Composition and nanoparticle suitable for use in the delivery of a therapeutic agent

Abstract

There is provided a nanoparticle for delivery of a therapeutic agent, the nanoparticle comprising a surface structure which comprises a cationic lipid and an ionizable lipid in a ratio that falls in the range of 1 -5 : 5-1.

Description

[0001] COMPOSITION AND NANOPARTICLE SUITABLE FOR USE IN THE DELIVERY OF A THERAPEUTIC AGENT

[0002] TECHNICAL FIELD

[0003] The present disclosure relates broadly to a composition and a nanoparticle for delivery of a therapeutic agent, related methods, and uses.

[0004] BACKGROUND

[0005] Lipid nanoparticles (LNPs) have recently emerged as promising vehicles for delivery of nucleic acid therapeutics. Patisiran, an LNP-based formulation of transthyretin-targeted small interfering ribonucleic acid (siRNA), has been proven efficacious for the treatment of hereditary transthyretin-mediated amyloidosis. Additionally, LNPs have played a crucial role in the success of two COVID-19 messenger ribonucleic acid (mRNA) vaccines (BNT162b2 and mRNA-1273).

[0006] LNPs are mainly composed of 4 different lipids: an ionizable lipid bearing a tertiary amine headgroup, a helper phospholipid, cholesterol, and lipidpolyethylene glycol (PEG) conjugate. The ionizable lipid, such as ALC-0315 and SM-102 used in the formulation of BNT162b2 and mRNA-1273, respectively, has been considered a key component of LNP-based nucleic acid delivery systems. For example, the ionizable lipid is designed to have a near-neutral charge at extracellular fluid pH (~7.4), thus minimizing toxicity of LNPs. Moreover, the tertiary amine group of ionizable lipids becomes protonated at endosomal pH (5.5-6 5) and associates with anionic endosomal phospholipids, thereby facilitating the membrane fusion and favoring delivery of the nucleic acid payload into the cytoplasm of target cells.

[0007] Particularly, solid lipid nanoparticles (SLNs) are colloidal nanocarriers having a solid lipid core stabilized by an amphiphilic lipid shell layer. Recently, SLNs have gained increasing attention as gene delivery vehicles due to their ease of manufacture, high colloidal stability, and enhanced bioavailability. Over the last decade, a variety of cationic lipids have been incorporated in SLNs to endow them with the ability to condense anionic nucleic acid molecules into nanosized polyelectrolyte complexes for efficient transfection. However, this approach introduces a permanent positive charge on SLNs, resulting in increased toxicity and inflammation. In addition, the strong binding between cationic SLNs and anionic nucleic acid molecules can negatively affect transfection efficiency due to insufficient dissociation after cellular uptake. On the other hand, commercially available Lipofectamine-based gene transfection reagents such as LF300 and LFMax are extremely expensive. Their use is primarily limited to in vitro gene transfection reagents as they are highly positively charged and can cause toxicity and inflammation if used in vivo. Therefore, it is highly desirable to explore alternative approaches to improve transfection efficiency of SLNs while mitigating toxicity.

[0008] In view of the above, there is a need to address or at least ameliorate the above-mentioned problems. In particular, there is a need to provide a composition and / or a nanoparticle composition for a cost efficient, substantially safe and stable, and / or efficacious delivery of therapeutic, prophylactic, and / or biological agents.

[0009] SUMMARY

[0010] In one aspect, there is provided a composition for delivery of a therapeutic agent, the composition comprising a cationic lipid and an ionizable lipid in a ratio that falls in the range of 1 -5 : 5-1 .

[0011] In one embodiment, the cationic lipid is selected from the group consisting of 1 ,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1 ,2-dioleoyl- 3-trimethylammonium propane (DOTAP), dimethyldioctadecylammonium (DDAB), / V-(2-hydroxyethyl)- / V, / \ / -dimethyl-2,3-bis(oleoyloxy)propan-1 -aminium (DORI), 1 ,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC), 0,0’- ditetradecanoyl- / V-(a-trimethylammonioacetyl)diethanolamine (DC-6-14), 2,3- dioleyloxy- / V-[2-(sperminecarboxamido)ethyl]- / V, / V-dimethyl-1 -propanaminium (DOSPA), / V1-[2-((1 S)-1-[(3-aminopropyl)amino]-4-[di(3-amino- propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), and combinations thereof.

[0012] In one embodiment, the ionizable lipid is selected from the group consisting of (4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2- hexyldecanoate) (ALC-0315), 1 -octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-

[0013] (undecyloxy)hexyl]amino]-octanoate (SM-102), MC3 or (6Z,9Z,28Z,31Z)- Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (D-Lin-MC3- DMA), 1 ,1‘-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl) (2- hydroxydodecyl)amino)ethyl)piperazin-1 -yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione (CKK-E12), 9Z,12Z-octadecadienoic acid, 1 ,1',1",r"-[(3,6-dioxo-2,5- piperazinediyl)bis(4, 1 -butaned iy In itri lod i-2, 1 -ethanediyl)] ester (OF-Deg-Lin), ethyl 5,5-bis[(Z)-heptadec-8-enyl]-1-(3-pyrrolidin-1 -ylpropyl)-2H-imidazole-2- carboxylate (A2-iso5-2DC18), tetrakis(8-methylnonyl) 3,3',3",3"'- (((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate (3060i10), bis(2-(dodecyldisulfaneyl)ethyl) 3,3'-((3-methyl-9-oxo-10-oxa-13, 14- dithia-3,6-diazahexacosyl)azanediyl)dipropionate (BAMEA-O16B), N1 ,N3,N5- tris[3-(didodecylamino)propyl]-1 ,3,5-benzenetricarboxamide (TT3), 2-

[0014] (dioctylamino)ethyl nonyl hydrogen phosphate (9A1 P9), hexa(octan-3-yl) 9, 9', 9", 9"', 9"", 9"'"- ((((benzene-1 ,3,5-tricarbonyl)yris(azanediyl)) tris (propane- 3,1 -diyl)) tris(azanetriyl))hexanonanoate (FTT5), 1 ,2-dioleyloxy-3- dimethylaminopropane (DODMA), 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1 - aminium (DOBAQ), and combinations thereof.

[0015] In one embodiment, the cationic lipid is present in an amount of from 1 wt% to 50 wt% of the composition. In one embodiment, the ionizable lipid is present in an amount of from 1 wt% to 50 wt% of the composition.

[0016] In one embodiment, the total amount of the ionizable lipid and the cationic lipid present in the composition falls in the range of 30 wt% to 60 wt% of the composition.

[0017] In one embodiment, the composition further comprises one or more of the following components:

[0018] (i) glycerolipid;

[0019] (ii) esterified cholesterol;

[0020] (iii) phospholipid;

[0021] (iv) cholesterol or analogues thereof; and / or

[0022] (v) PEG-conjugate.

[0023] In one embodiment, the glycerolipid is present and comprises a triglyceride selected from the group consisting of triolein, tristearin, trielaidin, trilinolein, tripalmitin, tripalmitolein, trimyristin, trilaurin, tricaproin, trioctanoin, triarachidin, triarachidonin, and combinations thereof.

[0024] In one embodiment, the esterified cholesterol is present and comprises a cholesteryl ester selected from the group consisting of cholesteryl oleate, cholesteryl stearate, cholesteryl linoleate, cholesteryl palmitate, cholesteryl myristate, cholesteryl laurate, cholesteryl caproate, cholesteryl arachidate, cholesteryl arachidonate, and combinations thereof.

[0025] In one embodiment, the phospholipid is present and is selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), 1 -palmitoyl-2- oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1 ,2-dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE), 1 ,2-dilauroyl-sn-glycero-3- phosphoethanolamine (DLPE), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2-dipalmitoyl-sn- glycero-3-phosphatidylcholine (DPPC), 1 ,2-dimyristoyl-sn-glycero-3-dilauroyl-sn- glycero-3-phosphocholine (DLPC), 1 ,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1 -stearoyl- 2-oleoyl-sn-glycero-3-phosphocholine(SOPC), and combinations thereof.

[0026] In one embodiment, the cholesterol or analogues thereof is present and is selected from the group consisting of cholesterol, p-sitosterol, p-sitosterol- acetate, stigmastanol, campesterol, fucosterol, brassicasterol, ergosterol, 9,11 - dehydroergosterol, daucosterol, vitamin D2, vitamin D3, vitamin E, calcipotriol, betulin, lupeol, ursolic acid, oleanolic acid, derivatives thereof and combinations thereof.

[0027] In one embodiment, wherein the PEG conjugate is present and is selected from the group consisting of 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine- polyethylene glycol 2000 (DSPE-PEG), methoxypolyethyleneglycoloxy(2000)- N,N-ditetradecylacetamide (ALC-0159), 1 ,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG), 1 ,2-distearoyl-rac-glycero-3- methylpolyoxyethylene 2000 (DSG-PEG), 1 ,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-[(polyethylene glycol)-2000] (DPPE-PEG), 1 ,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000] (DMPE-PEG), 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000] (DOPE-PEG), N-palmitoyl-sphingosine-1 -

[0028] {succinyl[methoxy(polyethylene glycol)2000]} (C16 PEG ceramide), N-octanoyl- sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]} (C8 PEG2000 ceramide), and combinations thereof.

[0029] In one embodiment, the glycerolipid is present in an amount of from 0.10 wt% to 6 wt% of the composition.

[0030] In one embodiment, the esterified cholesterol is present at an amount of from 10 wt% to 30 wt% of the composition. In one embodiment, the phospholipid is present in an amount of from 10 wt% to 40 wt of the composition.

[0031] In one embodiment, the cholesterol and analogues thereof is present in an amount of from 0.10 wt% to 40 wt% of the composition.

[0032] In one embodiment, the PEG conjugate is present in an amount of from 0.10 mol% to 3 mol% of the composition.

[0033] In one embodiment, the total lipid concentration in the composition is from 1 mg / mL to 50 mg / mL.

[0034] In another aspect, there is provided a composition for delivery of a therapeutic agent, the composition comprising a cationic lipid in an amount of 5 to 50 wt% of the composition; an ionizable lipid in an amount of 5 to 50 wt% of the composition; a glycerolipid in an amount of 1 to 5 wt% of the composition; a phospholipid in an amount of 10 to 40 wt% of the composition; a cholesterol or analogues thereof in an amount of 1 to 30 wt% of the composition; optionally an esterified cholesterol in an amount of 10 to 30 wt% of the composition; and optionally a PEG-conjugate in an amount of from 0.10 mol% to 3 mol% of the composition, wherein the cationic lipid and ionizable lipid are present in a ratio that falls in the range of 1 -5 : 5-1 .

[0035] In one embodiment, the total amount of the ionizable lipid and the cationic lipid present in the composition falls in the range of 30 wt% to 60 wt% of the composition.

[0036] In another aspect, there is provided a nanoparticle comprising a core structure comprising glycerolipid and optionally an esterified cholesterol; a surface structure comprising an ionizable lipid, a cationic lipid, a phospholipid, a cholesterol or analogues thereof, and optionally a PEG- conjugate; and a therapeutic agent complexed with the surface structure, wherein the cationic lipid and ionizable lipid are present in a ratio that falls in the range of 1 -5 : 5-1 .

[0037] In one embodiment, the total amount of the ionizable lipid and cationic lipid present in the nanoparticle falls in the range of 30 wt% to 60 wt% of total lipid composition of the nanoparticle.

[0038] In one embodiment, the therapeutic agent comprises a nucleic acid.

[0039] In one embodiment, the nanoparticle is an ionizable lipid incorporated solid lipid nanoparticle or an ionizable lipid incorporated liquid lipid nanoparticle.

[0040] In one embodiment, the nanoparticle has an apparent acid dissociation constant (pKa) value in the pH range of 4.5 to 7.

[0041] In another aspect, there is provided a method of preparing the nanoparticle as disclosed herein, the method comprises, adding an aqueous solution to an composition comprising a cationic lipid and an ionizable lipid in an organic solvent, wherein the ratio of the cationic lipid to the ionizable lipid is in a ratio that falls in the range of 1 -5 : 5-1 , to form an immiscible mixture; agitating the immiscible mixture to obtain an oil-in-water (O / W) emulsion; evaporating the organic solvent from the O / W emulsion to obtain the nanoparticle in aqueous suspension; adding an aqueous solution of a therapeutic agent to the nanoparticle in aqueous suspension; and optionally incubating the mixture.

[0042] In one embodiment, the organic composition further comprises one or more of:

[0043] (i) glycerolipid;

[0044] (ii) esterified cholesterol;

[0045] (iii) phospholipid;

[0046] (iv) cholesterol or analogue thereof; and / or

[0047] (v) PEG-conjugate.

[0048] In one embodiment, the organic composition comprises an organic solvent selected from the group consisting of chloroform, ethanol, methanol, isopropanol, butanol, ethyl acetate, dichloromethane (DCM), and combinations thereof.

[0049] In one embodiment, the aqueous solution comprises an aqueous solvent selected from the group consisting of deionized water, normal saline, phosphate- buffered saline, Tris-buffered saline, sodium acetate buffer, sodium citrate buffer, and combinations thereof.

[0050] DEFINITIONS

[0051] The term “particle” as used herein broadly refers to a discrete entity or a discrete body. The particle described herein can include an organic, an inorganic, a composite particle or a biological particle. The particle used described herein may also be a macro-particle that is formed by an aggregate of a plurality of subparticles or a fragment of a small object. The particle of the present disclosure may be spherical, substantially spherical, or non-spherical, such as irregularly shaped particles or ellipsoidally shaped particles. The term “size” when used to refer to the particle broadly refers to the largest dimension of the particle. For example, the term “size” when used in the context of nanoparticle can refer to the diameter of the nanoparticle although it is not limited as such. 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 largest length of the particle.

[0052] The term "nano" as used herein is to be interpreted broadly to include dimensions in a nanoscale, / '.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 from about 1 nm to about 100 nm. Accordingly, the term “nanostructures”, “nanoparticles”, “nanomaterials” and the like as used herein may include structures that have at least one dimension in the range of no more than said range. The term “nanostructures”, “nanoparticles”, “nanomaterials” and the like as used herein may include structures that have at least one dimension that is no more than about 200 nm, no more than about 150 nm, no more than about 100 nm, no more than about 90 nm, no more than about 80 nm, no more than about 70 nm, no more than about 60 nm, no more than about 50 nm, no more than about 40 nm, no more than about 30 nm, no more than about 20 nm, or no more than about 10 nm.

[0053] The term "micro" as used herein is to be interpreted broadly to include dimensions from about 1 micron to about 1000 microns, about 1 micron to less than about 1000 microns, about 1 micron to about 900 microns, about 1 micron to about 800 microns, about 1 micron to about 700 microns, about 1 micron to about 600 microns, about 1 micron to about 500 microns, about 1 micron to about 400 microns, about 1 micron to about 300 microns, about 1 micron to about 200 microns, about 1 micron to about 100 microns, or from about 1 micron to about 5 microns. In various embodiments, particles of about 5 microns or lesser may be useful for intranasal spray delivery.

[0054] The term “treatment", "treat" and “therapy”, and synonyms thereof as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a medical condition, which includes but is not limited to diseases, symptoms and disorders. A medical condition also includes a body’s response to a disease or disorder, e.g., inflammation. Those in need of such treatment include those already with a medical condition as well as those prone to getting the medical condition or those in whom a medical condition is to be prevented.

[0055] As used herein, the term "therapeutically effective amount" of a compound is intended to refer to an amount that is sufficient or capable of preventing or at least slowing down (lessening) a medical condition, such as infectious diseases, respiratory illnesses (e g., coronavirus caused by the SARS-CoV-2 virus or flu caused by influenza virus). Dosages and administration of compounds, compositions and formulations of the present disclosure may be determined by one of ordinary skill in the art of clinical pharmacology or pharmacokinetics. An effective amount of the active agent of the present disclosure to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the patient. Accordingly, it may be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect.

