Lyophilized and liquid formulations of lipid nanoparticles

Abstract

The present invention provides a lyophilized formulation of lipid nanoparticles comprising a cationic lipid and a cryoprotectant combination comprising sucrose and a non-polar amino acid.

Description

Technical Field

[0001] This invention relates to novel formulations of lipid nanoparticles. In particular, this invention relates to lyophilized and liquid formulations of lipid nanoparticles. Background Technology

[0002] Lipid nanoparticles (LNPs) represent the most advanced nonviral gene delivery system in clinical practice. They safely and efficiently deliver nucleic acids, overcoming significant obstacles to the development and use of gene therapy. Lipid nanoparticles have been successfully used clinically for mRNA delivery. Lipid nanoparticles encapsulating mRNA have been widely used in COVID-19 vaccines during the pandemic. However, mRNA is sensitive to hydrolysis and has limited thermal stability. In most cases, long-term storage of mRNA-LNP vaccines requires cryogenic storage to slow mRNA degradation. However, cryogenic freezers are not suitable for some rural areas.

[0003] Lyophilization is believed to help extend the shelf life of mRNA-LNPs by removing moisture from the formulation. The dried formulation can be stored for several years at 2-8°C.

[0004] Therefore, there is a need to develop lyophilized formulations of mRNA-LNP.

[0005] However, as a fragile system, mRNA-LNP can be significantly affected by the physical stress of sublimation. In most cases, the particle size increases significantly, and a certain amount of mRNA leaks out during freeze-drying. This can affect the in vivo activity of mRNA vaccines.

[0006] Therefore, mRNA-LNP lyophilized formulations with relatively high encapsulation efficiency and suitable particle size are required. Summary of the Invention

[0007] The inventors have now discovered that specific combinations of cryoprotectants can protect mRNA-LNP from lyophilization stress. This invention is based on such an incidental discovery.

[0008] In a first aspect, the present invention provides a lyophilized formulation of lipid nanoparticles (also referred to herein as a "lyophilized LNP formulation") comprising, by weight relative to the total weight of the lyophilized formulation: (A) 0.2-20 wt.% of lipid nanoparticles containing cationic lipids; and (B) A cryoprotectant combination, the cryoprotectant combination comprising (i) 75-99 wt.% sucrose; and (ii) 0.1-10 wt.% of nonpolar amino acids.

[0009] The present invention also provides a liquid formulation of lipid nanoparticles (also referred to herein as "liquid LNP formulation"), which can be used to prepare the lyophilized LNP formulation disclosed herein.

[0010] In a second aspect, the present invention provides a liquid formulation of lipid nanoparticles, comprising, by weight relative to the total volume of the liquid formulation: (A) Lipid nanoparticles containing cationic lipids, ranging from 0.001% to 0.2% w / v; and (B) A cryoprotectant combination, the cryoprotectant combination comprising (i) 0.001-40% w / v sucrose, and (ii) 0.01-10% w / v nonpolar amino acids.

[0011] The inventors have now discovered that a specific combination of cryoprotectants containing sucrose and nonpolar amino acids can protect mRNA-LNPs from freeze-drying stress, so that the particle size of mRNA-LNPs does not change significantly, for example, the particle size change of mRNA-LNPs before and after freeze-drying is less than 10 nm.

[0012] The inventors have also discovered that when using a specific combination of cryoprotectants containing sucrose and nonpolar amino acids, the resulting mRNA-LNP lyophilized formulation has a suitable particle size, for example, 80-100 nm, and a relatively high encapsulation efficiency, for example, not less than 85%, which allows the in vivo biological activity of the mRNA to be maintained to a great extent.

[0013] The inventors have also discovered that when using a specific combination of cryoprotectants containing sucrose, nonpolar amino acids, and salts or Tris, the resulting mRNA-LNP lyophilized formulation has high-purity RNA and a reduced level of lipid adducts during storage.

[0014] Other features and advantages of the present invention will become more apparent from the following description and examples. Attached Figure Description

[0015] The present invention will be described and explained in detail below with reference to the accompanying drawings.

[0016] Figure 1 The size and polydispersity index of LNPs of CE.10-17 before and after lyophilization are shown.

[0017] Figure 2 The packaging efficiency of the LNP for CE.10-17 is shown.

[0018] Figure 3 The size and polydispersity index of LNPs before and after lyophilization for IE.16-20 are shown.

[0019] Figure 4 The packaging efficiency of LNPs for IE.12 and 16-19 is shown.

[0020] Figure 5 The sizes and polydispersity indices of LNPs before and after lyophilization for IE.12 and 20-21 are shown.

[0021] Figure 6 The packaging efficiency of LNPs for IE.12 and 20-21 is shown.

[0022] Figure 7 The in vivo hEPO expression levels (μg / ml) of the test group treated with mRNA LNP in Example 22 of the present invention were measured before and after lyophilization.

[0023] Figure 8 The figures show the in vivo hEPO expression levels (μg / ml) of the test groups treated with mRNA LNP before and after lyophilization in Examples 23-26 of the present invention.

[0024] Figure 9 The sizes of LNPs in IE.27-31 before and after lyophilization are shown.

[0025] Figure 10 The packaging efficiency of the LNP for IE.27-31 is shown.

[0026] Figure 11 The RNA purity (%) of LNPs from CE.19 and IE.32 after storage at 25°C for a period of time is shown.

[0027] Figure 12 The percentage of lipid adducts in LNPs of CE.19 and IE.32 after storage at 25°C for a period of time is shown.

[0028] Figure 13 The percentage of lipid adducts in LNPs of IE.33-36 and CE.20 after storage at 37°C for a period of time is shown. Detailed Implementation

[0029] Unless otherwise described, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict between the definitions of terms in this specification and their common understanding by one of ordinary skill in the art to which this invention pertains, the definitions set forth herein shall prevail.

[0030] As used herein, the term “comprising” should be interpreted as encompassing all specifically mentioned features as well as optional, additional, unspecified features.

[0031] As used herein, the use of the term “comprising” also discloses an implementation in which no features other than those specifically mentioned are present (i.e., “composed of”).

[0032] Unless otherwise stated, all numerical values ​​of quantities, etc., of all express components used in this specification and claims should be understood to be modified by the term "about". Therefore, unless indicated to the contrary, the numerical values ​​and parameters described herein are approximate and can be varied according to the desired purpose.

[0033] As used herein and unless otherwise stated, the term “lipid” refers to a group of organic compounds, including but not limited to esters of fatty acids, and is generally characterized by poor solubility in water but soluble in many nonpolar organic solvents. While lipids are generally poorly soluble in water, certain classes of lipids (e.g., lipids modified with polar groups, such as DMG-PEG2000) have limited water solubility and can be soluble in water under certain conditions. Known types of lipids include biomolecules such as fatty acids, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, and phospholipids. Lipids can be classified into at least three categories: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “complex lipids,” which include phospholipids and glycolipids (e.g., DMPE-PEG2000); and (3) “derived lipids,” such as steroids. In addition, as used herein, lipids also encompass lipid-like compounds. The term “lipid-like compound,” also simply “lipid,” refers to lipid-like compounds (e.g., amphiphilic compounds with lipid-like physical properties).

[0034] The term "lipid nanoparticle" or "LNP" refers to a particle having at least one nanometer (nm) scale size (e.g., 1 to 1,000 nm) containing one or more types of lipid molecules. The LNPs described herein may also contain at least one non-lipid payload molecule (e.g., one or more nucleic acid molecules). In some embodiments, the LNP comprises a non-lipid payload molecule partially or completely encapsulated within a lipid shell. Specifically, in some embodiments, 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. Without being bound by theory, it is anticipated that cationic lipids can interact with negatively charged payload molecules and facilitate payload incorporation and / or encapsulation into the LNP during LNP formation. Other lipids that can form part of an LNP as described herein include, but are not limited to, neutral and charged lipids, such as steroids, polymer-conjugated lipids, and various zwitterionic lipids.

[0035] The term "cationic lipid" refers to a lipid that carries a positive charge at any pH or hydrogen ion activity in its environment, or is capable of carrying a positive charge in response to the pH or hydrogen ion activity of its environment (e.g., the environment in which it is intended for use). Therefore, the term "cationic" encompasses both "permanent cation" and "cationizable." In some embodiments, the positive charge in the cationic lipid is caused by the presence of a quaternary nitrogen atom. In some embodiments, the cationic lipid comprises a zwitterionic lipid that carries a positive charge in the environment in which it is intended for use (e.g., at physiological pH). In a preferred embodiment, the cationic lipid is an ionizable cationic lipid. The term "ionizable cationic lipid" refers to an ionizable lipid that carries a positive charge at acidic pH to aggregate RNA into a composition (e.g., LNP), but is neutral at physiological pH to minimize toxicity.

[0036] The term "polymer-conjugated lipid" refers to a molecule that comprises both a lipid moiety and a polymer moiety. An example of a polymer-conjugated lipid is a polyethylene glycol-modified lipid (PEG-lipid), in which the polymer moiety comprises polyethylene glycol.

[0037] The term "neutral lipid" encompasses any lipid molecule present in an uncharged or neutral zwitterionic form at or within a selected pH value or range. In some embodiments, the selected useful pH value or range corresponds to the pH conditions of the environment in which the lipid is intended to be used, such as physiological pH. As a non-limiting example, neutral lipids that may be used in conjunction with this disclosure include, but are not limited to, phosphatidylcholines, such as 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), and 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC); phosphatidylethanolamines, such as 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE) and 2-((2,3-bis(oleoyloxy)propyl)dimethylammonium)ethyl hydrogen phosphate (DOCP); sphingomyelin (SM); ceramides; and steroids, such as sterols and their derivatives. Neutral lipids, as described herein, may be synthetic or derived from natural sources or compounds (isolated or modified).

[0038] The term "charged lipid" encompasses any lipid molecule present in a positively or negatively charged form at or within a selected pH range. In some embodiments, the selected pH value or range corresponds to the pH conditions of the environment in which the lipid is intended to be used, such as physiological pH. As a non-limiting example, charged lipids that may be used in conjunction with this disclosure include, but are not limited to, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylammonium propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, dimethylaminoethane carbamoyl sterol (e.g., DC-Chol), sodium 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine (DOPS-Na), sodium 1,2-dioleoyl-sn-glycerol-3-phosphate-(1'-racemic-glycerol) (DOPG-Na), and sodium 1,2-dioleoyl-sn-glycerol-3-phosphate (DOPA-Na). The charged lipids described herein may be synthetic or derived (isolated or modified) from natural sources or compounds.

[0039] As used herein and unless otherwise stated, the term “pharmaceuticalally acceptable carrier, diluent, or excipient” includes, but is not limited to, any adjuvant, carrier, excipient, gliding agent, sweetener, diluent, preservative, dye / coloring agent, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier that has been approved by the United States Food and Drug Administration for acceptable use in humans or livestock.

[0040] The term “composition” is intended to cover products containing optionally specified amounts of specified ingredients (e.g., lipid compounds and / or mRNA molecules provided herein).

[0041] As used interchangeably herein, the terms “polynucleotide” or “nucleic acid” refer to a polymer of nucleotides of any length and include, for example, DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogues, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and their analogues. Nucleic acids can be single-stranded or double-stranded. As used herein and unless otherwise stated, “nucleic acid” also includes nucleic acid mimics such as locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and morpholinonucleotides. As used herein, “oligonucleotide” refers to a short synthetic polynucleotide whose length is typically, but not necessarily, less than about 200 nucleotides. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The above description of polynucleotides applies equally and fully to oligonucleotides. Unless otherwise stated, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of addition from 5' to 3' of a newly generated RNA transcript is called the transcription direction; the region on the DNA strand that has the same sequence as the RNA transcript and is located at 5' relative to the 5' end of the RNA transcript is called the "upstream sequence"; the region on the DNA strand that has the same sequence as the RNA transcript and is located at 3' relative to the 3' end of the RNA transcript is called the "downstream sequence".

[0042] "Isolated nucleic acid" is a nucleic acid, such as RNA, DNA, or a mixture of nucleic acids, that is substantially isolated from other genomic DNA sequences naturally accompanying the native sequence, as well as proteins or complexes (such as ribosomes and polymerases). An "isolated" nucleic acid molecule is a nucleic acid molecule isolated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. Furthermore, "isolated" nucleic acid molecules (e.g., mRNA molecules) may be substantially free of other cellular material or culture media when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a particular embodiment, one or more nucleic acid molecules encoding antigens as described herein are isolated or purified. The term includes nucleic acid sequences that have been removed from their natural environment and includes recombinant or cloned DNA or RNA isolates, as well as chemically synthesized analogs or analogs biosynthesized from heterologous systems. A substantially pure molecule may include the isolated form of the molecule.

[0043] The term “encoding nucleic acid” or its grammatical equivalent, when used to refer to nucleic acid molecules, encompasses: (a) nucleic acid molecules that are native or, when manipulated by methods well known to those skilled in the art, can be transcribed to produce mRNA, which is then translated into peptides and / or polypeptides; and (b) the mRNA molecule itself. The antisense strand is the complement of such nucleic acid molecules, and the coding sequence can be inferred from it. The term “coding region” refers to the portion of a coding nucleic acid sequence that is translated into a peptide or polypeptide. The term “untranslated region” or “UTR” refers to the portion of a coding nucleic acid that is not translated into a peptide or polypeptide. Depending on the orientation of the UTR relative to the coding region of the nucleic acid molecule, a UTR located at the 5' end of the coding region is called a 5'-UTR, and a UTR located at the 3' end of the coding region is called a 3'-UTR.

[0044] As used herein, the term "mRNA" refers to a messenger RNA molecule containing one or more open reading frames (ORFs) that can be translated by a cell or organism having said mRNA to produce one or more peptide or protein products. The region containing one or more ORFs is referred to as the coding region of the mRNA molecule. In some embodiments, the mRNA molecule also contains one or more untranslated regions (UTRs).

[0045] As used herein, the term "functional nucleotide analog" refers to a modified form of a canonical nucleotide A, G, C, U, or T that (a) retains the base-pairing property of the corresponding canonical nucleotide and (b) contains at least one chemical modification of the corresponding native nucleotide's (i) nucleotide base, (ii) glycosyl group, (iii) phosphate ester group, or (iv) any combination of (i) to (iii). As used herein, base pairing encompasses not only canonical Watson-Crick adenine-thymine, adenine-uracil, or guanine-cytosine base pairs, but also base pairs formed between a canonical nucleotide and a functional nucleotide analog or between a pair of functional nucleotide analogs, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between the modified nucleotide base and a canonical nucleotide base or between two complementary modified nucleotide structures. For example, a functional analog of guanosine (G) retains the ability to pair with functional analogs of cytosine (C) or cytosine. An example of this type of non-canonical base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. As described herein, functional nucleotide analogs can be naturally occurring or non-natural. Therefore, nucleic acid molecules containing functional nucleotide analogs may have at least one modified nucleobase, glycosyl, and / or nucleoside linking. This document provides exemplary chemical modifications to nucleobase, glycosyl, or nucleoside linking in nucleic acid molecules.

[0046] All publications, patent applications, registry numbers, and other references cited in this specification are incorporated herein by reference in their entirety, as if each individual publication or patent application specifically and separately indicated to be incorporated by reference. The publications discussed herein are provided only for their disclosure prior to the filing date of this application. Nothing herein should be construed as an admission that the invention is not entitled to a prior art invention prior to such publications. Furthermore, the publication dates provided may differ from the actual publication dates, which require independent verification.

[0047] Lyophilized LNP formulations

[0048] In a first aspect, the present invention provides a lyophilized LNP formulation comprising, by weight relative to the total weight of the lyophilized formulation: (A) 0.2-20 wt.% of lipid nanoparticles containing cationic lipids; and (B) A cryoprotectant combination, the cryoprotectant combination comprising (i) 75-99 wt.% sucrose; and (ii) 0.1-10 wt.% of nonpolar amino acids.

[0049] lipid nanoparticles

[0050] In some embodiments, the lyophilized LNP formulation comprises 0.2-20 wt.% (e.g., 0.3-18 wt.%, 0.4-16 wt.%, 0.5-12 wt.%, 0.6-10 wt.%, or 0.2-1 wt.%, 0.2-0.8 wt.%, 0.25-0.7 wt.%) of lipid nanoparticles relative to the total weight of the lyophilized LNP formulation.

