WO2023198085A1 - 一种含有叔胺的氮支化非线性聚乙二醇化脂质及其应用 - Google Patents

一种含有叔胺的氮支化非线性聚乙二醇化脂质及其应用 Download PDF

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WO2023198085A1
WO2023198085A1 PCT/CN2023/087702 CN2023087702W WO2023198085A1 WO 2023198085 A1 WO2023198085 A1 WO 2023198085A1 CN 2023087702 W CN2023087702 W CN 2023087702W WO 2023198085 A1 WO2023198085 A1 WO 2023198085A1
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lipid
nitrogen
branched
lipids
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PCT/CN2023/087702
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English (en)
French (fr)
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翁文桂
刘超
陈丹丹
林昇
王爱兰
魏国华
林聪明
朱琦
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厦门赛诺邦格生物科技股份有限公司
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Priority to CN202380011318.7A priority Critical patent/CN117413004A/zh
Publication of WO2023198085A1 publication Critical patent/WO2023198085A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C235/10—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by nitrogen atoms not being part of nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule

Definitions

  • the invention belongs to the field of drug delivery, and generally relates to a PEGylated lipid, and specifically relates to a nitrogen-branched nonlinear PEGylated lipid containing a tertiary amine that can be used as a drug carrier component, and a PEGylated lipid containing the lipid.
  • Liposomes are widely used to deliver nucleic acid drugs, gene vaccines, anti-tumor drugs, small molecule drugs, peptide drugs or protein drugs. Among them, the development of vaccines against the new coronavirus has made lipid nanoparticles (LNP) a current hot topic. delivery technology. In addition to negatively charged mRNA, LNP also contains four components: cationic lipids, neutral lipids, steroid lipids and pegylated lipids. Among them, cationic lipids interact with negatively charged mRNA through electrostatic interaction.
  • Neutral lipids are generally phospholipids that prevent lipid oxidation or connect ligands to the surface of liposomes or LNPs or reduce lipid particle aggregation; steroid lipids have strong membrane fusion and promote intracellular mRNA Uptake and cytoplasmic entry; PEGylated lipids are located on the surface of lipid nanoparticles to improve hydrophilicity, avoid rapid clearance by the immune system, prevent particle aggregation, and increase stability.
  • Cationic lipids and PEGylated lipids are particularly critical components of LNP.
  • Cationic lipids include permanent cationic lipids and ionizable cationic lipids. Permanent cationic lipids can easily cause interference or even damage to biological membranes in systemic circulation, while ionizable cationic lipids can not ionize or weakly ionize under physiological conditions, reducing Little toxicity. After being taken up by cells, ionizable cationic lipids can be ionized under the acidic conditions of the endosomal cavity and become partially positively charged, increasing membrane permeability. At the same time, LNP is degraded or reorganized, promoting the escape of mRNA from the endosome and releasing it into the cytoplasm. Further translation, thereby completing the delivery and transfection of the mRNA molecule.
  • Cationic liposomes or LNPs without surface modification generally do not have sufficient systemic circulation stability.
  • Adding PEGylated lipids to liposome or LNP formulations can achieve the effect of covalently binding polyethylene glycol to the surface of liposomes or LNPs, reducing non-specific cellular uptake during drug delivery, and achieving liposome or The invisible effect of LNP improves the effectiveness of its pharmaceutical preparations.
  • the PEGylated lipids that are currently used and studied more are basically linear polyethylene glycol modifications. Typical examples include DMG-PEG2000 (PEG2k-DMG) and ALC-0159, while linear PEGylated lipids are rarely modified. There are applications. Polyethylene glycol modification can improve the stability and safety of the modified substance in the body, while also reducing the uptake efficiency of the drug by cells. Traditional PEGylated lipids are modified with linear polyethylene glycol. If the molecular weight is similar, if nonlinear polyethylene glycol is used, the umbrella structure has higher protective capabilities (Veronese et al., BioDrugs. 2008, 22, 315-329 ), resulting in a reduction in the cellular uptake rate of the modified substance.
  • the present invention provides a new type of nonlinear PEGylated lipid, especially a nitrogen-branched nonlinear PEGylated lipid containing a tertiary amine, which is applied to the preparation of lipid nanoparticles and pharmaceutical preparations thereof to achieve It has the advantages of excellent protective effect and systemic circulation stability, good cellular uptake efficiency, high biocompatibility, low toxicity, high encapsulation efficiency and high delivery efficiency.
  • a nitrogen-branched PEGylated lipid characterized in that its structure is as shown in general formula (1):
  • B 1 and B 2 are each independently a connecting bond or an alkylene group
  • L 1 , L 2 , L d and y L x are each independently a connecting bond or a divalent connecting group L;
  • R 1 and R 2 are each independently a C 1-50 aliphatic hydrocarbon group or a C 1-50 aliphatic hydrocarbon derivative residue containing 0-10 heteroatoms; the heteroatoms are B, O, N, Si, P or S;
  • N core is a multivalent group with a valence state of y+1; N core contains a trivalent nitrogen atom branched core connected to L d ;
  • y is 2, 3, 4, 5, 6, 7, 8, 9 or y ⁇ 10, preferably 2 or 3;
  • XPEG is the polyethylene glycol component.
  • a lipid composition comprising a pegylated lipid having a structure shown in formula (1).
  • a lipid pharmaceutical composition containing the aforementioned lipid composition and a drug.
  • a lipid pharmaceutical composition preparation containing the aforementioned lipid pharmaceutical composition and a pharmaceutically acceptable diluent or excipient.
  • the present invention has the following beneficial effects:
  • the present invention provides nitrogen-branched nonlinear PEGylated lipid compounds containing tertiary amines (nitrogen-branched PEGylated lipids for short), and also provides lipid combinations containing the nitrogen-branched PEGylated lipids. Drugs, lipid pharmaceutical compositions and preparations thereof. Compared with linear PEGylated lipids, the nitrogen-branched PEGylated lipids of the present invention can achieve better LNP surface modification with less molar dosage and/or shorter polyethylene glycol chains. Effectively, it plays a better protective role, prolongs the systemic circulation time of LNP, and reduces the biological toxicity of LNP to a greater extent.
  • the LNP of the present invention can be used as a carrier for targeted delivery of one or more bioactive agents.
  • introducing degradative groups at appropriate positions in the nitrogen-branched PEGylated lipid structure can enable cationic lipid nanoparticles carrying nucleic acid drugs to have better cellular uptake efficiency and/or transfection efficiency.
  • the present invention also provides lipid pharmaceutical compositions and preparations containing the nitrogen-branched PEGylated lipids, which have better drug efficacy, including improved drug targeting and nucleic acid drug transfection efficiency.
  • Figure 1 is a nuclear magnetic resonance spectrum ( 1 H NMR) of the nitrogen-branched PEGylated lipid (E1-5) prepared in Example 1.5 of the present invention.
  • the "selected from"/"preferred" of any two objects are independent of each other.
  • the selected from/preferred of any two objects may be of the same level or Different levels.
  • LA and LB are each independently selected from A, B, and C. It can mean that both L A and LB are A, or it can be that L A is A and LB is B 1 (B 1 is B's a subordinate situation).
  • LA is preferably A (level 1 preference), more preferably A 1 to A 3 (level 2 preference), most preferably A 11 to A 13 (level 3 preference), and LB is preferably B (1 Preferred grade), more preferably B 1 to B 3 (preferred grade 2), most preferably B 11 to B 13 (preferred grade 3), preferably A is A 1 to A 3 (preferred grade 2) and B B 11 to B 13 (3rd level preference), or both A and B may be 3rd level preference.
  • the "combination" of the listed items consists of any two or more than two of the previously listed items; and the number of items is not limited.
  • the number of any item It can be one or more than one.
  • the specific forms of any two items can be the same or different.
  • an interval marked in the form of an interval can represent the group of all integers and non-integers within the interval, and the range includes two endpoints.
  • the average number of EO units is selected from 22 to 100, and its selection range is not limited to integers within this interval, and can also be any non-integer.
  • an integer from 1 to 3 represents the group consisting of 1, 2, and 3.
  • -(CH 2 ) 1-4 - represents the group consisting of -CH 2 -, -(CH 2 ) 2 -, -(CH 2 ) 3 -, -(CH 2 ) 4 -.
  • the numerical range in the present invention includes, but is not limited to, the numerical range expressed by integers, non-integers, percentages, and fractions. Unless otherwise specified, both endpoints are included.
  • the polymer molecular weight generally refer to the numerical range of ⁇ 10%. In some cases, it can be enlarged to ⁇ 15%, but not more than ⁇ 20%. Based on the default value. For example, the deviations between 11 kDa, 12 kDa and 10 kDa are 10% and 20% respectively, and "about 10 kDa” includes but is not limited to 11 kDa and 12 kDa. For another example, when the molecular weight of a certain PEG component in the general formula is specified to be about 5kDa, the corresponding molecular weight or number average molecular weight is allowed to vary within the range of 5kDa ⁇ 10%, that is, 4500 to 5500Da.
  • “about” and “about” generally refer to the numerical range of ⁇ (0.5*0.1 N )% .
  • about 1% refers to the range of 1 ⁇ (0.5*0.1 0 )%, which is 0.5%-1.5%
  • about 2.2% refers to the range of 2.2 ⁇ (0.5*0.1 1 )%, which is 2.15%-2.25%.
  • about 2.33% refers to the range of 2.33 ⁇ (0.5*0.1 2 )%, that is, 2.335%-2.325%.
  • the divalent linking group in the present invention is not particularly limited. When connecting to other groups, either of the two connecting ends can be selected.
  • connection key such as Represents the group structure, specifically, means -CCH 3 (CH 2 CH 2 CH 3 ) 2 ; Represents -C(CH 2 CH 2 CH 3 ) 2 -. rather than in group form Then it means (CH 3 ) 2 C(CH 2 CH 2 CH 3 ) 2 .
  • a bond or group derived from a cyclic structure when a bond or group derived from a cyclic structure is not labeled with a specific ring-forming atom, but points to the inside of the ring, it means that the bond or group can be derived from any appropriate ring-forming atom.
  • the connecting bond or group when the marked ring is part of a paracyclic or fused ring structure, can be derived from any appropriate ring-forming atom in the paracyclic or fused ring structure.
  • Represented structures include but are not limited to wait.
  • the range of the number of carbon atoms in a group is marked in the subscript position of C, indicating the number of carbon atoms in the group. Unless otherwise specified, the number of carbon atoms does not include the contribution of substituents.
  • C 1-12 means “having 1 to 12 carbon atoms.”
  • C 1-10 alkylene represents any alkylene group with a number of carbon atoms in the range indicated by the subscript, namely C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , Any of C 8 , C 9 , and C 10 alkylene groups, including but not limited to linear C 1-10 alkylene groups (such as -(CH 2 ) 6 -) and branched C 1-10 alkylene groups.
  • Alkyl eg -(CH 2 ) 3 -CH(CH 3 )-(CH 2 ) 3 -).
  • substituted C 1-12 alkyl refers to C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 or C 12 alkyl
  • An alkyl group in which at least one hydrogen atom of the group is replaced by a substituent, and the number of carbon atoms and heteroatoms in the substituent is not particularly limited.
  • a structure with cis-trans isomers can be either a cis structure or a trans structure
  • a structure with E/Z isomers can be either an E structure or a Z structure
  • the described structure should have greater weight.
  • the molecular weight of polyethylene glycol and its derivatives refers to the average molecular weight by default, and unless otherwise specified, the "average molecular weight” generally refers to the "number average molecular weight” Mn .
  • the number average molecular weight may be either the molecular weight of a polydisperse block or substance or the molecular weight of a monodisperse block or substance. Unless otherwise specified, the measurement unit of molecular weight is Dalton (Da).
  • the "degree of polymerization” can also be used to characterize the molecular weight of the polyethylene glycol chain, specifically referring to the number of repeating units (oxyethylene units). Correspondingly, it is preferable to use “average degree of polymerization” and “number average degree of polymerization” to characterize the average value and number average value of the number of repeating units.
  • any suitable in “any suitable linking group”, “any suitable reactive group”, etc. refers to a structure that complies with the basic principles of chemical structure and enables the smooth implementation of the preparation method of the present invention. . Chemical structures described in this way can be considered to have clear, defined ranges.
  • degradable refers to the cleavage of the chemical bond of the invention, and the cleavage into at least two residues that are independent of each other. If the structure is changed after a chemical change, but the entire linker is still only a complete linker, then the linker is still classified as “stable.”
  • the degradable conditions are not particularly limited, and can be either in vivo physiological conditions or in vitro simulated physiological environments or other conditions, preferably in vivo physiological conditions and in vitro simulated physiological conditions.
  • the physiological conditions are not particularly limited, including but not limited to serum, heart, liver, spleen, lung, kidney, bone, muscle, fat, brain, lymph nodes, small intestine, gonads and other parts, and may refer to intracellular or extracellular In the matrix, it can refer to normal physiological tissues or diseased physiological tissues (such as tumors, inflammation, etc.).
  • the in vitro simulated environment is not particularly limited, including but not limited to physiological saline, buffer, culture medium, etc.
  • the degradable speed is not particularly limited. For example, it may be rapid degradation under the action of enzymes or slow hydrolysis under physiological conditions.
  • the physiological conditions in the body include physiological conditions during treatment, such as ultraviolet irradiation, hyperthermia, etc.
  • degradable under conditions such as light, heat, low temperature, enzymes, redox, acidity, alkalinity, physiological conditions, and in vitro simulated environments, preferably under conditions such as light, heat, enzymes, redox, acidity, and alkalinity.
  • Decomposable refers to degradation under stimulation under any of the above conditions.
  • the light conditions include but are not limited to visible light, ultraviolet light, infrared light, near-infrared light, mid-infrared light and other lighting conditions.
  • the thermal condition refers to a temperature condition higher than normal physiological temperature, usually higher than 37°C, and usually lower than 45°C, preferably lower than 42°C.
  • the acidic and alkaline conditions mainly refer to the pH conditions of normal tissues, diseased tissues, organs or tissues in the treatment period and other parts of the body.
  • the stomach has acidic conditions, and tumor sites are often acidic.
  • Degradable here means that it can be degraded through metabolic effects in the body (such as physiological effects, such as enzymes, such as redox, etc.), degraded by microenvironmental stimulation in specific parts of the body (such as acidic, alkaline), or stimulated by clinical treatments. Degradation occurs under conditions (such as light, heat, low temperature), etc.
  • “can exist stably” means that the linking group can maintain its existence as a complete linking group (a linking group is stably covalently connected to its adjacent group), then it is defined as “can exist stably”, where, Chemical changes are allowed to occur that maintain the integrity of the linker.
  • the chemical changes are not particularly limited, including but not limited to isomerization transformation, oxidation, reduction, ionization, protonation, deprotonation, substitution reaction, etc.
  • the conditions under which it can stably exist are not particularly limited, including but not limited to light, heat, low temperature, enzymes, redox, neutral, acidic, alkaline, physiological conditions, in vitro It can exist stably under conditions such as simulated environment, and preferably can exist stably under conditions such as light, heat, enzyme, redox, acidity, alkalinity, etc.
  • the stable existence here refers to the ability to maintain a stable connection in the metabolic cycle of the body without any special stimulation (such as the pH conditions of special parts, light, heat, low temperature during treatment, etc.) and will not cause damage due to bond breakage. Molecular weight is reduced (as long as integrity is maintained).
  • “can exist stably” is not an absolute concept.
  • amide bonds are much more stable than ester bonds under acidic or alkaline conditions.
  • “can exist stably” "The linker contains an amide bond.
  • a peptide bond, an amide bond formed by the dehydration and condensation of the ⁇ -carboxyl group of an amino acid molecule and the ⁇ -amino group of an amino acid molecule can be broken when encountering the action of a specific enzyme, so it is also included in the "degradable " in the connection base.
  • a stable linking group may be either a stable linking group or a degradable linking group. More generally, carbamate groups, thiocarbamate groups, etc. tend to degrade slowly, while amide bonds other than peptide bonds are stable during circulation in the body. Another example is that common ester bonds can be degraded under acid and alkali conditions, and ester bonds contained in special structures can also be degraded under ultraviolet light conditions. For another example, even if certain chemical bonds can be degraded under the action of a specific enzyme, if they are used clinically, if the circulation path does not pass through or basically does not pass through the specific enzyme environment (such as in the case of targeted drug administration), the corresponding chemical bond will It can still be considered as stable.
  • salts of the compounds of the general formula shall be understood to include salts of the compounds of the general formula.
  • the term "salt” used is selected from any one, any two, or a combination of two or more acid addition salts formed with inorganic and/or organic acids and base addition salts formed with inorganic and/or organic bases.
  • a basic moiety such as, but not limited to, pyridine or imidazole
  • an acidic moiety such as, but not limited to, carboxylic acid
  • zwitterions internal salts
  • Salt may be a pharmaceutically acceptable (ie, non-toxic, physiologically acceptable) salt or other salts.
  • the salts of the compounds of the general formula may be formed by reacting themselves with an amount (such as equivalents) of acid or base in a medium such as a salt precipitation or in an aqueous medium followed by lyophilization.
  • exemplary acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate, borate, butyrate Salt, citrate, camphorate, camphorsulfonate, cyclopentane propionate, digluconate, lauryl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate , Hemisulfate, Enanthate, Caproate, Hydrochloride, Hydrobromide, Hydroiodide, 2-Hydroxyethanesulfonate, Lactate, Maleate, Methanesulfonate, 2 -Naphthalene
  • Basic nitrogen-containing groups can be quaternized with reagents such as lower alkyl halides (e.g. methyl, ethyl, propyl and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g. dimethyl, diethyl, dibutyl and dipentyl sulfates), long chain halides (e.g. decyl, lauryl, tetradecyl and stearyl chlorides, bromides and iodides), Arylalkyl halides (eg, benzyl and phenethyl bromides) and others.
  • Both the acid addition salts and the base addition salts are preferably pharmaceutically acceptable salts and, for the purposes of this disclosure, are considered equivalent to the free forms of the corresponding compounds of the general formula.
  • heteroatoms in the present invention are not particularly limited, including but not limited to O, S, N, P, Si, F, Cl, Br, I, B, etc.
  • a group formed by losing some atoms or groups is also called a residue.
  • linking groups groups with a valence of 2 or more are collectively referred to as "linking groups".
  • the linking group can contain only one atom, such as ether group (-O-) and thioether group (-S-).
  • ether group -O-
  • thioether group -S-
  • connecting bond it means that the group may not exist and only plays a connecting role.
  • valence state of the group “multivalent” means that the valence state is at least 3.
  • connecting bond means that it only plays a connecting role and does not contain any atoms.
  • the "base" in the divalent linking group can be replaced by "bond” without changing the meaning.
  • a divalent ether group can also be called an ether bond (-O-)
  • a "linking base” cannot be called a "connecting bond.”
  • amino and amine group have the same meaning, including monovalent, divalent, trivalent, tetravalent neutral groups or cationic groups, substituted or unsubstituted.
  • -NH in CH3 - NH2 may be called “amino", "amine” or "primary amino”.
  • -NH- in CH 3 -NH-CH 3 can be called a “secondary amino group” or “secondary amino group”
  • -NH-CH 3 can also be understood as a methyl-substituted amino group.
  • amine group includes but is not limited to primary amine group, secondary amine group, tertiary amine group and quaternary ammonium ion.
  • amine group includes but is not limited to primary amine group, secondary amine group, tertiary amine group and quaternary ammonium ion.
  • -NR t R t and -N + R t R t R t where each R t is independently a hydrogen atom or any hydrocarbon structure.
  • the "hydrocarbon group” can be a monovalent hydrocarbon group, a divalent hydrocarbon group, a trivalent hydrocarbon group, ..., an n-valent hydrocarbon group, where n is the highest valence state that the hydrocarbon group can have.
  • hydrocarbylene group is a divalent hydrocarbon group.
  • “functional group”, that is, “functional group” is preferably a reactive group, a protected reactive group, a precursor of a reactive group, etc.
  • “Polymeric” means that the number of functional groups is at least 3, for example, polyols refer to compounds containing at least 3 hydroxyl groups, polythiols refer to compounds containing at least 3 mercapto groups, etc. It should be noted that it is allowed to contain other types of heterogeneous functional groups, such as tris(hydroxymethyl)aminomethane as a trihydric alcohol that also contains an amino group, and citric acid as a tricarboxylic acid that also contains a hydroxyl group.
  • the reactive group also includes its protected form, and the protected form can be deprotected in any appropriate step of the actual preparation process to obtain the corresponding active form.
  • ring-forming atoms are atoms that together constitute the ring skeleton.
  • the source of the amino acid in the present invention is not particularly limited unless otherwise specified. It can be a natural source, a non-natural source, or a mixture of the two.
  • the amino acid structure type in the present invention is not particularly limited unless otherwise specified. It can refer to either L-form or D-form, or a mixture of the two.
  • amino acid residues include amino acids in which hydrogen atoms are removed from the amino group and/or hydroxyl groups are removed from the carboxyl group and/or hydrogen atoms are removed from the thiol group and/or the amino group is protected and/or the carboxyl group is protected and/or the thiol group is protected. .
  • amino acid residues may be called amino acids.
  • carboxylic hydroxyl groups including all C-terminal carboxylic hydroxyl groups, including carboxylic hydroxyl groups on side groups such as aspartic acid and glutamic acid
  • hydrogen atoms on hydroxyl groups and hydrogen atoms on phenolic hydroxyl groups (casein amino acid)
  • the hydrogen atom on the sulfhydryl group such as cysteine
  • the hydrogen atom on the nitrogen atom including all N-terminal hydrogen atoms, and also includes the hydrogen atom in the amino group in the side group such as lysine, ornithine
  • the amino group on the amide such as asparagine, glutamine, etc.
  • guanidine A residue formed by the amino group in the side group or the hydrogen atom in the amino group.
  • Amino acid derivative residues refer to atoms or group parts that,
  • biologically related substances include but are not limited to documents CN104877127A, WO/2017/206540A, CN106967213A, CN108530637A, CN108530617A and the substances described, listed and cited in each cited document.
  • biologically relevant substances include, but are not limited to, the following substances: drugs, proteins, peptides, oligopeptides, protein mimetics, fragments and analogs, enzymes, antigens, antibodies and their fragments, receptors, small molecule drugs, nucleosides, Nucleotides, oligonucleotides, antisense oligonucleotides, polynucleotides, nucleic acids, aptamers, polysaccharides, proteoglycans, glycoproteins, steroids, steroids, lipids, hormones, vitamins, phospholipids, Glycolipids, dyes, fluorescent substances, targeting factors, targeting molecules, cytokines, neurotransmitters, extracellular matrix substances, plant or animal extracts, viruses, vaccines, cells, vesicles, liposomes, micelles, etc.
  • drugs drugs, proteins, peptides, oligopeptides, protein mimetics, fragments and analogs, enzymes, antigens, antibodies and their fragments
  • the biologically relevant substance can be the biologically relevant substance itself, or its precursor, activated state, derivatives, isomers, mutants, analogs, mimics, polymorphs, pharmaceutically acceptable salts, Fusion proteins, chemically modified substances, genetic recombinant substances, etc., can also be corresponding agonists, activators, activators, inhibitors, antagonists, modulators, receptors, ligands or ligands, antibodies and fragments thereof, Acting enzymes (such as kinases, hydrolases, lytic enzymes, oxygen reductases, isomerases, transferases, deaminase, deiminase, invertase, synthetase, etc.), substrates of enzymes (such as coagulation cascade proteases substrate, etc.) etc.
  • Acting enzymes such as kinases, hydrolases, lytic enzymes, oxygen reductases, isomerases, transferases, deaminase, deiminase, invertas
  • the derivatives include, but are not limited to, glycosides, nucleosides, amino acids, and polypeptide derivatives.
  • Chemical modification products that form new reactive groups that is, modifications resulting from modification of the reactive group to change the type, additional introduction of functional groups, reactive groups, amino acids or amino acid derivatives, polypeptides and other structures
  • Sexual products are all chemically modified substances of biologically related substances. Before or after the biorelevant substance is combined with the functionalized compound, it is also allowed to have a target molecule, accessory or delivery vehicle combined with it to form a modified biorelevant substance or a composite biorelevant substance.
  • the pharmaceutically acceptable salts may be inorganic salts, such as hydrochlorides, sulfates, phosphates, or organic salts, such as oxalates, malates, citrates, etc.
  • drug in the present invention includes any agent, compound, composition or mixture that provides physiological or pharmacological effects in vivo or in vitro, and often provides beneficial effects.
  • the types are not particularly limited, including but not limited to drugs, vaccines, antibodies, vitamins, foods, food additives, nutritional supplements, nutraceuticals and other agents that provide beneficial effects.
  • the scope of the physiological or pharmacological effects of the "drug” in the body is not particularly limited. It can have systemic effects or only local effects.
  • the activity of the "drug” is not particularly limited. It is mainly an active substance that can interact with other substances, or it can be an inert substance that does not interact; however, inert drugs can be converted into an active form through in vivo effects or certain stimulation. .
  • “small molecule drugs” are biologically relevant substances with a molecular weight of no more than 1000 Da, or small molecule mimics or active fragments of any biologically related substance.
  • oligopeptide refers to a peptide molecule formed of two or more amino acids. Preferably, the number of amino acid residues constituting the oligopeptide is 2 to 20. "Oligopeptide residues” may be monovalent, divalent, trivalent or higher.
  • monosaccharide group refers to a monosaccharide residue, that is, a monosaccharide skeleton, including open-chain monosaccharide groups and cyclic monosaccharide groups (such as furanose ring and pyranose ring). ).
  • the monosaccharide group in the present invention can be selected from the residues of any compound including but not limited to monosaccharides, sugar alcohols, deoxysugar, amino sugars, amino sugar derivatives (such as amide derivatives), sugar acids, and glycosides. , can be an open chain structure or a cyclic structure.
  • the amino residue formed by removing an amino hydrogen atom from amino sugar and the acyl group formed by removing the carboxyl hydroxyl group from sugar acid, etc.
  • the monosaccharides may include, but are not limited to, aldose (polyhydroxyaldehyde), ketose (polyhydroxyketone).
  • alkyl ether derivatives, methyl ether derivatives for example, lechonol.
  • the number of carbon atoms of the monosaccharide group in the present invention is not particularly limited, including but not limited to tetose, pentose, hexose, and heptose.
  • Pentose sugar and hexose sugar are preferred.
  • examples of tetroses, pentoses, hexoses, heptoses, sugar alcohols, deoxysugar, amino sugars, amide derivatives of amino sugars, sugar acids, glycosides, etc. include but are not limited to the structures disclosed in CN106967213A.
  • Micro-modification in the present invention refers to a chemical modification process that can be completed through a simple chemical reaction process.
  • the simple chemical reaction process mainly refers to chemical reaction processes such as protection, deprotection, salt complexation, decomplexation, ionization, protonation, deprotonation or changing of leaving groups.
  • micro-change form corresponds to "micro-modification” and refers to simple modifications such as protection, deprotection, salt complexation, decomplexation, ionization, protonation, deprotonation or changing of leaving groups.
  • the object can be formed The structural form of the labeled reactive group.
  • the said changing of the leaving group that is, the transformation of the leaving group, is for example but not limited to the transformation of the ester form to the acid chloride form.
  • the reaction process also involves the "protection" and "deprotection" processes of relevant groups.
  • the reactive group In order to prevent a reactive group from affecting the reaction, the reactive group is usually protected.
  • the target reactive group when there are two or more reactive groups, only the target reactive group is selectively allowed to react, thereby protecting other reactive groups.
  • the protecting group not only remains stable during the target reaction, but can also be removed by conventional technical means in the art if necessary.
  • deprotection and “deprotection” have the same meaning, and both refer to the process of converting a protected group from a protected form to an unprotected form.
  • the "hydroxyl protecting group” includes all groups that can be used as a general protecting group for hydroxyl groups.
  • the hydroxyl protecting group is preferably an alkanoyl group (such as acetyl, tert-butyryl), aralkanoyl (such as benzyl), benzyl, trityl, trimethylsilyl, tert-butyldimethylsilyl, alkenyl Propyl, acetal or ketal group.
  • metal sodium can also be used for reduction and cracking in ethanol or liquid ammonia; trityl group is generally removed through catalytic hydrogenation; trimethylsilyl group usually uses reagents containing fluoride ions (such as tetrabutylamine fluoride/anhydrous THF, etc.); tert-butyldisilyl ether is relatively stable and can withstand the ester hydrolysis conditions of alcoholic potassium hydroxide and mild reducing conditions (such as Zn/CH 3 OH, etc.), and can be used with fluoride ions (such as Bu 4 N + F - ) can be removed in tetrahydrofuran solution or with aqueous acetic acid at room temperature.
  • fluoride ions such as Bu 4 N + F -
  • dioxolanes, dioxanes, cyclic carbonates or cyclic borates are preferably formed.
  • the “mercapto protecting group” includes all groups that can be used as a general protecting group for mercapto groups. Similar to hydroxyl groups, thiol groups can be protected in the form of thioethers and thioesters. Thiol protecting group is preferably tert-butyl, benzyl, substituted benzyl, diphenylmethyl, substituted diphenylmethyl, trityl, acetyl, benzoyl, tert-butoxycarbonyl, benzyloxycarbonyl , thioacetal group or thioketal group.
  • the deprotection of thioether can be achieved by Na/NH 3 reduction under acid catalysis or reaction with heavy metal ions such as Ag + and Hg + , and then treated with hydrogen sulfide.
  • Some groups include S-diphenylmethyl, S-triphenylmethyl sulfide, S-2-tetrahydropyranyl, S-isobutoxymethyl semimercapto acetal, available (SCN) 2.
  • Iodine or thionyl chloride is oxidized to disulfide, and then reduced to mercaptan.
  • the formation and deprotection methods for thioesters are the same as for carboxylic acid esters.
  • the "carboxyl protecting group” refers to a protecting group that can be converted into a carboxyl group through hydrolysis and deprotection reaction of the carboxyl protecting group.
  • the carboxyl protecting group is preferably an alkyl group (such as methyl, ethyl, tert-butyl) or aralkyl group (such as benzyl), more preferably tert-butyl (tBu), methyl (Me) or ethyl (Et ).
  • protected carboxyl group refers to a group formed by a carboxyl group protected by a suitable carboxyl protecting group, preferably a methoxycarbonyl group, an ethoxycarbonyl group, a tert-butoxycarbonyl group, or a benzyloxycarbonyl group.
  • the carboxyl protecting group can be removed by hydrolysis under the catalysis of acid or alkali, and occasionally can be removed by pyrolysis reaction.
  • the tert-butyl group can be removed under mild acidic conditions
  • the benzyl group can be removed by hydrogenolysis.
  • the reagent for removing the carboxyl protecting group is selected from TFA, H 2 O, LiOH, NaOH, KOH, MeOH, EtOH and combinations thereof, preferably a combination of TFA and H 2 O, a combination of LiOH and MeOH, or a combination of LiOH and EtOH. .
  • the protected carboxyl group is deprotected to produce the corresponding free acid in the presence of a base that forms a pharmaceutically acceptable salt with the free acid formed by the deprotection.
  • amino protecting group is equivalent to “amino protecting group” and includes all groups that can be used as general protecting groups for amino/amine groups, such as aryl C 1-6 alkyl, C 1- 6 alkoxy C 1-6 alkyl, C 1-6 alkoxycarbonyl, aryloxycarbonyl, C 1-6 alkylsulfonyl, arylsulfonyl or silyl, etc.
  • the amino protecting group is preferably Boc (tert-butoxycarbonyl), Moz (p-methoxybenzyloxycarbonyl) and Fmoc (9-fluorenimethyleneoxycarbonyl).
  • Remove the amino protecting group Reagents include, but are not limited to, TFA, H 2 O, LiOH, MeOH, EtOH and combinations thereof, preferably a combination of TFA and H 2 O, a combination of LiOH and MeOH, or a combination of LiOH and EtOH.
  • the reagent for deprotecting Boc is TFA or HCl/EA; TFA is preferred.
  • the deprotecting agent used to remove Fmoc protection is N,N-dimethylformamide (DMF) solution containing 20% piperidine.
  • the "alkynyl protecting group” includes all groups that can be used as a general protecting group for alkynyl groups, and is preferably trimethylsilyl (TMS), triethylsilyl, or tert-butyldimethyl. Silyl (TBS) or biphenyldimethylsilyl.
  • TMS-protected alkynyl groups can be easily deprotected under alkaline conditions, such as K 2 CO 3 /MeOH or KOH/MeOH.
  • TBS-protected alkynyl groups can be deprotected in a solution of tetra-n-butylammonium fluoride in tetrahydrofuran (TBAF/THF).
  • the hydroxyl group protected by the hydroxyl protecting group is not particularly limited, and can be, for example, an alcoholic hydroxyl group, a phenolic hydroxyl group, etc.;
  • the amino/amine group protected by an amino protecting group is not particularly limited, and can be derived from, for example, primary amines, secondary amines, Hydrazine, amide, etc.
  • the amine groups in the present invention are not particularly limited, including but not limited to primary amine groups, secondary amine groups, tertiary amine groups, and quaternary ammonium ions.
  • the deprotection of the protected hydroxyl group is related to the type of hydroxyl protecting group.
  • the type of the hydroxyl protecting group is not particularly limited. Taking the protection of terminal hydroxyl groups by benzyl, silicon ether, acetal, and tert-butyl groups as examples, the corresponding deprotection methods include but are not limited to:
  • the hydrogenation reduction catalyst is not limited, but palladium and nickel are preferred.
  • the carrier is not limited, but alumina or carbon is preferred, and carbon is more preferred.
  • the amount of palladium used is 1 to 100 wt% of the protected hydroxyl compound, preferably 1 to 20 wt% of the protected hydroxyl compound.
  • the reaction solvent is not particularly limited as long as both the raw material and the product can be used as a solvent, but methanol, ethanol, ethyl acetate, tetrahydrofuran, and acetic acid are preferred; methanol is more preferred.
  • the hydrogen donor is not particularly limited, but hydrogen, cyclohexene, 2-propanol, ammonium formate, and the like are preferred.
  • the reaction temperature is preferably 25 to 40°C.
  • the reaction time is not particularly limited, and is inversely related to the amount of catalyst used, and is preferably 1 to 5 hours.
  • Preferred acetal or ketal compounds for such hydroxyl protection include ethyl vinyl ether, tetrahydropyran, acetone, 2,2-dimethoxypropane, benzaldehyde, and the like.
  • the deprotection of such acetals and ketals is achieved under acidic conditions, and the pH of the solution is preferably 0 to 4.
  • the acid is not particularly limited, but acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid, and nitric acid are preferred, and hydrochloric acid is more preferred.
  • the reaction solvent is not particularly limited as long as it can dissolve the reactants and products, and water is preferred.
  • the reaction temperature is preferably 0 to 30°C.
  • hydroxyl protection examples include trimethylsilyl ether, triethylsilyl ether, dimethyl tert-butylsilyl ether, tert-butyldiphenylsilyl ether, etc.
  • silicon ethers are deprotected by compounds containing fluoride ions, preferably tetrabutylammonium fluoride, tetraethylammonium fluoride, hydrofluoric acid, and potassium fluoride, and more preferably tetrabutylammonium fluoride and potassium fluoride.
  • the amount of fluorine-containing reagent is 5 to 20 times the molar equivalent of the protected hydroxyl group, preferably 8 to 15 times.
  • the reaction solvent is not particularly limited as long as it can dissolve the reactants and products.
  • An aprotic solvent is preferred, and tetrahydrofuran and dichloromethane are more preferred.
  • the reaction temperature is preferably 0 to 30°C. When the temperature is lower than 0°C, the reaction speed is slow and the protecting group cannot be completely removed.
  • the removal of tert-butyl group is carried out under acidic conditions, and the pH of the solution is preferably 0 to 4.
  • the acid is not particularly limited, but acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid, and nitric acid are preferred, and hydrochloric acid is more preferred.
  • the reaction solvent is not particularly limited as long as it can dissolve It suffices to decompose the reactants and products, preferably water.
  • the reaction temperature is preferably 0 to 30°C.
  • carboxy group activation refers to activating the carboxyl group with a carboxyl activator. Activation of the carboxyl group can promote the condensation reaction to proceed better, such as inhibiting the generation of racemic impurities in the condensation reaction, catalyzing the reaction speed, etc.
  • Carboxyl activating group is the residue of a carboxyl activator.
  • the carboxyl activator is N-hydroxysuccinimide (NHS), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), N-hydroxy-5 - A combination of one or more of norbornene-2,3-dicarboximide (HONb) and N,N-dicyclohexylcarbodiimide (DCC), preferably NHS/EDCI, NHS/DCC , the combination of HONb/DCC, most preferably the combination of NHS/EDCI or NHS/DCC.
  • NHS N-hydroxysuccinimide
  • EDCI 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride
  • HONb norbornene-2,3-dicarboximide
  • DCC N,N-dicyclohexylcarbodiimide
  • the condensing agent used in the reaction is not limited, but is preferably N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-(3-dimethylaminopropyl)carbonyldiimide Imine hydrochloride (EDC ⁇ HCl), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), benzotriazole Azole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), most preferably DCC.