[0056] The term “subject” is intended to broadly refer to any animal, such as a mammal, and including humans. Exemplary subjects include but are not limited to humans and non-human primates. The term “subject” as used herein also includes patients and non-patients. The term “patient” refers to individuals suffering or are likely to suffer from a medical condition such as infectious diseases (e.g., coronavirus caused by the SARS-CoV-2 virus), while “nonpatients” refer to individuals not suffering and are likely to not suffer from the medical condition. “Non-patients” include healthy individuals, non-diseased individuals and / or an individual free from the medical condition. As used herein, the term "mammal" includes vertebrate such as a human or a large veterinary mammal (e.g., horses, cattle, deer, sheep, llamas, goats, pigs). The terms "coupled" or "connected" as used in this description are intended to cover both directly connected or connected through one or more intermediate means, unless otherwise stated.

[0057] The term "associated with", used herein when referring to two elements refers to a broad relationship between the two elements. The relationship includes, but is not limited to a physical, a chemical or a biological relationship. For example, when element A is associated with element B, elements A and B may be directly or indirectly attached to each other or element A may contain element B or vice versa.

[0058] The term "and / or", e.g., "X and / or Y" is understood to mean either "X and Y" or "X or Y" and should be taken to provide explicit support for both meanings or for either meaning.

[0059] Further, in the description herein, the word “substantially” whenever used is understood to include, but not restricted to, "entirely" or “completely” and the like. In addition, terms such as "comprising", "comprise", and the like whenever used, are intended to be non-restricting descriptive language in that they broadly include elements / components recited after such terms, in addition to other components not explicitly recited. For example, when “comprising” is used, reference to a “one” feature is also intended to be a reference to “at least one” of that feature. Terms such as “consisting”, “consist”, and the like, may in the appropriate context, be considered as a subset of terms such as "comprising", "comprise", and the like. Therefore, in embodiments disclosed herein using the terms such as "comprising", "comprise", and the like, it will be appreciated that these embodiments provide teaching for corresponding embodiments using terms such as “consisting”, “consist”, and the like. Further, terms such as "about", "approximately" and the like whenever used, typically means a reasonable variation, for example a variation of + / - 5% of the disclosed value, or a variance of 4% of the disclosed value, or a variance of 3% of the disclosed value, a variance of 2% of the disclosed value or a variance of 1 % of the disclosed value. Furthermore, in the description herein, certain values may be disclosed in a range. The values showing the end points of a range are intended to illustrate a preferred range. Whenever a range has been described, it is intended that the range covers and teaches all possible sub-ranges as well as individual numerical values within that range. That is, the end points of a range should not be interpreted as inflexible limitations. For example, a description of a range of 1% to 5% is intended to have specifically disclosed sub-ranges 1 % to 2%, 1 % to 3%, 1 % to 4%, 2% to 3% etc., as well as individually, values within that range such as 1 %, 2%, 3%, 4% and 5%. The intention of the above specific disclosure is applicable to any depth / breadth of a range.

[0060] Additionally, when describing some embodiments, the disclosure may have disclosed a method and / or process as a particular sequence of steps. However, unless otherwise required, it will be appreciated that the method or process should not be limited to the particular sequence of steps disclosed. Other sequences of steps may be possible. The particular order of the steps disclosed herein should not be construed as undue limitations. Unless otherwise required, a method and / or process disclosed herein should not be limited to the steps being carried out in the order written. The sequence of steps may be varied and still remain within the scope of the disclosure.

[0061] Furthermore, it will be appreciated that while the present disclosure provides embodiments having one or more of the features / characteristics discussed herein, one or more of these features / characteristics may also be disclaimed in other alternative embodiments and the present disclosure provides support for such disclaimers and these associated alternative embodiments.

[0062] It will also be appreciated that where priority is claimed to an earlier application, the full contents of the earlier application is also taken to form part of the present disclosure and may serve as support for embodiments disclosed herein. DESCRIPTION OF EMBODIMENTS

[0063] Exemplary, non-limiting embodiments of a composition for delivery of a therapeutic agent, a method of preparing said composition, a nanoparticle comprising said composition related methods / uses thereto are disclosed hereinafter.

[0064] COMPOSITION

[0065] There is provided a composition for delivery of a therapeutic agent, and / or prophylactic agent, and / or biological agent. Advantageously, in various embodiments, the composition may be used as an encapsulation / loading agent, delivery vehicle / system, and / or transfection vehicle / system. In various embodiments, the composition is designed / configured to allow loading / encapsulation of one or more types of molecules or cargoes. In various embodiments, the composition is also designed / configured to allow the loaded / encapsulated agent to be released from said composition and / or subsequently delivered to a desired target (e g., cell, cytosol, tissue, or organ). The molecules / cargoes to be loaded / encapsulated onto / into / within the composition may include but is not limited to a therapeutic agent, a prophylactic agent, a biological agent, or the like. In various embodiments, the molecules / cargoes to be loaded / encapsulated comprises a nucleic acid. For example, the molecules / cargoes to be loaded / encapsulated may be a nucleic acid selected from ribonucleic acid (RNA), microRNA (miRNA), messenger ribonucleic acid (mRNA), small interfering ribonucleic acid (siRNA), deoxyribonucleic acid (DNA), plasmid deoxyribonucleic acid (pDNA), oligonucleotides such as antisense oligonucleotide or allele-specific oligonucleotides (ASO), the like, or combinations thereof. In various embodiments, the composition / nanoparticle may be used for the delivery of all types of nucleic acid therapeutics and optimal transfection efficiency may be achieved. In various embodiments, the molecules / cargoes to be loaded / encapsulated comprises therapeutics. For example, the molecules / cargoes to be loaded / encapsulated may be therapeutics selected from negatively charged therapeutics, drug molecule, vaccine (e.g., dengue vaccine, Covid-19 vaccine etc.), an anti-cancer agent, an anti-bacterial agent, an antifungal agent, an anti-viral agent, the like, or combinations thereof. Advantageously, the composition is suitable for use in encapsulating and / or delivering one or more therapeutic agent, prophylactic agent, and / or biological agent to a desired target (e.g., subject, cell, cytosol, tissue, or organ).

[0066] Accordingly, in various embodiments, there is also provided a carrier, nanocarrier, or delivery system / vehicle comprising the composition.

[0067] In various embodiments, the composition comprises a cationic lipid and an ionizable lipid that falls in the range of 1-5 : 5-1. For example, the composition comprises ionizable lipid and cationic lipid at weight ratio of about 1 :5, about 1:4, about 1 :3, about 1 :2, about 1 :1 , about 1 :0, about 2:1 , about 3:1 , about 4:1 , or about 5:1.

[0068] In various embodiments, the cationic lipid is selected from 1 ,2-di-O- octadecenyl-3-trimethylammonium propane (DOTMA), 1 ,2-dioleoyl-3- trimethylammonium propane (DOTAP), dimethyldioctadecylammonium (DDAB), / V-(2-hydroxyethyl)- / V, / V-dimethyl-2,3-bis(oleoyloxy)propan-1 -aminium (DORI), 1 ,2- dioleoyl-sn-glycero-3-ethylphosphocholine (EPC), 0, 0’-ditetradecanoyl- / \ / -(a- trimethylammonioacetyl)diethanolamine (DC-6-14), 2,3-dioleyloxy- / V-[2- (sperminecarboxamido)ethyl]- / V, / \ / -dimethyl-1 -propanaminium (DOSPA), A / 1 -[2- ((1 S)-1-[(3-aminopropyl)amino]-4-[di(3-amino- propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), the like, or combinations thereof. In various embodiments, the cationic lipid is capable of condensing anionic nucleic acid molecules into nanosized polyelectrolyte complexes for efficient transfection. In various embodiments, the ionizable lipid is selected from (4- hydroxybutyl)azanediyl]di(hexane-6, 1 -diyl) bis(2-hexyldecanoate) (ALC-0315), 1 -octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoate (SM-102), MC3 or (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31 -tetraen-19-yl 4- (dimethylamino)butanoate (D-Lin-MC3-DMA), 1 ,1 ‘-((2-(4-(2-((2-(bis(2- hydroxydodecyl)amino)ethyl) (2-hydroxydodecyl)amino)ethyl)piperazin-1 - yl)ethyl)azanediyl)bis(dodecan-2-ol) (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-piperazinediyl)bis(4, 1 - butanediylnitrilodi-2,1 -ethanediyl)] ester (OF-Deg-Lin), ethyl 5,5-bis[(Z)- heptadec-8-enyl]-1 -(3-py rrol id in-1 -ylpropyl)-2 / - / -imidazole-2-carboxylate (A2- iso5-2DC18), tetrakis(8-methylnonyl) 3,3',3",3"'-(((methylazanediyl)bis(propane-

[0069] 3.1-diyl))bis(azanetriyl))tetrapropionate (3060i10), bis(2-

[0070] (dodecyldisulfaneyl)ethyl) 3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6- diazahexacosyl)azanediyl)dipropionate (BAMEA-O16B), A / 1, / 3, A / 5-tris[3-

[0071] (didodecylamino)propyl]-1 ,3,5-benzenetricarboxamide (TT3), 2-

[0072] (dioctylamino)ethyl nonyl hydrogen phosphate (9A1 P9), hexa(octan-3-yl) 9, 9', 9", 9"', 9"", 9""'- ((((benzene-1 ,3,5-tricarbonyl)yris(azanediyl)) tris (propane-

[0073] 3.1 -diyl)) tris(azanetriyl))hexanonanoate (FTT5), 1 ,2-dioleyloxy-3- dimethylaminopropane (DODMA), 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP), A / -(4-carboxybenzyl)- / \ / , / \ / -dimethyl-2,3-bis(oleoyloxy)propan-1 - aminium (DOBAQ), the like, or combinations thereof. Advantageously, in various embodiments, the ionizable lipid exhibits a nearly neutral charge at the pH of extracellular fluids (~7.4), thereby mitigating nanoparticle toxicity attributed to the presence of the cationic lipid.

[0074] Advantageously, in various embodiments, the ionizable lipid contains tertiary amine groups that may e g., be protonated at endosomal pH levels between 5.5 and 6.5, facilitating their association with anionic endosomal phospholipids, thereby promoting membrane fusion and enhancing the delivery of a therapeutic agent, and / or prophylactic agent, and / or biological agent into the cytoplasm of target cells. In various embodiments, the composition comprises from more than about 0 wt%, more than about 0.1 wt%, more than about 0.2 wt%, more than about 0.3 wt%, more than about 0.4 wt%, more than about 0.5 wt%, more than about 0.6 wt%, more than about 0.7 wt%, more than about 0.8 wt%, more than about 0.9 wt%, from about 1 .00 wt% to about 50.00 wt, from about 2.00 wt% to about 50.00 wt, from about 3.00 wt% to about 50.00 wt, from about 4.00 wt% to about 50.00 wt, from about 5.00 wt% to about 50.00 wt, from about 2.00 wt% to about 49.00 wt%, from about 3.00 wt% to about 48.00 wt%, from about 4.00 wt% to about 47.00 wt%, from about 5.00 wt% to about 46.00 wt%, from about 6.00 wt% to about 45.00 wt%, from about 7.00 wt% to about 44.00 wt%, from about 8.00 wt% to about 43.00 wt%, from about 9.00 wt% to about 42.00 wt%, from about 10.00 wt% to about 41 .00 wt%, from about 1 1 .00 wt% to about 40.00 wt%, from about 12.00 wt% to about 39.00 wt%, from about 13.00 wt% to about 38.00 wt%, from about 14.00 wt% to about 37.00 wt%, from about 15.00 wt% to about 36.00 wt%, from about 16.00 wt% to about 35.00 wt%, from about 17.00 wt% to about 34.00 wt%, from about 18.00 wt% to about 33.00 wt%, from about 19.00 wt% to about 32.00 wt%, from about 20.00 wt% to about 31 .00 wt%, from about 21 .00 wt% to about 30.00 wt%, from about 22.00 wt% to about 29.00 wt%, from about 23.00 wt% to about 28.00 wt%, from about 24.00 wt% to about 27.00 wt%, from about 25.00 wt% to about 26.00 wt%, or about 25.50 wt% of the cationic lipid.

[0075] In various embodiments, the composition comprises more than about 0 wt%, more than about 0.1 wt%, more than about 0.2 wt%, more than about 0.3 wt%, more than about 0.4 wt%, more than about 0.5 wt%, more than about 0.6 wt%, more than about 0.7 wt%, more than about 0.8 wt%, more than about 0.9 wt%, from about 1.00 wt% to about 50.00 wt%, from about 2.00 wt% to about

[0076] 50.00 wt, from about 3.00 wt% to about 50.00 wt, from about 4.00 wt% to about 50.00 wt, from about 5.00 wt% to about 50.00 wt, from about 2.00 wt% to about 49.00 wt%, from about 3.00 wt% to about 48.00 wt%, from about 4.00 wt% to about 47.00 wt%, from about 5.00 wt% to about 46.00 wt%, from about 6.00 wt% to about 45.00 wt%, from about 7.00 wt% to about 44.00 wt%, from about 8.00 wt% to about 43.00 wt%, from about 9.00 wt% to about 42.00 wt%, from about 10.00 wt% to about 41 .00 wt%, from about 11 .00 wt% to about 40.00 wt%, from about 12.00 wt% to about 39.00 wt%, from about 13.00 wt% to about 38.00 wt%, from about 14.00 wt% to about 37.00 wt%, from about 15.00 wt% to about 36.00 wt%, from about 16.00 wt% to about 35.00 wt%, from about 17.00 wt% to about 34.00 wt%, from about 18.00 wt% to about 33.00 wt%, from about 19.00 wt% to about 32.00 wt%, from about 20.00 wt% to about 31 .00 wt%, from about 21 .00 wt% to about 30.00 wt%, from about 22.00 wt% to about 29.00 wt%, from about 23.00 wt% to about 28.00 wt%, from about 24.00 wt% to about 27.00 wt%, from about 25.00 wt% to about 26.00 wt%, or about 25.50 wt% of the ionizable lipid. In various embodiments, the optimal weight ratios and concentrations of the ionizable lipid in the composition may vary. In various embodiment, incorporation of the ionizable lipid at appropriate levels enhances gene expression and yields cytocompatibility.

[0077] In various embodiments, the total amount of the ionizable lipid and the cationic lipid present in the composition falls in the range of from about 30 wt% to about 60 wt%, from about 31 wt% to about 59 wt%, from about 32 wt% to about 58 wt%, from about 33 wt% to about 57 wt%, from about 34 wt% to about 56 wt%, from about 35 wt% to about 55 wt%, from about 36 wt% to about 54 wt%, from about 37 wt% to about 53 wt%, from about 38 wt% to about 52 wt%, from about 39 wt% to about 51 wt%, from about 40 wt% to about 50 wt%, from about 41 wt% to about 49 wt%, from about 42 wt% to about 48 wt%, from about 43 wt% to about 47 wt%, from about 44 wt% to about 46 wt%, or about 45 wt% of the composition.

[0078] In various embodiments, the composition further comprises one or more of the following components:

[0079] (i) glycerolipid;

[0080] (ii) esterified cholesterol;

[0081] (iii) phospholipid (e.g., helper phospholipid);

[0082] (iv) cholesterol or its analogues thereof; and / or (v) polyethylene glycol (PEG) (e.g., in the form of a PEG-conjugate such as lipid-PEG conjugate).

