Nanoparticle compositions and related methods

Abstract

Provided is a nanoparticle composition comprising a compound represented by general formula (1), or an ionized form thereof, where R1, R2 and R4 to R8 are each independently H, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group, R3 is an optionally substituted alkylene group, an optionally substituted alkenylene group, or an optionally substituted alkynyl group, and R4 is an optionally substituted alkyl group, an optionally substituted alkenylene group, or an optionally substituted alkynyl group. And R9 and R10 are each independently a hydrophobic tail or contain at least one of the groups as defined above for R4 to R8; therapeutic, prophylactic and / or biological agents encapsulated with a compound of general formula (1) to form nanoparticles; and a cryoprotectant. Methods of making the nanoparticle compositions are also provided.

Description

TECHNICAL FIELD

[0001] The present disclosure broadly relates to nanoparticle compositions, methods of making the compositions, and related uses. BACKGROUND

[0002] Nanoparticles (NPs) have been used as carriers to deliver therapeutic, prophylactic, and / or biological agents. In some applications, these NPs need to be further processed, for example, by freeze-drying or lyophilization, to extend their shelf-life for transportation or long-term storage.

[0003] One such example is the use of nanoparticles, for example, lipid nanoparticles, as non-viral nanocarriers to deliver messenger ribonucleic acid (mRNA). mRNA is a large molecule with a high negative charge that cannot enter cells to exert its biological function. Therefore, in the Pfizer / BioNTech and Moderna SARS-CoV-2 mRNA vaccines, lipids are used as carriers. The lipids in the Pfizer / BioNTech SARS-CoV-2 mRNA vaccine assemble with mRNA to form lipid nanoparticles (LNPs), which are taken up by immune cells through endocytosis to stimulate immune cells.

[0004] However, the thermal stability of these mRNA LNPs is challenging, which leads to expensive cold-chain logistics. For example, the current COVID-19 mRNA vaccines need to be stored and transported in ultra-low temperature conditions (the Pfizer / BioNTech and Moderna SARS-CoV-2 mRNA vaccines require -80°C and -20°C, respectively). Therefore, the cold-chain logistics problem can be alleviated by lyophilizing mRNA LNPs.

[0005] The basic principle of freeze-drying or lyophilization involves removing water from a frozen sample through sublimation and desorption processes. This requires freezing the sample, followed by primary drying through sublimation, and secondary drying through desorption. However, the freeze-drying process generates various stresses that can destabilize NPs. Examples of destabilization include NP aggregation (leading to size increase), reorganization of lipid components within LNPs, and reduction of LNP transfection efficiency.

[0006] In view of the above, there is a need to provide nanoparticle compositions and related methods that address or at least ameliorate the above problems. In particular, there is a need to provide nanoparticle compositions and related methods to enable the maintenance of the stability and potency required for the therapeutic, prophylactic, and / or biological agents contained in the nanoparticle compositions to be delivered even after freeze-drying or lyophilization. SUMMARY

[0007] In one aspect, a nanoparticle composition is provided for delivering therapeutic, preventative, and / or biological agents, the nanoparticle composition comprising:

[0008] Compounds or their ionized forms represented by general formula (1)

[0009]

[0010] in

[0011] R 1 R 2 and R 4 To R 8 Each of the following can be independently H, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group.

[0012] R 3 It can be an optionally substituted alkylene group, an optionally substituted alkenyl group, or an optionally substituted ynylene group, and

[0013] R 9 and R 10 Each is independently a hydrophobic tail or contains the above-mentioned R. 4 To R 8 At least one of the defined groups;

[0014] Therapeutic agents, preventative agents, and / or biological agents, which are encapsulated in compounds of general formula (1) to form nanoparticles; and

[0015] Cryoprotectant.

[0016] In one implementation, R 1 and R 2 Each is H; R 3 Contains alkylene groups; and

[0017] R 9 and R 10 Each of the components independently contains an alkyl group, wherein the alkyl group contains at least 5 carbon atoms.

[0018] In one implementation, R 4 To R 8 Each is represented by H.

[0019] In one embodiment, the compound is represented by formula (1A):

[0020] .

[0021] In one implementation, the therapeutic agent, preventive agent, and / or biological agent includes nucleic acids.

[0022] In one implementation, the nucleic acid includes messenger ribonucleic acid (mRNA).

[0023] In one embodiment, the nanoparticle composition further comprises an ionizable lipid that is different from a compound of general formula (1).

[0024] In one embodiment, the ionizable lipids that differ from compounds of general formula (1) include ALC-0315.

[0025] In one embodiment, the ratio of the compound of general formula (1) to an ionizable lipid different from the compound is 1:1 to 1:20.

[0026] In one embodiment, the composition further comprises neutral lipids / auxiliary lipids, sterols, and polyethylene glycol (PEG) modified lipids.

[0027] In one embodiment, the compound of general formula (1), neutral lipids / auxiliary lipids, sterols and PEG-modified lipids are mixed in a molar ratio of 1 to 70:1 to 20:10 to 60:1 to 20.

[0028] In one embodiment, the neutral lipid / auxiliary lipid is selected from 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycerol-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycerol-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), and 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine (18:0 Diether). PC), 1-oleoyl-2-cholesterol hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C 16Lyso PC), 1,2-dilinoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidyl-sn-glycerol-3-phosphate choline, 1,2-bis(docosahexaenoic acid)-sn-glycerol-3-phosphate choline, 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (ME 16.0 PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-disarachidyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenoic acid)-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-racemic-(1-glycerol) sodium salt (DOPG), sphingomyelin and combinations thereof.

[0029] In one embodiment, the sterol is selected from cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, alfalfa sterol, and combinations thereof.

[0030] In one embodiment, the PEG-modified lipid is selected from 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristoxypropyl-3-amine (PEG-c-DOMG), 3-N-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristoxypropylamine (PEG-S-DMG), PEG-DMPE (1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine-N-[(polyethylene glycol)-methoxy](sodium salt)), PEG-DPPC, PEG-DSPE lipids, and combinations thereof.

[0031] In one embodiment, the cryoprotectant comprises a carbohydrate source selected from at least one of maltose, sucrose, trehalose, mannitol, glucose, fructose, lactose, galactose, ribose, xylose, mannose, arabinose, and combinations thereof.

[0032] In one embodiment, the amount of cryoprotectant is from 1 w / v% to 30 w / v relative to the total volume of the composition.

[0033] In one embodiment, the nanoparticle composition is in lyophilized or freeze-thawed form.

[0034] In one implementation, the therapeutic and / or preventive and / or biological agents are encapsulated with an encapsulation rate of at least 60%.

[0035] In one embodiment, the zeta potential of the nanoparticles is from -10 mV to +10 mV.

[0036] In one embodiment, the polydispersity index (PDI) of the nanoparticles is from 0.01 to 0.5.

[0037] In one embodiment, the nanoparticles have a particle size of 50 nm to 1200 nm.

[0038] In one aspect, the nanoparticle compositions disclosed herein are provided for use in pharmaceuticals.

[0039] In one aspect, the use of the nanoparticle compositions disclosed herein in the manufacture of medicaments for inducing an immune response in subjects in need is provided.

[0040] In one aspect, nanoparticle compositions disclosed herein are provided for inducing an immune response in a subject in which such response is desired, wherein the nanoparticle composition or lyophilized nanoparticle composition is to be applied to the subject.

[0041] In one aspect, a method for inducing an immune response in a subject is provided, the method comprising the step of administering a therapeutically effective amount of the nanoparticle composition disclosed herein to the subject.

[0042] In one implementation, the immune response is specific to coronaviruses, cancer, or bacteria.

[0043] In one implementation, the coronavirus is the SARS-CoV-2 coronavirus.

[0044] In one aspect, a method for preparing the nanoparticle compositions disclosed herein is provided, the method comprising:

[0045] Prepare an aqueous composition comprising therapeutic and / or preventive and / or biological reagents;

[0046] The aqueous composition is mixed with a compound of general formula (1), an auxiliary lipid, a sterol, and a PEG-modified lipid to obtain nanoparticles encapsulating the therapeutic and / or preventive and / or biological reagents.

[0047] Add cryoprotectants to nanoparticles.

[0048] In one embodiment, the mixing step is carried out in the presence of other ionizable lipids that are different from those of the general formula (1).

[0049] In one embodiment, the compound of general formula (1) is mixed with an ionizable lipid different from the compound in a ratio of 1:1 to 1:20.

[0050] definition

[0051] As used herein, the term "particle" refers to a discrete entity or discrete body. The particles described herein can include organic particles, inorganic particles, composite particles, or biological particles. The particles used herein can also be large particles formed by the aggregation of multiple subparticles or fragments of small objects. The particles of this disclosure can be spherical, substantially spherical, or non-spherical, such as irregularly shaped or elliptical particles. When used to refer to particles, the term "size" refers to the maximum size of the particle. For example, in the context of nanoparticles, the term "size" can refer to the diameter of the nanoparticle, although not limited thereto. In various embodiments, when the particle is substantially spherical, the term "size" can refer to the diameter of the particle; or when the particle is substantially non-spherical, the term "size" can refer to the maximum length of the particle.

[0052] As used herein, the term "nano" should be interpreted broadly to include nanoscale dimensions, i.e., less than about 1000 nm, about 1 nm to less than about 1000 nm, about 1 nm to about 900 nm, about 1 nm to about 800 nm, about 1 nm to about 700 nm, about 1 nm to about 600 nm, about 1 nm to about 500 nm, about 1 nm to about 400 nm, about 1 nm to about 300 nm, about 1 nm to about 200 nm, or about 1 nm to about 100 nm. Therefore, as used herein, the terms "nanostructure," "nanoparticle," "nanomaterial," etc., can include structures with at least one dimension not exceeding the range described herein. The terms "nanostructure," "nanoparticle," "nanomaterial," etc., as used herein, can include structures having at least one dimension not exceeding about 100 nm, not exceeding about 90 nm, not exceeding about 80 nm, not exceeding about 70 nm, not exceeding about 60 nm, not exceeding about 50 nm, not exceeding about 40 nm, not exceeding about 30 nm, not exceeding about 20 nm, or not exceeding about 10 nm.

[0053] The term "micrometer" as used herein should be interpreted broadly, including sizes from about 1 micrometer to about 1000 micrometers, from about 1 micrometer to less than about 1000 micrometers, from about 1 micrometer to about 900 micrometers, from about 1 micrometer to about 800 micrometers, from about 1 micrometer to about 700 micrometers, from about 1 micrometer to about 600 micrometers, from about 1 micrometer to about 500 micrometers, from about 1 micrometer to about 400 micrometers, from about 1 micrometer to about 300 micrometers, from about 1 micrometer to about 200 micrometers, from about 1 micrometer to about 100 micrometers, or from about 1 micrometer to about 5 micrometers. In various embodiments, particles of about 5 micrometers or less can be used for intranasal spray delivery.

[0054] As used herein, the terms “treatment,” “management,” and “therapy,” and their synonyms, refer to therapeutic treatments and preventative or preventative measures aimed at preventing or alleviating a medical condition, which includes, but is not limited to, disease, symptoms, and disorders. Medical conditions also include the body’s response to a disease or disorder, such as an inflammatory response. Those who require such treatment include those who already have a medical condition, those who are susceptible to a medical condition, or those who need to prevent a medical condition.

[0055] As used herein, the term “therapeutic effective amount” for a compound is intended to refer to an amount sufficient to prevent or at least alleviate (mitigate) a medical condition, including, for example, infectious / contact-borne diseases, viral infections (i.e., diseases caused by viruses), bacterial infections (i.e., diseases caused by bacteria), fungal infections (i.e., diseases caused by fungi), parasitic infections (i.e., diseases caused by parasites), respiratory diseases, and combinations thereof.

[0056] The dosage and administration of the compounds, compositions, and formulations disclosed herein can be determined by those skilled in the art of clinical pharmacology or pharmacokinetics. The effective amount of the active agent disclosed herein for therapeutic use will depend, for example, on the target patient, route of administration, and patient condition. Therefore, clinicians need to perform dose titrations and modify the route of administration as needed to achieve optimal therapeutic effects.

[0057] The term “object” is intended to refer generally to any animal, such as a mammal, and includes humans. Exemplary objects include, but are not limited to, humans and non-human primates. As used herein, the term “object” also includes patients and non-patients. The term “patient” refers to an individual who is currently suffering from or may suffer from a medical condition, including, for example, infectious / contact-transmitted diseases, viral infections (i.e., diseases caused by viruses), bacterial infections (i.e., diseases caused by bacteria), fungal infections (i.e., diseases caused by fungi), parasitic infections (i.e., diseases caused by parasites), respiratory diseases, and combinations thereof. “Non-patient” refers to an individual who does not have a medical condition and is unlikely to have one. “Non-patient” includes healthy individuals, non-ill individuals, and / or individuals without a medical condition. As used herein, the term “mammal” includes vertebrates, such as humans or large veterinary mammals (e.g., horses, cattle, deer, sheep, llamas, goats, pigs).

[0058] The term "alkyl" as a group or part of a group refers to a straight-chain or branched aliphatic hydrocarbon group having 1 to 20 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. Examples of suitable straight-chain and branched alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, hexyl, pentyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 1,2, 2-Trimethylpropyl, 1,1,2-Trimethylpropyl, 2-Ethylpentyl, 3-Ethylpentyl, Heptyl, 1-Methylhexyl, 2,2-Dimethylpentyl, 3,3-Dimethylpentyl, 4,4-Dimethylpentyl, 1,2-Dimethylpentyl, 1,3-Dimethylpentyl, 1,4-Dimethylpentyl, 1,2,3-Trimethylbutyl, 1,1,2-Trimethylbutyl, 1,1,3-Trimethylbutyl, 5-Methylheptyl, 1-Methylheptyl, Octyl, Nonyl, Decyl, etc. The group can be a terminal group or a bridging group.