  • the general amount of condensing agent used is 1 to 20 times the molar equivalent of carboxylic acid, preferably 5 to 10 times.
  • An appropriate catalyst such as 4-dimethylaminopyridine (DMAP) can be added to this reaction.
  • the obtained product can be purified through extraction, recrystallization, adsorption treatment, precipitation, reverse precipitation, membrane dialysis or supercritical extraction and other purification methods.
  • the step/multi-step reaction can be completed in multiple practical operations by skilled personnel, or can be completed in one practical operation.
  • two reactive groups in a low molecular weight lipid compound undergo a coupling reaction with a single-chain polyethylene glycol derivative reagent to obtain a molecule with two single-chain PEG arms.
  • the entire process can also be regarded as the introduction of the polyethylene glycol component in steps/multiple steps.
  • the base used in the reaction can be an organic base (such as triethylamine, pyridine, 4-dimethylaminopyridine, imidazole or diisopropylethylamine, preferably triethylamine or pyridine), or it can be Inorganic bases (eg K 2 CO 3 ).
  • organic base such as triethylamine, pyridine, 4-dimethylaminopyridine, imidazole or diisopropylethylamine, preferably triethylamine or pyridine
  • Inorganic bases eg K 2 CO 3
  • the repeating unit of the polyethylene glycol component is an oxyethylene unit, that is, -CH 2 CH 2 O- or -OCH 2 CH 2 -, which is also recorded as an EO unit, and the number of repeating units is also recorded as an EO unit. number, the average number of repeating units is also recorded as the average number of EO units, and the number average is preferred.
  • the molecular weight/degree of polymerization of a single molecule of a compound, the number average molecular weight/number average degree of polymerization of the compound components in the macroscopic substance are "equal” or “the same” or “equal to” (including other forms Equivalent expression), unless otherwise specified, is not limited to being strictly equal in numerical value, but the index value is close or approximately equal, and the preferred deviation of the close or approximately equal value does not exceed ⁇ 10%, usually in The default value is the base.
  • the same or equal number of oxyethylene units in a single compound molecule and the general formula means that they are strictly numerically equal; for example, if the number of EO units of a certain PEG component is set to 11, it is equal to The set value of 12 does not fall within the set range; however, for macroscopic products obtained by using a certain preparation method in order to obtain compound components containing a set number of EO units, due to limitations of the preparation method and purification method, the macroscopic product may The product also contains other EO unit number components besides the target EO unit number component.
  • the molecular weight value is allowed to fall within a certain range of the given value (including the endpoints). , preferably within the range of ⁇ 10%); when the number of oxyethylene units is used to describe the preset molecular weight of the general formula of a monodisperse component, there is no range fluctuation and it is a discrete point, but the prepared product may be caused by uneven molecular weight.
  • the average number of EO units fluctuates within a certain range (not exceeding ⁇ 10% or ⁇ 1, preferably not exceeding ⁇ 5% or ⁇ 0.5).
  • the molecular weight of mPEG (methoxypolyethylene glycol unit) is 5kDa, which means that the molecular weight value of a single molecule in the general formula is between 4500 and 5500Da.
  • the average molecular weight of the corresponding components of the corresponding prepared product is 5kDa, that is, the average molecular weight
  • the product when the value is between 4500 and 5500Da is the target product, and only the components with a molecular weight within this range contribute to the content of the target component; and if mPEG is designed to have 22 oxyethylene units, then in the general formula
  • the number of EO units of all compound molecules should be strictly 22, but the prepared product may be a mixture of compounds with 20, 21, 22, 23, and 24 EO units, in which case the average number of EO units falls within the range of 22 ⁇ 2.2 (preferably Within the range of 22 ⁇ 1.1), the target component is deemed to be obtained, and the components whose molecular weight falls within this range can be regarded as the target component for calculation of purity.
  • mPEG is a methoxy-terminated polyethylene glycol segment, and its structural formula is: Among them, n i is the degree of polymerization of the polyethylene glycol chain, which is an integer selected from 1 to 1000.
  • the number of repeating units in the "single-chain component" is at least 2.
  • lipid is also called “lipid”, including but not limited to esters of fatty acids, and is characterized by generally poor solubility in water, but being soluble in many non-polar organic substances. .
  • lipids generally have poor solubility in water
  • certain classes of lipids e.g., lipids modified with polar groups such as DMG-PEG2000
  • lipids modified with polar groups such as DMG-PEG2000
  • Known types of lipids include biomolecules such as fatty acids, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, triglycerides and phospholipids.
  • lipids include simple esters, complex esters and derived lipids.
  • the simple esters are esters composed of fatty acids and alcohols, which can be divided into three categories: fats, oils and waxes.
  • the complex esters are "lipoid compounds", also known as “lipoids” or “lipoids”, including phospholipids, sphingolipids, glycolipids, steroids and sterols, and lipoproteins.
  • the derived lipids including simple lipid derivatives and complex lipid derivatives, have the general properties of lipids.
  • lipids may be synthetic or derived (isolated or modified) from natural sources or compounds.
  • liposome is a closed vesicle formed by lipid self-assembly and has one or more bilayer structures.
  • lipid nanoparticle refers to nanometer-sized (such as 1 nm to 1000 nm) particles containing one or more types of lipid molecules.
  • the LNP may further comprise at least one non-lipid payload molecule (eg, one or more nucleic acid molecules).
  • the LNPs comprise non-lipid payload molecules partially or completely encapsulated inside a lipid shell.
  • the payload is a negatively charged molecule (e.g., mRNA encoding a viral protein) and the lipid component of the LNP comprises at least one cationic lipid and at least one pegylated Lipids.
  • cationic lipids can interact with negatively charged payload molecules and facilitate payload incorporation and/or encapsulation into LNPs during LNP formation.
  • other lipids that can form part of the LNP include, but are not limited to, neutral lipids and steroid lipids.
  • cationic means that the corresponding structure is permanently or non-permanently able to bear a positive charge in response to certain conditions (such as pH).
  • cations include both permanent cations and cationizable ones.
  • a permanent cation is a corresponding compound or group or atom that is positively charged at any pH value or hydrogen ion activity of its environment load. Typically, a positive charge is generated by the presence of quaternary nitrogen atoms. When a compound carries multiple such positive charges, it may be called a polyvalent cation.
  • Cationizable means that a compound or group or atom is positively charged at lower pH and uncharged at the higher pH of its environment.
  • cationizable compounds, groups or atoms are positively charged at high hydrogen ion concentrations and uncharged at low hydrogen ion concentrations or activities. It depends on the individual properties of the cationizable or polycationizable compound, in particular the pKa of the corresponding cationizable group or atom, which is charged or uncharged at the stated pH or hydrogen ion concentration.
  • the fraction of positively charged cationizable compounds, groups or atoms can be estimated using the so-called Henderson-Hasselbalch equation, which is well known to those skilled in the art .
  • the entirety or portion of the cationizable compound is positively charged at physiological pH (eg, about 7.0-7.4).
  • the cationizable compound in whole or in part, is neutral at physiological pH (eg, about 7.0-7.4), but positively charged at lower pH (eg, about 5.5 to 6.5). charge.
  • the pKa of all or a portion of the cationizable compound preferably ranges from about 5 to about 7.
  • cationic lipid refers to a lipid that is positively charged at any pH value or hydrogen ion activity of its environment, or is capable of responding to the pH value of its environment (such as the environment in which it is intended to be used) or Positively charged lipids that are hydrogen ion active.
  • the term “cationic” encompasses the scope of “permanently cationic” and “cationizable”.
  • the positive charge in the cationic lipid results from the presence of quaternary nitrogen atoms.
  • cationic lipids include zwitterionic lipids, which are positively charged in the environment in which they are intended to be administered (eg, at endosomal pH).
  • a liposome or LNP contains a cationizable lipid, it is preferred that it is at a pH of about 1 to 9, preferably 4 to 9, 5 to 8, or 6 to 8 , most preferably at an endosome pH value (eg, about 5.5 to 6.5) in which about 1% to 100% of the cationizable lipids are cationized.
  • Cationic lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy) Propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 3-(didodecylamino) -N1,N1,4-tri-dodecyl-1-piperazineethylamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tri-decane Dialkyl-1,4-piperaz
  • PEGylated lipid refers to a molecule containing a lipid moiety and a polyethylene glycol moiety.
  • PEGylated lipids also include, but are not limited to, polyethylene glycol-1,2 dimyristate glyceryl (PEG-DMG), polyethylene glycol-dimyristate Stearoylphosphatidylethanolamine (PEG-DSPE), PEG-cholesterol, polyethylene glycol-diacylglycerol (PEG-DAG), polyethylene glycol-dialkoxypropyl (PEG-DAA), specifically include Polyethylene glycol 500-dipalmitoylphosphatidylcholine, polyethylene glycol 2000-dipalmitoylphosphatidylcholine, polyethylene glycol 500-distearoylphosphatidylethanolamine, polyethylene glycol 2000-distearoylcholine Fatty acylphosphatidy
  • neutral lipid refers to any one of many lipid substances that exist in the form of uncharged or neutral zwitterions at a selected pH, preferably phospholipids.
  • lipids include, but are not limited to, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (MPC), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl -sn- Glyceryl-3-phosphocholine (DSPC), 1,2-disundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine Base (POPC), 1,2-di-O-
  • steroid lipid is selected from the group consisting of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicesterol, tomatine, ursolic acid, and ⁇ -tocopherol. any one and combinations thereof.
  • the liposomes/lipid nanoparticles in the present invention can be called "PEGylated liposomes/lipid nanoparticles” when they contain PEGylated lipids, and they can be called “cationic lipids” when they contain cationic lipids.
  • Plasmids/lipid nanoparticles when containing both PEGylated lipids and cationic lipids, can be called either "PEGylated liposomes/lipid nanoparticles” or "cationic liposomes” /lipid nanoparticles”.
  • the "N/P ratio” refers to the molar ratio of nitrogen atoms in the cationic lipid to phosphoric acid in the nucleic acid.
  • nucleic acid refers to DNA or RNA or a modified form thereof, which contains the purine or pyrimidine bases (adenine “A”, cytosine “C”, guanine “G”, thymine) present in DNA. Pyrimidine “T”) or the purine or pyrimidine bases present in RNA (adenine "A”, cytosine "C”, guanine “G”, uracil “U”).
  • RNA refers to ribonucleic acid that may be naturally occurring or non-naturally occurring.
  • RNA may include modified and/or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers.
  • RNA may include cap structures, chain-terminating nucleosides, stem loops, polyadenylation sequences, and/or polyadenylation signals.
  • the RNA may have a nucleotide sequence encoding a polypeptide of interest.
  • the RNA may be messenger RNA (mRNA).
  • RNA may be selected from the non-limiting group consisting of: small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, single-stranded guide RNA (sgRNA), cas9mRNA, and mixtures thereof.
  • siRNA small interfering RNA
  • aiRNA asymmetric interfering RNA
  • miRNA microRNA
  • dsRNA Dicer-substrate RNA
  • shRNA small hairpin RNA
  • mRNA single-stranded guide RNA
  • cas9mRNA single-stranded guide RNA
  • antisense oligonucleotides or small interfering RNA can inhibit the expression of target genes and target proteins in vitro or in vivo.
  • FLuc mRNA can express luciferase protein, which emits biological light in the presence of a luciferin substrate, so FLuc is often used in mammalian cell culture to measure gene expression and cell activity.
  • inhibiting the expression of a target gene refers to the ability of a nucleic acid to silence, reduce or inhibit the expression of a target gene.
  • a test sample eg, a sample of cells in culture medium expressing the target gene
  • a control sample that has not been exposed to or administered the nucleic acid (eg, a sample of cells in culture medium expressing the target gene).
  • the expression of the target gene in the control sample can be assigned a value of 100%.
  • the target gene expression level is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50% relative to the target gene expression level in the control sample or control mammal , 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 0%, the expression of the target gene is inhibited.
  • methods for determining the expression level of target genes include, but are not limited to, dot blotting, northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme action and phenotypic assay.
  • transfection refers to the introduction of a species (eg, RNA) into a cell. Transfection can occur, for example, in vitro, ex vivo or in vivo.
  • a species eg, RNA
  • antigen typically means an antigen that is recognized by the immune system, preferably by the adaptive immune system, and is capable of triggering an antigen-specific immune response, for example by forming antibodies and/or antigens as part of an adaptive immune response.
  • the antigen may be or comprise a peptide or protein that may be presented to T cells by the MHC.
  • the antigen may be a translation product of the provided nucleic acid molecule, preferably mRNA as defined herein.
  • fragments, variants and derivatives of peptides and proteins containing at least one epitope are also understood to be antigens.
  • delivery refers to providing an entity to a target.
  • drugs and/or therapeutic and/or prophylactic agents are delivered to a subject, which is tissue and/or cells of a human and/or other animal.
  • pharmaceutically acceptable carrier refers to a diluent, adjuvant, excipient or vehicle that is administered with a therapeutic agent and is suitable for contact with humans and/or within the scope of reasonable medical judgment. Tissues from other animals without undue toxicity, irritation, allergic reactions, or other problems or complications commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions of the present invention include, but are not limited to, sterile liquids, such as water, and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is an exemplary carrier when the pharmaceutical composition is administered intravenously.
  • Physiological saline and aqueous glucose and glycerol solutions may also be used as liquid carriers, particularly for injections.
  • suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerin, propylene glycol, water, Ethanol etc.
  • the compositions may also, if desired, contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents.
  • Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.
  • excipients include but are not limited to anti-adhesive agents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), emollients, emulsifiers, fillers (diluents), Film-forming agents or coatings, flavoring agents, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and hydration water.
  • excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, dicalcium phosphate, calcium stearate, croscarmellose sodium, crospolyvinylpyrrolidone, citric acid, Crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methyl Thionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, phenylparaben, retinol palm Acid ester, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (alpha-tocopo
  • compositions of the invention may act systemically and/or locally.
  • they may be administered by a suitable route, for example by injection (eg intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular injection, including instillation) or transdermally; or by oral, buccal, transdermal Nasal, transmucosal, topical, in the form of ophthalmic preparations or by inhalation.
  • a suitable route for example by injection (eg intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular injection, including instillation) or transdermally; or by oral, buccal, transdermal Nasal, transmucosal, topical, in the form of ophthalmic preparations or by inhalation.
  • the pharmaceutical compositions of the present invention can be administered in suitable dosage forms.
  • a vaccine is a preventive or therapeutic material that provides at least one antigen or antigenic function. Antigens or antigen functions stimulate the body's adaptive immune system to provide an adaptive immune response.
  • treatment means the treatment and care of a patient for the purpose of combating a disease, disorder or condition, and is intended to include delaying the progression of the disease, disorder or condition, alleviating or alleviating symptoms and complications, and/or curing or eliminating the disease , disorder or illness.
  • the patient to be treated is preferably a mammal, especially a human.
  • a nitrogen-branched PEGylated lipid characterized in that its structure is as shown in general formula (1):
  • B 1 and B 2 are each independently a connecting bond or an alkylene group
  • L 1 , L 2 , L d and y L x are each independently a connecting bond or a divalent connecting group L;
  • R 1 and R 2 are each independently a C 1-50 aliphatic hydrocarbon group or a C 1-50 aliphatic hydrocarbon derivative residue containing 0-10 heteroatoms; the heteroatoms are B, O, N, Si, P or S;
  • N core is a multivalent group with a valence state of y+1; N core contains a trivalent nitrogen atom branched core connected to L d ;
  • y is 2, 3, 4, 5, 6, 7, 8, 9 or y ⁇ 10, preferably 2 or 3;
  • XPEG is the polyethylene glycol component.
  • the compound of general formula (1) can exist in an unsolvated or solvated form, including a hydrated form.
  • solvated forms with a pharmaceutically acceptable solvent such as water, ethanol, etc. are equivalent to the unsolvated forms for the purposes of this disclosure.
  • the compounds of general formula (1) and their salts and solvates can exist in their tautomeric forms, including keto-enol tautomers, amide-imidic acid tautomers, and lactam-lactam tautomers. transition, enamine-imine interconversion, proton transfer interconversion and valence interconversion.
  • the compound of general formula (1) is understood to include its salts, tautomers, stereoisomers and solvates.
  • any XPEG includes x RPEGs; y x are each independently selected from an integer of 1-4, preferably y x are all 1; when y x are greater than or equal to 2, Preferably all x are equal;
  • the structure of the RPEG is in,
  • n i is the degree of polymerization of the polyethylene glycol chain, which is an integer selected from 4 to 250, and i is an integer selected from 1 to 20; the polydispersity of the polyethylene glycol chain is polydispersity or monodispersity;
  • T is H, C 1-6 alkyl or R 01 -L 01 -;
  • L 01 is a connecting bond or a divalent linking group L
  • R 01 is a functional group that can interact with biologically relevant substances.
  • n 1 and n 2 are each independently selected from an integer from 4 to 100, preferably from an integer from 10 to 60, more preferably from an integer from 10 to 45, most preferably 10, 11, 12, 20, 21, Any one of 22, 23, 24, and 25.
  • the stability of all divalent linking groups is not particularly limited. Any divalent linking group or the divalent linking group composed of it and an adjacent heteroatom group is independently a stable connection. base or degradable linker.
  • the structures of all divalent linking groups are not particularly limited and can be linear structures, branched structures or cyclic structures; the number of non-hydrogen atoms of the divalent linking groups is not particularly limited and can be selected independently.
  • the non-hydrogen atom is any one of a combination of carbon atom and carbon atom, a combination of carbon atom and heteroatom, or a combination of heteroatom and heteroatom.
  • the alkylene group is -(CR c R c ) t -, wherein each occurrence of t is independently an integer from 1 to 12, preferably t is an integer from 1 to 4, and more preferably t is 1 or 2; R Each time c appears, it is independently any one of H, C 1-6 alkyl group, carbocyclic ring-containing group, heterocyclic ring-containing group, and side chain group of amino acid, preferably H, methyl, Any one of isopropyl, isobutyl and benzyl, more preferably H or benzyl, most preferably H.
  • each of the y L x's independently be in any of the following situations:
  • L x is a linking group containing main chain heteroatoms, preferably L x or -OL Thioester group, carbonate group, monothiocarbonate group, dithiocarbonate group, trithiocarbonate group, secondary amino group, amide group, urethane group, monothiocarbamate group , any one of dithiocarbamate group, imine group, triazole linker, 4,5-dihydroisoxazole linker, 2,5-dioxopyrrolidine linker, or any of them Any one, any two or more combinations with alkylene groups, more preferably L x or -OL x is an ether group, an ester group, a carbonate group, a disulfide bond, an amide group or a urethane group;
  • L x or -OL x contains a linker generated by coupling reaction: L x or -OL x is generated through alkylation reaction, amidation reaction, esterification reaction, thioesterification reaction, click reaction, The linking group generated by cycloaddition reaction, Diels-Alder addition reaction or 1,3-dipolar cycloaddition reaction, or a combination of any one or more of the above linking groups and a hydrocarbylene group.
  • L d is selected from the group consisting of connecting bonds, alkylene groups, or any one of the following categories, or a combination of any 1 to 4 species and alkylene groups:
  • B 1 and B 2 are each independently a connecting bond or a linear C 1-14 alkylene group; the C 1-14 alkylene group has 0-2 hydrogen atoms.
  • R q substitutions each occurrence of R q is independently -(CH 2 ) tq C[(CH 2 ) tq H] 3 , -(CH 2 ) tq O(CH 2 ) tq H, - (CH 2 ) tq S(CH 2 ) tq H or -(CH 2 ) tq N[(CH 2 ) tq H] 2 , where tq is independently an integer of 0-4 each time it appears; preferably R q each time Each occurrence is independently -OH or -CH 3 ;
  • R 1 and R 2 each independently contain 0-8 carbon-carbon double bonds and/or 0-8 carbon-carbon triple bonds, and each independently contains 0-10 hydrogen atoms.
  • Each is independently substituted by 0-10 R m ; each occurrence of R m is independently selected from linear C 1-6 alkyl, branched C 1-6 alkyl, C 3-12 cycloalkyl Any one of base, C 2-8 alkenyl, C 2-8 alkynyl, phenyl, and benzyl; preferably R m is methyl;
  • R 1 and R 2 are each independently selected from any one of RL , RB and R r .
  • R 1 and R 2 are each independently selected from RL or RB ;
  • R L is selected from any one of the following structures or any one of the cis and trans isomers:
  • R L is preferably any one of the following structures:
  • R B is more preferably any one of the following structures:
  • R r is a C 4-30 alkyl group or a C 4-30 heteroalkyl group containing a cyclic structure, preferably
  • R L is preferably a C 1-30 linear aliphatic hydrocarbon group containing 0-10 R m ; more preferably, it is a C 1-25 linear alkyl group, tridecane-8- Alkenyl, tetradec-9-enyl, pentadeca-8-enyl, hexadeca-9-enyl, heptadeca-5-enyl, heptadeca-8-enyl, heptadeca-9-enyl Carbon-10-alkenyl, heptadeca-8,11-dienyl, octadec-6-enyl, octadec-9-enyl, octadec-11-enyl, octadec- 9,12-dienyl, octadec-9,12,15-trienyl, octadec-8,11,14-trienyl, nonadeca-10-
  • the number average molecular weight of any two RPEGs is the same or different, and each RPEG Each is independently monodisperse or polydisperse.
  • the degree of polymerization of any two RPEGs is the same or different; preferably, the degree of polymerization of all RPEGs is the same.
  • each XPEG is independently a linear structure or a non-linear structure; the non-linear structure is selected from any of the following situations:
  • Case (1) Constructed from multivalent residues of any of the following small molecules: polyfunctionalized small molecules, polyfunctionalized small molecules containing a heterogeneous functional group, and amino acid small molecules;
  • Case (2) Constructed from any of the following non-linear combinations: branched structure, comb structure, tree structure, tree-like structure, ring structure, hyperbranched structure and any combination thereof;
  • the polyfunctionalized small molecules are preferably polyols, polythiols, polycarboxylic acids, polyvalent primary amines or polyvalent secondary amines;
  • the polyfunctionalized small molecule containing a heterogeneous functional group is preferably a polyhydric alcohol containing a heterogeneous functional group, a polythiol containing a heterogeneous functional group, a polycarboxylic acid containing a heterogeneous functional group, a polycarboxylic acid containing a heterogeneous functional group, Polymeric primary amines or polyvalent secondary amines containing a heterogeneous functional group;
  • the amino acid small molecules are preferably glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, and asparagine. , glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine, ornithine or citrulline;
  • T's in the same molecule have the same structure.
  • R 01 is a functional group that can interact with biologically relevant substances, wherein the reaction is selected from the group consisting of covalent bond formation, hydrogen bond formation, fluorescence effect and targeting effect. ; R 01 is selected from any one of reactive groups, variations of reactive groups, functional groups with therapeutic targeting, and fluorescent functional groups; wherein, the variations are selected from reactive Any one of the precursor of a group, an active form with a reactive group as a precursor, an active form in which the reactive group is substituted, or an inactive form in which the reactive group is protected; wherein, the reactive group
  • the precursor of the group means that it can be transformed into the reaction after at least one process of oxidation, reduction, hydration, dehydration, electron rearrangement, structural rearrangement, salt complexation and decomplexation, ionization, protonation, and deprotonation.
  • the structure of the sex group means that it can be transformed into the reaction after at least one process of oxidation, reduction, hydration, dehydration, electron rearrangement, structural rear
  • R 01 is any functional group selected from the following Class A to Class I or a variation thereof:
  • Class A active ester group, similar structure of active ester group; wherein, the active ester group is selected from succinimide active ester group, p-nitrobenzene active ester group, o-nitrobenzene active ester group, 1,3,5 -Trifluorobenzene active ester group, 1,3,5-trichlorobenzene active ester group, 1,3,5-tribromobenzene active ester group, 1,3,5-triiodobenzene active ester group, pentafluorobenzene Active ester group, imidazole active ester group, benzotriazole active ester group, thiazolidine-2-thione active ester group, tetrahydropyrrole-2-thione active ester group, 2-mercaptobenzothiazole active ester group, Any one of the 1-oxo-3-thioxoisoindoline active ester groups; wherein the similar structure of the active ester group is an active carboxylic acid ester group or an active
  • Class B Carboxyl group, protected carboxyl group, sulfonic acid group, sulfonate ester group, sulfinic acid group, sulfinate ester group, sulfenic acid group, ester group, thioester group, dithioester group, carbonic acid Ester group, thiocarbonate group, dithiocarbonate group, trisulfide group Carbonate group, xanthate group, tetrathiodiester group, sulfone group, sulfoxide group, methacryloyl group, hydroxamic acid group, thiohydroxamic acid group, sulfonyl halide group, thiocarboxylic group ;
  • Class C aldehyde group, hydrated aldehyde group, sulfide group, acid halide group, ketone group, hydrated ketone group, thione group, thione hydrate group, glyoxal group, acetal group, monoacetal group, Bisthiacetal group, ketal group, monothione group, bisthione group, hemiacetal group, thiohemiacetal group, hemiketal group, orthoacid group, protected orthoacid group, Orthoester group, cyanate group, thiocyanate group, isocyanate group, isothiocyanate group, oxazoline group, isoxazoline group;
  • Class D primary amino group, secondary amino group, protected amino group, hydroxylamine group, thiol group, disulfide group, halogen atom, haloacetamide group, ammonium salt, hydrazine group, tetramethylpiperidinyloxy group, dioxa Piperidinyloxy group, O-carbonylhydroxylamine group, amide group, imide group, hydrazide group, sulfonylhydrazine group, hydrazone group, imine group, enamine group, alkynamine group, carbamate group, a Thiocarbamate group, dithiocarbamate group;
  • Category E urea group, thiourea group, guanidine group and its protonated form, amidine group and its protonated form, anhydride group, squaryl acid group, squaryl acid ester group, semisquaryl acid group, semisquaryl acid ester group, imidazole -1-Carboxamide group, imide ester group, nitrone group, aldoxime group, ketoxime group;
  • Class F maleimide group, furan-protected maleimide group, acrylate group, N-acrylamide group, N-methacrylamide group, methacrylate group, maleamic acid group, 1,2,4-triazolin-3,5-dione group, linear azo compound group, cyclic azo compound group;
  • Class G alkenyl, alkenyl hydrocarbon, cycloalkenyl, alkynyl, alkynyl hydrocarbon, protected alkynyl, cycloalkynyl, linear conjugated diene, cyclic conjugated diene, hetero-containing Atomic cyclic conjugated diene group, epoxy group, 1,2,4,5-tetrazinyl group, azide group, nitrile oxide group, cyano group, isocyanate group, diazo group, diazonium ion , azo oxide group, nitrile imine group, N-oxyaldimine group, tetrazolium group, 4-acetyl-2-methoxy-5-nitrophenoxy group and its diazotized form, imidazole base, indolyl group; wherein, the cycloalkene group is selected from any one of cyclooctene group, norbornenyl group, norbornadienyl group, oxanorbornenyl group, and
  • Class H hydroxyl group, protected hydroxyl group, protected dihydroxyl group, siloxy group, trihydroxysilyl group, protected trihydroxysilyl group; where the hydroxyl group is selected from alcoholic hydroxyl group, phenolic hydroxyl group, enol hydroxyl group, semi-hydroxyl group Any of the acetal hydroxyl groups;
  • Class I Monosaccharide base, selected from allose, altrose, arabinose, cladinose, erythrose, erythrulose, fructose, fucose, fucosamine, fucose, ink Chlorose, galactosamine, galactosamine, N-acetyl-galactosamine, galactose, glucosamine, N-acetyl-glucosamine, glucosamine, glucose, glucose-6-phosphate, paleo Glyceraldehyde, L-glyceraldehyde, D-mannose, glycerin, glyceraldehyde, dihydroxyacetone, gulose, idose, lyxose, mannosamine, mannose, mannose-6-phosphate , Mannoheptulose, psicose, quinose, quinosamine, rhamnitol, rhamnosamine, rhamnose, ribose
  • the protected hydroxyl group is preferably any one of ether, silicon ether, ester, carbonate, and sulfonate;
  • the protected amino group is preferably carbamate, amide, imide, N-alkylamine, N-aryl Any one of amine, imine, enamine, imidazole, pyrrole, and indole;
  • the protected mercapto group is preferably any one of thioether, disulfide, silicon sulfide, and thioester;
  • the protected carboxyl group is preferably It is the form in which the carboxyl group is protected by any one of methyl, ethyl, tert-butyl and benzyl;
  • the protected alkynyl group is preferably in the form of the alkynyl group being protected by a silicon group;
  • the protected dihydroxyl group preferably has a protective group and two oxygen atoms constitute an acetal structure of a five-membered ring or a six
  • R 01 is selected from any functional group in the following categories A to I or variations thereof:
  • X 1 is a halogen atom, selected from any one of fluorine atom, chlorine atom, bromine atom and iodine atom;
  • Y 1 is selected from C 1-5 alkyl, vinyl, phenyl, benzyl, p-methylphenyl, 4-(trifluoromethoxy)phenyl, trifluoromethyl and 2,2,2 - any one of trifluoroethyl;
  • R d2 is an organic group, preferably each time it appears, it is independently selected from any one of C 1-5 alkyl, C 2-5 alkenyl, C 2-5 alkynyl and phenyl, and the alkyl Base, alkenyl, alkynyl and phenyl are each independently substituted or unsubstituted;
  • W is a leaving group, selected from any one of -F, -Cl, -Br, -I and -SPh;
  • M 5 is a ring-forming atom, selected from any one of carbon atoms, nitrogen atoms, phosphorus atoms and silicon atoms;
  • the cyclic structure in which M 5 is located is a 3-30-membered ring, preferably a 3-20-membered ring, and more preferably 3 to 16 membered rings, more preferably 5 to 16 membered rings;
  • the cyclic structure is preferably any one of the following groups, any of the substituted forms, or any of the hybrid forms: cyclohexane, furan Sugar ring, pyranose ring, benzene, tetrahydrofuran, pyrrolidine, thiazolidine, cyclohexene, tetrahydropyran, piperidine, 1,4-dioxane, pyridine, pyridazine, pyrimidine, pyrazine, 1 ,3,5-triazine, 1,4,7-triazacyclononane,
  • cyclic structures containing an acetal group, a disulfide bond, an amine group, an imide group, an anhydride group, an azo group, a carbon-carbon double bond, a carbon-carbon triple bond, and a conjugated diene on the ring skeleton.
  • the cyclic structure is selected from carbocycle, heterocycle, benzoheterocycle, substituted carbocycle, substituted heterocycle or substituted benzoheterocycle;
  • Q is an atom or substituent that contributes to the induction and conjugation effect of unsaturated bond electrons; when Q is on the ring, the number is one or more; when the number is multiple, it is the same structure, or A combination of two or more different structures; when it is a substituent, Q has a straight chain structure, a branched chain structure containing side groups, or a cyclic structure;
  • the modified form is selected from the group consisting of a precursor of a reactive group, an active form using a reactive group as a precursor, an active form in which the reactive group is substituted, and an inactive form in which the reactive group is protected.
  • the precursor of the reactive group refers to at least one of oxidation, reduction, hydration, dehydration, electron rearrangement, structural rearrangement, salt complexation and decomplexation, ionization, protonation, and deprotonation. process, the structure of the reactive group can be transformed.
  • the aforementioned R 01 is a hydroxyl group, a mercapto group, an active ester group, an active carbonate group, a sulfonate group, an amino group, a maleimide group, a succinimide group, a carboxyl group, an acid chloride group
  • the modified form is selected from the precursor of the reactive group, the active form using the reactive group as a precursor, the active form in which the reactive group is substituted, the reactive form Any inactive form in which the group is protected; wherein, the precursor of the reactive group refers to the precursor that has undergone oxidation, reduction, hydration, dehydration, electron rearrangement, structural rearrange
  • T is a hydrogen atom or a C 1-6 alkyl group, preferably a methyl group.
  • N core is a multivalent nuclear structure of any type such as atomic, branched, or cyclic, and the multivalent nuclei are trivalent nitrogen nuclei; when N core is the polyvalent When it is a valent branched chain core structure or a multivalent cyclic core structure, it contains at least one trivalent nitrogen atom nucleus, and is preferably any of the following:
  • Case (1) Constructed from multivalent residues of any of the following small molecules: polyfunctionalized small molecules, polyvalent heterofunctionalized small molecules, and amino acids;
  • Case (2) Constructed from any of the following nonlinear structures: branched structure, comb structure, tree structure, tree-like structure, ring structure, hyperbranched structure and their combinations;
  • the polyfunctionalized small molecules are preferably polyols, polythiols, polycarboxylic acids, polyvalent primary amines or polyvalent secondary amines;
  • the polyvalent heterofunctional small molecule is preferably a polyol containing a heterofunctional group, a polythiol containing a heterofunctional group, a polycarboxylic acid containing a heterofunctional group, a polyprimary amine containing a heterofunctional group, or a polyvalent primary amine containing a heterofunctional group.
  • the amino acids are preferably glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, and glutamine.
  • N core is an amino acid residue, a dimer amino acid residue or a polyamino acid residue.
  • the polyamino acid contains at least 3 amino acid units.
  • the amino acid units in the dimer amino acid and polyamino acid are each independent and The amino acid units between any two have the same or different structures.
  • N core is a trivalent core structure, preferably N core is any structure in any of the following categories:
  • Category (1) trivalent nitrogen nucleus structure; specifically the following structure:
  • Category (2) cyclic trivalent core structure containing nitrogen heteroatoms, wherein the cyclic structure is selected from any one of the following, any substituted form, or any hybrid form: pyrrolidine, piperazine Ridine, pyrazine, 1,4,7-triazacyclononane, cyclic tripeptide, indole, isoindole, purine, carbazole, iminodibenzyl, azadibenzocyclooctyne; preferably Any of the following structures:
  • Q is an electron-changing group, and its number is 0, 1, or greater than 1; when the number of Q is greater than 1, any two Qs have the same or different structures;
  • Category (3) trivalent residues of any of the following structures: trivalent alcohol, trivalent thiol, trivalent primary amine, trivalent secondary amine, trivalent carboxylic acid, trivalent sulfonate, trivalent isocyanate, Trivalent heterofunctional small molecule, amino acid; the trivalent heterofunctional small molecule contains two different functional groups, one of which has 1 number and the other has 2; wherein, the trivalent residue of the amino acid
  • the base is preferably any of the following structures:
  • u 1 , u 2 , and u 3 are each independently a connection key connected to L d or L x, and any two of u 1 , u 2 , and u 3 are not connected to L d or L x at the same time. L d or any L x are connected.
  • N core contains any type of tetravalent core structure in atoms, branched chains, or cyclic structures, or a combination of two trivalent core structures; preferably N
  • the structure of core is any of the following:
  • u 1 , u 2 , u 3 , and u 4 are all connecting keys.
  • u 1 , u 2 , u 3 , and u 4 are each independently connected to L d or any L x
  • u 1 , u 2 Any two of u 3 and u 4 are not connected to L d or any L x at the same time.
  • N core is selected from any one of comb structure, tree structure, branched structure, hyperbranched structure and cyclic structure;
  • the tree structure is selected from any of the following:
  • u 1 is the connecting bond connected to L d
  • the asterisk * in the structure is marked as the connecting bond connected to the polyethylene glycol component
  • d represents the algebra of the tree-like combination, and d is selected from 2, 3, 4, 5 or 6;
  • the branched structure, hyperbranched structure and comb structure are any one of trivalent core structure, tetravalent core structure, pentavalent core structure and combinations thereof, preferably any one of amino acids and their derivatives or More than one constituent polymer residue or its oxidized form;
  • N core is selected from any of the following structures:
  • u 1 , u 2 , and u 3 are each independently a connection key connected to L d or L x , and any two of u 1 , u 2 , and u 3 are not connected to the same L d or L x at the same time. ;
  • Q is an electron-changing group, and its number is 0, 1, or greater than 1; when the number of Q is greater than 1, any two Qs have the same or different structures.
  • the pegylated lipid is obtained by a preparation process involving a coupling reaction and/or a polymerization reaction;
  • each coupling reaction step is independently alkylation reaction, condensation reaction, amidation reaction, esterification reaction, thioesterification reaction, ring-opening reaction, ring-closing condensation reaction, addition reaction, cycloaddition reaction, ⁇ , ⁇ -Unsaturated bond addition reaction, alkynyl addition reaction, Schiff base reaction combined reduction reaction, click reaction, azide-alkyne addition reaction, 1,3-dipolar cycloaddition reaction, Diels-Alder addition reaction, thiol-yne reaction, thiol-ene reaction, thiol-vinyl reaction and condensation reaction Any one; the reaction conditions of the coupling reaction are related to the type of covalent linker generated by the reaction, and existing public technologies can be used; the valence state
  • the polymerization reaction undergoes at least the following two steps: deprotonation of a small molecule initiator and polymerization of ethylene oxide;
  • the small molecule initiator can be a directly obtained raw material or an intermediate in the preparation process;
  • the order of the coupling reaction and the polymerization reaction is not limited.