[0083] The term “lipid-PEG conjugate” may comprise and / or may be used interchangeably with the terms “PEG-modified lipid”, “PEGylated lipid” and “lipid modified with PEG”.

[0084] In various embodiments, the glycerolipid comprises a triglyceride such as triolein, tristearin, trielaidin, trilinolein, tripalmitin, tripalmitolein, trimyristin, trilaurin, tricaproin, trioctanoin, triarachidin, triarachidonin, the like, or combinations thereof.

[0085] In various embodiments, the glycerolipid or triglyceride exists in a liquid state at physiological temperature, e g., about 37 °C.

[0086] In various embodiments, the esterified cholesterol comprises cholesteryl ester such as cholesteryl oleate, cholesteryl stearate, cholesteryl linoleate, cholesteryl palmitate, cholesteryl myristate, cholesteryl laurate, cholesteryl caproate, cholesteryl arachidate, cholesteryl arachidonate, the like, or combinations thereof.

[0087] In various embodiments, the phospholipid is selected from dioleoylphosphatidylethanolamine (DOPE), 1 -palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE), 1 ,2-dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE), 1 ,2-dilauroyl-sn-glycero-3- phosphoethanolamine (DLPE), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2-dipalmitoyl-sn- glycero-3-phosphatidylcholine (DPPC), 1 ,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), 1 ,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1 ,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), 1 -palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine (POPC), 1 -stearoyl-2-oleoyl-sn-glycero-3- phosphocholine(SOPC), the like, or combinations thereof. Advantageously, in various embodiments, DOPE exhibits fusogenic properties and is capable of disrupting the endosomal membrane by inducing the formation of a non-bilayer hexagonal Hu phase under acidic pH conditions.

[0088] In various embodiments, the cholesterol or its analogues thereof is selected from cholesterol, p-sitosterol, p-sitosterol-acetate, stigmastanol, campesterol, fucosterol, brassicasterol, ergosterol, 9,11 -dehydroergosterol, daucosterol, vitamin D2, vitamin D3, vitamin E, calcipotriol, betulin, lupeol, ursolic acid, oleanolic acid, the like, derivatives thereof, or combinations thereof. In various embodiments, the composition comprises p-sitosterol. Advantageously, P-sitosterol may enhance mRNA translation efficiency of LNPs by preventing recognition by cholesterol transporters, which can trigger LNP exocytosis and decrease cellular retention.

[0089] In various embodiments, the PEG conjugate is selected from 1 ,2- distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 2000 (DSPE- PEG), methoxypolyethyleneglycoloxy(2000)- / V,A / -ditetradecylacetamide (ALC- 0159), 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG- PEG), 1 ,2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (DSG-PEG), 1 ,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine- / \ / -[(polyethylene glycol)- 2000] (DPPE-PEG), 1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine- / \ / - [(polyethylene glycol)-2000] (DMPE-PEG), 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine- / \ / -[(polyethylene glycol)-2000] (DOPE-PEG), / V-palmitoyl- sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]} (C16 PEG ceramide), / V-octanoyl-sphingosine-1 -{succinyl[methoxy(polyethylene glycol)2000]} (C8 PEG2000 ceramide), the like, or combinations thereof.

[0090] Advantageously, in various embodiments, the PEG conjugate is capable of effectively preventing the aggregation and the non-specific protein adsorption of the nanoparticles when exposed to serum components in cell culture media, thereby leading to improved nanoparticle stability and enhanced transfection efficiency of the therapeutic and / or prophylactic agent and / or biological agent. In various embodiments, the PEG-conjugate-incorporated composition (e.g., on the surface on the LNP) enhances gene transfection and allows for use in in vivo settings.

[0091] In various embodiments, the composition comprises from about 0.10 wt% to about 6.00 wt%, from about 0.20 wt% to about 5.00 wt%, from about 0.30 wt% to about 4.00 wt%, from about 0.40 wt% to about 3.90 wt%, from about 0.50 wt% to about 3.80 wt%, from about 0.60 wt% to about 3.70 wt%, from about 0.70 wt% to about 3.60 wt%, from about 0.80 wt% to about 3.50 wt%, from about 0.90 wt% to about 3.40 wt%, from about 1 .00 wt% to about 3.30 wt%, from about 1 .10 wt% to about 3.30 wt%, from about 1 .20 wt% to about 3.10 wt%, from about 1 .30 wt% to about 3.00 wt%, from about 1 .40 wt% to about 3.00 wt%, from about 1 .50 wt% to about 3. 00 wt%, from about 1 .00 wt% to about 5.00 wt%, from about 1 .50 wt% to about 4.50 wt%, from about 2.00 wt% to about 4.00 wt%, from about 2.50 wt% to about 3.50 wt%, about 2.00 wt%, about 2.10 wt%, about 2.20 wt%, about 2.30 wt%, about 2.40 wt%, about 2.50 wt%, about 2.60 wt%, about 2.70 wt%, about 2.90 wt%, or about 3 wt% of the glycerolipid.

[0092] In various embodiments, the composition comprises from about 10.00 wt% to about 30.00 wt%, from about 11 .00 wt% to about 29.00 wt%, from about 12.00 wt% to about 28.00 wt%, from about 13.00 wt% to about 27.00 wt%, from about 14.00 wt% to about 26.00 wt%, from about 15.00 wt% to about 25.00 wt%, from about 16.00 wt% to about 24.00 wt%, from about 17.00 wt% to about 23.00 wt%, from about 18.00 wt% to about 22.00 wt%, from about 19.00 wt% to about 21 .00 wt%, or about 20.00 wt% of the esterified cholesterol.

[0093] In various embodiments, the composition comprises from about 10.00 wt% to about 40.00 wt%, from about 11 .00 wt% to about 39.00 wt%, from about 12.00 wt% to about 38.00 wt%, from about 13.00 wt% to about 37.00 wt%, from about 14.00 wt% to about 36.00 wt%, from about 15.00 wt% to about 35.00 wt%, from about 16.00 wt% to about 34.00 wt%, from about 17.00 wt% to about 33.00 wt%, from about 18.00 wt% to about 32.00 wt%, from about 19.00 wt% to about 31 .00 wt%, from about 20.00 wt% to about 30.00 wt%, from about 21 .00 wt% to about 29.00 wt%, from about 22.00 wt% to about 28.00 wt%, from about 23.00 wt% to about 27.00 wt%, from about 24.00 wt% to about 26.00 wt%, from about 10.00 wt% to about 30.00 wt%, from about 11 .00 wt% to about 29.00 wt%, from about 12.00 wt% to about 28.00 wt%, from about 13.00 wt% to about 27.00 wt%, from about 14.00 wt% to about 26.00 wt%, from about 15.00 wt% to about 25.00 wt%, from about 16.00 wt% to about 24.00 wt%, from about 17.00 wt% to about 23.00 wt%, from about 18.00 wt% to about 22.00 wt%, from about 19.00 wt% to about 21 .00 wt%, or about 20.00 wt% of the phospholipid.

[0094] In various embodiments, the composition comprises from about 0.10 wt% to about 40.00 wt%, from about 1 .00 wt% to about 39.00 wt%, from about 2.00 wt% to about 38.00 wt%, from about 3.00 wt% to about 37.00 wt%, from about 4.00 wt% to about 36.00 wt%, from about 5.00 wt% to about 35.00 wt%, from about 6.00 wt% to about 34.00 wt%, from about 7.00 wt% to about 33.00 wt%, from about 8.00 wt% to about 32.00 wt%, from about 9.00 wt% to about 31.00 wt%, from about 10.00 wt% to about 30.00 wt%, from about 11.00 wt% to about 29.00 wt%, from about 12.00 wt% to about 28.00 wt%, from about 13.00 wt% to about 27.00 wt%, from about 14.00 wt% to about 26.00 wt%, from about 15.00 wt% to about 25.00 wt%, from about 16.00 wt% to about 24.00 wt%, from about 17.00 wt% to about 23.00 wt%, from about 18.00 wt% to about 22.00 wt%, from about 19.00 wt% to about 21.00 wt%, from about 0.10 wt% to about 6.00 wt%, from about 1.00 wt% to about 30.00 wt%, from about 2.00 wt% to about 29.00 wt%, from about 3.00 wt% to about 28.00 wt%, from about 4.00 wt% to about 27.00 wt%, from about 5.00 wt% to about 26.00 wt%, from about 6.00 wt% to about 25.00 wt%, from about 7.00 wt% to about 24.00 wt%, from about 8.00 wt% to about 23.00 wt%, from about 9.00 wt% to about 22.00 wt%, from about 10.00 wt% to about 21 .00 wt%, from about 11 .00 wt% to about 20.00 wt%, from about 12.00 wt% to about 19.00 wt%, from about 13.00 wt% to about 18.00 wt%, from about 14.00 wt% to about 17.00 wt%, from about 15.00 wt% to about 16.00 wt%, from about 0.10 wt% to 6.00 wt%, from about 0.20 wt% to about 5.00 wt%, from about 0.30 wt% to about 4.00 wt%, from about 0.40 wt% to about 3.90 wt%, from about 0.50 wt% to about 3.80 wt%, from about 0.60 wt% to about 3.70 wt%, from about 0.70 wt% to about 3.60 wt%, from about 0.80 wt% to about 3.50 wt%, from about 0.90 wt% to about 3.40 wt%, from about 1 .00 wt% to about 3.30 wt%, from about 1.10 wt% to about 3.30 wt%, from about 1.20 wt% to about 3.10 wt%, from about 1 .30 wt% to about 3.00 wt%, from about 1 .40 wt% to about 3.00 wt%, from about 1 .50 wt% to about 3. 00 wt%, about 2.00 wt%, about 2.10 wt%, about 2.20 wt%, about 2.30 wt%, about 2.40 wt%, about 2.50 wt%, about 2.60 wt%, about 2.70 wt%, about 2.90 wt%, or about 3 wt% of the cholesterol or its analogues thereof. In various embodiments, the cholesterol or its analogues thereof are different from the esterified cholesterol disclosed herein.

[0095] In various embodiments, the composition comprises from about 0.10 mol% to about 3.00 mol%, from about 0.20 mol% to about 2.90 mol%, from about 0.30 mol% to about 2.80 mol%, from about 0.40 mol% to about 2.70 mol%, from about 0.50 mol% to about 2.60 mol%, from about 0.60 mol% to about 2.50 mol%, from about 0.70 mol% to about 2.40 mol%, from about 0.80 mol% to about 2.30 mol%, from about 0.90 mol% to about 2.20 mol%, from about 1 .00 mol% to about 2.10 mol%, from about 1.10 mol% to about 2.00 mol%, from about 1.20 mol% to about 1.90 mol%, from about 1.30 mol% to about 1.80 mol%, from about 1.40 mol% to about 1.70 mol%, from about 1.50 mol% to about 1.60 mol%, or about 1.55 mol% of the PEG conjugate.

[0096] In various embodiments, the total lipid concentration is from about 1 mg / ml_ to about 50 mg / mL, from about 2 mg / mL to about 49 mg / mL, from about 3 mg / mL to about 48 mg / mL, from about 4 mg / mL to about 47 mg / mL, from about 5 mg / mL to about 46 mg / mL, from about 6 mg / mL to about 45 mg / mL, from about 7 mg / mL to about 44 mg / mL, from about 8 mg / mL to about 43 mg / mL, from about 9 mg / mL to about 42 mg / mL, from about 10 mg / mL to about 41 mg / mL, from about 11 mg / mL to about 40 mg / mL, from about 12 mg / mL to about 39 mg / mL, from about 13 mg / mL to about 38 mg / mL, from about 14 mg / mL to about 37 mg / mL, from about 15 mg / mL to about 36 mg / mL, from about 16 mg / mL to about 35 mg / mL, from about 17 mg / mL to about 34 mg / mL, from about 18 mg / mL to about 33 mg / mL, from about 19 mg / mL to about 32 mg / mL, from about 20 mg / mL to about 31 mg / mL, from about 21 mg / mL to about 30 mg / mL, from about 22 mg / mL to about 29 mg / mL, from about 23 mg / mL to about 28 mg / mL, from about 24 mg / mL to about 27 mg / mL, from about 25 mg / mL to about 26 mg / mL, or about 25.5 mg / mL. In various embodiments, the total lipid composition comprises cationic lipid, ionizable lipid, glycerolipid, phospholipid, cholesterol or its analogues thereof, optionally esterified cholesterol, and optionally PEG- conjugate. In various embodiments, the total lipid composition excludes the therapeutic, and / or prophylactic, and / or biological agent.

[0097] In various embodiments, the composition further comprises a therapeutic agent, and / or prophylactic agent, and / or biological agent.

[0098] In various embodiments, there is also provided a composition comprising / consisting essentially of / consisting of:

[0099] (i) a cationic lipid in an amount of 5 to 50 wt% of the composition;

[0100] (ii) an ionizable lipid in an amount of 5 to 50 wt% of the composition;

[0101] (iii) a glycerolipid in an amount of 1 to 5 wt% of the composition;

[0102] (iv) a phospholipid in an amount of 10 to 40 wt% of the composition;

[0103] (v) a cholesterol or analogues thereof in an amount of 1 to 30 wt% of the composition;

[0104] (vi) optionally an esterified cholesterol in an amount of 10 to 30 wt% of the composition;

[0105] (vii) optionally a PEG-conjugate in an amount of from 0.10 to 3 mol% of the composition; and

[0106] (viii) optionally a therapeutic agent, wherein the cationic lipid and ionizable lipid are present in a ratio that falls in the range of 1 -5 : 5-1 .

[0107] In some embodiments, the esterified cholesterol is present, while in other embodiments, the esterified cholesterol is absent. In some embodiments, cholesterol is present, while in other embodiments, a cholesterol analogue is used in place of cholesterol. For example, [3-sitosterol may be used in lieu of cholesterol to enhance mRNA translation efficiency of LNPs by avoiding recognition by cholesterol transporters that trigger LNP exocytosis and reduce cellular retention. In other embodiments, a combination of cholesterol and its analogues may be used. Cholesterol analogues include but are not limited to sterols such as plant sterols.

[0108] In various embodiments, the composition is suitable for producing high transfection efficiency and reduced toxicity. For example, administration of composition may produce high gene transfection efficiency in vitro.

[0109] In various embodiments, the presence of the PEG-conjugate (e.g., lipidpolyethylene glycol (PEG) conjugate) further allows gene transfection efficiency to be achieved in in vivo settings.

[0110] NANOPARTICLES

[0111] There is provided a nanoparticle (e.g., lipid nanoparticles) comprising the composition as disclosed herein. In various embodiments, the nanoparticle may also be considered as a composition itself.

[0112] Advantageously, in various embodiments, the composition allows for the condensation and encapsulation / loading of molecules / cargoes into embodiments of the composition, thereby forming nanoparticles. In other words, embodiments of the composition are capable of forming nanoparticles. In various embodiments, in the presence of a composition comprising molecules / cargoes (e.g., therapeutic agent, prophylactic agent, and / or biological agent), the cationic lipid in the composition condenses and encapsulates / loads the molecules / cargoes to form nanoparticles (e.g., lipid nanoparticles (LNPs)) in the composition. The term “nanoparticles” may comprise and / or may be used interchangeably with the terms “lipid nanoparticles’’, “encapsulated lipid nanoparticles”, “loaded lipid nanoparticles”, “LNPs”, or the like.

[0113] In various embodiments, the nanoparticle has a core-shell structure. In various embodiments, the core is a non-polar core. In various embodiments, the core comprises the glycerolipid and the esterified cholesterol as disclosed herein. In various embodiments, the shell structure (e.g., the amphiphilic lipid shell layer) comprises one or more of the ionizable lipid, the cationic lipid, the phospholipid, the cholesterol or analogues thereof, and / or the PEG conjugates as disclosed herein.

