nanoparticles

Abstract

Lipid-based nanoparticles containing lipids, phenolic compounds, and, optionally, metal ions are provided. In certain forms, the nanoparticles adopt non-lamellar, lyotropic, liquid crystalline structures. Bioactive lipid-based nanoparticles containing one or more active agents are also provided as are methods of treating diseases or conditions with said bioactive lipid-based nanoparticles and formulations thereof.

Description

Field of the disclosure

[0001] The present disclosure is directed to lipid-based nanoparticles containing lipids, phenolic compounds, and, optionally, metal ions. The present disclosure also relates to bioactive lipid-based nanoparticles containing one or more active agents and to methods of treating diseases or conditions with said bioactive lipid-based nanoparticles and formulations thereof. Related application

[0002] This application claims the benefit of priority from Australian provisional application no. 2024900331 filed 13 February 2024, the entire disclosure of which is hereby incorporated by reference. Background of the disclosure

[0003] Lipid-based nanoparticles are a key class of drug delivery system, and some of the formulations have been approved by US Food and Drug Administration (FDA), for example, Doxil® (doxorubicin-encapsulated liposomes for cancer treatment), Onpattro® (siRNA-encapsulated lipid nanoparticles (LNPs) for polyneuropathy treatment), as well as BioNTech / Pfizer’s BNT162b2 and Moderna’s mRNA-1273 COVID-19 vaccines (mRNA-encapsulated LNPs).

[0004] Despite the clinical success achieved with lipid-based nanoparticles having lamellar phases, research interests are moving towards utilizing lyotropic liquid crystalline lipid nanoparticles (LCNPs) with non-lamellar phases, including cubosomes, hexosomes, and micellarsomes, for drug delivery. Compared to the lipid-based nanoparticles with lamellar phase, cubosomes with bicontinuous inverse cubic phase, hexosomes with inverse hexagonal phase and micellarsomes with discontinuous micellar cubic phases exhibit more exotic ordered non-lamellar nanostructures with higher membrane surface areas. These unique nanostructures enable the encapsulation of diverse cargos, including hydrophilic, hydrophobic and amphiphilic molecules, and their non-lamellar phases with high membrane curvature play important roles in membrane fusion, fission and remodelling processes. Among the different LCNPs, cubosomes stand out as potential next-generation smart lipid nanoparticles, due to their high internal interface areas, high cargo loading capacities and controlled diffusion and release of cargoes, benefiting from their internal bicontinuous cubic nanostructures.

[0005] Cubosomes are formed by the self-assembly of certain types of non-lamellar lipids, with monoolein (MO) and phytantriol (Phy) being the most commonly used lipids, in combination with stabilizers to prevent aggregation. Cubosomes can exhibit three different morphologies, i.e., Ia3d (gyroid lattice (G-surface, Qiig)), Pn3m (doublediamond lattice (D-surface, Qiid)), and Im3m (primitive lattice (P-surface, Qiip)).

[0006] In recent years, much effort has been paid to applying lipid-based nanoparticles for RNA delivery, and the inclusion of permanent / ionizable cationic lipids into the lipid-based nanoparticles is necessary for the efficient encapsulation of the highly negatively charged RNA and in aiding in the intracellular RNA release via endosomal escape. The structural transition of the ionizable cationic lipids at acidic conditions into non-lamellar inverse hexagonal and cubic structures is attributed to promoting endosomal escape of the LNPs. However, permanent / ionizable cationic lipids are the key components in causing immune responses and potential long-term toxicity. Additionally, it is challenging for LCNPs to retain the nanostructures after incorporating permanent / ionizable cationic lipids and large RNA molecules.

[0007] There is a need to provide new lipid-based nanoparticles and methods for their production.

[0008] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and / or combined with other pieces of prior art by a skilled person in the art. Summary of the disclosure

[0009] In one aspect the present disclosure provides a lipid-based nanoparticle comprising one or more lipids and one or more phenolic compounds.

[0010] In embodiments, the one or more phenolic compounds has a molecular weight from about 110 to about 3,000 Daltons.

[0011] In embodiments, the one or more phenolic compounds comprise one or more 3,4,5-trihydroxyphenyl groups.

[0012] In embodiments, the one or more phenolic compounds comprise three or more ring-bonded hydroxy groups.

[0013] In embodiments, the one or more phenolic compounds comprise one or more galloyl groups, optionally with one or more catechol groups.

[0014] Non-limiting examples of the one or more phenolic compounds include one or more of apigenin, catechin, curcumin, luteolin, quercetin, kaempferol, genistein, gossypol, daidzein, fisetin, myricetin, naringenin, hesperetin, delphinidin, cyanidin, procyanidin, chalconaringenin, phloretin, tannic acid, (-)-epigallocatechin-3-gallate, gallocatechin, catechin-3-gallate, epigallocatechin, epicatechin-3-gallate, resveratrol, gallic acid, ellagic acid, and dopamine.

[0015] In embodiments, the one or more phenolic compounds comprise one or more of tannic acid and (-)-epigallocatechin-3-gallate, preferably tannic acid.

[0016] In embodiments, the one or more phenolic compounds comprise resveratrol.

[0017] In embodiments, the one or more lipids comprise one or more PEGylated lipids, phospholipids, structural lipids, and cationic or ionisable lipids.

[0018] In embodiments, the one or more lipids does not comprise one or more cationic or ionisable lipids.

[0019] In embodiments, the one or more lipids does not comprise monoolein or phytantriol.

[0020] In embodiments, the PEGylated lipid is functionalised.

[0021] In embodiments, the PEGylated lipid is non-functionalised.

[0022] In embodiments, the one or more lipids have a molecular weight from about 100 to about 100,000 Dalton.

[0023] In embodiments, the one or more lipids have a molecular weight from about 100 to about 3,000 Dalton.

[0024] In embodiments, the one or more lipids have a molecular weight of less than 3,000 Dalton.

[0025] Non-limiting examples of the one or more lipids include one or more of 1,2-dimyristoyl-rac-glycero-3-methoxy poly(ethylene glycol) (DMG-PEG); polyethylene glycol 2000-ester-stearic acid (PEG-SA); polyethylene glycol 2000-ester-myristic acid (PEG-MA); 2-[(polyethylene glycol)-N,N-ditetradecylacetamide (ALC-0159); polyethylene glycol-carbamate-1,2-dimyristoyl-sn-glycerol (PEG-c-DMG); 1,2-dimyristoyl-sn-glycero-3-succinyl-N-polysarcosine-25 (pSar-DMG); 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (ammonium salt) (DOPE-PEG 5000); methoxy polyethylene glycol 20k-distearoyl phosphatidyl ethanolamine (DSPE-PEG 20k), cholesterol-polyethylene glycol 5000 (CLS-PEG 5000), and cholesterol-polyethylene glycol 20k (CLS-PEG 20k); 1,2-dioleoyl-s / ?-glycero-3-phosphocholine (DOPC); 1,2-dioleoyl-s / ?-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DOPE-PEG); A / -(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-s / ?-glycero-3-phosphoethanolamine (sodium salt) (DSPE-PEG); 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP); and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (chloride salt) (EPC).

[0026] In embodiments, the one or more lipids comprise one or more of 1,2-dimyristoyl-rac-glycero-3-methoxy poly(ethylene glycol) (DMG-PEG); polyethylene glycol 2000-ester-stearic acid (PEG-SA); polyethylene glycol 2000-ester-myristic acid (PEG-MA); 2-[(polyethylene glycol)-N,N-ditetradecylacetamide (ALC-0159); polyethylene glycol-carbamate-1,2-dimyristoyl-sn-glycerol (PEG-c-DMG); 1,2-dimyristoyl-sn-glycero-3-succinyl-N-polysarcosine-25 (pSar-DMG); 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (ammonium salt) (DOPE-PEG 5000); 1,2-dioleoyl-s / ?-glycero-3-phosphocholine (DOPC); and 1,2-dioleoyl-s / ?-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DOPE-PEG).

[0027] In embodiments, the one or more lipids comprise one or more of 1,2-dimyristoyl-rac-glycero-3-methoxy poly(ethylene glycol) (DMG-PEG); polyethylene glycol 2000-ester-stearic acid (PEG-SA); 2-[(polyethylene glycol)-N,N-ditetradecylacetamide (ALC-0159); polyethylene glycol-carbamate-1,2-dimyristoyl-sn-glycerol (PEG-c-DMG); and 1,2-dimyristoyl-sn-glycero-3-succinyl-N-polysarcosine-25 (pSar-DMG).

[0028] In embodiments, the one or more lipids comprise one or more of 1,2-dimyristoyl-rac-glycero-3-methoxy poly(ethylene glycol) (DMG-PEG); polyethylene glycol 2000-ester-stearic acid (PEG-SA); and 2-[(polyethylene glycol)-N,N-ditetradecylacetamide (ALC-0159); polyethylene glycol-carbamate-1,2-dimyristoyl-sn-glycerol (PEG-c-DMG).

[0029] In embodiments, the one or more lipids comprise one or more of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (ammonium salt) (DOPE-PEG 5000); 1,2-dioleoyl-s / ?-glycero-3-phosphocholine (DOPC); and 1,2-dioleoyl-s / ?-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DOPE-PEG).

[0030] In embodiments, the one or more lipids are essentially the same lipids (e.g. the lipids are not a mixture of different lipids).

[0031] In embodiments, the lipid-based nanoparticle comprises a single type of lipid.

[0032] In embodiments, the lipid-based nanoparticle does not comprise a mixture of different lipids.

[0033] In embodiments, the molar ratio of the one or more phenolic compounds to the one or more lipids is from about 0.5:1 to about 200:1.

[0034] In embodiments, the molar ratio of the one or more phenolic compounds to the one or more lipids is from about 0.5:1 to about 50:1.

[0035] In embodiments, the molar ratio of the one or more phenolic compounds to the one or more lipids is from about 2:1 to about 200:1.

[0036] In embodiments, the polydispersity index of the lipid-based nanoparticle is less than about 0.3, or less than about 0.2, or less than about 0.1, or less than about 0.05.

[0037] In embodiments, the lipid-based nanoparticle has a mean particle size from about 5 nm to about 1 micron, or from about 10 nm to about 500 nm.

[0038] In embodiments, the zeta potential of the lipid-based nanoparticle is less than about -5 mV, or less than about -30 mV.

[0039] In embodiments, the zeta potential of the lipid-based nanoparticle is less than about -5 mV, or less than about -20 mV.

[0040] In embodiments, the zeta potential of the lipid-based nanoparticle is less than about -5 mV, or less than about -10 mV.

[0041] In embodiments, the lipid-based nanoparticle comprises a non-lamellar lyotropic liquid crystalline phase structure.

[0042] In embodiments, the structure comprises a cubosome, a hexosome, or a micellarsome.

[0043] In embodiments, the structure comprises a cubosome with bicontinuous inverse cubic phase.

[0044] In embodiments, the structure comprises a hexosome with inverse hexagonal phase.

[0045] In embodiments, the structure comprises a micellarsome with discontinuous micellar cubic phase.

[0046] In embodiments, the lipid-based nanoparticle further comprises one or more metal ions.

[0047] Non-limiting examples of the one or more metal ions include one or more of Cu(ll), Fe(lll), Zr(IV), Zn (II), Ni (II), Co(ll), Na(l), Ca(ll), Ti(IV), Mn(ll), Ir(IV), Ta(V), W(VI), Bi(lll), Zn(ll), Mn(ll), Gd(lll), Pt(IV), Nb(V), and Ga(lll).

[0048] In embodiments, the one or more metal ions comprise one or more of Zn(ll), Cu(ll), Fe(lll), Zr(IV), Co(ll) and Ni(ll), preferably Fe(lll).

[0049] In embodiments, the lipid-based nanoparticle comprises a single type of metal ion.

[0050] In embodiments, the molar ratio of one or more phenolic compounds to one or more metal ions is from about 200:1 to about 1:5.

[0051] In embodiments, the molar ratio of one or more phenolic compounds to one or more metal ions is from about 10:1 to about 1:5.

[0052] In embodiments, the molar ratio of one or more phenolic compounds to one or more metal ions is from about 5:1 to about 1:5.

[0053] In embodiments wherein the one or more phenolic compounds comprise resveratrol, preferably the lipid-based nanoparticle further comprises one or more metal ions, more preferably Fe(lll) metal ions.

[0054] In embodiments wherein the one or more lipids comprise DOPC, DOPE-PEG, DOPE-PEG5000 or PEG-MA, preferably the lipid-based nanoparticle further comprises one or more metal ions, more preferably Fe(lII) metal ions.

[0055] In another aspect the present disclosure provides a bioactive lipid-based nanoparticle comprising the lipid-based nanoparticle according to any one of the herein disclosed embodiments and one or more active agents.

[0056] In embodiments, the one or more active agents comprise one or more small molecules or biomacromolecules.

[0057] Non-limiting examples of biomacromolecules include one or more of proteins, peptides, nucleic acid molecules and enzymes.

[0058] In embodiments, the nucleic acid molecule is a therapeutic agent.

[0059] In embodiments, the nucleic acid molecule comprises DNA molecule or an RNA molecule. In some embodiments, the nucleic acid molecule comprises cDNA, mRNA, miRNA, siRNA, sgRNA, modified RNA, antagomir, antisense molecule, guide RNA molecule, CRISPR guide RNA molecule or any combination thereof.

[0060] In embodiments, the mRNA encodes one or more antigens. In some embodiments, the antigen comprises at least one viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, an influenza antigen, a tumor-associated antigen, a tumor-specific antigen, or any combination thereof.

[0061] In embodiments, the nucleic acid molecule comprises a promoter or regulatory sequence.

[0062] The amount of nucleic acid in the bioactive nanoparticle composition as described may depend on the size, sequence, and other characteristics of the nucleic acid. The amount of nucleic acid in the nanoparticle composition may also depend on the size, desired target, and other characteristics of the bioactive nanoparticle composition.

[0063] In embodiments, the one or more active agents are at least partly encapsulated within the lipid-based nanoparticle.

[0064] In embodiments, the one or more active agents are fully encapsulated within the lipid-based nanoparticle.

[0065] In embodiments, the biomacromolecule comprises an RNA molecule.

[0066] In embodiments, the biomacromolecule comprises an mRNA molecule or siRNA molecule.

[0067] In another aspect the present disclosure provides a pharmaceutical composition comprising the bioactive lipid-based nanoparticle according to any one of the herein disclosed embodiments, and a pharmaceutically acceptable carrier, diluent, and / or excipient.

[0068] In another aspect the present disclosure provides a method of producing a lipid-based nanoparticle according to any one of the herein disclosed embodiments comprising contacting one or more lipids with one or more phenolic compounds.

[0069] In another aspect the present disclosure provides a method of producing a metal ion containing lipid-based nanoparticle comprising contacting one or more lipids with one or more phenolic compounds, and one or more metal ions.

[0070] In another aspect the present disclosure provides a method of producing a bioactive lipid-based nanoparticle, optionally comprising one or more metal ions, comprising contacting one or more lipids with one or more phenolic compounds and one or more active agents, and, optionally, one or more metal ions.

[0071] In another aspect the present disclosure provides a method of producing a lipid-based nanoparticle comprising a non-lamellar lyotropic liquid crystalline phase structure according to any one of the herein disclosed embodiments comprising contacting one or more lipids with one or more phenolic compounds, thereby producing lipid-based nanoparticle comprising a non-lamellar lyotropic liquid crystalline phase structure.

[0072] In another aspect the present disclosure provides a method of producing a metal ion containing lipid-based nanoparticle comprising a non-lamellar lyotropic liquid crystalline phase structure contacting one or more lipids with one or more phenolic compounds, and one or more metal ions, thereby producing metal ion containing lipid-based nanoparticle comprising a non-lamellar lyotropic liquid crystalline phase structure.

[0073] In another aspect the present disclosure provides a method of producing a bioactive lipid-based nanoparticle comprising a non-lamellar lyotropic liquid crystalline phase structure, optionally comprising one or more metal ions, comprising contacting one or more lipids with one or more phenolic compounds and one or more active agents, and, optionally, one or more metal ions, thereby producing a bioactive lipid-based nanoparticle comprising a non-lamellar lyotropic liquid crystalline phase structure, optionally comprising one or more metal ions.

[0074] In embodiments of the methods of the present invention, the structure comprises a cubosome, a hexosome, or a micellarsome.

[0075] In embodiments of the methods of the present invention, the structure comprises a cubosome with bicontinuous inverse cubic phase.

[0076] In embodiments of the methods of the present invention, the structure comprises a hexosome with inverse hexagonal phase.

[0077] In embodiments of the methods of the present invention, the structure comprises a micellarsome with discontinuous micellar cubic phase.

[0078] In embodiments of the methods of the present invention, the one or more phenolic compounds, and optionally one or more metal ions, are provided in an aqueous solution, such as water, or a non-aqueous solution, such as an alcohol, preferably ethanol.

[0079] In embodiments of the methods of the present invention, the one or more lipids are provided in an aqueous solution, such as water, or a non-aqueous solution, such as an alcohol, preferably ethanol.

[0080] In embodiments of the methods of the present invention, the solution partially or fully solubilises the one or more phenolic compounds.

[0081] In embodiments of the methods of the present invention, the solution partially or fully solubilises the one or more lipids.

[0082] In embodiments of the methods of the present invention, the solution of one or more phenolic compound, optionally with one or more metal ions, and the solution of one or more lipids are admixable with each other.

[0083] In embodiments of the method of the present invention, the one or more lipids are not in a non-lamellar lyotropic liquid crystalline phase structure, prior to contacting with the one or more phenolic compounds.

[0084] In embodiments of the methods of the present invention, the lipid-based nanoparticle, metal ion containing lipid-based nanoparticle or bioactive lipid-based nanoparticle, optionally comprising one or more metal ions, is produced in a single step by contacting a solution comprising one or more phenolic compounds, optionally with one or more metal ions, with a solution comprising one or more lipids.

[0085] In embodiments, the solution comprises an alcohol, preferably ethanol.