  • the raw materials used in each preparation method can be purchased or synthesized by oneself.
  • the monodisperse raw material containing the polyethylene glycol component can be replaced with the polydisperse raw material of the same component to obtain the corresponding polydisperse product.
  • the polydisperse raw material containing polyethylene glycol component can be replaced with the monodisperse raw material of the same component to obtain the corresponding monodisperse product.
  • polyethylene glycol raw materials including but not limited to linear/nonlinear polyethylene glycol and its derivatives, can be prepared by referring to the methods in CN108530637B, CN110591079A, CN108659227A, CN108530617B, CN1243779C or CN101029131A. .
  • the intermediates and final products prepared in the present invention can be purified by purification methods including but not limited to extraction, recrystallization, adsorption treatment, precipitation, reverse precipitation, membrane dialysis or supercritical extraction.
  • purification methods including but not limited to extraction, recrystallization, adsorption treatment, precipitation, reverse precipitation, membrane dialysis or supercritical extraction.
  • characterization methods including but not limited to nuclear magnetic resonance, electrophoresis, UV-visible spectrophotometer, FTIR, AFM, GPC, HPLC, MALDI-TOF, circular dichroism spectrometry, etc. can be used.
  • the actual preparation process may also include any necessary micro-modification, protection/deprotection, intermediate preparation, post-treatment or purification processes familiar to those skilled in the art that are mentioned or not mentioned in the present invention.
  • the compound of formula (A-1) and the compound of formula (A-2) undergo 1 to 2 steps of reaction, and the protecting group is optionally removed according to the actual situation to obtain the nitrogen-branched lipid compound of formula (A-3).
  • n is 1 or 2
  • B 1 , B 2 , L 1 , L 2 , R 1 and R 2 are defined the same as in general formula (1)
  • W is a leaving group, selected from -Cl, -Br, - I, any one of -OMs and -OTs
  • W in WB 1 -L 1 -R 1 and WB 2 -L 2 -R 2 is the same or different group;
  • F G1 is a reactive group or its protected form, selected from any one of amino, hydroxyl, thiol, carboxyl, halogen group, sulfonate group, aldehyde group, acid halide group, activated carboxyl group, active ester group or Its protected form is preferably any one of -NH 2 , -NHBoc, -NHFmoc, -NHCbz, -OH, -OTBS, -OMs, and -OTs; in formula (A-1) and formula (A-3)
  • the F G1 are of the same or different structures.
  • Method 1 The synthesis method is the same as the aforementioned compound of formula (A-3). At this time, L m1 is the same as L m2 , F G1 is the same as F G3 , and the structure of the compound of formula (A-5) corresponds to formula (A-3).
  • Method 2 Perform one or multiple consecutive coupling reactions between the compound of formula (A-3) and the bifunctional small molecule compound of formula (A-4), and optionally remove the protecting group according to the actual situation to obtain formula (A -5)
  • SM is a small molecule residue
  • the definitions of FG i and FG 3 are the same as FG 1 , and the specific form is based on the smooth implementation of the reaction; when two or more compounds of formula (A-4) continuously participate in the reaction, SM has the same or different structure every time it appears, and FG i has the same or different structure every time it appears;
  • the compound of formula (A-5) reacts with the trifunctional small molecule compound of formula (A-6) to obtain a compound of formula (A-7) containing two reactive groups and at least two nitrogen branched structures.
  • the definitions of F G4 , F G5 and F G6 are the same as FG 1 , and the specific form is based on the smooth implementation of the reaction; the definitions of L 1A , L 1B and L 1A are all the same as L m1 , and the structure of any two is Identical or different; the divalent linking group produced by the reaction of F G4 and F G3 together with L m2 and L 1A constitutes L d .
  • the compound of formula (B-1) contains an exposed amino group, and reacts with the compound of formula (C-1) containing XPEG in 1 to 2 steps to obtain a two-arm nitrogen-branched PEGylated lipid compound represented by formula (3).
  • W is a leaving group, selected from any one of -Cl, -Br, -I, -OMs and -OTs.
  • the compound of formula (A-7) reacts with two identical or different compounds of formula (C-2) to obtain a two-arm nitrogen-branched PEGylated lipid compound represented by formula (3).
  • the definition of L 1D is the same as that of L m1 ;
  • the definition of F Gi is the same as that of F 1 , and the specific form is based on the smooth implementation of the reaction; an F Gi reacts with F G5 , and the obtained divalent linking group forms a linker with L 1B and L 1D L x , another F Gi reacts with F G6 , and the obtained divalent linking group forms another L x with L 1C and L 1D .
  • Method 3 Introduce the nitrogen-branched nonlinear polyethylene glycol component at one time:
  • the compound of formula (A-5) reacts with the compound of formula (C-3) to obtain the nitrogen-branched PEGylated lipid compound of general formula (1).
  • F G2 and F G3 are both reactive groups, each independently selected from amino group, hydroxyl group, mercapto group, carboxyl group, halogen group, sulfonate group, aldehyde group, acid halide group, activated carboxyl group, and active ester group. species; the divalent linker generated by the reaction of F G2 and F G3 together with L m2 and L m3 constitutes L d .
  • the end of the nitrogen-branched PEGylated lipid has a functional group R 01 .
  • the R 01 is unprotected or protected through an active polyethylene glycol derivative. way to introduce.
  • the raw material includes active polyethylene glycol derivatives.
  • the structure of the active polyethylene glycol derivative can be selected from any of the specific structures of monofunctional nonlinear polyethylene glycol described in documents CN108530637B, CN110591079A, CN108659227A, CN108530617B and CN1243779C.
  • the PEGylated lipid satisfying the general formula (1) or the general formula (2) is further terminally functionalized, and the obtained end-functionalized structure still satisfies the general formula (1) or the general formula (2).
  • the method of terminal functionalization is not particularly limited and is related to the type of the final functional group or its protected form. It mainly includes the functionalization of the terminal hydroxyl group and the functionalization of the target functional group or its protected form based on the reactive group. Changing forms of protection.
  • Method 1 Direct modification, based on direct modification of reactive groups, obtains the target functional group or its protected form.
  • the transformation of a carboxyl group into an acid halide, hydrazide, ester, thioester, and dithioester such as the transformation of a hydroxyl group, a mercapto group, an alkynyl group, an amino group, a carboxyl group, etc. into the corresponding protected structure, etc.
  • Another example is the modification of hydroxyl groups, amino groups, etc. by acid anhydrides.
  • Method 2 Coupling reaction between two reactive groups, using a heterofunctional reagent containing one reactive group and the target functional group or its protected form as raw material, through one of the reactive groups
  • the reaction between the group and the reactive group at the end of RPEG introduces the target functional group or its protected form.
  • the reaction mode and method between two reactive groups are not particularly limited.
  • the reaction conditions are related to the type of divalent linker generated by the reaction, and existing public technologies can be used. Such as alkylation, alkenyl addition reaction, alkynyl addition reaction, Schiff base reaction combined reduction reaction, condensation reaction, etc.
  • the alkylation reaction is preferably a reaction based on the alkylation of a mercapto group or an amino group, which in turn corresponds to the formation of a thioether bond, a secondary amino group or a tertiary amino group.
  • the condensation reaction includes but is not limited to the condensation reaction that generates ester group, thioester group, amide group, imine bond, hydrazone bond, urethane group, etc.
  • Another example is to use groups containing azide, alkynyl, alkenyl, trithioester, mercapto, dienyl, furyl, 1,2,4,5-tetrazinyl, cyanate and other groups with the target functional group Or its protected form of heterofunctional reagent is used as raw material, and the target functional group or its protected form is introduced through click reaction.
  • the reaction between two reactive groups is accompanied by the formation of new bonds.
  • Typical representatives of the newly generated divalent linking groups are amide bonds, urethane bonds, ester groups, secondary amine bonds, thioether bonds, and triazole groups. wait.
  • Method 3 Obtain the target functional group or its protected form through a combination of direct modification and coupling reaction.
  • the polyethylene glycol component has a two-arm structure:
  • the nitrogen-branched PEGylated lipid is selected from any of the following:
  • the polyethylene glycol component has a multi-arm structure:
  • Lipid compositions lipid pharmaceutical compositions and preparations thereof, liposomes, and lipid nanoparticles
  • a lipid composition characterized by comprising any of the aforementioned nitrogen-branched pegylated lipids.
  • the lipid composition also contains one or more of phospholipids, steroid lipids and pegylated lipids, selected from any one of the following situations:
  • Case (2) Also contains steroid lipids
  • Case (3) Also contains PEGylated lipids
  • Case (4) Also contains phospholipids and steroid lipids;
  • Case (5) Also contains phospholipids and PEGylated lipids;
  • Case (6) Also contains steroid lipids and pegylated lipids;
  • Case (7) Also contains phospholipids, steroid lipids and pegylated lipids;
  • lipids Preferably, it also contains three types of lipids: phospholipid, steroid lipid and pegylated lipid.
  • the phospholipid contained in the lipid composition is selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl -sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palm Acyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2
  • the steroid lipid contained in the lipid composition is selected from the group consisting of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomato Any one of alkali, ursolic acid, alpha-tocopherol and combinations thereof.
  • the cationic lipid contained in the lipid composition is selected from N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl -N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N -(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy Propylamine (DODMA), 3-(Didodecylamino)-N1,N1,4-tri-dodecyl-1-piperazineethylamine (KL10), N1-[2-(Didodecyl Amino)ethyl]-N1,N4,
  • the molar percentage of each of the PEGylated lipids, cationic lipids, phospholipids, and steroid lipids contained in the lipid composition to the total lipids is not particularly limited; wherein,
  • the molar percentage of PEGylated lipids in total lipids is 0.5-5%, preferably 1-3%, more preferably 1.5%, 1.6%, 1.7%, 1.8%, 1.9%;
  • the molar percentage of cationic lipids in total lipids is 30-65%, preferably 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 55%;
  • the molar percentage of phospholipids in total lipids is 7.5-13%, preferably 8%, 9%, 10%, 11%, or 12%;
  • the molar percentage of steroid lipids in total lipids is 35-50%, preferably 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% .
  • Lipid compositions of the present invention may be used to deliver biologically active ingredients to one or more of the following in a patient: liver or liver cells (e.g., hepatocytes), kidney or kidney cells, tumors or tumor cells, CNS or CNS cells ( central nervous system (e.g., brain and/or spinal cord), PNS or PNS cells (peripheral nervous system), lung or lung cells, blood vessels or vascular cells, skin or skin cells (e.g., dermal cells and/or follicular cells), eye or Eye cells (eg, macula, fovea, cornea, retina), ear or ear cells (eg, inner ear, middle ear, and/or outer ear cells).
  • liver or liver cells e.g., hepatocytes
  • CNS or CNS cells central nervous system (e.g., brain and/or spinal cord), PNS or PNS cells (peripheral nervous system)
  • lung or lung cells blood vessels or vascular cells
  • skin or skin cells e.g., dermal
  • a lipid pharmaceutical composition characterized in that it contains any of the aforementioned lipid compositions and drugs, and the drugs are selected from any one of nucleic acid drugs, gene vaccines, anti-tumor drugs, small molecule drugs, polypeptide drugs and protein drugs. kind.
  • the drug contained in the lipid pharmaceutical composition is a nucleic acid drug, selected from any one of DNA, RNA, antisense nucleic acid, plasmid, interfering nucleic acid, aptamer, antagomir and ribozyme,
  • the RNA is selected from any one of mRNA, saRNA, circRNA, miRNA and siRNA; preferably, it is any one of DNA, mRNA, miRNA and siRNA.
  • the drugs contained in the lipid pharmaceutical composition include but are not limited to doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, streptozotocin, Uzozocin, actinomycin D, vincristine, Vinblastine, cytosine arabinoside, anthracycline, nitrogen mustard, thiotepa, chlorambucil, laxithromycin, melphalan, carmustine, rolimustine, busulfan, Bromomannitol, mitomycin C, cisdichlorodiamine platinum (II), methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine, di bucaine, chlorpromazine, propranolol, dimoxanol, labetalol, clonidine, hydrala
  • the drug contained in the lipid pharmaceutical composition is a nucleic acid drug; the N/P ratio of cationic lipids and nucleic acids in the composition is (0.1 ⁇ 100):1, more preferably (0.2 ⁇ 30):1, most preferably (0.5 ⁇ 20):1.
  • the lipid pharmaceutical composition is used as a drug, and the drug is selected from any one of the following: cancer treatment drugs, anti-infective agents, antibiotic agents, antiviral agents, antifungal agents, vaccines .
  • the lipid pharmaceutical composition is an LNP-pharmaceutical composition, an LPP-pharmaceutical composition or a PNP-pharmaceutical composition; preferably it is an LNP-pharmaceutical composition, and more preferably it is an LNP-nucleic acid pharmaceutical composition.
  • the composition is more preferably an LNP-mRNA composition.
  • the lipid pharmaceutical composition can form different structures; among them, the "LNP-pharmaceutical composition” forms the structure of lipid nanoparticles (LNP), and the “LPP-pharmaceutical composition” forms the structure of lipid polymers.
  • the drug contained is a nucleic acid drug; among which, the "LNP-mRNA composition” is a type of LNP-nucleic acid drug composition, and the nucleic acid drug contained is mRNA.
  • a liposome or lipid nanoparticle is characterized by containing any of the aforementioned lipid compositions.
  • a lipid pharmaceutical composition preparation characterized in that it contains any of the aforementioned lipid pharmaceutical compositions and a working fluid, the working fluid is a pharmaceutically acceptable diluent or excipient, and the diluent or excipient
  • the agent is preferably any one of deionized water, ultrapure water, phosphate buffer and physiological saline, more preferably phosphate buffer or physiological saline, most preferably physiological saline; wherein, the lipid composition: The ratio is preferably (0.05-20)g:100mL, more preferably (0.1-10)g:100mL, and most preferably (0.2-5)g:100mL.
  • the preparation of lipid pharmaceutical composition preparation includes the following steps:
  • the equilibrium time is 0.1 ⁇ 12h, preferably 0.2 ⁇ 6h, more preferably 0.5 ⁇ 3h; preferably, the compounding time is 0.1 ⁇ 12h, preferably 0.2 ⁇ 5h, more preferably 0.5 ⁇ 2h.
  • the preparation of LNP-nucleic acid pharmaceutical composition includes the following steps:
  • the organic solvent is preferably methanol, ethanol, propanol, tert-butanol, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrrolidone. any one or a mixture of more than one solvent; slow
  • the flushing liquid is preferably a citrate buffer, further preferably the concentration is 5-80mM and the pH is 2-6, more preferably the concentration is 10-50mM and the pH is 3-5; the volumes of the organic phase solution and the aqueous phase solution The ratio is preferably 1:1-10, more preferably 1:2 or 1:3.
  • the lipid pharmaceutical composition forms lipid nanoparticles (LNP-pharmaceutical composition) containing the drug, and ultrasonic, extrusion or microfluidic devices are used to control the particle size of the lipid nanoparticles.
  • the particle size is 1 ⁇ 1000nm, preferably 20 ⁇ 500nm, more preferably 60 ⁇ 200nm, most preferably 60 ⁇ 150nm.
  • nitrogen-branched PEGylated lipids as well as lipid compositions, lipid pharmaceutical compositions and preparations of nitrogen-branched PEGylated lipids, and nitrogen-containing branched PEGylated lipids will be further described below in conjunction with some specific examples. describe. Specific examples are provided to further illustrate the present invention in detail, but do not limit the scope of the present invention. Among them, in the example of preparing nitrogen-branched PEGylated lipids, the structure of the final product was characterized by nuclear magnetic resonance, and the molecular weight was confirmed by GPC or MALDI-TOF.
  • Example 1 Nitrogen-branched PEGylated lipids with/without ester bonds in the lipid part
  • Example 1.1 Nitrogen-branched PEGylated lipid E1-1 without ester bonds between L 1 and L 2
  • B 1 and B 2 are both connecting bonds, L 1 and L 2 are both connecting bonds, R 1 and R 2 are both tetradecyl groups; L d is -CH 2 CH 2 -; both L x are -CH 2 CH 2 -; T is methyl.
  • the molecular weight of each PEG chain is approximately 1 kDa, corresponding to n 1 ⁇ n 2 ⁇ 22.
  • the preparation method is as follows:
  • Step a Dissolve 1-bromotetradecane (S1-2, 6.09g, 22.0mmol) in 80 mL of N,N-dimethylformamide (DMF), and add ethylenediamine (S1) containing Boc protected amino group -1, 1.60g, 10.0mmol) and potassium carbonate (K 2 CO 3 , 3.64g, 26.4mmol), stir and react at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and 80 mL of methylene chloride was poured into the mixture.
  • DMF N,N-dimethylformamide
  • Example 1.2 Nitrogen-branched PEGylated lipid E1-2 containing ester bonds between L 1 and L 2
  • the molecular weight of each PEG chain is approximately 1 kDa, corresponding to n 1 ⁇ n 2 ⁇ 22.
  • the preparation method is as follows:
  • Step a Under an argon atmosphere, add 2-hexyldecanoic acid (S1-6, 7.68g, 30.0mmol) and 6-bromohexanol (S1-7, 5.97g) dissolved in dichloromethane (100mL). , 33.0mmol) and DMAP (0.92g, 7.5mmol) were added to a round-bottom flask with DCC (9.27g, 45.0mmol) and reacted at room temperature for 16h. After the reaction, the precipitate was removed by filtration. The filtrate was concentrated, and the obtained residue was purified by silica gel column chromatography to obtain compound S1-8 (10.55g).
  • Example 1.3 Nitrogen-branched PEGylated lipid E1-3 containing ester bonds between L 1 and L 2
  • B 1 is pentylene
  • B 2 is heptylene
  • R 1 is undecane.
  • R 2 is L d is -CH 2 CH 2 -
  • both L x are -CH 2 CH 2 -
  • T is methyl.
  • the molecular weight of each PEG chain is approximately 1 kDa, corresponding to n 1 ⁇ n 2 ⁇ 22.
  • the preparation method is as follows:
  • Step a Under an argon atmosphere, add 2-hexyldecanoic acid (S1-10, 4.91g, 22.0mmol) and 6-bromohexanol (S1-11, 6.20g) dissolved in dichloromethane (80mL). , 24.2mmol) and DMAP (0.67g, 5.5mmol) were added to a round-bottom flask with DCC (6.80g, 33.0mmol) and reacted at room temperature for 16h. After the reaction, the precipitate was removed by filtration. The filtrate was concentrated, and the obtained residue was purified by silica gel column chromatography to obtain compound S1-12 (8.64g).
  • Step c Dissolve S1-13 (5.41g, 10.0mmol) in dry THF (60mL), slowly add NaH (60%, 4.00g, 100.0mmol) in an ice bath, and react in an ice bath for 1 hour. After the reaction is completed, add 6-bromocaproic acid undecyl ester (S1-14, 4.19g, 12.0mmol, where S1-14 is prepared by the reaction of 6-bromocaproic acid and 1-undecanol, refer to this Example step a): After the reaction was stirred in an ice bath for 1 hour, the reaction solution was slowly returned to room temperature and allowed to react overnight.
  • Step d Referring to the reaction conditions and feed ratio of steps c to d in Example 1.1, S1-15 (5.10g, 7.2mmol) and S1-4 were subjected to two alkylation reactions (the first time 1.2mmol, the second time 0.8 mmol), and purified by column chromatography to obtain nitrogen-branched PEGylated lipid E1-3 (0.74 g).
  • Example 1.4 Nitrogen-branched PEGylated lipid E1-4 without ester bonds for L 1 and L 2
  • the molecular weight of each PEG chain is approximately 1 kDa, corresponding to n 1 ⁇ n 2 ⁇ 22.
  • the preparation method is as follows:
  • Step a Dissolve myristic acid (S1-16, 1.82g, 8.0mmol) in 40mL anhydrous dichloromethane, add N-hydroxysuccinimide (NHS, 1.38g, 12.0mmol), and then add DCC ( 2.47g, 12.0mmol).
  • DMAP (0.20 g, 1.6 mmol) was added to a solution of tetradecylamine (S1-17, 2.04 g, 9.6 mmol) in 40 mL of dichloromethane. After mixing the two aforementioned solutions, the reaction was stirred at room temperature for 24 hours. After the reaction is complete. The insoluble matter was removed by filtration, concentrated under reduced pressure, and purified by column chromatography to obtain S1-18 (2.85 g).
  • Step b Under nitrogen protection, dissolve S1-18 (2.12g, 5.0mmol) in 1M BH 3 /THF solution (40mL), heat to 80°C and stir for 24 hours. Cool to room temperature, remove THF by rotary evaporation, dissolve the residue in 4N hydrochloric acid methanol solution, and reflux under nitrogen protection for 4 hours. Remove the methanol, dissolve the residue in 50 mL of water, wash with ether (25 mL*3), and then adjust the pH to 11 with 20% NaOH solution.
  • Example 1.5 Nitrogen-branched PEGylated lipid E1-5 without ester bonds for L 1 and L 2
  • the molecular weight of each PEG chain is approximately 1 kDa, corresponding to n 1 ⁇ n 2 ⁇ 22.
  • the preparation method is as follows:
  • Example 2 L d Nitrogen-branched PEGylated lipid containing amide bond and/or ester bond
  • the molecular weight of each PEG chain is approximately 1 kDa, corresponding to n 1 ⁇ n 2 ⁇ 22.
  • the preparation method is as follows:
  • the molecular weight of each PEG chain is approximately 1 kDa, corresponding to n 1 ⁇ n 2 ⁇ 22.
  • the preparation method is as follows:
  • Step a Dissolve S1-2 (3.05g, 11.0mmol) in 40mL of N,N-dimethylformamide (DMF), add ethanolamine (S2-2, 0.88g, 5.0mmol) containing TBS protected hydroxyl group and potassium carbonate (K 2 CO 3 , 1.82g, 13.2mmol), and the reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and 40 mL of methylene chloride was poured into the mixture.
  • DMF N,N-dimethylformamide
  • Example 2.3 L d Nitrogen-branched PEGylated lipid E2-3 containing amide bond and ester bond
  • B 1 and B 2 are both connecting bonds, L 1 and L 2 are both connecting bonds, R 1 and R 2 are both tetradecyl groups;
  • T is methyl.
  • the molecular weight of each PEG chain is approximately 1 kDa, corresponding to n 1 ⁇ n 2 ⁇ 22.
  • the preparation method is as follows:
  • Step a Under an argon atmosphere, combine 3-aminopropionic acid (S2-4, 1.13g, 6.0mmol) containing Boc-protected amino groups, lipid compound S2-3 (2.18g, 4.8mmol) containing exposed hydroxyl groups and DMAP (0.18g, 1.5mmol) was dissolved in dichloromethane (30mL) in turn, DCC (1.85g, 9.0mmol) was added, and the reaction was carried out at room temperature for 16h. After the reaction, the precipitate was removed by filtration. Concentrate the filtrate, use TFA/DCM mixed solution (1:1v/v) to remove Boc protection, wash with purified water, and then extract with dichloromethane. Combine the organic phases, dry over anhydrous sodium sulfate, filter, concentrate, and pass Column chromatography purified the lipid compound S2-5 (2.07g) containing exposed amino groups.
  • DMAP 24 mg, 0.2 mmol was added to a solution of S2-5 (1.58 g, 3.0 mmol) in 15 mL of dichloromethane. After mixing the two aforementioned solutions, the reaction was stirred at room temperature for 24 hours. After the reaction is complete.
  • the molecular weight of each PEG chain is approximately 1 kDa, corresponding to n 1 ⁇ n 2 ⁇ 22.
  • the preparation method is as follows:
  • Step a Dissolve Boc-protected amino-containing glycine (S2-6, 1.05g, 6.0mmol) in 15mL anhydrous dichloromethane, add NHS (1.04g, 9.0mmol), and then add DCC (1.85g, 9.0mmol) ).
  • DMAP (0.15 g, 1.2 mmol) was added to a solution of secondary amine compound S1-19 (1.97 g, 4.8 mmol) in 15 mL of dichloromethane. After mixing the two aforementioned solutions, the reaction was stirred at room temperature for 24 hours. After the reaction is completed. The insoluble matter was removed by filtration, concentrated under reduced pressure, and purified by column chromatography to obtain S2-7 (2.02 g).
  • NHS 0.17g, 1.5mmol
  • DCC 0.31g, 1.5mmol
  • DMAP 24 mg, 0.2 mmol
  • S2-7 1.40 g, 3.0 mmol
  • the molecular weight of each PEG chain is approximately 1 kDa, corresponding to n 1 ⁇ n 2 ⁇ 22.
  • the preparation method is as follows:
  • R 2 is nonyl;
  • T is methyl.
  • the molecular weight of each PEG chain is approximately 1 kDa, corresponding to n 1 ⁇ n 2 ⁇ 22.
  • the preparation method is as follows:
  • Step a Dissolve S2-9 (3.08g, 9.6mmol, S2-9 is obtained by the esterification reaction of 6-bromohexanol and nonanoic acid under DMAP/DCC conditions) in 50 mL of DMF, and add S2-2 (1.40g, 8.0mmol) and potassium carbonate (K 2 CO 3 , 1.59g, 11.5mmol), stir and react at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and 50 mL of methylene chloride was poured into the mixture.
  • Step b Dissolve S2-10 (2.08g, 5.0mmol) in dry THF (40mL), slowly add NaH (60%, 2.00g, 50.0mmol) in an ice bath, and react in an ice bath for 1 hour. After the reaction is completed, the brominated alkane S2-11 (1.84g, 6.0mmol) containing an ether bond is added, where S2-11 is sulfonated with 6-bromohexanol, and then mixed with 1-nonanol in diphenylmethyl. (obtained by alkylation reaction under conditions of potassium base/THF). After stirring and reacting in an ice bath for 1 hour, the reaction solution slowly returned to room temperature and reacted overnight.
  • the molecular weight of each PEG chain is approximately 1 kDa, corresponding to n 1 ⁇ n 2 ⁇ 22.
  • the preparation method is as follows:
  • Example 3.1 L x Nitrogen-branched PEGylated lipid E3-1 containing ester bonds
  • the molecular weight of each PEG chain is approximately 1 kDa, corresponding to n 1 ⁇ n 2 ⁇ 22.
  • the preparation method is as follows:
  • Example 3.2 L x Nitrogen-branched PEGylated lipid E3-2 containing ester bonds
  • the molecular weight of each PEG chain is approximately 1 kDa, corresponding to n 1 ⁇ n 2 ⁇ 22.
  • the preparation method is as follows:
  • Step a Under an argon atmosphere, combine N,N-dihydroxyethylglycine (S3-3, 0.78g, 2.0mmol) containing two TBS-protected hydroxyl groups, and lipid compound S2-3 (1.09) containing exposed hydroxyl groups. g, 2.4mmol) and DMAP (61mg, 0.5mmol) were dissolved in dichloromethane (12mL) in sequence, DCC (0.62g, 3.0mmol) was added, and the reaction was carried out at room temperature for 16h. After the reaction, the precipitate was removed by filtration. Dry over anhydrous sodium sulfate, filter, and concentrate the filtrate.
  • lipid compound S3-4 (0.83g) containing two exposed hydroxyl groups.
  • Example 4 Nitrogen-branched PEGylated lipid containing 1,4,7-triazacyclononane branched core (E4-1)
  • the molecular weight of each PEG chain is approximately 1 kDa, corresponding to n 1 ⁇ n 2 ⁇ 22.
  • the preparation method is as follows:
  • Step a Dissolve NOTA-di-tert-butyl ester (S4-1, 0.83g, 2.0mmol) in 15mL anhydrous dichloromethane, add NHS (0.35g, 3.0mmol), and then add DCC (0.62g, 3.0mmol) ).
  • DMAP 49 mg, 0.4 mmol was added to a solution of S1-3 (1.09 g, 2.4 mmol) in 15 mL of dichloromethane. After mixing the two aforementioned solutions, the reaction was stirred at room temperature for 24 hours. After the reaction is completed.
  • Example 5 Nitrogen-branched PEGylated lipid containing carbazole branched core (E5-1)
  • the molecular weight of each PEG chain is approximately 1 kDa, corresponding to n 1 ⁇ n 2 ⁇ 22.
  • the preparation method is as follows:
  • Step a Dissolve the carbazole derivative S5-2 (1.86g, 4.0mmol) containing two Cbz protected amino groups in dry THF (30mL), and slowly add NaH (60%, 1.60g, 40.0mmol) under ice bath ), react in an ice bath for 1 hour. After the reaction, S5-1 (1.08g, 4.8mmol, obtained by sulfonate esterification of 3-hydroxypropionic acid with methylsulfonyl chloride) was added. After stirring in an ice bath for 1 hour, the reaction solution slowly returned to room temperature. Reaction was allowed to take place overnight.
  • Step b Under an argon atmosphere, add S5-3 (1.08g, 2.0mmol), S2-3 (1.09g, 2.4mmol) and DMAP (61mg, 0.5mmol) dissolved in dichloromethane (15mL). Add DCC (0.62g, 3.0mmol) to the round-bottomed flask and react at room temperature for 16h. After the reaction, the precipitate was removed by filtration. Concentrate the filtrate, dissolve it in methanol, add 10% Pd/C catalyst, bubble hydrogen and stir at room temperature for 14 hours. After the reaction is completed, filter out the catalyst with diatomaceous earth, concentrate the reaction solution, and purify through column chromatography to obtain lipid compound S5. -4(1.20g).
  • DMAP 61 mg, 0.5 mmol
  • B 1 and B 2 are both connecting bonds, L 1 and L 2 are both connecting bonds, R 1 and R 2 are both tetradecyl groups;
  • T is methyl.
  • the molecular weight of each PEG chain is approximately 1 kDa, corresponding to n 1 ⁇ n 2 ⁇ 22.
  • the preparation method is as follows:
  • Step a Under an argon atmosphere, combine the glycine dipeptide containing Boc-protected amino groups (S6-1, 1.39g, 6.0mmol), the lipid compound S2-3 containing exposed hydroxyl groups (2.18g, 4.8mmol) and DMAP ( 0.18g, 1.5mmol) was dissolved in dichloromethane (30mL), DCC (1.85g, 9.0mmol) was added, and the reaction was carried out at room temperature for 16h. After the reaction, the precipitate was removed by filtration. Concentrate the filtrate, use TFA/DCM mixed solution (1:1v/v) to remove Boc protection, wash with purified water, and then extract with dichloromethane. Combine the organic phases, dry over anhydrous sodium sulfate, filter, concentrate, and pass Column chromatography purified the lipid compound S6-2 (2.37g) containing exposed amino groups.
  • B 1 is pentylene
  • B 2 is heptylene
  • R 1 is undecane.
  • N core is a trivalent nitrogen nucleus
  • T is methyl.
  • the molecular weight of each PEG chain is approximately 1 kDa, corresponding to n 1 ⁇ n 2 ⁇ 22.
  • the preparation method is as follows:
  • Step a Under an argon atmosphere, combine the phenylalanine-glycine dipeptide (S6-3, 1.93g, 6.0mmol) containing Boc-protected amino groups and the lipid compound S6-4 (3.41g, 4.8mmol, refer to the implementation Preparation method of Example 1.3, synthesized using S2-2, S1-14 and S1-12 as raw materials) and DMAP (0.18g, 1.5mmol) were dissolved in dichloromethane (50mL) in sequence, and DCC (1.85g, 9.0mmol) was added , react at room temperature for 16h. After the reaction, the precipitate was removed by filtration.
  • Example 7 Nitrogen-branched PEGylated lipid (E7-1) in which L x , L 1 , and L 2 all contain ester bonds
  • the molecular weight of each PEG chain is approximately 1 kDa, corresponding to n 1 ⁇ n 2 ⁇ 22.
  • the preparation method is as follows:
  • Example 8 Folic acid-conjugated nitrogen-branched pegylated lipid (E8-1)
  • B 1 and B 2 are both connecting bonds, L 1 and L 2 are both connecting bonds, R 1 and R 2 are both tetradecyl groups; L d is -CH 2 CH 2 -; both L x are -CH 2 CH 2 -; T is R 01 -L 01 -, R 01 is a folic acid residue, and L 01 is -NHCH 2 CH 2 -.
  • the molecular weight of each PEG chain is approximately 1 kDa, corresponding to n 1 ⁇ n 2 ⁇ 22.
  • the preparation method is as follows:
  • Step b Dissolve folic acid (S8-3, 0.88g, 2.0mmol) in 15mL anhydrous dichloromethane, add NHS (0.35g, 3.0mmol), and then add DCC (0.62g, 3.0mmol).
  • an LNP-mRNA pharmaceutical composition (LNP/Fluc-mRNA) containing Fluc-mRNA is prepared.
  • the phospholipids it contains are all DSPC, the sterol lipids it contains are cholesterol, and the cationic lipids it contains are all CL-1 is different from PEGylated lipids; the CL-1 is obtained by referring to the preparation method disclosed in CN113402405A, and its structure is as follows:
  • the preparation method of LNP/Fluc-mRNA is as follows:
  • the ethanol phase solution and the aqueous phase solution were mixed (1:3v/v) to prepare cationic lipid nanoparticles LNP/Fluc-mRNA, and washed through multiple DPBS ultrafiltration to remove ethanol and free molecules, and finally passed through 0.2 ⁇ m sterile filter Filter, spare.
  • Example 10 Biological activity test of lipid pharmaceutical composition
  • nucleic acid complexing ability Use gel electrophoresis experiment to examine the nucleic acid complexing ability of LNP/Fluc-mRNA. Weigh 0.8g of agarose and dissolve it in 40mL of TAE solution. Heat it in a microwave to completely dissolve the agarose particles. Cool it and add 5 ⁇ L of nucleic acid dye GelGreen to the cooled agarose gel. Add the gel to the gel tank. Allow to dry naturally. Add the mixture of LNP/Fluc-mRNA and 2 ⁇ L of LoadingBuffer into the agarose gel well, set the electrophoresis voltage to 90V for the electrophoresis experiment, and electrophores for 10 minutes at room temperature. The results show that there is basically no free Fluc-mRNA in both the experimental group and the control group, indicating that the lipid composition containing the nitrogen-branched PEGylated lipid of the present invention has good nucleic acid complexing ability.
  • encapsulation efficiency Use an ultracentrifuge to ultracentrifuge LNP/Fluc-mRNA (4°C, 60000rpm, 1h), use a nucleic acid quantifier to detect the concentration of unencapsulated Fluc-mRNA in the supernatant, and calculate the effect of LNP on Fluc-mRNA
  • the encapsulation rate the results are summarized in Table 1, show that the cationic lipid nanoparticles of the present invention have a high encapsulation rate for nucleic acid drugs, in which the nitrogen-branched PEGylated lipid single-arm molecular weight is about 1kDa, The encapsulation rate of Fluc-mRNA in the 2 kDa experimental group was greater than 87%, while the experimental group (L1-1-500) with a nitrogen-branched pegylated lipid single-arm molecular weight of about 0.5 kDa encapsulated Fluc-mRNA.
  • the closure rate reached 83%.
  • the experimental group L1-1-half contains nitrogen-branched PEGylated lipid E1-1 with a single-arm molecular weight of approximately 1 kDa
  • the control group L-0 contains a linear PEGylated lipid PEG2k-DMG with a molecular weight of approximately 2 kDa.
  • Particle size measurement According to the literature (Hassett et al., J. Controlled Release 2021, 335, 237-246), it is known that the particle size of LNP preparations containing nucleic acid drugs can exert better drug effects when the particle size is 60 to 150 nm.
  • the particle size of LNP/Fluc-mRNA was measured by dynamic light scattering (DLS). The measured LNP/Fluc-mRNA size uniformity is high, and its PDI is less than 0.3.
  • Experimental results show that the particle size of LNP/Fluc-mRNA modified with the nitrogen-branched PEGylated lipid of the present invention is in the range of about 65nm to about 112nm, which is within the particle size range that can achieve better drug efficacy. diameter range.
  • Cationic lipids play an important role in LNPs used to deliver nucleic acid drugs.
  • the nitrogen-branched PEGylated lipids of the present invention can impart a "stealth effect" to LNPs.
  • the above LNP/Fluc-mRNA (1 control group L-0, 24 experimental groups L1-1 to L1-1-d) was added to the culture medium containing 10% fetal bovine serum (FBS), and incubated at 37°C. Stir and take samples regularly to measure the particle size changes of LNP/Fluc-mRNA, and analyze the serum stability of nucleic acid drug preparations by testing the particle size changes.
  • the experimental results show that within 7 days, the particle size of the experimental group L1-1-500 with a single-arm molecular weight of polyethylene glycol component of about 0.5kDa changed slightly (9%), and the particle sizes of all other experimental groups and the control group changed slightly.
  • the change is less than 6%; in particular, the particle size change of L1-1-half is about 5%, which is not significantly different from the particle size changes of L-0 and L1-1 (about 4% and 2% respectively), while L1
  • the amount of PEGylated lipid used in -1-half is only half that of L-0 and L1-1.
  • the LNP-nucleic acid pharmaceutical composition prepared from nitrogen-branched PEGylated lipids with a single-arm molecular weight of about 1 kDa has good serum stability; when the PEGylated lipids have similar molecular weights, such as linear in L-0
  • the molecular weight of the PEG component of the nonlinear nitrogen-branched PEGylated lipid E1-1 in the PEGylated lipids DMG and L1-1 is both about 2kDa.
  • the nonlinear structure of E1-1 can achieve better results. Improved serum stability of lipid pharmaceutical compositions; non-linear PEGylated lipids can achieve better serum stability of lipid pharmaceutical compositions with less dosage.
  • the cells used were HeLa cells, and the method used was the CCK-8 kit to measure cell viability.
  • the commercial transfection reagent Lipofectamine 2000 (L2K) was used to prepare the complex L2K/Fluc-mRNA according to the method of Example 9.
  • HeLa was inoculated into a 96-well plate at a ratio of 6000 pieces/well and 100 ⁇ L per well, and divided into control group (blank control group), L2K/Fluc-mRNA group (positive control group) and LNP/Fluc-mRNA group (experimental group). , L1-1 ⁇ L7-1), incubate at 37°C and 5% CO2 . After the cells were incubated for 24 hours, 10 ⁇ L of PBS solution was added to the blank control group, 3.3 ⁇ g/mL L2K/Fluc-mRNA (10 ⁇ L) and 3.3 ⁇ g/mL LNP/Fluc-mRNA (10 ⁇ L) were added to the positive control group and experimental group respectively.
  • the results of three repeated tests were averaged.
  • the cell survival rate of L2K/Fluc-mRNA in the positive control group was 92%.
  • the cell survival rates of all experimental groups were greater than 88%.
  • the cell survival rate of L2-1, L2-4, L2-7, and 7-1 is 97% and above.
  • Luciferase bioluminescence was used for testing. Dissolve the LNP/Fluc-mRNA composition preparation in the culture medium to prepare the required dose, use HeLa cells as the cell model, seed the cell suspension at a density of 6000 cells/well, and inoculate 100 ⁇ L/well of the cell suspension into a black-edged transparent bottom in 96-well plates. After inoculation, incubate in a cell culture incubator for 24 hours, and then administer a dose of 0.2ug mRNA per well. Add the corresponding dose of free Fluc-mRNA to the blank control group.
  • the LNP/Fluc-mRNA composition prepared by the present invention has excellent in vitro transfection effect, that is, the LNPs in the control group and the experimental group are effective delivery carriers, and the PEGylated lipid contains degradable linkers.
  • the transfection effects of the experimental group were mostly better than those of the control group.
  • the linker between the polyethylene glycol component and the lipid component contains oligopeptide residues L6-1 and L6-2, and Lx , L1 , and L2 all contain ester bonds for L7-1.
  • the staining effect is significantly better than L-0 whose PEGylated lipid is DMG.
  • the nitrogen-branched PEGylated lipid containing intracellular degradable groups of the present invention is more conducive to making the lipid nucleic acid pharmaceutical composition better exert its medicinal effect after being taken up by cells.
  • This example examines the targeting ability of LNP containing E8-1 with folic acid-modified polyethylene glycol end, and uses E1-1, which has the closest structure except the PEG end, as a comparison.
  • Step a Dissolve DSPC (4g) and cholesterol (667mg) in 60mL of methylene chloride, add 20mL of aqueous solution containing 80mg of oxaliplatin, stir for 30min, sonicate for 20min, remove the organic solvent by rotary evaporation at 40°C, and wait until the gel collapses. After that, add 50mL of aqueous solution containing E1-1 (0.50g, 0.2mmol) and poloxamer F68 (400mg) according to the components, continue to evaporate for 30min, homogenize under high pressure at 400bar for 5min, adjust the volume to 100mL with water, and then use cutting Separate free oxaliplatin by lateral flow ultrafiltration.
  • Step b Use the same preparation method as the above experimental group, using E8-1 (0.68g, 0.2mmol) instead of E1-1 to prepare folic acid-coupled nitrogen-branched PEGylated lipid nanoparticles/oxalidine Platinum composition formulation LP-2.
  • Blank control group 6 animals, 5% glucose injection was administered once from the tail vein and then the tumor tissue was removed;
  • Control group 1 10 mg/kg oxaliplatin injection, administered once, 12 animals;
  • Control group 2 10 mg/kg LP-1 preparation, administered once, 12 animals;
  • control group 1, control group 2 and experimental group were administered from the tail vein, and were killed at different time points at 0.05, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 8 and 48 hours after administration.
  • the nude mouse tumor pieces were stored in a -20°C refrigerator, and then the drug concentration was detected by HPLC.
  • the results showed that compared with oxaliplatin injection, LP-1 did not show higher targeting; the targeting of LP-2 was significantly enhanced, and the peak plasma concentration was significantly increased (increased by about 102%), indicating that this
  • the invention uses targeting groups to functionally modify the polyethylene glycol terminals of nitrogen-branched pegylated lipids, which can improve the targeting efficiency of lipid nanoparticles.