[0114] In various embodiments, there is provided a nanoparticle comprising / consisting essentially of / consisting of

[0115] (i) a core structure comprising glycerolipid and optionally an esterified cholesterol;

[0116] (ii) a surface structure comprising a cationic lipid, an ionizable lipid, a phospholipid, a cholesterol or analogues thereof, and optionally a PEG-conjugate; and

[0117] (iii) a therapeutic agent complexed with the surface structure, wherein the cationic lipid and ionizable lipid are present in a ratio that falls in the range of 1 -5 : 5-1 .

[0118] Advantageously, in various embodiments, the cationic lipid is capable of condensing nucleic acid therapeutics onto the surface of the nanoparticles, and the ionizable lipid is capable of promoting endosomal release of the nucleic acid therapeutics for effective transfection. This may allow the nanoparticles disclosed herein to mediate high gene transfection efficiency, comparable to the commercially available lipofectamine-based reagents, while being more cytocompatible. In various embodiments, the incorporation of lipid-PEG in the form of a PEG-conjugate allows for greater stability, thus enhancing gene transfection. The presence of PEG on the surface of nanoparticles may also allow for in vivo application.

[0119] In various embodiments, the nanoparticle is an ionizable lipid-solid lipid nanoparticle (iSLN). The iSLN may comprise the chemical composition of (i). cholesteryl ester (cholesteryl oleate) and triglyceride (triolein), (ii). cationic lipid (DOTMA), (iii). ionizable lipid (ALC-0315), (iv). phospholipids (DOPE), and (v). cholesterol. In various embodiments, component (i). makes up the core of the iSLN while components (ii)-(v) make up the surface of the iSLN.

[0120] In various embodiments, the nanoparticle is an ionizable liquid lipid nanoparticles (iLLNs) with a similar composition of the iSLN but without cholesteryl ester (cholesteryl oleate).

[0121] In some embodiments, the esterified cholesterol is present in the nanoparticles, while in other embodiments, the esterified cholesterol is absent.

[0122] In some embodiments, cholesterol is present, while in other embodiments, a cholesterol analogue is used in place of cholesterol. For example, p-sitosterol may be used in lieu of cholesterol to enhance mRNA translation efficiency of LNPs by avoiding recognition by cholesterol transporters that trigger LNP exocytosis and reduce cellular retention. In other embodiments, a combination of cholesterol and its analogues may be used. Cholesterol analogues include but are not limited to sterols such as plant sterols.

[0123] In various embodiments, the therapeutic agent is bound to the surface of the nanoparticle.

[0124] In various embodiments, the nanoparticle comprises ionizable lipid and cationic lipid at weight ratio of about 1 :5, about 1 :4, about 1 :3, about 1 :2, about 1 :1 , about 1 :0, about 2:1 , about 3:1 , about 4:1 , or about 5:1. In various embodiments, the total amount of the ionizable lipid and cationic lipid present in the nanoparticle falls in the range of from about 30 wt% to about 60 wt%, from about 31 wt% to about 59 wt%, from about 32 wt% to about 58 wt%, from about 33 wt% to about 57 wt%, from about 34 wt% to about 56 wt%, from about 35 wt% to about 55 wt%, from about 36 wt% to about 54 wt%, from about 37 wt% to about 53 wt%, from about 38 wt% to about 52 wt%, from about 39 wt% to about 51 wt%, from about 40 wt% to about 50 wt%, from about 41 wt% to about 49 wt%, from about 42 wt% to about 48 wt%, from about 43 wt% to about 47 wt%, from about 44 wt% to about 46 wt%, or about 45 wt% of the total lipid composition of the nanoparticle. In various embodiments, the total lipid composition comprises cationic lipid, ionizable lipid, glycerolipid, phospholipid, cholesterol or its analogues thereof, optionally esterified cholesterol, and optionally PEG- conjugate. In various embodiments, the total lipid composition excludes the therapeutic, and / or prophylactic, and / or biological agent.

[0125] In various embodiments, the therapeutic, and / or prophylactic agent, and / or biological agent is coupled / bonded / linked / tagged to the nanoparticle surface and / or shell structure instead of being encapsulated in the core of the LNP. In various embodiments, the LNP surface may be modulated via tagging the nucleic acid therapeutics to enhance transfection and reduced specific toxicity.

[0126] In various embodiments, the nanoparticle is a solid or liquid lipid nanoparticle such as an ionizable lipid-incorporated solid or liquid lipid nanoparticle. In various embodiments, the nanoparticle may be formulated as a medicament suitable for inhalation and administered through the respiratory tract, such as for nasal, trachea, and / or pulmonary administration. For example, the medicament may be in the form of a spray. In various embodiments, liquid core LNPs may effectively bypass the mucus barrier and traverse the nasal mucosa, facilitating mRNA delivery beyond the underlying epithelium, making them suitable candidates for nasal vaccines. In various embodiments, the nanoparticle has a N:P or N / P ratio (i.e., molar ratio of ionizable (in the physiological pH range) nitrogen atoms in a nanoparticle / composition to phosphate groups in the therapeutic and / or prophylactic agent and / or biological agent (e.g., nucleic acid) is from about 1 :1 to about 20: 1 , from about 1 : 1 to about 19:1 , from about 1 : 1 to about 18:1 , from about 1 :1 to about 17:1 , from about 1 :1 to about 16:1 , from about 1 :1 to about 15:1 , from about 1 :1 to about 14:1 , from about 1 :1 to about 13:1 , from about 1 :1 to about 12:1 , from about 1 :1 to about 11 :1 , from about 1 :1 to about 10:1 , from about 1 :1 to about 9:1 , from about 1 :1 to about 8:1 , from about 1 :1 to about 7:1 , from about 1 : 1 to about 6: 1 , from about 1 : 1 to about 5: 1 , from about 1 : 1 to about 4: 1 , from about 1 :1 to about 3:1 , or from about 1 :1 to about 2:1. It will be appreciated that in various embodiments, the optimal N / P ratio is dependent on the type of therapeutic, and / or prophylactic agent, and / or biological agent (e.g., a nucleic acid such as mRNA, siRNA, pDNA and oligonucleotides). For example, the optimal N / P ratio may be different for siRNA, pDNA, and oligonucleotides.

[0127] In various embodiments, the in vitro nucleic acid transfection efficiency of the therapeutic, and / or prophylactic agent, and / or biological agent in the nanoparticle is greater than that of a commercially available agent (e.g., Lipofectamine 3000 (LF3000) reagent, and Lipofectamine™ MessengerMAX (LFMax) reagent).

[0128] In various embodiments, the nanoparticle has an in vitro nucleic acid transfection efficiency that is no less or higher than that of a commercially available agent under similar conditions. For example, the pDNA transfection efficiency may be at least 0 time, 1 time, 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times higher than that of a commercially available agent under similar conditions.

[0129] It will be appreciated that in various embodiments, the in vitro nucleic acid transfection efficiency of the nanoparticle may vary depending on the experimental conditions during nucleic acid transfection such as nucleic acid cargoes, cell types, incubation time, and culture media.

[0130] It will be appreciated that in various embodiments, the nanoparticle may still be useful for in vivo therapeutic and / or prophylactic applications even when its in vitro transfection efficiency is lower than that of a commercially available agent.

[0131] In various embodiments, the nanoparticle has an average particle size (or diameter) of from about 50 nm to about 150 nm, from about 60 nm to about 140 nm, from about 70 nm to about 130 nm, from about 80 nm to about 120 nm, from about 90 nm to about 110 nm, or about 100 nm.

[0132] In various embodiments, the nanoparticle has a polydispersity index (PDI) of from about 0.05 to about 0.30, from about 0.06 to about 0.29, from about 0.07 to about 0.28, from about 0.08 to about 0.27, from about 0.09 to about 0.26, from about 0.10 to about 0.25, from about 0.11 to about 0.24, from about 0.12 to about 0.23, from about 0.13 to about 0.22, from about 0.14 to about 0.21 , from about 0.15 to about 0.20, from about 0.16 to about 0.19, from about 0.17 to about 0.18, or about 0.175. In various embodiments, the nanoparticle has a narrow particle size distribution (PDI < 0.30), and / or the nanoparticle composition is relatively / substantially homogenous.

[0133] In various embodiments, the nanoparticle in normal saline or a 0.90% w / v NaCI solution has a zeta potential of from about 0 mV to about +50 mV, from about +1 mV to about +49 mV, from about +2 mV to about +48 mV, from about +3 mV to about +47 mV, from about +4 mV to about +46 mV, from about +5 mV to about +45 mV, from about +6 mV to about +44 mV, from about +7 mV to about +43 mV, from about +8 mV to about +42 mV, from about +9 mV to about +41 mV, from about +10 mV to about +40 mV, from about +11 mV to about +39 mV, from about +12 mV to about +38 mV, from about +13 mV to about +37 mV, from about +14 mV to about +36 mV, from about +15 mV to about +35 mV, from about +16 mV to about +34 mV, from about +17 mV to about +33 mV, from about +18 mV to about +32 mV, from about +19 mV to about +31 mV, from about +20 mV to about +30 mV, from about +21 mV to about +29 mV, from about +22 mV to about +28 mV, from about +23 mV to about +27 mV, from about +24 mV to about +26 mV, or about +25 mV. Advantageously, in various embodiments, the nanoparticle of any one of the preceding AS may form a positively charged surface at an acidic pH (e.g., pH 5.5) within endosomes, thereby inducing endosomal disruption and accelerating the release of the therapeutic, and / or prophylactic agent, and / or biological agent into the cytoplasm. Advantageously, in various embodiments, the nanoparticle has a substantially neutral surface charge, making the nanoparticle suitable / desirable for in vivo applications.

[0134] In various embodiments, the nanoparticle has an acid dissociation constant (pKa) of from about 4.5 to about 7.0, from about 4.6 to about 6.9, from about 4.7 to about 6.8, from about 4.8 to about 6.7, from about 4.9 to about 6.6, from about 5.0 to about 6.5, from about 5.1 to about 6.4, from about 5.2 to about 6.3, from about 5.3 to about 6.2, from about 5.4 to about 6.1 , from about 5.5 to about 6.0, from about 5.6 to about 5.9, from about 5.7 to about 5.8, or about 5.75. Advantageously, in various embodiments, the pKa of the nanoparticle is closely aligned with the endosomal pH range (5.55 to 6.5), enabling the formation of a more cationic surface charge within endosomes at acidic pH, which results in endosomal disruption and facilitates the release of the therapeutic, and / or prophylactic agent, and / or biological agent into the cytoplasm.

[0135] In various embodiments, the composition / nanoparticle is biocompatible and cytocompatible, i.e., the composition / nanoparticle is compatible with biological systems or parts of the biological systems without substantially or significantly eliciting an adverse physiological response such as a toxic reaction / response (e.g., cytotoxicity), an immune reaction / response, an injury or the like when used on the human or animal body. In various embodiments, the composition / nanoparticle is substantially devoid of substances that elicit an adverse physiological response.

[0136] In various embodiments, the composition / nanoparticle may be stored at a temperature of from about -10 °C to about 10 °C, from about -9 °C to about 9 °C, from about -8 °C to about 8 °C, from about -7 °C to about 7 °C, from about -6 °C to about 6 °C, from about -5 °C to about 5 °C, from about -4 °C to about 4 °C, from about -3 °C to about 3 °C, from about -2 °C to about 2 °C, from about -1 °C to about 1 °C, or about 0 °C.

[0137] In various embodiments, when the composition / nanoparticle is to be stored in a frozen state (i.e., at a temperature below 0 °C), suitable cryoprotectants may be added.

[0138] In various embodiments, the composition / nanoparticle may have a storage stability of at least about 20 weeks, at least about 19 weeks, at least about 18 weeks, at least about 17 weeks, at least about 16 weeks, at least about 15 weeks, at least about 14 weeks, at least about 13 weeks, at least about 12 weeks, at least about 11 weeks, at least about 10 weeks, at least about 9 weeks, at least about 8 weeks, at least about 7 weeks, at least about 6 weeks, at least about 5 weeks, at least about 4 weeks, at least about 3 weeks, at least about 2 weeks, at least about 1 week without an appreciable loss in desired properties. In various embodiments, the composition / nanoparticle may exhibit storage stability and retain activity for over 3 months when stored at 4 °C.

[0139] In various embodiments, the presence of both ionizable lipid and cationic lipid may induce the formation of highlight crystalline lipid phase in the nanoparticle dispersion, enhancing its storage stability. METHODS OF PREPARING COMPOSITION AND NANOPARTICLES

[0140] There is provided a method of preparing the composition. In various embodiments, the method comprises providing one or more of the various ingredients as disclosed herein in an organic solvent.

[0141] There is also provided method of preparing the nanoparticle as disclosed herein. In various embodiments, the method comprises

[0142] (a) adding an aqueous solution to the organic solvent comprising the composition as disclosed herein to form an immiscible mixture;

[0143] (b) agitating the immiscible mixture to obtain an oil-in-water (O / W) emulsion / suspension of the composition as disclosed herein;

[0144] (c) evaporating the organic solvent from the O / W emulsion / suspension to obtain the nanoparticle in aqueous suspension;

[0145] (d) optionally adding an aqueous solution of the therapeutic and / or prophylactic agent and / or biological agent as disclosed herein to the nanoparticle in aqueous suspension; and

[0146] (e) optionally incubating the mixture.

[0147] In various embodiments, the organic solvent may be an organic solvent such as chloroform, ethanol, methanol, isopropanol, butanol, ethyl acetate, dichloromethane (DCM), or the like or combinations thereof.

[0148] In various embodiments, the aqueous solution may be free from any components that react unfavourably with the therapeutic or biological substance. For example, when the therapeutic, prophylactic or biological agent / substance comprises nucleic acids, any nuclease-free aqueous solution / solvent such as nuclease-free water or nuclease-free deionized water, normal saline, phosphate- buffered saline, Tris-buffered saline, sodium acetate buffer, sodium citrate buffer, or the like or combinations thereof may be used. In various embodiments, the organic solvent may be evaporated from the oil-in-water emulsion / suspension of the composition at a temperature of from about 40°C to about 80°C, from about 41 °C to about 79°C, from about 42°C to about 78°C, from about 43°C to about 77°C, from about 44°C to about 76°C, from about 45°C to about 75°C, from about 46°C to about 74°C, from about 47°C to about 73°C, from about 48°C to about 72°C, from about 49°C to about 71 °C, from about 50°C to about 70°C, from about 51 °C to about 69°C, from about 52°C to about 68°C, from about 53°C to about 67°C, from about 54°C to about 66°C, from about 55°C to about 65°C, from about 56°C to about 64°C, from about 57°C to about 63°C, from about 58°C to about 62°C, from about 59°C to about 61 °C, or about 60°C for a period of from about 5 minutes to about 30 minutes, from about 6 minutes to about 29 minutes, from about 7 minutes to about 28 minutes, from about 8 minutes to about 27 minutes, from about 9 minutes to about 26 minutes, from about 10 minutes to about 25 minutes, from about 11 minutes to about 24 minutes, from about 12 minutes to about 23 minutes, from about 13 minutes to about 22 minutes, from about 14 minutes to about 21 minutes, from about 15 minutes to about 20 minutes, from about 16 minutes to about 19 minutes, from about 17 minutes to about 18 minutes, or about 17 minutes and 30 seconds.