[0086] In embodiments, the solution comprises water.

[0087] In another aspect the present disclosure provides a method of delivering an active agent to a cell or tissue, the method comprising contacting the bioactive lipid-based nanoparticle or pharmaceutical composition according to any one of the herein disclosed embodiments with the cell or tissue, thereby introducing the active agent into the cell or tissue.

[0088] In another aspect, the present disclosure provides a method of producing a polypeptide of interest in a cell, preferably a mammalian cell, the method comprising contacting the bioactive lipid-based nanoparticle or pharmaceutical composition according to any one of the herein disclosed embodiments with the cell or tissue, wherein the active agent is an mRNA encoding the polypeptide of interest, and wherein the mRNA is capable of being translated in the cell to produce the polypeptide of interest.

[0089] In embodiments, the cell or tissue is present in vivo.

[0090] In embodiments, the cell or tissue is present in vitro.

[0091] In embodiments, the cell or tissue is a mammalian cell or tissue, preferably a human cell or tissue.

[0092] In embodiments, the active agent comprises one or more small molecules or biomacromolecules.

[0093] In embodiments, the biomacromolecule is an RNA, for example an mRNA or siRNA.

[0094] In embodiments, an RNA transfection efficiency is greater than 25%, or greater than 50%, or greater than 75%, or greater than 85%, or greater than 95%.

[0095] In embodiments, the cell is a 2D or 3D cultured cell.

[0096] In another aspect the present disclosure provides a method of in vivo delivery of an active agent, the method comprising administering the bioactive lipid-based nanoparticle or pharmaceutical composition to a subject, thereby delivering the active agent to the subject.

[0097] In another aspect the present disclosure provides a method of preventing, treating or ameliorating an infection, disease, disorder, or condition in a subject in need thereof, the method comprising administering to the subject the bioactive lipid-based nanoparticle or pharmaceutical composition according to any one of the herein disclosed embodiments, thereby treating or preventing an infection, disease, disorder or condition in a subject in need thereof.

[0098] In another aspect, the present disclosure provides a method of treating or preventing a cancer in a subject, comprising administering to the subject a bioactive nanoparticle composition according to any one of the herein disclosed embodiments, thereby treating or preventing cancer.

[0099] In embodiments, the cancer is a solid tumour or a soft tumour, such as a leukemia or lymphoma.

[0100] In embodiments, the cancer is selected from malignant neoplasms of lip, oral cavity and pharynx, malignant neoplasms of digestive organs, malignant neoplasms of respiratory and intrathoracic organs, malignant neoplasms of bone and articular cartilage, melanoma and other malignant neoplasms of skin, malignant neoplasms of mesothelial and soft tissue, malignant neoplasm of breast, malignant neoplasms of female genital organs, malignant neoplasms of male genital organs, malignant neoplasms of urinary tract, malignant neoplasms of eye, brain and other parts of central nervous system, malignant neoplasms of thyroid and other endocrine glands, malignant neoplasms of lymphoid, haematopoietic and related tissue, malignant neoplasms of ill-defined, secondary and / or unspecified sites.

[0101] In embodiments, the cancer is breast cancer.

[0102] In another aspect, the present disclosures provides a use of a bioactive nanoparticle composition according to any one of the herein disclosed embodiments in the manufacture of a medicament for preventing, treating or ameliorating an infection, disease, disorder, or condition in a subject in need thereof.

[0103] In another aspect, the present disclosures provides a use of a bioactive nanoparticle composition according to any one of the herein disclosed embodiments in the manufacture of a medicament for treating or preventing a cancer in a subject.

[0104] In another aspect, the present disclosures provides a bioactive nanoparticle composition according to any one of the herein disclosed embodiments for use in preventing, treating or ameliorating an infection, disease, disorder, or condition in a subject in need thereof.

[0105] In another aspect, the present disclosures provides a bioactive nanoparticle composition according to any one of the herein disclosed embodiments for use in treating or preventing a cancer in a subject.

[0106] In another aspect, the present disclosure provides a bioactive lipid-based nanoparticle or pharmaceutical composition according to any one of the herein disclosed embodiments in the manufacture of a medicament for treating or preventing a disease or condition in a subject in need thereof.

[0107] In another aspect, the present disclosure provides a bioactive lipid-based nanoparticle or a pharmaceutical composition according to any one of the herein disclosed embodiments for use in the treatment or prevention of a disease or condition in a subject in need thereof.

[0108] In another aspect, the present disclosure provides a method of delivering an RNA into a cell, preferably a mammalian cell, the method comprising administering to a subject a bioactive lipid-based nanoparticle, wherein an active agent is an RNA, thereby delivering an RNA into a cell.

[0109] In any aspect or embodiment the RNA is an mRNA or siRNA.

[0110] Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.

[0111] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and processes are clearly within the scope of the disclosure, as described herein.

[0112] Further aspects of the present disclosure and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings. Brief description of the drawings

[0113] Figure 1 shows size distributions of TA / DMG-PEG cubosomes assembled by TA and DMG-PEG (2 mM) with the molar ratios of TA to DMG-PEG from 0:1 to 10:1.

[0114] Figure 2 shows (A) 1D diffraction SAXS patterns of TA / DMG-PEG cubosomes assembled by TA and DMG-PEG (2 mM) with the molar ratios of TA to DMG-PEG from 0:1 to 10:1, (B) zeta-potential and (C) 1D diffraction SAXS patterns of TA / DMG-PEG prepared by admixing water solubilised TA with water solubilised DMG-PEG. Data are shown as mean ± standard deviation (SD) (n = 3 independent replicates)

[0115] Figure 3 shows cryo-TEM images and 2D SAXS diffraction patterns of DMG-PEG nanoparticles (e) and TA / DMG-PEG cubosomes (f-h) assembled from TA and DMG-PEG of different molar ratios (1:1 (f,h) and 10:1 (g)). The concentration of DMG-PEG for the cubosome preparation was 2 (f,g) and 10 mM (h). Scale bars are 200 nm.

[0116] Figure 4 shows 1D SAXS diffraction patterns of TA / DMG-PEG assembled from TA and DMG-PEG (10 mM), the molar ratios of TA to DMG-PEG are from 0:1 to 3:1.

[0117] Figure 5 shows 1D SAXS diffraction patterns of TA-FeHI / DMG-PEG with different ratios of TA to FeIH.

[0118] Figure 6 shows 1D SAXS diffraction patterns of DMG-PEG-based cubosomes (Im3m) assembled by different types of phenolic compounds and metal ions, including EGCG, EGCG-Fe1", TA-Zrlv, TA-Co", TA-Ni", TA-Cu", and TA-Zn".

[0119] Figure 7 shows 1D SAXS diffraction patterns of DOPC-based hexosomes (H2) assembled by TA and TA-FeIH and embedded cryo-TEM image of the TA-Fe^ / DOPC hexosome (scale bar is 100 nm).

[0120] Figure 8 shows 1D SAXS diffraction patterns of DOPE-PEG-based micellarsomes (Fd3m) assembled by TA and TA-FeIH and embedded cryo-TEM image of the TA-FeIH / DOPE-PEG micellar cubosome (scale bar is 100 nm).

[0121] Figure 9 shows cryo-TEM images of the siRNA-loaded and mRNA-loaded TA / DMG-PEG cubosomes (scale bars are 200 nm).

[0122] Figure 10 shows RNA (siRNA and mRNA) loading efficiencies of TA / DMG-PEG cubosomes and the mean particle sizes of RNA-loaded cubosomes.

[0123] Figure 11 shows 1D SAXS diffraction pattern of siRNA-loaded TA / DMG-PEG cubosomes of different molar ratios of TA to DMG-PEG.

[0124] Figure 12 shows 1D SAXS diffraction pattern of mRNA-loaded TA / DMG-PEG cubosomes of different molar ratios of TA to DMG-PEG.

[0125] Figure 13 shows downregulation efficiency of luciferase siRNA-loaded TA / DMG-PEG cubosomes assembled from TA and DMG-PEG of different molar ratios (0:1,2:1,4:1, and 8:1) in PC3-luc2 cells at the siRNA concentration of 20 nM after 48 h transfection. Statistical significance was determined by one-way ANOVA: **** p < 0.0001, *** p < 0.001 and ns not significantly different. Error bars represent the standard deviation of three independent experiments.

[0126] Figure 14 shows transfection efficiency of the mRNA-loaded TA / DMG-PEG cubosomes, in terms of percentage in HEK293T cells at the concentration of 500 ng mRNA / 1 x105 cells after 24 h transfection. Statistical significance was determined by one-way ANOVA: **** p < 0.0001, *** p < 0.001 and ns not significantly different. Error bars represent the standard deviation of three independent experiments

[0127] Figure 15 shows transfection efficiency of the mRNA-loaded TA / DMG-PEG cubosomes, in terms of median fluorescence intensity (MFI) in HEK293T cells at the concentration of 500 ng mRNA / 1 x105 cells after 24 h transfection. Statistical significance was determined by one-way ANOVA: **** p < 0.0001, *** p < 0.001 and ns not significantly different. Error bars represent the standard deviation of three independent experiments.

[0128] Figure 16 shows intracellular trafficking of siRNA-loaded cubosomes with endo / lysosomes in PC3-luc2 cells. Cubosomes (red) are labelled by cy5-siRNA, endo / lysosomes (green) are stained with LysoTracker Green, and nuclei (blue) are stained with Hoechst 33342. Scale bars are 20 pm.

[0129] Figure 17. 1D diffraction SAXS patterns of DMG-PEG-based MPN cubosomes prepared from TA and metal ions, Zn2+(A), Fe3+(B), or Zr4+(C) of different TA / metal ions molar ratios.

[0130] Figure 18. 1D diffraction SAXS patterns of DMG-PEG-based MPN cubosomes prepared from EGCG and metal ions, Zn2+(A), Fe3+(B), or Zr4+(C) of different TA / metal ions molar ratios.

[0131] Figure 19. 1D diffraction SAXS patterns of P-LCNPs prepared from DMG-PEG and resveratrol and resveratrol-Fe3+. Resveratrol / DMG-PEG displayed no ordered nanostructures, while resveratrol-Fe3+ / DMG-PEG displayed cubic nanostructures.

[0132] Figure 20. 1D diffraction SAXS patterns of P-LCNPs prepared from PEG-SA with TA (TA / PEG-SA), and PEG-SA with TA-Fe3+ (TA-Fe3+ / PEG-SA), PEG-SA only was prepared as the negative control. Both TA / PEG-SA and TA-Fe3+ / PEG-SA P-LCNPs are cubic nanostructures.

[0133] Figure 21. 1D diffraction SAXS patterns of P-LCNPs prepared from PEG-MA with TA (TA / PEG-MA), and PEG-MA with TA-Fe3+ (TA-Fe3+ / PEG-MA), PEG-MA only was prepared as the negative control. TA / PEG-MA P-LCNPs didn’t display ordered nanostructures, while TA-Fe3+ / PEG-MA showed ordered nanostructures, the specific phase of which needs to be further confirmed by cryo-EM.

[0134] Figure 22. 1D diffraction SAXS patterns of P-LCNPs prepared from ALC-0159 (A) and PEG-c-DMG (B). ALC-0159 or PEG-c-DMG only was prepared as the negative control. TA / ALC-0159, TA-Fe3+ / ALC-0159, TA / PEG-c-DMG, and TA-Fe3+ / PEG-c-DMG are all cubic nanostructures.

[0135] Figure 23. 1D diffraction SAXS patterns of P-LCNPs prepared from DOPEPEG 5000 with TA (TA / DOPE-PEG 5000), and DOPE-PEG 5000 with TA-Fe3+ (TA-Fe3+ / DOPE-PEG 5000), DOPE-PEG 5000 only was prepared as the negative control. TA / DOPE-PEG 5000 displayed no ordered nanostructures. TA-Fe3+ / DOPE-PEG 5000 P-LCNPs are cubic nanostructures.

[0136] Figure 24. 1D diffraction SAXS patterns of P-LCNPs prepared from pSar-DMG with TA (TA / pSar-DMG), and pSar-DMG with TA-Fe3+ (TA-Fe3+ / pSar-DMG), pSar-DMG only was prepared as the negative control. TA / pSar-DMG and TA-Fe3+ / pSar-DMG showed ordered nanostructures, the specific phase of which needs to be further confirmed by cryo-EM.

[0137] Figure 25. 1D diffraction SAXS patterns (A) and size distribution (B) of TA / DMG-PEG cubosomes (10:1 TA:DMG-PEG, 2 mM DMG-PEG) after storage at (i) 4 °C for 1 year or (ii) 22 °C for 1 week or 1 year.

[0138] Figure 26. Characterization of solid-state-like 3D periodic assemblies from MD simulations. (A) Atomistic snapshots (top) and molecular density profiles (bottom) of the TA:DMG-PEG4oo morphologies show the effects of increasing TA concentration on the structure of the assembled systems. (B) Atomistic snapshots illustrating the TA:DMG-PEG2000 (2.7:1) system that experimentally forms cubosomes. The primitive unit cell (center), 3x3x3 supercell (top left), and 1.5 nm thick molecular slices along Cartesian axes reveal a cubic porous structure with distinct molecular regions. Lipids aggregate into bicelle-like particles, surrounded by a connected PEG network embedded within a TA scaffold. (C) MD-generated 2D mass density plots of the TA:DMG-PEG2ooo (2.7:1) system. (D) Radial distribution functions, g(f), show persistent specific interactions in the assembled TA:DMG-PEG2ooo (2.7:1) system structure. Hydrogen bond acceptors: PEG ether (OPEG); TA ester / carbonyl, pyranose ether (OTA); and water (Ow). Hydrogen bond donors: TA phenol (HTA) and water (Hw).

[0139] Figure. 27. Phenolic LC-LNPs assembled using different building blocks. (A, B) Library of phenolic LC-LNPs with different nanostructures assembled from different lipids (A) or MPNs composed of different polyphenols (TA or EGCG) and metal ions (Zn2+, Fe3+, or Zr4+) (B). TA-Fe3+ represents the MPN complex formed from TA and Fe3+. (C) Cryo-electron microscopy image of TA-Fe3+ / DMG-PEG cubosomes. Scale bar is 200 nm. Inset shows fast Fourier transform image with assigned miller indices for the section marked with a red frame. (D) UV-vis spectra of TA solution, TA-Fe3+ solution, DMG-PEG solution, and TA / DMG-PEG cubosomes and TA-Fe3+ / DMG-PEG cubosomes. TA-Fe3+ / DMG-PEG cubosomes featured a ligand-to-metal charge transfer (LMCT) band within the range of 450-650 nm, suggesting MPN formation in the cubosomes. (E) 1D SAXS patterns of TA / DMG-PEG cubosomes and TA-Fe3+ / DMG-PEG cubosomes in pH 3 and pH 5 buffers.

[0140] Figure 28. P-LCNPs loaded with cargos. (A) Schematic of P-LCNPs loaded with diverse cargos: small-molecule drug (DOX), polypeptide (PLA), protein (HRP), polysaccharide (chitosan), and nucleic acid (mRNA). (B) 1D SAXS patterns of cargo-loaded phenolic cubosomes. The peak ratios in all the SAXS patterns are V2:V4:V6. (C) Viability of MDA-MB-468 cells after incubation with free DOX or DOX@EGCG-Fe3+ / DMG-PEG cubosomes at different drug dosages. Cell viability (%) was normalized to the cell only control group. Data are shown as mean ± SD (n = 3 independent replicates) and analyzed by one-way analysis of variance (ANOVA), ****p < 0.0001. (D) ^-potential of PLA@TA / DMG-PEG and chitosan@TA / DMG-PEG cubosomes assembled using different mass ratios of TA and PLA / chitosan at different pHs. Data are shown as mean ± SD (n = 3 independent replicates). (E) Time-dependent absorbance changes upon oxidation of amplex red by different catalytic systems: free HRP, TA / DMG-PEG cubosomes, and HRP@TA / DMG-PEG cubosomes. (F) mCherry mRNA transfection efficiency, in terms of percentage of transfected cells, in HEK 293T cells treated with mCherry mRNA (mRNA), mCherry mRNA-lipofectamine MessengerMAX (Lipofectamine-mRNA), EGCG mixed with mRNA (EGCG-mRNA), and mCherry mRNA-loaded cubosomes (mRNA@EGCG-Zn2+ / DMG-PEG cubosomes). Data are shown as mean ± SD (n = 3 independent replicates) and analyzed by one-way ANOVA, **p < 0.01 and ****p < 0.0001.

[0141] Figure 29. (A) Stability (as measured by changes in particle size) of TA / DMG-PEG cubosomes (10:1 TA:DMG-PEG, 2 mM DMG-PEG) in different solutions over time. Data are shown as mean ± SD (n = 3 independent replicates). (B) 1D diffraction SAXS patterns of TA / DMG-PEG cubosomes (1:1 TA:DMG-PEG, 10 mM DMG-PEG) after incubation with NaCI, urea, or Tween 20.

[0142] Figure 30. Summary of systems modeled by MD. (A) Phase diagram highlighting the range of compositions investigated by varying the relative concentrations of TA, DMG-PEG, and water. Data points are relative to the weight percentage (wt%) of each component in the ternary plot, whereas ratio labels indicate TA:DMG-PEG molar ratios. (B) Initial conformations and chemical structures of the constituent molecules simulated in the TA-lipid assemblies. The DMG-PEG400 lipids (9 PEG monomers, 414 atoms) and DMG-PEG2000 lipids (45 PEG monomers, 162 atoms) have a TT-helical conformation along their principal molecular axis. The TA molecules (174 atoms) feature galloyl arms approximately planar with the central |3-D-glucose moiety. TA is modeled with galloyl moieties linked via meta-depside bonds. Atomistic space-filling representations depict carbon in grey, oxygen in red, and hydrogen in white. Detailed description of the embodiments

[0143] It will be understood that the disclosure described and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the disclosure. Definitions

[0144] For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0145] As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

[0146] "About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, in some instances ±5%, in some instances ±1%, and in some instances ±0.1% from the specified value, as such variations are appropriate to perform the disclosed processes.

