-alanine tBu (988 mg) were added and the reaction was carried out at 50°C. １ After confirming by 1H NMR that an average of three benzyloxy groups in the intermediate (I-6) were replaced with β-alanine tBu, dialysis was performed using tetrahydrofuran. Three repeating units B are formed from the three repeating units in which the benzyloxy groups were replaced with β-alanine tBu through a deprotection reaction described later.

[0201] The dialysis solution was concentrated under reduced pressure, and the resulting concentrate was dissolved in tetrahydrofuran (1.5 mL). Then, 2-hydroxypyridine (360 mg) and DET-CartBu (1.314 g) were added, and the reaction was carried out at 50°C for 20 hours to replace the remaining 27 benzyloxy groups in intermediate (I-6) with DET-CartBu. From the 27 repeating units in which the benzyloxy groups were replaced with DET-CartBu, 27 repeating units A were formed by a deprotection reaction described later.

[0202] The yellow transparent solution obtained after the above reaction was concentrated under reduced pressure at 50°C for 2 hours. Then, 6M hydrochloric acid was added to the resulting concentrate to adjust the pH to 1.0. This reaction mixture was heated at 50°C for 15 hours to carry out a deprotection reaction, after which a deprotection rate of 99.9% or higher was confirmed. The pH was adjusted to 3.4-3.6 using a 5M sodium hydroxide aqueous solution, and by dialysis using water and freeze-drying, a pH-responsive lipid derivative H was obtained in a yield of 0.40 g. １H NMR (bihydrochloric acid + heavy water, 400MHz): δ 4.30 to 4.50 (m, average degree of polymerization x 1H), 3.20 to 3.80 (m, average degree of polymerization x 9.2H), 2.85 to 3.00 (t, average degree of polymerization x 1.8H), 2.35 -2.70 (m, average degree of polymerization x 2.2H), 1.85 - 2.25 (m, average degree of polymerization x 2H), 1.10 - 1.30 (m, 60H), 0.84 - 0.91 (m, 6H).

[0203] The average degree of polymerization and number-average molecular weight of the obtained pH-responsive lipid derivative H were calculated using the method described in (Calculation of Average Degree of Polymerization 2), and it was found that the average degree of polymerization was 30 and the number-average molecular weight was 9,100. In addition, the number of each repeating unit was calculated using the method described in (Calculation of Number of Each Repeating Unit), and it was found that the R of pH-responsive lipid derivative H ２ It was found that the molecule was in the form of a random copolymer having, on average, 27 repeating units A and 3 repeating units B.

[0204] [Examples 1-9: pH-responsive lipid derivative I (R in formula (1)) １ : Base (4a), L in formula (1) １ [Composition of: *-NH-*, Repeating unit A: Unit (2), Number of repeating units A (average): 24, Repeating unit B: Unit (3a), Number of repeating units B (average): 6]

[0205]

[0206] To the intermediate (I-1) (300 mg) obtained in the same manner as in Example 1-1, tetrahydrofuran (1.5 mL) was added and dissolved. 2-hydroxypyridine (360 mg), β-alanine tBu (988 mg), and DET-CartBu (1.314 g) were added, and the reaction was carried out at 50°C for 20 hours to replace the benzyloxy group in intermediate (I-1) with β-alanine tBu or DET-CartBu.

[0207] The yellow transparent solution obtained after the above reaction was concentrated under reduced pressure at 50°C for 2 hours. Then, 6M hydrochloric acid was added to the resulting concentrate to adjust the pH to 1.0. This reaction mixture was heated at 50°C for 15 hours to carry out a deprotection reaction, after which a deprotection rate of 99.9% or higher was confirmed. The pH was adjusted to 3.4-3.6 using a 5M sodium hydroxide aqueous solution, and pH-responsive lipid derivative I was obtained in a yield of 0.42 g by dialysis using water and freeze-drying. １ H NMR (bihydrochloric acid + heavy water, 400MHz): δ 4.30 to 4.50 (m, average degree of polymerization x 1H), 3.20 to 3.80 (m, average degree of polymerization x 8.4H), 2.85 to 3.00 (t, average degree of polymerization x 1.6H), 2.35 to 2.70 (m, average degree of polymerization x 2.4H), 1.85 to 2.25 (m, d, average degree of polymerization x 2H), 1.10 to 1.30 (m, 64H), 0.84 to 0.91 (m, 6H).