Abstract

本发明公开一种如式(1)所示的含有叔胺的氮支化非线性聚乙二醇化脂质，其中，B1、B2为连接键或亚烷基，L1、L2、Ld和Lx为连接键或二价连接基L，R1、R2为含有0-10个杂原子的C1-50脂肪烃基或C1-50脂肪烃衍生物残基，Ncore为价态为y+1的多价基团且含有一个与Ld相连的三价氮原子支化核，y为2、3、4、5、6、7、8、9或y≥10，XPEG为聚乙二醇组分。本发明提供的氮支化聚乙二醇化脂质与线性聚乙二醇化脂质相比，能实现更好的LNP表面修饰。本发明还提供含有所述聚乙二醇化脂质的脂质药物组合物及其制剂，可提高药物靶向性以及核酸药物转染效率。

Description

一种含有叔胺的氮支化非线性聚乙二醇化脂质及其应用 技术领域
本发明属于药物递送领域，总体上涉及一种聚乙二醇化脂质，具体涉及一种可作为药物载体组分的含有叔胺的氮支化非线性聚乙二醇化脂质，以及包含该脂质的脂质组合物、脂质药物组合物及其制剂和应用。
背景技术
脂质体被广泛用于递送核酸药物、基因疫苗、抗肿瘤药物、小分子药物、多肽药物或蛋白质药物，其中，针对新冠病毒的疫苗研发使得脂质纳米粒(lipid nanoparticle，LNP)成为当下热门的递送技术。LNP除了含有带负电荷的mRNA外，还含有阳离子脂质、中性脂质、类固醇脂质和聚乙二醇化脂质四种成分，其中，阳离子脂质通过静电与携带负电荷的mRNA相互作用；中性脂质一般为磷脂起到防止脂质氧化或将配体连接至脂质体或LNP表面或减少脂质颗粒聚集的作用；类固醇脂质有较强的膜融合性，促进mRNA胞内摄入和胞质进入；聚乙二醇化脂质位于脂质纳米粒表面，改善亲水性，避免被免疫系统快速清除，防止颗粒聚集，增加稳定性。
阳离子脂质和聚乙二醇化脂质是LNP尤为关键的组分。阳离子脂质涵盖永久阳离子脂质和可电离阳离子脂质，其中永久阳离子脂质容易在体循环中对生物膜造成干扰甚至破坏，而可电离阳离子脂质可以在生理条件下不电离或微弱电离，减小毒性。可电离阳离子脂质被细胞摄入后，在内体腔的酸性条件下能电离而带部分正电荷，增加膜渗透性，同时LNP降解或重组，促进mRNA从内体逃逸，释放到细胞质中的mRNA进一步翻译，从而完成mRNA分子的递送和转染。
没有进行表面修饰的阳离子脂质体或LNP通常不具备足够的体循环稳定性。在脂质体或LNP配方中加入聚乙二醇化脂质，可以实现脂质体或LNP表面共价结合聚乙二醇的效果，减少药物递送过程中的非特异性细胞摄取，实现脂质体或LNP的隐形效应，提高其药物制剂的效果。
目前得到应用以及研究较多的聚乙二醇化脂质基本为线性聚乙二醇修饰，典型例子包括DMG-PEG2000(PEG2k-DMG)和ALC-0159，而非线性聚乙二醇化脂质则鲜有应用。聚乙二醇修饰可以提高被修饰物在体内的稳定性和安全性，同时也会降低细胞对药物的摄取效率。传统的PEG化脂质采用线性聚乙二醇修饰，在分子量相近的情况下若改用非线性聚乙二醇，其雨伞构造有更高的保护能力(Veronese等，BioDrugs.2008,22,315-329)，从而导致被修饰物的细胞摄取率降低。此外，许多研究指出聚乙二醇化脂质会逐渐从脂质体或LNP表面脱离的现象，导致脂质体或LNP在体循环过程中逐渐部分或完全地失去隐形性，这种情况在血浆蛋白和血液剪切力作用下有所加剧。单链长度接近的情况下，非线性聚乙二醇化脂质比线性聚乙二醇化脂质更容易脱落，因其单个脂质分子上的聚乙二醇分子量更高，所受剪切力更大(Mastrotto等，Mol.Pharmaceutics 2020,17,472-487)。
为解决上述问题，需要开发一种新的非线性聚乙二醇化脂质。
发明概述
本发明提供新型的非线性聚乙二醇化脂质，尤其是一种含有叔胺的氮支化非线性聚乙二醇化脂质，将其应用于脂质纳米粒及其药物制剂的制备，实现优良的保护作用和体循环稳定性、较好的细胞摄取效率、高生物相容性、低毒性、高封装效率和高递送效率等优势。
本发明的上述目的通过如下技术方案予以实现，
本发明的一种实施方案：
一种氮支化聚乙二醇化脂质，其特征在于，结构如通式(1)所示：
其中，
B1、B2各自独立地为连接键或亚烷基；
L1、L2、Ld和y个Lx各自独立地为连接键或二价连接基L；
R1、R2各自独立地为含有0-10个杂原子的C1-50脂肪烃基或C1-50脂肪烃衍生物残基；所述杂原子为B、O、N、Si、P或S；
Ncore为价态为y+1的多价基团；Ncore含有一个与Ld相连的三价氮原子支化核；
y为2、3、4、5、6、7、8、9或y≥10，优选为2或3；
XPEG为聚乙二醇组分。
本发明还提供了另外一种实施方案：
一种脂质组合物，包含结构如式(1)所示的聚乙二醇化脂质。
本发明还提供了另外一种实施方案：
一种脂质药物组合物，含有前述脂质组合物和药物。
本发明还提供了另一种实施方案：
一种脂质药物组合物制剂，含有前述的脂质药物组合物和药学上可接受的稀释剂或赋形剂。
与现有技术相比，本发明具有如下有益效果：
本发明提供含有叔胺的氮支化非线性聚乙二醇化脂质化合物(简称氮支化聚乙二醇化脂质)，还提供含有所述氮支化聚乙二醇化脂质的脂质组合物、脂质药物组合物及其制剂。与线性聚乙二醇化脂质相比，本发明的氮支化聚乙二醇化脂质能够在摩尔用量更少和/或聚乙二醇链更短的情况下，实现更好的LNP表面修饰效果，起到更好的保护作用，延长LNP的体循环时间，更大程度地减少LNP的生物毒性。特别地，通过在聚乙二醇末端偶联靶向基团，可以将本发明的LNP用作一种或多种生物活性剂靶向递送的载体。特别地，在氮支化聚乙二醇化脂质结构中的合适位置引入降解性基团，可以使运载核酸药物的阳离子脂质纳米粒具备更好的细胞摄取效率和/或转染效率。本发明还提供含有所述氮支化聚乙二醇化脂质的脂质药物组合物及其制剂，具有较好药效，包括药物靶向性以及核酸药物转染效率的提升。
附图说明
图1为本发明实施例1.5所制备的氮支化聚乙二醇化脂质(E1-5)的核磁共振谱图(1H NMR)。
1.发明详述
本发明对于具体的实施方式作出了详细描述，然而，应理解的是，其仅以说明性方式而非限制性方式给出，在本发明的范围内的各种变化和修改对于所属领域的技术人员将是显而易见的。
本发明的引用文献中的描述与本发明的描述不同的，以本发明为准；这个原则针对 说明书全文的所有引用文献。
1.1术语说明
在本发明中，除非另有描述，否则本文中使用的所有技术和科学术语具有与本领域普通技术人员通常所理解的相同的含义。本文引用的所有专利和其他出版物的公开内容通过引用的方式整体并入本文。在本文术语的任何描述阐释与通过引用并入本文的任何文件相冲突的情况下，以下述术语的描述与阐释为准。除非另外指明，否则各术语具有以下含义。
本发明中，除非特别说明，任两个对象的“选自”/“优选”各自独立，当具有多级的选自/优选情况时，任两个对象的选自/优选可以为同级或不同级。例如，“LA、LB各自独立地选自A、B、C”，可以是LA、LB均为A，也可以是LA为A而LB为B1(B1为B的一种下级情形)。又例如，“LA优选为A(1级优选)，更优选为A1～A3(2级优选)，最优选为A11～A13(3级优选)，LB优选为B(1级优选)，更优选为B1～B3(2级优选)，最优选为B11～B13(3级优选)”，优选可以是A为A1～A3(2级优选)而B为B11～B13(3级优选)，也可以是A、B均为3级优选。
本发明中，“各自独立地为/选自/优选”不仅可以指不同类目可以各自独立地为/选自/优选定义里的任一选项，还可以在加上“每次出现时”表示同一类目在不同位置或不同时间出现时每次独立地为/选自/优选定义里的任一选项，例如，“二价连接基为-NRcC(＝O)-、-C(＝O)NRc-、-NRcC(＝O)NRc-、-OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc-、-NRcC(＝O)S-、-C(Rc)＝N-NRc-或-NRc-N＝C(Rc)-；其中，Rc每次出现时各自独立地为氢原子或C1-12烷基”这样的说明指出，在“-NRc-N＝C(Rc)-”中，两个Rc基团可以相同或不同(例如，一个Rc为甲基，另一个Rc为氢原子或乙基)，且“-NRcC(＝O)NRc-”的任一个Rc可以和“-NRcC(＝O)O-”中的Rc相同或不同。
本发明中，当列举了至少两项时，所列举的项的“组合”由前述列举的项中任两种或任两种以上构成；且对项的数量不做限定，任一项的数量可以为一个或大于一个，同种项的数量大于1时，任两个该项的具体形式可以相同或不同。例如，“二价连接基选自-CH2-、-O-、-S-、-C(＝O)-、-NRc-及其组合中的任一种；其中，Rc每次出现时各自独立地为氢原子或C1-5烷基”，所述二价连接基可以为-CH2-、-O-、-S-、-C(＝O)-或-NRc-，也可以为组合，例如-CH2-NH-CH2-(其中，-CH2-的数量为2，-NRc-的数量为1)、-(CH2)2-NH-(CH2)4-N(CH3)-(其中，-CH2-的数量为6，-NRc-的数量为2且具体形式不同)、-(CH2)2-NHC(＝O)-(其中，-CH2-的数量为2，-NRc-的数量为1，-C(＝O)-的数量为1)等。特别地，由连接键与任意连接基构成的组合依然为所述连接基本身，本发明中“连接基”默认含有至少一个原子。
本发明中，除非特别说明，否则术语“包括”、“包含”和“含有”以及类似的表述应在本说明书和权利要求书中以开放性和包含性的含义解释为“包括但不限于”或“非限制性地包括”。
本发明中，“包括但不限于”某范围，指所述范围内的项可选，但不限定为所述范围的项，且并非所述范围内的所有结构都适用，尤其是本发明明确排除的项不在候选之列，以本发明顺利实施为筛选标准。
本发明中，数值区间的释义，既包括短横线标记的数值区间(如1-6)，也包括波浪线标记的数值区间(如1～6)，还包括“至/到”标记的数值区间(如，1至6、1到6)。在没有特别说明的情况下，以区间形式标记的区间均可表示该区间范围内所有整数与非整数构成的组，且该范围包括两个端点。例如，EO单元平均数选自22～100，其选择范围并不限于该区间内的整数，还可以是任意的非整数。又如，“1-3中的整数”表示1、2、3构成的组。又如，-(CH2)1-4-表示-CH2-、-(CH2)2-、-(CH2)3-、-(CH2)4-构成的组。 又如，表示构成的组。
本发明中的数值范围，包括但不限于整数、非整数、百分数、分数表示的数值范围，如无特别说明，均包括两个端点。
本发明中，对于聚合物分子量而言，“约”、“左右”一般指±10％的数值范围，部分情况可放大到±15％，但不超过±20％。以预设数值为基数。例如，11kDa、12kDa与10kDa之间的偏差分别为10％、20％，“约10kDa”包括但不限于11kDa和12kDa。又如，指定通式中某个PEG组分的分子量约5kDa时，允许相应的分子量或数均分子量在5kDa±10％，也即4500～5500Da的范围内变化。
本发明中，对于百分数而言，当给出的数值(不含百分号)精确到小数点后第N位时，“约”、“左右”一般指±(0.5*0.1N)％的数值范围。例如，约1％指的是1±(0.5*0.10)％即0.5％-1.5％的范围，约2.2％指的是2.2±(0.5*0.11)％即2.15％-2.25％的范围，约2.33％指的是2.33±(0.5*0.12)％即2.335％-2.325％的范围。
本发明中，聚乙二醇链的聚合度用ni表示(i选自1、2、3、…的自然数)，除非特别说明，聚合度之间用“≈”表示相同或相等。当聚合物为单分散性时，其聚合度还可以用“＝”表示相同或相等。同一结构中取值相同的ni可互换表示，例如，n2≈n3时，可将n2用n3表示，也可将n3用n2表示。
本发明中的二价连接基，没有特别限定的情况下，其连接其它基团时可选两个连接端中的任一个，例如在GroupA和GroupB之间以酰胺键作为二价连接基时，可以为GroupA-C(＝O)NH-GroupB或GroupA-NHC(＝O)-GroupB。
本发明的结构式中，涉及相连接基团的原子归属问题时，采用来标记连接键，如以表示基团结构，具体地，表示-CCH3(CH2CH2CH3)2；表示-C(CH2CH2CH3)2-。而非基团形式的则表示(CH3)2C(CH2CH2CH3)2。
本发明中，对于从环状结构引出的连接键或基团，当未标记于特定的成环原子，而是指向环内部时，表示该连接键或基团可以从合适的任意成环原子引出，且当被标记的环为并环或稠环结构的一部分时，该连接键或基团可以从所述并环或稠环结构中合适的任意成环原子引出。如表示的结构包括但不限于等。
本发明中，基团中的碳原子数范围以下标形式标注在C的下标位置，表示该基团具有的碳原子数，除非特别说明，所述碳原子数不含取代基的贡献。例如，C1-12表示“具有1至12个碳原子”。又如，C1-10亚烷基表示碳原子数在下标所示范围中的任一种亚烷基，即C1、C2、C3、C4、C5、C6、C7、C8、C9、C10亚烷基中的任一种，包括但不限于直链的C1-10亚烷基(例如-(CH2)6-)和支化的C1-10亚烷基(例如-(CH2)3-CH(CH3)-(CH2)3-)。又如，“取代的C1-12烷基”指C1、C2、C3、C4、C5、C6、C7、C8、C9、C10、C11或C12烷基的至少一个氢原子被取代基替换得到的烷基，所述取代基中的碳原子数和杂原子数无特别限制。
本发明中，当涉及到的结构具有同分异构体时，没有特别指定的情况下，可以为其中任一种异构体。例如对于存在顺反异构体的结构，既可以为顺式结构也可以为反式结构；存在E/Z异构体的结构，既可以为E结构也可以为Z结构；有旋光性时可以为左旋 或右旋。
本发明中，若本文所描述的结构与该结构的名称之间存在差异，则所描述的结构应具有更大的权重。
本发明中，聚乙二醇及其衍生物的分子量默认指平均分子量，且没有特别规定时，“平均分子量”一般指“数均分子量”Mn。对于数均分子量，既可以为多分散性嵌段或物质的分子量，也可以为单分散性嵌段或物质的分子量。没有特别写明时，分子量的计量单位为道尔顿(Da)。还可以用“聚合度”表征聚乙二醇链的分子量大小，具体指其中重复单元(氧化乙烯基单元)的数量。相应地，优选用“平均聚合度”、“数均聚合度”来表征重复单元数量的平均值、数均值。
本发明中，“任意合适的连接基”、“任意合适的反应性基团”等中的“任意合适的”是指符合化学结构的基本原则，且能够使本发明的制备方法顺利实施的结构。用此方式描述的化学结构可视为具有清楚的、确定的范围。
本发明中基团的“可稳定存在”和“可降解”是一对相对的概念。
本发明中，“可降解”指发明化学键的断裂，且断裂为彼此独立的至少两个残基。如果经化学变化后改变了结构，但整个连接基仍仅为一个完整的连接基，那么该连接基仍归到“可稳定存在”的范畴。所述可降解的条件没有特别限制，既可为体内生理条件，也可为体外模拟生理环境或其它条件，优选在体内生理条件及体外模拟生理条件。所述生理条件没有特别限制，包括但不限于血清、心、肝、脾、肺、肾、骨骼、肌、脂肪、脑、淋巴结、小肠、生殖腺等部位，可以指细胞内，也可指细胞外基质中，可以指正常生理组织，也可以指病变生理组织(如肿瘤、炎症等)。所述体外模拟环境没有特别限制，包括但不限于生理盐水、缓冲液、培养基等。所述可降解的速度没有特别限制，例如既可以为酶作用下的快速降解，也可以指生理条件下的缓慢水解等。所述的体内生理条件包括治疗时的生理条件，如紫外照射、热疗等情况。包括但不限于在光、热、低温、酶、氧化还原、酸性、碱性、生理条件、体外模拟环境等条件下可降解，优选在光、热、酶、氧化还原、酸性、碱性等条件下可降解。所述可降解指在上述任一条件下的刺激下发生降解。所述光条件包括但不限于可见光、紫外光、红外光、近红外光、中红外光等光照条件。所述热条件指高于正常生理温度，通常指高于37℃的温度条件，且通常低于45℃，优选低于42℃。所述低温条件指低于人体生理温度，优选低于25℃，更优选≤10℃，具体举例如冷藏温度、冷冻温度、液氮治疗温度、2～10℃、4～8℃、4℃、0℃、-20±5℃等。所述酶条件没有特别限制，生理条件下可生成的酶均包含在内，作为举例，如肽酶、蛋白酶、裂解酶等。所述氧化还原条件没有特别限制，如巯基与二硫键之间的氧化还原转变、氢化还原转变。所述酸性、碱性条件主要指正常组织、病变组织、处于治疗期的器官或组织等体内部位的pH条件，比如胃为酸性条件，肿瘤部位也往往偏酸性。这里的可降解指可通过体内代谢作用发生降解(如生理作用、如酶、如氧化还原等)、在体内特定部位因微环境刺激而发生降解(如酸性、碱性)、或在临床治疗刺激下发生降解(如光、如热、如低温)等。需要说明的是，有机化学中相对于生物体而言的一些极端条件，如强酸、强碱、高温(如100℃以上)等条件下的键断裂，并不包括在本发明的可降解条件的范畴。又如，虽然醚键可在如氢溴酸的强酸条件下发生断裂，但本发明中始终将其归为可稳定存在的连接基。
本发明中，“可稳定存在”指连接基能保持作为一个完整的连接基存在(一个连接基与其相邻的基团稳定地共价相连接)，则定义为“可稳定存在”，其中，允许发生能保持连接基完整性的化学变化。所述化学变化没有特别限制，包括但不限于异构化转变、氧化、还原、离子化、质子化、去质子化、取代反应等。可稳定存在的条件没有特别限制，包括但不限于光、热、低温、酶、氧化还原、中性、酸性、碱性、生理条件、体外 模拟环境等条件下可稳定存在，优选在光、热、酶、氧化还原、酸性、碱性等条件下可稳定存在。这里的稳定存在指不进行特殊刺激(如特殊部位的pH条件、治疗时的光、热、低温等)的条件下，在体内代谢循环中可保持稳定的连接，不因发生键的断裂而导致分子量降低(只要仍能保持整体性)。
本发明中，对同一个连接基而言，“可稳定存在”并非绝对的概念，比如酰胺键在酸性或碱性条件下相比于酯键要稳定地多，本发明中的“可稳定存在”的连接基包含了酰胺键。但是，比如肽键，由一分子氨基酸的α-羧基和一分子氨基酸的α-氨基脱水缩合形成的一种酰胺键，当遇到特定酶作用时，则可以断裂，因此也包括在“可降解”的连接基中。同样地，氨基甲酸酯基、硫代氨基甲酸酯基等既可以为可稳定存在的连接基，也可以为可降解的连接基。更普遍地，氨基甲酸酯基、硫代氨基甲酸酯基等更倾向会发生缓慢降解，而非肽键的酰胺键则在体内循环过程中可稳定存在。又如常见的酯键可在酸、碱条件下降解，而包含在特殊结构中的酯键还可在紫外光条件下发生降解。又比如，即使某些化学键在特定酶作用下能发生降解，但如果其在临床使用时，如果循环路径不经过或者基本不经过该特定酶环境(比如定点给药的情况下)，相应的化学键仍可以视为是可稳定存在的。
本发明中，所有通式化合物应被理解为包括该通式化合物的盐。所用术语“盐”选自与无机和/或有机酸形成的酸加成盐和与无机和/或有机碱形成的碱加成盐中的任一种、任二种或者任二种以上的组合。当所述通式化合物含有碱性部分(例如但不限于吡啶或咪唑)和酸性部分(例如但不限于羧酸)，可以形成两性离子(“内盐”)并包括在所用术语“盐”中。“盐”可以是药学上可接受的(即，无毒、生理学上可接受的)盐，也可以是其他盐。所述通式化合物的盐可通过其自身与一定量(诸如当量)的酸或碱在诸如盐沉淀的介质中或在水性介质中反应然后冻干而形成。示例性的酸加成盐包括醋酸盐、己二酸盐、藻酸盐、抗坏血酸盐、天冬氨酸盐、苯甲酸盐、苯磺酸盐、硫酸氢盐、硼酸盐、丁酸盐、柠檬酸盐、樟脑酸盐、樟脑磺酸盐、环戊烷丙酸盐、二葡萄糖酸盐、十二烷基硫酸盐、乙磺酸盐、延胡索酸盐、葡庚酸盐、甘油磷酸盐、半硫酸盐、庚酸盐、己酸盐、盐酸盐、氢溴酸盐、氢碘酸盐、2-羟基乙磺酸盐、乳酸盐、马来酸盐、甲磺酸盐、2-萘磺酸盐、烟酸盐、硝酸盐、草酸盐、果胶酸盐、过硫酸盐、3-苯丙酸盐、磷酸盐、苦味酸盐、新戊酸盐、丙酸盐、水杨酸盐、琥珀酸盐、硫酸盐、磺酸盐、酒石酸盐、硫氰酸盐、甲苯磺酸盐、十一烷酸盐等；示例性碱加成盐包括铵盐、碱金属盐(诸如钠盐、锂盐和钾盐)、碱土金属盐(诸如钙盐和镁盐)、具有有机碱(例如有机胺)的盐以及具有氨基酸(诸如精氨酸或赖氨酸)的盐。碱性含氮基团可用以下试剂进行季铵化，诸如低级烷基卤化物(例如甲基、乙基、丙基和丁基氯化物、溴化物和碘化物)、二烷基硫酸盐(例如二甲基、二乙基、二丁基和二戊基硫酸盐)、长链卤化物(例如，癸基、月桂基、十四烷基和硬脂基氯化物、溴化物和碘化物)、芳基烷基卤化物(例如，苄基和苯乙基溴化物)及其他。所述酸加成盐和碱加成盐均优选为药学上可接受的盐，并且出于本公开的目的，均被认为等同于对应的通式化合物的游离形式。
本发明中的杂原子没有特别限定，包括但不限于O、S、N、P、Si、F、Cl、Br、I、B等。
本发明中，相对于化合物，失去部分原子或基团后形成的基团也称为残基。
本发明中，价态大于等于2的基团统称为“连接基”。连接基可以只含有一个原子，如醚基(-O-)、硫醚基(-S-)。特别地，当某个基团的定义包含“连接键”时，表示该基团可以不存在且仅起连接作用。
本发明中，关于基团的价态，“多价”指价态至少为3。
本发明中，除非特别说明，“连接键”指只起连接作用，不含有任何原子。
本发明中，二价连接基中的“基”可以替换为“键”，而不改变意义。例如，二价醚基也可称为醚键(-O-)，二价的酯基也可以称为酯键(-OC(＝O)-或-C(＝O)O-)，二价的氨基甲酸酯基也可称为氨基甲酸酯键(-OC(＝O)NH-或-NHC(＝O)O-)。特别地，“连接基”不能称为“连接键”。
本发明中，当取代基所含原子数为1时，也可称为“取代原子”。
本发明中，所述化合物或基团为“取代的”时，意指所述化合物或所述基团含有一个或多个取代基。
本发明中，除非特别说明，“氨基”与“胺基”意义相同，包括一价、二价、三价、四价的中性基团或阳离子基团，取代的或未取代的。例如，CH3-NH2中的-NH2可以称为“氨基”、“胺基”或“伯胺基”。又如，CH3-NH-CH3中的-NH-可以称为“仲胺基”或“仲氨基”，其中的-NH-CH3也可以理解为甲基取代的胺基。
本发明中，“胺基”包括但不限于伯胺基、仲胺基、叔胺基和季铵离子。例如，-NRtRt和-N+RtRtRt，其中每个Rt各自独立地为氢原子或任意烃基结构。
本发明中，未指明价态时，“烃基”可以为一价烃基、二价烃基、三价烃基、…、n价烃基，其中n为所述烃基能具备的最高价态。
本发明中，“亚烃基”为二价烃基。
本发明中的仲胺键、联氨键指“-NH-”或“-NH-NH-”两端均被烃基封端，如-CH2-NH-CH2-和-CH2-NH-NH-CH2-；而如-C(＝O)-NH-则称为酰胺键，不视为含有仲胺键。
本发明中，“官能团”也即“功能性基团”，优选反应性基团、被保护的反应性基团、反应性基团的前体等等。“多元”指官能团的数量至少为3，如多元醇指至少含3个羟基的化合物，多元硫醇指至少含3个巯基的化合物，等。需要说明的是，允许还含有异质的其他类型的官能团，比如三(羟甲基)氨基甲烷为还含有一个氨基的三元醇，柠檬酸为还含有一个羟基的三元羧酸。
本发明的制备方法，除非特别说明，反应性基团还包含其被保护形式，所述被保护形式可在实际制备过程的任一合适步骤中进行脱保护得到相应的活性形式。
本发明中，成环原子为共同构成环骨架的原子。
本发明中的氨基酸的来源，在没有特别指明的情况下没有特别限制，既可以为天然来源，也可以是非天然来源，还可以为两者的混合。本发明中的氨基酸结构类型，在没有特别指明的情况下没有特别限制，既可以指L-型，也可以指D-型，还可以为两者的混合。
本发明中，参考文献CN104877127A、WO/2016/206540A、CN106967213A、CN108530637A、CN108530617A及各引用文献中有关氨基酸骨架、氨基酸衍生物骨架、环状单糖骨架的定义与举例亦作为参考纳入本发明中。除非特别说明，所述骨架也即残基。其中，氨基酸残基，包括从氨基上除去氢原子和/或从羧基上除去羟基和/或从巯基上除去氢原子和/或氨基被保护和/或羧基被保护和/或巯基被保护的氨基酸。不严密地说，氨基酸残基可以被称为氨基酸。具体举例，例如失去羧羟基(包括所有的C端羧羟基，还包括如天冬氨酸、谷氨酸中侧基上的羧羟基)、羟基上的氢原子、酚羟基上的氢原子(酪氨酸)、巯基上的氢原子(如半胱氨酸)、氮原子上的氢原子后(包括所有的N端氢原子，还包括侧基中氨基中的氢原子如赖氨酸、鸟氨酸上的ε-氨基上的氢原子、组氨酸及色氨酸的侧基环上的氨基中的氢原子等)、酰胺上的氨基(如天冬氨酰胺、谷氨酰胺等)、胍基侧基中的氨基或氨基中的氢原子形成的残基。氨基酸衍生物残基指除具有氨基酸基本特征外，还具有非氨基酸基本特征的原子或基团部分。
本发明中，“生物相关物质”包括但不限于文献CN104877127A、WO/2016/206540A、 CN106967213A、CN108530637A、CN108530617A及各引用文献中所描述、列举及引用的物质。概括地，生物相关物质包括但不仅限于以下物质：药物、蛋白质、多肽、寡肽、蛋白模拟物、片段及类似物、酶、抗原、抗体及其片段、受体、小分子药物、核苷、核苷酸、寡核苷酸、反义寡核苷酸、多核苷酸、核酸、适配体、多糖、蛋白多糖、糖蛋白、类固醇、甾类化合物、脂类化合物、激素、维生素、磷脂、糖脂、染料、荧光物质、靶向因子、靶向分子、细胞因子、神经递质、细胞外基质物质、植物或动物提取物、病毒、疫苗、细胞、囊泡、脂质体、胶束等。所述生物相关物质可以为生物相关物质自身，也可以其前体、激活态、衍生物、异构体、突变体、类似物、模拟物、多晶型物、药物学上可接受的盐、融合蛋白、化学改性物质、基因重组物质等，还可以为相应的激动剂、激活剂、活化剂、抑制剂、拮抗剂、调节剂、受体、配体或配基、抗体及其片段、作用酶(如激酶、水解酶、裂解酶、氧还原酶、异构酶、转移酶、脱氨酶、脱亚胺酶、转化酶、合成酶等)、酶的底物(如凝血级联蛋白酶底物等)等。所述衍生物包括但不限于甙类、核苷类、氨基酸类、多肽类衍生物。形成新的反应性基团的化学修饰产物，即对反应性基团进行改性而改变类型、额外引入功能性基团、反应性基团、氨基酸或氨基酸衍生物、多肽等结构后生成的改性产物，均属于生物相关物质的化学改性物质。生物相关物质在与官能化化合物结合之前或之后，还允许有与其结合的目标分子、附属物或递送载体，形成改性的生物相关物质或复合的生物相关物质。其中，所述药物学上可接受的盐，既可以为无机盐，如盐酸盐、硫酸盐、磷酸盐，也可以为有机盐，如草酸盐、苹果酸盐、柠檬酸盐等。其中，本发明中的“药物”包括在体内或体外提供生理或药理作用的任何药剂、化合物、组合物或混合物，且往往提供的是有益效果。其种类没有特别限制，包括但不限于药物、疫苗、抗体、维生素、食品、食品添加剂、营养剂、营养保健品及其它提供有益效果的药剂。所述“药物”在体内产生生理或药理作用的范围没有特别限制，可以为全身效果，也可以只在局部产生效果。所述“药物”的活性没有特别限制，主要为能够与其它物质发生相互作用的活性物质，也可以为不发生相互作用的惰性物质；但惰性的药物可通过体内作用或一定刺激转变为活性形式。其中，“小分子药物”为分子量不超过1000Da的生物相关物质，或任一生物相关物质的小分子拟态物或活性片段。
本发明中，“寡肽”指两个或两个以上氨基酸形成的肽类分子，优选构成寡肽的氨基酸残基数目为2至20个。“寡肽残基”可以为一价、二价、三价或更高价态。
本发明中，除非特别说明，“单糖基”即单糖残基，也即单糖骨架，包括开链式单糖基、也包括环状单糖基(如呋喃糖环、吡喃糖环)。
本发明中的单糖基，可以选自包括但不限于单糖、糖醇、脱氧糖、氨基糖、氨基糖衍生物(如酰胺衍生物)、糖酸、糖苷中任一种化合物的残基，可以为开链结构或环状结构。如氨基糖脱除一个氨基氢原子后形成的氨基残基，又如糖酸脱除羧羟基后形成的酰基等。所述单糖可以包括但不限于醛糖(多羟基醛)、酮糖(多羟基酮)。如烷基醚衍生物，甲基醚衍生物，举例如白雀木醇。本发明中单糖基的碳原子数无特别限制，包括但不限于丁糖、戊糖、己糖、庚糖。优选戊糖、己糖。其中，丁糖、戊糖、己糖、庚糖、糖醇、脱氧糖、氨基糖、氨基糖的酰胺衍生物、糖酸、糖苷等的举例包括但不限于CN106967213A中公开的结构。
本发明中的“微修饰”，指经过简单的化学反应过程即可完成的化学修饰过程。所述简单的化学反应过程主要指保护、脱保护、盐络合、解络合、离子化、质子化、去质子化或改变离去基团等化学反应过程。
本发明中，“微变化形式”与“微修饰”相对应，指经历保护、脱保护、盐络合、解络合、离子化、质子化、去质子化或改变离去基团等简单的化学反应过程后能形成目 标反应性基团的结构形式。所述改变离去基团，即离去基团的转变，例如但不限于酯形式向酰氯形式的转变。
本发明中的一些具体实施方案中，反应过程中还涉及相关基团的“保护”和“脱保护”过程。为防止某反应性基团对反应产生影响，通常对该反应性基团进行保护。本发明中的一些具体实施方案中，反应性基团为2个以上时，选择性地仅使目标反应性基团进行反应，因此对其他反应性基团进行保护。保护基不仅在目标反应进行过程中保持稳定，根据需要，可以通过本领域常规技术手段去除。
本发明中，反应性基团的“保护”，意指通过特定试剂将所要保护的反应性基团可逆地转化为惰性基团(非反应性基团)的策略。被保护的基团中，区别于未保护形式的部分称为“保护基”。例如，-OTBS是羟基(-OH)的一种被保护形式，其中-TBS基团为羟基的保护基。
本发明中，“脱保护”和“去保护”意义相同，皆指将被保护的基团从被保护形式转变为未保护形式的过程。
本发明中，“羟基保护基”包含可作为通常的羟基的保护基而使用的所有的基团。羟基保护基，优选为烷酰基(例如乙酰基、叔丁酰基)、芳烷酰基(例如苄酰基)、苄基、三苯甲基、三甲基硅基、叔丁基二甲硅基、烯丙基、缩醛基或缩酮基。乙酰基的脱去一般在碱性条件下进行，最常用的是NH3/MeOH的氨解和甲醇阴离子催化的甲醇解；苄基在中性溶液中室温下钯催化氢解很容易除去苄基，也可用金属纳在乙醇或液氨中还原裂去；三苯甲基一般是通过催化氢解除去；三甲基硅基通常使用含氟离子的试剂(如四丁基氟化胺/无水THF等)除去；叔丁基二甲硅醚较为稳定，能够承受醇性氢氧化钾的酯水解条件以及温和的还原条件(如Zn/CH3OH等)，可用氟离子(如Bu4N+F-)在四氢呋喃溶液中脱去，也可用含水乙酸于室温下脱去。二醇的保护，优选形成二氧戊环、二氧六环、环状碳酸酯或环状硼酸酯。
本发明中，“巯基保护基”包含可作为通常的巯基的保护基而使用的所有的基团。与羟基类似，巯基可以硫醚和硫酯的形式来保护。巯基保护基，优选为叔丁基、苄基、取代的苄基、二苯甲基、取代的二苯甲基、三苯甲基、乙酰基、苯甲酰基、叔丁氧羰基、苄氧羰基、硫代缩醛基或硫代缩酮基。硫醚的去保护可在酸催化下用Na/NH3还原或用重金属离子如Ag+、Hg+反应，再用硫化氢处理即可。有些基团包括S-二苯基甲基、S-三苯基甲基硫醚、S-2-四氢吡喃基、S-异丁氧甲基的半琉基缩醛，可用(SCN)2、碘或亚硫酰氯氧化成二硫醚，接着再还原成硫醇。硫酯的形成及去保护方法与羧酸酯相同。
本发明中，“羧基保护基”是指能通过水解、羧基保护基的去保护反应而转化为羧基的保护基。羧基保护基，优选为烷基(例如甲基、乙基、叔丁基)或芳烷基(例如苄基)，更优选为叔丁基(tBu)、甲基(Me)或乙基(Et)。本发明中，“被保护的羧基”是指羧基被适合的羧基保护基保护后所形成的基团，优选为甲氧羰基、乙氧羰基、叔丁氧羰基、苄氧羰基。所述羧基保护基可以在酸或碱的催化下水解除去，偶尔也可用热解反应消去，例如叔丁基可以在温和的酸性条件下除去，苄基可以通过氢解脱去。脱除羧基保护基的试剂选自TFA、H2O、LiOH、NaOH、KOH、MeOH、EtOH及其组合，优选为TFA和H2O的组合、LiOH和MeOH的组合、或LiOH和EtOH的组合。被保护的羧基脱保护，从而产生相应的游离酸，所述脱保护在碱存在下进行，所述碱和由所述脱保护形成的所述游离酸形成药学可接受的盐。
本发明中，“氨基保护基”等同于“胺基保护基”，包含可作为通常的氨基/胺基的保护基而使用的所有的基，例如芳基C1-6烷基、C1-6烷氧基C1-6烷基、C1-6烷氧基羰基、芳基氧基羰基、C1-6烷基磺酰基、芳基磺酰基或甲硅烷基等。氨基保护基优选为Boc(叔丁氧羰基)、Moz(对甲氧基苄氧羰基)及Fmoc(9-芴亚甲氧羰基)。脱除氨基保护基的 试剂包括但不限于TFA、H2O、LiOH、MeOH、EtOH及其组合，优选为TFA和H2O的组合、LiOH和MeOH的组合、或LiOH和EtOH的组合。脱除Boc保护的试剂为TFA或HCl/EA；优选TFA。脱除Fmoc保护所用的脱保护剂为含20％哌啶的N,N-二甲基甲酰胺(DMF)溶液。
本发明中，“炔基保护基”包含可作为通常的炔基的保护基而使用的所有的基，优选为三甲基硅基(TMS)、三乙基硅基、叔丁基二甲基硅基(TBS)或联苯基二甲基硅基。TMS保护的炔基在碱性条件下容易完成脱保护，如K2CO3/MeOH或KOH/MeOH。TBS保护的炔基在四正丁基氟化铵的四氢呋喃溶液中(TBAF/THF)可脱除保护基。
本发明中，被羟基保护基保护的羟基没有特别限制，例如可以为醇羟基、酚羟基等的羟基；被氨基保护基保护的氨基/胺基没有特别限制，例如可以来自伯胺、仲胺、联胺、酰胺等。本发明中胺基没有特别限制，包括但不限于伯胺基、仲胺基、叔胺基、季铵离子。
本发明中，被保护羟基的脱保护与羟基保护基的类型有关。所述羟基保护基的类型没有特别限制，以苄基、硅醚、缩醛、叔丁基对末端羟基进行保护为例，相应的脱保护方法包括但不限于：
A：苄基的脱除
苄基脱除可以利用氢化还原剂和氢供体的氢化作用来实现，在这个反应体系中的含水量应小于1％，反应才能顺利进行。
氢化还原催化剂没有限制，优选为钯和镍，但是并不限制载体，但优选氧化铝或碳，更优选碳。钯的用量为含被保护羟基化合物的1至100wt％，优选为含被保护羟基化合物的1至20wt％。
反应溶剂没有特别的限制，只要原料和产物均可以溶剂即可，但优选甲醇、乙醇、乙酸乙酯、四氢呋喃，乙酸；更优选甲醇。并不特别限制氢供体，但优选氢气、环己烯、2-丙醇、甲酸铵等。反应温度优选为25至40℃。反应时间没有特别限制，反应时间与催化剂的用量成负相关，优选为1至5个小时。
B：缩醛、缩酮的脱保护
用于这类羟基保护的缩醛或缩酮化合物优选乙基乙烯基醚、四氢吡喃、丙酮、2,2-二甲氧基丙烷、苯甲醛等。而这类缩醛、缩酮的脱保护通过在酸性条件下实现，溶液pH优选0至4。酸没有特别限制，但优选乙酸、磷酸、硫酸、盐酸、硝酸，更优选盐酸。反应溶剂没有特别的限制，只要能够溶解反应物和产物即可，优选水。反应温度优选0至30℃。
C：硅醚的脱保护
用于这类羟基保护的化合物包括三甲基硅醚、三乙基硅醚、二甲基叔丁基硅醚、叔丁基二苯基硅醚等。而这类硅醚的脱保护通过含氟离子的化合物，优选四丁基氟化铵、四乙基氟化铵、氢氟酸、氟化钾，更优选四丁基氟化铵、氟化钾。含氟试剂的用量在被保护羟基的摩尔当量的5至20倍，优选8至15倍，如果含氟的用量小于5倍被保护羟基的摩尔当量，会导致脱保护不完全；当脱保护试剂的用量大于20倍被保护羟基的摩尔当量，过量的试剂或化合物给纯化带来麻烦，可能混入后续步骤，从而引起副反应。反应溶剂没有特别的限制，只要能够溶解反应物和产物即可，优选非质子性溶剂，更优选四氢呋喃、二氯甲烷。反应温度优选0至30℃，当温度低于0℃，反应速度较慢，不能完全脱除保护基。
D：叔丁基的脱除
叔丁基的脱除在酸性条件下进行，溶液pH优选0至4。酸没有特别限制，但优选乙酸、磷酸、硫酸、盐酸、硝酸，更优选盐酸。反应溶剂没有特别的限制，只要能够溶 解反应物和产物即可，优选水。反应温度优选0至30℃。
本发明中，“羧基活化”是指用羧基活化剂对羧基进行活化处理，羧基活化后能够促进缩合反应更好的进行，如：抑制缩合反应中消旋杂质的产生、催化加快反应速度等。“羧基活化基”是羧基活化剂的残基。所述羧基活化剂为N-羟基丁二酰亚胺(NHS)、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDCI)、N-羟基-5-降冰片烯-2,3-二甲酰亚胺(HONb)和N,N-二环己基碳二亚胺(DCC)中一种或多种的组合，优选为NHS/EDCI、NHS/DCC、HONb/DCC的组合，最优选为NHS/EDCI或NHS/DCC的组合。
本发明中，反应用到的缩合剂并不限制，但优选N,N’-二环己基羰二亚胺(DCC)，1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC·HCl)，2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU)，苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐(HBTU)，最优选为DCC。而一般缩合剂的用量为羧酸摩尔当量的1至20倍，优选为5-10倍，这个反应可以加入适当的催化剂(如4-二甲基氨基吡啶(DMAP))。
本发明中，得到的产物可通过萃取、重结晶、吸附处理、沉淀、反沉淀、薄膜透析或超临界提取等纯化方法加以纯化。
本发明中，分步/多步反应可以在技术人员的多个实际操作中完成，也可以在一个实际操作中完成。例如，在一个实际操作的反应体系中，一分子低分子量脂质化合物中的两个反应性基团都和单链聚乙二醇衍生物试剂发生偶合反应，获得具有两个单链PEG臂的非线性PEG化脂质化合物，考虑到两个偶合反应实际发生有先后顺序，整个过程也可以视为分步/多步引入聚乙二醇组分。
本发明中，反应的溶剂可以是无溶剂或非质子性溶剂，非质子性溶剂包括甲苯、苯、二甲苯、乙腈、乙酸乙酯、乙醚、甲基叔丁基醚、四氢呋喃、氯仿、二氯甲烷、二甲基亚砜、二甲基甲酰胺或二甲基乙酰胺，优选四氢呋喃、二氯甲烷、二甲基亚砜、二甲基甲酰胺。
本发明中，反应用到的碱可以为有机碱(如三乙胺、吡啶、4-二甲基氨基吡啶、咪唑或二异丙基乙基胺，优选三乙胺、吡啶)，也可以为无机碱(例如K2CO3)。
本发明中，聚乙二醇组分的重复单元为氧化乙烯基单元，即-CH2CH2O-或-OCH2CH2-，也记为EO单元，重复单元的数量也记为EO单元数，重复单元的平均数也记为EO单元平均数，且优选数均平均数。
本发明中，对于多分散性情况，化合物单个分子的分子量/聚合度、宏观物质中化合物组分的数均分子量/数均聚合度的“相等”或“相同”或“等于”(包括其他形式的等价表达)，在没有特别指定的情况下，并不限定在数值上严格相等，而是指数值相接近或近似相等，所述相接近或近似相等优选偏差不超过±10％，通常以预设数值为基数。
本发明中，对于单分散性情况，单个化合物分子及通式中氧化乙烯基单元数相同或相等是指在数值上严格相等；例如设定某个PEG组分的EO单元数为11，则等于12的设定值不落在设定范畴；但对于为了获得含设定EO单元数的化合物组分，而采用一定制备方法获得的宏观产物，由于制备方法、纯化方法的限制，可能导致该宏观产物中还含有除目标EO单元数组分之外的其他EO单元数组分，此时当EO单元平均数偏离预设的EO单元数不超过±5％(基数≥10)或者不超过±0.5(基数<10)时，视为获得了含目标组分的单分散性宏观产物；此外，当符合EO单元数或EO单元平均数范围的组分含量达到一定百分比时(优选≥90％，更优选>95％，更优选大于96％，更优选大于98％，更优选99％～100％)，也视为获得了含目标组分的单分散性宏观产物；即使未达到上述的含量比例，只要采用了本发明的制备方法或采用基本相同的制备思路的类似方法，因故获得的含量不足的产品、以主产品、联产品或副产品形式出现的组分，不论是否进行分离纯化，均在本发明的范围内。
本发明中，当用Da、kDa、重复单元数、EO单元数描述多分散组分的化合物通式的分子量时，对于单个化合物分子，分子量数值允许落在给定数值的一定范围内(包括端点，优选±10％范围内)；用氧化乙烯基单元数描述单分散组分的化合物通式的预设分子量时，则无范围波动，为离散点，但其制备产物可能因分子量不均一而使EO单元平均数在一定范围范围内波动(不超过±10％或±1，优选不超过±5％或±0.5)。例如mPEG(甲氧基聚乙二醇单元)的分子量为5kDa，指通式中单个分子的分子量数值在4500～5500Da之间，对应的制备产物相应组分的平均分子量为5kDa，也即平均分子量的数值在4500～5500Da之间时的产物为目标产物，且分子量落在该范围的组分才对目标组分的含量有贡献；又如设计mPEG具有22个氧化乙烯基单元，则通式中所有化合物分子的EO单元数均应当严格为22，但制备产物可能是20、21、22、23、24个EO单元的化合物的混合物，此时EO单元的平均数落在22±2.2范围内(优选在22±1.1范围内)时则视为获得目标组分，而分子量落在该数值范围内的组分均可视为目标组分用以计算纯度。
本发明中，除非特别说明，“mPEG”为甲氧基封端的聚乙二醇链段，其结构式为其中，ni为聚乙二醇链的聚合度，选自1～1000的整数。
本发明中，产物PDI<1.005即可视为单分散性，可记为PDI＝1。
本发明中，“单链组分”中的重复单元个数至少为2。
本发明中，“脂质”又称“脂类”(lipid)，包括但不限于脂肪酸的酯，并且以通常在水中有较差的溶解性，但可溶于许多非极性有机物中为特征。尽管脂质通常在水中具有较差的溶解度，但是某些类别的脂质(例如，被极性基团修饰的脂质如DMG-PEG2000)具有有限的水溶性，并且在某些条件下可以溶解于水中。脂质的已知类型包括生物分子，例如脂肪酸、蜡、固醇、脂溶性维生素(如维生素A、D、E和K)、单酸甘油酯、二酸甘油酯、三酸甘油酯和磷脂。
本发明中，脂质包括单纯酯类、复合酯类和衍生脂质。所述单纯酯类为脂肪酸与醇所组成的酯类，它又可分为脂、油及腊三小类。所述复合酯类即“类脂质化合物”，也称为“类脂质”或“类脂”(lipoid)，包括磷脂、鞘脂类、糖脂、类固醇及固醇、脂蛋白类。所述衍生脂质，包括简单脂质衍生物和复合脂质衍生物，具有脂质的一般性质。
本发明中，脂质可以是合成的或衍生(分离或修饰)自天然来源或化合物。
本发明中，“脂质体”(liposome)是由脂质自组装形成的封闭囊泡，具有一个或多个双分子层结构。
本发明中，“脂质纳米粒”或“LNP”(lipid nanoparticle)是指包含一种或多种类型的脂质分子的纳米量级(如1nm至1000nm)颗粒。本发明中，LNP可以进一步包含至少一种非脂质有效载荷分子(如，一种或多种核酸分子)。在一些实施方案中，LNP包含部分或完全包封在脂质壳内部的非脂质有效载荷分子。特别地，在一些实施方案中，其中有效载荷是带负电荷的分子(如，编码病毒蛋白的mRNA)，并且LNP的脂质组分包含至少一种阳离子脂质和至少一种聚乙二醇化脂质。可以预期的是，阳离子脂质可以与带负电荷的有效负载分子相互作用，并在LNP形成过程中促进有效负载掺入和/或封装到LNP中。如本文提供的，可以形成LNP的一部分的其他脂质包括但不限于中性脂质和类固醇脂质。
本发明中，“阳离子”是指相应的结构永久地、或非永久地能响应某些条件(例如pH)而带有正电荷。因此，阳离子既包括永久性阳离子，也包括可阳离子化的。永久性阳离子是指相应的化合物或基团或原子在其环境的任何pH值或氢离子活性下均带正电 荷。典型地，因季氮原子的存在而产生正电荷。当化合物携带多个这样的正电荷时，它可以被称为多价阳离子。可阳离子化的是指化合物或基团或原子在较低pH下带正电荷并且在其环境的较高pH下不带电荷。另外，在不能测定pH值的非水性环境中，可阳离子化的化合物、基团或原子在高氢离子浓度下带正电荷并且在低氢离子浓度或活性下不带电荷。它取决于可阳离子化的或可聚阳离子化的化合物的各个性质，特别是相应的可阳离子化基团或原子的pKa，在所述pH或氢离子浓度下它带电荷或不带电荷。在稀释的水性环境中，可以使用所谓的海森巴赫(Henderson-Hasselbalch)方程来估计带有正电荷的可阳离子化的化合物、基团或原子的分率，该方程是本领域技术人员公知的。在一些实施方案中，可阳离子化的化合物的整体或部分在生理pH值(例如约7.0-7.4)下带正电荷。在一些优选的实施方案中，可阳离子化的化合物的整体或部分在生理pH值(例如约7.0-7.4)下是中性的，但在较低pH值(例如约5.5至6.5)下带正电荷。在一些实施方案中，可阳离子化的化合物的整体或部分的pKa优选范围是约5至约7。
本发明中，“阳离子脂质”是指在其所处环境的任何pH值或氢离子活性下带正电荷的脂质，或能够响应其所处环境(例如其预期使用环境)的pH值或氢离子活性而带正电荷的脂质。因此，术语“阳离子”涵盖“永久阳离子”和“可阳离子化的”的范围。在一些实施方案中，阳离子脂质中的正电荷源自季氮原子的存在。在一些实施方案中，阳离子脂质包括两性离子脂质，该两性离子脂质在其预期施用的环境中(例如，在内体pH下)带正电荷。在一些实施方案中，如果某脂质体或LNP中含有可阳离子化脂质，则优选的是，它在约1至9，优选地4至9、5至8或6至8的pH值下，最优选地在内体(endosome)pH值(例如约5.5至6.5)下，其中约1％至100％的可阳离子化脂质发生阳离子化。阳离子脂质包括但不限于N,N-二油基-N,N-氯化二甲铵(DODAC)、N,N-二硬脂基-N,N-溴化二甲铵(DDAB)、N-(1-(2,3-二油酰氧基)丙基)-N,N,N-氯化三甲铵(DOTAP)、N-(1-(2,3-二油基氧基)丙基)-N,N,N-氯化三甲铵(DOTMA)、N,N-二甲基-2,3-二油基氧基丙胺(DODMA)、3-(双十二烷基氨基)-N1,N1,4-三-十二烷基-1-哌嗪乙胺(KL10)、N1-[2-(双十二烷基氨基)乙基]-N1,N4,N4-三-十二烷基-1,4-哌嗪二乙胺(KL22)、14,25-双十三烷基-15,18,21,24-四氮杂-三十八烷(KL25)、1,2-二亚油基氧基-N,N-二甲基氨基丙烷(DLin-DMA)、2,2-二亚油基-4-二甲基氨基甲基-[1,3]-二氧杂环戊烷(DLin-K-DMA)、4-(二甲基氨基)丁酸三十七碳-6,9,28,31-四烯-19-基酯(DLin-MC3-DMA)、2,2-二亚油基-4-(2-二甲基氨基乙基)-[1,3]-二氧杂环戊烷(DLin-KC2-DMA)、((4-羟基丁基)氮杂二烷基)双(己烷-6,1-二基)双(2-己基癸酸酯)(ALC-0315)和十七烷-9-基-8-((2-羟乙基)(6-氧代-6-((十一烷氧基)己基)氨基)辛酸酯)(SM102)、CN113402405A公开的阳离子脂质中任一种及其混合物。
本发明中，“聚乙二醇化脂质”指包含脂质部分和聚乙二醇部分的分子。聚乙二醇化脂质除了本发明结构通式(1)所示的，还包括但不限于聚乙二醇-1,2二肉豆蔻酸甘油酯(PEG-DMG)、聚乙二醇-二硬脂酰基磷脂酰乙醇胺(PEG-DSPE)、PEG-胆固醇、聚乙二醇-二酰基甘油(PEG-DAG)，聚乙二醇-二烷氧基丙基(PEG-DAA)，具体地包括聚乙二醇500-二棕榈酰磷脂酰胆碱、聚乙二醇2000-二棕榈酰磷脂酰胆碱、聚乙二醇500-二硬脂酰磷脂酰乙醇胺、聚乙二醇2000-二硬脂酰磷脂酰乙醇胺、聚乙二醇500-1,2-油酰基磷脂酰乙醇胺、聚乙二醇2000-1,2-油酰基磷脂酰乙醇胺和聚乙二醇2000-2,3-二肉豆蔻酰甘油(PEG-DMG)等。
本发明中，“中性脂质”指在选定的pH下以无电荷或中性两性离子形式存在的许多脂质物质中的任一种，优选为磷脂。此类脂质包括但不限于1,2-二亚油酰基-sn-甘油-3-磷酸胆碱(DLPC)、1,2-二肉豆蔻酰基-sn-甘油-磷酸胆碱(MPC)、1,2-二油酰基-sn-甘油-3-磷酸胆碱(DOPC)、1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二硬脂酰基-sn- 甘油-3-磷酸胆碱(DSPC)、1,2-双十一烷酰基-sn-甘油-磷酸胆碱(DUPC)、1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸胆碱(POPC)、1,2-二-O-十八碳烯基-sn-甘油-3-磷酸胆碱(18:0Diether PC)、1-油酰基-2-胆固醇基半琥珀酰基-sn-甘油-3-磷酸胆碱(OChemsPC)、1-十六烷基-sn-甘油-3-磷酸胆碱(C16Lyso PC)、1,2-二亚麻酰基-sn-甘油-3-磷酸胆碱、1,2-二花生四烯酰基-sn-甘油-3-磷酸胆碱、1,2-双二十二碳六烯酰基-sn-甘油-3-磷酸胆碱、1,2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)、1,2-二植烷酰基-sn-甘油-3-磷酸乙醇胺(ME16.0PE)、1,2-二硬脂酰基-sn-甘油-3-磷酸乙醇胺、1,2-二亚油酰基-sn-甘油-3-磷酸乙醇胺、1,2-二亚麻酰基-sn-甘油-3-磷酸乙醇胺、1,2-二花生四烯酰基-sn-甘油-3-磷酸乙醇胺、1,2-双二十二碳六烯酰基-sn-甘油-3-磷酸乙醇胺、1,2-二油酰基-sn-甘油-3-磷酸-rac-(1-甘油)钠盐(DOPG)、二油酰基磷脂酰丝氨酸(DOPS)、二棕榈酰基磷脂酰甘油(DPPG)、棕榈酰基油酰基磷脂酰乙醇胺(POPE)、二硬脂酰基-磷脂酰-乙醇胺(DSPE)、二棕榈酰基磷脂酰乙醇胺(DPPE)、二肉豆蔻酰基磷酸乙醇胺(DMPE)、1-硬脂酰基-2-油酰基-硬脂酰乙醇胺(SOPE)、1-硬脂酰基-2-油酰基-磷脂酰胆碱(SOPC)、鞘磷脂、磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰肌醇、磷脂酸、棕榈酰基油酰基磷脂酰胆碱、溶血磷脂酰胆碱和溶血磷脂酰乙醇胺(LPE)中任一种及其组合物。中性脂质可以是合成的或天然来源的。
本发明中，“类固醇脂质”选自胆固醇、粪固醇、谷固醇、麦角固醇、菜油固醇、豆固醇、菜籽固醇、番茄碱、熊果酸、α-生育酚中任一种及其组合物。
本发明中的脂质体/脂质纳米粒，含有聚乙二醇化脂质时可以称为“聚乙二醇化脂质体/脂质纳米粒”，含有阳离子脂质时可称为“阳离子脂质体/脂质纳米粒”，同时含有聚乙二醇化脂质和阳离子脂质时，既可称为“聚乙二醇化脂质体/脂质纳米粒”也可称为“阳离子脂质体/脂质纳米粒”。
本发明中，“N/P比”是指阳离子脂质中的氮原子与核酸中磷酸的摩尔比。
本发明中，“核酸”是指DNA或RNA或其修饰的形式，其包含在DNA中存在的嘌呤或嘧啶碱基(腺嘌呤“A”，胞嘧啶“C”，鸟嘌呤“G”，胸腺嘧啶“T”)或在RNA中存在的嘌呤或嘧啶碱基(腺嘌呤“A”，胞嘧啶“C”，鸟嘌呤“G”，尿嘧啶“U”)。
本发明中，“RNA”是指可能天然存在或非天然存在的核糖核酸。例如，RNA可以包括修饰过的和/或非天然存在的组分，如一个或多个核碱基、核苷、核苷酸或连接子。RNA可以包括帽结构、链终止核苷、茎环、聚腺苷酸序列和/或聚腺苷酸化信号。RNA可以具有编码所关注多肽的核苷酸序列。例如，RNA可以是信使RNA(mRNA)。翻译编码特定多肽的mRNA，例如在哺乳动物细胞内部体内翻译mRNA可以产生编码的多肽。RNA可以选自由以下组成的非限制性组：小干扰RNA(siRNA)、不对称干扰RNA(aiRNA)、微RNA(miRNA)、Dicer-底物RNA(dsRNA)、小发夹RNA(shRNA)、mRNA、单链向导RNA(sgRNA)、cas9mRNA及其混合物。
本发明中，反义寡核苷酸或小干扰RNA(siRNA)可以在体外或体内抑制靶基因和靶蛋白质的表达。
本发明中，FLuc mRNA能表达荧光素酶蛋白，其在萤光素底物的存在下发射出生物光，所以FLuc常用于哺乳动物细胞培养以测量基因表达和细胞活度。
本发明中，“抑制靶基因的表达”指核酸沉默、减少或抑制靶基因表达的能力。为检验基因沉默的程度，使测试样品(例如，表达靶基因的培养基中的细胞样品)接触抑制靶基因表达的核酸。将测试样品或测试动物中的靶基因的表达与未接触或未施用核酸的对照样品(例如，表达靶基因的培养基中的细胞样品)中的靶基因的表达相比较。对照样品中的靶基因的表达可以指定为100％的值。在特定的实施方案中，当测试样品中 的靶基因表达水平相对于对照样品或对照哺乳动物中的靶基因表达水平为约95％、90％、85％、80％、75％、70％、65％、60％、55％、50％、45％、40％、35％、30％、25％、20％、15％、10％、5％或0％时，即实现了抑制靶基因的表达。
本发明中，确定靶基因表达水平的方法包括不限于斑点印迹、northern印迹、原位杂交、ELISA、免疫沉淀、酶作用以及表型测定。
本发明中，“转染”是指将一个物种(例如RNA)引入细胞中。转染可以例如在体外、离体或体内发生。
本发明中，“抗原”典型地是指可以被免疫系统识别，优选地被适应性免疫系统识别，并且能够触发抗原特异性免疫应答，例如通过作为适应性免疫应答的一部分形成抗体和/或抗原特异性T细胞的物质。典型地，抗原可以是或可以包含可以由MHC呈递给T细胞的肽或蛋白。在本发明的意义上，抗原可以是所提供的核酸分子(优选地如本文所定义的mRNA)的翻译产物。在此上下文中，包含至少一个表位的肽和蛋白的片段、变体和衍生物也被理解为抗原。
本发明中，“递送”是指将实体提供至目标。例如，将药物和/或治疗剂和/或预防剂递送至受试者，所述受试者为人类和/或其它动物的组织和/或细胞。
本发明中“药学上可接受的载体”是指与治疗剂一同给药的稀释剂、辅剂、赋形剂或媒介物，并且其在合理的医学判断的范围内适于接触人类和/或其它动物的组织而没有过度的毒性、刺激、过敏反应或与合理的益处/风险比相应的其它问题或并发症。在本发明的药物组合物中可使用的药学上可接受的载体包括但不限于无菌液体，例如水和油，包括那些石油、动物、植物或合成来源的油，例如花生油、大豆油、矿物油、芝麻油等。当所述药物组合物通过静脉内给药时，水是示例性载体。还可以使用生理盐水和葡萄糖及甘油水溶液作为液体载体，特别是用于注射液。适合的药物赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽糖、白垩、硅胶、硬脂酸钠、单硬脂酸甘油酯、滑石、氯化钠、脱脂奶粉、甘油、丙二醇、水、乙醇等。所述组合物还可以视需要包含少量的湿润剂、乳化剂或pH缓冲剂。口服制剂可以包含标准载体，如药物级的甘露醇、乳糖、淀粉、硬脂酸镁、糖精钠、纤维素、碳酸镁等。具体地，例如赋形剂包括但不限于抗黏附剂、抗氧化剂、黏合剂、包衣、压缩助剂、崩解剂、染料(色素)、缓和剂、乳化剂、填充剂(稀释剂)、成膜剂或包衣、调味剂、香料、助流剂(流动增强剂)、润滑剂、防腐剂、印刷墨水、吸附剂、悬浮剂或分散剂、甜味剂以及水合用水。更具体地赋形剂包括但不限于丁基化羟基甲苯(BHT)、碳酸钙、磷酸氢二钙、硬脂酸钙、交联羧甲基纤维素钠、交联聚乙烯吡咯烷酮、柠檬酸、交联聚维酮(crospovidone)、半胱氨酸、乙基纤维素、明胶、羟丙基纤维素、羟丙基甲基纤维素、乳糖、硬脂酸镁、麦芽糖醇、甘露糖醇、甲硫氨酸、甲基纤维素、对羟基苯甲酸甲酯、微晶纤维素、聚乙二醇、聚乙烯吡咯烷酮、聚维酮、预胶化淀粉、对羟基苯甲酸苯酯、视黄醇棕榈酸酯、虫胶、二氧化硅、羧甲基纤维素钠、柠檬酸钠、羟基乙酸淀粉钠、山梨糖醇、淀粉(玉米)、硬脂酸、蔗糖、滑石、二氧化钛、维生素A、维生素E(α-生育酚)、维生素C、木糖醇。
本发明的药物组合物可以系统地作用和/或局部地作用。为此目的，它们可以适合的途径给药，例如通过注射(如静脉内、动脉内、皮下、腹膜内、肌内注射，包括滴注)或经皮给药；或通过口服、含服、经鼻、透粘膜、局部、以眼用制剂的形式或通过吸入给药。对于这些给药途径，可以适合的剂型给药本发明的药物组合物。所述剂型包括但不限于片剂、胶囊剂、锭剂、硬糖剂、散剂、喷雾剂、乳膏剂、软膏剂、栓剂、凝胶剂、糊剂、洗剂、软膏剂、水性混悬剂、可注射溶液剂、酏剂、糖浆剂。
本发明中，疫苗为提供至少一种抗原或抗原功能的预防性或治疗性材料。抗原或抗原功能可以刺激身体的适应性免疫系统提供适应性免疫应答。
本发明，治疗，是指为了抵御疾病、障碍或病症而对患者进行的处理和护理，意在包括延迟疾病、障碍或病症的进展，减轻或缓和症状和并发症，和/或治愈或消除疾病、障碍或病症。待治疗的患者优选哺乳动物，尤其是人。
1.2氮支化聚乙二醇化脂质
本发明的一种实施方案：
一种氮支化聚乙二醇化脂质，其特征在于，结构如通式(1)所示：
其中，
B1、B2各自独立地为连接键或亚烷基；
L1、L2、Ld和y个Lx各自独立地为连接键或二价连接基L；
R1、R2各自独立地为含有0-10个杂原子的C1-50脂肪烃基或C1-50脂肪烃衍生物残基；所述杂原子为B、O、N、Si、P或S；
Ncore为价态为y+1的多价基团；Ncore含有一个与Ld相连的三价氮原子支化核；
y为2、3、4、5、6、7、8、9或y≥10，优选为2或3；
XPEG为聚乙二醇组分。
本发明中，通式(1)化合物能够以非溶剂化或溶剂化形式存在，包括水合形式。一般来讲，出于本公开的目的，具有药学上可接受的溶剂(诸如水、乙醇等)的溶剂化形式等同于非溶剂化形式。
本发明中，通式(1)化合物及其盐、溶剂化物可以它们的互变异构形式存在，包括酮-烯醇互变、酰胺-亚胺酸互变、内酰胺-内酰亚胺互变、烯胺-亚胺互变、质子转移互变和价互变。
本发明中，通式(1)化合物应理解为包含其盐、互变异构体、立体异构体和溶剂化物。
本发明的一种具体实施方案中，任一个XPEG包含x个RPEG；y个x各自独立地选自1-4的整数，优选y个x均为1；当y个x均大于等于2时，优选所有的x均相等；
所述RPEG的结构为其中，
ni为聚乙二醇链的聚合度，选自4～250的整数，i选自1-20的整数；所述聚乙二醇链多分散性为或单分散性；
T为H、C1-6烷基或R01-L01-；
L01为连接键或二价连接基L；
R01为能与生物相关物质相互反应的功能性基团。
本发明的一种具体实施方案中，前述氮支化聚乙二醇化脂质的结构如通式(2)所示：
其中，n1、n2各自独立地选自4-100的整数，优选选自10-60的整数，更优选选自10-45的整数，最优选为10、11、12、20、21、22、23、24、25中任一种。
1.2.1.连接基
本发明中，除非特别说明，所有二价连接基的稳定性没有特别限制，任一个二价连接基或其与相邻杂原子基团组成的二价连接基各自独立地为可稳定存在的连接基或可降解的连接基。
本发明中，除非特别说明，所有二价连接基的结构没有特别限制，为直链结构、支链结构或含环状结构；二价连接基的非氢原子数没有特别限制，各自独立地选自0～20的整数，优选选自1～10的整数；其中，非氢原子为C、O、S、N、P、Si或B；非氢原子的个数大于1时，非氢原子的种类为1种，或2种，或2种以上，非氢原子为碳原子与碳原子组合、碳原子与杂原子组合、杂原子与杂原子组合中的任一种。
本发明的一种具体实施方案中，二价连接基L为亚烷基、-C(＝O)-、-OC(＝O)-、-C(＝O)O-、-OC(＝O)O-、-O-、-S-、-S-S-、-C(＝O)S-、-SC(＝O)-、-NRc-、-NRcC(＝O)-、-C(＝O)NRc-、-NRcC(＝O)NRc-、-OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc-、-NRcC(＝O)S-及其组合中的任一种；氮支化聚乙二醇化脂质含有两个或两个以上L时，任两个L为相同或不同结构；
所述亚烷基为-(CRcRc)t-，其中t每次出现时各自独立地为1-12的整数，优选t为1-4的整数，更优选t为1或2；Rc每次出现时各自独立地为H、C1-6烷基、含碳环的基团、含杂环的基团、氨基酸的侧链基团中任一种，优选为H、甲基、异丙基、异丁基、苄基中任一种，更优选为H或苄基，最优选为H。
本发明的一种具体实施方案中，L1、L2各自独立地为连接键、-O-、-C(＝O)-、-OC(＝O)-、-C(＝O)O-、-NHC(＝O)-、-C(＝O)NH-、-OC(＝O)O-、-OC(＝O)NH-、-NHC(＝O)O-中任一种。
本发明的一种具体实施方案中，Lx各自独立地为连接键、-(CH2)t-、-(CH2)tC(＝O)(CH2)t-、-(CH2)tC(＝O)O(CH2)t-、-(CH2)tOC(＝O)(CH2)t-、-(CH2)tC(＝O)NH(CH2)t-、-(CH2)tNHC(＝O)(CH2)t-中任一种；其中t为1-4的整数，t优选为1或2；优选Lx各自独立地为-CH2CH2-、-CH2C(＝O)-、-C(＝O)CH2-、-C(＝O)CH2CH2-、-CH2CH2C(＝O)OCH2CH2-、-C(＝O)NH-中任一种。
本发明的一种具体实施方案中，任一Lx与XPEG中的二价连接基-O-共同构成二价连接基-O-Lx；
优选y个Lx各自独立地为以下任一种情形：
情形(1)：Lx为连接键；
情形(2)：Lx为碳链连接基，且选自亚烃基、羰基、侧基含杂原子的碳链连接基及其组合中的任一种；
情形(3)：Lx为含有主链杂原子的连接基，优选Lx或-O-Lx为醚基、硫醚基、二硫键、二硒键、酯基、一硫代酯基、二硫代酯基、碳酸酯基、一硫代碳酸酯基、二硫代碳酸酯基、三硫代碳酸酯基、仲氨基、酰胺基、氨基甲酸酯基、一硫代氨基甲酸酯基、二硫代氨基甲酸酯基、亚胺基、三氮唑连接基、4,5-二氢异恶唑连接基、2,5-二氧代吡咯烷连接基中任一种，或其中任一种、任两种或两种以上与亚烃基的组合，更优选Lx或-O-Lx为醚基、酯基、碳酸酯基、二硫键、酰胺基或氨基甲酸酯基；
情形(4)：Lx或-O-Lx中含有经偶合反应生成的连接基：Lx或-O-Lx为通过烷基化反应、酰胺化反应、酯化反应、硫酯化反应、click反应、环加成反应、Diels-Alder加成反应或1,3-偶极环加成反应生成的连接基，或为所述连接基中的任一种或一种以上与亚烃基的组合。
本发明的一种具体实施方案中，Ld为连接键、-(CRcRc)t-、-C(＝O)-、-O-、-NH-及其组合中的任一种，其中t为1或2，Rc为H或氨基酸的侧链基团；
优选Ld为-CH2CH2-、-CH2C(＝O)-、-CH2CH2C(＝O)-、-CH2C(＝O)NHCH2CH2-、-CH2C(＝O)OCH2CH2-、-CH2CH2C(＝O)OCH2CH2-、-CH2C(＝O)NHCH2C(＝O)-、 -CH2C(＝O)NHCH2CH2C(＝O)OCH2CH2-、-CH2CH2C(＝O)NHCH2C(＝O)-、-CH2C(＝O)-Gly-Gly-OCH2CH2-、-CH2C(＝O)-Phe-Gly-OCH2CH2-中任一种，且左端与Ncore相连；其中，Gly为甘氨酸残基，结构为-NHCH2C(＝O)-；Phe为苯丙氨酸残基，结构为
本发明的一种具体实施方案中，Ld选自连接键、亚烷基或以下任一类别中的任一种，或任1-4种与亚烷基的组合：
(i)由氨基、羟基、羧基、巯基、二硫键、二硒连键、醛基、酮羰基、胍基、酰胺基、叠氮基、炔基、烯基、咪唑基、吲哚基中任一种活性基团参与偶合反应而生成的共价连接基；
(ii)酰胺基、仲氨基、二价叔氨基、氨基甲酸酯基、硫代氨基甲酸酯基、亚胺基、烯胺基、氨基胍基、氨基脒基、亚氨酸酯基、亚氨酸硫酯基、酯基、羰基、苄氧羰基、硫代酯基、酸酐连接基、酰肼基、酰腙基、酰亚胺连接基、醚基、硫醚基、二硫键、二硒键、含胍基的连接基、含咪唑基的连接基、含三氮唑基的连接基、4,5-二氢异恶唑连接基、乙烯醚键、碳酸酯基、硫代碳酸酯基、二硫代碳酸酯基、三硫代碳酸酯基、二硫代氨基甲酸酯基、缩醛基、环缩醛基、缩硫醛基、二硫代缩醛基、半缩醛基、硫代半缩醛基、缩酮基、缩硫酮基、肟键、硫肟醚基、半卡巴腙键、硫代半卡巴腙键、硫代碳酰肼基、偶氮羰酰肼基、硫代偶氮羰酰肼基、肼基甲酸酯基、肼基硫代甲酸酯基、卡巴肼基、硫代卡巴肼基、脲基甲酸酯基、硫脲基甲酸酯基、胍基、脒基、磺酸基、磺酸酯基、亚磺酸酯基、原酸酯基、磷酸酯基、亚磷酸酯基、次磷酸酯基、膦酸酯基、磷硅烷酯基、硅烷酯基、肼基、脲键、硫脲键、异脲键、异硫脲键、硫代酰胺基、磺酰胺基、磷酰胺基、亚磷酰胺基、焦磷酰胺基、环磷酰胺基、异环磷酰胺基、硫代磷酰胺基、氮杂缩醛基、氮杂环缩醛基、氮硫杂缩醛基、氮杂半缩醛基、氮杂缩酮基、氮杂环缩酮基、氮硫杂缩酮基，以及其中任一种或一种以上与亚烷基的组合。
1.2.2.脂质部分
本发明的一种具体实施方案中，B1、B2各自独立地为连接键或直链的C1-14亚烷基；所述C1-14亚烷基有0-2个氢原子被0-2个Rq取代，Rq每次出现时各自独立地为-(CH2)tqC[(CH2)tqH]3、-(CH2)tqO(CH2)tqH、-(CH2)tqS(CH2)tqH或-(CH2)tqN[(CH2)tqH]2，其中tq每次出现时各自独立地为0-4的整数；优选Rq每次出现时各自独立地为-OH或-CH3；
优选B1、B2各自独立地为连接键或-(CH2)1-10-，更优选B1、B2各自独立地为连接键、-(CH2)2-、-(CH2)3-、-(CH2)4-、-(CH2)5-、-(CH2)6-、-(CH2)7-、-(CH2)8-中任一种。
本发明的一种具体实施方案中，R1、R2各自独立地含有0-8个碳碳双键和/或0-8个碳碳三键，且各自独立地有0-10个氢原子各自独立地被0-10个Rm取代；所述Rm每次出现时各自独立地为选自直链C1-6烷基、支化C1-6烷基、C3-12环烷基、C2-8烯基、C2-8炔基、苯基、苄基中任一种；优选Rm为甲基；
所述R1、R2各自独立地选自RL、RB、Rr中任一种，优选R1、R2各自独立地为RL或RB；
其中，RL选自以下结构中任一种或任一种的顺反异构体：