[0059] The term "alkenyl" as a group or part of a group refers to an aliphatic hydrocarbon group containing at least one carbon-carbon double bond, and it can be straight-chain or branched, having 2 to 20 carbon atoms, 2 to 10 carbon atoms, 2 to 6 carbon atoms, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms in the chain. The group can contain multiple double bonds, and the configuration around each double bond is independently E or Z. Exemplary alkenyl groups include, but are not limited to, ethenyl, vinyl, allyl, 1-methylvinyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1,3-pentadienyl, 2, 4-Pentadienyl, 1,4-pentadienyl, 3-methyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 2-methylpentenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, etc. The group can be a terminal group or a bridging group.

[0060] The term "alkynyl" as a group or part of a group refers to an aliphatic hydrocarbon group containing at least one carbon-carbon triple bond. It can be straight-chain or branched, having 2 to 20 carbon atoms, 2 to 10 carbon atoms, 2 to 6 carbon atoms, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms in the chain. The group can contain multiple triple bonds. Exemplary alkynyl groups include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 5-hexynyl, 1-hepynyl, 2-hepynyl, 6-hepynyl, 1-octyynyl, 2-octyynyl, 7-octyynyl, 1-nonynyl, 2-nonynyl, 8-nonynyl, 1-decynyl, 2-decynyl, 9-decynyl, etc. The group can be a terminal group or a bridging group.

[0061] The term "amino" is intended to refer generally to groups containing –NR2, where R is independently hydrogen or an organic group. The group can be a terminal group or a bridging group.

[0062] When used to describe a chemical structure or moiety, the term "optionally substituted" means that one or more hydrogen atoms in that structure or moiety are optionally substituted by a chemical moiety or functional group, such as alcohol, alkoxy, alkanoyloxy, alkoxycarbonyl, alkenyl, alkyl (e.g., methyl, ethyl, propyl, tert-butyl), alkynyl, alkylcarbonyloxy (–OC(O)alkyl), amide (–C(O)NH–alkyl– or –alkylNHC(O)alkyl), amino (e.g., alkylamino, arylamino, aralkylamino), aryl, aryloxy, azo, carbamoyl (–NHC(O)O–alkyl– or –OC(O)NH-alkyl), carbamoyl (e.g., CONH2, and CONH–alkyl, CONH–aryl and CONH-aralkyl), carboxyl, carboxylic acid, cyano, ester, ether (e.g., methoxy, ethoxy), halogen, haloalkyl (e.g., –CCl3, –CF3, –C(CF3)3), heteroalkyl, isocyanate, isothiocyanate, nitrile, nitro, phosphate diester, sulfide, sulfonamide (e.g., SO2NH2), sulfone, sulfonyl (including alkylsulfonyl, arylsulfonyl and aralkylsulfonyl), sulfoxide, thiol (e.g., mercapto, thioether) or urea (–NHCONH-alkyl–).

[0063] The terms “coupled” or “connected” used in this specification are intended to cover direct connections or connections through one or more intermediate means, unless otherwise stated.

[0064] The term “and / or”, for example, “X and / or Y”, should be understood to mean “X and Y” or “X or Y”, and should be regarded as providing explicit support for both meanings or either meaning.

[0065] Furthermore, in the description herein, terms such as “comprising” and “including” are intended, whenever used, to be non-restrictive descriptive language, as they broadly encompass the elements / components listed after these terms, except for other components not explicitly enumerated. For example, when “comprising” is used, a reference to “one” feature is also intended to be a reference to “at least one” of that feature. Terms such as “composed of” and “consisting of” can be considered, in appropriate context, as subsets of terms such as “comprising” and “including”. Therefore, in embodiments of this disclosure using terms such as “comprising” and “including”, it should be understood that these embodiments provide instruction for corresponding embodiments using terms such as “composed of” and “consisting of”. Additionally, terms such as “about” and “approximately”, whenever used, generally refer to reasonable variations, such as a deviation of + / -5% from the disclosed value, or a deviation of 4% from the disclosed value, or a deviation of 3%, 2%, or 1% from the disclosed value.

[0066] Furthermore, certain values ​​may be disclosed within a range in this description. Values ​​representing the endpoints of a range are intended to illustrate a preferred range. Whenever a range is described, it is intended that the range encompasses and teaches all possible subranges as well as individual values ​​within that range. That is, the endpoints of a range should not be interpreted as immutable limitations. For example, a range description of 1% to 5% is intended to specifically disclose subranges of 1% to 2%, 1% to 3%, 1% to 4%, 2% to 3%, etc., and individual values ​​within that range, such as 1%, 2%, 3%, 4%, and 5%. The above intention to specifically disclose ranges of any depth / breadth applies.

[0067] Furthermore, in describing some embodiments, this disclosure may have already disclosed methods and / or processes in a specific order of steps. However, unless otherwise required, it should be understood that the methods or processes should not be limited to the specific order of steps disclosed. Other orders of steps are permissible. The specific order of steps in this disclosure should not be construed as an undue limitation. Unless otherwise required, the methods and / or processes of this disclosure should not be limited to steps performed in the order they are written. The order of steps may be changed and still remain within the scope of this disclosure.

[0068] Furthermore, it should be understood that while this disclosure provides embodiments having one or more features / characteristics discussed herein, one or more of these features / characteristics may be omitted in other alternative embodiments, and this disclosure provides support for such omissions and these related alternative embodiments.

[0069] It should also be understood that, in cases where priority is claimed in an earlier application, the entire contents of the earlier application are also considered to constitute part of this disclosure and may serve as support for implementation of this disclosure. Detailed Implementation

[0070] The following discloses exemplary, non-limiting embodiments of nanoparticle compositions and related methods / uses.

[0071] Nanoparticle compositions

[0072] Nanoparticle compositions for delivering therapeutic, preventative, and / or biological agents are provided, the nanoparticle compositions comprising:

[0073] Compounds or their ionized forms represented by general formula (1),

[0074]

[0075] in

[0076] R 1 R 2 and R 4 To R 8 Each of the following can be independently H, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group.

[0077] R 3 It can be an optionally substituted alkylene group, an optionally substituted alkenyl group, or an optionally substituted ynylene group, and

[0078] R 9 and R 10 Each is independently a hydrophobic tail / chain / group or contains the above-mentioned R. 4 To R 8 At least one of the defined groups;

[0079] Therapeutic agents, preventative agents, and / or biological agents, which are encapsulated in compounds of general formula (1) to form nanoparticles; and

[0080] Cryoprotectant.

[0081] In various embodiments, the compound of general formula (1) is a portion of the ionizable lipid component of the composition. In various embodiments, the compound of general formula (1) is a portion of the total lipid / lipid derivative component / content in the composition.

[0082] In various implementation schemes, R 1 and R 2 Each is independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl. For example, R 1and R 2 It can be selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, hexyl, pentyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 1,2,2-trimethylpropyl, 1,1,2 - Trimethylpropyl, 2-ethylpentyl, 3-ethylpentyl, heptyl, 1-methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2,3-trimethylbutyl, 1,1,2-trimethylbutyl, 1,1,3-trimethylbutyl, 5-methylheptyl, 1-methylheptyl, octyl, nonyl, decyl, etc., or combinations thereof.

[0083] In various implementation schemes, R 1 and R 2 All are H. In this type of implementation, –NR 1 R 2 It is –NH2 or a primary amino group, and the compound contains a primary (1°) amino group (e.g., an ionizable primary amino group).

[0084] In various implementation schemes, R 1 Is it H or R? 2 It is H. In such implementations, –NR 1 R 2 Yes – NHR 2 Or –NR 1 H (i.e., secondary amino group), and the compound contains a secondary (2°) amino group (e.g., an ionizable secondary amino group).

[0085] In various implementation schemes, R 1 and R 2 None of them are H. In various implementation schemes, R 1 and R 2 Each is independently selected from optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl groups, or combinations thereof. In such embodiments, –NR 1 R 2 It is a tertiary amino group, and the compound contains a tertiary (3°) amino group (e.g., an ionizable tertiary amino group).

[0086] In various embodiments, the compound comprises one or more ionizable and / or capable of being ionized amino groups. The amino group can be a primary (1°) amino group, i.e., when R... 1 and R 2All are H. Therefore, in various embodiments, the compound is ionizable and / or capable of being ionized and / or exists in an ionized form, for example, at physiological pH. Advantageously, the ionizable nature of the compound (due to the presence of ionizable amino groups) allows embodiments of the compound to be used as encapsulating agents / loading agents, delivery carriers / delivery systems, and / or transfection carriers / transfection systems. In various embodiments, the compound is designed / configured to allow loading / encapsulation of one or more types of molecules or cargo. In various embodiments, the compound is also designed / configured to allow the loaded / encapsulated reagent to be released from the compound and / or subsequently delivered to a desired target (e.g., cells, cytosol, tissue, or organ). The molecules / cargo to be loaded / encapsulated onto / into / in the compound can include, but are not limited to, therapeutic agents, preventative agents, biological agents, etc. In various embodiments, the molecules / cargo to be loaded / encapsulated include nucleic acids. For example, the molecule / cargo to be loaded / encapsulated may be a nucleic acid selected from ribonucleic acid (RNA), messenger ribonucleic acid (mRNA), small interfering ribonucleic acid (siRNA), deoxyribonucleic acid (DNA), plasmid deoxyribonucleic acid (pDNA), oligonucleotides such as antisense oligonucleotides (ASO), and combinations thereof. In various embodiments, the molecule / cargo to be loaded / encapsulated includes therapeutic agents. For example, the molecule / cargo to be loaded / encapsulated may be a therapeutic agent selected from negatively charged therapeutic agents, drug molecules, vaccines (e.g., “Severe Acute Respiratory Syndrome Coronavirus 2” (SARS-CoV-2) vaccines, etc.), and combinations thereof. Advantageously, the compound is suitable for encapsulating and / or delivering one or more therapeutic agents, prophylactic agents, and / or biological agents to a desired target (e.g., object, cell, cytosol, tissue, or organ).

[0087] In various embodiments, the compound represented by general formula (1) is capable of being ionized (e.g., protonated) at pH 3 to physiological pH (or neutral pH), such that the nanoparticle composition encapsulates therapeutic and / or preventative and / or biological agents coupled / bonded / linked / bound to the nanoparticle composition. In various embodiments, NR 1 R 2 This refers to a group that is ionizable or capable of being ionized at pH 3 to physiological pH. In various embodiments, the compound represented by general formula (1) is capable of being ionized (e.g., protonated) at physiological pH (or neutral pH), such that the composition encapsulates therapeutic and / or preventive and / or biological agents coupled / bonded / linked / bound to the nanoparticle composition. The therapeutic and / or preventive and / or biological agents may be coupled / bonded / linked / bound to the nanoparticle composition via electrostatic interactions and / or other physical interactions. In various embodiments, the therapeutic and / or preventive and / or biological agents are coupled / bonded / linked / bound to the nanoparticle composition via electrostatic interactions and / or physical means.

[0088] Advantageously, depending on the type or properties of one or more amino groups, the compound is designed / configured to be ionizable at a pH of about 3 to about physiological pH. In various embodiments, the compound can be ionized at pH values ​​of about 3.0 to about 7.8, about 3.1 to about 7.7, about 3.2 to about 7.6, about 3.3 to about 7.5, about 3.4 to about 7.4, about 3.5 to about 7.3, about 3.6 to about 7.2, about 3.7 to about 7.1, about 3.8 to about 7.0, about 3.9 to about 6.9, about 4.0 to about 6.8, about 4.1 to about 6.7, about 4.2 to about 6.6, about 4.3 to about 6.5, about 4.4 to about 6.4, about 4.5 to about 6.3, about 4.6 to about 6.2, about 4.7 to about 6.1, about 4.8 to about 6.0, about 4.9 to about 5.9, about 5.0 to about 5.8, about 5.1 to about 5.7, about 5.2 to about 5.6, about 5.3 to about 5.5, or about 5.4. In various embodiments in which the compound comprises a primary amino group, the compound is capable of being ionized at a physiological pH of about 7.00 to about 7.80, about 7.05 to about 7.75, about 7.10 to about 7.70, about 7.15 to about 7.65, about 7.20 to about 7.60, about 7.25 to about 7.55, about 7.30 to about 7.50, about 7.35 to about 7.45, about 7.36, about 7.37, about 7.38, about 7.39, about 7.40, about 7.41, about 7.42, about 7.43, about 7.44, or about 7.45.

[0089] In various embodiments, the compound is in an ionized form, wherein –NR 1 R 2 It has been ionized into a positively charged group. In various implementations, –NR 1 R 2 It is ionized / protonated at pH values ​​of approximately 3 to physiological pH (or approximately neutral pH), becoming a positively charged group / ion / cation. This occurs in compounds containing primary amines (i.e., –NR). 1 R 2 In various implementations of (primary amino) –NR 1 R 2 It ionizes into positively charged groups / ions / cations at physiological pH (or approximately neutral pH). This is especially true when the compound contains secondary and / or tertiary amino groups (e.g., –NR). 1 R 2 In various embodiments (where the amino group is a secondary amino group and / or a tertiary amino group), –NR 1 R 2 It is ionized into positively charged groups / ions / cations at a pH of about 3 to about 5 or about pH 4. In various embodiments, –NR 1 R2 Protonated to become –NR 1 R 2 H + For example, when R 1 and R 2 When both are H, then –NR 1 R 2 It can be protonated to become –NH3 + .

[0090] In various implementations, for those containing a primary amine (e.g., when R...), 1 and R 2 Compounds (e.g., lipid compounds) that readily protonate in water to form positively charged molecules can aggregate molecules / cargo (e.g., nucleic acids such as mRNA) into lipid nanoparticles (LNPs) through electrostatic interactions and / or other physical interactions, forming encapsulated LNPs (e.g., mRNA LNPs). For example, after being taken up by a cell via endocytosis, mRNA LNPs can enter endolysosomes, where they fuse with the endolysosomal membrane, resulting in the release of the encapsulated mRNA into the cytosol for transfection (e.g., gene transfection).

[0091] Advantageously, the nanoparticle composition is suitable for encapsulating, delivering and / or transfecting one or more therapeutic, preventative and / or biological agents to, for example, desired targets (such as objects, cells, cytosols, tissues or organs).