[0149] In various embodiments, the mixture in step (e) is allowed to be incubated at a temperature of from about 20°C to about 30°C, from about 21 °C to about 29°C, from about 22°C to about 28°C, from about 23°C to about 27°C, from about 24°C to about 26°C, or about 25°C for a period of from about 5 minutes to about 30 minutes, from about 6 minutes to about 29 minutes, from about 7 minutes to about 28 minutes, from about 8 minutes to about 27 minutes, from about 9 minutes to about 26 minutes, from about 10 minutes to about 25 minutes, from about 11 minutes to about 24 minutes, from about 12 minutes to about 23 minutes, from about 13 minutes to about 22 minutes, from about 14 minutes to about 21 minutes, from about 15 minutes to about 20 minutes, from about 16 minutes to about 19 minutes, from about 17 minutes to about 18 minutes, or about 17 minutes and 30 seconds. In various embodiments, step (d) comprises mixing the aqueous nanoparticle suspension with the aqueous solution of the therapeutic and / or prophylactic agent and / or biological agent at a volume ratio of from about 5:1 to about 1 :1 , from about 4: 1 to about 1 :1 , from about 3: 1 to about 1 : 1 , or from about 2:1 to about 1 :1.

[0150] TRANSFECTION METHOD

[0151] There is provided a method of transfecting a cell with a nucleic acid therapeutic agent (e g., mRNA, siRNA, DNA, etc.). In various embodiments, the method comprises contacting the cell of with the nanoparticle containing the nucleic acid therapeutic agent as disclosed herein.

[0152] In various embodiments, the cell may be from a human cell line, wherein the human cell line may be human embryonic kidney (HEK) 293 cell line or its variants such as HEK293T, HEK293F, HEK293FT, HEK293S, HEK293FTM, HEK293SG, HEK293SGGD, HEK293H, HEK293E, HEK EBNA1 -6E, HEK293MSR, or HEK 293A.

[0153] In various embodiments, the contacting step is carried out in the presence of a culture media which may comprise fetal bovine serum, penicillin, streptomycin, the like, or combinations thereof.

[0154] In various embodiments, the cell is allowed to be cultured for a period of from about 20 hours to 40 hours, from about 21 hours to about 39 hours, from about 22 hours to about 38 hours, from about 23 hours to about 37 hours, from about 24 hours to about 36 hours, from about 25 hours to about 35 hours, from about 26 hours to about 34 hours, from about 27 hours to about 33 hours, from about 28 hours to about 32 hours, from about 29 hours to about 31 hours, or about 30 hours. In various embodiments, the method further comprises incubating the cell and the nanoparticle for a period of from about 30 hours to 60 hours, from about 31 hours to about 59 hours, from about 32 hours to about 58 hours, from about

[0155] 33 hours to about 57 hours, from about 34 hours to about 56 hours, from about

[0156] 35 hours to about 55 hours, from about 36 hours to about 54 hours, from about

[0157] 37 hours to about 53 hours, from about 38 hours to about 52 hours, from about

[0158] 39 hours to about 51 hours, from about 40 hours to about 50 hours, from about

[0159] 41 hours to about 49 hours, from about 42 hours to about 48 hours, from about

[0160] 43 hours to about 47 hours, from about 44 hours to about 46 hours, or about 45 hours to allow transfection to take place.

[0161] In various embodiments, the cost of transfecting a therapeutic, and / or prophylactic agent, and / or biological agent in the nanoparticle as disclosed herein is lower than that in a commercially available reagent (e.g., LP3000, LPMax, etc ). In various embodiments, the cost of transfecting a therapeutic, and / or prophylactic agent , and / or biological agent in the nanoparticle as disclosed herein is lower than that in lipofectamine reagent by at least about 100 folds, at least about 90 folds, at least about 80 folds, at least about 70 folds, at least about 60 folds, at least about 50 folds, at least about 40 folds, at least about 30 folds, at least about 20 folds, or at least about 10 folds.

[0162] In various embodiments, there is also provided a carrier, nanocarrier, or delivery system / vehicle comprising the composition / nanoparticles as disclosed herein.

[0163] In various embodiments, there is also provided a vaccine composition comprising the composition / nanoparticles as disclosed herein.

[0164] In various embodiments, there is also provided a carrier, a nanocarrier, a delivery system / vehicle, a nanoparticle composition, nanoparticles (or lipid nanoparticles) disclosed herein for use in medicine (e g., for the treatment or prophylaxis of one or more of the diseases, disorders, or conditions mentioned herein).

[0165] In various embodiments, there is also provided a carrier, a nanocarrier, a delivery system / vehicle, a nanoparticle composition, nanoparticles (or lipid nanoparticles) disclosed herein for use in the treatment or prophylaxis of a disease, disorder, or condition, the use of said carrier, a nanocarrier, a delivery system / vehicle, a nanoparticle composition, nanoparticles (or lipid nanoparticles) in the manufacture of a medicament for the treatment or prophylaxis of a disease, disorder, or condition and / or a method of treatment or prophylaxis of a disease, disorder, or condition, comprising a step of administering (e.g. , in a therapeutically effective amount of) said carrier, a nanocarrier, a delivery system / vehicle, a nanoparticle composition, nanoparticles (or lipid nanoparticles) to a subject (e g., vertebrate such as a human or a large veterinary mammal (e g., horses, cattle, deer, sheep, llamas, goats, pigs) in need thereof. The disease, disorder, or condition may be selected from the group consisting of infectious / contagious diseases, viral infections (i.e. , diseases caused by virus), bacterial infections (i.e., diseases caused by bacteria), fungal infections (i.e., diseases caused by fungi), respiratory diseases, the like, or combinations thereof. In various embodiments, the disease, disorder, or condition is mediated by an influenza virus (e.g., influenza A, B, C, and / or D virus). For example, the disease may be influenza A, B, C, or D such as H1 N1 , H3N2). In various embodiments, the disease, disorder, or condition is mediated by a coronavirus (e.g., severe acute respiratory syndrome coronavirus such as SARS-CoV-2 or SARS-CoV-1 ). For example, the disease, disorder, or condition may be SARS-CoV-2 coronavirus disease.

[0166] In various embodiments, there is also provided a carrier, a nanocarrier, a delivery system / vehicle, a nanoparticle composition, nanoparticles (or lipid nanoparticles) disclosed herein for use in encapsulating and / or delivering a therapeutic, prophylactic, and / or biological agent to a subject, cell, cytosol, tissue or organ (e.g., a mammalian cell, cytosol, tissue, or organ), the use of said carrier, a nanocarrier, a delivery system / vehicle, a nanoparticle composition, nanoparticles (or lipid nanoparticles) in the manufacture of a medicament for encapsulating and / or delivering a therapeutic, prophylactic, and / or biological agent to a subject, cell, cytosol, tissue or organ (e.g., a mammalian cell, cytosol, tissue, or organ), and / or a method of delivering a therapeutic, prophylactic and / or biological agent to a subject, cell, cytosol, tissue or organ (e.g., a mammalian cell, cytosol, tissue, or organ), comprising a step of administering (e.g., in a therapeutically effective amount of) said carrier, a nanocarrier, a delivery system / vehicle, a nanoparticle composition, nanoparticles, (or lipid nanoparticles) to a subject (e g., vertebrate such as a human or a large veterinary mammal (e.g., horses, cattle, deer, sheep, llamas, goats, pigs) in need thereof.

[0167] In various embodiments, there is also provided a carrier, a nanocarrier, a delivery system / vehicle, a nanoparticle composition, nanoparticles (or lipid nanoparticles) disclosed herein for use in inducing an immune response in a subject (e.g., vertebrate such as a human or a large veterinary mammal (e.g., horses, cattle, deer, sheep, llamas, goats, pigs), the use of said carrier, a nanocarrier, a delivery system / vehicle, a nanoparticle composition, nanoparticles (or lipid nanoparticles) in the manufacture of a medicament for inducing an immune response in a subject, and / or a method of inducing an immune response in a subject, comprising a step of administering (e.g., in a therapeutically effective amount of) said carrier, a nanocarrier, a delivery system / vehicle, a nanoparticle composition, nanoparticles (or lipid nanoparticles) to a subject in need thereof. In various embodiments, an immune response in the subject is to be induced through the administration of a nanoparticle composition, nanoparticles (or lipid nanoparticles) thereto. In various embodiments, by inducing an immune response in the subject, the subject is protected against various diseases, disorders, or conditions e.g., infectious / contagious diseases, viral infections (i.e., diseases caused by virus), bacterial infections (i.e. , diseases caused by bacteria), fungal infections (i.e., diseases caused by fungi), respiratory diseases or the like, or combinations thereof as mentioned herein. The carrier, nanocarrier, delivery system / vehicle, nanoparticle composition, nanoparticles may be delivered to a subject in the form of or as a component of a vaccine.

[0168] In various embodiments, the disease, disorder or condition is mediated by a coronavirus (e g., severe acute respiratory syndrome coronavirus such as SARS-CoV-2 or SARS-CoV-1 ). For example, the disease, disorder, or condition may be SARS-CoV-2 coronavirus disease.

[0169] In various embodiments, the carrier, nanocarrier, delivery system / vehicle, nanoparticle composition, nanoparticles prepared from embodiments of the method disclosed herein comprises one or more of the following characteristics or properties: broad applicability (e g., can be used to encapsulate, deliver and / or transfect a wide range of therapeutic, prophylactic and / or biological reagents), nanosized, substantially neutral surface charge, high encapsulation efficiency, high transfection efficiency, high stability, low toxicity (e g., low cytotoxicity), low production / synthesis cost, therefore making them suitable for in vivo applications that require efficient cellular uptake and / or gene transfection.

[0170] BRIEF DESCRIPTION OF FIGURES

[0171] FIG. 1 shows the Z-average size and polydispersity index (PDI) of the ionizable lipid-incorporated solid lipid nanoparticle (iSLN), and cationic solid lipid nanoparticle (cSLN) formulations synthesized in accordance with various embodiments disclosed herein. The of iSLN (iSLN-1 to iSLN-8) and cSLN (cSLN- 1 and cSLN-2) formations were formulated at a total lipid concentration of 10 or 20 mg / mL using various weight ratios and concentrations of ALC-0315 and 1 ,2- dioleoyl-3-trimethylammonium-propane (DOTMA) as disclosed in Table 2. The values reported are the average (mean) values ± the standard deviation (SD), based on three independent measurements (n = 3).

[0172] FIG. 2 shows the 6-(p-Toluidino)-2-naphthalenesulfonic acid sodium salt (TNS) fluorescence measurement of the iSLN formulations (iSLN-1 to iSLN-8) synthesized in accordance with various embodiments disclosed herein as a function of measured pH. The apparent pKa value was determined as the pH value at which half of the maximum fluorescence signal was reached.

[0173] FIG. 3 shows the TNS fluorescence measurement of the cSLN (cSLN-1 and cSLN-2) formulations synthesized in accordance with various embodiments disclosed herein as a function of measured pH. The apparent pKa values could not be determined because the data points were poorly fitted to a four-parameter logistic function (R-squared value < 0.9).

[0174] FIG. 4 shows the apparent pKa values of the iSLN (iSLN-1 to iSLN-8) and cSLN (cSLN-1 and cSLN-2) formulations synthesized in accordance with various embodiments disclosed herein. The gray region indicates the range of endosomal pH (5 5-6.5). N D.: not detectable.

[0175] FIG. 5 shows the zeta potential values of the iSLN (iSLN-1 to iSLN-8) and cSLN (cSLN-1 and cSLN-2) formulations synthesized in accordance with various embodiments disclosed herein. The zeta potential values were measured in normal saline (pH 5.5). The values reported are the average (mean) values ± the standard deviation (SD), based on three independent measurements (n = 3).

[0176] FIG. 6 shows the Luciferase expression level and the viability of HEK293T cells transfected for 48 hours with iSLN / pDNA, cSLN / pDNA, or LF3000 / pDNA (positive control) complexes prepared in accordance with various embodiments disclosed herein. Untreated HEK293T cells were used as the negative control. Cell viability was determined as a percentage of the fluorescence intensity of analyzed cells relative to untreated controls. The values reported are the average (mean) values ± the standard deviation (SD), based on six independent measurements ( / ? = 6); ***P < 0.001 ; ****P < 0.0001 .

[0177] FIG. 7 shows the evaluation of storage stability. After storage at 4 °C for the specified time, iSLN-4 or cSLN-1 was complexed with FLuc pDNA (100 ng) and then used to treat HEK293T cells for 48 hours in accordance with various embodiments disclosed herein. The values reported are the average (mean) values ± the standard deviation (SD), based on six independent measurements (n = 6).

[0178] FIG. 8 shows the fluorescence microscopic images of HepG2-GFP cells transfected for 72 hours with iSLN / siRNA, cSLN / siRNA, or LF3000 / siRNA (positive control) complexes prepared in accordance with various embodiments disclosed herein. Untreated HepG2-GFP cells were used as the negative control. Scale bars represent 50 pm.

[0179] FIG. 9 shows the GFP expression level and viability of HepG2-GFP cells transfected for 72 hours with iSLN / siRNA, cSLN / siRNA, or LF3000 / siRNA (positive control) complexes prepared in accordance with various embodiments disclosed herein. Untreated HepG2-GFP cells were used as the negative control. The values reported are the average (mean) values ± the standard deviation (SD), based on six independent measurements (n = 6); ***P < 0.001.

[0180] FIG. 10 shows the Luciferase expression level and viability of HEK293T cells transfected for 48 hours with iSLN / mRNA, cSLN / mRNA, or LFMax / mRNA (positive control) complexes at N / P ratio of 2 prepared in accordance with various embodiments disclosed herein. Untreated HEK293T cells were used as the negative control. The values reported are the average (mean) values ± the standard deviation (SD), based on six independent measurements (n = 6).

[0181] FIG. 11 shows the Luciferase expression level and viability of HEK293 cells transfected for 48 hours with PEG-iSLN-4 / mRNA, iSLN-4 / mRNA, or LFMax / mRNA (positive control) complexes at N / P ratios of 2, 4, and 6 prepared in accordance with various embodiments disclosed herein. Untreated HEK293 cells were used as the negative control. The values reported are the average (mean) values ± the standard deviation (SD), based on six independent measurements (n - 6); **P < 0.01 ; ****P < 0.0001 . FIG. 12 shows the Luciferase expression level and viability of A549 cells transfected for 48 hours with ALC-LNP, PEG-iLLN / mRNA, or PEG-cLLN- 1 / mRNA complexes at a N / P ratio of 6 prepared in accordance with various embodiments disclosed herein. Untreated A549 cells were used as the negative control. The values reported are the average (mean) values ± the standard deviation (SD), based on six independent measurements (n = 6); ****P < 0.0001.

[0182] FIG. 13 shows the whole-body luminescence images and total flux values of the nasal cavity of BALB / c mice at 4 hours after intranasal administration of ALC-LNP, PEG-iLLN-2 / mRNA or PEG-cLLN-1 / mRNA complexes prepared in accordance with various embodiments disclosed herein. The values reported are the average (mean) values ± the standard deviation (SD), based on four (n = 4) or six (n = 2) independent measurements for the treatment groups and the control groups respectively; **P < 0.01.

[0183] FIG. 14 shows the ex vivo luminescence images and total flux values of the excised major organs of BALB / c mice at 4 hours after intranasal administration of ALC-LNP, PEG-iLLN-2 / mRNA, or PEG-cLLN-1 / mRNA complexes synthesized in accordance with various embodiments disclosed herein. The values reported are the average (mean) values ± the standard deviation (SD), based on four (n = 4) or six (n = 2) independent measurements for the treatment groups and the control groups respectively; *P < 0.05.

[0184] EXAMPLES

[0185] Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following examples, tables and if applicable, in conjunction with the figures. It should be appreciated that other modifications related to structural, biological, and / or chemical changes may be made without deviating from the scope of the invention. Example embodiments are not necessarily mutually exclusive as some may be combined with one or more embodiments to form new example embodiments. The example embodiments should not be construed as limiting the scope of the disclosure.