[0051] The LNP particle size in the lyophilized LNP formulation is about 80 nm to about 100 nm, preferably about 80 nm to about 95 nm, for example about 82 nm to about 95 nm.

[0052] As used in this article, particle size refers to the average size, which can be determined by dynamic light scattering using the Malvern Zetasizer Nano ZS (Malvern UK) in 173° backscatter detection mode.

[0053] The lipid nanoparticles contain cationic lipids.

[0054] The lipid nanoparticles may also include one or more of the following: structural lipids, phospholipids, and polymer-conjugated lipids.

[0055] As used herein, unless otherwise stated, the molar percentage of lipids is calculated based on the total number of moles of all lipids present in the nanoparticles.

[0056] Cationic lipids include at least one of the following 01-06 series compounds (and their sub-formulas).

[0057] 01 series compounds

[0058] In some embodiments, the cationic lipids of this disclosure comprise at least one of those disclosed in International Application Publication No. WO2021204175, the entire teachings of which are incorporated herein by reference. Specifically, the cationic lipids comprise compounds represented by formula (01-I): (01-I), Or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein: G 1 and G 2 Each is an independent key, C2-C 12 Alkylene or C2-C 12 The alkenyl group, wherein one or more of the -CH2- groups in the alkylene group or alkenyl group are optionally replaced by -O-; L 1 It is -OC(=O)R 1 -C(=O)OR 1 -OC(=O)OR 1 -C(=O)R 1 -OR 1 -S(O) x R 1 -S-SR 1 -C(=O)SR 1 -SC(=O)R 1 -NR a C(=O)R 1 -C(=O)NR b R c -NR a C(=O)NR b R c -OC(=O)NR b R c -NR a C(=O)OR 1 -SC(=S)R 1 -C(=S)SR 1 -C(=S)R 1 -CH(OH)R 1 -P(=O)(OR) b(OR) c -(C6-C) 10 (Aspartic)-R 1 -(6 to 10 yuan of heteroaryl)-R 1 or R 1 ; L 2 It is -OC(=O)R 2 -C(=O)OR 2 -OC(=O)OR 2 -C(=O)R 2 -OR 2 -S(O) x R 2 -S-SR 2 -C(=O)SR 2 -SC(=O)R 2 -NR d C(=O)R 2 -C(=O)NR e R f -NR d C(=O)NR e R f -OC(=O)NR e R f -NR d C(=O)OR 2 -SC(=S)R 2 -C(=S)SR 2 -C(=S)R 2 -CH(OH)R 2 -P(=O)(OR) e (OR) f -(C6-C) 10 (Aspartic)-R 2 -(6 to 10 yuan of heteroaryl)-R 2 or R 2 ; R 1 and R 2 Each is independently C6-C 32 Alkyl or C6-C 32 alkenyl; R a R b R d and R e Each is independently H, C1-C 24 Alkyl or C2-C 24 alkenyl; R c and R f Each is independently C1-C32 Alkyl or C2-C 32 alkenyl; G 3 It is C2-C 24 Alkylene, C2-C 24 alkenyl, C3-C8 cycloalkyl or C3-C8 cycloalkyl; R 3 It is -N(R) 4 )R 5 ; R 4 It is a C3-C8 cycloalkyl, C3-C8 cycloalkenyl, 4- to 8-membered heterocyclic group or C6-C 10 aryl; or R 4 G 3 or G 3 A portion of them, together with the nitrogen they are attached to, forms a ring-like portion; R 5 It is C1-C 12 Alkyl or C3-C8 cycloalkyl; or R 4 R 5 Together with the nitrogen atoms they are attached to, they form a ring-like structure; x is 0, 1, or 2; and Each of the alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclic, aryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroarylene, and cyclic moiety is independently and optionally substituted.

[0059] In one embodiment, the cationic lipid comprises a (01-10) compound: (01-IO), Where y and z are each an independent integer from 2 to 12. s is an integer from 2 to 24. t is an integer from 1 to 12, and R 1 and R 2 Each is independently C6-C 32 Alkyl or C6-C 32 alkenyl; R 4 It is a C3-C8 cycloalkyl, C3-C8 cycloalkenyl, 4- to 8-membered heterocyclic group or C6-C 10 Aryl; R 6 It is hydrogen or hydroxyl. Or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0060] In one embodiment, the cationic lipid comprises a compound from Table 01-1 or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0061] Table 01-1

[0062] 02 series compounds

[0063] In one embodiment, the cationic lipids contained in the compositions, nanoparticle compositions, or nanoparticles provided herein are the cationic lipids described in International Patent Publication No. WO2023138611A1, the entire contents of which are incorporated herein by reference.

[0064] In some embodiments, the cationic lipids of the present invention comprise compounds of formula (02-I): (02-I), Or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein: G 1 and G 2 Each is independently C2-C 12 Alkylene or C2-C 12 alkenyl groups, in which G 1 and G 2 One or more of the -CH2- in the form are optionally replaced by -O-, -C(=O)O- or -OC(=O)-; Each L 1 Independently is -OC(=O)R 1 -C(=O)OR 1 -OC(=O)OR 1 -C(=O)R 1 -OR 1 -S(O) x R 1 -S-SR 1 -C(=O)SR 1 -SC(=O)R 1 -NR a C(=O)R 1 -C(=O)NR b R c -NR a C(=O)NR b R c -OC(=O)NR b R c -NR a C(=O)OR 1 -SC(=S)R 1-C(=S)SR 1 -C(=S)R 1 -CH(OH)R 1 -P(=O)(OR) b (OR) c -NR a P(=O)(OR b (OR) c ); Each L 2 Independently is -OC(=O)R 2 -C(=O)OR 2 -OC(=O)OR 2 -C(=O)R 2 -OR 2 -S(O) x R 2 -S-SR 2 -C(=O)SR 2 -SC(=O)R 2 -NR d C(=O)R 2 -C(=O)NR e R f -NR d C(=O)NR e R f -OC(=O)NR e R f -NR d C(=O)OR 2 -SC(=S)R 2 -C(=S)SR 2 -C(=S)R 2 -CH(OH)R 2 -P(=O)(OR) e (OR) f -NR d P(=O)(OR e (OR) f ); R 1 and R 2 Each is independently C6-C 24 Alkyl or C6-C 24 alkenyl; R a R b R d and R e Each is independently H, C1-C 24 Alkyl or C2-C 24 alkenyl; Rc and R f Each is independently C1-C 24 Alkyl or C2-C 24 alkenyl; G 3 It is C2-C 24 Alkylene, C2-C 24 alkenyl, C3-C8 cycloalkyl or C3-C8 cycloalkyl; R 3 It is -N(R) 4 )R 5 or -OR 6 ; R 4 It is C1-C 12 Alkyl, C2-C 12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 Aryl or 4- to 8-membered heterocyclic alkyl groups; R 5 It is hydrogen, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 Aryl or 4- to 8-membered heterocyclic alkyl groups; R 6 It is hydrogen, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl or C6-C 10 Aryl; x is 0, 1, or 2; and Each of the alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene, and cycloalkenyl groups is independently and optionally substituted.

[0065] In one embodiment, the cationic lipid comprises a compound of formula (O2-V): (02-V), Where X 1 and X 2 Each is independently a bond, -O-, -C(=O)O-, or -OC(=O)-; z is an integer from 2 to 12; Each L 1 Independently is -OC(=O)R 1 -C(=O)OR 1 -OC(=O)OR 1 -C(=O)R 1 -OR 1 -S(O) x R 1 -S-SR1 、 -C(=O)SR 1 、 -SC(=O)R 1 、 -NR a C(=O)R 1 、 -C(=O)NR b R c 、 -NR a C(=O)NR b R c 、 -OC(=O)NR b R c 、 -NR a C(=O)OR 1 、 -SC(=S)R 1 、 -C(=S)SR 1 [[ID=3​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​d P(=O)(OR e (OR) f ); R 3 It is -N(R) 4 )R 5 or -OR 6 ; R 4 It is C1-C 12 Alkyl, C2-C 12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 Aryl or 4- to 8-membered heterocyclic alkyl groups; R 5 It is hydrogen, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 Aryl or 4- to 8-membered heterocyclic alkyl groups; R 6 It is hydrogen, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl or C6-C 10 Aryl; When X 1 When it is -O-, -C(=O)O-, or -OC(=O)-, x2 is an integer from 2 to 5, and when X 1 When x2 is a key, it is an integer from 2 to 6; When X 2 When it is -O-, -C(=O)O-, or -OC(=O)-, y2 is an integer from 2 to 5, and when X 2 When y is a key, y2 is an integer from 2 to 6; G 4 and G 5 Each is independently a C2-C6 alkylene group; Or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0066] In one embodiment, the cationic lipid comprises a compound from Table 02-1 or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0067] Table 02-1

[0068] 03 series compounds

[0069] In some embodiments, the cationic lipids of this disclosure comprise at least one of those disclosed in International Application Publication No. WO2022152109A2, the entire teachings of which are incorporated herein by reference. Specifically, the cationic lipids comprise compounds represented by formula (03-I): (03-I), Or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein: G 1 and G 2 Each is an independent key, C2-C 12 Alkylene or C2-C 12 alkenyl groups, in which G 1 and G 2 One or more of the -CH2- in the form are optionally replaced by -O-; Each L 1 Independently is -OC(=O)R 1 -C(=O)OR 1 -OC(=O)OR 1 -C(=O)R 1 -OR 1 -S(O) x R 1 -S-SR 1 -C(=O)SR 1 -SC(=O)R 1 -NR a C(=O)R 1 -C(=O)NR b R c -NR a C(=O)NR b R c -OC(=O)NR b R c -NR a C(=O)OR 1 -SC(=S)R 1 -C(=S)SR 1 -C(=S)R 1 -CH(OH)R 1 -P(=O)(OR) b (OR) c -NR a P(=O)(OR b (OR) c -(C6-C) 10 (Aspartic)-R 1 -(6 to 10 yuan of heteroaryl)-R 1-(4- to 8-membered heterocyclic group)-R 1 or R 1 ; Each L 2 Independently is -OC(=O)R 2 -C(=O)OR 2 -OC(=O)OR 2 -C(=O)R 2 -OR 2 -S(O) x R 2 -S-SR 2 -C(=O)SR 2 -SC(=O)R 2 -NR d C(=O)R 2 -C(=O)NR e R f -NR d C(=O)NR e R f -OC(=O)NR e R f -NR d C(=O)OR 2 -SC(=S)R 2 -C(=S)SR 2 -C(=S)R 2 -CH(OH)R 2 -P(=O)(OR) e (OR) f -NR d P(=O)(OR e (OR) f -(C6-C) 10 (Aspartic)-R 2 -(6 to 10 yuan of heteroaryl)-R 2 -(4- to 8-membered heterocyclic group)-R 2 or R 2 ; R 1 and R 2 Each is independently C6-C 24 Alkyl or C6-C 24 alkenyl; R a R b R d and R e Each is independently H, C1-C 24 Alkyl or C2-C 24 alkenyl; Rc and R f Each is independently C1-C 24 Alkyl or C2-C 24 alkenyl; G 3 It is C2-C 12 Alkylene or C2-C 12 alkenyl groups, wherein the alkylene group or all of the alkenyl group is optionally composed of a C3-C8 cycloalkylene group, a C3-C8 cycloalkylene group, a C3-C8 cycloalkynyl group, a 4- to 8-membered heterocyclic group, or a C6-C... 10 Aryl or 5- to 10-member heteroaryl substitution; R 3 It is hydrogen, C1-C 12 Alkyl, C2-C 12 alkenyl, C2-C 12 Alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C3-C8 cycloalkynyl, 4- to 8-membered heterocyclic groups, C6-C 10 aryl or 5- to 10-membered heteroaryl; or R 3 G 1 or G 1 A portion of them, together with the nitrogen they are attached to, forms a cyclic portion; or R 3 G 3 or G 3 A portion of them, together with the nitrogen they are attached to, forms a ring-like portion; R 4 It is C1-C 12 Alkyl or C3-C8 cycloalkyl; x is 0, 1, or 2; n is 1 or 2; m is 1 or 2; and Each of the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclic, aryl, heteroaryl, alkylene, alkenyl, cycloalkylene, cycloalkenyl, cycloalkynyl, heterocyclic, aryl, heteroaryl and cyclic moieties is independently optionally substituted.

[0070] In one embodiment, the cationic lipid comprises a compound of formula (O3-II-D): (03-II-D), Or its pharmaceutically acceptable salt, prodrug, or stereoisomer. Among them G 1 G 2 G 3 L 1 L 2 R 3 and R 4 It is as defined above.

[0071] In one embodiment, the cationic lipid comprises a compound from Table 03-1 or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0072] Table 03-1.

[0073] 04 series compounds

[0074] In some embodiments, the cationic lipids of this disclosure comprise at least one of those disclosed in International Application Publication No. WO2010144740, the entire teachings of which are incorporated herein by reference. For example, the cationic lipids comprise compounds represented by formula (04-I): (04-I) 05 series compounds

[0075] In some embodiments, the cationic lipids of this disclosure comprise at least one of those disclosed in U.S. Patent Nos. US10442756B2, US9868691B2, and US9868692B2, the entire teachings of which are incorporated herein by reference. For example, the cationic lipids comprise compounds represented by formula (05-I): (05-I), or its salts or isomers, wherein: l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M1 is a bond or M′; R4 is unsubstituted C. 1-3 Alkyl or -(CH2) n Q, where Q is OH, -NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R )R8, -NHC(═NR9)N(R)2, -NHC(═CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N (OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N (R)2, -N(OR)C(═NR9)N(R)2, -N(OR)C(═CHR9)N(R)2 or heteroaryl, and n is selected from 1, 2, 3, 4 or 5; M and M′ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R′)-, -P(O)(OR′)O-, -SS-, aryl, and heteroaryl; and R2 and R3 are both C 1-14 Alkyl or C 2-14 Alkenyl, R8 is selected from the group consisting of: C 3-6 Carbon rings and heterocycles; R9 is selected from the following groups: H, CN, NO2, C 1-6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2-6 alkenyl, C 3-6 Carbon rings and heterocycles; Each R is independently selected from the following groups: C 1-3 Alkyl, C 2-3 Alkenyl and H; and R′ is a straight-chain alkyl group.

[0076] In some implementations, R' is a straight-chain C 1-18 Alkyl groups, such as C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C 10 Alkyl, C 11 Alkyl, C 12 Alkyl, C 13 Alkyl, C 14 Alkyl, C 15 Alkyl, C 16 Alkyl, C 17 Alkyl and C 18 Alkyl groups, preferably C9 alkyl groups and C6 alkyl groups. 11 alkyl.

[0077] In some embodiments, the cationic lipid comprises at least one of compounds of formula (A) and (B): Or its salts or isomers.

[0078] 06 series compounds

[0079] In some embodiments, the cationic lipids of this disclosure comprise at least one of those disclosed in U.S. Patent No. US10166298B2, the entire teachings of which are incorporated herein by reference. For example, the cationic lipids comprise compounds represented by formula (06-I): (06-I) Or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein: L 1 or L2 One of them is -O(C═O)-, -(C═O)O-, -C(═O)-, -O-, -S(O) x -, -SS-, -C(═O)S-, SC(═O)-, -NR a C(═O)-、-C(═O)NR a -、NR a C(═O)NR a -、-OC(═O)NR a -or-NR a C(═O)O-, and L 1 or L 2 The other two are -O(C═O)-, -(C═O)O-, -C(═O)-, -O-, and -S(O). x -, -SS-, -C(═O)S-, SC(═O)-, -NR a C(═O)-、-C(═O)NR a -、NR a C(═O)NR a -、-OC(═O)NR a -or-NR a C(═O)O- or direct bond; G 1 and G 2 Each is an unsubstituted C1-C. 12 Alkylene or C1-C 12 alkenyl; G 3 It is C1-C 24 Alkylene, C1-C 24 C3-C8 cycloalkylene, C3-C8 cycloalkylene; R a Is it H or C1-C? 12 alkyl; R 1 and R 2 Each is independently C6-C 24 Alkyl or C6-C 24 alkenyl; R 3 Is it H or OR? 5 CN, -C(═O)OR 4 -OC(═O)R 4 or -NR 5 C(═O)R 4 ; R 4 It is C1-C 12 alkyl; R 5It is an H or C1-C6 alkyl group; and x is 0, 1, or 2.