RL优选为以下结构中任一种：
其中，RB结构为其中，X为CH或N；t为0-5的整数；Be、Bf各自独立地为连接键或C1-10亚烷基；Le、Lf各自独立地为连接键、-O-、-OC(＝O)-、-C(＝O)O-、-NHC(＝O)-、-C(＝O)NH-中任一种；Re、Rf各自独立地为C1-12烷基；
RB优选为以下结构中任一种：
RB更优选为以下结构中任一种：
其中，Rr为含环状结构的C4-30烷基或C4-30杂烷基，优选为
本发明的一种具体实施方案中，RL优选含0-10个Rm的C1-30直链状脂肪烃基；更优选为C1-25直链状烷基、十三碳-8-烯基、十四碳-9-烯基、十五碳-8-烯基、十六碳-9-烯基、十七碳-5-烯基、十七碳-8-烯基、十七碳-10-烯基、十七碳-8,11-二烯基、十八碳-6-烯基、十八碳-9-烯基、十八碳-11-烯基、十八碳-9,12-二烯基、十八碳-9,12,15-三烯基、十八碳-8,11,14-三烯基、十九碳-10-烯基、十九碳-10,13-二烯基、二十碳-11-烯基、二十碳-11,14-二烯基、2,6,10-三甲基十一碳-1,5,9-三烯基、3,7,11-三甲基十二碳-2,6,10-三烯基和3,7,11,15-四甲基十六碳-2,6,10,14-四烯基中任一种。
1.2.3.聚乙二醇组分
本发明的一种具体实施方案中，氮支化聚乙二醇化脂质的结构如通式(1)或通式(2)所示。
本发明的一种具体实施方案中，任两个RPEG的数均分子量相同或不同，每个RPEG 各自独立地为单分散性或多分散性。
本发明的一种具体实施方案中，任两个RPEG的聚合度相同或不同；优选所有RPEG的聚合度相同。
本发明的一种具体实施方案中，RPEG的数均分子量选自0.5kDa～20kDa；优选为0.5kDa～5kDa；更优选为0.5kDa、1kDa、2kDa或5kDa。
本发明的一种具体实施方案中，XPEG各自独立地为线性结构或非线性结构；所述非线性结构选自以下任一情形：
情形(1)：由以下任一种小分子的多价残基构建：多元官能化小分子、含有一个异质官能团的多元官能化小分子、氨基酸小分子；
情形(2)：由以下任一种非线性组合构建：支化结构、梳状结构、树状结构、类树状结构、环状结构、超支化结构及其组合中的任一种；
其中，多元官能化小分子优选多元醇、多元硫醇、多元羧酸、多元伯胺或多元仲胺；
其中，含有一个异质官能团的多元官能化小分子优选为含有一个异质官能团的多元醇、含有一个异质官能团的多元硫醇、含有一个异质官能团的多元羧酸、含有一个异质官能团的多元伯胺或含有一个异质官能团的多元仲胺；
其中，氨基酸小分子优选甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、色氨酸、酪氨酸、天冬氨酸、组氨酸、天冬酰胺、谷氨酸、赖氨酸、谷氨酰胺、甲硫氨酸、精氨酸、丝氨酸、苏氨酸、半胱氨酸、鸟氨酸或瓜氨酸；
优选任一个XPEG包含2个RPEG，进一步优选所述非线性结构由以下任一种结构的三价残基构建：三元醇、三元硫醇、三元伯胺、三元仲胺、三元羧酸、三元磺酸酯、三元异氰酸酯、含有一个异质官能团的三元官能化小分子、氨基酸小分子。
本发明的一种具体实施方案中，同一分子中的T具有相同结构。
本发明的一种具体实施方案中，L01为连接键、-(CH2)t-、-NH(CH2)t-、-NH(CH2)tC(＝O)NH(CH2)t-、-O(CH2)t-、-NH(CH2)tC(＝O)O(CH2)t-、-OC(＝O)(CH2)t-、-OC(＝O)O(CH2)t-、-OC(＝O)(CH2)tC(＝O)-、-(CH2)tC(＝O)NH(CH2)t中任一种，且左端与R01连接，其中t为1-4的整数，t优选为1或2。
本发明的一种具体实施方案中，R01为能与生物相关物质相互反应的功能性基团，其中，所述反应选自共价键的形成、氢键的形成、荧光作用和靶向作用；R01选自反应性基团、反应性基团的变化形式、具有治疗靶向性的功能性基团、荧光性功能性基团中任一种；其中，所述变化形式选自反应性基团的前体、以反应性基团作为前体的活性形式、反应性基团被取代的活性形式、反应性基团被保护的非活性形式中任一种；其中，所述反应性基团的前体指经过氧化、还原、水合、脱水、电子重排、结构重排、盐络合与解络合、离子化、质子化、去质子化中至少一个过程，可转变为所述反应性基团的结构。
本发明的一种具体实施方案中，前述R01为选自下述类A～类I中的任一种功能性基团或其变化形式：
类A：活性酯基、活性酯基的类似结构；其中，活性酯基选自琥珀酰亚胺活性酯基、对硝基苯活性酯基、邻硝基苯活性酯基、1,3,5-三氟苯活性酯基、1,3,5-三氯苯活性酯基、1,3,5-三溴苯活性酯基、1,3,5-三碘苯活性酯基、五氟苯活性酯基、咪唑活性酯基、苯并三唑活性酯基、噻唑烷-2-硫酮活性酯基、四氢吡咯-2-硫酮活性酯基、2-巯基苯并噻唑活性酯基、1-氧代-3-硫氧代异吲哚啉活性酯基中任一种；其中，活性酯基的类似结构为活性羧酸酯基或活性酰基；
类B：羧基、被保护的羧基、磺酸基、磺酸酯基、亚磺酸基、亚磺酸酯基、次磺酸基、酯基、硫代酯基、二硫代酯基、碳酸酯基、硫代碳酸酯基、二硫代碳酸酯基、三硫 代碳酸酯基、黄原酸基、四硫双酯基、砜基、亚砜基、甲基丙烯酰基、异羟肟酸基、硫代异羟肟酸基、磺酰卤基、硫代羧基；
类C：醛基、水合醛基、硫醛基、酰卤基、酮基、水合酮基、硫酮基、硫酮水合物基、乙二醛基、缩醛基、单缩硫醛基、双缩硫醛基、缩酮基、单缩硫酮基、双缩硫酮基、半缩醛基、硫代半缩醛基、半缩酮基、原酸基、被保护的原酸基、原酸酯基、氰酸酯基、硫氰酸酯基、异氰酸酯基、异硫氰酸酯基、噁唑啉基、异噁唑啉基；
类D：伯氨基、仲氨基、被保护的氨基、羟胺基、巯基、双硫化合物基、卤原子、卤代乙酰胺基、铵盐、肼基、四甲基哌啶氧基、二氧杂哌啶氧基、O-羰基羟胺基、酰胺基、酰亚胺基、酰肼基、磺酰肼基、腙基、亚胺基、烯胺基、炔胺基、氨基甲酸酯基、一硫代氨基甲酸酯基、二硫代氨基甲酸酯基；
类E：脲基、硫脲基、胍基及其质子化形式、脒基及其质子化形式、酸酐基、方酸基、方酸酯基、半方酸基、半方酸酯基、咪唑-1-甲酰胺基、亚胺酸酯基、硝酮基、醛肟基、酮肟基；
类F：马来酰亚胺基、呋喃保护的马来酰亚胺基、丙烯酸酯基、N-丙烯酰胺基、N-甲基丙烯酰胺基、甲基丙烯酸酯基、马来酰胺酸基、1,2,4-三唑啉-3,5-二酮基、线形的偶氮化合物基、环状的偶氮化合物基；
类G：烯基、烯基烃基、环烯烃基、炔基、炔基烃基、被保护的炔基、环炔烃基、线性的共轭二烯烃基、环状的共轭二烯烃基、含杂原子的环状的共轭二烯烃基、环氧基、1,2,4,5-四嗪基、叠氮基、氧化腈基、氰基、异氰基、重氮基、重氮鎓离子、氧化偶氮基、腈亚胺基、N-氧化醛亚胺基、四氮唑基、4-乙酰基-2-甲氧基-5-硝基苯氧基及其重氮化形式、咪唑基、吲哚基；其中，环烯烃基选自环辛烯烃基、降冰片烯基、降冰片二烯基、氧杂降冰片烯基、氧杂降冰片二烯基中任一种；
类H：羟基、被保护的羟基、被保护的双羟基、硅氧基、三羟基硅基、被保护的三羟基硅基；其中，羟基选自醇羟基、酚羟基、烯醇式羟基、半缩醛羟基中任一种；
类I：单糖基，选自阿洛糖、阿卓糖、阿拉伯糖、克拉定糖、赤藓糖、赤藓酮糖、果糖、岩藻糖醇、岩藻糖胺、岩藻糖、墨角藻糖、半乳糖胺、半乳糖胺醇、N-乙酰-半乳糖胺、半乳糖、葡糖胺、N-乙酰-葡糖胺、葡糖胺醇、葡萄糖、葡萄糖-6-磷酸、古洛糖甘油醛、L-甘油-D-甘露-庚糖、甘油、甘油醛、二羟基丙酮、古洛糖、艾杜糖、来苏糖、甘露糖胺、甘露糖、甘露糖-6-磷酸、甘露庚酮糖、阿洛酮糖、奎诺糖、奎诺糖胺、鼠李糖醇、鼠李糖胺、鼠李糖、核糖、核酮糖、脱氧核糖、景天庚酮糖、山梨糖、塔格糖、塔罗糖、酒石酸、苏糖、木糖、木酮糖中任一种或其功能性衍生物的残基；所述单糖基为D-构型或L-构型，为环状或链状结构，为取代的或未取代的；
其中，被保护的羟基优选醚、硅醚、酯、碳酸酯、磺酸酯中任一种；被保护的氨基优选氨基甲酸酯、酰胺、酰亚胺、N-烷基胺、N-芳基胺、亚胺、烯胺、咪唑、吡咯、吲哚中任一种；被保护的巯基优选硫醚、二硫醚、硅基硫醚、硫代酯中任一种；被保护的羧基优选为羧基被甲基、乙基、叔丁基、苄基中任一种保护的形式；被保护的炔基优选为炔基被硅基保护的形式；被保护的双羟基优选其保护基与两个氧原子构成五元环或六元环的缩醛结构；所述双羟基的保护基优选为亚甲基或取代的亚甲基，更优选为亚甲基、1-甲基亚甲基、1,1-二甲基亚甲基、1,1-亚环戊烷基、1,1-亚环己烷基、1-苯基亚甲基、3,4-二甲基苯基亚甲基中任一种。
本发明的一种具体实施方案中，前述R01选自下述类A～类I中的任一种功能性基团或其变化形式：
类A：
类B：
类C：
类D：
类E：
类F：
类G：