[0147] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1,2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0148] As used herein, the term “non-lamellar lyotropic liquid crystalline nanoparticle” refers to a self-assembled non-lamellar liquid crystalline phase nanoparticle formed from one or more lipids and one or more phenolic compounds and which has a two and / or three-dimensional mesophase structure which is capable of carrying one or more active agents.

[0149] The present disclosure provides a lipid-based nanoparticle comprising one or more lipids and one or more phenolic compounds.

[0150] Lipid-based nanoparticles comprising a “single type of metal ion” refers to nanoparticles comprising essentially the same metal ions. For example the lipid-based nanoparticles comprises one or more metal ions, wherein the metal ions consist essentially of Fe(llI) metal ions.

[0151] Lipid-based nanoparticles comprising a “single type of lipid” refers to nanoparticles comprising essentially the same lipids. For example the lipid-based nanoparticles comprises one or more lipids, wherein the lipids consist essentially of DMG-PEG.

[0152] In embodiments of the disclosure it has been discovered that phenolic compounds (e.g., tannic acid (TA)), as well as phenolic compound-metal ion compositions can mediate the self-assembly phase of lamellar lipids from lamellar liquid crystal nanoparticles to a variety of non-lamellar liquid crystal nanoparticles, including cubosomes, hexosomes, and micellarsomes. This may address the limitation of using certain types of non-lamellar lipids for forming non-lamellar liquid crystal nanoparticles. The phenolic compounds not only act as the mediator of lipid assembly, but also mediate the encapsulation of biomolecules such as an RNA. Due to the high binding affinity of the phenolic compounds with RNA molecules and the cubic nanostructures, the phenolic compound-mediated cubosomes achieve successful RNA (siRNA and mRNA) loading without cubic phase transition and in vitro transfection without the addition of any cationic lipids, which are commonly associated with in vivo toxicity.

[0153] Owing to the ability of phenolic compounds to interact with diverse molecules, the phenolic LC-LNPs can encapsulate different cargos, including small-molecule drugs, polypeptides, proteins, polysaccharides, and mRNA, fordrug delivery, catalysis, and protein expression, while maintaining a cubic or hexagonal nanostructure.

[0154] Advantages of the presently disclosed lipid-based nanoparticles may include one or more of: (i) improved encapsulation of active agents such as an RNA; (ii) improved transfection of an RNA; (iii) reduced toxicity of the active agent compared to administration of the free active agent; (iv) the cubic structure of lipid nanoparticles promotes cellular uptake and intracellular endosomal escape, which leads to improved RNA transfection and small molecule drug delivery efficacy, (v) the ability to remain both colloidal stable and nanostructure stable under storage for more than a year and (vi) applicability to a wide range of lipids and phenolic compounds. Lipid-based nanoparticles

[0155] In embodiments, the lipid-based nanoparticle of the present disclosure has a mean particle size from about 5 nm to about 1 micron, or from about 10 nm to about 500 nm. The mean particle size of the lipid-based nanoparticle may be determined by, for example, dynamic light scattering (DLS).

[0156] In embodiments, the mean particle size of the lipid-based nanoparticle may be from about 10 nm to about 500 nm, about 10 nm to about 400 nm, or from about 10 nm to about 350 nm, or from about 10 nm to about 300 nm, or from about 10 nm to about 250 nm, or from about 50 nm to about 400 nm, or from about 50 nm to about 350 nm, or from about 50 nm to about 300 nm, or from about 50 nm to about 250 nm.

[0157] In embodiments, the mean particle size of the lipid-based nanoparticle may be greater than, or equal to, about 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205 nm, 210 nm, 215 nm, 220 nm, 225 nm, 230 nm, 235 nm, 240 nm, 245 nm, 250 nm, 255 nm, 260 nm, 265 nm, 270 nm, 275 nm, 280 nm, 285 nm, 290 nm, 295 nm, 300 nm, 305 nm, 310 nm, 315 nm, 320 nm, 325 nm, 330 nm, 335 nm, 340 nm, 345 nm, or 350 nm.

[0158] The lipid-based nanoparticles of the present disclosure may be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of a lipid-based nanoparticle composition, e.g., the particle size distribution of the lipid-based nanoparticle composition. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. The polydispersity index of a lipid-based nanoparticle composition may be determined by, for example, dynamic light scattering.

[0159] In embodiments, the polydispersity index of the lipid-based nanoparticle composition is less than about 0.3, or less than about 0.2, or less than about 0.1, or less than about 0.05.

[0160] In embodiments, the polydispersity index of the lipid-based nanoparticle composition is from about 0.2 to about 0.01.

[0161] The lipid-based nanoparticle composition may have a polydispersity index of about 0.01,0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11,0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21,0.22, 0.23, 0.24, or 0.25.

[0162] As used herein, the "zeta potential" is the electrokinetic potential of a lipid, e.g., in a nanoparticle.

[0163] The zeta potential of a nanoparticle may be used to indicate the electrokinetic potential of the particle. For example, the zeta potential may describe the surface charge of a nanoparticle. Nanoparticles with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. However, existing lipid nanoparticles have unusually high negative zeta potential.

[0164] In some embodiments, the zeta potential of a nanoparticle may be from about -50 mV to about 0 mV, preferably about -30mV to about -5mV. In some embodiments, the zeta potential of a nanoparticle may be from -50 mV to +0 mV, preferably -10mV to -5mV. Further, in some embodiments, the zeta potential of a nanoparticle may be from about -20mV to about -5mV, from about -15mV to about -5mV, from about -10mV to about -5mV, from about -20mV to about -10mV. Further, in some embodiments, the zeta potential of a nanoparticle may be from -20mV to -5mV, from -15mV to -5mV, from -10mV to -5mV, from -20mV to -10mV. Further, in some embodiments, the zeta potential of a nanoparticle may be about -5mV, about -1 OMV, about -15mV or about -20mV. Further, in some embodiments, the zeta potential of a nanoparticle may be -5mV, -10mV, -15mV or -20mV. Preferably, the bioactive nanoparticles have a zeta potential as described herein at a pH of about 4 to about 9.

[0165] In embodiments, the zeta potential of the lipid-based nanoparticle is less than about -5 mV, or less than about -10 mV, or from about -5 mV to about -35 mV, or from about -10 mV to about -35 mV.

[0166] In some embodiments, the zeta potential of a nanoparticle may be about -5 mV, or about -10 mV, or about -15 mV, or about -20 mV, or about -25 mV, or about -30 mV.

[0167] In embodiments, the presently disclosed lipid-based nanoparticle may have a non-crystalline structure.

[0168] In embodiments, the presently disclosed lipid-based nanoparticle may have a crystalline structure.

[0169] In embodiments, the presently disclosed lipid-based nanoparticle may comprise a non-lamellar lyotropic liquid crystalline phase structure.

[0170] In embodiments, the non-lamellar liquid crystalline phase comprises two and / or three-dimensional mesophase structures which are capable of carrying an active agent.

[0171] The term “non-lamellar” refers to the lyotropic liquid crystalline phase which is not a liposome (or La phase). Accordingly, a “non-lamellar” structure does not comprise a planar lipid bilayer structure as is found in a liposome structure.

[0172] In embodiments, non-lamellar lyotropic liquid crystalline phase structures include one or more phases comprising hexagonal (normal and reversed), cubic (normal discrete, reversed discrete, reversed bicontinuous - including primitive, gyroid and diamond - and reversed discontinuous), and other ‘intermediate phases’ including ribbon, mesh, or non-cubic ‘sponge’ bicontinuous phases.

[0173] In embodiments, the structure comprises a cubosome with bicontinuous inverse cubic phase.

[0174] In embodiments, the structure comprises a hexosome with inverse hexagonal phase.

[0175] In embodiments, the structure comprises a micellarsome with discontinuous micellar cubic phase.

[0176] See, for example, Silvestrini etaL, Progress and challenges of lytropic liquid crystalline nanoparticles for innovative therapies, International Journal of Pharmaceutics, 628 (2022) 122299, which is incorporated herein by reference in its entirety.

[0177] In embodiments, the lattice parameter of the non-lamellar lyotropic liquid crystalline phase structure is from about 20 to about 300 A, or from about 20 to about 250 A, or from about 20 to about 200 A, or from about 20 to about 190 A, or from about 20 to about 180 A, or from about 20 to about 170 A, or from about 20 to about 160 A, or from about 40 to about 300 A, or from about 40 to about 250 A, or from about 40 to about 200 A, or from about 40 to about 190 A, or from about 40 to about 180 A, or from about 40 to about 170 A, or from about 40 to about 160 A.

[0178] In embodiments, the lattice parameter of the non-lamellar lyotropic liquid crystalline phase structure is from about 80 to about 140 A, or from about 100 to about 130 A, or from about 40 to about 80 A, or from about 120 to about 170 A.

[0179] The lipid-based nanoparticle may be any one described herein including those listed in the Examples, e.g. Examples 1,2, 3, 5, 8, 9, 11 and 12. Phenolic compounds

[0180] In embodiments, the one or more phenolic compounds has a molecular weight from about 110 to about 3,000 Daltons.

[0181] In embodiments, the one or more phenolic compounds comprise one or more 3,4,5-trihydroxyphenyl groups.

[0182] In embodiments, the one or more phenolic compounds comprise three or more ring-bonded hydroxy groups.

[0183] In embodiments, the one or more phenolic compounds comprise one or more naturally occurring phenolic compounds.

[0184] In embodiments, the one or more phenolic compounds are partially or fully soluble in an aqueous solution, such as water, or a non-aqueous solution, such as an alcohol, preferably ethanol.

[0185] Phenolic compounds that are partially or fully soluble in water may include compounds comprising one or more of the following: - one or more 3,4,5-trihydroxyphenyl groups; - three or more ring-bonded hydroxy groups; and - one or more galloyl groups, optionally with one or more catechol groups.

[0186] In embodiments, the one or more phenolic compounds comprise one or more flavonoids. The one or more flavonoids may be selected from one or more of anthocyanins, chaicones, flavanones, flavones, flavonols, and isoflavonoids.

[0187] Non-limiting examples of flavonoids include apigenin, catechin, curcumin, luteolin, quercetin, kaempferol, genistein, gossypol, daidzein, fisetin, myricetin, naringenin, hesperetin, delphinidin, cyanidin, procyanidin, chalconaringenin, and phloretin.

[0188] Further non-limiting examples of the one or more phenolic compounds include one or more of tannic acid, (-)-epigallocatechin-3-gallate, gallocatechin, catechin-3-gallate, epigallocatechin, epicatechin-3-gallate, resveratrol, gallic acid, ellagic acid, and dopamine. Other phenolic compounds are contemplated.

[0189] In embodiments, the one or more phenolic compounds are one or more of tannic acid and (-)-epigallocatechin-3-gallate, preferably tannic acid. Lipids

[0190] In embodiments, the one or more lipids comprise one or more pegylated lipids, phospholipids, structural lipids, and cationic or ionisable lipids.

[0191] In embodiments, the one or more lipids are partially or fully soluble in a nonaqueous solution, such as an alcohol, preferably ethanol, or an aqueous solution, such as water. PEGylated lipid

[0192] The lipid component of the presently disclosed lipid-based nanoparticles may include one or more PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids. PEG-modified lipids or PEGylated lipid refer to a lipid comprising a polyethylene glycol component. A PEG lipid may be selected from the non-limiting group consisting of: PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof.

[0193] In an embodiment, the PEGylated lipid may be 1,2-dimyristoyl-rac-glycero-3-methoxy poly(ethylene glycol) (DMG-PEG).

[0194] In another embodiment, the PEGylated lipid may be methoxypolyethyleneglycoloxy(2000)-N,N-ditetradecylacetamide (ALC-0159).

[0195] In another embodiment, the PEGylated lipid may be 1,2-dioleoyl-s / ?-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DOPEPEG).

[0196] In another embodiment, the PEGylated lipid may be A / -(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-s / ?-glycero-3-phosphoethanolamine (sodium salt) (DSPE-PEG).

[0197] In another embodiment, the PEGylated lipid may be polyethylene glycol 2000-ester-stearic acid (PEG-SA); polyethylene glycol 2000-ester-myristic acid (PEG-MA); 2-[(polyethylene glycol).

[0198] In another embodiment, the PEGylated lipid may be polyethylene glycol-carbamate-1,2-dimyristoyl-sn-glycerol (PEG-c-DMG).

[0199] In another embodiment, the PEGylated lipid may be 1,2-dimyristoyl-sn-glycero-3-succinyl-N-polysarcosine-25 (pSar-DMG).

[0200] In another embodiment, the PEGylated lipid may be 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (ammonium salt) (DOPEPEG 5000).

[0201] In another embodiment, the PEGylated lipid may be methoxy polyethylene glycol 20k-distearoyl phosphatidyl ethanolamine (DSPE-PEG 20k).

[0202] In another embodiment, the PEGylated lipid may be cholesterol-polyethylene glycol 5000 (CLS-PEG 5000).

[0203] In another embodiment, the PEGylated lipid may be cholesterol-polyethylene glycol 5000 (CLS-PEG 5000).

[0204] In another embodiment, the PEGylated lipid may be cholesterol-polyethylene glycol 20k (CLS-PEG 20k).

[0205] The PEGylated lipid may have a PEG component that has a molecular weight from about 100 Dalton to about 100,000 Dalton or more. The PEG component may have a molecular weight from about 100 Da to about 10,000 Dalton, including but not limited to 10,000 Dalton, 9,000 Dalton, 8,000 Dalton, 7,000 Dalton, 6,000 Dalton, 5,000 Dalton, 4,000 Dalton, 3,000 Dalton, 2,000 Dalton, 1,000 Dalton, 900 Dalton, 800 Dalton, 700 Dalton, 600 Dalton, 500 Dalton, 400 Dalton, 300 Dalton, 200 Dalton, and 100 Dalton. In some embodiments, the PEG molecular weight is from about 100 Dalton to about 10,000 Dalton, from about 1000 Dalton to about 9,000 Dalton, from about 1000 Dalton to about 8,000 Dalton, from about 1000 Dalton to about 7,000 Dalton, from about 1000 Dalton to about 6,000 Dalton, from about 1000 Dalton to about 5,000 Dalton, from about 1000 Dalton to about 4,000 Dalton, from about 1000 Dalton to about 3,000 Dalton, or from about 1000 Dalton to about 2,000 Dalton. In some embodiments, the PEG molecular weight is from about 1,000 Dalton to about 5,000 Dalton. In some embodiments, the PEG molecular weight is from about 2,000 Dalton to about 5,000 Dalton. In some embodiments, the PEG molecular weight is from about 500 Dalton to about 3,000 Dalton. Phospholipids

[0206] The lipid component of the presently disclosed lipid-based nanoparticles may include one or more phospholipids.

[0207] As used herein, a "phospholipid" is a lipid that includes a phosphate moiety and one or more carbon chains. A phospholipid may include one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturations) in the one or more carbon chains.

[0208] A phospholipid moiety may be selected from the non-limiting group consisting of: phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.

[0209] A fatty acid moiety may be selected from the non-limiting group consisting of: lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0210] No limiting examples of phospholipids include lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Structural lipids

[0211] The lipid component of the presently disclosed lipid-based nanoparticles may include one or more structural lipids. Non-limiting examples of structural lipids include one or more of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, and alpha-tocopherol. Cationic or ionisable lipids

[0212] The lipid component of the presently disclosed lipid-based nanoparticles may include one or more cationic or ionisable lipids.

[0213] Cationic lipids which are useful in the present disclosure can be any of a number of lipid species which carry a net positive charge at physiological pH. Such lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioeoyloxy-3-(dimethylamino)propane (DODAP), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N—(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1 -(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3.beta.-oxy)-3'-oxapentoxy)-3-dimethy-1 -(cis,cis-9', 1 -2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 4-Hydroxybutyl)azanediyl)bis(hexane-6,1 -diyl) bis(2-hexyldecanoate) (ALC-0315), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid,1-octylnonyl ester (SM-102), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (chloride salt) (EPC), and mixtures thereof.

[0214] In embodiments, the molar ratio of the one or more phenolic compounds to the one or more lipids in the lipid-based nanoparticles is from about 0.5:1 to about 50:1, or about 1:1 to about 50:1.

[0215] In embodiments, the molar ratio of the one or more phenolic compounds to the one or more lipids in the lipid-based nanoparticles is about 0.5:1, or about 1:1, or about 2:1, or about 3:1, or about 4:1, or about 5:1, or about 6:1, or about 7:1, or about 8:1, or about 9:1, or about 10:1, or about 11:1, or about 12:1, or about 13:1, or about 14:1, or about 15:1, or about 16:1, or about 17:1, or about 18:1, or about 19:1, or about 20:1, or about 21:1, or about 22:1, or about 23:1, or about 24:1, or about 25:1, or about 26:1, or about 27:1, or about 28:1, or about 29:1, or about 30:1, or about 31:1, or about 32:1, or about 33:1, or about 34:1, or about 35:1, or about 36:1, or about 37:1, or about 38:1, or about 39:1, or about 40:1, or about 41:1, or about 42:1, or about 43:1, or about 44:1, or about 45:1, or about 46:1, or about 47:1, or about 48:1, or about 49:1, or about 50:1. Metal ions

[0216] In embodiments, the lipid-based nanoparticle of the present disclosure further comprises one or more metal ions.

[0217] Non-limiting examples of the one or more metal ions include one or more of Cu(ll), Fe(lll), Zr(IV), Zn(ll), Ni(ll), Co(ll), Na(l), Ca(ll), Ti(IV), Mn(ll), Ir(IV), Ta(V), W(VI), Bi(lll), Mn(l I), Gd(lll), Pt(IV), Nb(V), and Ga(l 11). Other metal ions are contemplated.

[0218] In embodiments, the one or more metal ions comprise one or more of Zn(ll), Cu(ll), Fe(lll), Zr(IV), Co(ll) and Ni(ll), preferably Fe(lll).

[0219] In embodiments, the lipid-based nanoparticle comprises a single type of metal ion.