[0208] The average degree of polymerization and number-average molecular weight of the obtained pH-responsive lipid derivative I were calculated using the method described in (Calculation of Average Degree of Polymerization 2), and it was found that the average degree of polymerization was 30 and the number-average molecular weight was 9,000. In addition, the number of each repeating unit was calculated using the method described in (Calculation of Number of Each Repeating Unit), and the R of pH-responsive lipid derivative I was found to be 30. ２ It was found that the molecule was in the form of a random copolymer having, on average, 24 repeating units A and 6 repeating units B.

[0209] [Comparative Example 1-1: Synthesis of pH-responsive lipid derivative J represented by the following formula (C-1) (In the following formula (C-1), C １８ H ３７ (This is an octadecyl group, and m1 is 30.)

[0210]

[0211] To the intermediate (I-1) (300 mg) obtained in the same manner as in Example 1-1, tetrahydrofuran (1.5 mL) was added and dissolved. Then, 2-hydroxypyridine (360 mg) and DET-CartBu (1.314 g) were added, and the reaction was carried out at 50°C for 20 hours. The yellow transparent solution was concentrated under reduced pressure at 50°C for 2 hours, and then 6 M hydrochloric acid was added to the obtained concentrate to adjust the pH to 1.0. After reacting this reaction mixture at 50°C for 15 hours, a deprotection rate of 99.9% or higher was confirmed. The pH was adjusted to 3.4-3.6 using 5 M sodium hydroxide, and by dialysis using water and freeze-drying, a pH-responsive lipid derivative J represented by the above formula (C-1) was obtained in a yield of 0.42 g. １ H NMR (bihydrochloric acid + heavy water, 400MHz): δ 4.30 to 4.50 (m, average degree of polymerization x 1H), 3.20 to 3.80 (m, 12H), 2.85 to 3.00 (t, average degree of polymerization x 2H), 2.35 to 2.55 (m, average degree of polymerization x 2H), 1.90 to 2.25 (m, d, average degree of polymerization x 2H), 1.10 to 1.30 (m, 64H), 0.84 to 0.91 (m, 6H).

[0212] The average degree of polymerization (i.e., m1 in formula (C-1)) and number-average molecular weight of the obtained pH-responsive lipid derivative J were calculated using the method described in (Calculation of Average Degree of Polymerization 2). The results showed that the average degree of polymerization was 30 and the number-average molecular weight was 9,200.

[0213] [Comparative Example 1-2: pH-responsive lipid derivative K (R in formula (1)) １ : Base (4a), L in formula (1) １ [Composition of: *-NH-*, Repeating unit A: Unit (2), Number of repeating units A (average): 29, Repeating unit B: Unit (3a), Number of repeating units B (average): 1]

[0214]

[0215] To the intermediate (I-1) (300 mg) obtained in the same manner as in Example 1-1, tetrahydrofuran (1.5 mL) was added and dissolved. 2-hydroxypyridine (360 mg) and β-alanine tBu (988 mg) were added and the reaction was carried out at 50°C. １After confirming by 1H NMR that an average of one benzyloxy group in intermediate (I-1) was replaced with β-alanine tBu, dialysis was performed using tetrahydrofuran. Furthermore, from the one repeating unit in which the benzyloxy group was replaced with β-alanine tBu, one repeating unit B is formed by a deprotection reaction described later.

[0216] The dialysis solution was concentrated under reduced pressure, and the resulting concentrate was dissolved in tetrahydrofuran (1.5 mL). Then, 2-hydroxypyridine (360 mg) and DET-CartBu (1.314 g) were added, and the reaction was carried out at 50°C for 20 hours to replace the remaining 29 benzyloxy groups in intermediate (I-1) with DET-CartBu. From the 29 repeating units in which the benzyloxy groups were replaced with DET-CartBu, 29 repeating units A were formed by a deprotection reaction described later.