[0080] In one embodiment, the cationic lipid is a compound in Table 06-1 or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0081] Table 06-1

[0082] 07 series compounds

[0083] In some embodiments, the cationic lipids of this disclosure comprise at least one of those disclosed in International Patent Application No. PCT / CN2022 / 094227, the entire contents of which are incorporated herein by reference. For example, the cationic lipids comprise compounds represented by formula (07-I): (07-I), Or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein: G 1 and G 2 Each is an independent key, C2-C 12 Alkylene or C2-C 12 alkenyl; L 1 It is -OC(=O)R 1 -C(=O)OR 1 -OC(=O)OR 1 -C(=O)R 1 -OR 1 -S(O) x R 1 -S-SR 1 -C(=O)SR 1 -SC(=O)R 1 -NR a C(=O)R 1 -C(=O)NR b R c -NR a C(=O)NR b R c -OC(=O)NR b R c -NR a C(=O)OR 1 -SC(=S)R 1 -C(=S)SR 1 -C(=S)R 1 -CH(OH)R1 -P(=O)(OR) b (OR) c -(C6-C) 10 (Aspartic)-R 1 -(6 to 10 yuan of heteroaryl)-R 1 or R 1 ; L 2 It is -OC(=O)R 2 -C(=O)OR 2 -OC(=O)OR 2 -C(=O)R 2 -OR 2 -S(O) x R 2 -S-SR 2 -C(=O)SR 2 -SC(=O)R 2 -NR d C(=O)R 2 -C(=O)NR e R f -NR d C(=O)NR e R f -OC(=O)NR e R f -NR d C(=O)OR 2 -SC(=S)R 2 -C(=S)SR 2 -C(=S)R 2 -CH(OH)R 2 -P(=O)(OR) e (OR) f -(C6-C) 10 (Aspartic)-R 2 -(6 to 10 yuan of heteroaryl)-R 2 or R 2 ; R 1 and R 2 Each is independently C5-C 32 Alkyl or C5-C 32 alkenyl; R a R b R d and R e Each is independently H, C1-C 24 Alkyl or C2-C 24 alkenyl; Rc and R f Each is independently C1-C 32 Alkyl or C2-C 32 alkenyl; R 0 It is C1-C 12 Alkyl, C2-C 12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 Aryl or 4- to 8-membered heterocyclic alkyl groups; G 3 It is C2-C 12 Alkylene or C2-C 12 alkenyl; R 4 It is C1-C 12 Alkyl, C2-C 12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 Aryl or 4- to 8-membered heterocyclic alkyl groups; R 5 It is C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 Aryl or 4- to 8-membered heterocyclic alkyl groups; x is 0, 1, or 2; s is 0 or 1; and Each of the alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, arylene, and heteroarylene groups is independently and optionally substituted.

[0084] In some embodiments, the cationic lipid is a compound of formula (07-III): (07-III), Or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein: R 1 and R 2 Each is independently C5-C 32 Alkyl or C5-C 32 alkenyl; R 0 It is C1-C 12 Alkyl, C2-C 12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 Aryl or 4- to 8-membered heterocyclic alkyl groups; G 3 It is C2-C 12 Alkylene or C2-C 12 alkenyl; G4 It is C2-C 12 Alkylene or C2-C 12 alkenyl; R 3 It is -N(R) 4 )R 5 or -OR 6 ; R 4 It is C1-C 12 Alkyl, C2-C 12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 Aryl or 4- to 8-membered heterocyclic alkyl groups; R 5 It is C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 Aryl or 4- to 8-membered heterocyclic alkyl; or R 4 and R 5 Together with the nitrogen atoms they are attached to, they form a ring-like structure; R 6 It is hydrogen, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl or C6-C 10 Aryl; and Each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, and cyclic moiety is independently and optionally substituted.

[0085] In some embodiments, the cationic lipid is a compound from Table 4 or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0086] Table 07-1

[0087] ● Structural lipids

[0088] Unbound by theory, it is anticipated that structural lipids can stabilize the amphiphilic structures of nanoparticles, such as, but not limited to, lipid bilayer structures of nanoparticles. Exemplary structural lipids that can be used in conjunction with this disclosure include, but are not limited to, steroids, such as cholesterol, coccidosterol, sitosterol, ergosterol, campesterol, stigmasterol, phytosterol, tomatine, tomatine glycoside, ursolic acid, α-tocopherol, and combinations thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid is selected from cholesterol, corticosteroids (such as prednisolone, dexamethasone, prednisolone, and hydrocortisone), and combinations thereof.

[0089] In one embodiment, the lipid nanoparticles used in this invention comprise a steroid or a steroid analogue. In one embodiment, the lipid nanoparticles comprise cholesterol. In one embodiment, the lipid nanoparticles comprise a steroid present at a concentration within the range of: 13 to 55 mol%, 20 to 50 mol%, 30 to 50 mol%, 32 to 50 mol%, 39 to 49 mol%, 40 to 46 mol%, 40 to 44 mol%, 40 to 42 mol%, 42 to 44 mol%, or 44 to 46 mol%. In one embodiment, the lipid nanoparticles comprise a steroid present at a concentration of 40, 41, 42, 43, 44, 45, or 46 mol%.

[0090] In one embodiment, the lipid nanoparticles comprise a steroid, and the molar ratio of cationic lipid to steroid is in the range of 0.6 to 2.75 or 1.0 to 1.5. In one embodiment, the lipid nanoparticles comprise a steroid (such as cholesterol), and the molar ratio of cationic lipid to cholesterol is in the range of about 1.0 to 1.5. In one embodiment, the lipid nanoparticles comprise a steroid, and the steroid is present at a concentration in the range of 20 to 50 molar percentages of steroid.

[0091] ● Phospholipids

[0092] Unbound by theory, it is expected that phospholipids can assemble into one or more lipid bilayer structures. Exemplary phospholipids that can form part of the lipid nanoparticles usable in this invention include, but are not limited to, 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-di(undecanoyl)-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), and 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 Diether 1,2-Oleoyl-2-cholesterolylhemisuccino-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicyl-sn-glycerol-3-phosphate choline, 1,2-bis(docohexanoyl)-sn-glycerol-3-phosphate choline, 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (ME16.0) The LNP comprises 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docohexanoyl)-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-racemic-(1-glycerol) sodium salt (DOPG), and sphingomyelin. In some embodiments, the LNP comprises DSPC. In some embodiments, the LNP comprises DOPE. In some embodiments, the LNP comprises both DSPC and DOPE.

[0093] Additional exemplary phospholipids include, for example, dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleiminomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearate-phosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), and 1,2-ditransoleoyl-sn-glycerol-3-phosphate ethanolamine (transDOPE). In one embodiment, the lipid nanoparticles comprise 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC). In one embodiment, the lipid nanoparticles comprise phospholipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

[0094] In one embodiment, the lipid nanoparticles comprise phospholipids selected from phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), and phosphatidylglycerol (PG).

[0095] In addition, phospholipids that can form part of the LNP of the present invention also include those described in WO2017 / 112865, the entire contents of which are hereby incorporated by reference.

[0096] In one embodiment, the lipid nanoparticles comprise phospholipids, and the phospholipids are present at a concentration ranging from 2 to 50 mol%, 5 to 40 mol%, 5 to 15 mol%, 5 to 10 mol%, 7 to 13 mol%, or 9 to 11 mol%. In another embodiment, the lipid nanoparticles comprise phospholipids, and the phospholipids are present at a concentration of about 9.5, 10, or 10.5 mol%.

[0097] In one embodiment, the lipid nanoparticles comprise phospholipids, and the molar ratio of cationic lipids to phospholipids is in the range of about 0.5 to about 11 or about 1.3 to about 6. In another embodiment, the lipid nanoparticles comprise phospholipids, and the molar ratio of cationic lipids to phospholipids is in the range of about 4 to about 7, about 4.5 to about 6, or about 4.5 to 5.5.

[0098] ● Polymer-conjugated lipids

[0099] In some embodiments, the lipid component of the LNP used in this invention may include one or more polymer-conjugated lipids, such as PEGylated lipids (PEG lipids). Without being bound by theory, it is anticipated that the polymer-conjugated lipid component in the LNP may improve the colloidal stability of the nanoparticles and / or reduce protein uptake. Exemplary polymer-conjugated lipids that may be used in conjunction with this disclosure include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and combinations thereof. For example, PEG lipids may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, ceramide-PEG2000, or Chol-PEG2000.

[0100] In one embodiment, the lipid nanoparticles comprise PEGylated lipids. For example, in some embodiments, the lipid nanoparticles comprise polymer-conjugated lipids selected from: PEGylated diacylglycerols (PEG-DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEGylated phosphatidylethanolamine (PEG-PE), and PEGylated succinate diacylglycerols (PEG-S-DAG). (Such as 4-O-(2',3'-di(tetradecyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG)), PEGylated ceramide (PEG-cer), PEG dialkoxypropyl carbamates (such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecyloxy)propyl)carbamate and 2,3-di(tetradecyloxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate). In one embodiment, the lipid nanoparticles comprise DMG-PEG. In one embodiment, the lipid nanoparticles comprise PEG-PE.

[0101] In one embodiment, the lipid nanoparticles comprise polymer-conjugated lipids present at a concentration ranging from 1.0 to 2.5 mol percent. In one embodiment, the lipid nanoparticles comprise polymer-conjugated lipids present at a concentration of about 1.7 mol percent. In one embodiment, the lipid nanoparticles comprise polymer-conjugated lipids present at a concentration of about 1.5 mol percent. In one embodiment, the lipid nanoparticles comprise polymer-conjugated lipids, and the molar ratio of cationic lipids to polymer-conjugated lipids is in the range of about 20 to about 100. In one embodiment, the lipid nanoparticles comprise polymer-conjugated lipids, and the molar ratio of cationic lipids to polymer-conjugated lipids is in the range of about 25 to about 50. In one embodiment, the lipid nanoparticles comprise polymer-conjugated lipids, and the molar ratio of cationic lipids to polymer-conjugated lipids is in the range of about 25 to about 40.

[0102] In one embodiment, the total number of moles of the lipid nanoparticles relative to all lipids present in the nanoparticles comprises: (a) Cationic lipids of approximately 30 mol% to approximately 55 mol%; (b) Approximately 20 mol% to approximately 50 mol% of structural lipids; (c) Phospholipids of about 5 mol% to about 40 mol%; and (d) Polymer-conjugated lipids of about 0.5 mol% to about 5 mol%.

[0103] In one embodiment, the total number of moles of the lipid nanoparticles relative to all lipids present in the nanoparticles comprises: (a) Cationic lipids of approximately 30 mol% to approximately 55 mol%; (b) Steroids of about 20 mol% to about 50 mol%; (c) Phospholipids of about 5 mol% to about 40 mol%; and (d) Polymer-conjugated lipids of about 0.5 mol% to about 3 mol%.

[0104] In one embodiment, the lipid nanoparticles comprise cationic lipids, DSPC, cholesterol, and PEG-lipids.

[0105] In one embodiment, the lipid nanoparticles comprise, relative to the total molar percentage of all lipids in the nanoparticles, 30-55 mol% cationic lipids, 5-40 mol% DSPC, 20-50 mol% cholesterol, and 0.5-3 mol% PEG-lipids. Preferably, the cationic lipids are selected from the following compounds: ; ; ; ; ; ;as well as .

[0106] In some embodiments, the lyophilized LNP formulation comprises, relative to the total weight of the lyophilized LNP formulation, 0.1-10 wt.%, preferably 0.1-1 wt.%, more preferably 0.2-0.7 wt.% (e.g., 0.35 wt.%) of cationic lipids selected from compounds of formula O6-I: (06-I) in L 1 and L 2 It is -O(C═O)-; G 1 and G 2 Each is independently an unsubstituted C4-C8 alkylene group; G 3 It is a C3-C8 alkylene group; R 1 and R 2 Each is C independently 12 -C 22 alkyl; R 3 It is H or OH. Preferably, the cationic lipid is a compound of the following formula: .

[0107] In some embodiments, the lyophilized LNP formulation comprises, relative to the total weight of the lyophilized LNP formulation, 0.1-10 wt.%, preferably 0.1-1 wt.%, more preferably 0.2-0.7 wt.% (e.g., 0.32 wt.% or 0.33 wt.%) of cationic lipids selected from compounds of formula 05-I: (05-I) in l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M1 is -C(O)O-; R4 is -(CH2) n OH, and n is selected from 1, 2, 3, 4 or 5; M is -OC(O)-; Both R2 and R3 are C. 6-10 Alkyl; and R' is a straight-chain alkyl group; Preferably, the cationic lipid is a compound of formula B: (Formula B).

[0108] In some embodiments, the lyophilized LNP formulation comprises, relative to the total weight of the lyophilized LNP formulation, 0.1-10 wt.%, preferably 0.1-1 wt.%, more preferably 0.2-0.7 wt.% (e.g., 0.29 wt.%) of the following cationic lipids: .

[0109] In some embodiments, the lyophilized LNP formulation comprises, relative to the total weight of the lyophilized LNP formulation, 0.1-10 wt.%, preferably 0.1-1 wt.%, more preferably 0.2-0.7 wt.% (e.g. 0.25 wt.%, 0.26 wt.%, 0.38 wt.%, or 0.39 wt.%) of cationic lipids selected from compounds of formula (01-10). (01-IO), Where y and z are each independent integers from 4 to 6. s is an integer from 2 to 4. t is an integer from 1 to 3, and R 1 and R 2 Each is C independently 12 -C 22 alkyl; R 4 It is a C3-C8 cycloalkyl group; R 6 It is hydrogen or hydroxyl. Preferably, the cationic lipid is a compound of the following formula: .

[0110] In some embodiments, the lyophilized LNP formulation comprises, relative to the total weight of the lyophilized LNP formulation, 0.1-10 wt.%, preferably 0.1-1 wt.%, more preferably 0.2-0.7 wt.% (e.g., 0.40 wt.%) of cationic lipids selected from compounds of formula (03-I): (03-I), in G 1 and G 2 Each is independently a C3-C8 alkylene group; Each L 1 Independently is -OC(=O)R 1 ; Each L 2 Independently is -C(=O)OR 2 ; R 1 Independently, it is C6-C 10 alkyl; R 2 C is independent 12 -C 22 alkyl; G 3 It is C2-C 12 Alkylene; R 3 It is a C3-C8 cycloalkyl group; R 4 It is a C1-C4 hydroxyalkyl group; n is 2; m is 1, Preferably, the cationic lipid is a compound of the following formula: .

[0111] In some embodiments, the lyophilized LNP formulation comprises, relative to the total weight of the lyophilized LNP formulation, 0.1-10 wt.%, preferably 0.1-1 wt.%, more preferably 0.2-0.7 wt.% (e.g., 0.33 wt.% or 0.35 wt.%) of cationic lipids selected from compounds of formula (07-III): (07-III), in R 1 and R 2 Each is independently C6-C 22 alkyl; R 0 It is a C3-C8 cycloalkyl group; G 3 It is a C2-C6 alkylene group; G 4 It is a C2-C6 alkylene group; R 3 Yes - OR 6 ; R 6 It is hydrogen; Preferably, the cationic lipid is a compound of the following formula: or .

[0112] cryoprotectant combination

[0113] The cryoprotectant combination in the lyophilized LNP formulation contains sucrose and nonpolar amino acids.

[0114] In some embodiments, the lyophilized LNP formulation comprises 78-99 wt.% (e.g., 80-98.5 wt.%, 82-98 wt.%, 84-97.5 wt.%, 85-97.5 wt.%, 87-97.5 wt.%, 89-97.5 wt.%, 91-97.5 wt.%, or 93-97.5 wt.%) of sucrose relative to the total weight of the lyophilized LNP formulation.