[0092] In various embodiments, the compound comprises a lipid compound. The term "compound" may be used interchangeably with the terms "lipid," "lipid compound," "ionizable lipid," "ionizable lipid compound," "cationic lipid compound," "ionizable cationic lipid compound," etc. In various embodiments, the compound is amphiphilic / amphiphilic and comprises a hydrophilic and a hydrophobic portion. In various embodiments, the lipid portion of the compound is hydrophobic, while the groups in the compound, such as amino and / or hydroxyl groups, are hydrophilic. In various embodiments, the compound contains amino groups (e.g., ionizable NR)... 1 R 2 The compound contains one or more hydrophilic moieties at R. In various embodiments, the compound contains one or more hydrophilic moieties at R. 9 and R 10 The region contains a hydrophobic portion / tail / chain / group. In various embodiments, R 9 and R 10Each hydrophobic tail / chain / group independently comprises an optionally substituted alkyl group. The alkyl group may have at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. For example, R 9 and R 10 Each can be independently designated as C. y H 2y+1 Where y ≥ 5, y ≥ 6, y ≥ 7, y ≥ 8, y ≥ 9, y ≥ 10, y ≥ 11, y ≥ 12, y ≥ 13, y ≥ 14, y ≥ 15, y ≥ 16, y ≥ 17, y ≥ 18, y ≥ 19 or y ≥ 20. Advantageously, in various embodiments, the presence of a hydrophobic portion / tail / chain / group in the compound helps to confer improved cell uptake and / or transfection, thereby leading to higher and / or better transfection efficiency.

[0093] In various implementation schemes, R 3 Contains alkylene groups. In various embodiments, for example, R 3 It can be selected from methylene, ethylene, n-propylene, isopropylene, n-butylene, isobutylene, secondary butylene, tert-butylene, hexylene, pentylene, 1,2-dimethylpropylene, 1,1-dimethylpropylene, pentylene, isopentylene, hexylene, 4-methylpentylene, 1-methylpentylene, 2-methylpentylene, 3-methylpentylene, 2,2-dimethylbutylene, 3,3-dimethylbutylene, 1,2-dimethylbutylene, 1,3-dimethylbutylene, 1,2,2-trimethylpropylene, 1, 1,2-Trimethylpropene, 2-Ethylpentane, 3-Ethylpentane, Heptane, 1-Methylhexane, 2,2-Dimethylpentane, 3,3-Dimethylpentane, 4,4-Dimethylpentane, 1,2-Dimethylpentane, 1,3-Dimethylpentane, 1,4-Dimethylpentane, 1,2,3-Trimethylbutylene, 1,1,2-Trimethylbutylene, 1,1,3-Trimethylbutylene, 5-Methylheptane, 1-Methylheptane, Octylene, Nonylene, Decylene, etc., or combinations thereof.

[0094] In various implementation schemes, R 4 To R 8 Each is represented by H.

[0095] In various implementation schemes, R 9 and R 10 They can be the same or different. In various implementation schemes, R... 9 and R 10 Not both of them are H, or neither of them is H. For example, R9 and R 10 Only one of them can be H (i.e., when R...). 9 When it is H, R 10 (Not H, or vice versa). In various implementation schemes, R 9 and R 10 Only one of them is a hydrophobic tail / chain / group. For example, when R 9 When it is H, R 10 It is a hydrophobic tail / chain / group, and vice versa. In various embodiments, R 9 and R 10 All are hydrophobic tails / chains / hydrophobic groups. For example, R 9 and R 10 Each of the components independently contains an alkyl group, wherein the alkyl group contains at least 5 carbon atoms. For example, R 9 and R 10 Each of the following can be selected independently: pentyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 1,2,2-trimethylpropyl, 1,1,2-trimethylpropyl, 2-ethylpentyl 5-methylhexyl, 3-ethylpentyl, heptyl, 1-methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2,3-trimethylbutyl, 1,1,2-trimethylbutyl, 1,1,3-trimethylbutyl, 5-methylheptyl, 1-methylheptyl, octyl, nonyl, decyl, etc., or combinations thereof.

[0096] In various embodiments, the compound is represented by formula (1A):

[0097]

[0098] In various embodiments, the nanoparticle composition further comprises an ionizable lipid that is different from a compound of general formula (1). In various embodiments, the ionizable lipid that is different from a compound of general formula (1) is part of the ionizable lipid component of the composition. In various embodiments, the ionizable lipid that is different from a compound of general formula (1) is part of the total lipid / lipid derivative content / component of the composition.

[0099] In various embodiments, the ionizable lipids include [(4-hydroxybutyl)azadiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoate-1-octylnonyl ester (SM-102), and (6Z,9Z,28Z,31Z)-heptadec-6,9,28,31-tetraen-19-yl-4-(dimethyl) (D-Lin-MC3-DMA) aminobutyrate, heptadecan-9-yl-(Z)-N-(((4-(dimethylamino)butyl)thio)carbonyl)-N-(2-(non-2-en-1-yloxy)-2-oxoethyl)glycine ester (Lipid-2,2-(8,8)-4C-CH3), bis(2-butyloctyl)-10-(N-(3-(dimethylamino)propyl)nonamido)nonadecanedioate (Lipid) A9), 5-(dimethylamino)-pentanoic acid-(6Z)-1,2-di-(4Z)-4-decen-1-yl-6-dodecen-1-yl ester (CL1), 9Z,12Z-octadecadienoic acid-3-[4,4-bis(octyloxy)-1-oxobutoxy]-2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl ester (LP01), ethyl-5,5-bis[(Z)-heptadec-8-enyl]-1-(3-pyrrolid-1-ylpropyl)- 2H-imidazolium-2-carboxylic acid ester (A2-Iso5-2DC18), bis(2-(dodecyldithio)ethyl)-3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexadecyl)azadiyl)dipropionate (BAME-O16B), 2-(dioctylamino)ethylnonylhydrophosphate (9A1P9), 1,1'-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl)amino]ethyl] [2-hydroxydodecyl]amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanool (C12-200), 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione (cKK-E12), 9Z,12Z-octadecadienoic acid-1,1',1'',1'''-[(3,6-dioxo-2,5-piperazindiyl)bis(4,1-butanediylaza-2,1-ethylenediyl)] ester (OF-De) g-Lin), N1,N3,N5-tris[3-(bis(dodecylamino)propyl]-1,3,5-benzenetricarboxamide (TT3), 9,9',9'',9''',9''',9'''',9''''-[1,3,5-benzenetriyltris(carbonylimino-3,1-propanediazine)]hexa(nonanoic acid)-1,1',1'',1''',1''',1'''',1''''-hexa(1-ethylhexyl) ester (FTT5), tetra(8-methylnonyl)-3,3',3'',3'''-(((methylazadiyl)bis(propane-3,1-diyl))bis(azatriyl))tetrapropionate (306O, i10 ).

[0100]

[0101] Chemical structure of ALC-0315.

[0102] In various embodiments, compounds of general formula (1) and ionizable lipids other than said compounds are present in molar ratios of 1:about 1 to 20, 1:about 1 to 19, 1:about 1 to 18, 1:about 1 to 17, 1:about 1 to 16, 1:about 1 to 15, 1:about 1 to 14, 1:about 1 to 13, 1:about 1 to 12, 1:about 1 to 11, 1:about 1 to 10, 1:about 1 to 9, 1:about 1 to 8, 1:about 1 to 7, 1:about 1 to 6, 1:about 1 to 5, 1:about 1 to 4, 1:about 1 to 3, 1:about 1 to 2, 1:1, 1:9 or 1:4.

[0103] In various embodiments, the nanoparticle composition comprises nanoparticles formed from a compound represented by general formula (1) or its ionized form.

[0104] The term "nanoparticle" may include and / or be used interchangeably with the terms "lipid nanoparticles," "encapsulated lipid nanoparticles," "loaded lipid nanoparticles," "LNP," etc.

[0105] In various embodiments, the cryoprotectant comprises a carbohydrate source. The carbohydrate source may be selected from at least one of maltose, sucrose, trehalose, mannitol, glucose, fructose, lactose, galactose, ribose, xylose, mannose, arabinose, and combinations thereof.

[0106] In various embodiments, the amount of cryoprotectant relative to the total volume of the composition is about 1 w / v% to 30 w / v%, about 2 w / v% to 29 w / v%, about 3 w / v% to 28 w / v%, about 4 w / v% to 27 w / v%, about 5 w / v% to 26 w / v%, about 6 w / v% to 25 w / v%, about 7 w / v% to 24 w / v%, about 8 w / v% to 23 w / v%, about 9 w / v% to 22 w / v%, about 10 w / v% to 21 w / v%, about 11 w / v% to 20 w / v%, about 12 w / v% to 19 w / v%, about 13 w / v% to 18 w / v%, about 14 w / v% to 17 w / v%, about 15 w / v% to 16 w / v, or about 15.5 w / v. Advantageously, in various embodiments, the concentration of the cryoprotectant used makes the composition suitable for in vivo administration. It should be understood that when the concentration of the cryoprotectant is too high relative to the total volume of the composition, the nanoparticle solution will be hypertonic and therefore unsuitable for in vivo application.

[0107] In various embodiments, the nanoparticle composition is in lyophilized / freeze-dried or freeze-thawed form. In various embodiments, the compound represented by general formula (1) can enhance the binding of therapeutic and / or preventive and / or biological agents to the nanoparticle composition to stabilize the therapeutic and / or preventive and / or biological agents during the lyophilization process.

[0108] In various embodiments, the nanoparticle composition further comprises:

[0109] Neutral lipids / helper lipids;

[0110] Sterols; and

[0111] Lipids modified with polyethylene glycol (PEG).

[0112] In various embodiments, neutral lipids / auxiliary lipids, sterols, and PEG-modified lipids are part of the total lipids / lipid derivative content / components in the composition.

[0113] The term “polyethylene glycol (PEG) modified lipids” may include and / or may be used interchangeably with the terms “PEGylated lipids” and “lipids modified with PEG”.

[0114] In various embodiments, compounds of general formula (1) or their ionized forms, neutral lipids / auxiliary lipids, sterols, and PEG-modified lipids are mixed / dissolved in an organic solvent. In various embodiments, any organic solvent that is effective as a medium for containing reaction mixture components (e.g., reactants / substrates) can be used in embodiments of the reaction mixture of this disclosure. In various embodiments, the organic solvent is capable of substantially dissolving the components present in the mixture. The organic solvent may comprise ethanol, isopropanol, acetonitrile, ethyl acetate, methanol, tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, etc., or combinations thereof.

[0115] In various embodiments, the compound represented by general formula (1) or its ionized form, neutral lipids / auxiliary lipids, sterols, and PEG-modified lipids are mixed / dissolved in a molar ratio of about 1 to 70: 1 to 20: 10 to 60: 1 to 20. In various embodiments, when an ionizable lipid other than the compound represented by general formula (1) is not present, the compound or its ionized form, neutral lipids / auxiliary lipids, sterols, and PEG-modified lipids may be mixed / dissolved in a molar ratio of about 40 to 60: about 2 to 20: about 30 to 50: about 1 to 5. In various embodiments, when an ionizable lipid other than the compound represented by general formula (1) is present, the ionizable lipid, the compound represented by general formula (1) or its ionized form, neutral lipids / auxiliary lipids, sterols, and PEG-modified lipids are mixed / dissolved in a molar ratio of about 30 to 50: about 1 to 10: about 2 to 20: about 30 to 50: about 1 to 5. In various embodiments, compounds of general formula (1) or their ionized forms, neutral lipids / auxiliary lipids, sterols, and PEG-modified lipids are mixed / dissolved in a molar ratio of approximately 54.6:7.9:36.1:1.4. In various embodiments, ionizable lipids other than those of general formula (1), compounds of general formula (1) or their ionized forms, neutral lipids / auxiliary lipids, sterols, and PEG-modified lipids are mixed / dissolved in a molar ratio of approximately 41.7:4.6:9.4:42.7:1.6. In various embodiments, ionizable lipids other than those of general formula (1), compounds of general formula (1) or their ionized forms, neutral lipids / auxiliary lipids, sterols, and PEG-modified lipids are mixed / dissolved in a molar ratio of approximately 37.0:9.3:9.4:42.7:1.6.

[0116] In various embodiments, the amount of the compound represented by general formula (1) contained in the nanoparticle composition is about 1 mol% to about 60 mol%, about 2 mol% to about 59 mol%, about 3 mol% to about 58 mol%, or about 4 mol% to about 57 mol%, about 5 mol% to about 56 mol%, about 6 mol% to about 55 mol%, about 7 mol% to about 54 mol%, about 8 mol% to about 53 mol%, about 9 mol% to about 52 mol%, about 10 mol% to about 51 mol%, about 11 mol% to about 50 mol%, about 12 mol% to about 49 mol%, about 13 mol% to about 50 mol%, relative to the total amount of lipids / lipid derivatives present (e.g., compounds of general formula (1), neutral lipids / auxiliary lipids, sterols, (PEG) modified lipids, and ionizable lipids optionally different from formula (1)). 1% to about 48 mol%, about 14 mol% to about 47 mol%, about 15 mol% to about 46 mol%, about 16 mol% to about 45 mol%, about 17 mol% to about 44 mol%, about 18 mol% to about 43 mol%, about 19 mol% to about 42 mol%, about 20 mol% to about 41 mol%, about 21 mol% to about 40 mol%, about 22 mol% to about 39 mol%, about 23 mol% to about 38 mol%, about 24 mol% to about 37 mol%, about 25 mol% to about 36 mol%, about 26 mol% to about 35 mol%, about 27 mol% to about 34 mol%, about 28 mol% to about 33 mol%, about 29 mol% to about 32 mol%, about 30 mol% to about 31 mol%, or about 30.5 mol.

[0117] In various embodiments, when ionizable lipids other than those represented by general formula (1) are absent, the amount of the compound represented by general formula (1) contained in the nanoparticle composition is about 40 mol% to about 60 mol%, about 41 mol% to about 59 mol%, about 42 mol% to about 58 mol%, about 43 mol% to about 57 mol%, about 44 mol% to about 56 mol%, about 45 mol% to about 55 mol%, about 46 mol% to about 54 mol%, about 47 mol% to about 53 mol%, about 48 mol% to about 52 mol%, about 49 mol% to about 51 mol%, about 50 mol%, or about 54.6 mol%, relative to the total amount of lipids / lipid derivatives present (e.g., compounds of general formula (1), neutral lipids / auxiliary lipids, sterols, (PEG) modified lipids).