[0220] In embodiments, the molar ratio of one or more phenolic compounds to one or more metal ions is from about 5:1 to about 1:5.

[0221] In embodiments, the molar ratio of phenolic compounds to one or more metal ions is about 5:1, or about 4:1, or about 3:1, or about 2:1, or about 1:1, or about 1:2, or about 1:3, or about 1:4, or about 1:5. Bioactive lipid-based nanoparticles

[0222] The present disclosure also provides a bioactive lipid-based nanoparticle comprising the lipid-based nanoparticle according to any one of the herein disclosed embodiments and one or more active agents, such as small molecules or biomacromolecules.

[0223] The use of bioactive lipid-based nanoparticle as described herein is contemplated for the introduction of the one or more bioactive agents into a host cell (in vitro, ex vivo or in vivo). In another aspect, the one or more bioactive agents may be associated with a phenolic ligand. The one or more bioactive agents associated with a phenolic ligand and / or metal ion may be encapsulated in the interior of a lipid-based nanoparticle, attached to a lipid-based nanoparticle via a linking molecule that is associated with phenolic ligand and bioactive agent, entrapped in a nanoparticle complexed with a nanoparticle, dispersed in a solution containing a phenolic ligand, mixed with a phenolic ligand, or combined with a phenolic ligand. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape.

[0224] Bioactive agents include biologically active substances and are alternately referred to as "active agents”, “therapeutic agents” and / or “prophylactic agents”, A therapeutic and / or prophylactic agent may be a substance that, once delivered to a cell or organ, brings about a desirable change in the cell, organ, or other bodily tissue or system. Such agents may be useful in the treatment of one or more diseases, disorders, or conditions. In some embodiments, a therapeutic and / or prophylactic agent is a small molecule drug useful in the treatment of a particular disease, disorder, or condition. Examples of drugs useful in the nanoparticle compositions include, but are not limited to, antineoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), antitumor agents (e.g., actinomycin D, vincristine, vinblastine, cystine arabinoside, anthracyclines, alkylative agents, platinum compounds, antimetabolites, and nucleoside analogs, such as methotrexate and purine and pyrimidine analogs), anti-infective agents, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blockers (e.g., propranolol, timolol, and labetolol), antihypertensive agents (e.g., clonidine and hydralazine), anti-depressants (e.g., imipramine, amitriptyline, and doxepim), anti-conversants (e.g., phenytoin), antihistamines (e.g., diphenhydramine, chlorphenirimine, and promethazine), antibiotic / antibacterial agents (e.g., gen-tamycin, ciprofloxacin, and cefoxitin), antifungal agents (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, vitamins, narcotics, and imaging agents.

[0225] Non-limiting examples of biomacromolecules include one or more of proteins, peptides, nucleic acid molecules and enzymes.

[0226] The present disclosure provides bioactive nanoparticle compositions comprising one or more bioactive agents, methods of making the bioactive nanoparticles, and methods of delivering and / or administering the bioactive nanoparticles (e.g., for the treatment of a disease or disorder).

[0227] Advantageously, the bioactive nanoparticle compositions as described herein are substantially non-toxic to mammals such as humans.

[0228] In some embodiments, the bioactive agent comprises a nucleic acid molecule. In certain instances, the nucleic acid is a DNA molecule, an RNA molecule or a DNA / RNA molecule. In certain other instances, the nucleic acid comprises singlestranded or double-stranded DNA, RNA, or a DNA / RNA hybrid.

[0229] The amount of nucleic acid molecule in a bioactive nanoparticle composition may depend on the size, sequence, and other characteristics of the mRNA. The amount of nucleic acid molecules in a bioactive nanoparticle compositions may also depend on the size, desired target, and other characteristics of the bioactive nanoparticle composition.

[0230] In other embodiments, the bioactive agent comprises a peptide or polypeptide. In certain instances, the peptide or polypeptide comprises an antibody such as, e.g., a polyclonal antibody, a monoclonal antibody, an antibody fragment; a humanized antibody, a recombinant antibody, a recombinant human antibody, or mixtures thereof. In certain other instances, the peptide or polypeptide comprises a cytokine, a growth factor, an apoptotic factor, a differentiation-inducing factor, a cell-surface receptor, a ligand, a hormone, a small molecule (e.g., small organic molecule or compound), or mixtures thereof.

[0231] In some embodiments, the bioactive agent is a therapeutic agent, or a salt or derivative thereof. Therapeutic agent derivatives may be therapeutically active themselves or they may be prodrugs, which become active upon further modification. Thus, in one embodiment, a therapeutic agent derivative retains some or all of the therapeutic activity as compared to the unmodified agent, while in another embodiment, a therapeutic agent derivative is a prodrug that lacks therapeutic activity, but becomes active upon further modification.

[0232] In embodiments, the one or more small molecules or biomacromolecules are at least partly encapsulated within the lipid-based nanoparticle.

[0233] In embodiments, the active agent is fully encapsulated within the lipid-based nanoparticle such that the active agent in the lipid-based nanoparticle is resistant in aqueous solution to enzymatic degradation.

[0234] In some embodiments, the biomacromolecule comprises a nucleic acid molecule. The nucleic acid may comprise single-stranded or double-stranded DNA, RNA, or a DNA / RNA hybrid.

[0235] In embodiments, the biomacromolecule comprises an RNA.

[0236] In embodiments, the biomacromolecule comprises an mRNA or siRNA.

[0237] The amount of RNA in a lipid-based nanoparticle may depend on the size, sequence, and other characteristics of the RNA. The amount of RNA in a lipid-based nanoparticle may also depend on the size, composition, desired target, and other characteristics of the lipid-based nanoparticle. The relative amounts of RNA and other elements (e.g., lipids) may also vary.

[0238] In some embodiments, the wt / wt ratio of the lipid component to an RNA in a nanoparticle composition may be from about 5:1 to about 50:1, such as 5:1,6:1,7:1, 8:1,9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1,20:1,25:1,30:1,35:1, 40:1,45:1, and 50:1.

[0239] For example, the wt / wt ratio of the lipid component to an RNA may be from about 10:1 to about 40:1. The amount of RNA in a nanoparticle composition may, for example, be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

[0240] In some embodiments, the wt / wt ratio of the lipid component to the RNA in the nanoparticle composition is from about 5:1 to about 50:1. In certain embodiments, the wt / wt ratio is from about 10:1 to about 40:1.

[0241] As used herein, "loading efficiency" refers to the amount of active agent, such as an RNA, that becomes part of a nanoparticle composition, relative to the initial total amount of active agent used in the preparation of a nanoparticle composition. For example, if 90 mg of active agent is loaded in a nanoparticle composition out of a total 100 mg of active agent initially provided to the composition, the loading efficiency may be given as 90%. As used herein, loading may refer to encapsulation, such encapsulation being complete, substantial, or partial enclosure, confinement, surrounding, or encasement.

[0242] In embodiments of the present disclosure the loading efficiency of active agent may be greater than 50%, or greater than 55%, or greater than 60%, or greater than 65%, or greater than 70%, or greater than 75%, or greater than 80%, or greater than 85%, or greater than 90%, or greater than 95%.

[0243] In embodiments, the active agent may comprise one or more therapeutic proteins, polypeptides, or small organic molecules or compounds. Non-limiting examples of such therapeutically effective agents or drugs include oncology drugs (e.g., chemotherapy drugs, hormonal therapeutic agents, immunotherapeutic agents, radiotherapeutic agents, etc.), lipid-lowering agents, anti-viral drugs, anti-inflammatory compounds, antidepressants, stimulants, analgesics, antibiotics, birth control medication, antipyretics, vasodilators, anti-angiogenics, cytovascular agents, signal transduction inhibitors, cardiovascular drugs such as anti-arrhythmic agents, hormones, vasoconstrictors, and steroids. These active agents may be administered alone in the lipid-based nanoparticles of the present disclosure, or in combination (e.g., coadministered) with the lipid-based nanoparticles of the present disclosure comprising, for example, a nucleic acid such as mRNA. Methods for producing lipid-based nanoparticles

[0244] The present disclosure also provides a method for producing a lipid-based nanoparticle according to any one of the herein disclosed embodiments comprising contacting one or more lipids with one or more phenolic compounds.

[0245] In embodiments of the method an solution of phenolic compound is contacted with a non-aqueous solution of lipid. In embodiments, the solution of phenolic compound is contacted with a non-aqueous solution of lipid are independently selected from an aqueous solution, such as water, or a non-aqueous solution such as an alcohol, preferably ethanol.

[0246] In certain embodiments, comprising an aqueous solution of phenolic compound and a non-aqueous solution of lipid, the ratio of aqueous solution to nonaqueous solution may be in the range from about 1:0.5 to about 1:5 by volume, preferably from about 1:2 to about 1:4 by volume.

[0247] In embodiments of the methods, the solution of phenolic compound and the solution of lipid as described herein are admixable with each other. The skilled person will appreciate and be able to select solutions known in the art which are compatible for admixing according to the methods of the present invention. For example, the solution of phenolic compound comprises water, and the solution of lipid comprises ethanol. Alternatively, the solution of phenolic compound comprises ethanol, and the solution of lipid comprises water. Alternatively, yet again, solution of phenolic compound comprises water, and the solution of lipid comprises water.

[0248] The molar ratio of the one or more phenolic compounds to the one or more lipids may be from about 0.5:1 to about 50:1, or about 1:1 to about 50:1.

[0249] In embodiments, the molar ratio of the one or more phenolic compounds to the one or more lipids is about 0.5:1, or about 1:1, or about 2:1, or about 3:1, or about 4:1, or about 5:1, or about 6:1, or about 7:1, or about 8:1, or about 9:1, or about 10:1, or about 11:1, or about 12:1, or about 13:1, or about 14:1, or about 15:1, or about 16:1, or about 17:1, or about 18:1, or about 19:1, or about 20:1, or about 21:1, or about 22:1, or about 23:1, or about 24:1, or about 25:1, or about 26:1, or about 27:1, or about 28:1, or about 29:1, or about 30:1, or about 31:1, or about 32:1, or about 33:1, or about 34:1, or about 35:1, or about 36:1, or about 37:1, or about 38:1, or about 39:1, or about 40:1, or about 41:1, or about 42:1, or about 43:1, or about 44:1, or about 45:1, or about 46:1, or about 47:1, or about 48:1, or about 49:1, or about 50:1.

[0250] The lipid-based nanoparticles may be produced in batch mode by contacting the solutions and mixing, for example using a vortex mixer.

[0251] The lipid-based nanoparticles may also be produced in a microfluidic system.

[0252] Without being bound by theory, it is envisaged that the formation of the non-lamellar lyotropic liquid crystalline phase structure as described herein may be assisted by using phenolic compounds with stronger intermolecular interaction, such as having increased hydrogen bonding and tt-tt interactions, or phenolic compounds that more easily undergo oxidative polymerisation, during the production of said structures.

[0253] Further, without being bound by theory, it is envisaged that phenolic compounds mainly interact with the lipid hydrophilic heads to form the non-lamellar lyotropic liquid crystalline phase structures as described herein. Methods for producing lipid-based nanoparticles comprising metal ions

[0254] The present disclosure also provides a method for producing a lipid-based nanoparticle comprising metal ions according to any one of the herein disclosed embodiments comprising contacting one or more lipids with one or more phenolic compounds and one or more metal ions.

[0255] In embodiments of the method an solution of phenolic compound is contacted with one or more metal ions and the resulting solution contacted with a solution of lipid. In embodiments, the solution of phenolic compound is contacted with a non-aqueous solution of lipid are independently selected from an aqueous solution, such as water, or a non-aqueous solution such as an alcohol, preferably ethanol.

[0256] In certain embodiments, comprising an aqueous solution of phenolic compound and a non-aqueous solution of lipid, the ratio of aqueous solution to nonaqueous solution may be in the range from about 1:0.5 to about 1:5 by volume, preferably from about 1:2 to about 1:4 by volume.

[0257] The source of metal ion is not critical and various metal salts, such as metal halides, metal nitrates, etc., may be utilised.

[0258] The molar ratio of the one or more phenolic compounds to the one or more lipids may be similar to those utilised to prepare the lipid-based nanoparticles without metal ions.

[0259] The molar ratio of one or more phenolic compounds to one or more metal ions may be from about 5:1 to about 1:5.

[0260] The molar ratio of one or more phenolic compounds to one or more metal ions may be about 5:1, or about 4:1, or about 3:1, or about 2:1, or about 1:1, or about 1:2, or about 1:3, or about 1:4, or about 1:5. Methods for producing lipid-based nanoparticles containing active agents

[0261] The present disclosure also provides method for producing a bioactive lipid-based nanoparticle according to any one of the herein disclosed embodiments comprising contacting one or more lipids, one or more phenolic compounds, optionally one or metal ions, and one or more active agents, such as bioactive small molecules or biomacromolecules.

[0262] In embodiments of the method the active agent may be first contacted with the one or more phenolic compounds in solution and the resulting solution contacted with a solution of one or more lipids. Pharmaceutical compositions

[0263] The presently disclosed lipid-based nanoparticles and bioactive lipid-based nanoparticles may be formulated in whole or in part as pharmaceutical compositions. For example, a pharmaceutical composition may include one or more nanoparticles including one or more different therapeutic and / or prophylactic agents. Pharmaceutical compositions may further include one or more pharmaceutically acceptable excipients or accessory ingredients. General guidelines for the formulation and manufacture of pharmaceutical compositions and agents are available, for example, in Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Conventional excipients and accessory ingredients may be used in any pharmaceutical composition, except insofar as any conventional excipient or accessory ingredient may be incompatible with one or more components of the lipid-based nanoparticles. An excipient or accessory ingredient may be incompatible with a component of a lipid-based nanoparticle or bioactive lipid-based nanoparticle if its combination with the component may result in any undesirable biological effect or otherwise deleterious effect.

[0264] In some embodiments, one or more excipients or accessory ingredients may make up greater than 50% of the total mass or volume of a pharmaceutical composition including a lipid-based nanoparticle or bioactive lipid based nanoparticle. For example, the one or more excipients or accessory ingredients may make up 50%, 60%, 70%, 80%, 90%, or more of a pharmaceutical composition. In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0265] Relative amounts of lipid-based nanoparticles or bioactive lipid-based nanoparticles and one or more pharmaceutically acceptable excipients, and / or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, a pharmaceutical composition may comprise between 0.1% and 100% (wt / wt) of one or more lipid-based nanoparticles or bioactive lipid based nanoparticles.

[0266] In certain embodiments, the lipid-based nanoparticles, bioactive lipid-based nanoparticles and / or pharmaceutical compositions of the present disclosure are refrigerated or frozen for storage and / or shipment (e.g., being stored at a temperature of 4° C. or lower, such as a temperature from about -150° C. and about 0° C. or from about -80° C. and about -20° C. (e.g., about -5° C., -10° C., -15° C., -20° C., -25° C., -30° C., -40° C., -50° C., -60° C., -70° C., -80° C., -90° C., -130° C. or -150° C.)

[0267] Lipid-based nanoparticles, bioactive lipid-based nanoparticles and / or pharmaceutical compositions may be administered to any patient or subject, including those patients or subjects that may benefit from a therapeutic effect provided by the delivery of a therapeutic and / or prophylactic to one or more particular cells, tissues, organs, or systems or groups thereof. Although the descriptions provided herein of lipid-based nanoparticles, bioactive lipid-based nanoparticles and pharmaceutical compositions are principally directed to compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other mammal. Modification of compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the compositions is contemplated include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals such as cattle, pigs, hoses, sheep, cats, dogs, mice, and / or rats.

[0268] A pharmaceutical composition including one or more lipid-based nanoparticles or bioactive lipid-based nanoparticles may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include bringing the active ingredient into association with an excipient and / or one or more other accessory ingredients, and then, if desirable or necessary, dividing, shaping, and / or packaging the product into a desired single- or multi-dose unit.

[0269] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient (e.g., lipid-based nanoparticles or bioactive lipid-based nanoparticles). The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. Application and Administration

[0270] As used herein, the term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, to which the compositions and methods of the present disclosure are administered.

[0271] Pharmaceutical compositions may be prepared in a variety of forms suitable for a variety of routes and methods of administration. For example, pharmaceutical compositions may be prepared in liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable forms, solid dosage forms (e.g., capsules, tablets, pills, powders, and granules), dosage forms for topical and / or transdermal administration (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and patches), suspensions, powders, and other forms.

[0272] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to active ingredients, liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions can include additional therapeutic and / or prophylactics, additional agents such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and / or perfuming agents. In certain embodiments for parenteral administration, compositions are mixed with solubilizing agents such as Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof.

[0273] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and / or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and / or emulsions in nontoxic parenterally acceptable diluents and / or solvents, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.

[0274] Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing compositions with suitable non-irritating excipients such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.

[0275] Solid dosage forms for oral administration include capsules, tablets, pills, films, powders, and granules. In such solid dosage forms, an active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and / or fillers or extenders (e.g. starches, lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g., carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g., glycerol), disintegrating agents (e.g., agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate), solution retarding agents (e.g., paraffin), absorption accelerators (e.g., quaternary ammonium compounds), wetting agents (e.g., cetyl alcohol and glycerol monostearate), absorbents (e.g., kaolin and bentonite clay, silicates), and lubricants (e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate), and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may comprise buffering agents.

[0276] Dosage forms for topical and / or transdermal administration of a composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and / or patches. Generally, an active ingredient is admixed under sterile conditions with a pharmaceutically acceptable excipient and / or any needed preservatives and / or buffers as may be required. Additionally, the present disclosure contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms may be prepared, for example, by dissolving and / or dispensing the compound in the proper medium. Alternatively or additionally, rate may be controlled by either providing a rate controlling membrane and / or by dispersing the compound in a polymer matrix and / or gel.