[0115] In some embodiments, the lyophilized LNP formulation comprises, by weight relative to the total weight of the lyophilized LNP formulation, 93.39 wt.%, 93.85 wt.%, 94.20 wt.%, 94.22 wt.%, 94.25 wt.%, 94.27 wt.%, 94.3 wt.%, 94.36 wt.%, 94.41 wt.%, 94.44 wt.%, 94.55 wt.%, 94.62 wt.%, 94.64 wt.%, 94.89 wt.%, 95.14 wt.%, 95.40 wt.%, 95.96 wt.%, 96.48 wt.%, 96.58 wt.%, 96.78 wt.%, 97.04 wt.%, 97.08 wt.%, 97.31 wt.%, 97.58 wt.%, 97.68 wt.% Sucrose at wt.%, 97.84 wt.%, 98.16 wt.%, or 98.73 wt.%.

[0116] In some embodiments, the lyophilized LNP formulation contains 0.2-9 wt.% (e.g., 0.4-9 wt.%, 0.6-9 wt.%, 0.8-8 wt.%, 1-7 wt.%, 1.5-6 wt.%, 1.8-5 wt.%, or 2-4.8 wt.%) of nonpolar amino acids relative to the total weight of the lyophilized LNP formulation.

[0117] nonpolar amino acids

[0118] The nonpolar amino acids used in this invention are selected from... (Alanine, Ala) (Isoleucine, Ile) (Leucine, Leu) (Valine) (proline, Pro) (glycine, Gly) and their combinations.

[0119] In some embodiments, the lyophilized LNP formulation contains 0.4-6 wt.%, preferably 1-4 wt.%, or 1-3 wt.% alanine relative to the total weight of the lyophilized LNP formulation. In some embodiments, the lyophilized LNP formulation contains 1.70 wt.%, 1.74 wt.%, or 3.42 wt.% alanine relative to the total weight of the lyophilized LNP formulation.

[0120] In some embodiments, the lyophilized LNP formulation contains 0.6-9 wt.%, preferably 1-5 wt.%, of isoleucine relative to the total weight of the lyophilized LNP formulation. In some embodiments, the lyophilized LNP formulation contains 2.48 wt.%, or 2.48 wt.%, of isoleucine relative to the total weight of the lyophilized LNP formulation.

[0121] In some embodiments, the lyophilized LNP formulation contains 0.6-9 wt.%, preferably 1-5 wt.%, of leucine relative to the total weight of the lyophilized LNP formulation. In some embodiments, the lyophilized LNP formulation contains 2.48 wt.%, 2.54 wt.%, 4.95 wt.%, or 4.96 wt.%, of leucine relative to the total weight of the lyophilized LNP formulation.

[0122] In some embodiments, the lyophilized LNP formulation contains 0.2-9 wt.% (e.g., 0.5-8 wt.% or 1.5-6 wt.%) of valine relative to the total weight of the lyophilized LNP formulation. In some embodiments, the lyophilized LNP formulation contains 0.38 wt.%, 0.58 wt.%, 0.76 wt.%, 1.15 wt.%, 1.51 wt.%, 2.22 wt.%, 2.24 wt.%, 2.27 wt.%, 2.97 wt.%, 4.38 wt.%, 4.40 wt.%, 4.42 wt.%, 4.43 wt.%, or 4.45 wt.% of valine relative to the total weight of the lyophilized LNP formulation.

[0123] In some embodiments, the lyophilized LNP formulation contains 0.5-8 wt.%, preferably 1-5 wt.%, of proline relative to the total weight of the lyophilized LNP formulation. In some embodiments, the lyophilized LNP formulation contains 2.24 wt.%, of proline relative to the total weight of the lyophilized LNP formulation.

[0124] In some embodiments, the lyophilized LNP formulation contains 0.3-5 wt.%, preferably 0.8-3 wt.%, of glycine relative to the total weight of the lyophilized LNP formulation. In some embodiments, the lyophilized LNP formulation contains 1.47 wt.% or 2.90 wt.% of glycine relative to the total weight of the lyophilized LNP formulation.

[0125] In some embodiments, the lyophilized LNP formulation comprises 1.5-4 wt.% valine and 1-3 wt.% alanine relative to the total weight of the lyophilized LNP formulation. In some embodiments, the lyophilized LNP formulation comprises 2.24 wt.% valine and 1.70 wt.% alanine relative to the total weight of the lyophilized LNP formulation.

[0126] In some embodiments, the lyophilized LNP formulation comprises 1.5-4 wt.% valine and 1.5-4 wt.% leucine relative to the total weight of the lyophilized LNP formulation. In some embodiments, the lyophilized LNP formulation comprises 2.22 wt.% valine and 2.48 wt.% leucine relative to the total weight of the lyophilized LNP formulation.

[0127] In some embodiments, the lyophilized LNP formulation comprises 1.5-4 wt.% valine and 1.5-4 wt.% isoleucine relative to the total weight of the lyophilized LNP formulation. In some embodiments, the lyophilized LNP formulation comprises 2.22 wt.% valine and 2.48 wt.% isoleucine relative to the total weight of the lyophilized LNP formulation.

[0128] In some embodiments, the lyophilized LNP formulation comprises 95-98.5 wt.% sucrose and 1-4 wt.% alanine relative to the total weight of the lyophilized LNP formulation.

[0129] In some embodiments, the lyophilized LNP formulation comprises 95-98.5 wt.% sucrose and 1-3 wt.% alanine relative to the total weight of the lyophilized LNP formulation.

[0130] In some embodiments, the lyophilized LNP formulation comprises 93-98 wt.% sucrose and 1.5-4 wt.% isoleucine relative to the total weight of the lyophilized LNP formulation.

[0131] In some embodiments, the lyophilized LNP formulation comprises 93-98 wt.% sucrose and 1.5-4 wt.% leucine relative to the total weight of the lyophilized LNP formulation.

[0132] In some embodiments, the lyophilized LNP formulation comprises 93-99 wt.% sucrose and 0.3-6 wt.% valine relative to the total weight of the lyophilized LNP formulation.

[0133] In some embodiments, the lyophilized LNP formulation comprises 93-99 wt.% sucrose and 0.4-6 wt.% valine relative to the total weight of the lyophilized LNP formulation.

[0134] In some embodiments, the lyophilized LNP formulation comprises 96-98 wt.% sucrose and 1.5-3 wt.% proline relative to the total weight of the lyophilized LNP formulation.

[0135] In some embodiments, the lyophilized LNP formulation comprises 97-98.5 wt.% sucrose and 1-2 wt.% glycine relative to the total weight of the lyophilized LNP formulation.

[0136] In some embodiments, the lyophilized LNP formulation comprises 94-96 wt.% sucrose, 1.5-3 wt.% valine, and 1-2.5 wt.% alanine relative to the total weight of the lyophilized LNP formulation.

[0137] In some embodiments, the lyophilized LNP formulation comprises 93-95 wt.% sucrose, 1.5-3 wt.% valine, and 1.5-3 wt.% leucine relative to the total weight of the lyophilized LNP formulation.

[0138] In some embodiments, the lyophilized LNP formulation comprises 93-95 wt.% sucrose, 1.5-3 wt.% valine, and 1.5-3 wt.% isoleucine relative to the total weight of the lyophilized LNP formulation.

[0139] Salt or Tris

[0140] The lyophilized LNP formulation may also contain salt. In some embodiments, the lyophilized LNP formulation contains 0.01-3 wt.% (e.g., 0.1-3 wt.%, 0.15-2 wt.%, 0.2-1.5 wt.%, or 0.2-1.3 wt.%) of salt relative to the total weight of the lyophilized LNP formulation.

[0141] In some embodiments, the lyophilized LNP formulation contains 0.1-3 wt.% (e.g., 0.15-2 wt.%, 0.2-1.5 wt.%, or 0.2-1.3 wt.%) of salt relative to the total weight of the lyophilized LNP formulation.

[0142] Representative salts that can be used in this invention include, but are not limited to, tris-HCl, KCl, NaCl, K2HPO4, KH2PO4, sodium citrate, sodium acetate, and combinations thereof.

[0143] In some embodiments, the salt is selected from tris-HCl, KCl, NaCl, sodium citrate, sodium acetate, and combinations thereof.

[0144] In some embodiments, the salt is selected from tris-HCl, KCl, NaCl, K2HPO4, KH2PO4, sodium citrate, sodium acetate, and combinations thereof.

[0145] In some embodiments, the lyophilized LNP formulation contains 0.1-3 wt.% (e.g., 0.28 wt.%, 0.55 wt.%, 0.74 wt.%, 0.75 wt.%, 0.76 wt.%, or 1.10 wt.%) of NaCl relative to the total weight of the lyophilized LNP formulation.

[0146] In some embodiments, the lyophilized LNP formulation contains 0.1-3 wt.% (e.g., 0.35 wt.% or 0.70 wt.%) of KCl relative to the total weight of the lyophilized LNP formulation.

[0147] In some embodiments, the salt is selected from KCl, NaCl, and combinations thereof.

[0148] In some embodiments, the salt is selected from KCl, NaCl, K2HPO4, KH2PO4, and combinations thereof.

[0149] In some embodiments, the lyophilized LNP formulation comprises, by weight relative to the total weight of the lyophilized LNP formulation, 0.03 wt.% K₂HPO₄, 0.66 wt.% KH₂PO₄, and 0.74 wt.% NaCl.

[0150] In some embodiments, the lyophilized LNP formulation comprises, by weight relative to the total weight of the lyophilized LNP formulation, 0.03 wt.% K₂HPO₄, 0.67 wt.% KH₂PO₄, and 0.75 wt.% NaCl.

[0151] In some embodiments, the lyophilized LNP formulation comprises, by weight relative to the total weight of the lyophilized LNP formulation, 0.03 wt.% K₂HPO₄, 0.68 wt.% KH₂PO₄, and 0.76 wt.% NaCl.

[0152] The lyophilized LNP formulation may also contain Tris.

[0153] As used in this article, “Tris” refers to tris(hydroxymethyl)aminomethane compounds.

[0154] In some embodiments, the lyophilized LNP formulation contains 0.1-3 wt.% (e.g., 0.9-2 wt.%, 1.2-1.8 wt.%, or 1.4-1.7 wt.%) of Tris relative to the total weight of the lyophilized LNP formulation. In some embodiments, the lyophilized LNP formulation contains 1.58 wt.% of Tris relative to the total weight of the lyophilized LNP formulation.

[0155] In some embodiments, the nonpolar amino acid is combined with a salt or Tris to form a combinatorial system.

[0156] In one embodiment, the lyophilized LNP formulation contains 97.58 wt.% sucrose and 1.74 wt.% alanine by weight relative to the total weight of the lyophilized formulation.

[0157] In one embodiment, the lyophilized LNP formulation comprises 96.78 wt.% sucrose and 2.54 wt.% isoleucine by weight relative to the total weight of the lyophilized formulation.

[0158] In one embodiment, the lyophilized LNP formulation contains 96.78 wt.% sucrose and 2.54 wt.% leucine by weight relative to the total weight of the lyophilized formulation.

[0159] In one embodiment, the lyophilized LNP formulation comprises 97.04 wt.% sucrose and 2.27 wt.% valine by weight relative to the total weight of the lyophilized formulation.

[0160] In one embodiment, the lyophilized LNP formulation contains 97.08 wt.% sucrose and 2.24 wt.% proline by weight relative to the total weight of the lyophilized formulation.

[0161] In one embodiment, the lyophilized LNP formulation contains 97.84 wt.% sucrose and 1.47 wt.% glycine by weight relative to the total weight of the lyophilized formulation.

[0162] In one embodiment, the lyophilized LNP formulation comprises 98.73 wt.% sucrose and 0.58 wt.% valine by weight relative to the total weight of the lyophilized formulation.

[0163] In one embodiment, the lyophilized LNP formulation comprises 98.16 wt.% sucrose and 1.15 wt.% valine by weight relative to the total weight of the lyophilized formulation.

[0164] In one embodiment, the lyophilized LNP formulation contains 94.89 wt.% sucrose and 4.45 wt.% valine by weight relative to the total weight of the lyophilized formulation.

[0165] In one embodiment, the lyophilized LNP formulation comprises 94.55 wt.% sucrose, 4.43 wt.% valine, and 0.35 wt.% KCl by weight relative to the total weight of the lyophilized formulation.

[0166] In one embodiment, the lyophilized LNP formulation comprises 94.22 wt.% sucrose, 4.42 wt.% valine, and 0.70 wt.% KCl by weight relative to the total weight of the lyophilized formulation.

[0167] In one embodiment, the lyophilized LNP formulation comprises 94.62 wt.% sucrose, 4.43 wt.% valine, and 0.28 wt.% NaCl by weight relative to the total weight of the lyophilized formulation.

[0168] In one embodiment, the lyophilized LNP formulation comprises 94.36 wt.% sucrose, 4.42 wt.% valine, and 0.55 wt.% NaCl by weight relative to the total weight of the lyophilized formulation.

[0169] In one embodiment, the lyophilized LNP formulation comprises 93.85 wt.% sucrose, 4.40 wt.% valine, and 1.10 wt.% NaCl by weight relative to the total weight of the lyophilized formulation.

[0170] In one embodiment, the lyophilized LNP formulation comprises 94.25 wt.% sucrose, 4.42 wt.% valine, and 0.70 wt.% KCl by weight relative to the total weight of the lyophilized formulation.

[0171] In one embodiment, the lyophilized LNP formulation comprises 94.27 wt.% sucrose, 4.42 wt.% valine, and 0.70 wt.% KCl by weight relative to the total weight of the lyophilized formulation.

[0172] In one embodiment, the lyophilized LNP formulation comprises 94.3 wt.% sucrose, 4.42 wt.% valine, and 0.70 wt.% KCl by weight relative to the total weight of the lyophilized formulation.

[0173] In one embodiment, the lyophilized LNP formulation comprises 94.20 wt.% sucrose, 4.4 wt.% valine, and 0.70 wt.% KCl by weight relative to the total weight of the lyophilized formulation.

[0174] In one embodiment, the lyophilized LNP formulation comprises 95.40 wt.% sucrose, 2.24 wt.% valine, and 1.70 wt.% alanine by weight relative to the total weight of the lyophilized formulation.

[0175] In one embodiment, the lyophilized LNP formulation comprises 94.64 wt.% sucrose, 2.22 wt.% valine, and 2.48 wt.% leucine by weight relative to the total weight of the lyophilized formulation.

[0176] In one embodiment, the lyophilized LNP formulation comprises 94.64 wt.% sucrose, 2.22 wt.% valine, and 2.48 wt.% isoleucine by weight relative to the total weight of the lyophilized formulation.

[0177] In one embodiment, the lyophilized LNP formulation contains 95.96 wt.% sucrose and 3.42 wt.% alanine by weight relative to the total weight of the lyophilized formulation.

[0178] In one embodiment, the lyophilized LNP formulation comprises 96.48 wt.% sucrose and 2.897 wt.% glycine by weight relative to the total weight of the lyophilized formulation.

[0179] In one embodiment, the lyophilized LNP formulation comprises 94.41 wt.% sucrose and 4.95 wt.% leucine by weight relative to the total weight of the lyophilized formulation.

[0180] In one embodiment, the lyophilized LNP formulation comprises 96.48 wt.% sucrose and 2.90 wt.% glycine by weight relative to the total weight of the lyophilized formulation.

[0181] In one embodiment, the lyophilized LNP formulation comprises 94.4 wt.% sucrose and 4.96 wt.% leucine by weight relative to the total weight of the lyophilized formulation.

[0182] In one embodiment, the lyophilized LNP formulation comprises 93.39 wt.% sucrose, 4.38 wt.% valine, and 1.58 wt.% Tris by weight relative to the total weight of the lyophilized formulation.

[0183] In one embodiment, the lyophilized LNP formulation comprises, by weight relative to the total weight of the lyophilized formulation, 95.14 wt.% sucrose, 2.97 wt.% valine, 0.03 wt.% K₂HPO₄, 0.66 wt.% KH₂PO₄ and 0.74 wt.% NaCl.