[0118] In various embodiments, when ionizable lipids other than those represented by general formula (1) are present, the amount of ionizable lipids other than those represented by general formula (1) contained in the nanoparticle composition is about 1 mol% to about 10 mol%, about 2 mol% to about 9 mol%, about 3 mol% to about 8 mol%, about 4 mol% to about 7 mol%, about 5 mol% to about 6 mol%, about 5.5 mol%, ... 4.6 mol% or about 9.3 mol% of compounds of general formula (1), and about 30 mol% to about 50 mol%, about 31 mol% to about 49 mol%, about 32 mol% to about 48 mol%, about 33 mol% to about 47 mol%, about 34 mol% to about 46 mol%, about 35 mol% to about 45 mol%, about 36 mol% to about 44 mol%, about 37 mol% to about 43 mol%, about 38 mol% to about 42 mol%, about 39 mol% to about 41 mol%, about 40 mol%, about 41.7 mol%, or about 37 mol%.

[0119] In various embodiments, compounds represented by general formula (1) and ionizable lipids other than those represented by general formula (1) are the main components of the nanoparticle composition.

[0120] In various embodiments, the neutral lipid / auxiliary lipid is selected from 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycerol-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycerol-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), and 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine (18:0 Diether). PC), 1-oleoyl-2-cholesterol hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C 16Lyso PC), 1,2-dilinoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidyl-sn-glycerol-3-phosphate choline, 1,2-bis(docosahexaenoic acid)-sn-glycerol-3-phosphate choline, 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (ME 16.0 PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-disarachidyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenoic acid)-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-racemic-(1-glycerol) sodium salt (DOPG), sphingomyelin, and combinations thereof.

[0121] In various embodiments, the amount of neutral lipids / auxiliary lipids contained in the nanoparticle composition is about 1 mol% to about 20 mol%, about 2 mol% to about 19 mol%, about 3 mol% to about 18 mol%, about 4 mol% to about 17 mol%, about 5 mol% to about 16 mol%, about 6 mol% to about 15 mol%, about 7 mol% to about 14 mol%, about 8 mol% to about 13 mol%, about 9 mol% to about 12 mol%, about 10 mol% to about 11 mol%, about 10.5 mol%, about 7.9 mol%, or about 9.4 mol%, relative to the total amount of lipids / lipid derivatives present (e.g., compounds of general formula (1), neutral lipids / auxiliary lipids, sterols, (PEG) modified lipids, and optionally ionizable lipids other than formula (1)).

[0122] In various implementation schemes, the sterols are selected from cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, alfalfa sterol, and combinations thereof.

[0123] In various embodiments, the amount of sterols contained in the nanoparticle composition is about 20 mol% to about 60 mol%, about 21 mol% to about 59 mol%, about 22 mol% to about 58 mol%, about 23 mol% to about 57 mol%, about 24 mol% to about 56 mol%, about 25 mol% to about 55 mol%, about 26 mol% to about 54 mol%, and about 27 mol% to about 53 mol% relative to the total amount of lipids / lipid derivatives present (e.g., compounds of general formula (1), neutral lipids / auxiliary lipids, sterols, (PEG) modified lipids, and optionally ionizable lipids other than formula (1)). %, about 28 mol% to about 52 mol%, about 29 mol% to about 51 mol%, about 30 mol% to about 50 mol%, about 31 mol% to about 49 mol%, about 32 mol% to about 48 mol%, about 33 mol% to about 47 mol%, about 34 mol% to about 46 mol%, about 35 mol% to about 45 mol%, about 36 mol% to about 44 mol%, about 37 mol% to about 43 mol%, about 38 mol% to about 42 mol%, about 39 mol% to about 41 mol%, about 39.5 mol%, about 36.7 mol% or about 36.1 mol%.

[0124] In various embodiments, the PEG-modified lipids are selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and combinations thereof. Examples of PEG-modified / PEGylated lipids include, but are not limited to: 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristoxypropyl-3-amine (PEG-c-DOMG), 3-N-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristoxypropylamine (PEG-S-DMG), PEG-DMPE (1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine-N-[(polyethylene glycol)-methoxy](sodium salt)), PEG-DPPC, PEG-DSPE lipids, and combinations thereof.

[0125] In various embodiments, the amount of PEG-modified lipids contained in the nanoparticle composition is about 1 mol% to about 5 mol%, about 1 mol% to about 4 mol%, about 1 mol% to about 3 mol%, about 1 mol% to about 2 mol%, about 1.6 mol%, or about 1.4 mol%, relative to the total amount of lipids / lipid derivatives present (e.g., compounds of general formula (1), neutral lipids / auxiliary lipids, sterols, (PEG)-modified lipids, and optionally ionizable lipids other than formula (1)).

[0126] In various implementations, the therapeutic, preventative, and / or biological agents are provided in an aqueous buffer solution. The aqueous buffer solution may be sodium acetate.

[0127] In various embodiments, the N:P or N / P ratio of the nanoparticles (i.e., the molar ratio of ionizable nitrogen atoms in a compound (e.g., an ionizable lipid compound) to phosphate groups in a therapeutic, preventative, and / or biological reagent (e.g., nucleic acid) is from about 2:1 to about 40:1. The N:P or N / P ratio of the nanoparticles can be from about 2:1 to about 40:1, from about 3:1 to about 39:1, from about 4:1 to about 38:1, from about 5:1 to about 37:1, from about 6:1 to about 36:1, from about 7:1 to about 35:1, from about 8:1 to about 34:1, from about 9:1 to about 33:1, from about 10:1 to about 32:1, from about 11:1 to about 31:1, from about 12:1 to about 30:1, from about 13:1 to about 29:1, from about 14:1 to about 28:1, from about 15:1 to about 27:1, from about 16:1 to about 26:1, from about 17:1 to about 25:1, from about 18:1 to about 24:1, from about 19:1 to about 23:1, from about 20:1 to about 22:1 or about 21:1.

[0128] In various implementations, the therapeutic and / or preventive and / or biological agents are encapsulated at an encapsulation rate of at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%.

[0129] In various embodiments, the nanoparticles have a zeta potential of about -10 mV to about +10 mV, about -10 mV to about 10 mV, about -9 mV to about 9 mV, about -8 mV to about 8 mV, about -7 mV to about 7 mV, about -6 mV to about 6 mV, about -5 mV to about 5 mV, about -4 mV to about 4 mV, about -3 mV to about 3 mV, about -2 mV to about 2 mV, about -1 mV to about 1 mV, or about 0 mV in various embodiments. Advantageously, in various embodiments, the nanoparticles have a substantially neutral surface charge, which makes the nanoparticles suitable for / suitable for in vivo administration.

[0130] In various embodiments, the polydispersity index (PDI) of the nanoparticles is about 0.01 to about 1.0, about 0.05 to about 0.95, about 0.1 to about 0.9, about 0.15 to about 0.85, about 0.2 to about 0.8, about 0.25 to about 0.75, about 0.3 to about 0.7, about 0.35 to about 0.65, about 0.4 to about 0.6, about 0.45 to about 0.55, or about 0.5.

[0131] In various embodiments, the nanoparticles have a particle size (diameter) of about 50 nm to about 1200 nm, about 100 nm to about 1150 nm, about 150 nm to about 1100 nm, about 200 nm to about 1050 nm, about 250 nm to about 1000 nm, about 300 nm to about 950 nm, about 350 nm to about 900 nm, about 400 nm to about 850 nm, about 450 nm to about 800 nm, about 500 nm to about 750 nm, about 550 nm to about 700 nm, about 600 nm to about 650 nm, or about 625 nm. Advantageously, in various embodiments, larger-sized nanoparticles can be used as nasal spray formulations.

[0132] In various implementations, the cellular translation efficiency using FLuc mRNA is at least about 40.0%, at least about 50.0%, at least about 60.0%, at least about 70.0%, at least about 80.0%, at least about 90.0%, at least about 95.0%, at least about 96.0%, at least about 97.0%, at least about 98.0%, at least about 99.0%, at least about 99.5%, at least about 99.9%, or about 100% of the cellular translation efficiency mediated by uncryopyrohydrated LNP.

[0133] In various embodiments, the cell transfection efficiency of the nanoparticles is comparable to, or not less than, or greater than, the cell transfection efficiency of the corresponding nanoparticles using only ALC-0315 as an ionizable lipid under similar conditions. For example, the cell transfection efficiency may be at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% to about 800%, at least about 150% to about 750%, at least about 200% to about 700%, at least about 250% to about 650%, at least about 300% to about 600%, at least about 350% to about 550%, at least about 400% to about 500%, or at least about 450%.

[0134] In various embodiments, the nanoparticle composition is biocompatible, meaning that when used in humans or animals, it is compatible with or partially compatible with a biological system and does not substantially or significantly cause adverse physiological reactions, such as toxic reactions / responses (e.g., cytotoxicity), adverse immune reactions / responses, damage, etc. In various embodiments, the nanoparticle composition is substantially free of substances that would cause adverse physiological reactions. Advantageously, the nanoparticles (e.g., lipid nanoparticles) in the nanoparticle composition are capable of effectively binding therapeutic agents, prophylactic agents, and / or biological agents (e.g., RNA) and / or providing high transfection efficiency without causing / inducing substantial or any cytotoxicity.

[0135] Process for preparing compounds of general formula (1)

[0136] A method for preparing compounds represented by general formula (1) of this disclosure is provided, the method comprising:

[0137] (ai) React an amine compound represented by general formula (2) with a cyclic anhydride represented by general formula (3) to obtain a first intermediate compound represented by general formula (4) containing a carboxylic acid ester group:

[0138] ;

[0139] (a-ii) In the presence of a coupling agent, the first intermediate compound represented by general formula (4) is reacted with N-hydroxysuccinimide (NHS) to obtain the second intermediate compound represented by general formula (5) containing an amide group:

[0140] ;

[0141] (a-iii) React the second intermediate compound represented by general formula (5) with an amine compound represented by general formula (6) to obtain the compound represented by general formula (1):

[0142]

[0143] Where R 1 To R 10 It contains one or more features and / or shares one or more properties similar to those described above (e.g., as defined in general formula (1)); and

[0144] (a-iv) Optionally –NR 1 R 2 It ionizes into positively charged groups.

[0145] Advantageously, the embodiments of this method are easy to implement and have low production / manufacturing costs (i.e., cost-effective) because they can be carried out in just three synthetic / reaction steps. It should be understood that currently available or known methods in the art require at least five or six synthetic / reaction steps to produce ionizable lipid compounds. Advantageously, the embodiments of this method are scalable and / or have substantially high scalability.

[0146] In various implementations, the cyclic anhydrides represented by general formula (3) include succinic anhydride, etc.

[0147] In various embodiments, the coupling agent comprises a carbodiimide. For example, the coupling agent may be 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), or combinations thereof.

[0148] In various embodiments, the amine compounds represented by general formula (6) include ethylenediamine (EDA), N,N-dimethyldipropylenetriamine, or combinations thereof.

[0149] In various embodiments, steps (a-iii) include reacting the amine compound represented by general formula (6) with the second intermediate compound represented by general formula (5) in a molar ratio of about 1:1 to about 5:1. Steps (a-iii) may include reacting the amine compound represented by general formula (6) with the second intermediate compound represented by general formula (5) in a molar ratio of about 1:1 to about 5:1, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1. It should be understood that the amine compound represented by general formula (6) may be provided in excess (e.g., slightly excess) to ensure that the desired molar ratio is achieved between the amine compound represented by general formula (6) and the second intermediate compound represented by general formula (5).

[0150] In various embodiments, reaction steps (a-iii) include adding the second intermediate compound represented by general formula (5) to the amine compound represented by general formula (6) dropwise.

[0151] In various implementations, reaction steps (a-i), (a-ii), and / or (a-iii) include the following steps: dispersion, mixing, stirring, dissolving, sonication, and / or ultrasonic treatment.

[0152] In various embodiments, reaction steps (a-i), (a-ii), and / or (a-iii) are carried out in the presence of an organic solvent. In various embodiments, any organic solvent that can be effectively used as a medium containing the components of the reaction mixture (e.g., reactants / substrates) can be used in embodiments of the reaction mixture of this disclosure. In various embodiments, the organic solvent is capable of substantially dissolving the components present in the reaction mixture. The organic solvent can be a dry organic solvent or an anhydrous organic solvent, such as dry dichloromethane (DCM) or anhydrous dichloromethane (DCM).

[0153] In various embodiments, reaction steps (a-i), (a-ii), and / or (a-iii) are carried out in an inert gas environment. For example, the steps of dispersion, mixing, and / or stirring may be carried out in the presence of an inert gas such as argon or nitrogen, or in the absence of a reactive gas such as oxygen (e.g., dissolved oxygen).

[0154] In various implementations, the duration of reaction steps (ai), (a-ii) and / or (a-iii) is from about 1 hour to about 72 hours, from about 2 hours to about 60 hours, from about 3 hours to about 48 hours, from about 4 hours to about 36 hours, from about 5 hours to about 24 hours, or from about 6 hours to about 12 hours.

[0155] In various embodiments, reaction steps (ai) and / or (a-ii) are optionally carried out at room temperature, for example, about 20°C to about 30°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, or about 30°C.

[0156] In various embodiments, reaction steps (a-iii) are optionally carried out at temperatures of about -30°C to about -80°C, about -35°C to about -75°C, about -40°C to about -70°C, about -45°C to about -65°C, about -50°C to about -60°C, or about -55°C, for example, to control reaction kinetics. For example, the reaction steps may be carried out in a dry ice bath.

[0157] In various implementation schemes, the method also includes:

[0158] (bi) The step of separating the first intermediate compound after step (ai);

[0159] (b-ii) The step of separating the second intermediate compound after step (a-ii); and

[0160] (b-iii) The step of separating the compound represented by general formula (1) after step (a-iii).