[0277] Formulations suitable for topical administration include, but are not limited to, liquid and / or semi liquid preparations such as liniments, lotions, oil in water and / or water in oil emulsions such as creams, ointments and / or pastes, and / or solutions and / or suspensions. Topically-administrable formulations may, for example, comprise from about 1% to about 10% (wt / wt) active ingredient, although the concentration of active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

[0278] A pharmaceutical composition may be prepared, packaged, and / or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder and / or using a self-propelling solvent / powder dispensing container such as a device comprising the active ingredient dissolved and / or suspended in a low-boiling propellant in a sealed container. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

[0279] Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (wt / wt) and as much as 100% (wt / wt) of active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition may be prepared, packaged, and / or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets and / or lozenges made using conventional methods, and may, for example, 0.1% to 20% (wt / wt) active ingredient, the balance comprising an orally dissolvable and / or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder and / or an aerosolized and / or atomized solution and / or suspension comprising active ingredient. Such powdered, aerosolized, and / or aerosolized formulations, when dispersed, may have an average particle and / or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein. Methods of treating diseases or disorders

[0280] The lipid-based nanoparticles or bioactive lipid-based nanoparticles of the present disclosure may be useful for treating a disease, disorder, or condition. For example, a lipid-based nanoparticle comprising an active agent, such as mRNA may be administered or delivered to a cell. Subsequent translation of the mRNA may produce a polypeptide, thereby reducing or eliminating an issue caused by the absence of or aberrant activity caused by the polypeptide. A therapeutic and / or prophylactic included in a nanoparticle composition may also be capable of altering the rate of transcription of a given species, thereby affecting gene expression.

[0281] Diseases, disorders, and / or conditions for which a composition may be administered include, but are not limited to, rare diseases, infectious diseases (as both vaccines and therapeutics), cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardio-and reno-vascular diseases, and metabolic diseases. Multiple diseases, disorders, and / or conditions may be characterized by missing (or substantially diminished such that proper protein function does not occur) protein activity. Such proteins may not be present, or they may be essentially non-functional.

[0282] The present disclosure provides a method for treating such diseases, disorders, and / or conditions in a subject by administering a bioactive lipid-based nanoparticle of the present disclosure including an RNA, wherein the RNA may be an mRNA encoding a polypeptide that antagonizes or otherwise overcomes an aberrant protein activity present in the cell of the subject.

[0283] The present disclosure provides methods involving administering lipid-based nanoparticles including one or more therapeutic and / or prophylactic active agents and pharmaceutical compositions including the same. The terms therapeutic and prophylactic can be used interchangeably herein with respect to features and embodiments of the present disclosure. Therapeutic compositions, or imaging, diagnostic, or prophylactic compositions thereof, may be administered to a subject using any reasonable amount and any route of administration effective for preventing, treating, diagnosing, or imaging a disease, disorder, and / or condition and / or any other purpose. The specific amount administered to a given subject may vary depending on the species, age, and general condition of the subject; the purpose of the administration; the particular composition; the mode of administration; and the like. Compositions in accordance with the present disclosure may be formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of a bioactive lipid-based nanoparticle or pharmaceutical composition of the present disclosure will be decided by an attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or otherwise appropriate dose level (e.g., for imaging) for any particular patient will depend upon a variety of factors including the severity and identify of a disorder being treated, if any; the one or more therapeutic and / or prophylactic agents employed; the specific composition employed; the age, body weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific pharmaceutical composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific pharmaceutical composition employed; and like factors well known in the medical arts.

[0284] A bioactive lipid-based nanoparticle according to the present disclosure including one or more therapeutic and / or prophylactic active agents may be administered by any route. In some embodiments, compositions, including prophylactic, diagnostic, or imaging compositions including one or more bioactive lipid-based nanoparticles described herein, are administered by one or more of a variety of routes, including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, trans- or intra-dermal, interdermal, rectal, intravaginal, intraperitoneal, intraocular, subretinal, intravitreal, topical (e.g. by powders, ointments, creams, gels, lotions, and / or drops), mucosal, nasal, buccal, enteral, vitreal, intratumoral, sublingual, intranasal; by intratracheal instillation, bronchial instillation, and / or inhalation; as an oral spray and / or powder, nasal spray, and / or aerosol, and / or through a portal vein catheter. In some embodiments, a composition may be administered intravenously, intramuscularly, intradermally, intra-arterially, intratumorally, subcutaneously, intraocularly, subretinally, intravitreally, or by inhalation. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the nanoparticle composition including one or more therapeutic and / or prophylactics (e.g., its stability in various bodily environments such as the bloodstream and gastrointestinal tract), the condition of the patient (e.g., whether the patient is able to tolerate particular routes of administration), etc.

[0285] In certain embodiments, compositions in accordance with the present disclosure may be administered at dosage levels sufficient to deliver from about 0.0001 mg / kg to about 10 mg / kg, from about 0.001 mg / kg to about 10 mg / kg, from about 0.005 mg / kg to about 10 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.05 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 10 mg / kg, from about 2 mg / kg to about 10 mg / kg, from about 5 mg / kg to about 10 mg / kg, from about 0.0001 mg / kg to about 5 mg / kg, from about 0.001 mg / kg to about 5 mg / kg, from about 0.005 mg / kg to about 5 mg / kg, from about 0.01 mg / kg to about 5 mg / kg, from about 0.05 mg / kg to about 5 mg / kg, from about 0.1 mg / kg to about 5 mg / kg, from about 1 mg / kg to about 5 mg / kg, from about 2 mg / kg to about 5 mg / kg, from about 0.0001 mg / kg to about 2.5 mg / kg, from about 0.001 mg / kg to about 2.5 mg / kg, from about 0.005 mg / kg to about 2.5 mg / kg, from about 0.01 mg / kg to about 2.5 mg / kg, from about 0.05 mg / kg to about 2.5 mg / kg, from about 0.1 mg / kg to about 2.5 mg / kg, from about 1 mg / kg to about 2.5 mg / kg, from about 2 mg / kg to about 2.5 mg / kg, from about 0.0001 mg / kg to about 1 mg / kg, from about 0.001 mg / kg to about 1 mg / kg, from about 0.005 mg / kg to about 1 mg / kg, from about 0.01 mg / kg to about 1 mg / kg, from about 0.05 mg / kg to about 1 mg / kg, from about 0.1 mg / kg to about 1 mg / kg, from about 0.0001 mg / kg to about 0.25 mg / kg, from about 0.001 mg / kg to about 0.25 mg / kg, from about 0.005 mg / kg to about 0.25 mg / kg, from about 0.01 mg / kg to about 0.25 mg / kg, from about 0.05 mg / kg to about 0.25 mg / kg, or from about 0.1 mg / kg to about 0.25 mg / kg of a therapeutic and / or prophylactic agent (e.g., an mRNA) in a given dose, where a dose of 1 mg / kg (mpk) provides 1 mg of a therapeutic and / or prophylactic per 1 kg of subject body weight. In some embodiments, a dose of about 0.001 mg / kg to about 10 mg / kg of a therapeutic and / or prophylactic (e.g., mRNA) of a nanoparticle composition may be administered. In other embodiments, a dose of about 0.005 mg / kg to about 2.5 mg / kg of a therapeutic and / or prophylactic may be administered. In certain embodiments, a dose of about 0.1 mg / kg to about 1 mg / kg may be administered. In other embodiments, a dose of about 0.05 mg / kg to about 0.25 mg / kg may be administered. A dose may be administered one or more times per day, in the same or a different amount, to obtain a desired level of mRNA expression and / or therapeutic, diagnostic, prophylactic, or imaging effect. The desired dosage may be delivered, for example, three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). In some embodiments, a single dose may be administered, for example, prior to or after a surgical procedure or in the instance of an acute disease, disorder, or condition.

[0286] Bioactive lipid-based nanoparticles of the invention including one or more therapeutic and / or prophylactic active agents may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. By “in combination with,” it is not intended to imply that the agents must be administered at the same time and / or formulated for delivery together, although these methods of delivery are within the scope of the present disclosure. For example, one or more lipid-based nanoparticles including one or more different therapeutic and / or prophylactic agents may be administered in combination. Bioactive lipid-based nanoparticles can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and / or on a time schedule determined for that agent. In some embodiments, the present disclosure encompasses the delivery of bioactive lipid-based nanoparticles, compositions, or imaging, diagnostic, or prophylactic compositions thereof in combination with agents that improve their bioavailability, reduce and / or modify their metabolism, inhibit their excretion, and / or modify their distribution within the body. Examples

[0287] Tannic acid (TA, ACS reagent), epigallocatechin gallate (EGCG, >95%), urea (99.0-100.5%), sodium chloride (NaCI, >99.5%), Tween-20 (BioXtra, viscous liquid), iron(lll) chloride hexahydrate (FeCl3'6H2O, >98.0%), cobalt(ll) chloride (C0CI2, >98.0%), nickel(ll) chloride (NiCl2, 98.0%), copper(ll) chloride dihydrate (CUCI2 2H2O, ACS reagent, >99.0%), zinc(ll) chloride (ZnCh, >98.0%), zirconium(IV) chloride (ZrCI4, >99.5%), glycine (>99.0%), 3-(N-Morpholino)propanesulfonic acid (MOPS, >99.5%), hydrochloric acid (HCI, 36.5-38.0%), sodium hydroxide (NaOH, >98%), dimethylsulfoxide-d6 (DMSO-d6), Dulbecco’s phosphate-buffered saline (DPBS), 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP) and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (chloride salt) (EPC) were purchased from Sigma-Aldrich. Slide-A-Lyzer MINI Dialysis Devices (20K molecular weight cutoff) and Opti-MEM were purchased from Thermo Fisher Scientific. 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DOPE-PEG2000) were purchased from Avanti Polar Lipids. Methoxypolyethyleneglycoloxy(2000)-N,N-ditetradecylacetamide (ALC-0159) and PEG-carbamate-1,2-dimyristoyl-s / ?-glycerol (PEG-c-DMG) were purchased from MedChemExpress. NE-Glo™ EX luciferase assay system was purchased from Promega. 1,1'-Dioctadecyl-3,3,3',3'- Tetramethylindodicarbocyanine, 4-Chlorobenzenesulfonate Salt (DiD) was purchased from ThermoFisher Scientific. High-purity water with a resistivity of 18.2 MO cm was obtained from an inline Millipore RiOs / Origin water purification system. All solutions were freshly prepared for immediate use in each experiment. PC3-luc2 and HEK293T cells were purchased from the American Type Culture Collection (USA). Lipofectamine RNAiMAX transfection reagent, lipofectamine MesengerMAX, Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), Roswell Park Memorial Institute Medium (RPMI), and 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt (XTT) were purchased from Life Technologies. mCherry mRNA and cyanine5 (cy5)-labeled green fluorescent protein (GFP) mRNA were purchased from Messenger Bio. MDA-MB-231, MDA-MB-468, and HEK293T cells were purchased from the American Type Culture Collection (USA). Example 1: Preparation of TA / lipid nanoparticles

[0288] Liquid crystalline nanoparticles were formulated using a NanoAssemblr platform (Ignite System) (Precision NanoSystems, Canada). The total flow rate ratio was set as 12 mL min-1 and the flow rate ratio of the aqueous phase to ethanol phase was set as 4:1 unless specified.

[0289] To prepare TA / DMG-PEG cubosomes of low concentration, DMG-PEG (2 mM, dissolved in ethanol or water) and TA (1.1,3.4, 5.7, and 11.3 mg / mL, dissolved in MillQ water) were mixed in the designed cartridges of NanoAssemblr. As a comparison, nanoparticles were also generated in the absence of TA. The total flow rate was set as 12 mL / min, and the flow rate ratio of aqueous phase to ethanol phase was 3:1 to achieve a molar ratio between TA and DMG-PEG of 0:1, 1:1,3:1,5:1, and 10:1. To prepare TA / DMG-PEG cubosomes of high concentration, DMG-PEG (10 mM, dissolved in ethanol) and TA (1.9, 2.8, 3.4, 5.7, 11.3, and 17 mg / mL, dissolved in MillQ water) were mixed in the designed cartridges of NanoAssemblr. Again, as a comparison, nanoparticles were also generated in the absence of TA. The total flow rate was set as 12 mL / min, and the flow rate ratio of aqueous phase to ethanol phase was 3:1 to achieve a molar ratio between TA and DMG-PEG of 0:1, 1:3, 1:2, 3:5, 1:1,2:1, and 3:1. The TA / DMG-PEG cubosomes were also prepared by directly adding the TA aqueous solution to the DMG-PEG ethanol solution in a 1.7 mL Eppendorf tube on a vortex mixer. After formulation, the cubosomes were purified through dialysis against MilliQ water using 10 kDa dialysis membrane for 24 h. Table 1 collects mean particle size, polydispersity and zeta potential data of the nanoparticles. Table 1 Molar Ratio of TA to DMG- PEG Size (nm) Polydispersity Index Zeta Potential (mV) 2 mM DMG-PEG 0:1 13 ±3 0.662 -4 ±2 1:1 94 ±28 0.072 -18 ± 1 3:1 130 ±36 0.046 -25 ±3 5:1 168 ±40 0.016 -27 ±1 10:1 195 ±58 0.088 -30 ± 1 10 mM DMG-PEG 1:1 262 ± 47 0.088 -21 ± 1

[0290] The effects of the dynamic mixing parameters during nanoparticle preparation in the NanoAssemblr platform, including total flow rate (TFR) and flow rate ratio (FRR), on the size and polydispersity of cubosomes were studied. It was found that at a molar ratio of TA to DMG-PEG of 10:1, the size distribution of the nanoparticles did not significantly change within the total flow rate range from 3 to 18 ml / min. However, the size distribution was affected by the flow rate ratio of aqueous to ethanol phases within the range of 1:1 to 1:4 and a ratio of 1:3 was selected for further study as was a total flow rate of 12 ml / min.

[0291] TA was found to mediate the mesophase of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG) from non-ordered vesicle phase to ordered Im3m cubic phase (Qiip).

[0292] Dynamic light scattering (DLS) indicated that when mixing DMG-PEG (2 mM) with MilliQ water, DMG-PEG self-assembled into highly polydispersed (PDI=0.66) aggregates of ~13 nm (Figure 1). When keeping the concentration of DMG-PEG at 2 mM, the inclusion of TA into the aqueous phase led to the formation of TA / DMG-PEG cubosomes with mean particle sizes from 90 to 200 nm as the molar ratio of TA to DMG-PEG increased from 1:1 to 10:1, and these cubosomes remained highly monodispersed with PDI below 0.1 (Figure 1).

[0293] It was likely that more TA molecules were incorporated into the TA / DMG-PEG cubosomes as the molar ratio of TA to DMG-PEG increased, as evidenced by the decreasing zeta potential of the TA / DMG-PEG cubosomes (Table 1). Nuclear magnetic resonance (NMR) analysis of the purified cubosomes also evidenced an increasing amount of TA as the TA / DMG-PEG molar ratio increased.

[0294] Inverse primitive Im3m cubic phases of the TA / DMG-PEG cubosomes were indicated by the diffraction peaks in 1D SAXS patterns with the relative positions in the spacing ratios of V2:V4:V6:V8 while no ordered phases were observed in 1D SAXS pattern in the absence of TA (Figure 2A). From the SAXS diffraction results, the lattice parameters (LP) of the TA-DMG-PEG cubosomes slightly increased from 117.3 to 121.2 A, as the molar ratio between TA to DMG-PEG increased from 1:1 to 10:1 (Table 2), because of more TA molecules built in the cubic structures. Increasing the TA-to-DMG-PEG molar ratio from 0:1 to 10:1 resulted in the decrease in the ^-potentials of NPs from -4 to -30 mV, suggesting the presence of TA on the cubosome surface (Figure 2B). TA / DMG-PEG also formed cubosomes when both TA and DMG-PEG were dissolved in water (Figure 2C). Table 2 Sample name Ordered phase Lattice parameter (A) TA / DMG-PEG nanoparticles 1:1 TA / 2 mM DMG-PEG cubic 117.3 3:1 TA / 2 mM DMG-PEG cubic 117.3 5:1 TA / 2 mM DMG-PEG cubic 119.2 10:1 TA / 2 mM DMG-PEG cubic 121.2 1:3 TA / 10 mM DMG-PEG No multilamellar 52.1 1:2 TA / 10 mM DMG-PEG No multilamellar 59.9 3:5 TA / 10 mM DMG-PEG cubic 115.5 1:1 TA / 10 mM DMG-PEG cubic 115.5 1:1 TA / 10 mM DMG-PEG in Urea cubic 117.4 1:1 TA / 10 mM DMG-PEG in NaCI cubic 115.1 1:1 TA / 10 mM DMG-PEG in pH 7 cubic 115.5 1:1 TA / 10 mM DMG-PEG in pH 9 cubic 115.5 1:1 TA / 10 mM DMG-PEG at 4 °C cubic 115.5 1:1 TA / 10 mM DMG-PEG at 37 °C cubic 115.5 2:1 TA / 10 mM DMG-PEG cubic 115.5 3:1 TA / 10 mM DMG-PEG cubic 117.3

[0295] The morphologies of representative TA / DMG-PEG cubosomes prepared with molar ratios of TA to DMG-PEG of 1:1 (1:1 TA:Lipid (low con.)) and 10:1 (10:1 TA:Lipid (low con.)) were further characterized by cryo-TEM images and the embedded SAXS 2D patterns (Figure 3). From the cryo-TEM images, the 1:1 and 10:1 TA / DMG-PEG cubosomes showed a mean particle size of around 90 and 200 nm, which was consistent with DLS measurements. While no clear lattices were observed in the TA / DMG-PEG cubosomes prepared from the low concentrations of DMG-PEG (2 mM) (Figure 3), the 1:1 TA / DMG-PEG cubosomes prepared from a higher concentration of DMG-PEG (10 mM) showed a larger mean particle size of around 260 nm and exhibited clear square lattices indicating their cubic structures. Fast Fourier Transform (FFT) analysis of the cubosome displayed the characteristic square reflection planes of the Im3m cubic phase (Figure 3). These results suggested that the formation of cubosomes was influenced by the concentrations of both TA and DMG-PEG.