[0184] In one embodiment, the lyophilized LNP formulation comprises, by weight relative to the total weight of the lyophilized formulation, 96.58 wt.% sucrose, 1.51 wt.% valine, 0.03 wt.% K₂HPO₄, 0.67 wt.% KH₂PO₄ and 0.75 wt.% NaCl.

[0185] In one embodiment, the lyophilized LNP formulation comprises, by weight relative to the total weight of the lyophilized formulation, 97.31 wt.% sucrose, 0.76 wt.% valine, 0.03 wt.% K₂HPO₄, 0.68 wt.% KH₂PO₄ and 0.76 wt.% NaCl.

[0186] In one embodiment, the lyophilized LNP formulation comprises, by weight relative to the total weight of the lyophilized formulation, 97.68 wt.% sucrose, 0.38 wt.% valine, 0.03 wt.% K₂HPO₄, 0.68 wt.% KH₂PO₄ and 0.76 wt.% NaCl.

[0187] Therapeutic agents and / or preventative agents

[0188] The lyophilized LNP formulation of the present invention may also contain therapeutic agents and / or preventive agents in the lipid nanoparticles.

[0189] For example, the weight ratio of lipids to therapeutic and / or preventative agents in the lyophilized LNP formulation is from about 10:1 to about 60:1, or from about 2:1 to about 30:1, for example, or from 20:1 to about 30:1.

[0190] The lyophilized formulation may be stored and diluted before or during administration. For example, the formulation for administration has a therapeutic and / or prophylactic concentration of about 0.01 mg / mL to about 2 mg / mL (e.g., about 0.01 mg / mL, about 0.025 mg / mL, about 0.05 mg / mL, about 0.075 mg / mL, about 0.1 mg / mL, about 0.3 mg / mL, about 0.5 mg / mL, about 1 mg / mL, about 1.5 mg / mL, about 2 mg / mL, about 0.025-1 mg / mL, or about 0.05-1 mg / mL, or about 0.5-1 mg / mL).

[0191] In certain embodiments, the therapeutic agent comprises a vaccine composition as described herein (e.g., a gene vaccine). In some embodiments, the therapeutic agent comprises a compound capable of inducing immunity against one or more target diseases or illnesses. In some embodiments, the target disease is associated with or caused by a pathogen infection, such as coronaviruses (e.g., 2019-nCoV), influenza, measles, human papillomavirus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis, and tuberculosis. In some embodiments, the therapeutic agent comprises a nucleic acid sequence (e.g., mRNA) encoding a pathogenic protein specific to the pathogen or an antigenic fragment or epitope thereof. Upon administration to a vaccinated subject, the vaccine allows expression of the encoded pathogenic protein (or an antigenic fragment or epitope thereof), thereby inducing immunity against the pathogen in the subject.

[0192] In some embodiments, the target disease is associated with or caused by the proliferation of cells, such as cancer. In some embodiments, the therapeutic agent comprises a nucleic acid sequence (e.g., mRNA) encoding a tumor-associated antigen (TAA) specific to cancer or an antigenic fragment or epitope thereof. After administration to a vaccinated subject, the vaccine allows expression of the encoded TAA (or an antigenic fragment or epitope thereof), thereby inducing immunity against TAA-expressing proliferation cells in the subject.

[0193] The therapeutic and / or preventive agents may be, for example, nucleic acids, such as ribonucleic acid (RNA), such as mRNA.

[0194] For example, the mRNA is at least 30 nucleotides long (e.g., at least 300 nucleotides long).

[0195] In some embodiments, the mRNA is selected from rabies mRNA, respiratory syncytial virus mRNA, human erythropoietin mRNA, varicella-zoster virus mRNA, all of which are optionally modified with 1-N-methyl-pseuuridine, and combinations thereof.

[0196] In some embodiments, the mRNA is selected from rabies mRNA, respiratory syncytial virus mRNA modified with 1-N-methyl-pseudouridine, rabies mRNA modified with 1-N-methyl-pseudouridine, human erythropoietin mRNA, varicella-zoster virus mRNA modified with 1-N-methyl-pseudouridine, and combinations thereof.

[0197] In some embodiments, the mRNA is selected from rabies mRNA, respiratory syncytial virus mRNA modified with 1-N-methyl-pseuuridine, rabies mRNA modified with 1-N-methyl-pseuuridine, human erythropoietin mRNA, and combinations thereof.

[0198] The lyophilized LNP formulations of the present invention, comprising therapeutic and / or preventive agents, have relatively high encapsulation efficiency, for example, at least 85%, which allows the in vivo bioactivity of the therapeutic and / or preventive agents to be maintained to a great extent.

[0199] As used herein, “encapsulation efficiency” means the weight percentage of a therapeutic and / or preventative agent encapsulated in lipids relative to the total weight of the therapeutic and / or preventative agent.

[0200] In some embodiments, the encapsulation efficiency of the lyophilized LNP formulation is at least 87%, at least 90%, at least 92%, or at least 94%.

[0201] Preparation of lyophilized LNP formulations

[0202] The lyophilized LNP formulation can be prepared using methods known in the art.

[0203] Taking a lyophilized LNP formulation containing mRNA as an example, the lipids are dissolved in a solvent (such as ethanol). The mRNA is diluted in 10 to 50 mM citrate buffer (pH = 3-5). The lipid solution and the aqueous mRNA solution are mixed at a volume ratio of 1:3 using a microfluidic device (total flow rate in the range of 9-30 mL / min) to prepare LNPs at a total lipid:mRNA weight ratio of approximately 10:1 to 30:1. The solvent (such as ethanol) is then removed and replaced with PBS by dialysis. After dialysis, the LNPs are exchanged with buffer in a cryoprotectant buffer optionally containing salt or Tris to obtain a liquid LNP formulation, which is then filtered.

[0204] Lyophilization can be carried out in the glass chamber of a Pilot freeze dryer (Christ Epsilon 2-10D LSCplus freeze dryer). LNP is frozen at -40 to -60°C for 3 hours, followed by a drying cycle at -25 to -35°C / 20 to 100 mTorr for 48 to 60 hours. Then, during a second drying cycle, the LNP is heated to 5 to 25°C / 20 to 100 mTorr and held for 12 to 24 hours. The vials are then sealed under vacuum with stoppers, and additionally capped with aluminum caps, and transferred to 2 to 8°C for long-term storage.

[0205] Liquid LNP formulation

[0206] In a second aspect, the present invention provides a liquid formulation of lipid nanoparticles (also referred to as a "liquid LNP formulation"), comprising, by weight relative to the total volume of the liquid formulation: (A) Lipid nanoparticles containing cationic lipids, ranging from 0.001% to 0.2% w / v; and (B) A cryoprotectant combination, the cryoprotectant combination comprising (i) 0.001-40% w / v sucrose, and (ii) 0.01-10% w / v nonpolar amino acids.

[0207] The liquid LNP formulation can be used to prepare the lyophilized LNP formulation of the first aspect of the present invention.

[0208] The particle size of the LNP in the liquid LNP formulation is from about 50 nm to about 140 nm, preferably from about 60 nm to about 120 nm.

[0209] The definitions of cationic lipids and nonpolar amino acids are the same as those for lyophilized LNP formulations mentioned above.

[0210] In some embodiments, the liquid LNP formulation comprises 0.001-0.2% w / v (e.g., 0.001-0.1% w / v, 0.1-0.2% w / v, 0.1-0.15% w / v, 0.001-0.05% w / v, 0.001-0.03% w / v, 0.001-0.01% w / v, 0.01-0.1% w / v, or 0.05-0.1% w / v) of lipid nanoparticles by weight relative to the total volume of the liquid LNP formulation.

[0211] The lipid nanoparticles may also include one or more of the following: structural lipids, phospholipids, and polymer-conjugated lipids.

[0212] The definitions of structural lipids, phospholipids, and polymer-conjugated lipids are the same as those for lyophilized LNP formulations.

[0213] If any of the structural lipids, phospholipids, and polymer-conjugated lipids are present, their molar ratio to cationic lipids is the same as that for lyophilized LNP formulations.

[0214] In some embodiments, the liquid LNP formulation comprises, by weight relative to the total volume of the liquid LNP formulation, 0.01-1% w / v, preferably 0.01-0.1% w / v, more preferably 0.02-0.07% w / v (e.g., 0.038% w / v) of cationic lipids selected from compounds of formula O6-I: (06-I) in L 1 and L 2 It is -O(C═O)-; G 1 and G 2 Each is independently an unsubstituted C4-C8 alkylene group; G 3 It is a C3-C8 alkylene group; R 1 and R 2 Each is C independently 12 -C 22 alkyl; R 3 It is H or OH. Preferably, the cationic lipid is a compound of the following formula: .

[0215] In some embodiments, the liquid LNP formulation comprises, by weight relative to the total volume of the liquid LNP formulation, 0.01-1% w / v, preferably 0.01-0.1% w / v, more preferably 0.02-0.07% w / v (e.g., 0.034% w / v or 0.035% w / v) of cationic lipids selected from compounds of formula 05-I: (05-I) in l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M1 is -C(O)O-; R4 is -(CH2) n OH, and n is selected from 1, 2, 3, 4 or 5; M is -OC(O)-; Both R2 and R3 are C. 6-10 Alkyl; and R' is a straight-chain alkyl group; Preferably, the cationic lipid is a compound of formula B: (Formula B).

[0216] In some embodiments, the liquid LNP formulation comprises, by weight relative to the total volume of the liquid LNP formulation, 0.01-1% w / v, preferably 0.01-0.1% w / v, more preferably 0.02-0.07% w / v (e.g., 0.032% w / v) of the following cationic lipid: .

[0217] In some embodiments, the liquid LNP formulation comprises, by weight relative to the total volume of the liquid LNP formulation, 0.01-1% w / v, preferably 0.01-0.1% w / v, more preferably 0.02-0.07% w / v (e.g., 0.04% w / v or 0.041% w / v) of cationic lipids selected from compounds of formula (01-10): (01-IO), Where y and z are each independent integers from 4 to 6. s is an integer from 2 to 4. t is an integer from 1 to 3, and R 1 and R 2 Each is C independently 12 -C 22 alkyl; R 4It is a C3-C8 cycloalkyl group; R 6 It is hydrogen or hydroxyl. Preferably, the cationic lipid is a compound of the following formula: .

[0218] In some embodiments, the liquid LNP formulation comprises, by weight relative to the total volume of the liquid LNP formulation, 0.01-1% w / v, preferably 0.01-0.1% w / v, more preferably 0.02-0.07% w / v (e.g., 0.043% w / v) of cationic lipids selected from compounds of formula (03-I): (03-I), in G 1 and G 2 Each is independently a C3-C8 alkylene group; Each L 1 Independently is -OC(=O)R 1 ; Each L 2 Independently is -C(=O)OR 2 ; R 1 Independently, it is C6-C 10 alkyl; R 2 C is independent 12 -C 22 alkyl; G 3 It is C2-C 12 Alkylene; R 3 It is a C3-C8 cycloalkyl group; R 4 It is a C1-C4 hydroxyalkyl group; n is 2; m is 1, Preferably, the cationic lipid is a compound of the following formula: .

[0219] In some embodiments, the liquid LNP formulation comprises, by weight relative to the total volume of the liquid LNP formulation, 0.01-1% w / v, preferably 0.01-0.1% w / v, more preferably 0.02-0.07% w / v (e.g., 0.034% w / v or 0.037 w / v) of cationic lipids selected from compounds of formula (07-III): (07-III), in R 1 and R 2 Each is independently C6-C 22 alkyl; R 0 It is a C3-C8 cycloalkyl group; G 3 It is a C2-C6 alkylene group; G 4 It is a C2-C6 alkylene group; R 3 Yes - OR 6 ; R 6 It is hydrogen; Preferably, the cationic lipid is a compound of the following formula: or .

[0220] In some embodiments, the liquid LNP formulation contains 0.01-35% w / v (e.g., 0.1-30% w / v, 1-30% w / v, 2-25% w / v, 3-20% w / v, 4-15% w / v, 5-14% w / v, 6-13% w / v, 7-12% w / v, 8-11% w / v, 9-10% w / v, or 9.3-10% w / v) of sucrose by weight relative to the total volume of the liquid LNP formulation.

[0221] In some embodiments, the liquid LNP formulation contains 10% w / v sucrose by weight relative to the total volume of the liquid LNP formulation.

[0222] In some embodiments, the liquid LNP formulation contains 0.04-1% w / v (e.g., 0.06-0.9% w / v, 0.08-0.8% w / v, 0.1-0.7% w / v, 0.15-0.6% w / v, 0.18-0.5% w / v, or 0.2-0.5% w / v) of nonpolar amino acids by weight relative to the total volume of the liquid LNP formulation.

[0223] In some embodiments, the liquid LNP formulation contains 0.04-0.6% w / v, preferably 0.1-0.3% w / v, of alanine relative to the total volume of the liquid LNP formulation. In some embodiments, the liquid LNP formulation contains 0.18% w / v, 0.23% w / v, or 0.36% w / v of alanine relative to the total volume of the liquid LNP formulation.

[0224] In some embodiments, the liquid LNP formulation contains 0.06-0.9% w / v, preferably 0.1-0.5% w / v, of isoleucine relative to the total volume of the liquid LNP formulation. In some embodiments, the liquid LNP formulation contains 0.26% w / v of isoleucine relative to the total volume of the liquid LNP formulation.

[0225] In some embodiments, the liquid LNP formulation contains 0.06-0.9% w / v, preferably 0.1-0.5% w / v, of leucine based on the total volume of the liquid LNP formulation. In some embodiments, the liquid LNP formulation contains 0.26% w / v or 0.52% w / v of leucine based on the total volume of the liquid LNP formulation.

[0226] In some embodiments, the liquid LNP formulation contains 0.05-0.8% w / v, preferably 0.15-0.6% w / v, of valine relative to the total volume of the liquid LNP formulation. In some embodiments, the liquid LNP formulation contains 0.06% w / v, 0.12% w / v, 0.23% or 0.47% w / v of valine relative to the total volume of the liquid LNP formulation.

[0227] In some embodiments, the liquid LNP formulation contains 0.05-0.8% w / v, preferably 0.1-0.5% w / v, proline based on the total volume of the liquid LNP formulation. In some embodiments, the liquid LNP formulation contains 0.23% w / v, proline based on the total volume of the liquid LNP formulation.

[0228] In some embodiments, the liquid LNP formulation contains 0.03-0.5% w / v, preferably 0.08-0.3% w / v, of glycine relative to the total volume of the liquid LNP formulation. In some embodiments, the liquid LNP formulation contains 0.15% w / v or 0.30% w / v of glycine relative to the total volume of the liquid LNP formulation.

[0229] In some embodiments, the liquid LNP formulation contains 0.15-0.4% w / v valine and 0.1-0.3% w / v alanine by weight relative to the total volume of the liquid LNP formulation. In some embodiments, the liquid LNP formulation contains 0.23% w / v valine and 0.18% w / v alanine by weight relative to the total volume of the liquid LNP formulation.

[0230] In some embodiments, the liquid LNP formulation contains 0.15-0.4% w / v valine and 0.15-0.4% w / v leucine by weight relative to the total volume of the liquid LNP formulation. In some embodiments, the liquid LNP formulation contains 0.23% w / v valine and 0.26% w / v leucine by weight relative to the total volume of the liquid LNP formulation.

[0231] In some embodiments, the liquid LNP formulation comprises 0.15-0.4% w / v valine and 0.15-0.4% w / v isoleucine relative to the total volume of the liquid LNP formulation. In some embodiments, the liquid LNP formulation comprises 0.23% w / v valine and 0.26% w / v isoleucine relative to the total volume of the liquid LNP formulation.

[0232] In some embodiments, the liquid LNP formulation contains 9.5-10% w / v sucrose and 0.1-0.4% w / v alanine by weight relative to the total volume of the liquid LNP formulation.

[0233] In some embodiments, the liquid LNP formulation contains 9.5-10% w / v sucrose and 0.1-0.3% w / v alanine by weight relative to the total volume of the liquid LNP formulation.

[0234] In some embodiments, the liquid LNP formulation contains 9.3-10% w / v sucrose and 0.15-0.4% w / v isoleucine by weight relative to the total volume of the liquid LNP formulation.