[0161] In various embodiments, the separation step includes one or more of the following steps: redissolving, purifying, centrifuging, quenching, washing, precipitating, and / or recrystallizing the first intermediate compound, the second intermediate compound, and / or the compound represented by general formula (1). The purification, centrifugation, quenching, and / or washing steps may be repeated at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least fifteen times, or at least twenty times using a washing medium. In various embodiments, the separation step is performed to remove byproducts from the first intermediate compound, the second intermediate compound, and / or the compound represented by general formula (1). In various embodiments, the washing medium includes an aqueous medium / aqueous solution, such as a salt solution or deionized water. The salt solution may be a bicarbonate such as sodium bicarbonate, or a chloride such as sodium chloride (salt water). In various embodiments, the salt solution includes a high-concentration salt solution / saturated salt solution.

[0162] In various embodiments, the method further includes one or more of the following post-reaction steps: drying the first intermediate compound, the second intermediate compound, and / or the compound represented by general formula (1) under vacuum, optionally at low temperature (e.g., freeze-drying). The drying step may be carried out in the presence of a desiccant such as magnesium sulfate, sodium sulfate, calcium chloride, or combinations thereof.

[0163] In various implementations, steps (a-iv) are present and steps (a-iv) are performed at a pH of about 3 to about physiological pH. In various embodiments, steps (a-iv) are performed at pH values ​​of about 3.0 to about 7.8, about 3.1 to about 7.7, about 3.2 to about 7.6, about 3.3 to about 7.5, about 3.4 to about 7.4, about 3.5 to about 7.3, about 3.6 to about 7.2, about 3.7 to about 7.1, about 3.8 to about 7.0, about 3.9 to about 6.9, about 4.0 to about 6.8, about 4.1 to about 6.7, about 4.2 to about 6.6, about 4.3 to about 6.5, about 4.4 to about 6.4, about 4.5 to about 6.3, about 4.6 to about 6.2, about 4.7 to about 6.1, about 4.8 to about 6.0, about 4.9 to about 5.9, about 5.0 to about 5.8, about 5.1 to about 5.7, about 5.2 to about 5.6, about 5.3 to about 5.5, or about 5.4.

[0164] Process for preparing nanoparticle compositions

[0165] A method for preparing the nanoparticle composition of this disclosure is provided, the method comprising:

[0166] Prepare an aqueous composition comprising therapeutic and / or preventive and / or biological reagents;

[0167] Aqueous compositions are mixed with compounds of general formula (1), auxiliary lipids, sterols and PEG-modified lipids to obtain nanoparticles encapsulating therapeutic and / or preventive and / or biological reagents.

[0168] Add cryoprotectants to nanoparticles.

[0169] In various embodiments, the step of preparing the aqueous composition includes mixing the therapeutic and / or preventive and / or biological reagents in an aqueous buffer solution. The aqueous buffer solution may be sodium acetate (10 mM).

[0170] In various implementations, the mixing step is carried out at a pH of about 2 to about 6, about 2.5 to about 5.5, about 3 to about 5, about 3.5 to about 4.5, or about 4.

[0171] In various embodiments, the compositions disclosed herein comprise an organic phase (e.g., ethanol). In various embodiments, the aqueous compositions comprise an aqueous phase. In various embodiments, the step of mixing the aqueous composition with the composition comprises mixing the aqueous composition with the composition described herein at a volume ratio of about 10:1 to about 1:1 of aqueous phase to organic phase. For example, the aqueous phase and organic phase may be mixed at a volume ratio of about 10:1 to about 1:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, or about 2:1.

[0172] In various embodiments, the step of mixing the aqueous composition with the composition includes injecting (e.g., direct injection) the composition into the aqueous composition.

[0173] In various embodiments, the step of mixing the aqueous composition with the composition is carried out in the presence of another ionizable lipid different from the compound of general formula (1). In various embodiments, the compound of general formula (1) and the ionizable lipid different from said compound are mixed together in a molar ratio of 1:about 1 to 20, 1:about 1 to 19, 1:about 1 to 18, 1:about 1 to 17, 1:about 1 to 16, 1:about 1 to 15, 1:about 1 to 14, 1:about 1 to 13, 1:about 1 to 12, 1:about 1 to 11, 1:about 1 to 10, 1:about 1 to 9, 1:about 1 to 8, 1:about 1 to 7, 1:about 1 to 6, 1:about 1 to 5, 1:about 1 to 4, 1:about 1 to 3, 1:about 1 to 2, 1:1, 1:9, or 1:4.

[0174] In various embodiments, the step of mixing the aqueous composition with the composition includes micromixing, for example, microfluidic mixing using a microfluidic device. Micromixing can be performed passively using a passive micromixer, such as a T-shaped or Y-shaped microfluidic mixer, a parallel lamination mixer, a sequential mixer, a focused enhancement mixer, or a droplet micromixer. Micromixing can also be performed actively using external forces, such as pressure fields, electro-driven mixing, dielectrophoresis, electrowetting, magnetohydrodynamic mixing, or ultrasound. Advantageously, since microfluidic mixing involves mixing two compositions (i.e., the aqueous composition and the composition of this disclosure) in a controlled manner and / or at a specified / fixed / controlled mixing ratio, the interaction between the two compositions (e.g., between ionizable lipids and therapeutic, preventative, and / or biological agents) is modulated, resulting in nanoparticles with smaller particle size and / or narrower size distribution or uniformity (e.g., smaller PDI).

[0175] In various embodiments, the method further includes removing the organic phase (e.g., ethanol) and unencapsulated components after nanoparticle formation following step (c-ii). For example, removing the organic phase may include diluting the resulting LNP in 0.9% saline, PBS buffer, or Tris buffer, and subsequently centrifuging and ultrafiltration the resulting LNP to remove any residual organic solvent present. Alternatively, removing the organic phase may include dialysis of the nanoparticles to remove any residual organic solvent present. Advantageously, removing the organic phase by dialysis can improve the encapsulation efficiency of therapeutic and / or preventative and / or biological reagents.

[0176] In various embodiments, carriers, nanocarriers, or delivery systems / delivery carriers comprising the nanoparticle compositions of the present disclosure are also provided.

[0177] In various embodiments, vaccine compositions comprising the nanoparticle composition of the present disclosure are also provided.

[0178] In various embodiments, carriers, nanocarriers, delivery systems / delivery carriers, and nanoparticle compositions of this disclosure are also provided for use in pharmaceuticals (e.g., for the treatment or prevention of one or more diseases, disorders, or conditions mentioned herein).

[0179] In various embodiments, carriers, nanocarriers, delivery systems / carriers, and nanoparticle compositions of this disclosure are also provided for the treatment or prevention of diseases, disorders, or conditions; the use of said carriers, nanocarriers, delivery systems / carriers, and nanoparticle compositions in the manufacture of medicaments for the treatment or prevention of diseases, disorders, or conditions; and / or methods for treating or preventing diseases, disorders, or conditions, said methods comprising the step of administering (e.g., in a therapeutically effective amount) said carriers, nanocarriers, delivery systems / carriers, and nanoparticle compositions to an object in need (e.g., vertebrates such as humans or large veterinary mammals (e.g., horses, cattle, deer, sheep, llamas, goats, pigs)). Diseases, disorders, or conditions may be selected from infectious diseases / contact-transmitted diseases, viral infections (i.e., diseases caused by viruses), bacterial infections (i.e., diseases caused by bacteria), fungal infections (i.e., diseases caused by fungi), cancer, respiratory diseases, etc., or combinations thereof. In various embodiments, the type of disease may vary depending on the therapeutic, preventative, and / or biological goods being packaged. In various implementations, the disease, impairment, or symptom is mediated by a coronavirus (e.g., severe acute respiratory syndrome coronaviruses such as SARS-CoV-2 or SARS-CoV-1). For example, the disease, impairment, or symptom could be SARS-CoV-2 coronavirus disease.

[0180] In various embodiments, carriers, nanocarriers, delivery systems / delivery carriers, and nanoparticle compositions of this disclosure are also provided for encapsulating and / or delivering therapeutic, preventative, and / or biological agents to subjects, cells, cytosols, tissues, or organs (e.g., mammalian cells, cytosols, tissues, or organs). These carriers, nanocarriers, delivery systems / delivery carriers, and nanoparticle compositions are prepared for encapsulating and / or delivering therapeutic, preventative, and / or biological agents to subjects, cells, cytosols, tissues, or organs (e.g., mammalian cells, cytosols, tissues, or organs). Use in pharmaceuticals (animal cells, cytosols, tissues, or organs), and / or methods of delivering therapeutic, preventative, and / or biological agents to a subject, cell, cytosol, tissue, or organ (e.g., mammalian cells, cytosols, tissues, or organs), said methods comprising the steps of administering (e.g., in a therapeutically effective amount) the carrier, nanocarrier, delivery system / delivery carrier, or nanoparticle composition to a subject (e.g., a vertebrate, such as a human or a large veterinary mammal (e.g., a horse, cattle, deer, sheep, llama, goat, or pig)) in a desired manner.

[0181] In various embodiments, carriers, nanocarriers, delivery systems / carriers, and nanoparticle compositions of this disclosure are also provided for inducing an immune response in a subject (e.g., a vertebrate, such as a human or a large veterinary mammal (e.g., a horse, cattle, deer, sheep, llama, goat, pig)), use of the carriers, nanocarriers, delivery systems / carriers, and nanoparticle compositions in the preparation of a medicament for inducing an immune response in a subject, and / or a method for inducing an immune response in a subject, the method comprising the step of administering (e.g., in a therapeutically effective amount) the carriers, nanocarriers, delivery systems / carriers, and nanoparticle compositions to a subject in need. In various embodiments, an immune response in a subject is induced by administering the nanoparticle composition to the subject. In various embodiments, by inducing an immune response in a subject, the subject is protected from various diseases, barriers, or conditions, such as infectious diseases / contact-transmitted diseases, viral infections (i.e., diseases caused by viruses), bacterial infections (i.e., diseases caused by bacteria), fungal infections (i.e., diseases caused by fungi), cancer, respiratory diseases, etc., or combinations thereof, as mentioned herein. In various implementation schemes, the type of disease may vary depending on whether the packaged goods are therapeutic, prophylactic, and / or biological. Carriers, nanocarriers, delivery systems / carriers, and nanoparticle compositions may be delivered to the recipient in the form of a vaccine or as a component of a vaccine.

[0182] In various implementations, the disease, impairment, or symptom is mediated by a coronavirus (e.g., severe acute respiratory syndrome coronaviruses such as SARS-CoV-2 or SARS-CoV-1). For example, the disease, impairment, or symptom could be SARS-CoV-2 coronavirus disease.

[0183] In various embodiments, the carriers, nanocarriers, delivery systems / delivery carriers, compounds or their ionized forms, and nanoparticle compositions prepared by embodiments of the methods of this disclosure include one or more of the following features or properties: broad applicability (e.g., for encapsulation, delivery, and / or transfection of a variety of therapeutic, preventative, and / or biological agents), nanoscale size, substantially neutral surface charge, high encapsulation efficiency (e.g., >20%), high transfection efficiency, high stability, low toxicity (e.g., low cytotoxicity), and low production / synthesis cost, thus making them suitable for in vivo administration where efficient cellular uptake and / or gene transfection are required. Attached Figure Description

[0184] Figure 1Three-phase diagrams of water under different temperature and pressure conditions are shown to illustrate the principle of freeze-drying / lyophilization. In the freeze-drying / lyophilization step, water evaporates from the sample through sublimation and desorption processes. This step involves freezing the sample, followed by primary and secondary drying, as described in Section 1.5 of Example 1.

[0185] Figure 2 An experimental apparatus 100 for a freeze-drying / lyophilization process according to various embodiments of the present disclosure is shown. A sample 102 is connected to a freeze dryer 106 via a vacuum pump line 104 and immersed in a dry ice tank 108 to maintain its temperature during the process.

[0186] Figure 3 Freeze-dried / lyophilized mRNA LNP formulations with different formulations according to various embodiments of this disclosure are shown. All samples contain 20% sucrose and exhibit a spongy matrix appearance after freeze-drying.

[0187] Figure 4 This is a gel electrophoresis diagram showing the electrophoretic mobility of mRNA in LNPs prepared by ALC-0315 before freeze-drying, according to various embodiments disclosed herein, without sucrose and with concentrations of 5%, 10%, and 20% sucrose. Electrophoresis was performed at 100V for 20 minutes. The gel contains a DNA ladder, a RiboRuler high-range RNA ladder (Thermo Scientific), and free CleanCap. ® Firefly luciferase mRNA (300 ng) was used as a control.

[0188] Figure 5 This is a gel electrophoresis diagram showing the electrophoretic mobility of mRNA in LNPs prepared by ALC-0315 after 4 days of freeze-drying (Groups 2 to 5), including sucrose-free, sucrose-containing, 5%, 10%, and 20% sucrose concentrations. Electrophoretic mobility of mRNA in undelvulsified LNPs prepared by ALC-0315 is also included in lane 3 (Group 1). Electrophoresis was performed at 100V for 20 minutes. The gels contained RiboRuler high-range RNA Ladder (Thermo Scientific) and free CleanCap. ® Firefly luciferase mRNA (300 ng) was used as a control.

[0189] Figure 6This is a gel electrophoresis diagram showing the electrophoretic mobility of mRNA in sucrose-containing LNPs prepared by DTD-NH2 prior to freeze-drying, according to various embodiments of this disclosure. Electrophoresis was performed at 100V for 20 minutes. The gel contained DNA ladders and free CleanCaps. ® Firefly luciferase mRNA (1000 ng and 500 ng) served as controls.

[0190] Figure 7 This is a gel electrophoresis diagram showing the electrophoretic mobility of mRNA in LNPs prepared by DTD-NH2, including those without sucrose and those containing 5%, 10%, and 20% sucrose, after 4 days of freeze-drying (Groups 2 to 5). The electrophoretic mobility of mRNA in unfreezed LNPs prepared by DTD-NH2 is also included in lane 3 (Group 1). Electrophoresis was performed at 100V for 20 minutes. The gel contains DNA ladders and free CleanCap. ® Firefly luciferase mRNA (300 ng) was used as a control.