[0186] It will be appreciated by a person skilled in the art that other variations and / or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included etc. or the like across different exemplary embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.

[0187] Example 1 : Materials and Methods

[0188] 1.1. Materials

[0189] ALC-0315, ALC-0159, 1 ,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1 ,2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), cholesteryl oleate, triolein and 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 2000 (DSPE-PEG) were purchased from MedChem Express (New Jersey, USA). Cholesterol was purchased from Sigma-Aldrich (Minnesota, USA). fi- Sitosterol was a product of Abeam (Cambridge, UK). 6-(p-Toluidino)-2- naphthalenesulfonic acid sodium salt (TNS) was purchased from Santa Cruz Biotechnology (Dallas, USA). Plasmid DNA encoding firefly luciferase (FLuc pDNA, PGL4.51 [luc2 / CMV / Neo] vector), Luciferase Assay System reagent, Passive Lysis Buffer, ONE-Glo Luciferase Assay reagent, VivoGlo luciferin, and nuclease-free water were purchased from Promega Corporation (Madison, USA). Small interfering RNA targeting green fluorescent protein (GFP siRNA) was obtained from Lonza (Gampel, Switzerland). mRNA encoding firefly luciferase (FLuc mRNA) was purchased from OZ Biosciences (San Diego, USA). 5- Methoxyuridine-modified firefly luciferase (FLuc) mRNA was produced by TriLink BioTechnologies (San Diego, CA, USA). Pierce detergent-compatible Bradford assay kit and Quant-iT RiboGreen RNAassay kit were purchased from Thermo Fisher Scientific (Waltham, USA). AlamarBlue cell viability assay reagent (Life Technologies, USA), CellTiter 96 AQueous One cell proliferation assay reagent (Promega, USA), Lipofectamine 3000 (LF3000, Invitrogen, USA), and Lipofectamine MessengerMax (LFMax, Invitrogen, USA) were used per the manufacturer’s protocol. All other chemicals and reagents were of analytical grade.

[0190] 1.2. Preparation of ionizable lipid-incorporated solid lipid nanoparticles (iSLNs) and cationic solid lipid nanoparticles (cSLNs)

[0191] An emulsification / solvent evaporation technique was used to produce iSLNs and cSLNs. The core structure lipids (cholesteryl oleate and triolein) and the surface structure lipids (ALC-0315, DOTMA, DOPE, and cholesterol) were co-dissolved at various weight ratios (Table 2) in 1 mL of chloroform / ethanol mixture (4:1 , v / v) in a 15-mL conical tube. The total lipid concentration was set to 10 mg / mL for iSLN-1 to iSLN-4 and cSLN-1 , whereas it was set to 20 mg / mL for iSLN-5 to iSLN-8 and cSLN-2. After 5 mL of nuclease-free water was added, the mixture was vortexed for 10 sec and then sonicated for 2 minutes using a HTU Soni-130 ultrasonic homogenizer (20 kHz, 130 Watt). The oil-in-water emulsion was transferred to a 100 mL round-bottom flask and the solvents were evaporated using a Hei-VAP rotary evaporator (Heidolph, Germany) for 10 minutes at 60 °C. The resultant iSLNs or cSLNs in a form of suspension in nuclease-free water were stored at 4 °C until use.

[0192] 1.3. Characterization of iSLNs and cSLNs

[0193] The hydrodynamic diameter, polydispersity index (PDI), and zeta potential of iSLNs and cSLNs were examined by dynamic light scattering using a Zetasizer Ultra Red (Malvern Panalytical, UK). For zeta potential measurement, each sample was diluted 10-fold with normal saline (pH 5.5) and measured at 25 °C in triplicate.

[0194] 1.4. TNS binding assay

[0195] The acid dissociation constant (pKa) values of iSLNs and cSLNs were measured by the TNS binding assay, as described previously. First, a series of buffer solutions with pH values of 3-10 with 0.5 increments were prepared by titrating a master buffer stock (10 mM sodium phosphate, 10 mM sodium borate, 10 mM sodium citrate and 150 mM sodium chloride) using 0.1 M HCI or NaOH. Next, 2 pL of TNS solution (300 pM in DMSO) and 2.6 pL of either iSLN or cSLN were added to 90 pL of each buffer solution in a black-bottomed 96-well plate. The final concentration of TNS and ALC-0315 in each well was fixed to 6.3 pM and 873 pM, respectively. Fluorescence intensity was then measured using a Spark 10M microplate reader (Tecan Group, Switzerland) with an excitation wavelength of 321 nm and an emission wavelength of 445 nm. The pH of each well was measured using the Orion ROSS micro pH electrode (Thermo Fisher Scientific, USA). The fluorescence intensity was plotted against the measured pH and fitted to a four-parameter logistic function using the GraphPad Prism 8.0.1 software (GraphPad Software, USA). The apparent pKa value was determined as the pH value at which half of the maximum fluorescence signal was reached.

[0196] 1.5. Formation of iSLN / pDNA and cSLN / pDNA complexes iSLN in nuclease-free water (5 pL) was mixed with 800 ng of FLuc pDNA in 5 pL of nuclease-free water at various N / P ratios (i.e., the molar ratios of cationic / ionizable nitrogen groups of iSLN to phosphate groups of pDNA). The volume ratio of iSLN suspension and pDNA solution was set to 1 :1. The mixture was incubated for 10 minutes at 25 °C to form iSLN / pDNA complexes. For comparison, cSLN / pDNA complexes were produced in the same manner. 1.6. Evaluation of pDNA transfection efficacy and cell viability

[0197] Human embryonic kidney HEK293T cell line was purchased from the American Type Culture Collection (Rockville, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1 % penicillin / streptomycin. The cells were seeded in a black-walled 96-well plate at a density of 104cells per well and cultured for 24 h. Then, the cells were treated with the culture media containing iSLN / pDNA or cSLN / pDNA complexes formulated with 100 ng of FLuc pDNA at an N / P ratio of 12. As a positive control, LF3000 was complexed with 100 ng of FLuc pDNA and then treated to the cells according to the manufacturer’s protocol. After 48 hours of incubation, each well was rinsed with 100 pL of phosphate-buffered saline (PBS, pH 7.4) and lysed with 30 pL of Passive Lysis Buffer for 20 minutes at 25 °C. After 100 pL of Luciferase Assay System reagent was added, relative luminescence unit (RLU) of the cell lysate was measured on a Spark 10M microplate reader (Tecan Group, Switzerland). The results were standardized for protein content using the detergent-compatible Bradford assay kit and expressed as RLU / mg protein. To assess the cell viability, a separate group of cells were treated for 48 hours with iSLN / pDNA, cSLN / pDNA or LF3000 / pDNA complexes, as described above. Then, 100 pL of 20% (v / v) AlamarBlue reagent was added to each well and incubated for 2 h. Fluorescence intensity was then measured using a Spark 10M microplate reader (Tecan Group, Switzerland) with an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Cell viability was determined as a percentage of the fluorescence intensity of analyzed cells relative to untreated controls.

[0198] 1.7. Evaluation of storage stability

[0199] The as-prepared iSLN or cSLN formulations were distributed to 15-mL conical tubes and then stored at 4 °C. The tubes were sealed with Parafilm M (Heathrow Scientific, USA) to prevent evaporation. After storage for 4, 8 or 12 weeks, iSLN or cSLN was complexed with 100 ng of FLuc pDNA at an N / P ratio of 12 and then treated to HEK293T cells for 48 h. The pDNA transfection efficacy and cell viability were evaluated as described above.

[0200] 1.8. Formation of iSLN / siRNA and cSLN / siRNA complexes iSLN in 5 pL of nuclease-free water was mixed with 100 ng of GFP siRNA in 5 pL of nuclease-free water at an N / P ratio of 12. The volume ratio of iSLN suspension and siRNA solution was set to 1 :1 . The mixture was incubated for 10 minutes at 25 °C to form iSLN / siRNA complexes. For comparison, cSLN / siRNA complexes were produced in the same manner.

[0201] 1.9. Evaluation of GFP gene silencing effect and cell viability

[0202] GFP-expressing HepG2 (HepG2-GFP) cell line was purchased from the AcceGen (Fairfield, USA) and maintained in DMEM supplemented with 10% fetal bovine serum and 1 % penicillin / streptomycin. The cells were seeded in a blackwalled 96-well plate at a density of 104cells per well and cultured for 24 h. Then, the cells were treated with the culture media containing iSLN / siRNA or cSLN / siRNA complexes formulated with 100 ng of GFP siRNA at an N / P ratio of 12. As a positive control, LF3000 was complexed with 100 ng of GFP siRNA and then used to treat the cells according to the manufacturer’s protocol. After 72 hours of incubation, GFP fluorescence intensity was measured on a Spark 10M microplate reader (Tecan Group, Switzerland) with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The cellular fluorescence was observed under an Olympus inverted fluorescence IX83 microscope (Olympus, Japan). To assess cell viability, 20 pL of Cel ITiter 96 AQueous One cell proliferation assay reagent was added to each well and incubated for 2 h. Absorbance at 490 nm was then measured using the Spark 10M microplate reader. Cell viability was determined as a percentage of the absorbance of analyzed cells relative to untreated controls. 1.10. Formation of iSLN / mRNA and cSLN / mRNA complexes iSLN in 5 pL of nuclease-free water was mixed with 100 ng of FLuc mRNA in 5 pL of nuclease-free water at a specified N / P ratio. The volume ratio of iSLN suspension and mRNA solution was set to 1 :1. The mixture was incubated for 10 minutes at 25 °C to form iSLN / mRNA complexes. For comparison, cSLN / mRNA complexes were produced in the same manner.

[0203] 1.11. Evaluation of mRNA transfection efficacy and cell viability in HEK293T cells

[0204] HEK293T cells were seeded in a white-walled 96-well plate at a density of 104cells per well and cultured for 24 h. Then, the cells were treated with the culture media containing iSLN / mRNA or cSLN / mRNA complexes formulated with 100 ng of FLuc mRNA at an N / P ratio of 2. As a positive control, LFMax was complexed with 100 ng of FLuc mRNA and then used to treat the cells according to the manufacturer’s protocol. After 48 hours of incubation, 100 pL of ONE-Glo Luciferase Assay reagent was added and incubated for 5 minutes. After incubation, RLU of the cell lysate was measured on a Spark 10M microplate reader (Tecan Group, Switzerland). The results were standardized for protein content using the detergent-compatible Bradford assay kit and expressed as RLU / mg protein. To assess the cell viability, a separate group of cells was seeded in a black-walled 96-well plate and treated for 48 hours with iSLN / mRNA, cSLN / mRNA or LFMax / mRNA complexes, as described above. Then, 100 pL of 20% (v / v) AlamarBlue reagent was added to each well and incubated for 2 h. Fluorescence intensity was then measured using the Spark 10M microplate reader with an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Cell viability was determined as a percentage of the fluorescence intensity of analyzed cells relative to untreated controls. 1.12. Formation of PEG-iSLN / mRNA complexes

[0205] To produce PEG-iSLN, the core structure lipids (cholesteryl oleate and triolein) and the surface structure lipids (ALC-0315, DOTMA, DOPE, cholesterol and DSPE-PEG) were co-dissolved at various weight ratios (Table 3) in 1 mL of chloroform / ethanol mixture (4:1 , v / v) in a 15-mL conical tube. After 5 mL of nuclease-free water was added, the mixture was vortexed for 10 sec and then sonicated for 2 minutes using a HTU Soni-130 ultrasonic homogenizer (20 kHz, 130 Watt). The oil-in-water emulsion was transferred to a 100 mL round-bottom flask and the solvent was evaporated using a Hei-VAP rotary evaporator (Heidolph, Germany) for 10 minutes at 60 °C. The resultant PEG-iSLN was mixed with 100 ng of FLuc mRNA in 10 pL of nuclease-free water at a specified N / P ratio. The mixture was incubated for 10 minutes at 25 °C to form PEG- iSLN / mRNA complexes.

[0206] 1.13. Evaluation of mRNA transfection efficacy and cell viability in HEK293 cells.

[0207] Human embryonic kidney HEK293 cell line was purchased from the American Type Culture Collection (Rockville, USA) and maintained in DMEM supplemented with 10% fetal bovine serum and 1 % penicillin / streptomycin. The cells were seeded in a white-walled 96-well plate at a density of 104cells per well and cultured for 24 hours. Then, the cells were treated with the culture media containing PEG-iSLN / mRNA or iSLN / mRNA complexes formulated with 100 ng of FLuc mRNA at N / P ratios of 2, 4, and 6. As a positive control, LFMax was complexed with 100 ng of FLuc mRNA and then used to treat the cells according to the manufacturer’s protocol. After 48 hours of incubation, 100 pL of ONE-Glo Luciferase Assay reagent was added and incubated for 5 minutes After incubation, RLU of the cell lysate was measured on a Spark 10M microplate reader (Tecan Group, Switzerland). The results were standardized for protein content using the detergent-compatible Bradford assay kit and expressed as RLU / mg protein. To assess the cell viability, a separate group of cells were seeded in a black-walled 96-well plate and treated for 48 hours with PEG- iSLN / mRNA or iSLN / mRNA complexes, as described above. Then, 100 pL of 20% (v / v) AlamarBlue reagent was added to each well and incubated for 2 hours. Fluorescence intensity was then measured using the Spark 10M microplate reader with an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Cell viability was determined as a percentage of the fluorescence intensity of analyzed cells relative to untreated controls.

[0208] 1.14. Formation of PEG-iLLN / mRNA complexes

[0209] To prepare PEG-iLLN, the core structure lipids (triolein) and the surface structure lipids (ALC-0315, DOTMA, DOPE, / 3-sitosterol, and DSPE-PEG) were co-dissolved at specific weight ratios (Table 5) in 1 mL of chloroform / ethanol solution (4:1 , v / v) in a 15-mL conical tube. The total lipid concentration was set at 10 mg / mL. After the addition of 5 mL nuclease-free water, the mixture was vortexed for 10 sec and sonicated for 2 minutes using a HTU Soni-130 ultrasonic homogenizer (20 kHz, 130 Watt). The oil-in-water emulsion was transferred to a 100 mL round-bottom flask and the solvents were evaporated for 10 minutes at 60 °C using a Hei-VAP rotary evaporator (Heidolph, Germany). The resultant PEG-iLLN and PEG-cLLN-1 formulations were then mixed with 100 ng of FLuc mRNA in 5 pL of nuclease-free water at a N / P ratio of 6. The mixture was incubated at 25 °C for 10 minutes to form PEG-iLLN / mRNA and PEG-cLLN- 1 / mRNA complexes.

[0210] 1.15. Preparation of ALC-LNP mRNA was encapsulated in ALC-LNP using the same lipid composition as the BNT162b2 vaccine. Briefly, mRNA was prepared in 10 mM sodium acetate buffer (pH 4.8) to form the aqueous phase. Lipids were dissolved in ethanol at a molar ratio of 46.3:1.6:9.4:42.7 (ALC-0315:ALC-0159:DSPC:cholesterol) to create the organic phase. The aqueous and organic phases (N / P ratio = 6) were mixed at a volume ratio of 3:1 with a flow rate of 12 mL / min using the NanoAssemblr Ignite microfluidics platform (Precision Nanosystems, Canada). The resultant ALC-LNP was buffer-exchanged with normal saline and concentrated using a Vivaspin 20 centrifugal filter (Mwcut-off = 30 kDa). The encapsulated mRNA content was quantified using the Quant-iT RiboGreen RNA assay kit, as previously reported.