[0235] In some embodiments, the liquid LNP formulation comprises 9.3-10% w / v sucrose and 0.15-0.4% w / v leucine relative to the total weight of the liquid LNP formulation.

[0236] In some embodiments, the liquid LNP formulation contains 9.3-10% w / v sucrose and 0.03-0.6 wt.% valine by weight relative to the total volume of the liquid LNP formulation.

[0237] In some embodiments, the liquid LNP formulation contains 9.3-10% w / v sucrose and 0.04-0.6% w / v valine by weight relative to the total volume of the liquid LNP formulation.

[0238] In some embodiments, the liquid LNP formulation contains 9.6-10% w / v sucrose and 0.15-0.3% w / v proline by weight relative to the total volume of the liquid LNP formulation.

[0239] In some embodiments, the liquid LNP formulation contains 9.7-10% w / v glycine and 0.1-0.2% w / v glycine by weight relative to the total volume of the liquid LNP formulation.

[0240] In some embodiments, the liquid LNP formulation comprises, by weight relative to the total volume of the liquid LNP formulation, 9.4-10% w / v sucrose, 0.15-0.3% w / v valine and 0.1-0.25% w / v alanine.

[0241] In some embodiments, the liquid LNP formulation comprises, by weight relative to the total volume of the liquid LNP formulation, 9.3-10% w / v sucrose, 0.15-0.3% w / v valine and 0.15-0.3% w / v leucine.

[0242] In some embodiments, the liquid LNP formulation comprises, by weight relative to the total volume of the liquid LNP formulation, 9.3-10% w / v sucrose, 0.15-0.3% w / v valine and 0.15-0.3% w / v isoleucine.

[0243] The liquid LNP formulation may also contain salt.

[0244] The salt is the same as defined above for the lyophilized LNP formulation.

[0245] In some embodiments, the liquid LNP formulation contains 0.001-0.3% w / v (e.g., 0.01-0.3% w / v, 0.015-0.2% w / v, 0.02-0.15% w / v, or 0.02-0.13% w / v) of salt by weight relative to the total volume of the liquid LNP formulation.

[0246] In some embodiments, the liquid LNP formulation contains 0.01-0.3% w / v (e.g., 0.015-0.2% w / v, 0.02-0.15% w / v, or 0.02-0.13% w / v) of salt by weight relative to the total volume of the liquid LNP formulation.

[0247] In some embodiments, the liquid LNP formulation contains 0.01-0.3% w / v (e.g., 0.03% w / v, 0.06% w / v, 0.117% w / v, or 0.12% w / v) of NaCl by weight relative to the total volume of the liquid LNP formulation.

[0248] In some embodiments, the liquid LNP formulation contains 0.01-0.3% w / v (e.g., 0.04% w / v or 0.07% w / v) KCl by weight relative to the total volume of the liquid LNP formulation.

[0249] In some embodiments, the liquid LNP formulation comprises, by weight relative to the total volume of the liquid LNP formulation, 0.005% w / v K2HPO4, 0.10% w / v KH2PO4 and 0.117% w / v NaCl.

[0250] The liquid LNP formulation may also contain Tris.

[0251] In some embodiments, the liquid LNP formulation contains 0.01-0.3% w / v (e.g., 0.015-0.25% w / v, 0.12-0.22% w / v, or 0.15-0.20% w / v) of Tris relative to the total volume of the liquid LNP formulation. In some embodiments, the liquid LNP formulation contains 0.17% w / v of Tris relative to the total volume of the liquid LNP formulation.

[0252] In one embodiment, the liquid LNP formulation contains 10% w / v sucrose and 0.18% w / v alanine by weight relative to the total weight of the liquid LNP formulation.

[0253] In one embodiment, the liquid LNP formulation comprises 10% w / v sucrose and 0.26% w / v isoleucine by weight relative to the total weight of the liquid LNP formulation.

[0254] In one embodiment, the liquid LNP formulation comprises 10% w / v sucrose and 0.26% w / v leucine by weight relative to the total weight of the liquid LNP formulation.

[0255] In one embodiment, the liquid LNP formulation contains 10% w / v sucrose and 0.23% w / v valine by weight relative to the total weight of the liquid LNP formulation.

[0256] In one embodiment, the liquid LNP formulation comprises 10% w / v sucrose and 0.23% w / v proline by weight relative to the total weight of the liquid LNP formulation.

[0257] In one embodiment, the liquid LNP formulation contains 10% w / v sucrose and 0.15% w / v glycine by weight relative to the total weight of the liquid LNP formulation.

[0258] In one embodiment, the liquid LNP formulation comprises 10% w / v sucrose and 0.06% w / v valine by weight relative to the total weight of the liquid LNP formulation.

[0259] In one embodiment, the liquid LNP formulation contains 10% w / v sucrose and 0.12% w / v valine by weight relative to the total weight of the liquid LNP formulation.

[0260] In one embodiment, the liquid LNP formulation contains 10% w / v sucrose and 0.23% w / v valine by weight relative to the total weight of the liquid LNP formulation.

[0261] In one embodiment, the liquid LNP formulation contains 10% w / v sucrose and 0.47% w / v valine by weight relative to the total weight of the liquid LNP formulation.

[0262] In one embodiment, the liquid LNP formulation comprises 10% w / v sucrose, 0.47% w / v valine, and 0.04% w / v KCl by weight relative to the total weight of the liquid LNP formulation.

[0263] In one embodiment, the liquid LNP formulation comprises 10% w / v sucrose, 0.47% w / v valine, and 0.07% w / v KCl by weight relative to the total weight of the liquid LNP formulation.

[0264] In one embodiment, the liquid LNP formulation comprises 10% w / v sucrose, 0.47% w / v valine, and 0.03% w / v NaCl by weight relative to the total weight of the liquid LNP formulation.

[0265] In one embodiment, the liquid LNP formulation comprises 10% w / v sucrose, 0.47% w / v valine, and 0.06% w / v NaCl by weight relative to the total weight of the liquid LNP formulation.

[0266] In one embodiment, the liquid LNP formulation comprises 10% w / v sucrose, 0.47% w / v valine, and 0.12% w / v NaCl by weight relative to the total weight of the liquid LNP formulation.

[0267] In one embodiment, the liquid LNP formulation contains 10% w / v sucrose and 0.23% w / v valine by weight relative to the total weight of the liquid LNP formulation.

[0268] In one embodiment, the liquid LNP formulation comprises 10% w / v sucrose, 0.23% w / v valine, and 0.18% w / v alanine by weight relative to the total weight of the liquid LNP formulation.

[0269] In one embodiment, the liquid LNP formulation comprises 10% w / v sucrose, 0.23% w / v valine, and 0.26% w / v leucine by weight relative to the total weight of the liquid LNP formulation.

[0270] In one embodiment, the liquid LNP formulation comprises 10% w / v sucrose, 0.23% w / v valine, and 0.26% w / v isoleucine by weight relative to the total weight of the liquid LNP formulation.

[0271] In one embodiment, the liquid LNP formulation contains 10% w / v sucrose and 0.36% w / v alanine by weight relative to the total weight of the liquid LNP formulation.

[0272] In one embodiment, the liquid LNP formulation comprises 10% w / v sucrose and 0.30% w / v glycine by weight relative to the total weight of the liquid LNP formulation.

[0273] In one embodiment, the liquid LNP formulation comprises 10% w / v sucrose and 0.52% w / v leucine by weight relative to the total weight of the liquid LNP formulation.

[0274] In one embodiment, the liquid LNP formulation comprises 10% w / v sucrose, 0.47% w / v valine, and 0.17% w / v Tris by weight relative to the total weight of the liquid LNP formulation.

[0275] In one embodiment, the liquid LNP formulation comprises, by weight relative to the total weight of the liquid LNP formulation, 10% w / v sucrose, 0.47% w / v valine, 0.005% w / v K2HPO4, 0.10% w / v KH2PO4 and 0.117% w / v NaCl.

[0276] In one embodiment, the liquid LNP formulation comprises, by weight relative to the total weight of the liquid LNP formulation, 10% w / v sucrose, 0.23% w / v valine, 0.005% w / v K2HPO4, 0.10% w / v KH2PO4, and 0.117% w / v NaCl.

[0277] In one embodiment, the liquid LNP formulation comprises, by weight relative to the total weight of the liquid LNP formulation, 10% w / v sucrose, 0.12% w / v valine, 0.005% w / v K2HPO4, 0.10% w / v KH2PO4, and 0.117% w / v NaCl.

[0278] In one embodiment, the liquid LNP formulation comprises, by weight relative to the total weight of the liquid LNP formulation, 10% w / v sucrose, 0.06% w / v valine, 0.005% w / v K2HPO4, 0.10% w / v KH2PO4, and 0.117% w / v NaCl.

[0279] The liquid LNP formulation of the present invention may also contain therapeutic agents and / or preventive agents in the lipid nanoparticles.

[0280] The therapeutic and / or preventive agents are the same as those defined above for the lyophilized LNP formulation.

[0281] For example, the weight ratio of lipids to therapeutic and / or preventative agents in the lyophilized LNP formulation is from about 10:1 to about 60:1, or from about 2:1 to about 30:1, for example, or from 20:1 to about 30:1.

[0282] In some embodiments, the liquid formulations disclosed herein comprise a therapeutic and / or prophylactic agent (e.g., mRNA) at a concentration of about 0.025 mg / mL to about 4 mg / mL (e.g., about 0.025 mg / mL, about 0.04 mg / mL, about 0.05 mg / mL, about 0.075 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.25 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.75 mg / mL, about 1 mg / mL, about 1.5 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 0.025-0.4 mg / mL or about 0.05-0.2 mg / mL, about 0.05-0.1 mg / mL, about 0.25-2 mg / mL or about 0.5-2 mg / mL, or about 0.5-1 mg / mL), for example prior to lyophilization.

[0283] The liquid LNP formulation containing the therapeutic and / or preventive agents of the present invention has an encapsulation efficiency of at least 75%.

[0284] In some embodiments, the encapsulation efficiency of the liquid LNP formulation is at least 77%, at least 79%, at least 81%, or at least 83%.

[0285] The following examples are given as non-limiting illustrations of the present invention.

[0286] experiment

[0287] Embodiments (IE) 1-6 and Comparative Embodiments (CE) 1-17 of the present invention

[0288] Lyophilized nanoparticles encapsulating rabies mRNA (SEQ ID NO: 2) according to Examples 1-6 and Comparative Examples 1-17 of the present invention were prepared as follows.

[0289] The specified amounts of lipid components (approximately 30 to 55 mol percent of compound C1, i.e., compound 01-1 in Table 01-1, as a cationic lipid; approximately 5 to 40 mol percent of DSPC; approximately 20 to 50 mol percent of cholesterol; and DMG-PEG) were dissolved in ethanol. In the following examples, unless otherwise stated, the lipid components and their molar ratios are the same.

[0290] The rabies mRNA was diluted in 10 to 50 mM citrate buffer (pH = 3-5). In the following examples, the mRNA is identical unless otherwise stated.

[0291] LNPs were prepared by mixing an ethanol lipid solution and an aqueous mRNA solution at a volume ratio of 1:3 using a microfluidic device (total flow rate in the range of 9-30 mL / min), resulting in a total lipid:mRNA weight ratio of approximately 10:1 to 30:1. In all embodiments, unless otherwise stated, the concentration of mRNA in the liquid LNP formulation was approximately 0.003% w / v relative to the total volume of the liquid LNP formulation. The ethanol was then removed and replaced with PBS using dialysis. After dialysis, the LNPs were exchanged with a buffer solution for cryoprotectant buffer (see Tables 1-3) to obtain the liquid LNP formulation, and then filtered through a 0.2 μm sterile filter. The LNPs were loaded into Type I glass vials.

[0292] Lyophilization was performed in the glass chamber of a Pilot freeze dryer (Christ Epsilon 2-10D LSCplus freeze dryer). LNP was frozen at -40 to -60°C for 3 hours, followed by a drying cycle of -25 to -35°C / 20 to 100 mTorr for 48 to 60 hours. Then, during a second drying cycle, the LNP was heated to 5 to 25°C / 20 to 100 mTorr and held for 12 to 24 hours. The vials were capped under vacuum with a stopper and additionally capped with an aluminum cap, and then transferred to 2 to 8°C for long-term storage. In all embodiments, unless otherwise stated, the concentration of mRNA in the lyophilized LNP formulation was approximately 0.03 wt.% relative to the total weight of the lyophilized LNP formulation.

[0293] The particle size, polydispersity index (PDI), and encapsulation efficiency of LNPs are characterized as follows.

[0294] The size of lipid nanoparticles was determined by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern UK) in 173° backscatter detection mode. To measure the size of the lipid nanoparticles and the PDI, the formulation was diluted 20-fold in PBS and 1 mL was transferred to a measuring cuvette.

[0295] Following the manufacturer's instructions, the encapsulation efficiency (EE%) of the lipid nanoparticles was determined using the Quant-it Ribogreen RNA Quantification Kit (ThermoFisher Scientific, UK). To determine the fluorescence intensity of free RNA and total RNA, the LNP formulation was diluted to 0.5 µg / mL in Tris-EDTA and 0.1% Triton, respectively.

[0296] The Ribogreen reagent was diluted 200-fold with Tris-EDTA buffer and mixed in the same volume as the diluted LNP formulation. Fluorescence intensity was measured at room temperature using excitation and emission wavelengths of 488 nm and 525 nm on a Molecular Devices Spectramax iD3 spectrometer. Encapsulation efficiency was calculated based on the ratio of encapsulated RNA to total RNA fluorescence intensity.

[0297] The results obtained are summarized in Tables 1 and 2, and Figure 1 and Figure 2 middle.

[0298] Table 1

[0299] ΔS = Dimensional change after freeze-drying.

[0300] In Comparative Examples (CE) 1-6, the concentration of cationic lipids in the liquid LNP formulation was about 0.04% w / v relative to the total volume of the liquid LNP formulation, and the concentration of cationic lipids in the lyophilized LNP formulation was about 0.38-0.39 wt.% relative to the total volume of the lyophilized LNP formulation.

[0301] As can be seen from Table 1, conventional salts and cryoprotectants (e.g., Tris / sucrose, HEPES / sucrose, MES / sucrose) produced different size variations and encapsulation efficiencies of less than 85%.

[0302] Table 2

[0303] In Examples (IE) 1-6 and Comparative Examples (CE) 7-9 of the present invention, the concentration of cationic lipids in the liquid LNP formulation is about 0.04% w / v relative to the total volume of the liquid LNP formulation, and the concentration of cationic lipids in the lyophilized LNP formulation is about 0.38-0.39 wt.% relative to the total volume of the lyophilized LNP formulation.

[0304] As shown in Table 2, nonpolar amino acids (e.g., Val, Ala, Leu, Ile, Pro, and Gly), when combined with sucrose, minimized particle size variation (Δ size ≤ 10 nm) and maintained high mRNA encapsulation (>90%) after lyophilization. In contrast, basic amino acids (e.g., Arg, Lys, and His) and sucrose resulted in a significant increase in size and low EE%, indicating that not all amino acids are good cryoprotectants for mRNA-LNPs.

[0305] Table 3

[0306] Compound C1 is compound 01-1 in Table 01-1.

[0307] Figure 1 The sizes and polydispersity indices of LNPs of CE.10-17 obtained with the cryoprotectants in Table 3 before and after lyophilization are shown.

[0308] Figure 2 The encapsulation efficiency of LNPs for CE.10-17 obtained using the cryoprotectants in Table 3 is shown.

[0309] from Figure 1 and Figure 2 It can be seen that sugars other than sucrose or polyols (such as trehalose, maltose, lactose or mannitol) cannot protect mRNA-LNP from freeze-drying stress.

[0310] Embodiments (IE) 7-10 and Comparative Embodiment (CE) 18 of the present invention

[0311] According to the above method, lyophilized nanoparticles encapsulating hEPO mRNA (SEQ ID NO: 1) according to Examples 7-10 and Comparative Example 18 of the present invention were prepared using the cryoprotectants in Table 4.

[0312] The particle size and encapsulation efficiency of the LNPs before and after freeze-drying were characterized. The results are summarized in Table 4.