类H：
类I：
其中，X1为卤素原子，选自氟原子、氯原子、溴原子和碘原子中任一种；
其中，Y1选自C1-5烷基、乙烯基、苯基、苄基、对甲基苯基、4-(三氟甲氧基)苯基、三氟甲基和2,2,2-三氟乙基中任一种；
其中，Rd2为有机基团，优选每次出现时各自独立地选自C1-5烷基、C2-5烯基、C2-5炔基和苯基中任一种，所述烷基、烯基、炔基和苯基各自独立地为取代的或未取代的；
其中，W为离去基团，选自-F、-Cl、-Br、-I和-SPh中任一种；
其中，M5为成环原子，选自碳原子、氮原子、磷原子和硅原子中任一种；M5所在的环状结构为3～30元环，优选3～20元环，更优选3～16元环，更优选5～16元环；所述环状结构优选以下组中任一种、任一种的被取代形式、或任一种的被杂化形式：环己烷、呋喃糖环、吡喃糖环、苯、四氢呋喃、吡咯烷、噻唑烷、环己烯、四氢吡喃、哌啶、1,4-二氧六环、吡啶、哒嗪、嘧啶、吡嗪、1,3,5-三嗪、1,4,7-三氮杂环壬烷、环三肽、茚、二氢化茚、吲哚、异吲哚、嘌呤、萘、二氢蒽、氧杂蒽、硫代氧杂蒽、二氢菲、10,11-二氢-5H-二苯并[a,d]环庚烷、二苯并环庚烯、5-二苯并环庚烯酮、喹啉、异喹啉、芴、 咔唑、亚氨基二苄、萘乙环、二苯并环辛炔、氮杂二苯并环辛炔；
其中， 分别为环骨架上含有缩醛基、二硫键、胺基、酰亚胺基、酸酐基、偶氮基、碳碳双键、碳碳三键、共轭二烯的环状结构，所述环状结构选自碳环、杂环、苯并杂环、取代的碳环、取代的杂环或取代的苯并杂环；
其中，Q是有助于不饱和键电子的诱导、共轭效应的原子或取代基；当Q处于环上时，数量是一个或多个；当数量为多个时，为相同结构，或为两种或两种以上不同结构的组合；当为取代基时，Q具有直链结构、含侧基的支链结构或含环状结构；
所述变化形式选自反应性基团的前体、以反应性基团作为前体的活性形式、反应性基团被取代的活性形式、反应性基团被保护的非活性形式中任一种；其中，所述反应性基团的前体指经过氧化、还原、水合、脱水、电子重排、结构重排、盐络合与解络合、离子化、质子化、去质子化中至少一个过程，可转变为所述反应性基团的结构。
本发明的一种具体实施方案中，前述R01为羟基、巯基、活性酯基、活性碳酸酯基、磺酸酯基、氨基、马来酰亚胺基、琥珀酰亚胺基、羧基、酰氯基、醛基、叠氮基、氰基、烯基、炔基、环氧烷基、罗丹明基、叶酸残基、生物素残基、单糖基和多糖基中任一种反应性基团或其变化形式，所述变化形式选自所述反应性基团的前体、以所述反应性基团作为前体的活性形式、所述反应性基团被取代的活性形式、所述反应性基团被保护的非活性形式中任一种；其中，所述反应性基团的前体指经过氧化、还原、水合、脱水、电子重排、结构重排、盐络合与解络合、离子化、质子化、去质子化中至少一个过程，可转变为所述反应性基团的结构；优选R01选自以下结构中任一种：
本发明的一种具体实施方案中，T为氢原子或C1-6烷基，优选为甲基。
1.2.4.Ncore
本发明的一种具体实施方案中，Ncore为原子、支链状、环状中任一种类型的多价核结构，所述多价原子核为三价氮原子核；当Ncore为所述多价支链状核结构或多价环状核结构时，其含有至少一个三价氮原子核，且优选为以下任一情形：
情形(1)：由以下任一种小分子的多价残基构建：多元官能化小分子、多元异官能化小分子、氨基酸；
情形(2)：由以下任一种非线性结构构建：支化结构、梳状结构、树状结构、类树状结构、环状结构、超支化结构及其组合；
其中，多元官能化小分子优选多元醇、多元硫醇、多元羧酸、多元伯胺或多元仲胺；
其中，多元异官能化小分子优选为含有一个异质官能团的多元醇、含有一个异质官能团的多元硫醇、含有一个异质官能团的多元羧酸、含有一个异质官能团的多元伯胺或含有一个异质官能团的多元仲胺；
其中，氨基酸优选甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、色氨酸、酪氨酸、天冬氨酸、组氨酸、天冬酰胺、谷氨酸、赖氨酸、谷氨酰胺、甲硫氨酸、精氨酸、丝氨酸、苏氨酸、半胱氨酸、鸟氨酸或瓜氨酸，更优选为天冬氨酸、谷氨酸、赖氨酸、鸟氨酸或甘氨酸；
更优选Ncore为氨基酸残基、二聚氨基酸残基或多聚氨基酸残基，所述多聚氨基酸含有至少3个氨基酸单元，所述二聚氨基酸、多聚氨基酸中的氨基酸酸单元各自独立且任意两个之间氨基酸酸单元为相同或不同结构。
本发明的一种具体实施方案中，y为2，Ncore为三价核结构，优选Ncore为以下任一类中的任一种结构：
类(1)：三价氮原子核结构；具体为以下结构：
类(2)：含氮杂原子的环状三价核结构，其中环状结构选自以下任一种、任一种的被取代形式、或任一种的被杂化形式：吡咯烷、哌啶、吡嗪、1,4,7-三氮杂环壬烷、环三肽、吲哚、异吲哚、嘌呤、咔唑、亚氨基二苄、氮杂二苯并环辛炔；优选为以下任一种结构：
Q为电子改变基团，其数量为0、1或大于1；当Q的数量大于1时，任两个Q为相同或不同结构；
类(3)：以下任一种结构的三价残基：三元醇、三元硫醇、三元伯胺、三元仲胺、三元羧酸、三元磺酸酯、三元异氰酸酯、三元异官能化小分子，氨基酸；所述三元异官能化小分子含有两种不同官能团，其中一种的数量为1个，另一种的数量为2个；其中，氨基酸的三价残基优选为以下任一种结构：

类(1)～类(3)中，u1、u2、u3各自独立地为与Ld或Lx相连的连接键，且u1、u2、u3中任两个不同时与Ld或任一个Lx相连接。
本发明的一种具体实施方案中，y为3，Ncore含有原子、支链状、环状结构中任一种类型的四价核结构，或包含两个三价核结构的组合；优选Ncore的结构为以下任一种：

其中，u1、u2、u3、u4均为连接键，u1、u2、u3、u4各自独立地与Ld或任一个Lx相连接，且u1、u2、u3、u4中任两个不同时与Ld或任一个Lx相连接。
本发明的一种具体实施方案中，y≥4，Ncore的价态≥5，Ncore选自梳状结构、树状结构、支化结构、超支化结构、环状结构中任一种；
所述树状结构选自以下任一种：
其中，u1为连接至Ld的连接键，结构里的星号*标记为连接至聚乙二醇组分的连接键；d表示树状组合方式的代数，且d选自2、3、4、5或6；
所述支化结构、超支化结构、梳状结构为三价核结构、四价核结构、五价核结构及其组合中的任一种，优选为氨基酸及其衍生物中的任一种或一种以上构成的多聚体残基或其被氧化形式；
所述环状结构选自以下任一种：环肽或其衍生物的残基、1,4,7,10-四氮杂环十二烷的骨架。
本发明的一种具体实施方案中，Ncore选自以下任一种结构：
其中，u1、u2、u3各自独立地为与Ld或Lx相连的连接键，且u1、u2、u3中任两个不同时与同一个Ld或Lx相连接；
Q为电子改变基团，其数量为0、1或大于1；当Q的数量大于1时，任两个Q为相同或不同结构。
1.2.5.制备方法
本发明的一种具体实施方案中，聚乙二醇化脂质通过含有偶合反应和/或聚合反应的制备过程获得；
所述偶合反应，供选择范围没有特别限制，只要两个相同或不同的反应性基团经该反应能够形成共价连接基即可；同一制备过程中可含有单步或分步的偶合反应，优选每一步偶合反应各自独立地为烷基化反应、缩合反应、酰胺化反应、酯化反应、硫酯化反应、开环反应、关环缩合反应、加成反应、环加成反应、α,β-不饱和键加成反应、炔基加成反应、席夫碱反应联合还原反应、click反应、叠氮-炔加成反应、1,3-偶极环加成反应、Diels-Alder加成反应、thiol-yne反应、thiol-ene反应、thiol-vinyl反应和缩合反应中 任一种；所述偶合反应的反应条件与反应生成的共价连接基类型有关，可采用现有公开技术；经所述偶合反应生成的共价连接基的价态可以为二价或三价，优选为二价；经所述偶合反应可生成稳定的基团，也可生成可降解的基团；
所述聚合反应，至少经历以下两个步骤：小分子引发剂去质子化、环氧乙烷的聚合；所述小分子引发剂可以是直接获得的原料，也可以是制备过程中的中间体；
所述制备过程中，当偶合反应和聚合反应同时存在时，偶合反应、聚合反应的先后顺序没有限制。
本发明中，各制备方法中用到的原料可以购买获得或者自行合成获得。
本发明中，单分散性聚乙二醇化脂质的制备方法中，可以将含聚乙二醇组分的单分散性原料替换为相同组分的多分散性原料，获得相应的多分散性产物；同样地，多分散性聚乙二醇化脂质的制备方法中，可以将含聚乙二醇组分的多分散性原料替换为相同组分的单分散性原料，获得相应的单分散性产物。
本发明中的一些具体方案中，聚乙二醇原料，包括但不限于线性/非线性聚乙二醇及其衍生物，可以参考CN108530637B、CN110591079A、CN108659227A、CN108530617B、CN1243779C或CN101029131A中的方法制备得到。
本发明中制备的中间体、终产物都可通过包括但不限于萃取、重结晶、吸附处理、沉淀、反沉淀、薄膜透析或超临界提取等的纯化方法加以纯化。对终产物的结构、分子量的表征确认，可采用包括但不限于核磁、电泳、紫外-可见分光光度计、FTIR、AFM、GPC、HPLC、MALDI-TOF、圆二色谱法等表征方法。
本发明中，实际制备过程还可包括本发明中提及或未提及的本领域技术人员熟悉的任何必要的微修饰、保护/脱保护、中间体制备、后处理或纯化等过程。
式(A-3)化合物的合成：
式(A-1)化合物与式(A-2)化合物经过1～2步反应，根据实际情况可选地脱除保护基，获得式(A-3)氮支化脂质化合物。
其中，n为1或2，B1、B2、L1、L2、R1、R2定义与通式(1)相同；W为离去基团，选自-Cl、-Br、-I、-OMs和-OTs中任一种；W-B1-L1-R1和W-B2-L2-R2中的W为相同或不同基团；
Lm1为二价连接基，选自连接键、亚烷基、-C(＝O)-、-OC(＝O)-、-C(＝O)O-、-OC(＝O)O-、-O-、-S-、-S-S-、-C(＝O)S-、-SC(＝O)-、-NRc-、-NRcC(＝O)-、-C(＝O)NRc-、-NRcC(＝O)NRc-、-OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc-、-NRcC(＝O)S-及其组合中的任一种，所述组合优选含有亚烷基；所述亚烷基为-(CRcRc)1-12-，优选为-(CH2)1-12-，更优选为-(CH2)1-4-，最优选为-CH2-或-(CH2)2-；Rc每次出现时各自独立地为H、C1-6烷基、碳环基、杂环基中任一种，优选为H；
FG1为反应性基团或其被保护形式，选自氨基、羟基、巯基、羧基、卤素基团、磺酸酯基、醛基、酰卤基、活化羧基、活性酯基中任一种或其被保护形式，优选为-NH2、-NHBoc、-NHFmoc、-NHCbz、-OH、-OTBS、-OMs、-OTs中任一种；式(A-1)与式(A-3)中的FG1为相同或不同结构。
具体例子如实施例1.1中S1-3的合成：
式(A-5)化合物的合成：
方法一、与前述式(A-3)化合物的合成方法相同，此时Lm1与Lm2相同、FG1与FG3相同，式(A-5)化合物结构对应式(A-3)。
方法二、通过式(A-3)化合物与式(A-4)双官能化小分子化合物进行一次或连续多次的偶联反应，根据实际情况可选地脱除保护基，获得式(A-5)化合物：
其中，SM为小分子残基；FGi和FG3的定义均与FG1相同，具体形式以反应能够顺利实施为依据；当两个或多个式(A-4)化合物连续参与反应时，SM每次出现时为相同或不同结构，FGi每次出现时为相同或不同结构；
其中，Lm2为二价连接基，选自亚烷基、-C(＝O)-、-OC(＝O)-、-C(＝O)O-、-OC(＝O)O-、-O-、-S-、-S-S-、-C(＝O)S-、-SC(＝O)-、-NRc-、-NRcC(＝O)-、-C(＝O)NRc-、-NRcC(＝O)NRc-、-OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc-、-NRcC(＝O)S-及其组合中的任一种，所述组合优选含有亚烷基；所述亚烷基为-(CRcRc)1-12-，优选为-(CH2)1-12-，更优选为-(CH2)1-4-，最优选为-CH2-或-(CH2)2-；Rc每次出现时各自独立地为H、C1-6烷基、碳环基、杂环基中任一种，优选为H。
具体例子如实施例2.3中S2-5的合成：
式(A-7)化合物的合成：
式(A-5)化合物与式(A-6)三官能化小分子化合物反应，得到含两个反应性基团和至少两个氮支化结构的式(A-7)化合物。
其中，FG4、FG5、FG6的定义均与FG1相同，具体形式以反应能够顺利实施为依据；L1A、L1B、L1A定义均与Lm1相同，且任两个的结构为相同或不同；FG4与FG3反应产生的二价连接基与Lm2、L1A共同构成Ld。
具体例子如实施例3.2中S3-4的合成：
聚乙二醇组分的引入：
方法一、基于一个氨基引入两个聚乙二醇组分：
式(B-1)化合物含有一个裸露氨基，与含有XPEG的式(C-1)化合物经过1～2步反应，得到式(3)所示的两臂氮支化聚乙二醇化脂质化合物。其中，W为离去基团，选自-Cl、-Br、-I、-OMs和-OTs中任一种。
具体例子如实施例1.1中E1-1的合成：
方法二、基于两个反应性基团引入两个聚乙二醇组分：
式(A-7)化合物与两个相同或不同的式(C-2)化合物反应，得到式(3)所示的两臂氮支化聚乙二醇化脂质化合物。其中，L1D定义与Lm1相同；FGi定义与F1相同，具体形式以反应能够顺利实施为依据；一个FGi与FG5反应，得到的二价连接基与L1B、L1D构成一个Lx，另一个FGi与FG6反应，得到的二价连接基与L1C、L1D构成另一个Lx。
具体例子如实施例3.2中E3-2的合成：
方法三、一次性引入氮支化非线性聚乙二醇组分：
式(A-5)化合物与式(C-3)化合物反应，得到通式(1)氮支化聚乙二醇化脂质化合物。
其中，Lm3为二价连接基，选自连接键、亚烷基、-C(＝O)-、-OC(＝O)-、-C(＝O)O-、-OC(＝O)O-、-O-、-S-、-S-S-、-C(＝O)S-、-SC(＝O)-、-NRc-、-NRcC(＝O)-、-C(＝O)NRc-、-NRcC(＝O)NRc-、-OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc-、-NRcC(＝O)S-及其组合中的任一种，所述组合优选含有亚烷基；所述亚烷基为-(CRcRc)1-12-，优选为-(CH2)1-12-，更优选为-(CH2)1-4-，最优选为-CH2-或-(CH2)2-；Rc每次出现时各自独立地为H、C1-6烷基、碳环基、杂环基中任一种，优选为H；
FG2、FG3均为反应性基团，各自独立地选自氨基、羟基、巯基、羧基、卤素基团、磺酸酯基、醛基、酰卤基、活化羧基、活性酯基中任一种；FG2与FG3反应生成的二价连接基与Lm2、Lm3共同构成Ld。
具体例子如实施例2.1中E2-1的合成：
又如实施例2.3中E2-3的合成：
RPEG末端官能化：
本发明中的一种具体实施方案中，氮支化聚乙二醇化脂质的末端具有功能性基团R01，优选所述R01通过活性聚乙二醇衍生物以未保护或被保护的方式引入。
本发明中的一种具体实施方案中，原料包括活性聚乙二醇衍生物。所述活性聚乙二醇衍生物的结构可以选自文献CN108530637B、CN110591079A、CN108659227A、CN108530617B和CN1243779C中记载的单官能化非线性聚乙二醇具体结构中的任一种。
本发明的一种具体实施方案中，对满足通式(1)或通式(2)的聚乙二醇化脂质进一步末端功能化，所得末端功能化后的结构仍满足通式(1)或通式(2)。所述末端功能化的方法没有特别限制，与最终的功能性基团或其被保护形式的类型相关，主要包括末端羟基的功能化和基于反应性基团的向目标功能性基团或其被保护形式的转变。
本发明中，末端羟基的功能化的具体制备方法包括但不限于如文献CN104530417A中段落[0960]段到[1205]段记载的。
本发明中，基于反应性基团向目标功能性基团或其被保护形式的转变，可以通过以 下任一种方式实现：
方式一：直接修饰，基于反应性基团的直接修饰，得到目标功能性基团或其被保护形式。作为举例，如羧基向酰卤、酰肼、酯、硫酯、二硫代酯的转变，如羟基、巯基、炔基、氨基、羧基等向相应的被保护结构的转变等等。又如酸酐对羟基、氨基等的修饰等。
方式二：两个反应性基团之间的偶联反应，以含有1种反应性基团及目标功能性基团或其被保护形式的异官能化试剂为原料，通过其中一种反应性基团与RPEG末端的反应性基团之间的反应，引入目标功能性基团或其被保护形式。两个反应性基团之间的反应方式、方法没有特别限制，其反应条件与反应生成的二价连接基类型有关，可采用现有公开技术。如烷基化、烯基加成反应、炔基加成反应、席夫碱反应联合还原反应、缩合反应等。其中，烷基化反应优选为基于巯基或氨基的烷基化的反应，依次对应于硫醚键、仲氨基或叔氨基的形成。其中缩合反应包括但不限于生成酯基、硫酯基、酰胺基、亚胺键、腙键、氨基甲酸酯基等的缩合反应。又如以含叠氮、炔基、烯基、三硫酯基、巯基、二烯基、呋喃基、1,2,4,5-四嗪基、氰酸根等基团与目标功能性基团或其被保护形式的异官能化试剂为原料，通过click反应引入目标功能性基团或其被保护形式。两个反应性基团之间的反应伴随新键的生成，新生成的二价连接基的典型代表为酰胺键、尿烷键、酯基、仲胺键、硫醚键、三氮唑基团等。
方式三：通过直接修饰与偶联反应的组合，获得目标功能性基团或其被保护形式。
1.2.6.具体结构举例
聚乙二醇组分为二臂结构：
本发明的一种具体方案中，氮支化聚乙二醇化脂质选自以下任一种：