[0296] Further insights into the cubosome assembly process were obtained by decreasing the molar ratio of TA to DMG-PEG below 1:1. When this ratio was 1:3 and 1:2, the TA / DMG-PEG nanoparticles showed a single broad peak in 1D SAXS pattern (Figure 4), indicating multi-lamellar structures. Additionally, the intensities and position of the SAXS peak suggested that the proportion and the LP of the multi-lamellar structures in the TA / DMG-PEG nanoparticles gradually increased as the molar ratio between TA to DMG-PEG increased from 1:3 to 1:2, and then the phase transferred into the cubic phase and showed the co-existence of multi-lamellar and cubic phases as the molar ratio of TA to DMG-PEG reached 3:5 (Figure 4). Only when the ratio of TA to DMG-PEG was equal to or higher than 1:1 did the SAXS diffraction peaks corresponding to the primitive Im3m cubic phase appear (Figure 4).

[0297] To investigate how different interactions governed the assembly of the TA / DMG-PEG cubosomes, the cubosomes were incubated with Tween 20 (50 mM), urea (50 mM), or sodium chloride (NaCI, 10 mM), which could effectively disrupt hydrophobic interactions, hydrogen bonding, and ionic interactions, respectively. It was observed that the addition of Tween 20 disrupted the cubic nanostructures, as lipid assembly is primarily driven by hydrophobic forces. DLS measurements on DMG-PEG nanoparticles and TA / DMG-PEG cubosomes also showed that the mean particle size of both of the nanoparticles dropped to 5 nm in Tween 20. The SAXS intensity of the cubosomes was reduced significantly by NaCI, but was not affected by urea, indicating that the ionic interactions destabilized the cubic nanostructures. pH stability experiments showed that cubosomes were stable in neutral and alkaline environments, but lost their structures in acidic conditions. Large aggregates were observed in the cubosomes suspended in pH 3 and pH 5 buffers, suggesting intensified interactions between TA and DMG-PEG in acidic conditions, which could originate from the enhanced hydrogen bonding between TA and PEG chains in DMG-PEG in acidic conditions.

[0298] Nanoparticle formation was also studied using ALC-0159 in place of DMG-PEG. No ordered phases were observed in the 1D SAXS pattern in the absence of TA. However, in the presence of TA (approximately 9:1 molar ratio of TA to ALC-1059), cubosomes were indicated by the diffraction peaks in 1D SAXS pattern by the relative positions in the spacing ratios of V2:V4:V6. Example 2: Preparation of phenolic compound / lipid / metal ion nanoparticles

[0299] For the preparation of metal ion-based liquid crystalline nanoparticles, an aqueous solution was first prepared by mixing the phenolic compound (TA or EGCG) (1.5 mg / mL) and metal ion sources (FeCl3'6H2O, C0CI2, NiCh, CUCI22H2O, ZnCh, ZrCk) (1 mg / mL) to yield the certain molar ratio of phenolic compound to metal ions (4:1 to 2:3 for TA-Fe111, 1:1 for TA-Co", TA-Ni", and TA-Cu", 2:1 for TA-Zrlv, and 1:1 for EGCG-Fe111). Then the aqueous phase and the lipid in ethanol phase (DMG-PEG for cubosome preparation, DOPC for hexosome preparation, and DOPE-PEG for micellarsome preparation, the concentration of all the lipids was 2 mM) were mixed in the designed cartridges of NanoAssemblr. The total flow rate ratio was set as 12 mL / min, and the flow rate ratio of aqueous phase to ethanol phase was 3:1. The flow rate ratio of the aqueous phase to ethanol phase was changed to 5:1 for EGCG-based LC-LNPs. The metal ion-based liquid crystalline nanoparticles could also be prepared by directly adding the metal ion / phenolic compound aqueous solution to the lipid ethanol solution in a 1.7 mL Eppendorf tube in a vortex mixer. After formulation, the liquid crystalline nanoparticles were purified through dialysis against MilliQ water using 10 kDa dialysis membrane for 24 h. Details of the formulations are collected in Table 3. Table 3 Sample Name Size (nm) Polydispersity Index 4:1 TA-Fe(lll) / DMG-PEG 22.7 ±7.0 0.178 2:1 TA-Fe(lll) / DMG-PEG 91.7 ±23.7 0.037 4:3 TA-Fe(lll) / DMG-PEG 123.5 ±42.0 0.126 1:1 TA-Fe(lll) / DMG-PEG 183.2 ±49.4 0.039 2:3 TA-Fe(lll) / DMG-PEG 335.7 ±64.6 0.355 TA / DOPC 120.8 ±43.9 0.153 TA-Fe(lll) / DOPC 136.3 ±66.8 0.179 TA / DOPE-PEG 76.6 ±19.5 0.071 TA-Fe(lll) / DOPE-PEG 117.9 ±28.3 0.019

[0300] When mixing TA and Fe111 with DMG-PEG, the assembled TA-FeIH / DMG-PEG liquid crystalline nanoparticles showed the phase as TA / DMG-PEG cubosomes (Figure 5). The ratio between phenolic compound and metal ions was found to influence the mean particle size and the degree of order of the MPN / DMG-PEG cubosomes. When keeping the concentration of TA and DMG-PEG constant, the mean particle size of the TA-FeIH / DMG-PEG cubosomes increased from 22.7 to 335.7 nm as the molar ratio of TA to Fe111 increased from 4:1 to 2:3 (Table 3). The 1:1 TA-FeHI / DMG-PEG cubosomes showed the characteristic ligand-to-metal charge transfer (LMCT) band of TA / FeHI MPN at ~565 nm, indicating the presence of MPN in the cubosomes.

[0301] SAXS diffraction patterns showed that more ordered cubic phases were formed in the TA-FeHI / DMG-PEG cubosomes prepared from 1:1 and 2:3 TA to FeIH, while at the lower concentration of FeIH (molar ratio of TA to FeIH of 4:1,2:1, and 4:3), the cubosomes showed only one diffraction peak in SAXS patterns (Figure 5).

[0302] More ordered cubic nanostructures were observed in the cryo-TEM images of the cubosomes assembled from 1:1 TA: FeIH (1:1 TA-FeHI / DMG-PEG) than 4:3 TA: FeIH (4:3 TA-FeIH / DMG-PEG), which were consistent with the SAXS results. NMR analysis of the TA-Fe / DMG-PEG cubosomes assembled from different molar ratios of TA to FeIH showed that the molar ratio of TA to DMG-PEG in the purified cubosomes was around 1.6:1, regardless of the feeding ratio of TA to FeIH. Therefore, it could be concluded that when keeping the concentration of TA and DMG-PEG constant, increased FeIH concentration led to a stronger network built by phenolic compounds and metal ions to penetrate the lipid bilayer structure and form the interconnected channel, and also led to an increase of the lattice parameter from 112.8 (4:1 TA: FeIH) to 117.4 A (1:1 TA: FeIH) (Table 4). Table 4 Sample name Ordered phase Lattice parameter (A) TA-Metal ion / DMG-PEG nanoparticles 4:1 TA:FeIH / DMG-PEG cubic 112.8 2:1 TA:FeHI / DMG-PEG cubic 112.8 4:3 TA:FeHI / DMG-PEG cubic 116.3 1:1 TA:FeHI / DMG-PEG cubic 117.4 2:3 TA:FeHI / DMG-PEG cubic 119.8 1:1 TA:Co" / DMG-PEG cubic 117.4 1:1 TA:Ni" / DMG-PEG cubic 115.5 1:1 TA:Cu" / DMG-PEG cubic 118.6 1:1 TA:Zrlv / DMG-PEG cubic 115.5 200:1 TA:Zn2+ cubic 113.5 50:1 TA:Zn2+ cubic 111.6 10:1 TA:Zn2+ cubic 119.6 2:1 TA:Zn2+ cubic 113.5 1:1 TA: Zn2+ cubic 111.6 200:1 TA:Fe3+ cubic 113.5 50:1 TA:Fe3+ cubic 113.5 10:1 TA:Fe3+ cubic 113.5 2:1 TA:Fe3+ cubic 113.5 1:1 TA:Fe3+ cubic 117.5 200:1 TA:Zr4+ cubic 111.6 50:1 TA:Zr4+ cubic 113.5 10:1 TA:Zr4+ cubic 113.5 2:1 TA:Zr4+ cubic 113.5 EGCG(-Metal ion) / DMG-PEG nanoparticles EGCG-DMG-PEG cubic 121.1 EGCG-Fe'"-DMG-PEG cubic 119.8 200:1 EGCG:Zn2+ cubic 119.6 50:1 EGCG:Zn2+ cubic 119.6 Table 4 Sample name Ordered phase Lattice parameter (A) 10:1 EGCG:Zn2+ cubic 119.6 200:1 EGCG:Fe3+ cubic 119.6 50:1 EGCG:Fe3+ cubic 119.6 10:1 EGCG:Fe3+ cubic 121.8 200:1 EGCG:Zr4+ cubic 119.6 50:1 EGCG:Zr4+ cubic 119.6 10:1 EGCG:Zr4+ cubic 117.5 DOPC-based nanoparticles TA / DOPC multilamellar 50.5 TA-Fe / DOPC hexagonal 63.0 DOPE-PEG-based nanoparticles TA / DOPE-PEG-2k Amorphous N / A TA-Fe / DOPE-PEG-2k micellar cubic 154.7 TA / DOPE-PEG-5k Amorphous N / A TA-Fe3+ / DOPE-PEG-5k cubic 151.4

[0303] SAXS patterns on the TA-FeIH / DMG-PEG (1:1 TA: FeIH) cubosomes in buffers of different pH indicated that the phase of TA-FeHI / DMG-PEG cubosomes remained stable from pH 3 to 9. Various nanoparticle formulations with different phenolic compounds (TA and epigallocatechin gallate (EGCG)) and metal ions (FeIH, Zrlv, Co", Ni", Cu", Zn") were able to form the same Im3m cubic phase as the TA-FeHI / DMG-PEG cubosomes (Figure 6). In contrast, Resveratrol / DMG-PEG displayed no ordered nanostructions while resveratrol-Fe3+ / DMG-PEG displayed cubic nanostructures (Figure 19).

[0304] While changing the building blocks from phenolic compound to phenolic compound plus metal ion did not influence the phase of the liquid crystalline nanoparticles, lipid type was found to be associated with the self-assembled nanostructures of the nanoparticles. Phospholipid (1,2-Dioleoyl-sn-glycero-3-phosphocholine, DOPC) and TA-FeIH (1:1 TA: FeIH) assembled into hexosomes, characterized by the spacing ratios of Vl: V3: V4 of the diffraction peaks in SAXS patterns and the cryo-TEM image, as well as the DLS size distributions (Figure 7, Table 3). Another type of PEG-lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DOPE-PEG), formed ~120 nm Fd3m micellarsomes after mixing with TA-FeIH (1:1 TA: FeIH) (Table 3), the micellar cubic phase of which were shown by the spacing ratios of V3:V8:V11 of the diffraction peaks in SAXS patterns and cryo-TEM image (Figure 8).

[0305] Nanoparticle formation with the cationic lipids EPC and DOTAP was also studied. As for the non-ionic lipids, in the absence of phenolic compound, no ordered phases were observed in the 1D SAXS pattern. However, in contrast to the non-ionic lipids examined, in the presence of TA (approximately 4.5:1 molar ratio of TA to lipid), and optionally metal ion (1:1 molar ratio of TA to Fe(lll)), no ordered phases were observed and a multi-lamellar structure was observed in the 1D SAXS.

[0306] It was also observed that the yield of liquid crystalline nanoparticles increased in the presence of metal ions (Fe(l 11)), at the same concentration of TA and lipid (DOPC or DMG-PEG). Table 5 collects the relevant synthesis data and the resulting particle concentration per pL. Table 5 Sample name Aqueous phase Ethanol phase Particle concentration ( / pL) TA / DOPC 800 pL 1.5 mg / mLTA 200 pL DOPC (1 mM) 6.14 x 107 TA-Fe"' / DOPC 800 pL 1.5 mg / mL TA + FeCh The molar ratio between TA and FeIH is 4:3 200 pL DOPC (1 mM) 2.13 x 108 TA / DMG-PEG 800 pL 1.5 mg / mLTA 200 pL DMG-PEG (2 mM) 3.33 x 107 TA-FeHI / DMG- PEG 800 pL 1.5 mg / mL TA + FeCh The molar ratio between TA and FeIH is 4:3 200 pL DMG-PEG (2 mM) 9.54 x 108 Example 3: Preparation of RNA-TA / DMG-PEG cubosomes

[0307] For the preparation of RNA-TA / DMG-PEG cubosomes, RNA was first added into TA aqueous solution and mixed for a few seconds on a vortex mixer to form the aqueous phase, which was then mixed with the DMG-PEG in ethanol phase in the designed cartridges of NanoAssemblr. The total flow rate ratio was set at 12 mL / min, and the flow rate ratio of aqueous phase to ethanol phase was 3:1. Specifically, for the preparation of siRNA-TA / DMG-PEG cubosomes, 30 pL 50 pM luciferase siRNA was mixed with 150 pL TA aqueous solution of 0, 5, 10, and 20 mg / mL to form the aqueous phase and then it was mixed with 60 pL DMG-PEG (4 mM) in the NanoAssemblr to afford a molar ratio between TA and DMG-PEG of 0:1,2:1,4:1 and 8:1, respectively.

[0308] For the preparation of mRNA-TA / DMG-PEG cubosomes, 20 pL mCherry mRNA (1 pg / pL) was mixed with 160 pL TA aqueous solution of 0, 10, 20, 40, and 60 mg / mL to form the aqueous phase which was then mixed with 60 pL DMG-PEG (4 mM) in the NanoAssemblr to afford a molar ratio between TA and DMG-PEG of 0:1,4:1,8:1, 16:1 and 24:1. After formulation, the cubosomes were purified through dialysis against MilliQ water using 10 kDa dialysis membrane for 24 h.

[0309] To measure the molar ratio of TA to DMG-PEG in the assembled TA / DMG-PEG cubosomes, TA-FeIH / DMG-PEG cubosomes, or mRNA-TA / DMG-PEG cubosomes cubosomes, the cubosomes were centrifuged at 2,000 g for 10 mins and then washed two times with MilliQ water. The cubosomes were then freeze-dried and redispersed in dimethylsulfoxide-d6 (DMSO-d6) for NMR measurements.

[0310] To study the role of TA on the RNA encapsulation and lipid phase transition, the mass ratio between DMG-PEG to RNA was fixed at 30:1 and the molar ratio between TA to DMG-PEG was varied from 0 to 8:1 and 24:1 for siRNA-cubosomes and mRNA-cubosomes, respectively. The formulations are summarised in Table 6. Table 6 Sample name Ordered phase Lattice parameter (A) siRNA-loaded TA / DMG-PEG nanoparticles 2:1 TA / DMG-PEG cubic 117.3 4:1 TA / DMG-PEG cubic 117.3 Table 6 Sample name Ordered phase Lattice parameter (A) 8:1 TA / DMG-PEG cubic 117.3 mRNA-loaded TA / DMG-PEG nanoparticles 8:1 TA / DMG-PEG cubic 119.2 16:1 TA / DMG-PEG cubic 117.3 24:1 TA / DMG-PEG cubic 119.2

[0311] Figure 9 shows cryo-TEM images of siRNA-loaded and mRNA-loaded TA / DMG-PEG cubosomes indicating that TA / DMG-PEG cubosomes could load RNA (siRNA and mRNA) without phase change.

[0312] Increasing the molar ratio of TA to DMG-PEG increased the RNA loading efficiency (Figure 10). Additionally, increasing the molar ratio of TA to DMG-PEG increased the mean particle size and degree of order of the RNA-cubosomes (Figures 11 and 12). Example 4: Stability and cytotoxicity

[0313] The stability of the TA / DMG-PEG cubosomes in cell medium and the cytotoxicity of the DMG-PEG nanoparticles and TA / DMG-PEG cubosomes on cells were examined.

[0314] PC3-luc2 cell viability by the cell proliferation kit II (XTT) assay: PC3-luc2 cells were seeded in a 96-well plate (Costar, Corning, USA) at a density of 10,000 cells per well in 100 pL RMPI supplied with 10% FBS overnight. Then, the culture media was removed and replaced with fresh media (for cell control) or fresh media containing the siRNA-TA / DMG-PEG cubosomes at a siRNA concentration of 20, 50, 100, and 200 nM for 24 h. After the treatment, the media was replaced with fresh media containing activated XTT (9 mL of 0.2 mg mL-1 XTT in complete RPMI could be activated by adding 22.5 pL of 0.6 mg mL-1 PMS in DPBS), and cells were incubated for 3 h. Finally, cells were screened on an Infinite M200 microplate reader (Tecan, Switzerland). Absorbance readings were measured at 475 nm and a reference wavelength of 675 nm was used. Cell viability was expressed as a percentage by normalizing the absorbance to untreated cells. All experiments were performed in triplicates, and data were presented as mean ± standard deviation.

[0315] HEK293T cell viability by the XTT assay. HEK293 cells were seeded in a 96-well plate at a density of 10,000 cells per well in 100 pL DMEM supplied with 10% FBS overnight. Then, the culture media was removed and replaced with fresh media (for cell control) or fresh media containing the mRNA-TA / DMG-PEG cubosomes at a mRNA concentration of 20, 50, 100, 200, and 300 ng for 24 h. After the treatment, the media was replaced with fresh media containing activated XTT (9 mL of 0.2 mg mL-1 XTT in complete DMEM could be activated by adding 22.5 pL of 0.6 mg mL-1 PMS in DPBS), and cells were incubated for 3 h. Finally, cells were screened on an Infinite M200 microplate reader. Absorbance readings were measured at 475 nm and a reference wavelength of 675 nm was used. Cell viability was expressed as a percentage by normalizing the absorbance to untreated cells. All experiments were performed in triplicates, and data were presented as mean ± standard deviation.