[0313] Table 4 As can be seen from Table 4, when the molar concentration of valine, by weight relative to the total volume of the liquid LNP formulation, is less than 0.8% w / v, particularly 0.06-0.5% w / v, the mRNA-LNP size and EE can be adequately maintained.

[0314] Embodiments of the present invention (IE) 11-15

[0315] According to the above method, freeze-dried nanoparticles encapsulating rabies mRNA according to Examples 11-15 of the present invention were prepared using the salts and cryoprotectants in Table 5.

[0316] The particle size and encapsulation efficiency of the LNPs before and after freeze-drying were characterized. The results are summarized in Table 5.

[0317] Table 5

[0318] In embodiments (IE) 10-15 of the present invention, the concentration of cationic lipids in the liquid LNP formulation is 0.041% w / v relative to the total volume of the liquid LNP formulation, and the concentration of cationic lipids in the lyophilized LNP formulation is 0.38 wt.% relative to the total volume of the lyophilized LNP formulation.

[0319] As shown in Table 4, adding salts (such as KCl or NaCl) to the valine system does not affect the particle size and EE%. In addition, salts can provide a certain amount of ionic strength and better stability to the sample during the process.

[0320] Embodiments of the present invention (IE) 16-19

[0321] According to the above method, lyophilized nanoparticles encapsulating rabies mRNA according to Examples 16-19 of the present invention were prepared using cationic lipids and cryoprotectants that replace compound C1 in Table 6.

[0322] Table 6

[0323] ALC0315 is compound 06-1 in Table 06-1.

[0324] SM102 is compound B in the 05 series of compounds.

[0325] MC3 is compound 04-I in the 04 series of compounds.

[0326] Compound C1 is compound 01-1 in Table 01-1.

[0327] Compound C2 is compound 03-135 in Table 03-1.

[0328] The particle size, polydispersity index, and encapsulation efficiency of LNPs before and after freeze-drying were characterized.

[0329] Figure 3 The particle size and polydispersity index of LNPs obtained using the lipids and cryoprotectants in Table 6 are shown before and after lyophilization.

[0330] Figure 4 The encapsulation efficiency of LNPs obtained using the lipids and cryoprotectants in Table 6 is shown before and after lyophilization.

[0331] from Figure 3 and Figure 4 It can be seen that LNPs with different ionizable lipids can be adequately protected.

[0332] Embodiments of the present invention (IE) 20-21

[0333] According to the above method, lyophilized nanoparticles encapsulating different mRNAs according to Examples 20-21 of the present invention were prepared using lipids, mRNAs and cryoprotectants listed in Table 7.

[0334] Table 7

[0335] Rabies N1-methyl mRNA (SEQ ID NO: 3): Rabies mRNA modified with 1-N-methyl-pseuuridine.

[0336] RSV N1-methyl mRNA (SEQ ID NO: 4): Respiratory syncytial virus mRNA modified with 1-N-methyl-pseudouridine.

[0337] In embodiments (IE) 20-21 of the present invention, the concentration of cationic lipids in the liquid LNP formulation is 0.041% w / v relative to the total volume of the liquid LNP formulation, and the concentration of cationic lipids in the lyophilized LNP formulation is 0.38 wt.% relative to the total volume of the lyophilized LNP formulation.

[0338] The particle size, polydispersity index, and encapsulation efficiency of LNPs before and after freeze-drying were characterized.

[0339] Figure 5 The particle size and polydispersity index of LNPs obtained before and after lyophilization using the lipids, mRNAs and cryoprotectants listed in Table 7 are shown.

[0340] Figure 6 The encapsulation efficiency of LNPs obtained before and after lyophilization using the lipids, mRNA, and cryoprotectants listed in Table 7 is shown.

[0341] from Figure 5 and Figure 6 It can be seen that nonpolar amino acids and sucrose have wide applicability as cryoprotectants in different mRNA-LNPs.

[0342] Embodiment 22 of the present invention (IE)

[0343] According to the above method, lyophilized nanoparticles encapsulating human erythropoietin (hEPO) mRNA were prepared using the lipids and cryoprotectants listed in Table 8, as described in Example 22 of the present invention.

[0344] Table 8

[0345] In embodiment (IE) 22 of the present invention, the concentration of cationic lipids in the liquid LNP formulation is 0.041% w / v relative to the total volume of the liquid LNP formulation, and the concentration of cationic lipids in the lyophilized LNP formulation is 0.38 wt.% relative to the total volume of the lyophilized LNP formulation.

[0346] LNP, both before and after lyophilization, was administered systemically to 6-8 week old female ICR mice (Xipuer-Bikai, Shanghai) via tail vein injection at a dose of 0.4 mg / kg. Six hours after administration, the mice were euthanized by CO2 overdose, and blood samples were collected for hEPO measurement. Specifically, serum was separated from whole blood by centrifugation at 5000g for 10 minutes at 4°C, rapidly frozen, and stored at -80°C for analysis.

[0347] Serum hEPO levels were measured using an ELSA assay with a commercial kit (DEP00, R&D systems) according to the manufacturer's instructions. hEPO expression levels (μg / ml) measured before and after lyophilization in the test group treated with mRNA LNP were plotted on [plot]. Figure 7 middle.

[0348] Statistically, from Figure 7 As can be seen, no significant difference was observed between protein expression levels before and after LNP lyophilization, indicating that the cryoprotectant combination had no effect on the in vivo activity of mRNA-LNP.

[0349] Embodiments of the present invention (IE) 23-26

[0350] According to the above method, lyophilized nanoparticles encapsulating human erythropoietin (hEPO) mRNA, as described in Examples 23-26 of the present invention, were prepared using the lipids and cryoprotectants listed in Table 8.

[0351] The particle size and encapsulation efficiency of the LNPs before and after freeze-drying were characterized. The results are summarized in Table 9.

[0352] Table 9

[0353] In embodiments (IE) 23-26 of the present invention, the concentration of cationic lipids in the liquid LNP formulation is about 0.04% w / v relative to the total volume of the liquid LNP formulation, and the concentration of cationic lipids in the lyophilized LNP formulation is about 0.38-0.39 wt.% relative to the total volume of the lyophilized LNP formulation.

[0354] As shown in Table 9, valine and other nonpolar amino acids, together with sucrose, can protect mRNA-LNPs from freeze-drying stress. LNP characterization parameters were maintained after freeze-drying.

[0355] LNP was administered systemically to 6-8 week old female ICR mice (Xipuer-Bikai, Shanghai) via tail vein injection, both before and after lyophilization. Six hours after administration, the mice were euthanized by CO2 overdose, and blood samples were collected for hEPO measurement, as mentioned above in Example 22 of this invention. The hEPO expression levels (μg / ml) of the test group treated with mRNA LNP before and after lyophilization were plotted on [plotting data]. Figure 8 middle.

[0356] from Figure 8 It can be seen that the hEPO expression level (μg / ml) was not negatively affected by any cryoprotectant.

[0357] Embodiments of the present invention (IE) 27-31

[0358] According to the above method, lyophilized nanoparticles encapsulating rabies mRNA according to Examples 27-31 of the present invention were prepared using ionizable lipids and cryoprotectants that replace compound C1 in Table 10.

[0359] Table 10

[0360] Compound C3 is compound 07-92 in Table 07-1.

[0361] Compound C4 is compound 07-86 in Table 07-1.

[0362] SM102 is compound B in the 05 series of compounds.

[0363] The particle size and encapsulation efficiency of LNPs before and after freeze-drying were characterized.

[0364] Figure 9 The particle size of LNPs obtained using the lipids and cryoprotectants in Table 10 is shown before and after lyophilization.

[0365] Figure 10 The encapsulation efficiency of LNPs obtained using the lipids and cryoprotectants in Table 10 is shown before and after lyophilization.

[0366] from Figure 9 and Figure 10 It can be seen that LNPs with different ionizable lipids can be adequately protected.

[0367] Embodiments (IE) 32-36 and Comparative Embodiments (CE) 19-20 of the present invention.

[0368] According to the above method, freeze-dried nanoparticles encapsulating mRNA according to Examples 32-36 and Comparative Examples 19-20 of the present invention were prepared using the cryoprotectants in Table 11.

[0369] Table 11

[0370] Compound C1 is compound 01-1 in Table 01-1.

[0371] VZV N1-methyl mRNA (SEQ ID NO: 5): Varicella-zoster virus mRNA modified with 1-N-methyl-pseuuridine.

[0372] Rabies N1-methyl mRNA: Rabies mRNA modified with 1-N-methyl-pseuuridine.

[0373] After being stored at a certain temperature for a certain period of time, the RNA purity and lipid adduct levels of the lyophilized LNP were characterized.

[0374] For Examples 32 and Comparative Example 19 of the present invention, lyophilized LNPs were stored at 25°C for up to 4 weeks, and samples were collected at weeks 0, 1, 2, and 4 to measure lipid adducts and RNA purity. For Examples 33 to 36 of the present invention and Comparative Example 20, lyophilized LNPs were stored at 37°C for up to 7 days, and samples were collected at days 0, 3, and 7 to measure lipid adducts.

[0375] To measure RNA purity and lipid adduct levels, mRNA was extracted from the mRNA-LNP formulation by isopropanol precipitation. The mRNA-LNP was diluted 10-fold with isopropanol containing ammonium acetate, briefly vortexed, and centrifuged for 15 min. The aggregated particles were washed, dried under vacuum, and resuspended in 100 µL of RNase-free water at room temperature.

[0376] To measure lipid adducts, extracted mRNA samples were separated on a DNAPac reverse-phase column (Thermo Fisher Scientific) with 4-µm particles and a size of 2.1 × 100 mm. Mobile phase A consisted of 50 mM dibutylacetate and 10 mM triethylacetate, while mobile phase B consisted of 50% acetonitrile, 50 mM dibutylacetate, and 100 mM triethylacetate. Separation was achieved using a step gradient, with an initial 1.5 min holding at 25% B, a 1 min gradient of 25–45% B, a 12.5 min gradient of 45–100% B, and a 0.5 min gradient holding at 100% B. Approximately 2 µg of mRNA was loaded onto the column. mRNA was detected by UV at 260 nm. Lipid adducts were quantified as a percentage of late peak area relative to the total chromatographic peak area.

[0377] To measure RNA purity, extracted mRNA samples were tested on an Agilent Technologies fragment analyzer, an automated capillary electrophoresis system equipped with an LED light source and a charge-coupled device detector. An RNA analysis kit (Agilent Technologies DNF-489-0500) was required for the experiments. Extracted mRNA samples were denatured at 70°C for 2 min and immediately cooled on ice, remaining on ice prior to analysis. The denatured RNA samples were electrophoretically injected at 5 kV for 4 s and electrophoresed at 8 kV for 45 min. RNA bands were analyzed as a calibrator for nucleotide size. Data were analyzed using PROSize 2.0 software (Agilent Technologies).

[0378] Figure 11 The RNA purity of lyophilized LNPs obtained with lipids and cryoprotectants from Example 32 of the present invention and Comparative Example 19 is shown after further storage at 25°C for 0 to 4 weeks.

[0379] from Figure 11 It can be seen that, compared with using Tris / sucrose alone, nonpolar amino acids, together with Tris and sucrose, may slow down RNA degradation during storage at 25°C.

[0380] Figure 12 The lipid adduct levels of lyophilized LNPs obtained with lipids and cryoprotectants from Example 32 of the present invention and Comparative Example 19 are shown after further storage at 25°C for 0 to 4 weeks.

[0381] from Figure 12It can be seen that, compared with using Tris / sucrose alone, nonpolar amino acids, together with Tris and sucrose, may minimize the formation of lipid adducts during storage at 25°C.

[0382] Figure 13 The lipid adduct levels of lyophilized LNPs obtained with lipids and cryoprotectants from Examples 33 to 36 of the present invention and Comparative Example 20 are shown after further storage at 37°C for 0 to 7 days.

[0383] from Figure 13 It can be seen that the addition of valine to the KH2PO4 / K2HPO4 / NaCl / sucrose buffer helps reduce lipid adduct formation during storage at 37°C. The lipid adduct level depends on the valine concentration. When the molar concentration of valine, by weight relative to the total volume of the liquid LNP formulation, is 0.06-0.5% w / v, the lipid adduct level can be kept low. Higher valine concentrations result in lower lipid adduct levels.

[0384] Several embodiments and examples of the invention have been described. However, it should be understood that various modifications can be made without departing from the spirit and scope of the invention. Therefore, the descriptions in the experimental sections and examples are intended to illustrate, and not limit, the scope of the invention as described in the claims.

[0385] sequence

[0386] In the following sequences, unless otherwise stated, " " indicates "m1 (1-Methyl-pseudouridine). The abbreviation is for clarity of the sequence.

[0387] SEQ ID NO: 1 (hEPO mRNA) GGGGAUCCAAGCUUAGCUUGCUUGUUCUUUUUGCAGAAGCUCAGAAUAAACGCUCAACUUUGGCGCCGCCACCAUGGGUGUGCAUGAGUGUCCCGCUUGGCUUUGGCUGUUAUUAUCAUUACUUUCCUUACCUCUUGGCCUGCCUGUCUUAGGCGCCCCUCCUCGUUUAAUCUGUGAUAGCCGUGUGUUAGAGAGAUACUUGCUUGAGGCUAAAGAGGCCGAGAAUAUUACUACCGGUUGUGCUGAACAUUGCUCGCUGAACGAGAACAUUACCGUGCCCGACACCAAAGUUAACUUCUAUGCUUGGAAACGUAUGGAGGUGGGUCAGCAGGCCGUUGAGGUAUGGCAGGGUUUGGCCCUUCUUUCGGAGGCCGUGUUACGUGGUCAGGCUCUCUUGGUUAACUCUUCUCAGCCUUGGGAGCCUUUACAGUUACAUGUUGAUAAAGCCGUUAGCGGCUUGAGAAGUUUGACCACUUUGCUUCGGGCUCUGGGUGCUCAAAAAGAGGCUAUUUCACCCCCUGAUGCUGCUAGCGCUGCUCCACUUAGAACCAUCACUGCCGACACUUUUCGUAAAUUGUUUCGUGUUUAUAGCAAUUUCUUACGUGGCAAACUGAAAUUAUACACUGGCGAGGCUUGCAGAACUGGUGACAGAUGAUAAGAAUUCUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCACUAGUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0388] SEQ ID NO: 2 (Rabies mRNA)

[0389] SEQ ID NO: 3 (Rabies N1-methyl mRNA)

[0390] SEQ ID NO: 4 (RSV N1-methylmRNA)

[0391] SEQ ID NO: 5 (VZV N1-methylmRNA)

Claims

1. A lyophilized formulation of lipid nanoparticles, comprising, by weight relative to the total weight of the lyophilized formulation: (A) 0.2-20 wt.% of lipid nanoparticles containing cationic lipids; and (B) A cryoprotectant combination, the cryoprotectant combination comprising (i) 75-99 wt.% sucrose; and (ii) 0.1-10 wt.% of nonpolar amino acids.

2. The lyophilized formulation of claim 1, wherein the lipid nanoparticles have a particle size of 80 nm to 100 nm, preferably 80 nm to 95 nm or 82 nm to 95 nm.

3. The lyophilized formulation of claim 1, wherein the total molar number of the lipid nanoparticles relative to all lipids present in the nanoparticles comprises: (a) Cationic lipids of approximately 30 mol% to approximately 55 mol%; (b) Approximately 20 mol% to approximately 50 mol% of structural lipids; (c) Phospholipids of about 5 mol% to about 40 mol%; and (d) Polymer-conjugated lipids of approximately 0.5 mol% to approximately 5 mol%; Preferably, The total number of moles of lipids in the nanoparticles is 30-55 mol% cationic lipids, 5-40 mol% DSPC, 20-50 mol% cholesterol, and 0.5-3 mol% PEG-lipids.