聚乙二醇组分为多臂结构：
示例结构M-1：
M-1的合成路线如下：
示例结构M-2：
M-2的合成路线如下：
示例结构M-3：
参考M-2的合成，以二臂PEG中间产物代替线性PEG作为起始原料，获得结构如下所示的四臂聚乙二醇胺衍生物，并继续参考上述方法获得含五臂PEG组分的氮支化聚乙二醇化脂质M-3。
1.3.脂质组合物、脂质药物组合物及其制剂、脂质体、脂质纳米粒
本发明的一种实施，
一种脂质组合物，其特征在于，包含任意前述的氮支化聚乙二醇化脂质。
本发明的一种具体实施方案中，脂质组合物还含有磷脂、类固醇脂质和聚乙二醇化脂质中的一种或者一种以上，选自以下情形中任一种：
情形(1)：还含有磷脂；
情形(2)：还含有类固醇脂质；
情形(3)：还含有聚乙二醇化脂质；
情形(4)：还含有磷脂和类固醇脂质；
情形(5)：还含有磷脂和聚乙二醇化脂质；
情形(6)：还含有类固醇脂质和聚乙二醇化脂质；
情形(7)：还含有磷脂、类固醇脂质和聚乙二醇化脂质；
优选还同时含有磷脂、类固醇脂质和聚乙二醇化脂质三种脂质。
本发明的一种具体实施方案中，脂质组合物所含的磷脂选自1,2-二亚油酰基-sn-甘油-3-磷酸胆碱(DLPC)、1,2-二肉豆蔻酰基-sn-甘油-磷酸胆碱(DMPC)、1,2-二油酰基-sn-甘油-3-磷酸胆碱(DOPC)、1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DSPC)、1,2-双十一烷酰基-sn-甘油-磷酸胆碱(DUPC)、1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸胆碱(POPC)、1,2-二-O-十八碳烯基-sn-甘油-3-磷酸胆碱(18:0 Diether PC)、1-油酰基-2-胆固醇基半琥珀酰基-sn-甘油-3-磷酸胆碱(OChemsPC)、1-十六烷基-sn-甘油-3-磷酸胆碱(C16Lyso PC)、1,2-二亚麻酰基-sn-甘油-3-磷酸胆碱、1,2-二花生四烯酰基-sn-甘油-3-磷酸胆碱、1,2-双二十二碳六烯酰基-sn-甘油-3-磷酸胆碱、1,2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)、1,2-二植烷酰基-sn-甘油-3-磷酸乙醇胺(ME 16.0PE)、1,2-二硬脂酰基-sn-甘油-3-磷酸乙醇胺、1,2-二亚油酰基-sn-甘油-3-磷酸乙醇胺、1,2-二亚麻酰基-sn-甘油-3-磷酸乙醇胺、1,2-二花生四烯酰基-sn-甘油-3-磷酸乙醇胺、1,2-双二十二碳六烯酰基-sn-甘油-3-磷酸乙醇胺、1,2-二油酰基-sn-甘油-3-磷酸-rac-(1-甘油)钠盐(DOPG)、二油酰基磷脂酰丝氨酸(DOPS)、二棕榈酰基磷脂酰甘油(DPPG)、棕榈酰基油酰基磷脂酰乙醇胺(POPE)、二硬脂酰基-磷脂酰-乙醇胺(DSPE)、二棕榈酰基磷脂酰乙醇胺(DPPE)、二肉豆蔻酰基磷酸乙醇胺(DMPE)、1-硬脂酰基-2-油酰基-硬脂酰乙醇胺(SOPE)、1-硬脂酰基-2-油酰基-磷脂酰胆碱(SOPC)、鞘磷脂、磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰肌醇、磷脂酸、棕榈酰基油酰基磷脂酰胆碱、溶血磷脂酰胆碱和溶血磷脂酰乙醇胺(LPE)中任一种及其组合物；所述磷脂可以是合成的或天然来源的。
本发明的一种具体实施方案中，脂质组合物所含的类固醇脂质选自胆固醇、粪固醇、谷固醇、麦角固醇、菜油固醇、豆固醇、菜籽固醇、番茄碱、熊果酸、α-生育酚中任一种及其组合物。
本发明的一种具体实施方案中，脂质组合物所含的阳离子脂质选自N,N-二油基-N,N-氯化二甲铵(DODAC)、N,N-二硬脂基-N,N-溴化二甲铵(DDAB)、N-(1-(2,3-二油酰氧基)丙基)-N,N,N-氯化三甲铵(DOTAP)、N-(1-(2,3-二油基氧基)丙基)-N,N,N-氯化三甲铵(DOTMA)、N,N-二甲基-2,3-二油基氧基丙胺(DODMA)、3-(双十二烷基氨基)-N1,N1,4-三-十二烷基-1-哌嗪乙胺(KL10)、N1-[2-(双十二烷基氨基)乙基]-N1,N4,N4-三-十二烷基-1,4-哌嗪二乙胺(KL22)、14,25-双十三烷基-15,18,21,24-四氮杂-三十八烷(KL25)、1,2-二亚油基氧基-N,N-二甲基氨基丙烷(DLin-DMA)、2,2-二亚油基-4-二甲基氨基甲基-[1,3]-二氧杂环戊烷(DLin-K-DMA)、4-(二甲基氨基)丁酸三十七碳-6,9,28,31-四烯-19-基酯(DLin-MC3-DMA)、2,2-二亚油基-4-(2-二甲基氨基乙基)-[1,3]-二氧杂环戊烷(DLin-KC2-DMA)、((4-羟基丁基)氮杂二烷基)双(己烷-6,1-二基)双(2-己基癸酸酯)(ALC-0315)和十七烷-9-基-8-((2-羟乙基)(6-氧代-6-((十一烷氧基)己基)氨基)辛酸酯)(SM102)、
中任一种及其组合物。
本发明的一种更具体的实施方案中，脂质组合物所含的聚乙二醇化脂质、阳离子脂质、磷脂、类固醇脂质各自占总脂质的摩尔百分比没有特别限制；其中，
聚乙二醇化脂质占总脂质的摩尔百分比为0.5-5％，优选为1-3％，更优选为1.5％、1.6％、1.7％、1.8％、1.9％；
阳离子脂质占总脂质的摩尔百分比为30-65％，优选为35％、40％、45％、46％、47％、48％、49％、50％、55％；
磷脂占总脂质的摩尔百分比为7.5-13％，优选为8％、9％、10％、11％、12％；
类固醇脂质占总脂质的摩尔百分比为35-50％，优选为40％、41％、42％、43％、44％、45％、46％、47％、48％、49％、50％。
本发明的脂质组合物可以用于将生物活性成分传递至患者中的以下一或多种：肝脏或肝脏细胞(例如肝细胞)、肾脏或肾脏细胞、肿瘤或肿瘤细胞、CNS或CNS细胞(中枢神经系统，例如脑和/或脊髓)、PNS或PNS细胞(外周神经系统)、肺或肺细胞、血管或血管细胞、皮肤或皮肤细胞(例如真皮细胞和/或滤泡细胞)、眼或眼细胞(例如黄斑、中央凹、角膜、视网膜)、耳或耳细胞(例如内耳、中耳和/或外耳细胞)。
本发明的一种实施方案，
一种脂质药物组合物，其特征在于，含有任意前述的脂质组合物和药物，所述药物选自核酸药物、基因疫苗、抗肿瘤药物、小分子药物、多肽药物和蛋白质药物中任一种。
本发明的一种具体实施方案中，脂质药物组合物所含的药物为核酸药物，选自DNA、RNA、反义核酸、质粒、干扰核酸、适体、antagomir和核酶中任一种，所述RNA选自mRNA、saRNA、circRNA、miRNA和siRNA中任一种；优选为DNA、mRNA、miRNA和siRNA中任一种。
本发明的一种具体实施方案中，脂质药物组合物所含的药物包括但不限多柔比星、米托蒽醌、喜树碱、顺铂、博莱霉素、环磷酰胺、链脲佐菌素、放线菌素D、长春新碱、 长春碱、胞嘧啶阿拉伯糖苷、蒽环霉素、氮芥、噻替哌、苯丁酸氮芥、拉奇霉素、美法兰、卡莫司汀、罗莫司丁、白消安、二溴甘露醇、丝裂霉素C、顺二氯二胺络铂(II)、甲氨蝶呤、6-巯基嘌呤、6-硫鸟嘌呤、阿糖胞苷、5-氟尿嘧啶达卡巴嗪、地布卡因、氯丙嗪、普萘洛尔、第莫洛、拉贝洛尔、可乐定、肼酞嗪、丙咪嗪、阿米替林、多虑平、苯妥英、苯海拉明、氯苯那敏、异丙嗪、庆大霉素、环丙沙星、头孢西丁、咪康唑、特康唑、益康唑、异康唑、布康唑、克霉唑、伊曲康唑、制霉菌素、奈替芬、两性霉素B、抗寄生虫剂、激素、激素拮抗剂、免疫调节剂、神经递质拮抗剂、抗青光眼药、维生素、镇静剂以及成像剂、紫杉酚、细胞松弛素B、短杆菌肽D、溴化乙锭、依米丁、丝裂霉素、依托泊苷、替尼泊苷、秋水仙碱、柔红霉素、二羟基蒽二酮、光辉霉素、1-去氢睾酮、糖皮质激素、普鲁卡因、丁卡因、利多卡因、嘌呤霉素、类美登素、奥沙利铂。
本发明的一种具体实施方案中，脂质药物组合物所含的药物为核酸药物；组合物中阳离子脂质与核酸的N/P比为(0.1～100):1，更优选为(0.2～30):1，最优选为(0.5～20):1。
本发明的一种具体实施方案中，脂质药物组合物作为药物使用，所述药物选自以下任一种：治疗癌症的药物、抗感染剂、抗生素剂、抗病毒剂、抗真菌剂、疫苗。
本发明的一种具体实施方案中，脂质药物组合物为LNP-药物组合物、LPP-药物组合物或PNP-药物组合物；优选为LNP-药物组合物，更优选为LNP-核酸药物组合物，更优选为LNP-mRNA组合物。
本发明中，脂质药物组合物可以形成不同的结构；其中，“LNP-药物组合物”形成脂质纳米粒(lipid nanoparticle,LNP)的结构，“LPP-药物组合物”形成脂质多聚复合物(lipopolyplex,LPP)的结构，“PNP-药物组合物”形成多肽纳米颗粒(polypeptide nanoparticle,PNP)的结构；其中，“LNP-核酸药物组合物”形成脂质纳米粒的结构，且所含药物为核酸类药物；其中，“LNP-mRNA组合物”为LNP-核酸药物组合物的一种，且所含核酸类药物为mRNA。
本发明的一种实施方案：
一种脂质体或者脂质纳米粒，其特征在于，含有任意前述的脂质组合物。
本发明的一种实施方案，
一种脂质药物组合物制剂，其特征在于，含有任意前述的脂质药物组合物和工作液，所述工作液为药学上可接受的稀释剂或赋形剂，所述稀释剂或赋形剂优选为去离子水、超纯水、磷酸盐缓冲液和生理盐水中任一种，更优选为磷酸盐缓冲液或生理盐水，最优选为生理盐水；其中，脂质组合物:工作液的比例优选为(0.05～20)g:100mL，更优选为(0.1～10)g:100mL，最优选为(0.2～5)g:100mL。
本发明中的一种具体实施方案中，脂质药物组合物制剂的制备，包含下述步骤：
(1)将脂质组分在稀释剂或赋形剂中平衡；
(2)将药物加入平衡后的脂质体或脂质纳米粒与稀释剂或赋形剂的混合物中进行复合；
其中，优选地，所述平衡时间为0.1～12h，优选为0.2～6h，更优选为0.5～3h；优选地，所述复合时间为0.1～12h，优选为0.2～5h，更优选为0.5～2h。
本发明中的一种具体实施方案中，LNP-核酸药物组合物的制备，包含下述步骤：
(1)将脂质组分溶于有机溶剂，获得有机相溶液；
(2)将核酸药物加到缓冲液，获得水相溶液；
(3)将有机相溶液和水相溶液混合，获得LNP-核酸药物组合物，并通过超滤洗涤以除去有机溶剂和游离分子，最后通过无菌过滤器，备用；
其中，有机溶剂优选为甲醇、乙醇、丙醇、叔丁醇、乙腈、二甲基亚砜、N,N-二甲基甲酰胺、N,N-二甲基乙酰胺、N-甲基吡咯烷酮中任一种或任一种以上的混合溶剂；缓 冲液优选为柠檬酸盐缓冲液，进一步地，优选其浓度为5-80mM，pH为2-6，更优选浓度为10-50mM，pH为3-5；有机相溶液和水相溶液的体积比优选为1:1-10，更优选为1:2或1:3。
本发明中的一种具体实施方案中，脂质药物组合物形成包含药物的脂质纳米粒(LNP-药物组合物)，采用超声、挤出或微流控装置控制脂质纳米粒的粒径，所述粒径为1～1000nm，优选20～500nm，更优选为60～200nm，最优选为60～150nm。
2.具体实施方式
下面结合一些具体实施例对氮支化聚乙二醇化脂质，以及含氮支化聚乙二醇化脂质的脂质组合物、脂质药物组合物及其制剂的制备及生物活性测试做进一步描述。具体实施例为进一步详细说明本发明，非限定本发明的保护范围。其中，制备氮支化聚乙二醇化脂质的实施例中，终产物通过核磁表征结构，通过GPC或MALDI-TOF确认分子量。
2.1.氮支化聚乙二醇化脂质的制备
实施例1：脂质部分含/不含酯键的氮支化聚乙二醇化脂质
实施例1.1：L1和L2不含酯键的氮支化聚乙二醇化脂质E1-1
对应通式(2)，E1-1中，B1、B2均为连接键，L1、L2均为连接键，R1、R2均为十四烷基；Ld为-CH2CH2-；两个Lx均为-CH2CH2-；T为甲基。每条PEG链的分子量约为1kDa，对应n1≈n2≈22。
制备方法如下：
步骤a：将1-溴十四烷(S1-2,6.09g,22.0mmol)溶于80mL的N,N-二甲基甲酰胺(DMF)中，加入含Boc保护氨基的乙二胺(S1-1,1.60g,10.0mmol)和碳酸钾(K2CO3,3.64g,26.4mmol)，室温下搅拌反应过夜。反应结束后，将反应液减压浓缩，倒入80mL二氯甲烷。依次用10％柠檬酸(40mL*2)、盐水(40mL*2)洗涤后，有机相用无水硫酸钠干燥，过滤，减压浓缩后用TFA/DCM混合溶液(1:1v/v)脱除Boc保护后，用纯化水洗涤，再用二氯甲烷萃取，合并有机相，用无水硫酸钠干燥，过滤、浓缩，通过柱层析纯化得到脂质化合物S1-3(3.92g)。
步骤b：第一次烷基化反应：将线性甲氧基聚乙二醇甲磺酸酯衍生物S1-4(1.32g,1.2mmol,Mn≈1.1kDa,n1≈22,PDI＝1.01)溶于35mL二氯甲烷中，加入S1-3(3.26g,7.2mmol)，室温下反应24小时，反应完毕后将反应液浓缩，加入pH＝7.0的磷酸盐缓冲液搅拌16h，柱层析纯化得到含有仲氨基团的聚乙二醇衍生物S1-5(1.39g)。
步骤c：第二次烷基化反应：将S1-5(1.20g,0.8mmol,Mn≈1.5kDa,n1≈22,PDI＝1.01)与S1-4(0.88g,0.8mmol,Mn≈1.1kDa,n1≈22,PDI＝1.01)溶于10mL二氯甲烷中，室温下反应24小时，反应完毕后浓缩反应液，加入pH＝7.0的磷酸盐缓冲液搅拌16h，柱层析纯化得到氮支化聚乙二醇衍生物E1-1(0.76g)。1H NMR(400MHz,CDCl3)δ:3.75-3.42(m,PEG；4H,-OCH2CH2N<),3.33(s,6H,-OCH3),2.83-2.72(m,4H,-OCH2CH2N<),2.59-2.35(m,4H,>N(CH2)2N<；4H,-N[CH2(CH2)2-]2),1.48-0.98(m,44H,-CH2CH2CH2-；4H,-CH2CH3),0.82(t,6H,-CH2CH3)。GPC测试分子量约为2.5kDa，PDI＝1.02。
参考上述步骤a～c，采用分子量约为2.1kDa(n1≈45,PDI＝1.02)的线性甲氧基聚乙二醇甲磺酸酯衍生物为原料，获得单臂PEG分子量约为2kDa、结构式与E1-1相同、分子量约为4.5kDa的E1-1-2k(PDI＝1.02)，结构由核磁确定。
参考上述步骤a～c，采用分子量为639Da(n1＝11,PDI＝1)的线性甲氧基聚乙二醇甲磺酸酯衍生物为原料，获得单臂PEG分子量约为500Da、结构式与E1-1相同、分子量约为1539Da的E1-1-500(单分散性)，结构由核磁确定。
实施例1.2：L1和L2含酯键的氮支化聚乙二醇化脂质E1-2
对应通式(2)，E1-2中，B1、B2均为亚己基，L1、L2均为酯基-OC(＝O)-，R1、R2均为Ld为-CH2CH2-；两个Lx均为-CH2CH2-；T为甲基。每条PEG链的分子量约为1kDa，对应n1≈n2≈22。
制备方法如下：
步骤a：在氩气气氛下，向装有溶于二氯甲烷(100mL)的2-己基癸酸(S1-6,7.68g,30.0mmol)、6-溴己醇(S1-7,5.97g,33.0mmol)和DMAP(0.92g,7.5mmol)的圆底烧瓶加入DCC(9.27g,45.0mmol)，室温下反应16h。反应结束后，通过过滤将沉淀去除。浓缩滤液，得到的残余物通过硅胶柱层析纯化得到化合物S1-8(10.55g)。
步骤b：将S1-8(9.22g,22.0mmol)溶于100mL的N,N-二甲基甲酰胺(DMF)中，加入S1-1(1.60g,10.0mmol)和碳酸钾(K2CO3,3.64g,26.4mmol)，室温下搅拌反应过夜。反应结束后，将反应液减压浓缩，倒入100mL二氯甲烷。依次用10％柠檬酸(50mL*2)、盐水(50mL*2)洗涤后，有机相用无水硫酸钠干燥，过滤，减压浓缩后用TFA/DCM混合溶液(1:1v/v)脱除Boc保护后，用纯化水洗涤，再用二氯甲烷萃取，合并有机相，用无水硫酸钠干燥，过滤、浓缩，通过柱层析纯化得到脂质化合物S1-9(6.05g)。
步骤c：参考实施例1.1步骤c～d的反应条件和投料比，将S1-9(5.31g,7.2mmol)与S1-4进行两次烷基化反应(第一次1.2mmol，第二次0.8mmol)，柱层析纯化得到氮支化聚乙二醇化脂质E1-2(0.63g)。1H NMR(400MHz,CDCl3)δ:4.08(t,4H,-CH2OC(＝O)-),3.76-3.42(m,PEG；4H,-OCH2CH2N<),3.36(s,6H,-OCH3),2.85-2.70(m,4H,-OCH2CH2N<),2.58-2.36(m,4H,>N(CH2)2N<；4H,-N[CH2(CH2)2-]2),2.35-2.28(m,2H,-OC(＝O)CH<),1.71-1.08(m,56H,-CH2CH2CH2-；8H,-CH2CH3),0.88(t,12H,-CH2CH3)。GPC测试分子量约为2.8kDa，PDI＝1.02。
实施例1.3：L1和L2含酯键的氮支化聚乙二醇化脂质E1-3
对应通式(2)，E1-3中，B1为亚戊基，B2为亚庚基，L1、L2均为酯基-C(＝O)O-，R1为十一烷基，R2为Ld为-CH2CH2-；两个Lx均为-CH2CH2-；T为甲基。每条PEG链的分子量约为1kDa，对应n1≈n2≈22。
制备方法如下：
步骤a：在氩气气氛下，向装有溶于二氯甲烷(80mL)的2-己基癸酸(S1-10,4.91g,22.0mmol)、6-溴己醇(S1-11,6.20g,24.2mmol)和DMAP(0.67g,5.5mmol)的圆底烧瓶加入DCC(6.80g,33.0mmol)，室温下反应16h。反应结束后，通过过滤将沉淀去除。浓缩滤液，得到的残余物通过硅胶柱层析纯化得到化合物S1-12(8.64g)。
步骤b：将S1-12(8.32g,18.0mmol)溶于100mL的N,N-二甲基甲酰胺(DMF)中，加入S1-1(2.40g,15.0mmol)和碳酸钾(K2CO3,2.98g,21.6mmol)，室温下搅拌反应过夜。反应结束后，将反应液减压浓缩，倒入100mL二氯甲烷。依次用10％柠檬酸(50mL*2)、盐水(50mL*2)洗涤后，有机相用无水硫酸钠干燥，过滤，减压浓缩后用TFA/DCM混合溶液(1:1v/v)脱除Boc保护后，用纯化水洗涤，再用二氯甲烷萃取，合并有机相，用无水硫酸钠干燥，过滤、浓缩，通过柱层析纯化得到脂质化合物S1-13(6.64g)。
步骤c：将S1-13(5.41g,10.0mmol)溶于干燥的THF(60mL)中，冰浴下缓慢加入NaH(60％,4.00g,100.0mmol)，于冰浴下反应1小时。反应结束后，加入6-溴己酸十一烷基酯(S1-14,4.19g,12.0mmol，其中S1-14是由6-溴己酸和1-十一醇反应制备得到的，参考本实施例步骤a)，冰浴下搅拌反应1小时后，反应液慢慢恢复至室温反应过夜。反应结束后，将反应置于冰浴下，缓慢加入1mL水淬灭反应，搅拌反应30分钟后，旋蒸除去溶剂，加入水(60mL)搅拌混合，再用二氯甲烷(30mL*2)萃取两次，合并二氯甲烷有机相，用饱和氯化钠(30mL*2)洗涤。有机相减压浓缩后用TFA/DCM混合溶液(1:1v/v)脱除Boc保护后，用纯化水洗涤，再用二氯甲烷萃取，合并有机相，用无水硫酸钠干燥，过滤、浓缩，通过柱层析纯化得到脂质化合物S1-15(6.28g)。
步骤d：参考实施例1.1步骤c～d的反应条件和投料比，将S1-15(5.10g,7.2mmol)与S1-4进行两次烷基化反应(第一次1.2mmol，第二次0.8mmol)，柱层析纯化得到氮支化聚乙二醇化脂质E1-3(0.74g)。1H NMR(400MHz,CDCl3)δ:4.18-4.00(m,2H,-C(＝O)OCH2-；1H,-C(＝O)OCH<),3.75-3.41(m,PEG；4H,-OCH2CH2N<),3.36(s,6H, -OCH3),2.83-2.72(m,4H,-OCH2CH2N<),2.60-2.40(m,4H,>N(CH2)2N<；4H,-[NCH2(CH2)2-]2),2.35-2.24(m,4H,-CH2C(＝O)O-),1.69-1.11(m,56H,-CH2CH2CH2-；6H,-CH2CH3),0.85(t,9H,-CH2CH3)。GPC测试分子量约为2.8kDa，PDI＝1.02。
实施例1.4：L1和L2不含酯键的氮支化聚乙二醇化脂质E1-4
对应通式(2)，E1-4中，B1、B2均为连接键，L1、L2均为连接键，R1、R2均为十四烷基；Ld为-CH2CH2C(＝O)-；两个Lx均为-CH2CH2-；T为甲基。每条PEG链的分子量约为1kDa，对应n1≈n2≈22。
制备方法如下：
步骤a：将肉豆蔻酸(S1-16,1.82g,8.0mmol)溶于40mL无水二氯甲烷中，加入N-羟基琥珀酰亚胺(NHS,1.38g,12.0mmol)，再加入DCC(2.47g,12.0mmol)。将DMAP(0.20g,1.6mmol)加入十四烷基胺(S1-17,2.04g,9.6mmol)的40mL二氯甲烷溶液中。将前述两份溶液混合后，室温下搅拌反应24小时。反应完成后。过滤除去不溶物，减压浓缩，通过柱层析纯化得到S1-18(2.85g)。
步骤b：氮气保护下，将S1-18(2.12g,5.0mmol)溶于1M的BH3/THF溶液(40mL)，加热至80℃搅拌24小时。冷却至室温，旋蒸除去THF，残余物用4N盐酸甲醇溶液溶解，氮气保护下回流4小时。除去甲醇，残余物用50mL水溶解，用乙醚洗涤(25mL*3)，再用20％NaOH溶液调节pH至11。用二氯甲烷(30mL*3)萃取碱混合物，合并有机相，无水硫酸钠干燥，过滤，浓缩，柱层析纯化得到仲胺化合物S1-19(1.60g)。
步骤c：将对称氮支化聚乙二醇丙酸衍生物S1-20(2.10g,1.0mmol,Mn≈2.1kDa,n1≈n2≈22,PDI＝1.01,通过含Boc保护羧基的β-丙氨酸与S1-4进行两次烷基化后脱保护得到，参考实施例1.1步骤c～d)溶于15mL无水二氯甲烷中，加入NHS(0.17g,1.5mmol)，再加入DCC(0.31g,1.5mmol)。将DMAP(24mg,0.2mmol)加入S1-19(1.23g,3.0mmol)的15mL二氯甲烷溶液中。将前述两份溶液混合后，室温下搅拌反应24小时。反应完成后。过滤除去不溶物，减压浓缩，通过柱层析纯化得到氮支化聚乙二醇化脂质E1-4(1.07g)。1H NMR(400MHz,CDCl3)δ:3.77-3.42(m,PEG；4H, -OCH2CH2N<),3.37(s,6H,-OCH3),3.29-3.09(m,4H,-[NCH2(CH2)2-]2),2.88-2.71(m,4H,-OCH2CH2N<),2.64-2.21(m,4H,>N(CH2)2C(＝O)N<),1.59-1.02(m,44H,-CH2CH2CH2-；4H,-CH2CH3),0.84(t,6H,-CH2CH3)。GPC测试分子量约为2.5kDa，PDI＝1.01。
实施例1.5：L1和L2不含酯键的氮支化聚乙二醇化脂质E1-5
对应通式(2)，E1-5中，B1、B2均为连接键，L1、L2均为连接键，R1、R2均为十四烷基；Ld为-CH2C(＝O)-；两个Lx分别为-CH2CH2-和-CH2C(＝O)-；T为甲基。每条PEG链的分子量约为1kDa，对应n1≈n2≈22。
制备方法如下：
将不对称氮支化聚乙二醇乙酸衍生物S2-1(2.10g,1.0mmol,Mn≈2.1kDa,n1≈n2≈22,PDI＝1.01,参考CN1243779C合成)溶于15mL无水二氯甲烷中，加入NHS(0.17g,1.5mmol)，再加入DCC(0.31g,1.5mmol)。将DMAP(24mg,0.2mmol)加入S1-19(1.23g,3.0mmol)的15mL二氯甲烷溶液中。将前述两份溶液混合后，室温下搅拌反应24小时。反应完成后。过滤除去不溶物，减压浓缩，通过柱层析纯化得到E1-5(1.15g)。1H NMR(400MHz,CDCl3)δ:4.25(s,1H,-OCHaHbC(＝O)N<),4.20(s,1H,-OCHaHb-C(＝O)N<),4.13(s,1H,>NCHaHbC(＝O)N<),4.04(s,1H,>NCHaHbC(＝O)N<),3.77-3.35(m,PEG；4H,-OCH2CH2N<),3.31(s,6H,-OCH3),3.24-3.04(m,4H,>NCH2C(＝O)N[CH2(CH2)2-]2),1.56-0.96(m,44H,-CH2CH2CH2-；4H,-CH2CH3),0.81(t,6H,-CH2CH3)。GPC测试分子量约为2.5kDa，PDI＝1.01。
实施例2：Ld含酰胺键和/或酯键的氮支化聚乙二醇化脂质
实施例2.1：Ld含酰胺键的氮支化聚乙二醇化脂质E2-1
对应通式(2)，E2-1中，B1、B2均为连接键，L1、L2均为连接键，R1、R2均为十四烷基；Ld为-CH2C(＝O)NHCH2CH2-；两个Lx分别为-CH2CH2-和-CH2C(＝O)-；T为甲基。每条PEG链的分子量约为1kDa，对应n1≈n2≈22。
制备方法如下：
将S2-1(2.10g,1.0mmol,Mn≈2.1kDa,n1≈n2≈22,PDI＝1.01)溶于15mL无水二氯甲烷中，加入NHS(0.17g,1.5mmol)，再加入DCC(0.31g,1.5mmol)。将DMAP(24mg,0.2mmol)加入S1-3(1.36g,3.0mmol)的15mL二氯甲烷溶液中。将前述两份溶液混合后，室温下搅拌反应24小时。反应完成后。过滤除去不溶物，减压浓缩，通过柱层析纯化得到氮支化聚乙二醇化脂质E2-1(1.30g)。1H NMR(400MHz,CDCl3)δ:4.24(s,1H,-OCHaHbC(＝O)N<),4.18(s,1H,-OCHaHbC(＝O)N<),3.76-3.27(m,PEG；4H,-OCH2CH2N<；2H,>NCH2C(＝O)NH-；6H,-OCH3),3.22-2.74(m,4H,-C(＝O)NHCH2CH2N<；4H,-N[CH2(CH2)2-]2),1.46-1.03(m,44H,-CH2CH2CH2-；4H,-CH2CH3),0.85(t,6H,-CH2CH3)。GPC测试分子量约为2.6kDa，PDI＝1.01。
实施例2.2：Ld含酯键的氮支化聚乙二醇化脂质E2-2
对应通式(2)，E2-2中，B1、B2均为连接键，L1、L2均为连接键，R1、R2均为十四烷基；Ld为-CH2C(＝O)OCH2CH2-；两个Lx分别为-CH2CH2-和-CH2C(＝O)-；T为甲基。每条PEG链的分子量约为1kDa，对应n1≈n2≈22。
制备方法如下：
步骤a：将S1-2(3.05g,11.0mmol)溶于40mL的N,N-二甲基甲酰胺(DMF)中，加入含TBS保护羟基的乙醇胺(S2-2,0.88g,5.0mmol)和碳酸钾(K2CO3,1.82g,13.2mmol)，室温下搅拌反应过夜。反应结束后，将反应液减压浓缩，倒入40mL二氯甲烷。依次用10％柠檬酸(20mL*2)、盐水(20mL*2)洗涤后，有机相用无水硫酸钠干燥，过滤，减压浓缩后加入20mL四氢呋喃(THF)，再加入1M的20mL四正丁基氟化铵(TBAF)的四氢呋喃溶液，反应过夜，脱除TBS保护。反应完成后，浓缩、萃取、合并有机相，经无水硫酸钠干燥，过滤并浓缩，通过柱层析纯化得到脂质化合物S2-3(1.90g)。
步骤b：在氩气气氛下，向装有溶于二氯甲烷(15mL)的S2-1(2.10g,1.0mmol,Mn≈2.1kDa,n1≈n2≈22,PDI＝1.01)、S2-3(1.36g,3.0mmol)和DMAP(31mg,0.3mmol)的圆底烧瓶加入DCC(0.31g,1.5mmol)，室温下反应16h。反应结束后，通过过滤将沉淀去除。浓缩滤液，通过柱层析纯化得到氮支化聚乙二醇化脂质E2-2(1.14g)。1H NMR(400MHz,CDCl3)δ:4.22(s,1H,-OCHaHbC(＝O)N<),4.17(s,1H,-OCHaHbC(＝O)N<), 4.06(t,2H,-C(＝O)OCH2CH2N<),3.98(s,2H,>NCH2C(＝O)O-),3.76-3.30(m,PEG；4H,-OCH2CH2N<；6H,-OCH3),2.61(t,2H,-C(＝O)OCH2CH2N<),2.44-2.35(m,4H,>NCH2(CH2)2-),1.47-1.02(m,44H,-CH2CH2CH2-；4H,-CH2CH3),0.83(t,6H,-CH2CH3)。GPC测试分子量约为2.6kDa，PDI＝1.01。
实施例2.3：Ld含酰胺键和酯键的氮支化聚乙二醇化脂质E2-3
对应通式(2)，E2-3中，B1、B2均为连接键，L1、L2均为连接键，R1、R2均为十四烷基；Ld为-CH2C(＝O)NHCH2CH2C(＝O)OCH2CH2-；两个Lx分别为-CH2CH2-和-CH2C(＝O)-；T为甲基。每条PEG链的分子量约为1kDa，对应n1≈n2≈22。
制备方法如下：
步骤a：在氩气气氛下，将含有Boc保护氨基的3-氨基丙酸(S2-4,1.13g,6.0mmol)、含裸露羟基的脂质化合物S2-3(2.18g,4.8mmol)和DMAP(0.18g,1.5mmol)依次溶于二氯甲烷(30mL)，加入DCC(1.85g,9.0mmol)，室温下反应16h。反应结束后，通过过滤将沉淀去除。浓缩滤液，用TFA/DCM混合溶液(1:1v/v)脱除Boc保护后，用纯化水洗涤，再用二氯甲烷萃取，合并有机相，用无水硫酸钠干燥，过滤、浓缩，通过柱层析纯化得到含裸露氨基的脂质化合物S2-5(2.07g)。
步骤b：将S2-1(2.10g,1.0mmol,Mn≈2.1kDa,n1≈n2≈22,PDI＝1.01)溶于15mL无水二氯甲烷中，加入NHS(0.17g,1.5mmol)，再加入DCC(0.31g,1.5mmol)。将DMAP(24mg,0.2mmol)加入S2-5(1.58g,3.0mmol)的15mL二氯甲烷溶液中。将前述两份溶液混合后，室温下搅拌反应24小时。反应完成后。过滤除去不溶物，减压浓缩，通过柱层析纯化得到氮支化聚乙二醇化脂质E2-3(1.04g)。1H NMR(400MHz,CDCl3)δ:4.26(s,1H,-OCHaHbC(＝O)N<),4.20(s,1H,-OCHaHbC(＝O)N<),4.05(t,2H,-C(＝O)OCH2CH2N<),3.73-3.27(m,PEG；4H,-OCH2CH2N<；2H,>NCH2C(＝O)NH-；6H,-OCH3),3.22-2.93(m,2H,-C(＝O)NHCH2CH2C(＝O)O-；4H,-N[CH2(CH2)2-]2),2.60(t,2H,-C(＝O)OCH2CH2N<),2.47-2.32(m,2H,-C(＝O)NHCH2CH2C(＝O)O-),1.46-1.02(m,44H,-CH2CH2CH2-；4H,-CH2CH3),0.84(t,6H,-CH2CH3)。GPC测试分子量约为2.7kDa，PDI＝1.01。
实施例2.4：Ld含酰胺键的氮支化聚乙二醇化脂质E2-4
对应通式(2)，E2-4中，B1、B2均为连接键，L1、L2均为连接键，R1、R2均为十四烷基；Ld为-CH2CH2C(＝O)NHCH2C(＝O)-；两个Lx均为-CH2CH2-；T为甲基。每条PEG链的分子量约为1kDa，对应n1≈n2≈22。
制备方法如下：
步骤a：将含Boc保护氨基的甘氨酸(S2-6,1.05g,6.0mmol)溶于15mL无水二氯甲烷中，加入NHS(1.04g,9.0mmol)，再加入DCC(1.85g,9.0mmol)。将DMAP(0.15g,1.2mmol)加入仲胺化合物S1-19(1.97g,4.8mmol)的15mL二氯甲烷溶液中。将前述两份溶液混合后，室温下搅拌反应24小时。反应完成后。过滤除去不溶物，减压浓缩，通过柱层析纯化得到S2-7(2.02g)。
步骤b：将氮支化聚乙二醇丙酸衍生物S1-20(2.10g,1.0mmol,Mn≈2.1kDa,n1≈n2≈22,PDI＝1.01)溶于15mL无水二氯甲烷中，加入NHS(0.17g,1.5mmol)，再加入DCC(0.31g,1.5mmol)。将DMAP(24mg,0.2mmol)加入S2-7(1.40g,3.0mmol)的15mL二氯甲烷溶液中。将前述两份溶液混合后，室温下搅拌反应24小时。反应完成后。过滤除去不溶物，减压浓缩，通过柱层析纯化得到氮支化聚乙二醇化脂质E2-4(1.17g)。1H NMR(400MHz,CDCl3)δ:4.09(s,2H,-C(＝O)NHCH2C(＝O)N<),3.76-3.25(m,PEG；4H,-OCH2CH2N<；6H,-OCH3；2H,>NCH2CH2C(＝O)NH-),3.24-3.02(m,4H,-N[CH2(CH2)2-]2),2.81-2.73(m,4H,-OCH2CH2N<),2.48-2.31(m,2H,>NCH2CH2C(＝O)NH-),1.57-1.00(m,44H,-CH2CH2CH2-；4H,-CH2CH3),0.85(t,6H,-CH2CH3)。GPC测试分子量约为2.6kDa，PDI＝1.01。
实施例2.5：Ld含酯键的氮支化聚乙二醇化脂质E2-5
对应通式(2)，E2-5中，B1、B2均为亚戊基，L1、L2均为酯基-C(＝O)O-，R1、R2均为十一烷基；Ld为-CH2CH2C(＝O)OCH2CH2-；两个Lx均为-CH2CH2-；T为甲基。每条PEG链的分子量约为1kDa，对应n1≈n2≈22。
制备方法如下：
将氮支化聚乙二醇丙酸衍生物S1-20(2.10g,1.0mmol,Mn≈2.1kDa,n1≈n2≈22,PDI＝1.01)溶于15mL无水二氯甲烷中，加入NHS(0.17g,1.5mmol)，再加入DCC(0.31g,1.5mmol)。将DMAP(24mg,0.2mmol)加入S2-8(1.79g,3.0mmol,其中，S2-8由S1-14和S2-2合成，参考实施例2.2步骤a)的15mL二氯甲烷溶液中。将前述两份溶液混合后，室温下搅拌反应24小时。反应完成后。过滤除去不溶物，减压浓缩，通过柱层析纯化得到氮支化聚乙二醇化脂质E2-5(1.13g)。1H NMR(400MHz,CDCl3)δ:4.15-4.00(m,6H,-CH2OC(＝O)-),3.76-3.40(m,PEG；4H,-OCH2CH2N<),3.37-2.96(m,6H,-OCH3；4H,-N[CH2(CH2)2-]2),2.87-2.70(m,4H,-OCH2CH2N<；4H,>N(CH2)2C(＝O)-；2H,-C(＝O)OCH2CH2N<),2.39-2.21(m,4H,-(CH2)2CH2C(＝O)O-),1.70-1.05(m,42H,-CH2CH2CH2-；6H,-CH2CH3),0.85(t,6H,-CH2CH3)。GPC测试分子量约为2.7kDa，PDI＝1.01。
实施例2.6：Ld含酯键的氮支化聚乙二醇化脂质E2-6
对应通式(2)，E2-6中，B1、B2均为亚己基，L1为酯基-OC(＝O)-，L2为醚基-O-，R1为辛基，R2为壬基；Ld为-CH2CH2C(＝O)OCH2CH2-；两个Lx均为-CH2CH2-；T为甲基。每条PEG链的分子量约为1kDa，对应n1≈n2≈22。
制备方法如下：
步骤a：将S2-9(3.08g,9.6mmol,其中，S2-9由6-溴己醇和壬酸在DMAP/DCC条件下进行酯化反应获得)溶于50mL的DMF中，加入S2-2(1.40g,8.0mmol)和碳酸钾(K2CO3,1.59g,11.5mmol)，室温下搅拌反应过夜。反应结束后，将反应液减压浓缩，倒入50mL二氯甲烷。依次用10％柠檬酸(25mL*2)、盐水(25mL*2)洗涤后，有机相用无水硫酸钠干燥，过滤，减压浓缩后，通过柱层析纯化得到脂质化合物S2-10(2.70g)。
步骤b：将S2-10(2.08g,5.0mmol)溶于干燥的THF(40mL)中，冰浴下缓慢加入NaH(60％,2.00g,50.0mmol)，于冰浴下反应1小时。反应结束后，加入含醚键的溴代烷烃S2-11(1.84g,6.0mmol，其中S2-11是由6-溴己醇进行磺酸酯化后，与1-壬醇在二苯基甲基钾/THF条件下烷基化反应得到)，冰浴下搅拌反应1小时后，反应液慢慢恢复至室温反应过夜。反应结束后，将反应置于冰浴下，缓慢加入1mL水淬灭反应，搅拌反应30分钟后，旋蒸除去溶剂，加入水(40mL)搅拌混合，再用二氯甲烷(20mL*2)萃取两次，合并二氯甲烷有机相，用饱和氯化钠(20mL*2)洗涤。有机相用无水硫酸钠干燥，过滤，减压浓缩后加入20mL四氢呋喃(THF)，再加入1M的TBAF/THF(20mL)溶液，反应过夜，脱除TBS保护。反应完成后，浓缩、萃取、合并有机相，经无水硫酸钠干燥，过滤并浓缩，通过柱层析纯化得到脂质化合物S2-12(2.02g)。
步骤c：在氩气气氛下，向装有溶于二氯甲烷(20mL)的S1-20(2.10g,1.0mmol, Mn≈2.1kDa,n1≈n2≈22,PDI＝1.01)、S2-12(1.58g,3.0mmol)和DMAP(31mg,0.3mmol)的圆底烧瓶加入DCC(0.31g,1.5mmol)，室温下反应16h。反应结束后，通过过滤将沉淀去除。浓缩滤液，通过柱层析纯化得到氮支化聚乙二醇化脂质E2-6(1.27g)。1H NMR(400MHz,CDCl3)δ:4.15-4.03(m,4H,-CH2OC(＝O)-),3.80-3.40(m,PEG；4H,-OCH2CH2N<；4H,-(CH2)2CH2OCH2(CH2)2-),3.37-2.95(m,6H,-OCH3；4H,-N[CH2(CH2)2-]2),2.81-2.39(m,4H,-OCH2CH2N<；4H,>N(CH2)2C(＝O)-；2H,-C(＝O)OCH2CH2N<),2.32-2.24(m,2H,-(CH2)2CH2C(＝O)O-),1.76-1.12(m,36H,-CH2CH2CH2-；6H,-CH2CH3),0.87(t,6H,-CH2CH3)。GPC测试分子量约为2.7kDa，PDI＝1.01。
实施例2.7：Ld含酰胺键的氮支化聚乙二醇化脂质E2-7
对应通式(2)，E2-7中，B1、B2均为连接键，L1、L2均为连接键，R1、R2均为十四烷基；Ld为-CH2C(＝O)NHCH2C(＝O)-；两个Lx分别为-CH2CH2-和-CH2C(＝O)-；T为甲基。每条PEG链的分子量约为1kDa，对应n1≈n2≈22。
制备方法如下：
将S2-1(2.10g,1.0mmol,Mn≈2.1kDa,n1≈n2≈22,PDI＝1.01)溶于15mL无水二氯甲烷中，加入NHS(0.17g,1.5mmol)，再加入DCC(0.31g,1.5mmol)。将DMAP(24mg,0.2mmol)加入S2-7(1.40g,3.0mmol)的15mL二氯甲烷溶液中。将前述两份溶液混合后，室温下搅拌反应24小时。反应完成后。过滤除去不溶物，减压浓缩，通过柱层析纯化得到氮支化聚乙二醇化脂质E2-7(1.07g)。1H NMR(400MHz,CDCl3)δ:4.19(s,1H,-OCHaHbC(＝O)N<),4.14(s,1H,-OCHaHbC(＝O)N<),4.07(s,2H,-C(＝O)NHCH2C(＝O)N<),3.76-3.20(m,PEG；4H,-OCH2CH2N<；6H,-OCH3；4H,-N[CH2(CH2)2-]2；2H,>NCH2C(＝O)NH-),1.59-0.98(m,44H,-CH2CH2CH2-；4H,-CH2CH3),0.82(t,6H,-CH2CH3)。GPC测试分子量约为2.6kDa，PDI＝1.01。
实施例3：Lx含酯键的氮支化聚乙二醇化脂质
实施例3.1：Lx含酯键的氮支化聚乙二醇化脂质E3-1
对应通式(2)，E3-1中，B1、B2均为连接键，L1、L2均为连接键，R1、R2均为十四烷基；Ld为-CH2CH2-；两个Lx均为-C(＝O)CH2CH2-；T为甲基。每条PEG链的分子量约为1kDa，对应n1≈n2≈22。
制备方法如下：
参考实施例1.1步骤c～d的反应条件和投料比，将S1-3(3.26g,7.2mmol)与含酯键的线性聚乙二醇甲磺酸酯衍生物S3-1(Mn≈1.2kDa,n1≈22,PDI＝1.01)进行两次烷基化反应(第一次1.2mmol，第二次0.8mmol)，柱层析纯化得到氮支化聚乙二醇化脂质E3-1(0.55g)。1H NMR(400MHz,CDCl3)δ:4.26-4.05(m,4H,-OCH2CH2OC(＝O)-),3.75-3.42(m,PEG；4H,-OCH2CH2OC(＝O)-),3.36(s,6H,-OCH3),2.78-2.35(m,8H,-OC(＝O)CH2CH2N<；4H,>N(CH2)2N<；4H,-N[CH2(CH2)2-]2；),1.49-1.01(m,44H,-CH2CH2CH2-；4H,-CH2CH3),0.85(t,6H,-CH2CH3)。GPC测试分子量约为2.6kDa，PDI＝1.02。
实施例3.2：Lx含酯键的氮支化聚乙二醇化脂质E3-2
对应通式(2)，E3-2中，B1、B2均为连接键，L1、L2均为连接键，R1、R2均为十四烷基；Ld为-CH2CH2C(＝O)OCH2CH2-；两个Lx均为-CH2CH2C(＝O)OCH2CH2-；T为甲基。每条PEG链的分子量约为1kDa，对应n1≈n2≈22。
制备方法如下：
步骤a：在氩气气氛下，将含有两个TBS保护羟基的N,N-二羟乙基甘氨酸(S3-3,0.78g,2.0mmol)、含裸露羟基的脂质化合物S2-3(1.09g,2.4mmol)和DMAP(61mg,0.5mmol)依次溶于二氯甲烷(12mL)，加入DCC(0.62g,3.0mmol)，室温下反应16h。反应结束后，通过过滤将沉淀去除。经无水硫酸钠干燥，过滤，浓缩滤液，浓缩后加入8mL四氢呋喃(THF)，再加入1M的TBAF/THF(8mL)溶液，反应过夜，脱除TBS保护。反应完成后，浓缩、萃取、合并有机相，经无水硫酸钠干燥，过滤并浓缩，通过柱层析纯化得到含两个裸露羟基的脂质化合物S3-4(0.83g)。
步骤b：在氩气气氛下，向装有溶于二氯甲烷(30mL)的S3-5(5.50g,5.0mmol,Mn≈1.1kDa,n1≈22,PDI＝1.01)、S3-4(0.60g,1.0mmol)和DMAP(0.15g,1.3mmol)的圆底烧瓶加入DCC(1.55g,7.5mmol)，室温下反应16h。反应结束后，通过过滤将沉淀去除。浓缩滤液，通过柱层析纯化得到氮支化聚乙二醇化脂质E3-2(0.59g)。1H NMR(400MHz,CDCl3)δ:4.25-4.09(m,4H,-OCH2CH2C(＝O)O-),4.07(t,2H, -C(＝O)OCH2CH2N<),3.76-3.40(m,PEG；4H,-OCH2CH2C(＝O)O-),3.36(s,6H,-OCH3),3.32(s,2H,>NCH2C(＝O)O-),3.22-2.36(m,4H,-OCH2CH2C(＝O)N<；2H,-C(＝O)OCH2CH2N<；4H,-OCH2CH2C(＝O)O-；4H,-N[CH2(CH2)2-]2),1.49-1.03(m,44H,-CH2CH2CH2-；4H,-CH2CH3),0.88(t,6H,-CH2CH3)。GPC测试分子量约为2.7kDa，PDI＝1.02。
实施例4：含1,4,7-三氮杂环壬烷支化核的氮支化聚乙二醇化脂质(E4-1)
对应通式(2)，E4-1中，B1、B2均为连接键，L1、L2均为连接键，R1、R2均为十四烷基；Ld为-CH2C(＝O)NHCH2CH2-；Ncore为两个Lx均为-C(＝O)CH2-；T为甲基。每条PEG链的分子量约为1kDa，对应n1≈n2≈22。
制备方法如下：
步骤a：将NOTA-二叔丁酯(S4-1,0.83g,2.0mmol)溶于15mL无水二氯甲烷中，加入NHS(0.35g,3.0mmol)，再加入DCC(0.62g,3.0mmol)。将DMAP(49mg,0.4mmol)加入S1-3(1.09g,2.4mmol)的15mL二氯甲烷溶液中。将前述两份溶液混合后，室温下搅拌反应24小时。反应完成后。过滤除去不溶物，浓缩滤液，用TFA/DCM混合溶液(1:1v/v)脱除Boc保护后，用纯化水洗涤，再用二氯甲烷萃取，合并有机相，用无水硫酸钠干燥，过滤、浓缩，通过柱层析纯化得到S4-2(1.26g)。
步骤b：在氩气气氛下，向装有溶于二氯甲烷(20mL)的S4-2(0.74g,1.0mmol)、甲氧基聚乙二醇S4-3(3.00g,3.0mmol,Mn≈1.0kDa,n1≈22,PDI＝1.01)和DMAP(61mg,0.5mmol)的圆底烧瓶加入DCC(0.62g,3.0mmol)，室温下反应16h。反应结束后，通过过滤将沉淀去除。浓缩滤液，通过柱层析纯化得到氮支化聚乙二醇化脂质E4-1(0.74g)。1H NMR(400MHz,CDCl3)δ:4.18-4.08(m,4H,-OCH2CH2OC(＝O)-),3.74-3.31(m,PEG；4H,-OCH2CH2OC(＝O)-；6H,-OCH3),3.26-2.69(m,2H,-C(＝O)NHCH2CH2N<；4H,-OC(＝O)CH2N<；2H,>NCH2C(＝O)NH-；4H,-N[CH2(CH2)2-]2；12H,>N(CH2)2N<),1.46-1.01(m,44H,-CH2CH2CH2-；4H,-CH2CH3),0.85(t,6H,-CH2CH3)。GPC测试分子量约为2.7kDa，PDI＝1.01。
实施例5：含咔唑支化核的氮支化聚乙二醇化脂质(E5-1)
对应通式(2)，E5-1中，B1、B2均为连接键，L1、L2均为连接键，R1、R2均为十四烷基；Ld为-CH2CH2C(＝O)OCH2CH2-；Ncore为两个Lx均为-C(＝O)NH-；T为甲基。每条PEG链的分子量约为1kDa，对应n1≈n2≈22。
制备方法如下：
步骤a：将含有两个Cbz保护氨基的咔唑衍生物S5-2(1.86g,4.0mmol)溶于干燥的THF(30mL)中，冰浴下缓慢加入NaH(60％,1.60g,40.0mmol)，于冰浴下反应1小时。反应结束后，加入S5-1(1.08g,4.8mmol，由3-羟基丙酸用甲基磺酰氯进行磺酸酯化得到)，冰浴下搅拌反应1小时后，反应液慢慢恢复至室温反应过夜。反应结束后，将反应置于冰浴下，缓慢加入0.5mL水淬灭反应，搅拌反应30分钟后，旋蒸除去溶剂，加入水(30mL)搅拌混合，再用二氯甲烷(15mL*2)萃取两次，合并二氯甲烷有机相，用饱和氯化钠(15mL*2)洗涤。有机相减压浓缩后用TFA/DCM混合溶液(1:1v/v)脱除Boc保护后，用纯化水洗涤，再用二氯甲烷萃取，合并有机相，用无水硫酸钠干燥，过滤、浓缩，通过柱层析纯化得到S5-3(1.67g)。
步骤b：在氩气气氛下，向装有溶于二氯甲烷(15mL)的S5-3(1.08g,2.0mmol)、S2-3(1.09g,2.4mmol)和DMAP(61mg,0.5mmol)的圆底烧瓶加入DCC(0.62g,3.0mmol)，室温下反应16h。反应结束后，通过过滤将沉淀去除。浓缩滤液，用甲醇溶解，加入10％Pd/C催化剂，鼓泡氢气在室温下搅拌反应14h，反应完毕后用硅藻土滤除催化剂，浓缩反应液，通过柱层析纯化得到脂质化合物S5-4(1.20g)。
步骤c：将甲氧基聚乙二醇琥珀酰亚胺碳酸酯mPEG-SC(S13-4,3.30g,3.0mmol,Mn≈1.1kDa,n1≈22,PDI＝1.01)溶于50mL无水二氯甲烷，加入DMAP(61mg,0.5mmol)，搅拌混匀，冰浴条件下缓慢加入S5-4(0.71g,1.0mmol)的无水二氯甲烷溶液，混匀，25℃下搅拌反应16h。反应结束后，用碳酸氢钠溶液、3M氢氧化钠溶液和饱和食盐水洗涤，有机相浓缩，通过柱层析纯化得到氮支化聚乙二醇化脂质E5-1(1.21g)。1H NMR (400MHz,CDCl3)δ:8.22(s,2H,carbazolyl),7.49-7.41(m,4H,carbazolyl),4.61(t,2H,>NCH2CH2C(＝O)O-),4.24-4.20(m,4H,-OCH2CH2OC(＝O)NH-),4.08(t,2H,-C(＝O)OCH2CH2N<),3.77-3.40(m,PEG；4H,-OCH2CH2OC(＝O)NH-),3.37(s,6H,-OCH3),3.19-2.96(m,2H,-C(＝O)OCH2CH2N<；4H,-N[CH2(CH2)2-]2),2.83(t,2H,>NCH2CH2C(＝O)O-),1.49-1.06(m,44H,-CH2CH2CH2-；4H,-CH2CH3),0.84(t,6H,-CH2CH3)。GPC测试分子量约为2.8kDa，PDI＝1.01。
实施例6：Ld含肽键的氮支化聚乙二醇化脂质
实施例6.1：Ld含甘氨酸-甘氨酸肽键的氮支化聚乙二醇化脂质E6-1
对应通式(2)，E6-1中，B1、B2均为连接键，L1、L2均为连接键，R1、R2均为十四烷基；Ld为-CH2C(＝O)-Gly-Gly-OCH2CH2-，其中，Gly为甘氨酸残基-NHCH2C(＝O)-；Ncore为三价氮原子核，两个Lx分别为-CH2CH2-和-CH2C(＝O)-；T为甲基。每条PEG链的分子量约为1kDa，对应n1≈n2≈22。
制备方法如下：
步骤a：在氩气气氛下，将含有Boc保护氨基的甘氨酸二肽(S6-1,1.39g,6.0mmol)、含裸露羟基的脂质化合物S2-3(2.18g,4.8mmol)和DMAP(0.18g,1.5mmol)依次溶于二氯甲烷(30mL)，加入DCC(1.85g,9.0mmol)，室温下反应16h。反应结束后，通过过滤将沉淀去除。浓缩滤液，用TFA/DCM混合溶液(1:1v/v)脱除Boc保护后，用纯化水洗涤，再用二氯甲烷萃取，合并有机相，用无水硫酸钠干燥，过滤、浓缩，通过柱层析纯化得到含裸露氨基的脂质化合物S6-2(2.37g)。
步骤b：在氩气气氛下，向装有溶于二氯甲烷(30mL)的S2-1(2.10g,1.0mmol,Mn≈2.1kDa,n1≈n2≈22,PDI＝1.01)、S6-2(1.70g,3.0mmol)和DMAP(31mg,0.3mmol)的圆底烧瓶加入DCC(0.31g,1.5mmol)，室温下反应16h。反应结束后，通过过滤将沉淀去除。浓缩滤液，通过柱层析纯化得到氮支化聚乙二醇化脂质E6-1(1.32g)。1H NMR(400MHz,CDCl3)δ:4.16(s,1H,-OCHaHbC(＝O)N<),4.11(s,1H,-OCHaHbC(＝O)N<),4.06(t,2H,-C(＝O)OCH2CH2N<),3.97(s,2H,-C(＝O)NHCH2C(＝O)O-),3.74-3.29(m,PEG；4H,-OCH2CH2N<；6H,-OCH3；2H,>NCH2C(＝O)NH-；2H,-C(＝O)NHCH2C(＝O)NH-), 3.19-2.86(m,2H,-C(＝O)OCH2CH2N<；4H,-N[CH2(CH2)2-]2),1.49-0.99(m,44H,-CH2CH2CH2-；4H,-CH2CH3),0.84(t,6H,-CH2CH3)。GPC测试分子量约为2.7kDa，PDI＝1.01。
实施例6.2：Ld含苯丙氨酸-甘氨酸肽键的氮支化聚乙二醇化脂质E6-2
对应通式(2)，E6-2中，B1为亚戊基，B2为亚庚基，L1、L2均为酯基-C(＝O)O-，R1为十一烷基，R2为Ld为-CH2C(＝O)-Phe-Gly-OCH2CH2-，其中，Phe为苯丙氨酸残基，Gly为甘氨酸残基；Ncore为三价氮原子核，两个Lx分别为-CH2CH2-和-CH2C(＝O)-；T为甲基。每条PEG链的分子量约为1kDa，对应n1≈n2≈22。
制备方法如下：
步骤a：在氩气气氛下，将含有Boc保护氨基的苯丙氨酸-甘氨酸二肽(S6-3,1.93g,6.0mmol)、脂质化合物S6-4(3.41g,4.8mmol，参考实施例1.3的制备方法，以S2-2、S1-14和S1-12为原料合成)和DMAP(0.18g,1.5mmol)依次溶于二氯甲烷(50mL)，加入DCC(1.85g,9.0mmol)，室温下反应16h。反应结束后，通过过滤将沉淀去除。浓缩滤液，用TFA/DCM混合溶液(1:1v/v)脱除Boc保护后，用纯化水洗涤，再用二氯甲烷萃取，合并有机相，用无水硫酸钠干燥，过滤、浓缩，通过柱层析纯化得到含裸露氨基的脂质化合物S6-5(3.70g)。
步骤b：在氩气气氛下，向装有溶于二氯甲烷(50mL)的S2-1(2.10g,1.0mmol,Mn≈2.1kDa,n1≈n2≈22,PDI＝1.01)、S6-5(2.74g,3.0mmol)和DMAP(31mg,0.3mmol)的圆底烧瓶加入DCC(0.31g,1.5mmol)，室温下反应16h。反应结束后，通过过滤将沉淀去除。浓缩滤液，通过柱层析纯化得到氮支化聚乙二醇化脂质E6-2(1.27g)。1H NMR(400MHz,CDCl3)δ:7.30-7.19(m,5H,Ar)；4.50-4.44(m,1H,Phe-α-CH<),4.17(s,1H,-OCHaHbC(＝O)N<),4.13(s,1H,-OCHaHbC(＝O)N<),4.09(t,4H,-(CH2)2CH2OC(＝O)-),4.05(t,2H,-C(＝O)OCH2CH2N<),3.97(s,2H,-C(＝O)NHCH2C(＝O)O-),3.76-3.28(m,PEG；4H,-OCH2CH2N<；6H,-OCH3；2H,>NCH2C(＝O)NH-),3.19-2.83(m,2H,Phe-CH2-；2H,-C(＝O)OCH2CH2N<；4H,-[NCH2(CH2)2-]2),2.38-2.22(m,1H,-OC(＝O)CH<；2H,-(CH2)2CH2C(＝O)O-),1.73-1.16(m,56H,-CH2CH2CH2-；6H,-CH2CH3),0.88(t,9H,-CH2CH3)。GPC测试分子量约为3.0kDa，PDI＝1.01。
实施例7：Lx、L1、L2均含酯键的氮支化聚乙二醇化脂质(E7-1)
对应通式(2)，E7-1中，B1、B2均为亚乙基，L1、L2均为酯基-OC(＝O)-，R1、R2均为十三烷基；Ld为-CH2CH2-；两个Lx均为-C(＝O)CH2CH2-；T为甲基。每条PEG链的分子量约为1kDa，对应n1≈n2≈22。
制备方法如下：
步骤a：在氩气气氛下，向装有溶于二氯甲烷(60mL)的S1-16(3.42g,15.0mmol)、含Boc保护氨基的N,N-双(2-羟乙基)乙二胺(S7-1,1.49g,6.0mmol)和DMAP(0.46g,3.8mmol)的圆底烧瓶加入DCC(4.64g,22.5mmol)，室温下反应16h。反应结束后，通过过滤将沉淀去除。浓缩滤液，用TFA/DCM混合溶液(1:1v/v)脱除Boc保护后，用纯化水洗涤，再用二氯甲烷萃取，合并有机相，用无水硫酸钠干燥，过滤、浓缩，通过柱层析纯化得到S7-2(2.33g)。
步骤b：参考实施例1.1步骤c～d的反应条件和投料比，将S7-2(2.05g,3.6mmol)与S3-1(Mn≈1.2kDa,n1≈22,PDI＝1.01)进行两次烷基化反应(第一次0.6mmol，第二次0.4mmol)，柱层析纯化得到氮支化聚乙二醇化脂质E7-1(0.35g)。1H NMR(400MHz,CDCl3)δ:4.45-4.30(m,4H,>NCH2CH2OC(＝O)-),4.19-4.08(m,4H,-OCH2CH2OC(＝O)-),3.72-3.42(m,PEG；4H,-OCH2CH2OC(＝O)-；4H,>NCH2CH2OC(＝O)-)；3.34(s,6H,-OCH3),2.76-2.43(m,8H,-OC(＝O)(CH2)2N<；4H,>N(CH2)2N<),2.30(t,4H,-OC(＝O)CH2(CH2)2-)；1.73-1.02(m,40H,-CH2CH2CH2-；4H,-CH2CH3),0.85(t,6H,-CH2CH3)。GPC测试分子量约为2.7kDa，PDI＝1.02。
实施例8：叶酸偶联的氮支化聚乙二醇化脂质(E8-1)
对应通式(2)，E8-1中，B1、B2均为连接键，L1、L2均为连接键，R1、R2均为十四烷基；Ld为-CH2CH2-；两个Lx均为-CH2CH2-；T为R01-L01-，R01为叶酸残基，L01为-NHCH2CH2-。每条PEG链的分子量约为1kDa，对应n1≈n2≈22。
制备方法如下：
步骤a：参考实施例1.1步骤c～d的反应条件和投料比，将S1-3(6.52g,14.4mmol)与线性双官能化聚乙二醇衍生物S8-1(Mn≈1.3kDa,n1≈22,PDI＝1.02)进行两次烷基化反应(第一次2.4mmol，第二次1.6mmol)，再用TFA/DCM混合溶液(1:1v/v)脱除Boc保护，柱层析纯化得到氮支化聚乙二醇化脂质S8-2(1.44g)。
步骤b：将叶酸(S8-3,0.88g,2.0mmol)溶于15mL无水二氯甲烷中，加入NHS(0.35g,3.0mmol)，再加入DCC(0.62g,3.0mmol)。将DMAP(49mg,0.4mmol)加入S8-2(1.04g,0.4mmol,Mn≈2.6kDa,n1≈n2≈22,PDI＝1.02)的15mL二氯甲烷溶液中。将前述两份溶液混合后，室温下搅拌反应24小时。反应完成后。过滤除去不溶物，浓缩滤液，通过柱层析纯化得到E8-1(0.41g)。1H NMR(400MHz,CDCl3)δ:8.69(s,2H,pterinC7),7.65-7.62(d,4H,Ph),6.65(d,4H,Ph),4.48-4.45(d,4H,pterinC6-CH2NH-),4.33-4.21(m,2H,-CH(COOH)-),3.75-3.33(m,PEG；4H,-OCH2CH2N<；8H,-C(＝O)NH(CH2)2O-),2.86-2.68(m,4H,-OCH2CH2N<),2.52-2.35(m,4H,>NCH2(CH2)2-),2.34-1.93(m,4H,-CH(COOH)(CH2)2C(＝O)NH-),1.48-1.05(m,44H,-CH2CH2CH2-；4H,-CH2CH3),0.86(t,6H,-CH2CH3)。GPC测试分子量约为3.4kDa，PDI＝1.02。
2.2.脂质药物组合物
实施例9：LNP–mRNA药物组合物的制备
本实施例中制备了含有Fluc-mRNA的LNP-mRNA药物组合物(LNP/Fluc-mRNA)，其含有的磷脂都为DSPC，含有的甾醇类脂质都为胆固醇，含有的阳离子脂质都为CL-1，不同的是聚乙二醇化脂质；所述CL-1参考CN113402405A披露的制备方法获得，其结构如下：
LNP/Fluc-mRNA的制备方法如下：
移取一定量的CL-1、DSPC、胆固醇和聚乙二醇化脂质贮备液，将CL-1、DSPC、胆固醇和聚乙二醇化脂质以50:10:38:1.5的摩尔比溶于乙醇，获得乙醇相溶液(各组的脂质配方具体如实施例10的表1所示，对照组L-0的聚乙二醇化脂质为PEG2k-DMG(简称DMG)，实验组的聚乙二醇化脂质为本发明的非线性聚乙二醇化脂质)。将Fluc-mRNA加到10～50mM的柠檬酸盐缓冲液(pH＝4)中，获得水相溶液。将乙醇相溶液和水相溶液混合(1:3v/v)制备阳离子脂质纳米粒LNP/Fluc-mRNA，并通过多次DPBS超滤洗涤以除去乙醇和游离分子，最后通过0.2μm的无菌过滤器，备用。
采用上述步骤，各脂质的摩尔比更改为CL-1：DSPC：胆固醇：聚乙二醇化脂质＝50:10:38:0.75，其中聚乙二醇化脂质采用E1-1，其余条件不变，制备阳离子脂质纳米粒LNP/Fluc-mRNA(实验组L1-1-half)；
采用上述步骤，各脂质的摩尔比更改为CL-1：DSPC：胆固醇：聚乙二醇化脂质＝48:9:42:1.5，其中聚乙二醇化脂质采用E1-1，其余条件不变，制备阳离子脂质纳米粒LNP/Fluc-mRNA(实验组L1-1-d)。
实施例10：脂质药物组合物的生物学活性测试
(1)纳米粒尺寸与核酸复合能力测定
核酸复合能力测定：采用凝胶电泳实验考察LNP/Fluc-mRNA的核酸复合能力。称取0.8g的琼脂糖溶于40mL的TAE溶液中，在微波炉中加热使琼脂糖颗粒完全溶解，冷却，加入5μL的核酸染料GelGreen于冷却的琼脂糖凝胶中，凝胶加入凝胶槽，自然晾干。将LNP/Fluc-mRNA与2μL的LoadingBuffer的混合液加入到琼脂糖凝胶孔内，设置电泳电压为90V进行电泳实验，常温下电泳10min。结果显示，实验组和对照组均基本不存在游离的Fluc-mRNA，说明了含有本发明的氮支化聚乙二醇化脂质的脂质组合物有很好的核酸复合能力。
包封率测定：使用超速离心机对LNP/Fluc-mRNA超速离心(4℃,60000rpm,1h)，使用核酸定量仪检测上清中未被包封的Fluc-mRNA浓度，计算LNP对Fluc-mRNA的包封率，结果总结于表1中，显示本发明的阳离子脂质纳米粒对核酸药物有较高的包封率，其中，氮支化聚乙二醇化脂质单臂分子量约为1kDa、2kDa的实验组对Fluc-mRNA的包封率均大于87％，而氮支化聚乙二醇化脂质单臂分子量约为0.5kDa的实验组(L1-1-500)对Fluc-mRNA的包封率达到83％。特别地，实验组L1-1-half含有单臂分子量约1kDa的氮支化聚乙二醇化脂质E1-1，对照组L-0含有分子量约2kDa的线性聚乙二醇化脂质PEG2k-DMG，且L1-1-half中聚乙二醇化脂质E1-1的摩尔含量仅为L-0中PEG2k-DMG的一半，但L1-1-half的包封率甚至稍高于L-0，前者为93.3％，后者为92.3％，说明本发明的氮支化聚乙二醇化脂质在用量较少的情况下也不显著影响LNP对Fluc-mRNA的包封率。
粒径测定：根据文献(Hassett等人，J.Controlled Release 2021,335,237-246)可知，包载核酸药物的LNP制剂的粒径在60～150nm时能发挥较好的药效。本实施例中，通过动态光散射(DLS)测定LNP/Fluc-mRNA的粒径。所测定的LNP/Fluc-mRNA尺寸均匀性较高，其PDI均小于0.3。实验结果显示，采用本发明的氮支化聚乙二醇化脂质修饰的LNP/Fluc-mRNA粒径为约65nm～约112nm范围内，均处于能实现较好药效的粒 径范围内。
表1包封率与粒径测定
(2)血清稳定性评价
阳离子脂质在用于递送核酸药物的LNP起着重要作用。递送体内循环过程中存在大量带负电荷的血清蛋白，易与正电荷的阳离子脂质纳米粒发生吸附，形成大尺寸的聚集体而被单核吞噬系统清除，导致LNP基因转染效率降低。本发明的含有氮支化聚乙二醇化脂质可赋予LNP“隐形效应”。
将上述LNP/Fluc-mRNA(1个对照组L-0、24个实验组L1-1～L1-1-d)加入含有10％胎牛血清(FBS)的培养基中，在37℃条件下搅拌，定时取样测定LNP/Fluc-mRNA的粒径变化，通过测试其粒径变化来分析核酸类药物制剂的血清稳定性。实验结果显示，在7天内，聚乙二醇组分单臂分子量为0.5kDa左右的实验组L1-1-500的粒径变化稍大(9％)，其余所有实验组和对照组的粒径变化小于6％；特别地，L1-1-half的粒径变化约5％，与L-0和L1-1的粒径变化(分别约4％、2％)相比无显著差别，而L1-1-half的聚乙二醇化脂质用量仅为L-0和L1-1中的一半。这些结果说明，本发明中聚乙二醇 单臂分子量约1kDa的氮支化聚乙二醇化脂质制备的LNP-核酸药物组合物具有很好的血清稳定性；聚乙二醇化脂质分子量相近的情况下，例如L-0中的线性聚乙二醇化脂质DMG和L1-1中的非线性氮支化聚乙二醇化脂质E1-1，其PEG组分的分子量均约为2kDa，非线性结构的E1-1能实现更好的脂质药物组合物血清稳定性；非线性聚乙二醇化脂质可以在用量较少的情况下实现较好的脂质药物组合物血清稳定性。
(3)细胞毒性评价
所用细胞为HeLa细胞，选用的方法是CCK-8试剂盒测定细胞活力法。
用商用转染试剂Lipofectamine 2000(L2K)按照实施例9的方法制备复合物L2K/Fluc-mRNA。
HeLa按6000个/孔，每孔体积100μL的比例接种在96孔板中，分成control组(空白对照组)、L2K/Fluc-mRNA组(阳性对照组)和LNP/Fluc-mRNA组(实验组，L1-1～L7-1)，于37℃、5％CO2的条件下孵育。细胞孵育24小时后，空白对照组加入10μL的PBS溶液，阳性对照组和实验组分别加入3.3μg/mL的L2K/Fluc-mRNA(10μL)和3.3μg/mL的LNP/Fluc-mRNA(10μL)，于37℃、5％CO2的条件下继续孵育。孵育24小时后，在避光条件下，取出96孔板，吸去培养液，以每孔120μL的量加入已稀释好的CCK-8溶液。加毕，于37℃、5％CO2的条件下孵育l-4小时。
取出，用酶标仪测定每孔在450nm波长下的吸收值。
重复三次测试的结果取平均值，阳性对照组L2K/Fluc-mRNA的细胞存活率为92％，所有实验组的细胞存活率均大于88％，特别地，L1-1、L1-4、L1-5、L2-1、L2-4、L2-7、7-1的细胞存活率为97％及以上。
结果显示，与采用商用转染试剂Lipofectamine 2000的L2K/Fluc-mRNA相比，本发明的LNP/Fluc-mRNA同样对HeLa细胞没有明显的细胞毒性。
表2：细胞转染测试结果
(4)转染效果评价
为了考察本发明实施例9中制备的各组LNP/Fluc-mRNA组合物在细胞水平的mRNA转染率，采用Luciferase生物发光进行测试。将LNP/Fluc-mRNA组合物制剂溶解于培养基中配成所需剂量，用HeLa细胞作为细胞模型，以接种密度6000个细胞/孔，将细胞悬浮液100μL/孔接种到黑边透明底的96孔板中。接种之后，在细胞培养箱中孵育培养24h，然后按每孔0.2ug mRNA的剂量进行给药，空白对照组加入对应剂量的游离的Fluc-mRNA，转染24小时后，去掉旧培养基，换成含D-荧光素钠(1.5mg/mL)底物的新培养基，并孵育5分钟后，使用酶标仪检测生物发光，荧光越强表明转运进入细胞质且翻译出相应的荧光蛋白的Fluc-mRNA越多，实验结果如表2所示，其中，荧 光强度相对值为各组的荧光强度值与空白对照组的荧光强度的比值。结果表明本发明制备的LNP/Fluc-mRNA组合物都具有优异的体外转染效果，即对照组和实验组的LNP都是有效的递送载体，且聚乙二醇化脂质含可降解连接基的实验组的转染效果大多优于对照组。特别地，聚乙二醇组分和脂质组分间的连接基含寡肽残基的L6-1、L6-2以及Lx、L1、L2均含酯键的L7-1的转染效果都明显优于聚乙二醇化脂质为DMG的L-0。
结果表明，含有本发明的氮支化聚乙二醇化脂质的LNP能够有效的将Fluc-mRNA递送至细胞并转染。本发明含细胞内降解性基团的氮支化聚乙二醇化脂质更有助于使脂质核酸药物组合物在被细胞摄入后更好地发挥药效。
(5)靶向性实验
本实施例考察含叶酸修饰聚乙二醇末端的E8-1的LNP的靶向性，以除PEG末端外结构最为接近的E1-1作为对比。
一、制备氮支化聚乙二醇化脂质纳米粒/奥沙利铂组合物制剂：
步骤a：将DSPC(4g)、胆固醇(667mg)溶于60mL二氯甲烷中，加入20mL含80mg奥沙利铂的水溶液，搅拌30min，超声20min，40℃旋转蒸发除去有机溶剂，待凝胶塌陷后，按组分加入含E1-1(0.50g,0.2mmol)、泊洛沙姆F68(400mg)的水溶液50mL，继续蒸发30min，高压均质400bar、5min，用水定容至100mL，然后采用切向流超滤法分离游离奥沙利铂，用分子截留量为10K的切向流膜包进行超滤，用水作为置换液体，重复超滤3次，用超滤离心管法测其包封率和高效液相色谱法测定最后药物浓度，包封率为95.33％，粒径为124nm，加水调整LNP混悬液中药物浓度至500μg/mL即得氮支化聚乙二醇化脂质纳米粒/奥沙利铂组合物制剂LP-1。
步骤b：采用与上述实验组相同的制备方法，用E8-1(0.68g,0.2mmol)代替E1-1，制备得到叶酸偶联的氮支化聚乙二醇化脂质纳米粒/奥沙利铂组合物制剂LP-2。
二、进行HCT-116人结肠癌细胞株的复苏与培养，建立HCT-116人结肠癌荷瘤裸鼠模型，接种裸鼠自然生长，游标卡尺测量瘤体积，等肿瘤生长至50-75mm3后，按瘤体积随机分4组：
(1)空白对照组：6只，用5％葡萄糖注射液，从尾静脉给药一次后取出肿瘤组织；
(2)对照组1：用10mg/kg奥沙利铂注射液，给药一次，12只；
(3)对照组2：用10mg/kg的LP-1制剂，给药一次，12只；
(4)实验组：用10mg/kg的LP-2制剂，给药一次，12只；
其中，对照组1、对照组2和实验组从尾静脉给药，给药后按不同时间点0.05、0.5、1、1.5、2、2.5、3、3.5、4、6、8、48h处死，将裸鼠肿瘤块置-20℃冰箱保存，然后HPLC法检测药物浓度。结果显示，与奥沙利铂注射液相比，LP-1未体现出更高的靶向性；LP-2的靶向性明显增强，血浆峰浓度显著提高(提高约102％)，说明本发明用靶向基团对氮支化聚乙二醇化脂质的聚乙二醇末端进行功能化修饰，可以提高脂质纳米粒的靶向效率。
以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书内容所作的等效结构或等效流程变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。
对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，对此应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。