[0211] 1.16. Evaluation of FLuc mRNA transfection efficiency and cell viability in A549 cells

[0212] The human alveolar basal epithelial cell line A549 was maintained in DMEM media supplemented with 10% fetal bovine serum. The cells were seeded in a white-walled 96-well plate at a density of 104cells / well and cultured for 24 hours. Each well was then treated with PEG-iLLN / mRNA or PEG-cLLN-1 / mRNA complexes containing 100 ng of FLuc mRNA at a N / P ratio of 6. For comparison, other wells were treated with ALC-LNP containing same amount of FLuc mRNA (100 ng / well). After 48 h, the cells were rinsed with 100 pL of PBS, followed by the addition of 100 pL of ONE-Glo Luciferase Assay reagent per well. After 5 minutes, luminescence was measured using a Spark 10M microplate reader (Tecan Group, Switzerland) and expressed as relative luminescence unit per mg of protein (RLU / mg protein) normalized using the detergent-compatible Bradford assay kit. To assess cell viability, a separate group of cells was seeded in a blackwalled 96-well plate (104cells / well) and treated with PEG-iLLN / mRNA or PEG- cLLN-1 / mRNA complexes for 48 h, as described above. After rinsing with PBS, 100 pL of AlamarBlue reagent (10% in the culture media) was added and incubated for 2 hours. Fluorescence intensity (Fl) was measured with an excitation at 560 nm and emission at 590 nm using the Spark 10M microplate reader. Cell viability was determined as a percentage of Fl relative to untreated controls. 1.17. In vivo and ex vivo bioluminescence imaging

[0213] All animal procedures adhered to protocol 221681 , approved by the Institutional Animal Care and Use Committee (IACUC) at the Biological Resource Centre of A*STAR, Singapore. Female BALB / c mice (5-6 weeks old) were obtained from InVivos Pte Ltd (Singapore) and randomly allocated for different experimental groups: PEG-iLLN / mRNA complexes (n = 4), PEG-cLLN-1 / mRNA complexes (n = 4), ALC-LNP (n = 4) and mock control (n = 2). Mice were anesthetized with ketamine (75 mg / kg) / xylazine (5 mg / kg) and held by hand in an upright position. Each mouse received 60 pL of an isotonic glucose solution (5% w / v) containing the respective formulations (10 pg of FLuc mRNA / mouse) intranasally. Once every 10 pL was given dropwise into one nostril using a 10-pL pipette, the footpad was pinched to make the mouse take a deep breath, ensuring the instilled solution to reach the lower respiratory tract. After 4 hours, the mice were re-anesthetized and subjected to intranasal (50 pL) and intraperitoneal (150 pL) administration of VivoGlo luciferin (15 mg / mL in PBS). Following a 10-minute stabilization period, whole-body luminescence was captured using the IVIS Spectrum imaging system (PerkinElmer, USA). Organs (nose, lung, spleen, liver, kidney, heart) were excised and placed in 1 mL of VivoGlo luciferin (0.3 mg / mL in PBS) for ex vivo luminescence imaging. The total flux in each organ was quantified using the Living Image software (PerkinElmer, USA).

[0214] 1.18. Statistical analysis

[0215] All data are presented as mean ± standard deviation. Statistical analysis was conducted by an ordinary one-way ANOVA with Tukey's post hoc test using the GraphPad Prism 8.0.1 software (GraphPad Software, USA). Significance was determined at P values smaller than 0.05. Example 2: Experimental design for the development of iSLNs

[0216] Herein, the development of ionizable lipid-incorporated SLNs (iSLNs) as efficient and non-toxic vehicles for nucleic acid delivery is reported. By mixing an ionizable lipid with a cationic lipid at an optimal ratio, it was hypothesized it would be possible to formulate iSLNs exhibiting apparent acid dissociation constant (p / a) values in the endosomal pH range. These iSLN formulations are anticipated to form a more positively charged surface in the acidified endosome as the pH decreases below the p a, allowing their entry to the cytoplasm by disrupting the endosomal membrane. At cytosolic pH (7.2-7.4), the electrostatic interactions between iSLNs and nucleic acids would become weaken as the surface charge of iSLN decreases, ultimately promoting the complex dissociation and cargo release more efficiently. To test this hypothesis, a series of iSLN formulations were produced by mixing the ionizable lipid ALC-0315 with the cationic lipid DOTMA (1 ,2-dioleoyl-3-trimethylammonium propane) at varying weight ratios and investigated their applicability for delivery of diverse nucleic acid cargoes (pDNA, siRNA, and mRNA).

[0217] The iSLN formulations were designed to mimic the chemical composition of high-density lipoproteins (HDLs). Natural HDLs have a core-shell structure composed of a nonpolar lipid core (cholesteryl esters and triglycerides) sterically stabilized by a shell layer of polar phospholipids, cholesterol and apolipoproteins. In a healthy human, HDLs typically consist of 24 wt% cholesteryl esters, 3 wt% triglycerides, 45 wt% apolipoproteins, 25 wt% phospholipids and 3 wt% cholesterol. For iSLN formulations, 45 wt% apolipoproteins and 25 wt% phospholipids present in natural HDL were replaced with 45 wt% ALC- 0315 / DOTMA mixture and 25 wt% DOPE, respectively, while keeping the other constituents the same (Table 1). DSPE-PEG conjugate was also incorporated to enhance stability of iSLN. The fusogenic lipid DOPE was chosen to reconstitute the phospholipid portion because of its ability to destabilize the endosomal membrane via the formation of a non-bilayer hexagonal Hu phase at acidic pH. To prepare iSLN, the lipid components co-dissolved in chloroform / ethanol mixture were added into deionized water and then subjected to ultrasonication to form an oil-in-water (O / W) emulsion. Upon evaporation of the solvents, hydrophobic interactions between lipid molecules would lead to spontaneous self-assembly of SLNs with HDL-mimicking core-shell structure, where a nonpolar lipid core composed of cholesteryl oleate, and triolein is coated with a shell layer of ALC-0315 / DOTMA mixture, DOPE, and cholesterol.

[0218] Table 1. Chemical composition of natural HDL and iSLN formulations

[0219] Example 3: Results and Discussion for iSLN and cSLN formulations

[0220] 3.1. Preparation of iSLN and cSLN formulations

[0221] In this study, a series of iSLNs were formulated at various weight ratios and concentrations of ALC-0315 and DOTMA (Table 2). For example, 4 different formulations termed iSLN-1 to iSLN-4 were synthesized by adjusting the weight ratio of ALC-0315 and DOTMA, while keeping the total lipid concentration constant at 10 mg / mL. The other 4 formulations termed iSLN-5 to iSLN-8 were produced at a total lipid concentration of 20 mg / mL. For comparison, 2 different cationic SLN formulations termed cSLN-1 and -2 were prepared using DOTMA alone at total lipid concentration of 10 and 20 mg / mL, respectively. Table 2. Lipid composition of a series of iSLN and cSLN formulations.

[0222] 3.2. Z-average size and polydispersity index (PDI) of iSLN and cSLN formulations

[0223] Dynamic light scattering analysis revealed that Z-average size of iSLNs gradually decreased with raising the weight ratio of DOTMA to ALC-0315 (FIG. 1). The polydispersity index (PDI) was lower than 0.3 in all cases, indicating that iSLN population was homogenous in size. cSLNs made at the higher lipid concentration are larger than iSLNs. The Z-average size and PDI values of cSLNs were comparable to those of ISLNs prepared at the ALC-0315: DOTMA ratio of 1 :3.

[0224] 3.1. Apparent pKa values of iSLN and cSLN formulations

[0225] Next, the apparent p <a values of iSLN formulations were measured using TNS binding assay. In the case of iSLNs, TNS fluorescence increased steeply as the pH decreased below the p / a, indicative of the protonation of ionizable amine groups at acidic pH (FIG. 2). On the other hand, cSLNs lacking an ionizable lipid does not show a sharp pH transition in the TNS fluorescence curve (FIG. 3). As presented in FIG. 4, all the iSLN formulations had apparent p / <a values near the range of endosomal pH (5.5-6.5), suggesting that iSLNs would form a more cationic surface charge at acidic pH inside endosomes, causing endosomal disruption and releasing the payload to the cytoplasm.

[0226] 3.2. Zeta potential of iSLN and cSLN formulations

[0227] The finding was further corroborated by the zeta potential measurement in normal saline (pH 5.5) mimicking the acidic endosomal environment. As shown in FIG. 5, the zeta potential of iSLNs was found to increase from ~14 to ~35 mV when the weight ratio of ALC-0315: DOTMA was changed from 4:0 to 3:1. The observed increase in the zeta potential values was likely attributed to the existence of a quaternary amine moiety in DOTMA. A further increase in the DOTMA fraction had little influence on the zeta potential values at pH 5.5. These results revealed that all iSLN formulations were able to form a positively charged surface at acidic pH to accelerate their endosomal escape.

[0228] 3.3. Transfection studies of pDNA / iSLN and pDNA / cSLN formulations using HEK293T cells

[0229] Plasmid DNA encoding firefly luciferase reporter (FLuc pDNA) was mixed with iSLN for 10 min at 25 °C to form iSLN / pDNA complexes via electrostatic interactions. pDNA transfection efficacy and cytotoxicity of iSLN / pDNA complexes in human embryonic kidney HEK293T cells were investigated (FIG. 6). Given the fast-growing nature and relatively high protein productivity, HEK293T cell line has been widely used for large-scale production of human recombinant proteins and biopharmaceuticals. Interestingly, the weight ratio of ALC-0315 and DOTMA was found to have a significant impact on pDNA transfection efficiency of iSLNs. For instance, FLuc expression levels gradually increased when the weight ratio of ALC-0315 and DOTMA was changed from 4:0 to 1 :3. Of note, iSLN-3 and iSLN-4 prepared at the ALC-0315: DOTMA ratio of 2:2 and 1 :3, respectively, were much more efficient in mediating pDNA transfection than cSLN-1 bearing DOTMA alone (P < 0.001 for cSLN-1 vs. iSLN-3 or iSLN-4) and ISLN-1 bearing ALC-0315 alone (P < 0.0001 for ISLN-1 vs. iSLN-3 or iSLN- 4). This finding implies that the combination of ALC-0315 and DOTMA at an optimal ratio would be critical in achieving the efficient intracellular delivery of pDNA. Impressively, iSLN-4 exhibited significantly higher (P < 0.001 ) transfection efficiency than the commercially available Lipofectamine 3000 (LF3000) reagent. The enhanced transfection efficiency of iSLN-4 might be associated with its efficient endosome escape and dissociation after cellular uptake. iSLN-4 exhibiting the highest transfection efficiency was selected for further investigation. 3.4. Storage stability and cytotoxicity of pDNA / iSLN and pDNA / cSLN formulations using HEK293T cells

[0230] Stability is an important factor for practical use of nucleic acid delivery agents. The stability of iSLN-4 was compared with that of cSLN-1 bearing DOTMA alone during storage at a normal refrigerator temperature (FIG. 7). While iSLN-4 showed only a marginal reduction in transfection efficiency during storage at 4 °C for 12 weeks, a marked decline in transfection efficiency was observed from cSLN-1 under the same storage condition. There was no obvious change in the cytotoxicity for both iSLN-4 and cSLN-1 for 12 weeks. It has been reported that the crystalline state of solid lipid matrices can influence the storage stability of SLN dispersions. Hence, it is conceivable that the co-existence of ALC-0315 and DOTMA might contribute to the formation of SLN dispersions with a highly crystallized lipid phase and superior stability.

[0231] 3.5. Transfection and cytotoxicity studies of siRNA / iSLN and siRNA / cSLN formulations using HepG2-GFP cells

[0232] Next, the applicability of iSLN formulations for intracellular delivery of siRNA was evaluated. Green fluorescent protein-expressing HepG2 (HepG2- GFP) cells were used to examine the gene silencing effect of iSLN / GFP siRNA complexes. Fluorescence microscopy showed that ALC-0315 / DOTMA-modified iSLNs suppressed GFP expression more effectively than those modified with ALC-0315 alone (P < 0.001 for iSLN-5 vs. iSLN-6 or iSLN-7). (FIG. 8 and FIG. 9). All the tested iSLN formulations were more effective in inhibiting GFP expression than the commercially available LF3000 reagent. Cell viability assay revealed that iSLN-5 to -7 formulations were well tolerated in HepG2-GFP cells, whereas iSLN- 8 and cSLN-2 were highly toxic to the cells. Based on this finding, iSLN-6 and -7 are considered suitable for siRNA delivery applications. These results proved that SLNs bearing an optimal amount of the ionizable lipid ALC-0315 enabled efficient intracellular delivery of siRNA, while avoiding the toxicity associated with the cationic lipid DOTMA. 3.6. Transfection and cytotoxicity studies of mRNA / iSLN and mRNA / cSLN formulations using HEK293T cells

[0233] The potential use of iSLN formulations as mRNA delivery vehicles in HEK293T cells was further assessed. As shown in FIG. 10, the weight ratio of ALC-0315 and DOTMA had a substantial influence on mRNA transfection efficiency of iSLNs. For example, FLuc mRNA expression dramatically increased when the weight ratio of ALC-0315 and DOTMA was altered from 4:0 (iSLN-1 ) to 1 :3 (iSLN-4) at a fixed total lipid concentration of 10 mg / mL. When the total lipid concentration was set to 20 mg / mL, FLuc mRNA expression peaked at the ALC- 0315 and DOTMA weight ratio of 2:2 (iSLN-7). A further increase of DOTMA fraction negatively affected FLuc mRNA expression. This finding implies that combination of ALC-0315 and DOTMA at an optimal ratio would drive an enhancement of mRNA expression in a synergistic manner. Notably, iSLN-4 and iSLN-7 achieved comparable mRNA expression levels as the commercially available Lipofectamine MessengerMax (LFMax) did. Unlike LFMax, all the tested iSLN formulations were non-toxic to HEK293T cells. The above results demonstrated the capability of iSLN formulations for efficient delivery of mRNA with minimal cytotoxicity.

[0234] 3.7. PEGylation of iSLN-4 formulation (PEG-iSLN-4) and transfection studies of PEG-iSLN-4 / mRNA and iSLN-4 / mRNA using HEK293T cells

[0235] PEGylation, a process of polyethylene glycol (PEG) coating, has been extensively used to improve the stability and bioavailability of nanoparticles by avoiding aggregation, nonspecific protein adsorption and phagocytic clearance. iSLN-4 was chosen for PEGylation because it exhibited the highest FLuc mRNA expression in HEK293T cells PEGylated iSLN-4 (PEG-iSLN-4) was produced in the same manner as iSLN-4, except that 0.5 mol% of DSPE-PEG was substituted for 0.5 mol% of cholesterol (Table 3). Next, the mRNA delivery efficiency of PEG- iSLN-4 was compared with that of iSLN-4 in HEK293 cells at three different N / P ratios. As depicted in FIG. 11, PEG-iSLN-4 achieved a significantly higher FLuc mRNA expression than iSLN-4 at N / P ratios of 4 and 6. This finding suggests that PEGylation effectively prevented the aggregation and nonspecific protein adsorption of iSLNs upon exposure to serum components in the cell culture media, thus resulting in the augmented mRNA transfection efficacy.

[0236] Table 3. Comparison of the lipid compositions between iSLN-4 and PEG-iSLN- 4.