4. The lyophilized formulation according to any one of claims 1 to 3, wherein the lyophilized formulation comprises, by weight relative to the total weight of the lyophilized LNP formulation, 0.1-10 wt.%, preferably 0.1-1 wt.%, more preferably 0.2-0.7 wt.% of cationic lipids selected from: Compound of formula 06-I: (06-I) in L 1 and L 2 It is -O(C═O)-; G 1 and G 2 Each is independently an unsubstituted C4-C8 alkylene group; G 3 It is a C3-C8 alkylene group; R 1 and R 2 Each is C independently 12 -C 22 alkyl; R 3 It is H or OH. Preferably, the cationic lipid is a compound of the following formula: ； and / or Compound 05-I: (05-I) in l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M1 is -C(O)O-; R4 is -(CH2) n OH, and n is selected from 1, 2, 3, 4 or 5; M is -OC(O)-; Both R2 and R3 are C. 6-10 Alkyl; and R' is a straight-chain alkyl group; Preferably, the cationic lipid is a compound of formula B: (Formula B); and / or The following formula: ； and / or Compounds of formula (01-10): (01-IO), Where y and z are each independent integers from 4 to 6. s is an integer from 2 to 4. t is an integer from 1 to 3, and R 1 and R 2 Each is C independently 12 -C 22 alkyl; R 4 It is a C3-C8 cycloalkyl group; R 6 It is hydrogen or hydroxyl. Preferably, the cationic lipid is a compound of the following formula: ； and / or Compound of formula (03-I): (03-I)， in G 1 and G 2 Each is independently a C3-C8 alkylene group; Each L 1 Independently is -OC(=O)R 1 ; Each L 2 Independently is -C(=O)OR 2 ; R 1 Independently, it is C6-C 10 alkyl; R 2 C is independent 12 -C 22 alkyl; G 3 It is C2-C 12 Alkylene; R 3 It is a C3-C8 cycloalkyl group; R 4 It is a C1-C4 hydroxyalkyl group; n is 2; m is 1, Preferably, the cationic lipid is a compound of the following formula: ； and / or Compound of formula (07-III): (07-III), in R 1 and R 2 Each is independently C6-C 22 alkyl; R 0 It is a C3-C8 cycloalkyl group; G 3 It is a C2-C6 alkylene group; G 4 It is a C2-C6 alkylene group; R 3 is - OR 6 ; R 6 It is hydrogen; Preferably, the cationic lipid is a compound of the following formula: or 。 5. The lyophilized formulation according to any one of claims 1 to 4, wherein the lyophilized formulation comprises 78-99 wt.% (e.g., 80-98.5 wt.%, 82-98 wt.%, 84-97.5 wt.%, 85-97.5 wt.%, 87-97.5 wt.%, 89-97.5 wt.%, 91-97.5 wt.%, or 93-97.5 wt.%) of sucrose relative to the total weight of the lyophilized formulation.

6. The lyophilized formulation according to any one of claims 1 to 5, wherein the lyophilized formulation comprises 0.2-9 wt.% (e.g., 0.4-9 wt.%, 0.6-9 wt.%, 0.8-8 wt.%, 1-7 wt.%, 1.5-6 wt.%, 1.8-5 wt.%, or 2-4.8 wt.%) of the nonpolar amino acid relative to the total weight of the lyophilized formulation.

7. The lyophilized formulation according to any one of claims 1 to 6, wherein the nonpolar amino acid is selected from alanine, isoleucine, leucine, valine, proline, glycine, and combinations thereof.

8. The lyophilized formulation according to any one of claims 1 to 6, wherein the lyophilized formulation comprises 0.4-6 wt.% (e.g., 1-4 wt.% or 1-3 wt.%) of alanine relative to the total weight of the lyophilized formulation; and / or Isoleucine, at a concentration of 0.6-9 wt.%, preferably 1-5 wt.%, relative to the total weight of the lyophilized formulation; and / or Leucine, at a concentration of 0.6-9 wt.%, preferably 1-5 wt.%, relative to the total weight of the lyophilized LNP formulation; and / or Valine, 0.2-9 wt.% (e.g., 0.5-8 wt. or 1.5-6 wt.%) relative to the total weight of the lyophilized formulation; and / or 0.5-8 wt.%, preferably 1-5 wt.%, of proline relative to the total weight of the lyophilized formulation; and / or Glycine, 0.3-5 wt.%, preferably 0.8-3 wt.%, relative to the total weight of the lyophilized formulation; and / or Relative to the total weight of the lyophilized formulation, 1.5-4 wt.% valine and 1-3 wt.% alanine; and / or Relative to the total weight of the lyophilized formulation, 1.5-4 wt.% valine and 1.5-4 wt.% leucine; and / or The lyophilized formulation contains 1.5-4 wt.% valine and 1.5-4 wt.% isoleucine relative to its total weight.

9. The lyophilized formulation according to any one of claims 1 to 8, wherein the lyophilized formulation comprises 0.01-3 wt.%, 0.1-3 wt.%, 0.15-2 wt.%, 0.2-1.5 wt.%, or 0.2-1.3 wt.% of salt or Tris relative to the total weight of the lyophilized formulation.

10. The lyophilized formulation of claim 9, wherein the salt is selected from tris-HCl, KCl, NaCl, K2HPO4, KH2PO4, sodium citrate, sodium acetate, and combinations thereof.

11. The lyophilized formulation according to any one of claims 1 to 10, wherein the lyophilized formulation comprises The lyophilized formulation contains, by weight relative to its total weight, 80-98.5 wt.% sucrose, 0.2-9 wt.% valine, and 0.01-3 wt.% salt or Tris. For example, Based on the weight of the total weight of the lyophilized formulation, it comprises 93.39 wt.% sucrose, 4.38 wt.% valine, and 1.58 wt.% Tris; or Based on the weight of the total weight of the lyophilized formulation, it comprises 95.14 wt.% sucrose, 2.97 wt.% valine, 0.03 wt.% K₂HPO₄, 0.66 wt.% KH₂PO₄, and 0.74 wt.% NaCl; or Based on the total weight of the lyophilized formulation, it comprises 96.58 wt.% sucrose, 1.51 wt.% valine, 0.03 wt.% K₂HPO₄, 0.67 wt.% KH₂PO₄, and 0.75 wt.% NaCl; or Based on the total weight of the lyophilized formulation, it comprises 97.31 wt.% sucrose, 0.76 wt.% valine, 0.03 wt.% K₂HPO₄, 0.68 wt.% KH₂PO₄, and 0.76 wt.% NaCl; or The lyophilized formulation contains, by weight relative to its total weight, 97.68 wt.% sucrose, 0.38 wt.% valine, 0.03 wt.% K₂HPO₄, 0.68 wt.% KH₂PO₄ and 0.76 wt.% NaCl.

12. The lyophilized formulation according to any one of claims 1 to 11, wherein the lyophilized formulation further comprises a therapeutic agent and / or a preventive agent in the lipid nanoparticles.

13. The lyophilized formulation of claim 12, wherein the weight ratio of the lipid to the therapeutic agent and / or preventive agent is 10:1 to 60:1, or 2:1 to 30:1 or 20:1 to 30:

1.

14. The lyophilized formulation of claim 12 or 13, wherein the therapeutic and / or preventative agent is a nucleic acid. For example, Ribonucleic acid, Preferred is Messenger RNA, For example, Rabies mRNA, respiratory syncytial virus mRNA, human erythropoietin mRNA, varicella-zoster virus mRNA, all of which are optionally modified with 1-N-methyl-pseudouridine, and combinations thereof.

15. The lyophilized formulation according to any one of claims 1 to 14, wherein the encapsulation efficiency of the lyophilized formulation is at least 85%, at least 87%, at least 90%, at least 92%, or at least 94%.

16. A liquid formulation of lipid nanoparticles, comprising, by weight relative to the total volume of the liquid formulation: (A) Lipid nanoparticles containing cationic lipids, ranging from 0.001% to 0.2% w / v; and (B) A cryoprotectant combination, the cryoprotectant combination comprising (i) 0.001-40% w / v sucrose, and (ii) 0.01-10% w / v nonpolar amino acids.

17. The liquid formulation of claim 16, wherein the lipid nanoparticles have a particle size of about 50 nm to about 140 nm, preferably about 60 nm to about 120 nm.

18. The liquid formulation of claim 16 or 17, wherein the liquid formulation comprises, by weight relative to the total volume of the LNP formulation, 0.001-0.2% w / v, 0.001-0.1% w / v, 0.1-0.2% w / v, 0.1-0.15% w / v, 0.001-0.05% w / v, 0.001-0.03% w / v, 0.001-0.01% w / v, 0.01-0.1% w / v, or 0.05-0.1% w / v of the lipid nanoparticles.

19. The liquid formulation according to any one of claims 16 to 18, wherein the lipid nanoparticles comprise, relative to the total molar number of all lipids in the nanoparticles, 30-55 mol% cationic lipids, 20-50 mol% structural lipids, 5-40 mol% phospholipids, and 0.5-5 mol% polymer-conjugated lipids, preferably 30-55 mol% cationic lipids, 5-40 mol% DSPC, 20-50 mol% cholesterol, and 0.5-3 mol% PEG-lipids.

20. The liquid formulation of any one of claims 16 to 19, wherein the liquid formulation comprises, by weight relative to the total volume of the liquid LNP formulation, 0.01-1% w / v, preferably 0.01-0.1% w / v, more preferably 0.02-0.07% w / v of a cationic lipid selected from: Compound of formula 06-I: (06-I) in L 1 and L 2 It is -O(C═O)-; G 1 and G 2 Each is independently an unsubstituted C4-C8 alkylene group; G 3 It is a C3-C8 alkylene group; R 1 and R 2 Each is C independently 12 -C 22 alkyl; R 3 It is H or OH. Preferably, the cationic lipid is a compound of the following formula: ； and / or Compound 05-I: (05-I) in l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M1 is -C(O)O-; R4 is -(CH2) n OH, and n is selected from 1, 2, 3, 4 or 5; M is -OC(O)-; Both R2 and R3 are C. 6-10 Alkyl; and R' is a straight-chain alkyl group; Preferably, the cationic lipid is a compound of formula B: (Formula B); and / or The following formula: ； and / or Compounds of formula (01-10): (01-IO), Where y and z are each independent integers from 4 to 6. s is an integer from 2 to 4. t is an integer from 1 to 3, and R 1 and R 2 Each is C independently 12 -C 22 alkyl; R 4 It is a C3-C8 cycloalkyl group; R 6 It is hydrogen or hydroxyl. Preferably, the cationic lipid is a compound of the following formula: ； and / or Compound of formula (03-I): (03-I)， in G 1 and G 2 Each is independently a C3-C8 alkylene group; Each L 1 Independently is -OC(=O)R 1 ; Each L 2 Independently is -C(=O)OR 2 ; R 1 Independently, it is C6-C 10 alkyl; R 2 C is independent 12 -C 22 alkyl; G 3 It is C2-C 12 Alkylene; R 3 It is a C3-C8 cycloalkyl group; R 4 It is a C1-C4 hydroxyalkyl group; n is 2; m is 1, Preferably, the cationic lipid is a compound of the following formula: ； and / or Compound of formula (07-III): (07-III), in R 1 and R 2 Each is independently C6-C 22 alkyl; R 0 It is a C3-C8 cycloalkyl group; G 3 It is a C2-C6 alkylene group; G 4 It is a C2-C6 alkylene group; R 3 is - OR 6 ; R 6 It is hydrogen; Preferably, the cationic lipid is a compound of the following formula: or 。 21. The liquid formulation of any one of claims 16 to 20, wherein the liquid formulation comprises 0.01-35% w / v (e.g., 0.1-30% w / v, 1-30% w / v, 2-25% w / v, 3-20% w / v, 4-15% w / v, 5-14% w / v, 6-13% w / v, 7-12% w / v, 8-11% w / v, 9-10% w / v or 9.3-10% w / v) of sucrose by weight relative to the total volume of the liquid LNP formulation.

22. The liquid formulation of any one of claims 16 to 21, wherein the liquid formulation comprises 0.04-1% w / v (e.g., 0.06-0.9% w / v, 0.08-0.8% w / v, 0.1-0.7% w / v, 0.15-0.6% w / v, 0.18-0.5% w / v, or 0.2-0.5% w / v) of nonpolar amino acids by weight relative to the total volume of the liquid LNP formulation.

23. The liquid formulation according to any one of claims 16 to 22, wherein the nonpolar amino acid is selected from alanine, isoleucine, leucine, valine, proline, glycine, and combinations thereof.

24. The liquid formulation of any one of claims 16 to 22, wherein the liquid formulation comprises Alanine, by weight relative to the total volume of the liquid formulation, is 0.04-0.6% w / v (e.g., 0.1-0.4% w / v or 0.1-0.3% w / v). and / or Isoleucine, at a weight of 0.06-0.9% w / v, preferably 0.1-0.5% w / v, relative to the total volume of the liquid formulation; and / or Leucine, by weight relative to the total volume of the liquid formulation, is 0.06-0.9% w / v (e.g., 0.1-0.6% w / v or 0.1-0.5% w / v). and / or Valine, by weight relative to the total volume of the liquid formulation, is 0.05-0.8% w / v, preferably 0.15-0.6% w / v; and / or Proline, by weight relative to the total volume of the liquid formulation, is 0.05-0.8% w / v, preferably 0.1-0.5% w / v. and / or Glycine, by weight relative to the total volume of the liquid formulation, is 0.03-0.5% w / v, preferably 0.08-0.3% w / v; and / or Valine and alanine, by weight relative to the total volume of the liquid formulation, are 0.15-0.4% w / v. and / or Valine and leucine, by weight relative to the total volume of the liquid formulation, are 0.15-0.4% w / v. and / or Valine and isoleucine, by weight relative to the total volume of the liquid formulation, are 0.15-0.4% w / v.

25. The liquid formulation of any one of claims 16 to 24, wherein the liquid formulation comprises, by weight relative to the total weight of the liquid formulation, 0.001-0.3%, 0.01-0.3% w / v, 0.015-0.2% w / v, 0.02-0.15% w / v, or 0.02-0.13% w / v of a salt or Tris.

26. The liquid formulation of claim 25, wherein the salt is selected from tris-HCl, KCl, NaCl, K2HPO4, KH2PO4, sodium citrate, sodium acetate, and combinations thereof.

27. The liquid formulation of any one of claims 16 to 26, wherein the liquid formulation comprises The liquid formulation contains, by weight, 4-15% w / v sucrose, 0.05-0.8% w / v valine, and 0.001-0.3% w / v salt or Tris. For example, Based on the total weight of the liquid formulation, 10% w / v sucrose, 0.47% w / v valine, and 0.17% w / v Tris; or The liquid formulation contains, by weight relative to its total weight, 10% w / v sucrose, 0.47% w / v valine, 0.005% w / v K₂HPO₄, 0.10% w / v KH₂PO₄, and 0.117% w / v NaCl; or The liquid formulation contains, by weight relative to its total weight, 10% w / v sucrose, 0.23% w / v valine, 0.005% w / v K₂HPO₄, 0.10% w / v KH₂PO₄, and 0.117% w / v NaCl; or The liquid formulation contains, by weight relative to its total weight, 10% w / v sucrose, 0.12% w / v valine, 0.005% w / v K₂HPO₄, 0.10% w / v KH₂PO₄, and 0.117% w / v NaCl; or The liquid formulation contains, by weight, 10% w / v sucrose, 0.06% w / v valine, 0.005% w / v K₂HPO₄, 0.10% w / v KH₂PO₄, and 0.117% w / v NaCl.

28. The liquid formulation of any one of claims 16 to 27, wherein the liquid formulation further comprises a therapeutic agent and / or a preventive agent in the lipid nanoparticles.

29. The liquid formulation of claim 28, wherein the weight ratio of the lipid to the therapeutic and / or preventive agent is 10:1 to 60:1, or 2:1 to 30:1, or 20:1 to 30:

1.

30. The liquid formulation of claim 28 or 29, wherein the therapeutic and / or preventative agent is a nucleic acid. For example, Ribonucleic acid, Preferred is Messenger RNA, For example, Rabies mRNA, respiratory syncytial virus mRNA, human erythropoietin mRNA, varicella-zoster virus mRNA, all of which are optionally modified with 1-N-methyl-pseudouridine, and combinations thereof.

31. The liquid formulation according to any one of claims 16-30, wherein the encapsulation efficiency of the liquid formulation is at least 75%, at least 77%, at least 79%, at least 81%, or at least 83%.

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