Claims (27)

  1. 一种氮支化聚乙二醇化脂质，其特征在于，结构如通式(1)所示：
    其中，
    B1、B2各自独立地为连接键或亚烷基；
    L1、L2、Ld和y个Lx各自独立地为连接键或二价连接基L；
    R1、R2各自独立地为含有0-10个杂原子的C1-50脂肪烃基或C1-50脂肪烃衍生物残基；所述杂原子为B、O、N、Si、P或S；
    Ncore为价态为y+1的多价基团；Ncore含有一个与Ld相连的三价氮原子支化核；
    y为2、3、4、5、6、7、8、9或y≥10，优选为2或3；
    XPEG为聚乙二醇组分。
  2. 根据权利要求1所述的氮支化聚乙二醇化脂质，其特征在于，所述二价连接基L为亚烷基、-C(＝O)-、-OC(＝O)-、-C(＝O)O-、-OC(＝O)O-、-O-、-S-、-S-S-、-C(＝O)S-、-SC(＝O)-、-NRc-、-NRcC(＝O)-、-C(＝O)NRc-、-NRcC(＝O)NRc-、-OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc-、-NRcC(＝O)S-及其组合中的任一种；所述氮支化聚乙二醇化脂质含有两个或两个以上L时，任两个L为相同或不同结构；
    所述亚烷基为-(CRcRc)t-，其中t每次出现时各自独立地为1-12的整数，优选t为1-4的整数，更优选t为1或2；Rc每次出现时各自独立地为H、C1-6烷基、含碳环的基团、含杂环的基团、氨基酸的侧链基团中任一种，优选为H、甲基、异丙基、异丁基、苄基中任一种，更优选为H或苄基，最优选为H。
  3. 根据权利要求2所述的氮支化聚乙二醇化脂质，其特征在于，任一个XPEG包含x个RPEG；y个x各自独立地选自1-4的整数，优选y个x均为1；当y个x均大于等于2时，优选所有的x均相等；
    所述RPEG的结构为其中，
    ni为聚乙二醇链的聚合度，选自4～250的整数，i选自1-20的整数；所述聚乙二醇链多分散性为或单分散性；
    T为H、C1-6烷基或R01-L01-；
    L01为连接键或二价连接基L；
    R01为能与生物相关物质相互反应的功能性基团。
  4. 根据权利要求3所述的氮支化聚乙二醇化脂质，其特征在于，L01为连接键、-(CH2)t-、-NH(CH2)t-、-NH(CH2)tC(＝O)NH(CH2)t-、-O(CH2)t-、-NH(CH2)tC(＝O)O(CH2)t-、-OC(＝O)(CH2)t-、-OC(＝O)O(CH2)t-、-OC(＝O)(CH2)tC(＝O)-、-(CH2)tC(＝O)NH(CH2)t中任一种，且左端与R01连接，其中t为1-4的整数，t优选为1或2。
  5. 根据权利要求3所述的氮支化聚乙二醇化脂质，其特征在于，R01为羟基、巯基、活性酯基、活性碳酸酯基、磺酸酯基、氨基、马来酰亚胺基、琥珀酰亚胺基、羧基、酰氯基、醛基、叠氮基、氰基、烯基、炔基、环氧烷基、罗丹明基、叶酸残基、生物素残基、单糖基和多糖基中任一种反应性基团或其变化形式，所述变化形式选自所述反应性基团的前体、以所述反应性基团作为前体的活性形式、所述反应性基团被取代的活性形式、所述反应性基团被保护的非活性形式中任一种；其中，所述反应性基团的前体指经过氧化、还原、水合、脱水、电子重排、结构重排、盐络合与解络合、离子化、质子化、 去质子化中至少一个过程，可转变为所述反应性基团的结构；
    优选R01选自以下结构中任一种：
  6. 根据权利要求3所述的氮支化聚乙二醇化脂质，其特征在于，RPEG的数均分子量选自0.5kDa～20kDa；优选为0.5kDa～5kDa；更优选为0.5kDa、1kDa、2kDa或5kDa。
  7. 根据权利要求4-6中任一项所述的氮支化聚乙二醇化脂质，其特征在于，结构如通式(2)所示：
    其中，n1、n2各自独立地选自4-100的整数，优选选自10-60的整数，更优选选自10-45的整数，最优选为10、11、12、20、21、22、23、24、25中任一种。
  8. 根据权利要求7所述的氮支化聚乙二醇化脂质，其特征在于，T为甲基。
  9. 根据权利要求7所述的氮支化聚乙二醇化脂质，其特征在于，所述L1、L2各自独立地为连接键、-O-、-C(＝O)-、-OC(＝O)-、-C(＝O)O-、-NHC(＝O)-、-C(＝O)NH-、-OC(＝O)O-、-OC(＝O)NH-、-NHC(＝O)O-中任一种。
  10. 根据权利要求7所述的氮支化聚乙二醇化脂质，其特征在于，所述B1、B2各自独立地为连接键或直链的C1-14亚烷基；所述C1-14亚烷基有0-2个氢原子被0-2个Rq取代，Rq每次出现时各自独立地为-(CH2)tqC[(CH2)tqH]3、-(CH2)tqO(CH2)tqH、-(CH2)tqS(CH2)tqH或-(CH2)tqN[(CH2)tqH]2，其中tq每次出现时各自独立地为0-4的整数；优选Rq每次出现时各自独立地为-OH或-CH3；
    优选B1、B2各自独立地为连接键或-(CH2)1-10-，更优选B1、B2各自独立地为连接键、-(CH2)2-、-(CH2)3-、-(CH2)4-、-(CH2)5-、-(CH2)6-、-(CH2)7-、-(CH2)8-中任一种。
  11. 根据权利要求7所述的氮支化聚乙二醇化脂质，其特征在于，Lx各自独立地为连接键、-(CH2)t-、-(CH2)tC(＝O)(CH2)t-、-(CH2)tC(＝O)O(CH2)t-、-(CH2)tOC(＝O)(CH2)t-、-(CH2)tC(＝O)NH(CH2)t-、-(CH2)tNHC(＝O)(CH2)t-中任一种；其中t为1-4的整数，t优选为1或2；优选Lx各自独立地为-CH2CH2-、-CH2C(＝O)-、-C(＝O)CH2-、-C(＝O)CH2CH2-、-CH2CH2C(＝O)OCH2CH2-、-C(＝O)NH-中任一种。
  12. 根据权利要求7所述的氮支化聚乙二醇化脂质，其特征在于，Ld为连接键、-(CRcRc)t-、-C(＝O)-、-O-、-NH-及其组合中的任一种，其中t为1或2，Rc为H或氨基酸的侧链基团；
    优选Ld为-CH2CH2-、-CH2C(＝O)-、-CH2CH2C(＝O)-、-CH2C(＝O)NHCH2CH2-、-CH2C(＝O)OCH2CH2-、-CH2CH2C(＝O)OCH2CH2-、-CH2C(＝O)NHCH2C(＝O)-、-CH2C(＝O)NHCH2CH2C(＝O)OCH2CH2-、-CH2CH2C(＝O)NHCH2C(＝O)-、-CH2C(＝O)-Gly-Gly-OCH2CH2-、-CH2C(＝O)-Phe-Gly-OCH2CH2-中任一种，且左端与Ncore相连；其中，Gly为甘氨酸残基，结构为-NHCH2C(＝O)-；Phe为苯丙氨酸残基，结构为
  13. 根据权利要求7所述的氮支化聚乙二醇化脂质，其特征在于，R1、R2各自独立地含有0-8个碳碳双键和/或0-8个碳碳三键，且各自独立地有0-10个氢原子各自独立地被0-10个Rm取代；所述Rm每次出现时各自独立地为选自直链C1-6烷基、支化C1-6烷基、C3-12环烷基、C2-8烯基、C2-8炔基、苯基、苄基中任一种；优选Rm为甲基；
    所述R1、R2各自独立地选自RL、RB、Rr中任一种，优选R1、R2各自独立地为RL或RB；
    其中，RL选自以下结构中任一种或任一种的顺反异构体：
    RL优选为以下结构中任一种：
    其中，RB结构为其中，X为CH或N；t为0-5的整数；Be、Bf各自独立地为连接键或C1-10亚烷基；Le、Lf各自独立地为连接键、-O-、-OC(＝O)-、-C(＝O)O-、-NHC(＝O)-、-C(＝O)NH-中任一种；Re、Rf各自独立地为C1-12烷基；
    RB优选为以下结构中任一种：
    RB更优选为以下结构中任一种：

    其中，Rr为含环状结构的C4-30烷基或C4-30杂烷基，优选为
  14. 根据权利要求7所述的氮支化聚乙二醇化脂质，其特征在于，Ncore选自以下任一种结构：
    其中，u1、u2、u3各自独立地为与Ld或Lx相连的连接键，且u1、u2、u3中任两个不同时与同一个Ld或Lx相连接；
    Q为电子改变基团，其数量为0、1或大于1；当Q的数量大于1时，任两个Q为相同或不同结构。
  15. 根据权利要求7所述的氮支化聚乙二醇化脂质，其特征在于，其结构选自以下结构中任一种：


  16. 一种脂质组合物，其特征在于，包含权利要求1-15中任一项所述的氮支化聚乙二醇化脂质。
  17. 根据权利要求16所述的脂质组合物，其特征在于，还含有磷脂、类固醇脂质和聚乙二醇化脂质中的一种或者一种以上，选自以下情形中任一种：
    情形(1)：还含有磷脂；
    情形(2)：还含有类固醇脂质；
    情形(3)：还含有聚乙二醇化脂质；
    情形(4)：还含有磷脂和类固醇脂质；
    情形(5)：还含有磷脂和聚乙二醇化脂质；
    情形(6)：还含有类固醇脂质和聚乙二醇化脂质；
    情形(7)：还含有磷脂、类固醇脂质和聚乙二醇化脂质；
    优选还同时含有磷脂、类固醇脂质和聚乙二醇化脂质三种脂质。
  18. 根据权利要求17所述的脂质组合物，其特征在于，所述磷脂选自1,2-二亚油酰基-sn-甘油-3-磷酸胆碱、1,2-二肉豆蔻酰基-sn-甘油-磷酸胆碱、1,2-二油酰基-sn-甘油-3-磷酸胆碱、1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱、1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱、1,2-双十一烷酰基-sn-甘油-磷酸胆碱、1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸胆碱、1,2-二-O-十八碳烯基-sn-甘油-3-磷酸胆碱、1-油酰基-2-胆固醇基半琥珀酰基-sn-甘油-3-磷酸胆碱、1-十六烷基-sn-甘油-3-磷酸胆碱、1,2-二亚麻酰基-sn-甘油-3-磷酸胆碱、1,2-二花生四烯酰基-sn-甘油-3-磷酸胆碱、1,2-双二十二碳六烯酰基-sn-甘油-3-磷酸胆碱、1,2-二油酰基-sn-甘油-3-磷酸乙醇胺、1,2-二植烷酰基-sn-甘油-3-磷酸乙醇胺、1,2-二硬脂酰基-sn-甘油-3-磷酸乙醇胺、1,2-二亚油酰基-sn-甘油-3-磷酸乙醇胺、1,2-二亚麻酰基-sn-甘油-3-磷酸乙醇胺、1,2-二花生四烯酰基-sn-甘油-3-磷酸乙醇胺、1,2-双二十二碳六烯酰基-sn-甘油-3-磷酸乙醇胺、1,2-二油酰基-sn-甘油-3-磷酸-rac-(1-甘油)钠盐、二油酰基磷脂酰丝氨酸、二棕榈酰基磷脂酰甘油、棕榈酰基油酰基磷脂酰乙醇胺、二硬脂酰基-磷脂酰-乙醇胺、二棕榈酰基磷脂酰乙醇胺、二肉豆蔻酰基磷酸乙醇胺、1-硬脂酰基-2-油酰基-硬脂酰乙醇胺、1-硬脂酰基-2-油酰基-磷脂酰胆碱、鞘磷脂、磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰肌醇、磷脂酸、棕榈酰基油酰基磷脂酰胆碱、溶血磷脂酰胆碱和溶血磷脂酰乙醇胺中任一种及其组合物。
  19. 根据权利要求17所述的脂质组合物，其特征在于，所述类固醇脂质选自胆固醇、粪固醇、谷固醇、麦角固醇、菜油固醇、豆固醇、菜籽固醇、番茄碱、熊果酸、α-生育酚中任一种及其组合物。
  20. 根据权利要求17所述的脂质组合物，其特征在于，所述阳离子脂质选自N,N-二油基-N,N-氯化二甲铵、N,N-二硬脂基-N,N-溴化二甲铵、N-(1-(2,3-二油酰氧基)丙基)-N,N,N-氯化三甲铵、N-(1-(2,3-二油基氧基)丙基)-N,N,N-氯化三甲铵、N,N-二甲基-2,3-二油基氧基丙胺、3-(双十二烷基氨基)-N1,N1,4-三-十二烷基-1-哌嗪乙胺、N1-[2-(双十二烷基氨基)乙基]-N1,N4,N4-三-十二烷基-1,4-哌嗪二乙胺、14,25-双十三烷基-15,18,21,24-四氮杂-三十八烷、1,2-二亚油基氧基-N,N-二甲基氨基丙烷、2,2-二亚油基-4-二甲基氨基甲基-[1,3]-二氧杂环戊烷、4-(二甲基氨基)丁酸三十七碳-6,9,28,31-四烯-19-基酯、2,2-二亚油基-4-(2-二甲基氨基乙基)-[1,3]-二氧杂环戊烷、((4-羟基丁基)氮杂二烷基)双(己烷-6,1-二基)双(2-己基癸酸酯)和十七烷-9-基-8-((2-羟乙基)(6-氧代-6-((十一烷氧基)己基)氨基)辛酸酯)、


    中任一种及其组合物。
  21. 根据权利要求17所述的脂质组合物，其特征在于，
    聚乙二醇化脂质占总脂质的摩尔百分比为0.5-5％，优选为1-3％，更优选为1.5％、1.6％、1.7％、1.8％、1.9％；
    阳离子脂质占总脂质的摩尔百分比为30-65％，优选为35％、40％、45％、46％、47％、48％、49％、50％、55％；
    磷脂占总脂质的摩尔百分比为7.5-13％，优选为8％、9％、10％、11％、12％；
    类固醇脂质占总脂质的摩尔百分比为35-50％，优选为40％、41％、42％、43％、44％、45％、46％、47％、48％、49％、50％。
  22. 一种脂质药物组合物，其特征在于，含有权利要求16-21中任一项所述的脂质组合物和药物，所述药物选自核酸药物、基因疫苗、抗肿瘤药物、小分子药物、多肽药物和蛋白质药物中任一种。
  23. 根据权利要求22所述的脂质药物组合物，其特征在于，所述药物为核酸药物， 选自DNA、RNA、反义核酸、质粒、干扰核酸、适体、antagomir和核酶中任一种，所述RNA选自mRNA、saRNA、circRNA、miRNA和siRNA中任一种；优选为DNA、mRNA、miRNA和siRNA中任一种。
  24. 根据权利要求22所述的脂质药物组合物，其特征在于，所述脂质药物组合物作为药物使用，所述药物选自以下任一种：治疗癌症的药物、抗感染剂、抗生素剂、抗病毒剂、抗真菌剂、疫苗。
  25. 根据权利要求22所述的脂质药物组合物，其特征在于，所述脂质药物组合物为LNP-药物组合物、LPP-药物组合物或PNP-药物组合物；优选为LNP-药物组合物，更优选为LNP-核酸药物组合物，更优选为LNP-mRNA组合物。
  26. 一种脂质药物组合物制剂，其特征在于，含有权利要求22-25中任一项所述的脂质药物组合物和药学上可接受的稀释剂或赋形剂，所述稀释剂或赋形剂优选为去离子水、超纯水、磷酸盐缓冲液和生理盐水中任一种，更优选为磷酸盐缓冲液或生理盐水，最优选为生理盐水。
  27. 一种脂质体或者脂质纳米粒，其特征在于，含有权利要求16-21中任一项所述的脂质组合物。
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