[0217] The yellow transparent solution obtained after the above reaction was concentrated under reduced pressure at 50°C for 2 hours. Then, 6M hydrochloric acid was added to the resulting concentrate to adjust the pH to 1.0. This reaction mixture was heated at 50°C for 15 hours to carry out a deprotection reaction, after which a deprotection rate of 99.9% or higher was confirmed. The pH was adjusted to 3.4-3.6 using a 5M sodium hydroxide aqueous solution, and pH-responsive lipid derivative K was obtained in yield of 0.40 g by dialysis using water and freeze-drying. １ H NMR (bihydrochloric acid + heavy water, 400MHz): δ 4.30 to 4.50 (m, average degree of polymerization x 1H), 3.20 to 3.80 (m, average degree of polymerization x 9.6H), 2.85 to 3.00 (t, average degree of polymerization x 1.9H), 2.35 -2.70 (m, average degree of polymerization x 2.1H), 1.85 - 2.25 (m, average degree of polymerization x 2H), 1.10 - 1.30 (m, 64H), 0.84 - 0.91 (m, 6H).

[0218] The average degree of polymerization and number-average molecular weight of the obtained pH-responsive lipid derivative K were calculated using the method described in (Calculation of Average Degree of Polymerization 2). The results showed that the average degree of polymerization was 30 and the number-average molecular weight was 9,100. Furthermore, the number of each repeating unit was calculated using the method described in (Calculation of Number of Each Repeating Unit). The results showed that the R of pH-responsive lipid derivative K２ It was found to be a random copolymer having, on average, 29 repeating units A and 1 repeating unit B.

[0219] <Example 2 and Comparative Example 2: Preparation of Solid LNPs> [Example 2-1] Preparation of solid LNPs using pH-responsive lipid derivative A of Example 1-1 First, a 50 mM cholesterol solution, a 50 mM D-Lin-MC3-DMA solution, and a 20 mM DOPE solution were prepared using ethanol.

[0220] Next, using 10 mM acetate buffer, an aqueous solution of 1 mg / mL siRNA (siGL3, sense: 5'-CUUACGCUGAGAGUACUUCGAAdTdT-3' (SEQ ID NO: 1)) and an aqueous solution of 2 mM pH-responsive lipid derivative A were prepared, respectively.

[0221] Next, the cholesterol solution (38 μL), D-Lin-MC3-DMA solution (47 μL), DOPE solution (24 μL), and ethanol (341 μL) were mixed in a 1.5 mL tube to prepare a lipid solution.

[0222] A solution containing pH-responsive lipid derivative A and siRNA (hereinafter referred to as "nucleic acid solution") was prepared by mixing 70 μL of an aqueous solution of 1 mg / mL siRNA (siGL3, sense: 5'-CUUACGCUGAGUACUUCGAAdTdT-3' (SEQ ID NO: 1)), 24 μL of an aqueous solution of 2 mM pH-responsive lipid derivative A, and 606 μL of 10 mM acetate buffer.

[0223] The prepared lipid solution and nucleic acid solution were mixed using a microfluidic channel to form a solid LNP dispersion (content of lipid derivatives or lipids relative to the total lipids in the solid LNP; pH-responsive lipid derivative A = 1 mol%, cholesterol = 40 mol%, DOPE = 10 mol%, D-Lin-MC3-DMA = 49 mol%).

[0224] The obtained solid LNP dispersion was purified by ultrafiltration using HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) buffer (Merck Amicon Ultra-15 (MWCO 50kDA)) to obtain a solid LNP dispersion.

[0225] [Example 2-2] Preparation of solid LNP using pH-responsive lipid derivative B from Example 1-2 A solid LNP dispersion was prepared in the same manner as in Example 2-1, except that the aqueous solution of the pH-responsive lipid derivative used was changed to a 2.0 mM pH-responsive lipid derivative B aqueous solution (24 μL).

[0226] [Example 2-3] Preparation of solid LNP using pH-responsive lipid derivative C of Example 1-3 A solid LNP dispersion was prepared in the same manner as in Example 2-1, except that the aqueous solution of the pH-responsive lipid derivative used was changed to a 2.0 mM pH-responsive lipid derivative C aqueous solution (24 μL).

[0227] [Example 2-4] Preparation of solid LNP using pH-responsive lipid derivative D of Example 1-4 A solid LNP dispersion was prepared in the same manner as in Example 2-1, except that the aqueous solution of the pH-responsive lipid derivative used was changed to a 2.0 mM aqueous solution of pH-responsive lipid derivative D (24 μL).

[0228] [Example 2-5] Preparation of solid LNP using pH-responsive lipid derivative E of Example 1-5 A solid LNP dispersion was prepared in the same manner as in Example 2-1, except that the aqueous solution of the pH-responsive lipid derivative used was changed to a 2.0 mM pH-responsive lipid derivative E aqueous solution (24 μL).