[0237] 3.8. Cost-Effectiveness of iSLN formulations for transfection of nucleic acids in comparison with commercially available Lipofectamine reagents

[0238] To examine the commercial potential of iSLN formulations, the estimated cost for transfection per well of 96-well plates was calculated (Table 4). Of note, the best-performing iSLNs were much cheaper than the commercially available Lipofectamine reagents. For example, the cost of iSLN-4 needed for pDNA transfection per well of 96-well plates was only SS0.011 , which was 26.6-fold cheaper than the cost of LF3000 reagent (S$0.30). Additionally, iSLN-7 enabled mRNA transfection at an 86.3-fold lower cost than LFMax reagent. Collectively, this study manifested the use of iSLN formulations as cost-effective and efficient transfection agents for various nucleic acid cargoes. Table 4. The estimated cost for transfection of the cells in each well of 96-well plates

[0239] Example 4: Results and Discussion for PEGylated liquid lipid nanoparticle (PEG-LLN) formulations

[0240] 4.1. Preparation of PEGylated, ionizable lipid-incorporated liquid lipid nanoparticles (PEG-iLLN) and PEGylated, cationic lipid-incorporated liquid lipid nanoparticles (PEG-cLLN) formulations

[0241] PEGylated, ionizable lipid-incorporated liquid lipid nanoparticles (PEG- iLLNs) were prepared by removing cholesteryl oleate from the previous PEG- iSLN formulations. In contrast to PEG-iSLNs having a solid core, the core of PEG- iLLNs is composed of triolein with a low melting point, which exists as a liquid state at the physiological temperature. In these new series, cholesterol was replaced with / 3-sitosterol because it has shown to enhance mRNA translation efficiency of LNPs by avoiding recognition by cholesterol transporters that trigger LNP exocytosis and reduce cellular retention. A series of PEG-iLLNs were synthesized by varying the weight ratio of ALC-0315 to DOTMA (Table 5), while keeping the weight ratio among the other four components constant (DOPE / / 3- sitosterol / triolein / DSPE-PEG = 27 / 20 / 3 / 2).

[0242] Table 5. Lipid composition of a series of PEG-iLLN and PEG-cLLN formulations.

[0243] 4.2. Transfection studies of PEG-iLLN / mRNA and PEG-cLLN / mRNA formulations using A549 cells

[0244] To evaluate the transfection efficacy and the cytotoxicity of the PEG-iLLN series, PEG-iLLN / mRNA complexes were formulated with FLuc mRNA and tested in human alveolar epithelial A549 cells (FIG. 12). A549 is a widely studied lung cell line, known for its phenotypic similarity to human alveolar epithelial ceils. Similar to the previous iSLN series, the weight ratio of ALC-0315 to DOTMA had a significant impact on mRNA transfection efficacy of PEG-iLLNs. For instance, FLuc expression levels progressively increased as the ALC-0315 to DOTMA weight ratio shifted from 4:0 to 3:1. Notably, PEG-iLLN-2 prepared at ALC- 0315:DOTMA ratio of 3:1 demonstrated superior mRNA transfection efficacy over PEG-cSLN-1 bearing DOTMA alone and PEG-iLLN-1 bearing ALC-0315 alone. This suggests that the optimal combination of ALC-0315 and DOTMA plays a critical role in enhancing intracellular mRNA delivery for the PEG-iLLN series. Remarkably, PEG-iLLN-2 showed significantly higher (P < 0.0001 ) transfection efficiency than the benchmark ALC-LNP made of the same lipid composition used in BNT162b2 vaccine. All tested PEG-iLLN formulations were non-toxic to A549 cells.

[0245] 4.3. In vivo and ex vivo transfection studies of PEG-iLLN / mRNA and PEG- cLLN / mRNA formulations

[0246] Multiple studies have reported that the rigidity of nanoparticles can be altered to facilitate their mucus permeability. Owing to the more flexible structure, soft nanomaterials were found to move faster through mucus network than stiff ones. In this perspective, the liquid lipid core of PEG-iLLNs makes them an attractive candidate for the development of nasal mRNA vaccine vehicles capable of bypassing the mucus barrier. The in vivo FLuc mRNA transfection efficiency of PEG-iLLN-2 was compared with ALC-LNP and PEG-cLLN-1 following intranasal administration in BALB / c mice (FIG. 13). At 4 hours postadministration, a strong luminescence signal was observed in the nasal cavity of the mice dosed with PEG-iLLN-2 / mRNA complexes, suggesting that these complexes traversed the nasal mucosa and facilitated mRNA delivery beyond the underlying epithelium. A moderate level of luminescence was recorded in the mice administered with PEG-cLLN-1 / mRNA complexes, whereas only a faint spot was detected in the case of ALC-LNP. Total flux analysis further confirmed that PEG-iLLN-2 / mRNA complexes significantly (”P< 0.01 ) outperformed both ALC- LNP and PEG-cLLN-1 / mRNA complexes.

[0247] To further investigate biodistribution profiles, ex vivo luminescence imaging was performed on excised major organs (FIG. 14). Notably, FLuc mRNA expression was primarily localized to the nose and lung in mice treated with PEG- i LLN-2, while PEG-cLLN-1 predominantly transfected the nose and trachea. Total flux values in the nose and lung were the highest for PEG-iLLN-2, followed by PEG-cLLN-1 and ALC-LNP. Other organs, including spleen, liver, kidney and heart exhibited only marginal total flux values comparable to those of the mock controls, indicating minimal mRNA expression in these organs. Overall, these findings highlight the selective biodistribution and superior in vivo mRNA transfection performance of nasally administered PEG-iLLN-2 / mRNA complexes.

Claims

CLAIMS1. A composition for delivery of a therapeutic agent, the composition comprising a cationic lipid and an ionizable lipid in a ratio that falls in the range of 1 -5 : 5-1 .

2. The composition of claim 1 , wherein the cationic lipid is selected from the group consisting of 1 ,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1 ,2-dioleoyl-3-trimethylammonium propane (DOTAP), dimethyldioctadecylammonium (DDAB), / V-(2-hydroxyethyl)- / V, / V-dimethyl- 2,3-bis(oleoyloxy)propan-1 -aminium (DORI), 1 ,2-dioleoyl-sn-glycero-3- ethylphosphocholine (EPC), O,O’-ditetradecanoyl- / \ / -(a- trimethylammonioacetyl)diethanolamine (DC-6-14), 2,3-dioleyloxy- / V-[2- (sperminecarboxamido)ethyl]- / V, / \ / -dimethyl-1-propanaminium (DOSPA), W1 -[2-((1 S)-1 -[(3-aminopropyl)amino]-4-[di(3-amino- propyl)amino]butylcarboxamido)ethyl]-3, -di[oleyloxy]-benzamide (MVL5), and combinations thereof.

3. The composition of claim 1 or 2, wherein the ionizable lipid is selected from the group consisting of (4-hydroxybutyl)azanediyl]di(hexane-6,1 -diyl) bis(2- hexyldecanoate) (ALC-0315), 1 -octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl]amino]-octanoate (SM-102), MC3 or (6Z,9Z,28Z,31Z)- Heptatriaconta-6,9,28,31 -tetraen-19-yl 4-(dimethylamino)butanoate (D-Lin- MC3-DMA), 1 ,T-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl) (2- hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2- ol) (C12-200), 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5- piperazinedione (cKK-E12), 9Z,12Z-octadecadienoic acid, 1 ,T,T',T'r-[(3,6- dioxo-2,5-piperazinediyl)bis(4, 1 -buta ned iy In itri lod i-2, 1 -ethanediyl)] ester(OF-Deg-Lin), ethyl 5,5-bis[(Z)-heptadec-8-enyl]-1 -(3-pyrrolidin-1 -ylpropy I)- 2H-imidazole-2-carboxylate (A2-iso5-2DC18), tetrakis(8-methylnonyl) 3 ,3' ,3" ,3"'-(((methylazanediyl)bis(propane-3,1- diyl))bis(azanetriyl))tetrapropionate (3060i10), bis(2-(dodecyldisulfaneyl)ethyl) 3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6- diazahexacosyl)azanediyl)dipropionate (BAMEA-016B), N 1 , N3, N5-tris[3- (didodecylamino)propyl]-1 ,3,5-benzenetricarboxamide (TT3), 2-(dioctylamino)ethyl nonyl hydrogen phosphate (9A1 P9), hexa(octan-3-yl) 9, 9', 9", 9"', 9"", 9""'- ((((benzene-1 ,3,5-tricarbonyl)yris(azanediyl)) tris(propane-3,1 -diyl)) tris(azanetriyl))hexanonanoate (FTT5), 1 ,2-dioleyloxy-3- dimethylaminopropane (DODMA), 1 ,2-dioleoyl-3-dimethylammonium- propane (DODAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3- bis(oleoyloxy)propan-1-aminium (DOBAQ), and combinations thereof.

4. The composition of any one of the preceding claims, wherein the cationic lipid is present in an amount of from 1 wt% to 50 wt% of the composition.

5. The composition of any one of the preceding claims, wherein the ionizable lipid is present in an amount of from 1 wt% to 50 wt% of the composition.

6. The composition of any one of the preceding claims, wherein the total amount of the ionizable lipid and the cationic lipid present in the composition falls in the range of 30 wt% to 60 wt% of the composition.

7. The composition of any one of the preceding AS, wherein the composition further comprises one or more of the following components:(i) glycerolipid;(ii) esterified cholesterol;(iii) phospholipid;(iv) cholesterol or analogues thereof; and / or(v) PEG-conjugate.

8. The composition of claim 7, wherein the glycerolipid is present and comprises a triglyceride selected from the group consisting of triolein, tristearin, trielaidin, trilinolein, tripalmitin, tripalmitolein, trimyristin, trilaurin, tricaproin, trioctanoin, triarachidin, triarachidonin, and combinations thereof.

9. The composition of claims 7 or 8, wherein the esterified cholesterol is present and comprises a cholesteryl ester selected from the group consisting of cholesteryl oleate, cholesteryl stearate, cholesteryl linoleate, cholesteryl palmitate, cholesteryl myristate, cholesteryl laurate, cholesteryl caproate, cholesteryl arachidate, cholesteryl arachidonate, and combinations thereof.

10. The composition of any one of claims 7 to 9, wherein the phospholipid is present and is selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), 1 -palmitoyl-2-oleoyl-sn- glycero-3-phosphoethanolamine (POPE), 1 ,2-dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE), 1 ,2-dilauroyl-sn-glycero-3- phosphoethanolamine (DLPE), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2- dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1 ,2-dimyristoyl-sn- glycero-3-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1 ,2-dierucoyl-sn- glycero-3-phosphocholine (DEPC), 1 -palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), 1 -stearoyl-2-oleoyl-sn-glycero-3- phosphocholine(SOPC), and combinations thereof.

11. The composition of any one of claims 7 to 10, wherein the cholesterol or analogues thereof is present and is selected from the group consisting of cholesterol, [3-sitosterol, p-sitosterol-acetate, stigmastanol, campesterol, fucosterol, brassicasterol, ergosterol, 9,11 -dehydroergosterol, daucosterol, vitamin D2, vitamin D3, vitamin E, calcipotriol, betulin, lupeol, ursolic acid, oleanolic acid, derivatives thereof and combinations thereof.

12. The composition of any one of claims 7 to 11 , wherein the PEG conjugate is present and is selected from the group consisting of 1 ,2-distearoyl-sn- glycero-3-phosphoethanolamine-polyethylene glycol 2000 (DSPE-PEG), methoxypolyethyleneglycoloxy(2000)-N,N-ditetradecylacetamide (ALC- 0159), 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000(DMG-PEG), 1 ,2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (DSG-PEG), 1 ,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [(polyethylene glycol)-2000] (DPPE-PEG), 1 ,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[(polyethylene glycol)-2000] (DMPE-PEG), 1 ,2- dioleoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000] (DOPE-PEG), N-palmitoyl-sphingosine-1 -{succinyl[methoxy(polyethylene glycol)2000]} (C16 PEG ceramide), N-octanoyl-sphingosine-1 -{succinyl[methoxy(polyethylene glycol)2000]} (C8 PEG2000 ceramide), and combinations thereof.

13. The composition of any one of claims 7 to 12, wherein the glycerolipid is present in an amount of from 0.10 wt% to 6 wt% of the composition.

14. The composition of any one of claims 7 to 13, wherein the esterified cholesterol is present at an amount of from 10 wt% to 30 wt% of the composition.

15. The composition of any one of claims 7 to 14, wherein the phospholipid is present in an amount of from 10 wt% to 40 wt of the composition.

16. The composition of any one of claims 7 to 15, wherein the cholesterol and analogues thereof is present in an amount of from 0.10 wt% to 40 wt% of the composition.

17. The composition of any one of claims 7 to 16, wherein the PEG conjugate is present in an amount of from 0.10 mol% to 3 mol% of the composition.

18. The composition of any one of claims 7 to 17, wherein the total lipid concentration in the composition is from 1 mg / mL to 50 mg / mL.

19. A composition for delivery of a therapeutic agent, the composition comprisinga cationic lipid in an amount of 5 to 50 wt% of the composition; an ionizable lipid in an amount of 5 to 50 wt% of the composition; a glycerolipid in an amount of 1 to 5 wt% of the composition; a phospholipid in an amount of 10 to 40 wt% of the composition; a cholesterol or analogues thereof in an amount of 1 to 30 wt% of the composition; optionally an esterified cholesterol in an amount of 10 to 30 wt% of the composition; and optionally a PEG-conjugate in an amount of from 0.10 mol% to 3 mol% of the composition, wherein the cationic lipid and ionizable lipid are present in a ratio that falls in the range of 1 -5 : 5-1 .

20. The composition of claim 19, wherein the total amount of the ionizable lipid and the cationic lipid present in the composition falls in the range of 30 wt% to 60 wt% of the composition.21 . A nanoparticle comprising a core structure comprising glycerolipid and optionally an esterified cholesterol; a surface structure comprising an ionizable lipid, a cationic lipid, a phospholipid, a cholesterol or analogues thereof, and optionally a PEG- conjugate; and a therapeutic agent complexed with the surface structure, wherein the cationic lipid and ionizable lipid are present in a ratio that falls in the range of 1 -5 : 5-1 .

22. The nanoparticle of claim 21 , wherein the total amount of the ionizable lipid and cationic lipid present in the nanoparticle falls in the range of 30 wt% to 60 wt% of total lipid composition of the nanoparticle.

23. The nanoparticle of claim 19, wherein the therapeutic agent comprises a nucleic acid.

24. The nanoparticle of claim 19 or 20, wherein the nanoparticle is an ionizable lipid incorporated solid lipid nanoparticle or an ionizable lipid incorporated liquid lipid nanoparticle.

25. The nanoparticle of claim 21 , wherein the nanoparticle has an apparent acid dissociation constant (pKa) value in the pH range of 4.5 to 7.

26. A method of preparing the nanoparticle according to claim 21 , the method comprises, adding an aqueous solution to an composition comprising a cationic lipid and an ionizable lipid in an organic solvent, wherein the ratio of the cationic lipid to the ionizable lipid is in a ratio that falls in the range of 1 -5 : 5-1 , to form an immiscible mixture; agitating the immiscible mixture to obtain an oil-in-water (O / W) emulsion; evaporating the organic solvent from the O / W emulsion to obtain the nanoparticle in aqueous suspension; adding an aqueous solution of a therapeutic agent to the nanoparticle in aqueous suspension; and optionally incubating the mixture.

27. The method of claim 26, wherein the organic composition further comprises one or more of:(i) glycerolipid;(ii) esterified cholesterol;(iii) phospholipid;(iv) cholesterol or analogue thereof; and / or(v) PEG-conjugate.

28. The method of any one of claims 26 to 27, wherein the organic composition comprises an organic solvent selected from the group consisting of chloroform, ethanol, methanol, isopropanol, butanol, ethyl acetate, dichloromethane (DCM), and combinations thereof.

29. The method of any one of claims 26 to 28, wherein the aqueous solution comprises an aqueous solvent selected from the group consisting of deionized water, normal saline, phosphate-buffered saline, Tris-buffered saline, sodium acetate buffer, sodium citrate buffer, and combinations thereof.

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