[0316] After incubating the TA / DMG-PEG cubosomes with complete cell medium for 24 h, the mean particle size of the cubosomes did not show large variances (<10 nm), indicating they were stable in the complete medium. Cell viability results showed that the DMG-PEG nanoparticles and TA / DMG-PEG cubosomes had negligible cytotoxicity on the PC3-luc2 (for siRNA transfection experiments) and HEK293T cells (for mRNA transfection experiments) at the standard RNA feeding concentrations (20-100 nM siRNA and 100 ng mRNA / 104 cells). Example 5: RNA transfection

[0317] Luciferase siRNA transfection assay: PC3-Luc2 were seeded in a 96-well white plate with transparent bottom (Costar, Corning, MA, USA) at a density of 10,000 cells per well in RPMI supplied with 10% FBS (complete medium) (100 pL) overnight. The siRNA-lipofectamine construct (siRNA complexed with lipofectamine RNAiMax transfection agent; positive control) was incubated with cells in serum-free RMPI medium, and siRNA-TA / DMG-PEG cubosomes were incubated with cells in complete medium at the siRNA final concentration of 20 and 50 nM for 24 h. The medium was removed and replaced with complete RPMI medium. After 48 h, the percentage of gene downregulation was evaluated using a ONE-Glo Luciferase Kit (Promega) following the manufacturer’s protocol; luminescence readings were taken on an Infinite M200 microplate reader.

[0318] mCherry mRNA transfection assay: HEK293 cells were seeded in a 24-well plate at a density of 100,000 cells per well in 1 mL DMEM supplied with 10% FBS overnight. The mRNA-lipofectamine construct (mRNA complexed with lipofectamine MessengerMAX transfection agent; positive control) and mRNA-TA / DMG-PEG cubosomes were incubated with cells in serum-free DMEM medium with 500 ng mRNA for 6 h. The medium was removed and replaced with complete medium. After 18 h, The culture medium was then removed and cells were washed twice with PBS and detached with trypsin. The cells expressing mCherry were analyzed using a BD Accuri C6 flow cytometer.

[0319] Luciferase siRNA-loaded TA / DMG-PEG (siRNA-TA / DMG-PEG) and mCherry mRNA-loaded TA / DMG-PEG (mRNA-TA / DMG-PEG) cubosomes showed successful RNA transfection on PC3-luc2 and HEK293T cells, respectively, while DMG-PEG lipid alone could not encapsulate RNA and transfect the cells effectively.

[0320] The siRNA downregulation efficiency of the siRNA-loaded TA / DMG-PEG cubosomes was enhanced as the TA / DMG-PEG molar ratio increased, at the siRNA concentration of 20 and 50 nM (Figure 13), the trend of which was similar to the trend of siRNA loading efficiency (Figure 10).

[0321] TA / DMG-PEG cubosomes assembled from 24:1 TA: DMG-PEG achieved 90% mCherry mRNA encapsulation efficiency and showed comparable transfection efficiency with the positive control lipofectamine MessengerMax, confirmed by flow cytometry (Figures 14 and 15) and confocal microscope images.

[0322] Intracellular trafficking of the siRNA-Cyanine5 (cy5)-TA / DMG-PEG cubosomes in the PC3-luc cells showed that the loaded siRNA was not trapped and degraded in the endo / lysosomes (Figure 16), with a Pearson correlation coefficient (PCC) of the fluorescence from green and red channels of 0.26 ± 0.03. The high endosomal escape efficiency of the RNA-TA / DMG-PEG cubosomes may account for the high transfection efficiency of the RNA-TA / DMG-PEG cubosomes. Example 6: General characterization techniques Particle size measurements

[0323] Particle size measurements of the nanoparticles were performed on a Zetasizer Nano-ZS (Malvern Instruments, UK) instrument. Zeta-potential measurements of the nanoparticles were performed at pH 7.4 in phosphate buffer (5 mM) using a Zetasizer Nano-ZS (Malvern Instruments, UK). Nanoparticle concentrations

[0324] Nanoparticle concentrations were measured on a Malvern NanoSight NS300 instrument fitted with a 405 nm laser (65 mW output). NMR spectroscopy

[0325] Nuclear Magnetic Resonance (NMR) spectroscopy was conducted on a Varian Unity 400 MHz spectrometer at room temperature. Samples were dissolved in 0.65 mL DMSO-d6. UV-visible

[0326] UV-visible absorption measurements were performed on an Analytik Jena SPECORD 250 PL instrument. Synchrotron small angle X-ray scattering (SAXS)

[0327] Synchrotron SAXS experiments were conducted at the SAXS / WAXS beamline at the Australian Synchrotron, ANSTO. A wavelength of k = 1.128 A (11.0 keV) and a camera length of 1.6 m were employed. Samples were first loaded into a UV-clear 96 well plate (Greiner Bio-One, Germany) and screened at an exposure time of 2s. A Dectris-Pilatus 1 M detector was used to record the 2D X-ray diffraction images, which were subsequently integrated into 1D diffraction plots using the ScatterBrain (V 1.230) software. The scattering vector, q, is defined by q= 4irsin0 / A where 0 is the scattering angle and A is the wavelength. Subsequently, d spacing can be calculated using Bragg’s law, d=2n7q. The lattice parameters (LP) of the mesophases can be calculated according to LP = d(h2 + k2 + Z2)1 / 2 for cubic phases, LP = (2 / V3) * d(h2 +k2 + hk)1 / 2 for hexagonal phase, where (h,k,l) are Miller indices assigned to the mesophase. Cryo-TEM

[0328] An FEI Vitrobot was used to freeze the nanoparticle samples for cryo-TEM. The samples were dripped on carbon mesh grids and frozen rapidly in liquid ethane with a humidity of 95%, blot force of 1, and blot time of 4s. The samples were imaged using an FEI Tecnai F30 transmission electron microscope operating at 300 kV with a Gatan 626 cryo-transfer specimen holder, at a defocus level of -1 pm. Intracellular colocalization by confocal laser scanning microscopy

[0329] PC3-luc2 cells were seeded in the 8-well Lab-Tek-Chamber slide at a density of 40,000 cells per well in 400 pL of RPMI supplied with 10% FBS overnight. Fluorescently (DiD) labelled TA / DMG-PEG cubosomes were incubated with cells for 4 h. The samples were then gently washed twice with DPBS to remove excess particles, and LysoTracker Green was added to the medium with a final concentration of 50 nM and incubated for 1 h following the supplier’s protocol for endo / lysosome staining. Cells were gently washed three times with DPBS and incubated with Hoechst 33342 (1 pg / mL) for 10 min to stain the nucleus. Finally, the cells were live-imaged using a Nikon A1R confocal microscope with a 40x water immersion objective. PCC and colour scatter plots were obtained from WCIF Imaged software. Evaluation of cell viability by XTT assay

[0330] XTT assays were performed on MDA-MB-231 and MDA-MB-468 cells with passages less than 40 to assess the cell toxicity of free DOX, and EGCG-Fe3+ / DMG-PEG cubosomes before and after loading with DOX. The cells were seeded on a 96-well plate (Costar, Corning, USA) at a density of 10,000 cells per well in DMEM supplied with 10% FBS (100 pL) overnight. Then, the culture media was removed and replaced with fresh media containing free DOX or DOX@EGCG-Fe3+ / DMG-PEG cubosomes with DOX dosages of 0.025, 0.05, 0.1,0.25, 0.5, and 1 pg per well. The same amounts of EGCG-Fe3+ / DMG-PEG cubosomes without DOX loading were added to the cells as a negative control. The cells were further incubated for 24 h.

[0331] XTT assays were performed on HEK 293T cells with passages less than 20 to assess the cell toxicity of mRNA@EGCG-Zn2+ / DMG-PEG cubosomes. The cells were seeded on a 96-well plate (Costar, Corning, USA) at a density of 10,000 cells per well in DMEM supplied with 10% FBS (100 pL) overnight. Then, the culture media was removed and replaced with opti-MEM media (for cell control) or opti-MEM media containing the mRNA@EGCG-Zn2+ / DMG-PEG cubosomes at final mRNA concentrations of 0.2, 0.5, 1, 1.5, and 2 pg mL-1 for 6 h. After the treatment, the media was replaced with fresh media (DMEM supplied with 10% FBS) and the cells were further incubated for 18 h.

[0332] After incubation, fresh media containing activated XTT (9 mL of 0.2 mg mL-1 XTT in complete DMEM media was activated by adding 22.5 pL of 0.6 mg mL-1 N-methyl dibenzopyrazine methyl sulfate in DPBS) and cells were incubated for 3 h. Finally, cells were screened on an Infinite M200 microplate reader (Tecan, Switzerland). Absorbance readings were measured at 475 nm and a reference wavelength of 675 nm was used. Cell viability was expressed as a percentage by normalizing the absorbance to untreated cells. All experiments were performed in triplicates, and data are presented as mean ± SD. Catalytic activity studies

[0333] The catalytic activity of HRP-loaded cubosomes was evaluated by the H2O2-amplex red colorimetric reaction. Briefly, free HRP, cubosomes, or HRP@TA / DMG-PEG cubosomes were mixed with MOPS solution (10 mM, pH 7.4) containing H2O2 (20 mM) and amplex red (1 mg mL-1). Changes in the absorbance of the red oxidation product (resorufin) at 560 nm were monitored by UV-vis spectroscopy. The kinetic behaviour of HRP was studied by monitoring the absorbance at 560 nm at 10 s intervals by UV-vis spectroscopy. mRNA transfection study

[0334] HEK 293T cells with passages less than 20 were seeded on a 48-well plate at a density of 80,000 cells per well in DMEM supplied with 10% FBS (0.5 mL) overnight. Cells were incubated with mCherry mRNA (mRNA), mCherry mRNA-lipofectamine construct (Lipofectamine-mRNA), EGCG-mRNA mixture (EGCG-mRNA), or mRNA-loaded cubosomes (mRNA@EGCG-Zn2+ / DMG-PEG cubosomes) in opti-MEM media for 6 h, with a final mRNA concentration of 2 pg mL-1. The medium was removed and replaced with complete DMEM medium. After 18 h, the culture media was removed and cells were washed twice with PBS and detached using trypsin. The cells expressing mCherry were analyzed by flow cytometry (LSRFortessa, BD Bioscience). Example 7: Molecular dynamics (MD): model information

[0335] All-atom models were constructed to explore the spontaneous association of TA / lipid mixtures in water and to examine how the resultant supramolecular assemblies are influenced by three factors as summarised in Figure 30A, (i) the ratio of TA to lipid, (ii) the amount of hydration, and (iii) the length of the PEG chains. Each simulation contained a fixed number of particles with a specific TA / lipid composition (Table 7). The amount of water in the system was used to emulate a particular timepoint during the mesoscale self-assembly process. To represent the final “dry” structures formed experimentally, models were created under low hydration conditions that contain 15 water molecules per TA / lipid molecule (93.8 mol% water). This molar fraction of water is an approximate estimate of the amount of water in the 2:1 TA:DMG-PEG (10 mM) cubic assembly that has a final experimental TA-to-DMG-PEG molar ratio of 2.7:1. Systems modelled with 150 water molecules per TA / lipid molecule (99.3 mol% water) were used to represent moderately hydrated transient structures observed in the intermediary stages of assembly. Full hydration scenarios, with 900 water molecules per TA / lipid molecule (99.9 mol% water), were used to reflect TA / lipid seeds present in the initial (dilute) stages of self-assembly. Systems modelled with DMG-PEG400 (9 PEG monomers) were used to study the general effects of water and TA concentration, whereas the simulations with DMG-PEG2000 (45 PEG monomers) provided specific insights into a composition that is fully consistent with the 2:1 TA:DMG-PEG (10 mM) cubic assembly that has a final TA-to-DMG-PEG molar ratio of 2.7:1. This approach allowed for a systematic investigation of the fundamental interactions responsible for the formation of the solid-state-like, assembled materials. By varying the lipid-PEG chain length from 9 to 45 monomers, the influence of PEG chain length on the final structures was also assessed. Table 7 TA:lipid ratio Molecules per cell Number of atoms Cubic cell side length (nm) TA lipid H2O Shrinking3 Coolingb Ambient0 TA-lipid seeds in initial dilute stages (full hydration: 900 water per lipid / TA) 0.33Afh 25 75 90,000 286,500 — — 14.18 VAfh 50 50 90,000 286,800 - - 14.19 3Afh 75 25 90,000 287,100 - - 14.20 Table 7 TA:lipid ratio Molecules per cell Number of atoms Cubic cell side length (nm) TA lipid H2O Shrinking3 Coolingb Ambient0 Transient structures in intermediary stages (moderate hydration: 150 water per lipid / TA) 0:1mh 0 50 7,500 30,600 14.0-7.0 7.47 (0.11) 6.71 0.02:1""' 1 50 7,650 31,224 14.0-7.5 7.55 (0.03) 6.76 0.33:1""' 15 50 9,750 39,960 14.0-9.0 8.48 (0.24) 7.35 1 .^mh 50 50 15,000 61,800 17.0-9.5 9.41 (0.03) 8.52 3:1mh 150 50 30,000 124,200 20.0-11.5 11.77 (0.06) 10.77 2 135 50 27,750 127,440 25.0-12.0 11.87 (0.03) 10.84 “Dry” assembled structures in final stages (low hydration: 15 water per lipid / TA) 0:1 0 50 750 10,350 14.0-5.0 5.16 (0.04) 4.65 0.02:1 1 50 765 10,569 14.0-5.0 5.20 (0.05) 4.69 0.33:1 15 50 975 13,635 14.0-5.0 5.54 (0.13) 5.13 1:1 50 50 1,500 21,300 17.0-6.0 6.36 (0.08) 5.99 3:1 150 50 3,000 43,200 20.0-9.0 8.78 (0.12) 7.64 2.7:1* 135 50 2,775 52,515 25.0-9.0 8.77 (0.10) 8.09 alnitial and final cell lengths for 800K NVTshrinking, with sides decreasing at -0.1 nm ns-1. bAverage cell side length (± SD) for NPTcooling from 500-300 K at -0.5 K ns-1. Taken over the initial 200 ps at 500 K and 1 atm (20 datapoints, one every 10 ps), illustrating that cell lengths deviate less than 0.55 nm when ensembles first switching from NVTto NPT. c Ave rage cell side length for ambient NPTsimulations (303.15 K, 1 atm). Taken over the final, equilibrated 60 ns (6,000 datapoints, one every 10 ps), with SDs below 0.01 nm. *Systems containing DMG-PEG2000. All other systems are DMG-PEG400.

[0336] The Packmol program (version 20.14.2) (Sonora et al., J. Chern. Inf. Model. 2021; 13:777-785; Martinez et al., J. Comput. Chern. 2009; 30:2157-2164) was used to prepare models for MD. Lipid and TA molecules were randomly distributed with intermolecular distances of at least 1 nm. Water molecules were added at distances greater than 0.2 nm from other molecular components. The coordinates for DMG-PEG400 and DMG-PEG2000, used during packing, were generated by the CHARMM-GUI web server (Park et al., J. Chern. Inf. Model. 2021; 61:5192-5202) in a TT-helical conformation along the principal molecular axis. TA was constructed using the VMD 1.9.3 software (Humphrey et al., J. Mol. Graph, 1996; 14:33-38) with an initial conformation having galloyl arms planar to the central glucose moiety (Figure 30B). These structural features were artificially introduced to avoid steric clashes and excessively large periodic box sizes during model generation, and they dissipated rapidly into more energetically favorable conformations within the first few picoseconds of MD equilibration.

[0337] In total, 15 atomic models with explicit solvent were constructed and labeled based on TAJipid molar ratios ranging from 0:1 to 3:1 (Figure 30A and Table 7). To distinguish between systems with different hydration levels, subscripts fh (full hydration with 900 water molecules per TA / lipid) and mh (moderate hydration with 150 water molecules per TA / lipid) are used, whereas dehydrated models with 15 water molecules per TA / lipid have no subscript. The two systems simulated with DMG-PEG2000 that most closely represent the composition of 2:1 TA / DMG-PEG cubosomes have been labeled as 2.7:1 and 2.7:1mh. MD: Force field parameters, equilibration protocol, and simulation settings

[0338] All-atom classical MD simulations were performed using the graphical processing unit (GPU) accelerated NAMD (version 2.14) program (Phillips et al., J. Chern. Phys. 2020; 153:044130). The CHARMM36 lipid force field (Klauda et al., Journal of Physical Chemistry B, 2010; 114:7830-7843) CHARMM general force field (CGenFF) (version 4.0) (Vanommeslaeghe et al., J. Comput. Chern. 2010; 31: 671 -690), CHARMM carbohydrate force field (9), and CHARMM-modified version of the TIP3P water model (Jorgensen et al., J. Chern. Phys. 1983; 79:926-935) were all employed. In addition, parameters adapted from the recently improved CHARMM lignin parameter set of Vermaas et al. Green Chern, 2019; 21:109-122 were used for galloyl groups and galloyl-carbohydrate interlinkages.

[0339] Models were equilibrated from disordered mixtures to prevent assemblies from becoming kinetically trapped due to packing bias. Without a priori knowledge of the assembled material configurations, we employed a gentle equilibration protocol to ensure the final structures accurately represent equilibrium self-assembled structures. For the low and moderately hydrated systems, initial equilibration was performed in the canonical (A / V7) ensemble using cubic unit cells roughly 2-3 times larger than the target sizes (Table 7). The periodic unit cell volume was gradually compressed using 50-160 individual consecutive 1 ns NVTsimulations at 800 K, reducing the cell dimensions by 0.1 nm with each run until the atomic density became approximately uniform throughout. The high temperature allowed molecules sufficient kinetic energy to randomly mix during the unit cell compression process. Each simulation was minimized for 10,000 steps before running MD for 1,000,000 steps (1 ns). Once the target box size was reached (table S1), atomic velocities were reset using a Boltzmann-weighted ensemble at 500 K and NVTequilibration continued for 1 ns. For all NVTsimulations, velocities were reassigned to 800 or 500 K every 500 steps. Subsequently, the isothermal-isobaric (NPT) ensemble was used to equilibrate the pressure to 1 atm while slowly cooling the temperature from 500 to 300 K at a rate of 0.1 K every 0.2 ns. Following the initial shrinking and cooling equilibrations, NPTsimulations were conducted under ambient conditions of 1 atm and 303.15 K for 160 ns to collect data for analysis. For the highly hydrated systems, constituent molecules were placed in a 14 x 14x14 nm3 box and solvated with a water density of approximately 1 g cm-3. Production simulations began directly to equilibrate the assemblies and collect data, equivalent to the final step for the low and moderately hydrated models, under ambient NPTconditions of 1 atm and 303.15 K for 160 ns.