[0229] [Example 2-6] Preparation of solid LNP using pH-responsive lipid derivative F of Example 1-6 A solid LNP dispersion was prepared in the same manner as in Example 2-1, except that the aqueous solution of the pH-responsive lipid derivative used was changed to a 2.0 mM pH-responsive lipid derivative F aqueous solution (24 μL).

[0230] [Example 2-7] Preparation of solid LNP using pH-responsive lipid derivative G of Example 1-7 A solid LNP dispersion was prepared in the same manner as in Example 2-1, except that the aqueous solution of the pH-responsive lipid derivative used was changed to a 2.0 mM pH-responsive lipid derivative G aqueous solution (24 μL).

[0231] [Example 2-8] Preparation of solid LNP using pH-responsive lipid derivative H of Example 1-8 A solid LNP dispersion was prepared in the same manner as in Example 2-1, except that the aqueous solution of the pH-responsive lipid derivative used was changed to a 2.0 mM pH-responsive lipid derivative H aqueous solution (24 μL).

[0232] [Example 2-9] Preparation of solid LNP using pH-responsive lipid derivative I of Example 1-9 A solid LNP dispersion was prepared in the same manner as in Example 2-1, except that the aqueous solution of the pH-responsive lipid derivative used was changed to a 2.0 mM pH-responsive lipid derivative I aqueous solution (24 μL).

[0233] [Example 2-10] Preparation of solid LNP using pH-responsive lipid derivative A of Example 1-1 A solid LNP dispersion was prepared in the same manner as in Example 2-1, except that the siRNA used was changed to fluorescently labeled siRNA (Alexa647-siGL3, sense: 5'-CUUACGCUGAGAUUCUGAGAdTdT-3' (SEQ ID NO: 2), and antisense: 5'-Alexa647-UCGAAGUACUCAGGUAAGdTdT-3' (SEQ ID NO: 3)).

[0234] [Example 2-11] Preparation of solid LNPs using pH-responsive lipid derivative B of Example 1-2 A solid LNP dispersion was prepared in the same manner as in Example 2-1, except that the aqueous solution of the pH-responsive lipid derivative used was changed to a 2.0 mM aqueous solution of pH-responsive lipid derivative B (24 μL), and the siRNA used was changed to fluorescently labeled siRNA (Alexa647-siGL3, sense: 5'-CUUACGCUGAGAUUCUGAGAdTdT-3' (SEQ ID NO: 2), and antisense: 5'-Alexa647-UCGAAGUACUCAGGUAAGdTdT-3' (SEQ ID NO: 3)).

[0235] [Example 2-12] Preparation of solid LNPs using pH-responsive lipid derivative F of Example 1-6 A solid LNP dispersion was prepared in the same manner as in Example 2-1, except that the aqueous solution of the pH-responsive lipid derivative used was changed to a 2.0 mM aqueous solution of pH-responsive lipid derivative F (24 μL), and the siRNA used was changed to fluorescently labeled siRNA (Alexa647-siGL3, sense: 5'-CUUACGCUCAGUACUUCGAAdTdT-3' (SEQ ID NO: 2), and antisense: 5'-Alexa647-UCGAAGUACUCAGCGUAAGdTdT-3' (SEQ ID NO: 3)).

[0236] [Example 2-13] Preparation of solid LNPs using pH-responsive lipid derivative I of Example 1-9 A solid LNP dispersion was prepared in the same manner as in Example 2-1, except that the aqueous solution of the pH-responsive lipid derivative used was changed to a 2.0 mM aqueous solution of pH-responsive lipid derivative I (24 μL), and the siRNA used was changed to fluorescently labeled siRNA (Alexa647-siGL3, sense: 5'-CUUACGCUGAGAUUCUGAGAdTdT-3' (SEQ ID NO: 2), and antisense: 5'-Alexa647-UCGAAGUACUCAGGUAAGdTdT-3' (SEQ ID NO: 3)).

[0237] [Example 2-14] Preparation of solid LNPs using pH-responsive lipid derivative B of Example 1-2 A solid LNP dispersion was prepared in the same manner as in Example 2-1, except that the aqueous solution of the pH-responsive lipid derivative used was changed to a 2.0 mM aqueous solution of pH-responsive lipid derivative B (24 μL), and the siRNA used was changed to siPLK1 (sense: 5'-AGAuCACCCuCCUuAAAuAUU-3' (SEQ ID NO: 4), and antisense: 5'-UAUUUAAGGAGGGUGAuCUUU-3' (SEQ ID NO: 5)) which suppresses tumor growth. In the above siPLK1 sequences, those written in lowercase indicate that the base portion is the same, but the sugar portion has undergone 2'-O-methylation modification.