[0191] Figure 8 This diagram illustrates the encapsulation rates of mRNA in LNPs prepared from ALC-0315 or DTD-NH2 alone, without sucrose and containing 5%, 10%, and 20% sucrose, after a 4-day freeze-drying process, according to various embodiments of this disclosure. The encapsulation rate of mRNA in unfreezed LNPs prepared from ALC-0315 or DTD-NH2 alone is also shown as a control.

[0192] Figure 9 This diagram illustrates the cell viability of HeLa cells after 4 days of freeze-drying, according to various embodiments of the present disclosure, when incubated with mRNA from LNPs prepared by ALC-0315 only or TD-NH2 only, containing 5%, 10%, and 20% sucrose. Cell viability of untreated HeLa cells and HeLa cells incubated with mRNA from unfrozen LNPs prepared by ALC-0315 only or DTD-NH2 only are also shown as controls.

[0193] Figure 10This diagram illustrates the mRNA transfection of HeLa cells treated with LNPs prepared from ALC-0315 or DTD-NH2 alone (without sucrose and with concentrations of 5%, 10%, and 20% sucrose) four days after the freeze-drying process, according to various embodiments of this disclosure. mRNA transfection of untreated HeLa cells and mRNA transfection of HeLa cells treated with unfrozen LNPs prepared from ALC-0315 or DTD-NH2 alone are also shown as controls.

[0194] Figure 11 This diagram illustrates the encapsulation rates of mRNA in LNPs prepared from ALC-0315 alone or DTD-NH2 alone, with and without sucrose concentrations of 5%, 10%, and 20%, after one day of freeze-thaw or freeze-drying processes, according to various embodiments of this disclosure. The encapsulation rate of mRNA in unfreezed LNPs prepared from ALC-0315 alone or DTD-NH2 alone is also shown as a control.

[0195] Figure 12 This diagram illustrates the cell viability of HeLa cells, according to various embodiments of the present disclosure, after one day of freeze-thaw or freeze-drying processes, when incubated with mRNA from LNPs prepared by ALC-0315 only or DTD-NH2 only, containing 5%, 10%, and 20% sucrose. Cell viability of untreated HeLa cells and HeLa cells incubated with mRNA from unfreeze-dried LNPs prepared by ALC-0315 only or DTD-NH2 only are also shown as controls.

[0196] Figure 13 This diagram illustrates the mRNA transfection of HeLa cells treated with LNPs prepared from ALC-0315 or DTD-NH2 alone (without sucrose and with concentrations of 5%, 10%, and 20% sucrose) four days after the freeze-drying process, according to various embodiments of this disclosure. mRNA transfection of untreated HeLa cells and mRNA transfection of HeLa cells treated with unfrozen LNPs prepared from ALC-0315 or DTD-NH2 alone are also shown as controls.

[0197] Figure 14 This is a graph showing the particle size distribution of mRNA LNPs prepared from ALC-0315 alone before freeze-drying, according to various embodiments of the present disclosure.

[0198] Figure 15This is a graph showing the particle size distribution of mRNA LNPs prepared from ALC-0315 and DTD-NH2 in a molar ratio of 9:1 before freeze-drying, according to various embodiments of the present disclosure.

[0199] Figure 16 This is a graph showing the particle size distribution of mRNA LNPs prepared from ALC-0315 and DTD-NH2 in a molar ratio of 8:2 prior to freeze-drying, according to various embodiments of the present disclosure.

[0200] Figure 17 This is a graph showing the particle size distribution of sucrose-free mRNA LNPs prepared from ALC-0315 alone, without freeze-drying, according to various embodiments of the present disclosure.

[0201] Figure 18 This is a graph showing the particle size distribution of 20% sucrose-containing mRNA LNPs prepared from ALC-0315 alone, without freeze-drying, according to various embodiments of the present disclosure.

[0202] Figure 19 This is a graph showing the particle size distribution of 20% sucrose-containing mRNA LNPs prepared from ALC-0315 alone after freeze-thaw according to various embodiments of the present disclosure.

[0203] Figure 20 This is a graph showing the particle size distribution of mRNA LNPs prepared from ALC-0315 and DTD-NH2 in a 9:1 molar ratio after freeze-thaw according to various embodiments of the present disclosure.

[0204] Figure 21 This is a graph showing the particle size distribution of mRNA LNPs prepared from ALC-0315 and DTD-NH2 in a molar ratio of 8:2 after freeze-thaw according to various embodiments of the present disclosure.

[0205] Figure 22 This is a graph showing the particle size distribution of sucrose-free mRNA LNPs prepared from ALC-0315 alone after freeze-drying, according to various embodiments of the present disclosure.

[0206] Figure 23 This is a graph showing the particle size distribution of mRNA LNPs prepared from sucrose-free ALC-0315 and sucrose-free DTD-NH2 in a molar ratio of 9:1 after freeze-drying according to various embodiments of the present disclosure.

[0207] Figure 24 This is a graph showing the particle size distribution of sucrose-free mRNA LNPs prepared from ALC-0315 and DTD-NH2 in a molar ratio of 8:2 after freeze-drying according to various embodiments of the present disclosure.

[0208] Figure 25 This is a graph showing the particle size distribution of mRNA LNPs containing 20% ​​sucrose prepared from ALC-0315 alone after freeze-drying, according to various embodiments of the present disclosure.

[0209] Figure 26 This is a graph showing the particle size distribution of 20% sucrose-containing mRNA LNPs prepared from ALC-0315 and DTD-NH2 in a 9:1 molar ratio after freeze-drying according to various embodiments of the present disclosure.

[0210] Figure 27 This is a graph showing the particle size distribution of 20% sucrose-containing mRNA LNPs prepared from ALC-0315 and DTD-NH2 in a molar ratio of 8:2 after freeze-drying according to various embodiments of the present disclosure.

[0211] Figure 28 This diagram illustrates luciferase expression mediated by different mRNA LNP formulations in Balb / c mice according to various embodiments of this disclosure. In vivo studies were performed in Balb / c mice via intravenous injection (tail vein), with each mouse receiving a dose of 1 µg mRNA / 100 µL. Imaging was performed at 6 and 24 hours after mRNA LNP administration. The ALC-0315 group consisted of 6 mice before lyophilization (freeze-drying) and 7 mice after lyophilization, while the ALC-0315:DTD-NH2(9:1) group consisted of 7 mice after lyophilization. A "blank control" group served as a control group without any treatment.

[0212] Figure 29 The following are examples of DTD-NH2 prepared according to various embodiments of the method of this disclosure in CD3OD. 1 H NMR spectrum.

[0213] Example

[0214] Exemplary embodiments of this disclosure will be illustrated by the following examples, tables, and, if applicable, accompanying drawings. Figure 1 It is better understood and obvious to those skilled in the art.

[0215] As shown in Scheme 1, the freeze-drying / lyophilization process involves freezing the sample, followed by primary drying via sublimation and secondary drying via desorption to remove moisture. However, the freeze-drying process can generate various stresses that can compromise the stability of NPs. Examples of instability include NP aggregation (leading to increased size), remodeling of lipid components within LNPs, and reduced LNP transfection efficiency.

[0216]

[0217] Option 1. The basic principles, procedures, and potential drawbacks of freeze-drying.

[0218] The following examples demonstrate that nanoparticle compositions prepared according to various embodiments of the present disclosure can mitigate the negative effects typically experienced during freeze-drying / lyophilization, thereby providing freeze-dried / lyophilized compositions with enhanced stability.

[0219] It should be understood that other modifications related to structural, biological, and / or chemical changes may be made without departing from the scope of this disclosure. Exemplary embodiments are not necessarily mutually exclusive, as some embodiments may be combined with one or more other embodiments to form new exemplary embodiments. Exemplary embodiments should not be construed as limiting the scope of this disclosure.

[0220] Example 1: Materials and Methods

[0221] 1.1. Materials

[0222] Chemical reagents used for lipid synthesis were purchased from Sigma-Aldrich and used as is unless otherwise specified. Ditetradecylamine (DTDA) was purchased from Ambeed, Inc. (Arlington Heights, IL, USA). 1,2-Distearayoyl-sn-glycerol-3-phosphocholine (DSPC), cholesterol, ALC-0315, and ALC-0159 were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Sodium acetate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Agarose and Tris-acetate-EDTA were purchased from 1st Base (Singapore). GelStar nucleic acid gel staining reagent was purchased from Lonza (Basel, Switzerland). Triton®-X100 and Tris-EDTA were purchased from Promega (Madison, WI, USA). AlamarBlue, gel loading buffer, and Pierce firefly luciferase glow detection kit were purchased from Invitrogen (Waltham, MA, USA). The other reagents used were of analytical grade.

[0223] 1.2. Synthesis of DTD-NH2 (Scheme 2)

[0224] Scheme 2 provides an exemplary overall scheme for synthesizing compounds of general formula (1) or their ionized forms according to various embodiments of the present disclosure.

[0225] The synthesis method of DTD-NH2 is given below as a typical example.

[0226]

[0227] Chemical structure of DTD-NH2.

[0228]

[0229] Scheme 2. Chemical structure and synthesis steps of DTD-NH2.

[0230] Synthesis of DTD-COONa: In a 500 mL three-necked round-bottom flask, ditetradecylamine (DTDA, 3.44 g, 8 mmol), succinic anhydride (2.4 g, 24 mmol), and triethylamine (5.6 mL, 40 mmol) were dissolved in 300 mL of dry DCM, and the reaction solution was stirred overnight under N2 atmosphere. The solution was then transferred to a 1 L separatory funnel and washed three times with saturated sodium bicarbonate solution (100 mL). The organic phase was dried over MgSO4 for 4 to 5 hours. Finally, the solution was filtered by suction filtration, the filtrate was concentrated to dryness, and dried under vacuum to obtain DTD-COONa as a grayish-white solid (yield 93%). 1 H NMR (400MHz, CDCl3, 22℃): δ3.31(dt, 4H, -CON(CH2-)2), 2.67(s, 4H, -CH2CH2COONa), 1.53(m, br, 4H, -CON(CH2CH2-)2), 1.26(s, 44H, -(CH2) 11 CH3), 0.88 (t, 6H, -CH3).

[0231] Synthesis of DTD-NHS: In a 100 mL three-necked round-bottom flask, DTD-COONa (4.85 g, 9.13 mmol) and N-hydroxysuccinimide (NHS, 2.88 g, 25 mmol) were dissolved in 60 mL of dry DCM, followed by the addition of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC•HCl, 3.83 g, 20 mmol). The solution was stirred overnight under N2 atmosphere. It was then concentrated to dryness, and the residue was redissolved in a mixture of THF and deionized (DI) water (v / v = 1:1). An additional 300 mL of DI water was added, and the product was precipitated in an ice bath. The precipitate was centrifuged and washed three times with cold DI water. Finally, the wet solid was freeze-dried to obtain DTD-NHS as a white powder (yield 83%). 1H NMR (400MHz, CDCl3, 22°C): δ3.30 (dt, 4H, -CON(CH2-)2), 3.01 (t, 2H, -CH2- of NHS), 2.83 (s, 4H, -C OCH2CH2CON-), 2.72(t, 2H, -CH2- of NHS), 1.52(m, br, 4H, -CON(CH2CH2-)2), 1.26(s, 44H, -(CH2) 11 CH3), 0.88 (t, 6H, -CH3).

[0232] Synthesis of DTD-NH2: In a 100 mL single-necked round-bottom flask, ethylenediamine (EDA, 53 µL, g, 0.79 mmol) was dissolved in 15 mL of dry DCM, and the solution was cooled in a dry ice bath under N2 atmosphere for 30 min. DTD-NHS (0.455 g, 0.75 mmol) was dissolved in 15 mL of DCM, and this solution was added dropwise to the above EDA solution. The dry ice bath was then removed, and the reaction solution was stirred for another 2 hours. The mixture was transferred to a 250 mL separatory funnel, and 70 mL of DCM was added. The solution was washed three times with brine (20 mL) and dried overnight with MgSO4. Finally, the mixture was filtered by suction filtration, the filtrate was concentrated to dryness, and dried under vacuum to give DTD-NH2, which was a grayish-white viscous solid (yield 63%). 1 H NMR (400MHz, CD3OD, 22℃): δ2.80(t,2H,-CH2NH2), 2.69(m,2H,-NHCOCH2CH2CON-), 2.48(t,2H,-NHCOC H2CH2CON-), 1.62(m, br, 2H, -CON(CH2CH2-)2), 1.52(m, br, 2H, -CON(CH2CH2-)2), 1.29(s, 44H, -(CH2) 11 CH3), 0.90(t, 6H, -CH3).

[0233] 1.3. Nuclear magnetic resonance spectroscopy (NMR)

[0234] DTD-NH2 was characterized by nuclear magnetic resonance spectroscopy (NMR). DTD-NH2 and its precursors were recorded on a Bruker Advance 400 NMR spectrometer (400 MHz) under the following conditions: ambient temperature, acquisition time of 3.2 s, pulse repetition time of 2.0 s, pulse width of 30°, spectral width of 5208 Hz, and data point density of 32 K. 1 1H-NMR spectrum. Chemical shifts are based on their respective solvent peaks (δ = 3.31 ppm for CD3OD).

[0235] 1.4. Preparation of mRNA-loaded lipid nanoparticles (mRNA LNP)

[0236] CleanCap ® Firefly luciferase mRNA was purchased from Trilink Biotechnologies and encapsulated in all LNP formulations. LNPs were prepared using a controlled mixing process between an aqueous phase containing mRNA and an ethanol-lipid mixture at a 3:1 volume ratio and a flow rate of 12 mL / min using a NanoAssemblr Ignite microfluidic system (Precision NanoSystems Inc.). In the aqueous phase, the mRNA was diluted in 10 mM sodium acetate buffer at pH 4. The lipid mixture consisted of either the ionizable lipid ALC-0315 (MedChem Express) or DTD-NH2 (a self-made product; MW=551; structure as shown). Figure 1 The LNPs were composed of cholesterol (MedChem Express), the accessory lipid 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC; MedChemExpress), and the PEG-lipid ALC-0159 (MedChem Express), with molar ratios of 46.4:9.3:42.7:1.6 for ALC-0315 LNPs and 54.6:7.9:36.1:1.4 for DTD-NH2 LNPs. The N / P ratio of ionizable lipids to mRNA was 6 throughout the study. Ethanol and unencapsulated components were removed by diluting the resulting LNPs in 0.9% saline followed by centrifugation ultrafiltration using a Vivaspin ultrafiltration unit (MWCO: 30000 Da, PES; Satorius).