[0238] [Comparative Example 2-1] Preparation of Solid LNP using pH-responsive lipid derivative J of Comparative Example 1-1 A solid LNP dispersion was prepared in the same manner as in Example 2-1, except that the aqueous solution of the pH-responsive lipid derivative used was changed to a 2.0 mM pH-responsive lipid derivative J aqueous solution (24 μL).

[0239] [Comparative Example 2-2] Preparation of Solid LNP using pH-responsive lipid derivative K of Comparative Example 1-2 A solid LNP dispersion was prepared in the same manner as in Example 2-1, except that the aqueous solution of the pH-responsive lipid derivative used was changed to a 2.0 mM pH-responsive lipid derivative K aqueous solution (24 μL).

[0240] [Comparative Example 2-3] Preparation of Solid LNPs using pH-responsive lipid derivative J of Comparative Example 1-1 A solid LNP dispersion was prepared in the same manner as in Example 2-1, except that the aqueous solution of the pH-responsive lipid derivative used was changed to a 2.0 mM aqueous solution of pH-responsive lipid derivative J (24 μL), and the siRNA used was changed to a fluorescently labeled siRNA (Alexa647-siGL3, sense: 5'-CUUACGCUGAGAUACUUCGAAdTdT-3' (SEQ ID NO: 2), antisense: 5'-Alexa647-UCGAAGUACUCAGGUAAGdTdT-3' (SEQ ID NO: 3)).

[0241] [Comparative Example 2-4] Preparation of Solid LNPs using pH-responsive lipid derivative J of Comparative Example 1-1 A solid LNP dispersion was prepared in the same manner as in Example 2-1, except that the aqueous solution of the pH-responsive lipid derivative used was changed to a 2.0 mM aqueous solution of pH-responsive lipid derivative I (24 μL), and the siRNA used was changed to siPLK1 (sens: 5'-AGAuCACCCuCCUuAAAuAUU-3' (sequence number: 4), antisense: 5'-UAUUUAAGGAGGGUGAuCUUU-3' (sequence number: 5), which suppresses tumor growth. In the above siPLK1 sequences, those written in lowercase indicate that the base portion is the same, but the sugar portion has undergone 2'-O-methylation modification.

[0242] [Experimental Example 1] Measurement of Average Particle Size and Zeta Potential of Solid LNPs The average particle size of the solid LNPs obtained in Examples 2-1 to 2-9, and Comparative Examples 2-1 and 2-2, was measured by dynamic light scattering (DLS). In addition, the dispersion medium of the solid LNP dispersions obtained in Examples 2-1 to 2-9, and Comparative Examples 2-1 and 2-2 was replaced with 10 mM HEPES (pH 7.4) or 10 mM MES (2-morpholinoethanesulfonic acid) (pH 6.5), and the zeta potential of the solid LNPs in the dispersion medium at pH 7.4 or pH 6.5 was measured. The results are shown in Table 1.

[0243]

[0244] The zeta potentials of the solid LNPs in Examples 2-1 to 2-9 were -5.0 mV or less in a dispersion medium at pH 7.4, and 10.0 mV or more in a dispersion medium at pH 6.5. These results show that the solid LNPs containing the pH-responsive lipid derivative of the present invention have anionic surfaces at pH 7.4 and cationic surfaces at pH 6.5.

[0245] On the other hand, the solid LNPs of Comparative Example 2-1 and Comparative Example 2-2 had vesicles of 4.0 mV and 0 mV, respectively, in a dispersion medium at pH 7.4, indicating that their surfaces were cationic and neutral, respectively.

[0246] [Experimental Example 2] In vivo pharmacokinetic studies of siRNA were conducted using the solid LNP dispersions of Examples 2-10, 2-11, 2-12, 2-13, or Comparative Example 2-3. Tumor-bearing model mice were created by subcutaneous transplantation of the mouse colon cancer cell line CT26 into BALB / c mice (5 × 10⁻¹⁰ ５ cell / mouse). Tumor size 200 mm ３ After the cells had grown to a certain size, each solid LNP dispersion was administered via tail vein injection (dosage: 0.5 mg / kg siRNA equivalent).

[0247] Six hours after administration, blood and tumor were harvested. The blood and tumor were homogenized with Lysis Buffer and treated with a centrifuge. Then the supernatant was collected, and sodium lauryl sulfate and tert-butanol were added to the supernatant to adjust the concentration of sodium lauryl sulfate to 3% by weight and the concentration of tert-butanol to 60% by weight. The concentration of siRNA in the resulting solution was determined using a microplate reader (SPARK TKS01, manufactured by TECAN) (excitation wavelength: 630 nm, fluorescence wavelength: 690 nm), and the amount of nucleic acid in blood (%) and the amount of nucleic acid accumulated in tumor (%) were calculated by the following formulas. The results are shown in Table 2. Amount of nucleic acid in blood (%) = 100 × Amount of fluorescence-labeled siRNA remaining in blood / Amount of fluorescence-labeled siRNA administered to mice Amount of nucleic acid accumulated in tumor (%) = 100 × Amount of fluorescence-labeled siRNA accumulated in tumor / Amount of fluorescence-labeled siRNA administered to mice

[0248]

[0249] When the solid LNPs of Examples 2-10, 2-11, 2-12, or 2-13 were used, it was confirmed that the amount of nucleic acid in blood was higher and the blood retention of the solid LNPs of Examples 2-10, 2-11, 2-12, and 2-13 was better, compared with the case where the solid LNP of Comparative Example 2-3 was used.

[0250] It was confirmed that when the solid LNPs of Examples 2-10, 2-11, or 2-13 were used, the amount of nucleic acid accumulated in tumor was higher compared with the case where the solid LNPs of Example 2-12 or Comparative Example 2-3 were used.

[0251] From the above results, it was confirmed that the solid LNP containing the pH-responsive lipid derivative of the present invention exhibits stealth properties because its surface becomes anionic in blood components and normal tissues, which are neutral environments in vivo, and increases cationicity in response to minute pH changes around tumor tissues, thereby exhibiting excellent accumulation properties in tumor tissues.

[0252] It is considered that the pH-responsive lipid derivative F used in Example 2-12 has a long hydrophilic polymer moiety, which weakens the interaction with cells, and as a result, the tumor accumulation of the solid LNP of Example 2-12 was reduced.

[0253] [Experimental Example 3] Antitumor effect using siPLK1 The antitumor effect of siPLK1 was evaluated using solid LNPs from Example 2-14 or Comparative Example 2-4. A tumor-bearing model mouse was created by subcutaneously transplanting the human ovarian cancer cell line SKOV3-luccells into BAlB / c nude mice (5 × 10⁻¹⁰ ６ cell / mouse). Tumor size: 25 mm ３ When the mice reached a certain size, they were randomized and divided into three groups: a group administered the solid LNP dispersion of Example 2-14 by tail vein injection (hereinafter referred to as the "Example 2-14 group"), a group administered the solid LNP dispersion of Comparative Example 2-4 by tail vein injection (hereinafter referred to as the "Comparative Example 2-4 group"), and a control group administered phosphate-buffered saline instead of solid LNP by tail vein injection (hereinafter referred to as the "control group"). The single dose for the Example 2-14 group and the Comparative Example 2-4 group was 2.5 mg / kg in terms of siPLK1. All groups were administered once every two days (a total of nine times). Tumor volume was checked on the 19th day after the start of administration, and the antitumor effect was calculated using the following formula. The results are shown in Table 3. Antitumor effect = (Tumor volume of the control group on day 19 / Tumor volume of mice before administration) / (Tumor volume of groups 2-14 or comparative examples 2-4 / Tumor volume of mice before administration)

[0254]

[0255] Compared to the solid LNP administration group in Comparative Examples 2-4, the solid LNP administration group in Example 2-14 showed a higher antitumor effect and a slower increase in tumor volume. This is thought to be due to the high tumor accumulation by pH-responsive lipid derivative B, as demonstrated in Experimental Example 2.

[0256] The LNP containing the pH-responsive lipid derivative of the present invention has the property of becoming electrically anionic under neutral conditions (pH 7.4) and changing to cationic under weakly acidic conditions (pH 6.5) around tumor tissue. In the neutral environment of blood components or normal tissues in vivo, the LNP exhibits stealth properties because its surface becomes anionic. Around tumor tissue, in response to minute pH changes, the cationicity of its surface increases, improving the accumulation efficiency and uptake efficiency in tumor tissue. As a result, the LNP can efficiently introduce encapsulated small molecule drugs or nucleic acid drugs into the area around tumor tissue or into tumor cells.