[0237] 1.5. Preparation of lyophilized mRNA LNP with cryoprotectant and Reconstitution of lyophilized mRNA LNP

[0238] LNP was mixed with sucrose and / or maltose to achieve different %w / v (0%, 5%, 10%, and 20%) and then frozen overnight in glass vials at -80°C. The frozen LNP samples were lyophilized for 24 or 96 hours at -48°C and 0.021 mbar using a Labconco benchtop freeze dryer. The lyophilized LNP samples were reconstituted with nuclease-free water for 1 hour using the same volume as before lyophilization. On the same day as the reconstitution of the lyophilized LNP samples, the freeze-thawed LNP samples were thawed at 4°C. All LNP samples were stored at 4°C before use.

[0239] 1.6. mRNA encapsulation efficiency (EE) and concentration

[0240] The mRNA encapsulation efficiency and concentration in each LNP sample were determined using the Quant-iT RiboGreen RNA Detection Kit (Invitrogen). In short, LNPs were diluted with 1x TE buffer in the presence and absence of 5% Triton X-100 and incubated at 37°C for 20 minutes. Fluorescence intensity was measured at 485 nm excitation and 528 nm emission wavelengths using a Tecan Spark Multimode Microplate Reader. mRNA concentration was obtained using a standard curve plotted by linear regression of fluorescence intensity against a standard mRNA concentration. The mRNA encapsulation efficiency was determined using the following equation:

[0241] , of which (concentration) 总 This refers to the mRNA concentration in LNPs calculated in the presence of 5% Triton X-100. 未包封 This refers to the mRNA concentration in LNPs calculated in the absence of 5% Triton X-100.

[0242] Use GelStar TM The mRNA encapsulation of each LNP sample was visualized by electrophoresis on a 1% agarose gel stained with nucleic acid gel staining agent (Lonza) at 100V for 20 minutes. Each LNP sample was mixed with an equal volume of RNA gel loading dye (Thermo Scientific) and loaded onto the gel. The gel contained RiboRuler high-range RNA ladder (Thermo Scientific) and free CleanCap® firefly luciferase mRNA (300 ng) as controls.

[0243] 1.7. Dynamic light scattering (DLS)

[0244] LNP samples were diluted in 0.9% saline solution, and particle size and polydispersity index (PDI) were measured in polystyrene cuvettes using a Zetasizer Ultra (Malvern Panalytical) via dynamic light scattering. Measurements were obtained in three runs, with 20 readings per run. LNP diameter was reported as mean ± standard deviation (z-mean), and PDI as mean ± standard deviation (p-mean).

[0245] 1.8. Surface zeta potential

[0246] The surface zeta potential of LNP samples diluted with 0.9% saline was also measured using a Zetasizer Ultra in a disposable folded capillary cell (Malvern Panalytical). Zeta potential measurements were obtained by running the instrument three times, with 20 readings per run, and are reported as mean ± standard deviation.

[0247] 1.9. Cell culture and treatment with mRNA LNP and lyophilized mRNA LNP

[0248] HeLa cells were cultured in 1x Dulbecco modified Eagle medium (high D-glucose, L-glutamine, pyruvate-free; Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin / streptomycin (Gibco), and maintained in an incubator at 37°C and 5% CO2. For LNP treatment, HeLa cells were seeded at a density of 10,000 cells per well into 96-well microplates containing complete growth medium the day before treatment and incubated overnight at 37°C. The old medium in each well was replaced with fresh medium containing 100 ng mRNA per well for each LNP sample. The mRNA concentration of the LNP sample was measured before each cell treatment. The medium in the control wells was replaced with fresh medium without LNP. After LNP treatment, HeLa cells were incubated at 37°C and 5% CO2 for 48 h.

[0249] 1.10. In vitro cytotoxicity of mRNA LNP and lyophilized mRNA LNP

[0250] After 48 hours of LNP treatment, use alamarBlue. ® Cell viability was assessed using a cell viability reagent (Invitrogen). In short, after 48 hours of incubation, the medium containing LNP was discarded, and cells were stained with Alamar Blue reagent (diluted to 1x with complete growth medium) for 2 hours. Fluorescence intensity was measured using a Tecan Spark Multimode Microplate Reader at 570 nm excitation and 600 nm emission wavelengths. Cell viability was calculated using the following equation:

[0251] ,

[0252] Among them (fluorescence) 处理 The fluorescence intensity of cells treated with LNP (fluorescence). 对照 The fluorescence intensity refers to that of control cells (untreated with LNP). The calculated cell viability percentage is reported as mean ± standard deviation, with each experimental condition repeated 5 times.

[0253] 1.11. In vitro mRNA transfection efficiency

[0254] After LNP processing, use Pierce ® The Firefly Luciferase Glow Detection Kit (ThermoScientific) was used to determine the transfection efficiency of LNP-treated cells. The working reagent was prepared by mixing cell lysis buffer (diluted to 1× with 1× PBS, pH 7.4) with firefly glow detection buffer at a 1:1 v / v ratio, and adding D-luciferin to a final concentration of 0.3 mg / ml. The LNP-containing medium was discarded, and the working reagent was added to the cells, resulting in 30 µg of D-luciferin per well. The samples and working reagent were incubated at 37°C in the dark for 10 min, and after shaking for 20 s, the luminescence intensity was measured using a Tecan Spark Multimode Microplate Reader with an integration time of 1000 ms. The luminescence intensity is reported as mean ± standard deviation, and five replicates were performed for each experimental condition.

[0255] 1.12. Statistical analysis

[0256] Statistical analysis was performed using GraphPad Prism v.9.0.0 (GraphPad Software). Pairwise comparisons were performed using Welch's t-test to determine statistical significance.

[0257] 1.13. General experimental flow

[0258] As shown in Scheme 3 below, the experiments performed in Example 1 above can be summarized as follows. First, LNPs were prepared and buffered with 0.9% saline. Subsequently, LNPs loaded with mRNA were synthesized, divided into different equal portions, and cryoprotectants or lyophilization protectants were added. Before adding cryoprotectants or lyophilization protectants, the particle size, PDI, and zeta potential of the mRNA-loaded LNPs were characterized by gel electrophoresis and Quant-iTRiboGreen RNA detection. Afterward, the mixture was frozen overnight at -80°C and lyophilized for different times according to various embodiments of this disclosure. The lyophilized samples were then reconstituted with nuclease-free water to the original volume before lyophilization. After reconstitution, the samples were characterized as described above, and transfection efficiency and cell viability were assessed using HeLa cells.

[0259]

[0260] Scheme 3. Overall experimental procedure (NFW: nuclease-free water; FD: lyophilized).

[0261] Example 2: Preparation and characterization of mRNA-loaded lipid nanoparticles (mRNA LNPs) containing ALC-0315 or DTD-NH2 before and after lyophilization for one day or four days.

[0262] mRNA LNPs were prepared using a microfluidic device, and the preparation conditions are listed in Tables 1 and 2. The main component was ionizable lipids. The average sizes of the mRNA LNPs prepared from ALC-0315 and DTD-NH2 were 85.6 nm and 101.5 nm, respectively, with narrow size distributions and near-neutral zeta potentials (Tables 3 and 4), which are ideal for in vivo administration. After storage at 4°C for 6 days, the sizes of the mRNA LNPs prepared from ALC-0315 or DTD-NH2 remained unchanged (Tables 3 and 4). After a 4-day freeze-drying process, even with the use of 5% to 20% sucrose as a cryoprotectant, the size of mRNA LNPs prepared by ALC-0315 or DTD-NH2 significantly increased (ALC-0315 LNP: 85.6 nm vs. 556.6 nm to 846.3 nm; DTD-NH2 LNP: 101.5 nm vs. 413.5 nm to 885.7 nm), and the size distribution broadened, as evidenced by the increased PDI (Tables 3 and 4).

[0263] Table 1. Conditions for preparing mRNA LNP (total volume 4.0 mL) from ALC-0315.

[0264]

[0265] The total volume of the aqueous phase (3 mL) and organic phase (1 mL) is 4.0 mL.

[0266] N / P ratio = 6

[0267] • The amount of added fLuc mRNA (Trilink) = 0.2 mg

[0268] • Dilute mRNA with 10 mM sodium acetate (pH 4.0)

[0269] Table 2. Conditions for preparing mRNA LNPs from DTD-NH2 (total volume 4.0 mL).

[0270]

[0271] The total volume of the aqueous phase (3 mL) and the organic phase (1 mL) is 4.0 mL.

[0272] N / P ratio = 6

[0273] • Amount of added fLuc mRNA (Trilink) = 0.28 mg

[0274] • Dilute mRNA with 10 mM sodium acetate (pH 4.0)

[0275] Table 3. Particle size, size distribution, and zeta potential of mRNA LNPs prepared by ALC-0315 before and after 4 days of freeze-drying. Samples marked FD are freeze-dried samples, and the rest are non-freeze-dried samples.

[0276]

[0277] Table 4. Particle size, size distribution, and zeta potential of mRNA LNPs prepared from DTD-NH2 before and after 4 days of freeze-drying. Samples marked with FD are freeze-dried samples, and the rest are non-freeze-dried samples.

[0278]

[0279] The mRNA binding efficiency of mRNA LNPs before and after 4 days of lyophilization was evaluated using a gel retardation assay. Figure 4 As shown, mRNA migration arrest was observed in both sucrose-containing and sucrose-free ALC-0315 LNPs, indicating that the mRNA was well encapsulated in the LNPs. However, in Figure 5 In the study, free mRNA molecules were observed after 4 days of freeze-drying, indicating that the mRNA was not well encapsulated within the LNP after the freeze-drying process. After 8 days of storage at 4°C, some free mRNA molecules were also observed in the LNP. Figure 5 This indicates that mRNA stability was lost to some extent. These phenomena were not observed in the mRNA from LNP prepared by DTD-NH2. Figure 6 This demonstrates the stability of LNP mRNA after 4 days of freeze-drying or 6 days of storage at 4°C.

[0280] Notably, in the presence of 10% sucrose, 10% maltose, or a mixture of 10% sucrose and 10% maltose, the freeze-thaw process resulted in increased particle size and a wider size distribution of mRNA LNPs prepared by ALC-0315 (85 nm vs. 92.6 nm to 218 nm, PDI: 0.05 vs. 0.1 to 0.35, Table 5). Since 4 days of freeze-drying resulted in large particle size and a wide size distribution regardless of the use of cryoprotectants, the freeze-drying process was shortened to one day. After one day of freeze-drying without sucrose, the mRNA LNPs prepared by ALC-0315 or DTD-NH2 became larger and wider in size distribution (high PDI value) due to aggregation, while the zeta potential remained unchanged (Tables 5 and 6). However, the use of the cryoprotectant sucrose reduced the aggregation caused by one day of freeze-drying, resulting in smaller particle size and a narrower size distribution. The size of the lyophilized and redispersed mRNA LNPs in brine depends on the sucrose content used; increasing the sucrose content results in smaller sizes. A content of 5% to 20% is ideal for mRNA LNPs prepared from ALC-0315 or DTD-NH2, as the particle size is between 145 nm and 254 nm and the size distribution is narrow (PDI: 0.16 to 0.30) (Tables 5 and 6).

[0281] Table 5. Particle size, size distribution and zeta potential (FT: freeze-thaw; FD: freeze-drying) of mRNA LNPs prepared by ALC-0315 before and after 1 day of freeze-drying.

[0282]

[0283] Table 6. Particle size, size distribution and zeta potential (FT: freeze-thaw; FD: freeze-drying) of mRNA LNPs prepared by DTD-NH2 before and after 1 day of freeze-drying.

[0284]

[0285] The encapsulation efficiency (EE) of mRNA in ALC-0315 LNP and DTD-NH2 LNP was 85.6% and 94.0%, respectively, measured immediately after preparation of the mRNA LNP from the microfluidic device (Table 7). After purifying the mRNA LNP and removing ethanol, the mRNA EE of ALC-0315 LNP and DTD-NH2 LNP was 90.9% and 97.4%, respectively (Table 7). After freeze-thaw processing, the presence of 10% sucrose did not affect the stability of mRNA in ALC-0315 LNP, while the presence of 10% maltose reduced mRNA stability, leading to a decrease in the EE of mRNA in ALC-0315 LNP. After a one-day freeze-drying process, the EE of mRNA in ALC-0315 LNP decreased from 90.9% to 40.7% to 57.5% when using 5%, 10%, or 20% sucrose, while the EE of mRNA in DTD-NH2 LNP remained comparable before and after the one-day freeze-drying process (88% to 90% for 5% to 20% sucrose) (Table 7). The decrease in the EE of mRNA in ALC-0315 LNP may be due to mRNA degradation and / or the pulling of mRNA from the LNP core to the LNP surface during freeze-drying. In contrast, DTD-NH2 LNP was able to stabilize the mRNA during a one-day freeze-drying process in the presence of 5%, 10%, or 20% sucrose.

[0286] Table 7. Encapsulation efficiency (EE%) of mRNA in ALC-0315 LNP and DTD-NH2 LNP (FT: freeze-thaw; FD: freeze-dry).