[0257] This application is based on Japanese Patent Application No. 2025-28276, which is entirely contained herein.

Claims

1. Formula (1): R １ -L １ -R ２ (1) (wherein, R １ This is a lipid region, L １ is NH or O, and R ２ Equation (2): (In the formula, * indicates the bonding position.) The repeating unit A is represented by formula (3): (In the formula, a1 and c1 are independently 1 or 2, b1 is 0 or 1, X １ These are ester bonds, amide bonds, or thioester bonds, and Y １ A is a polymer moiety having a repeating unit B represented by ), wherein is a carboxyl group or a sulfo group, and * is the bonding position. A pH-responsive lipid derivative represented by ), wherein the amount of repeating unit B relative to the total of repeating unit A and repeating unit B is 6 to 50 mol%.

2. The pH-responsive lipid derivative according to claim 1, wherein the number-average molecular weight is 3,000 to 50,000.

3. In formula (3), both a1 and c1 are 2, b1 is 1, and X １ is an amide bond, and Y １ is a carboxy group. The pH-responsive lipid derivative according to claim 1.

4. R １ However, equation (4): (In the formula, R ３ and R ４ Each of these is independently an aliphatic hydrocarbon group having 8 to 24 carbon atoms, an acyl group having 8 to 24 carbon atoms, or a sterol residue, and M is a trivalent hydrocarbon group having 3 to 7 carbon atoms or *-N(CH ２ CH ２ -*)-* represents a trivalent nitrogen-containing hydrocarbon group, R ５ The pH-responsive lipid derivative according to claim 1, wherein a is a hydrogen atom, an alkali metal atom, or ammonium, a2, a3, b2, c2, and d1 are each independently 0 or 1, and * is a binding position.

5. A drug delivery carrier comprising a pH-responsive lipid derivative according to any one of claims 1 to 4.

6. The drug delivery carrier according to claim 5, which is a solid lipid nanoparticle.

7. The drug delivery carrier according to claim 5, wherein the average particle size is 10 to 250 nm.

8. The drug delivery carrier according to claim 5, further comprising at least one selected from the group consisting of phospholipids, ionic lipids, and sterols.

9. The drug delivery carrier according to claim 8, wherein, with respect to the total of the pH-responsive lipid derivative and lipids as needed in the drug delivery carrier, the content of the pH-responsive lipid derivative is 0.1 to 30 mol%, the content of the phospholipid is 5 to 60 mol%, the content of the ionic lipid is 5 to 60 mol%, and the content of the sterol is 5 to 60 mol%.

10. The drug delivery carrier according to claim 5, wherein the drug transported by the drug delivery carrier is at least one selected from the group consisting of nucleic acids, nucleic acid derivatives, peptides, small molecule drugs, and anticancer agents.

11. Formula (5): R １ -L １ -R ６ (5) (wherein, R １ This is a lipid region, L １ is NH or O, and R ６ Equation (6): (In the formula, a1 is 1 or 2, P １ is a protecting group for the carboxyl group, and * is the bond position. ) is a polymer moiety containing repeating units represented by ). ) and formula (7): X ２ - (CH ２ ) ｃ１ -Y ２ (7) (wherein c1 is 1 or 2, X ２ Y is a hydroxyl group, an amino group, or a sulfanyl group. ２ *-CO-OP ２ , sulfo group, or *-SO ２ -OP ３ P ２ P is a protecting group for the carboxyl group. ３ A is a protecting group for the sulfo group, and * indicates the bond position.) A compound represented by formula (8): H ２ N-CH ２ CH ２ -NH-CH ２ CH ２ -NH-CH ２ CH ２ - CO-OP ４ (8) (wherein, P ４ A method for producing a pH-responsive lipid derivative, comprising: step (A) reacting with any of the compounds represented by ) to obtain intermediate 1; step (B) reacting intermediate 1 obtained in step (A) with the remainder of the compound represented by formula (7) or formula (8) that was not used in step (A) to obtain intermediate 2 having a protected carboxyl group; and step (C) deprotecting intermediate 2 obtained in step (B) to obtain the pH-responsive lipid derivative according to claim 1, wherein b1 is 1.

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