[0287]

[0288] As shown in Table 8, using 20% ​​sucrose protected the mRNA stability of ALC-0315 LNPs after a one-day lyophilization process, maintaining 55% mRNA transfection efficiency, compared to unly lyophilized ALC-0315 LNPs. Using 10% sucrose during a one-day lyophilization process maintained 67% mRNA transfection efficiency for DTD-NH2 LNPs loaded with mRNA, compared to unly lyophilized ALC-0315 LNPs. When 5% sucrose was used during the lyophilization of DTD-NH2 LNPs loaded with mRNA, the transfection efficiency was similar to that of unly lyophilized DTD-NH2 LNPs loaded with mRNA (after lyophilization: 170514 vs. before lyophilization: 167626). These results demonstrate the importance of using sucrose as a cryoprotectant and a shorter lyophilization time for preparing mRNA-loaded LNPs from ALC-0315 or DTD-NH2. In addition, using DTD-NH2 during the freeze-drying process can better protect the mRNA in LNP.

[0289] Table 8. Cell viability and mRNA transfection efficiency of HeLa cells after incubation with various mRNA LNPs for 48 h (FT: freeze-thaw; FD: freeze-drying).

[0290]

[0291] Summary of Example 2

[0292] Using 5%, 10%, or 20% sucrose can protect the stability of mRNA in LNPs prepared by ALC-0315 or DTD-NH2 during a one-day freeze-drying process. After one day of freeze-drying, the particle size of the mRNA lipids remains less than 250 nm, depending on the amount of sucrose used. 10% to 20% sucrose results in smaller size of the freeze-dried mRNA LNPs. Furthermore, it was found that for ALC-0315 LNPs loaded with mRNA, the cryoprotectant sucrose (10% to 20%) better protected the stability of the mRNA during a one-day freeze-drying process, resulting in approximately 54% to 55% transfection efficiency compared to the unfreezed mRNA LNPs. When using 5% to 20% sucrose, the stability of the mRNA in LNPs prepared by DTD-NH2 was maintained after one day of freeze-drying, resulting in transfection efficiency on the same order of magnitude as the unfreezed mRNA LNPs.

[0293] After 4 days of lyophilization, despite the use of 5% to 20% sucrose as a cryoprotectant, the mRNA in ALC-0315 LNPs prepared using lipids from the Pfizer / BioNTech mRNA vaccine formulation still lost stability, resulting in no transfection efficiency. When lipid DTD-NH2 was used as an ionizable lipid during the 4-day lyophilization process, combined with 5% or 20% sucrose, the stability of the mRNA in DTD-NH2 LNPs was completely protected, and there was no loss of transfection efficiency. The 4-day lyophilization process resulted in an increase in particle size and a wider size distribution of the mRNA LNPs prepared from ALC-0315 or DTD-NH2, even in the presence of sucrose.

[0294] Therefore, it can be seen that combining compounds of general formula (1) (e.g., in the form of DTD-NH2) with cryoprotectants (e.g., in the form of sucrose) during the freeze-drying process provides better stability for mRNA.

[0295] Example 3: Preparation and characterization of mRNA-loaded lipid nanoparticles (mRNA LNPs) containing ALC-0315 or ALC-0315 and DTD-NH2 before and after lyophilization.

[0296] mRNA LNPs were prepared using a microfluidic device, and the preparation conditions are listed in Tables 9 to 11. Samples containing 20% ​​sucrose exhibited a sponge-like matrix appearance after lyophilization, such as... Figure 3 As shown in the table. All lyophilized samples were subsequently reconstituted to form well-dissolved milky white solutions. Table 12 presents the characteristics of the mRNA LNPs, including particle size, PDI, zeta potential, and final encapsulation efficiency. Figures 14 to 18 The particle size distribution of mRNA LNPs prepared from ALC-0315 or mixtures of ALC-0315 and DTD-NH in molar ratios of 9:1 and 8:2 is shown. Figures 14 to 16 As shown, the particle size distribution exhibits uniformity (unimodal) before freeze-drying / lyophilization. However, in the absence of a cryoprotectant, mRNA LNP aggregation occurred after lyophilization, as... Figures 22 to 24 The multi-peak pattern is shown in the figure. Conversely, in the presence of 20% sucrose, no aggregation was observed after freeze-drying, as shown in the figure. Figures 25 to 27 The single peak is shown in the figure.

[0297] Table 9. Conditions for preparing mRNA LNP (total volume 2.0 mL) from ALC-0315.

[0298]

[0299] The total volume of the aqueous phase (1.5 mL) and organic phase (0.5 mL) is 2.0 mL.

[0300] N / P ratio = 6

[0301] • The amount of added fLuc mRNA (Trilink) = 0.1 mg

[0302] • Dilute mRNA with 10 mM sodium acetate (pH 4.0)

[0303] • Prepare LNPs via microfluidic control

[0304] Table 10. Conditions for preparing mRNA LNP (total volume 2.0 mL) from ALC-0315 and DTD-NH2 at a molar ratio of 9:1.

[0305]

[0306] The total volume of the aqueous phase (1.5 mL) and organic phase (0.5 mL) is 2.0 mL.

[0307] N / P ratio = 6

[0308] • The amount of added fLuc mRNA (Trilink) = 0.1 mg

[0309] • Dilute mRNA with 10 mM sodium acetate (pH 4.0)

[0310] • Prepare LNPs via microfluidic control

[0311] Table 11. Conditions for preparing mRNA LNP (total volume 2.0 mL) from ALC-0315 and DTD-NH2 at a molar ratio of 8:2.

[0312]

[0313] The total volume of the aqueous phase (1.5 mL) and organic phase (0.5 mL) is 2.0 mL.

[0314] N / P ratio = 6

[0315] • Amount of added fLuc mRNA (Trilink) = 0.1 mg

[0316] • Dilute mRNA with 10 mM sodium acetate (pH 4.0)

[0317] • Prepare LNPs via microfluidic control

[0318] Table 12. Characterization of mRNA LNPs prepared using only ALC-0315 or ALC-0315 with DTD-NH2 at a molar ratio of 9:1 or 8:2 (total volume 2.0 mL) (particle size, PDI, zeta potential, final encapsulation efficiency).

[0319]

[0320] •FT: freeze-thaw; FD: freeze-dry; ALC: ALC-0315; DTD: DTD-NH2.

[0321] Furthermore, in in vitro studies using HeLa cells, Table 13 shows that the inclusion of sucrose as a cryoprotectant in the mRNA LNP and the lyophilization process did not affect cell viability. Additionally, Table 13 clearly demonstrates that mixing ALC-0315 with DTD-NH2 in the mRNA LNP helps maintain higher mRNA transfection efficiency. Specifically, the mRNA LNP containing 20% ​​sucrose as a cryoprotectant and both ALC-0315 and DTD-NH2 in molar ratios of 9:1 (4031 RLU) and 8:2 (6203 RLU), respectively, exhibited higher luminescence intensity after lyophilization than the mRNA LNP containing only ALC-0315 and 20% sucrose as a cryoprotectant (1267 RLU).

[0322] In in vivo studies in Balb / c mice via intravenous injection (tail vein), each mouse received a dose of 1 µg mRNA / 100 µL. Imaging was performed at 6 and 24 hours after mRNA LNP administration. The ALC-0315 group consisted of 6 mice before lyophilization (freeze-drying) and 7 mice after lyophilization. Additionally, the ALC-0315:DTD-NH2 (9:1) group consisted of 7 mice after lyophilization. A "blank control" group served as a control group without any treatment. It was observed that mixing ALC-0315 with DTD-NH2 in the mRNA LNP maintained significantly higher mRNA transfection efficiency in mice compared to using ALC-0315 alone. Specifically, as... Figure 28 As shown, the total light flux of mRNA LNP containing both ALC-0315 and DTD-NH2 in a molar ratio of 9:1 after lyophilization is higher than that of mRNA LNP containing only ALC-0315.

[0323] Table 13. Cell viability and mRNA transfection efficiency of HeLa cells after incubation with various mRNA LNPs for 48 h (FT: freeze-thaw; FD: lyophilization / freeze-drying).

[0324]

[0325] • FT: Freeze-thaw; FD: Freeze-drying

[0326] HeLa cells, incubated for 48 hours

[0327] Those skilled in the art will understand that other changes and / or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope broadly described herein. For example, features of different exemplary embodiments may be mixed, combined, interchanged, merged, adopted, modified, included, etc., among different exemplary embodiments in the description herein. Therefore, this embodiment should be considered illustrative rather than restrictive in all respects.

Claims

1. A nanoparticle composition for delivering therapeutic, preventative, and / or biological agents, said nanoparticle composition comprising: Compounds or their ionized forms represented by general formula (1) in R 1 R 2 and R 4 To R 8 Each of the following can be independently H, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group. R 3 It can be an optionally substituted alkylene group, an optionally substituted alkenyl group, or an optionally substituted ynylene group, and R 9 and R 10 Each is independently a hydrophobic tail or contains the above-mentioned R. 4 To R 8 At least one of the defined groups; Therapeutic agents, preventative agents, and / or biological agents, which are encapsulated in compounds of general formula (1) to form nanoparticles; and Cryoprotectant.

2. The nanoparticle composition according to claim 1, wherein... R 1 and R 2 Each is H; R 3 Contains alkylene groups; and R 9 and R 10 Each of the components independently contains an alkyl group, wherein the alkyl group contains at least 5 carbon atoms.

3. The nanoparticle composition according to claim 1 or 2, wherein R 4 To R 8 Each is represented by H.

4. The nanoparticle composition according to any one of the preceding claims, wherein the compound is represented by formula (1A): 。 5. The nanoparticle composition according to any one of the preceding claims, wherein the therapeutic agent, preventive agent, and / or biological agent comprises nucleic acid.

6. The nanoparticle composition according to claim 5, wherein the nucleic acid comprises messenger ribonucleic acid (mRNA).

7. The nanoparticle composition according to any one of the preceding claims, wherein the nanoparticle composition further comprises an ionizable lipid different from a compound of general formula (1).

8. The nanoparticle composition according to claim 7, wherein the ionizable lipid that is different from the compound of general formula (1) includes ALC-0315.

9. The nanoparticle composition according to any one of claims 7 to 8, wherein the ratio of the compound of general formula (1) to an ionizable lipid different from the compound is 1:1 to 1:

20.

10. The nanoparticle composition according to any one of the preceding claims, wherein the composition further comprises: Neutral lipids / helper lipids; Sterols; and Lipids modified with polyethylene glycol (PEG).

11. The nanoparticle composition according to claim 10, wherein the compound of general formula (1), the neutral lipid / auxiliary lipid, the sterol and the PEG-modified lipid are mixed in a molar ratio of 1 to 70:1 to 20:10 to 60:1 to 20.

12. The nanoparticle composition according to any one of claims 10 to 11, wherein the neutral lipid / auxiliary lipid is selected from 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-diundecanoyl-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 Diether 1,2-Oleoyl-2-cholesterol hemisuccino-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16LysoPC), 1,2-dilinolenoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicyl-sn-glycerol-3-phosphate choline, 1,2-bis(docosahexaenoyl-sn-glycerol-3-phosphate choline), 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (ME16.0) PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-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), sphingomyelin and combinations thereof.

13. The nanoparticle composition according to any one of claims 10 to 12, wherein the sterol is selected from cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicosterol, alfalfa sterol, and combinations thereof.

14. The nanoparticle composition according to any one of claims 10 to 13, wherein the PEG-modified lipid is selected from 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159), R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristoxypropyl-3-amine (PEG-c-DOMG), 3-N-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristoxypropylamine (PEG-S-DMG), PEG-DMPE (1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine-N-[(polyethylene glycol)-methoxy](sodium salt)), PEG-DPPC, PEG-DSPE lipid, and combinations thereof.

15. The nanoparticle composition according to any one of the preceding claims, wherein the cryoprotectant comprises a carbohydrate source selected from at least one of maltose, sucrose, trehalose, mannitol, glucose, fructose, lactose, galactose, ribose, xylose, mannose, arabinose, and combinations thereof.

16. The nanoparticle composition according to any one of the preceding claims, wherein the cryoprotectant is present in an amount of 1 w / v% to 30 w / v% relative to the total volume of the composition.

17. The nanoparticle composition according to any one of the preceding claims, wherein the nanoparticle composition is in a lyophilized or freeze-thawed form.

18. The nanoparticle composition of claim 17, wherein the therapeutic and / or preventive and / or biological agents are encapsulated with an encapsulation rate of at least 60%.

19. The nanoparticle composition according to any one of claims 17 to 18, wherein the zeta potential of the nanoparticles is from -10 mV to +10 mV.

20. The nanoparticle composition according to any one of claims 17 to 19, wherein the polydispersity index (PDI) of the nanoparticles is 0.01 to 0.

5.

21. The nanoparticle composition according to any one of claims 17 to 20, wherein the particle size of the nanoparticles is from 50 nm to 1200 nm.

22. A nanoparticle composition according to any one of the preceding claims, for use in a pharmaceutical.

23. Use of the nanoparticle composition according to any one of the preceding claims in the preparation of a medicament for inducing an immune response in a subject in which such response is desired.

24. A nanoparticle composition according to any one of the preceding claims, for inducing an immune response in a subject in which such response is desired, wherein the nanoparticle composition or lyophilized nanoparticle composition is to be applied to the subject.

25. A method for inducing an immune response in a subject, the method comprising the step of administering a therapeutically effective amount of the nanoparticle composition according to any one of the preceding claims to the subject.

26. The use according to claim 23, the nanoparticle composition according to claim 24, or the method according to claim 25, wherein the immune response is specific to coronaviruses, cancer, or bacteria.

27. The use, the nanoparticle composition, or the method according to claim 26, wherein the coronavirus is SARS-CoV-2 coronavirus.

28. A method for preparing the nanoparticle composition according to claim 1, the method comprising: Prepare an aqueous composition comprising therapeutic and / or preventive and / or biological reagents; The aqueous composition is mixed with a compound of general formula (1), an auxiliary lipid, a sterol, and a PEG-modified lipid to obtain nanoparticles encapsulating the therapeutic and / or preventive and / or biological reagents; A cryoprotectant was added to the nanoparticles.

29. The method of claim 28, wherein the mixing step is carried out in the presence of other ionizable lipids different from those of general formula (1).

30. The method according to claim 29, wherein the compound of general formula (1) is mixed with an ionizable lipid different from the compound in a ratio of 1:1 to 1:20.

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