[0340] All NVTequilibration was performed with an integration timestep of 1.0 fs, and all NPTsimulations were performed with an integration timestep of 2.0 fs. Nonbonded interactions were managed with a cutoff distance of 1.2 nm, a switching distance of 1.0 nm, and a pair list distance of 1.6 nm, updated every 10 steps. Electrostatics were handled using the Particle Mesh Ewald (PME) method (Darden et al., J. Chern. Phys. 1993; 98:10089-10092) with an interpolation order of 6 and a grid spacing of 0.1 nm. Rigid bonds for all hydrogen-involving bonds were enforced using the ShakeH algorithm of NAMD. Temperature was maintained using Langevin dynamics with a damping coefficient of 1.0 ps-1. For NPTsimulations, isotropic pressure coupling was controlled using the Nose-Hoover Langevin piston method (Martyna et al., J. Chern Phys. 1994; 101:4177-4189; Feller et al., J. Chem. Phys. 1995; 103:4613-4621), targeting a pressure of 1.01325 bar (1 atm) with a piston oscillation period of 50 fs and a piston decay time of 25 fs. Pressure was calculated using a hydrogen-group-based pseudo-molecular virial and kinetic energy approach, as this method results in less fluctuations and is required when using rigid ShakeH bonds.

[0341] The final 60 ns of the ambient NPTsimulations were used for data analysis after discarding the first 100 ns for equilibration. Frames were saved to a trajectory every 10 ps. Analysis focused on the solid-state low hydration systems with varying TA concentrations (Figure 26) to best reflect the experimentally characterized systems. The VMD 1.9.3 software (Humphrey et al., J. Mol. Graph. 1996; 14:33-38) was used for visualization, atomic rendering, calculating 1D (Figure 26A) and 2D mass densities via the Densitycalculator TCL code (Wang et al., Compute. Phys. Common. 2021; 266:108032). The MDAnalysis (Michaud-Agrawal et al., J. Comput. Chem. 2011; 32:3219-2327) and MDVWhole (Bruininks et aL, J. Chern. Inf. Model. 2023; 63:34483452) python packages were used for trajectory post-processing and analysis. Example 8: Structures of phenolic compound / lipid nanoparticles

[0342] P-LCNPs with different nanostructures were successfully assembled from different lipids with TA or TA-Fe3+ MPN using the methods described herein. The lipids used for engineering P-LCNPs include: polyethylene glycol 2000-ester-stearic acid (PEG-SA), polyethylene glycol 2000-ester-myristic acid (PEG-MA), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), polyethylene glycol-carbamate-1,2-dimyristoyl-sn-glycerol (PEG-c-DMG), 1,2-dimyristoyl-sn-glycero-3-succinyl-N-polysarcosine-25 (pSar-DMG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (ammonium salt) (DOPE-PEG 5000), methoxy polyethylene glycol 20k-distearoyl phosphatidyl ethanolamine (DSPE-PEG 20k), cholesterol-polyethylene glycol 5000 (CLS-PEG 5000), and cholesterol-polyethylene glycol 20k (CLS-PEG 20k).

[0343] SAXS diffraction results of P-LCNPs prepared from ALC-0159 or PEG-C-DMG with TA or TA-Fe3+ is shown in Table 8. The 1D diffraction SAXS patterns of P-LCNPs prepared from PEG-SA or PEG-MA with TA or TA-Fe3+ is shown in Figures 20 and 21, respectively. Table 8 Sample name Ordered phase Lattice parameter (A) ALC-0159-based cubosomes TA / ALC-0159 cubic 108.4 TA-Fe3+ / ALC-0159 cubic 106.7 PEG-c-DMG-based cubosomes TA / PEG-c-DMG cubic 122.3 TA-Fe3+ / PEG-c-DMG cubic 124.6

[0344] Exemplary particle size for P-LCNPs assembled using different lipids with TA or TA-Fe3+ MPN are shown in Table 9. Table 9 Sample Name Size (nm) TA / DSPE-PEG 20k 175.1 ±66.4 TA-Fe3+ / DSPE-PEG 20k 145.6 ±55.8 TA / CLS-PEG 5000 264.5 ±90.5 TA-Fe3+ / CLS-PEG 5000 161.9 ±44.5 TA / CLS-PEG 20k 239.6 ±64.5 TA-Fe3+ / CLS-PEG 20k 445.5 ±116.8 catechin-Zr4+ / DMG-PEG 425.1 ±118.2 Example 9: Preparation of cargo-loaded phenolic LC-LNPs

[0345] To prepare DOX@EGCG-Fe3+ / DMG-PEG cubosomes, 5 x 107 pL-1 EGCG-Fe3+ / DMG-PEG cubosomes (400 pL, EGCG-to-Fe3+ molar ratio was 10:1) were incubated with DOX aqueous solution (20 pL, 1 pg pL—1) in an Eppendorf thermomixer at 4 °C and 600 rpm for 2 h. The DOX-loaded cubosomes were then washed with Milli-Q water by centrifugation (1000 g, 5 min) and redispersed in Milli-Q water for cell viability experiments.

[0346] To prepare PLA@TA / DMG-PEG cubosomes, TA (1.5 mg mL-1 in Milli-Q water) was mixed with PLA (1 mg mL-1 in Milli-Q water) at TA-to-PLA mass ratios of 50:1,20:1, and 5:1. Then, the TA-PLA suspension was mixed with DMG-PEG (2 mM in ethanol) in the NanoAssemblr microfluidic device. The total flow rate ratio was set as 12 mL min-1, and the flow rate ratio of aqueous phase to ethanol phase was set as 4:1.

[0347] To prepare chitosan@TA / DMG-PEG cubosomes, TA (1.5 mg mL-1 in Milli-Q water) was mixed with chitosan (1 mg mL-1 in 1% acetic acid) at TA-to-chitosan mass ratios of 100:1,50:1,20:1, and 5:1. Then, the TA-chitosan suspension was mixed with DMG-PEG (2 mM in ethanol) in the NanoAssemblr microfluidic device. The total flow rate ratio was set as 12 mL min-1 and the flow rate ratio of the aqueous phase to ethanol phase was set as 4:1.

[0348] To prepare HRP@TA / DMG-PEG cubosomes, 5 x 107 pL-1 TA / DMG-PEG cubosomes (1 mL) (1:1 TA:DMG-PEG, 10 mM DMG-PEG) were incubated with HRP aqueous solution (50 pL, 1 pg pL-1) in an Eppendorf thermomixer at 4 °C and 600 rpm for 1 h. The HRP-loaded cubosomes were then washed with Milli-Q water by centrifugation (1000 g, 3 min) and redispersed in Milli-Q water for catalysis experiments.

[0349] mRNA@EGCG-Zn2+ / DMG-PEG cubosomes were prepared by incubating 2 x 108 pL-1 EGCG-Zn2+ / DMG-PEG cubosomes (100 pL, molar ratio between EGCG and Zn2+was 10:1) with mCherry mRNA (4 pL, 1 pg pL-1) dispersed in EGCG (100 pL, 3 mg mL-1) in an Eppendorf thermomixer at 4 °C and 600 rpm for 3 h. The samples were then purified by dialysis against Milli-Q water using a dialysis membrane (Slide-A-Lyzer MINI Dialysis Devices, 20K molecular weight cutoff) at 4 °C overnight for in vitro mRNA transfection experiments. Cy5-GFP mRNA was used for mRNA encapsulation measurements. The cubosomes were centrifuged at 6000 g for 10 min after mRNA loading and the supernatant was taken out for fluorescence intensity measurements. The encapsulation efficiency was calculated based on the standard curve of the fluorescence intensity of cy5-mRNA.

[0350] SAXS diffraction results of cargo-loaded P-LCNPs are shown in Table 10. Table 10 Sample name Ordered phase Lattice parameter (A) Cargo-loaded cubosomes DOX@EGCG-Fe3+ / DMG-PEG cubic 127.0 HRP@TA / DMG-PEG cubic 113.5 mRNA@EGCG-Zn2+ / DMG-PEG cubic 121.8 PLA@TA / DMG-PEG cubosomes (different mass ratios) 50:1 TA:PLA cubic 119.6 20:1 TA:PLA cubic 117.5 5:1 TA:PLA cubic 117.5 chitosan@TA / DMG-PEG cubosomes (different mass ratios) 100:1 TA:chitosan cubic 113.5 50:1 TA:chitosan cubic 113.5 20:1 TA:chitosan cubic 113.5 5:1 TA:chitosan cubic 113.5 Example 10: Molecular insights into the assembly of phenolic cubosomes

[0351] Molecular Dynamics (MD) simulations were employed to understand the role of the polyphenol and lipid components on the formation of the experimentally observed cubosome systems (Figure 30 and as described in Example 7). All-atom models were constructed to explore the spontaneous association of TA / lipid mixtures in water and to examine how the resultant supramolecular assemblies are influenced by the (i) TAJipid ratio, (ii) amount of hydration, or (iii) PEG chain length, as summarised in Figure 30 and Table 7. Representative structures with varying concentrations of TA, DMG-PEG, and water were constructed and simulated to equilibrium. Figure 26 show exemplar structures of solid-state-like three-dimensional (3D) assemblies with different ratios of components of the assemblies. The results indicate that higher TA concentrations instigate transition of the lipidic mesophase from lamellar bilayers to micelles (Figure 26A). The concentration profiles of the simulated 3D networks showed that DMG-PEG provided interfacial boundaries between TA and water (Figure 26A). Figure 30B shows the all-atom structure of the experimentally consistent network composition (molar ratio between TA and DMG-PEG was 2.7:1) and highlights the cubic porous periodic network observed experimentally. The ability of the amphipathic DMG-PEG to bind to both aqueous and TA media (via hydrophilic region PEG) and form lipid micelles (via hydrophobic region DMG) enabled the formation of these interfacial cubic systems, as evidenced by density profiles (Figure 26C) and specific interactions between the functional groups of the components (Figure 26D). The hydrophilic interactions, i.e., hydrogen bonding between PEG, TA, and water, are the major contributors to the formation of the observed structures (Figure 26D). The water molecules trapped in persistent positions are mostly within the TA scaffold, whereas dynamic water molecules that move within channels are mostly within the PEG region. Example 11: Changing the building blocks of phenolic LC-LNPs

[0352] The MD simulations revealed that the nanostructures of phenolic LC-LNPs are influenced by the interactions between polyphenols and lipids, offering the possibility of tuning the nanostructure by altering the assembly building blocks. Changing DMG-PEG to other types of PEG-conjugated di-tetradecyl lipids—i.e., 2-(PEG-2000)-N,N-ditetradecylacetamide (ALC-0159) and PEG-2000-carbamate-1,2-dimyristoyl-sn-glycerol (PEG-c-DMG)—resulted in the formation of Im3m cubosomes with different lattice parameters (108 and 122 A, respectively), after mixing with TA at a TA-to-lipid molar ratio of 1.8:1 (Figure 27A, Figure 22, Table 8). Further changing the lipid type to PEG-conjugated dioleoyl lipids—i.e., 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxyPEG-5000)] (DOPE-PEG) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)—led to the formation of liposomes with multilamellar nanostructures, after mixing with TA at a TA-to-lipid molar ratio of 1.8:1 (Figure 27A, Figure 23). These transitions could be due to the limited capabilities of TA to modulate the mesophases of lipids with long carbon chains into the micelle structures.

[0353] Incorporating metal ions (Fe3+) as the third building block component led to the formation of metal-phenolic network (MPN) in the LC-LNPs (Figure 27D), which had negligible influence on the phases and lattice parameters of DMG-PEG-, ALC-0159-, and PEG-c-DMG-based cubosomes (Figure 27A, C, Figure 22, Figure 17). However, a structural change was observed in DOPE-PEG- and DOPC-based LC-LNPs, from lamellar phase to cubic or hexagonal phase, respectively (Figure 27A, Figure 23). This could be due to the ability of TA-Fe3+ complex to more effectively modulate the mesophase of dioleoyl lipids from lamellar to micelle structures compared to free TA. Although changes in MPN composition, in terms of polyphenol (TA and epigallocatechin gallate (EGCG)), metal ion (Zn2+, Fe3+, and Zr4+), and the molar ratio between polyphenol and metal ions (from 200:1 to 1:1) did not influence the nanostructure of DMG-PEG-based cubosomes (Figure 27B, Figure 17, Figure 18, Table 2), the incorporation of metal ions increased the stability of the cubosomes under acid conditions (Figure 27E). Without being bound by theory, the inventors believe this increased stability is possibly due to the counterbalanced effects of reduced MPN coordination and enhanced tt-tt interactions of protonated TA (pKa ~6) in acid conditions. Example 12: Encapsulating cargos into phenolic LC-LNPs

[0354] Polyphenols display multiple interactions with diverse molecules through coordination, hydrogen bonding, it interactions, hydrophobic interactions, and electrostatic interactions. Accordingly, we evaluated the ability of the phenolic LC-LNPs for cargo loading (Figure 28A). The small-molecule anticancer drug doxorubicin (DOX) was loaded into the EGCG-Fe3+ / DMG-PEG cubosomes with up to 94% loading efficiency (data not shown), due to the affinity of DOX for both polyphenols and metal ions. The cubosomes showed minimal size changes after postloading of DOX (data not shown). Furthermore, the DOX-loaded EGCG-Fe3+ / DMG-PEG cubosomes (D0X@EGCG-Fe3+ / DMG-PEG cubosomes) showed comparable cytotoxicity with free DOX to breast cancer cells (MDA-MB-468) (Figure 28C) and MDA-MB-231 cells (data not shown), whereas the cubosomes without DOX loading exhibited negligible cytotoxicity (data not shown). Diverse types of biomolecules—including polypeptide (poly-L-arginine (PLA)), protein (horseradish peroxidase (HRP)), polysaccharide (chitosan), nucleic acid (mCherry mRNA)—were also incorporated into the phenolic LC-LNPs without disrupting the cubic nanostructure (Figure 28B). Variance in the doping ratio between TA and PLA or chitosan influenced the size of assembled cubosomes, which was tunable and ranged between 146 to 423 nm (data not shown). Furthermore, the PLA- and chitosan-loaded TA-based cubosomes (PLA@TA / DMG-PEG and chitosan@TA / DMG-PEG cubosomes) exhibited surface charge reversal properties, changing from negative or neutral (at pH 7) to positive (at pH 4) (Figure 28D). The TA / DMG-PEG cubosomes also enabled HRP loading with negligible size change (data not shown). The HRP-loaded TA-based cubosomes (HRP@TA / DMG-PEG cubosomes) showed comparable catalytic activity to free HRP in the oxidation of amplex red in the presence of H2O2 (Figure 28E). Postloading mRNA into cubosomes was achieved by incubating the EGCG-Zn2+ / DMG-PEG cubosomes with mRNA dispersed in EGCG solution. Moreover, the addition of EGCG during postloading enhanced the mRNA loading efficiency (data not shown). Without being bound by theory, the inventors believe that this may be due to the interactions between EGCG and phosphate backbone of mRNA via hydrogen bonds. The cubosomes showed negligible size change after mRNA loading (data not shown), and the mRNA-loaded cubosomes (mRNA@EGCG-Zn2+ / DMG-PEG cubosomes) showed ~75% transfection efficiency with negligible cytotoxicity (Figure 28F). Example 13: Stability of phenolic LC-LNPs

[0355] To evaluate the stability of the phenolic LC-LNPs under different conditions, the NPs were incubated in glycine-HCI (20 mM, pH 3.0), acetate acid buffer (20 mM, pH 5.0), phosphate buffer (20 mM, pH 7.0), MOPS buffer (20 mM, pH 9.0), or Milli-Q water for the desired times. To determine the possible driving forces for the assembly of phenolic LC-LNPs, the NPs were incubated in urea (50 mM), Tween 20 (50 mM), or NaCI (10 mM) for 24 h.

[0356] TA / DMG-PEG cubosomes disassembled in Tween 20 but remained stable in NaCI and urea solution (Figure 29), suggesting that the dominant interactions involved in the NP assembly are hydrophobic interactions. The low structural stability of cubosomes prepared using conventional formulations is a key concern for their medical applications. In contrast, the TA / DMG-PEG cubosomes prepared herein remain stable in aqueous environment for at least one year at room temperature and 4 °C without changes in size or structure (Figure 25, Table 11). Table 11 Sample name Ordered phase Lattice parameter (A) TA / DMG-PEG nanoparticles 10:1 TA:DMG-PEG (2 mM) at 4 °C for 1 year cubic 119.6 10:1 TA:DMG-PEG (2 mM) at 22 °C for 1 year cubic 117.5

Claims

1. A lipid-based nanoparticle comprising one or more lipids and one or more phenolic compounds.

2. The lipid-based nanoparticle according to claim 1, wherein the one or more phenolic compounds has a molecular weight from about 110 to about 3,000 Daltons.

3. The lipid-based nanoparticle according to claim 1 or 2, wherein the one or more phenolic compounds are partially or fully soluble in an aqueous solution, such as water.

4. The lipid-based nanoparticle according to claim 1 or 2, wherein the one or more phenolic compounds are partially or fully soluble in a non-aqueous solution, such as an alcohol, preferably ethanol.

5. The lipid-based nanoparticle according to any one of the preceding claims, wherein the one or more phenolic compounds comprise one or more selected from:- one or more 3,4,5-trihydroxyphenyl groups;- three or more ring-bonded hydroxy groups; and- one or more galloyl groups, optionally with one or more catechol groups6. The lipid-based nanoparticle according to claim 1 or claim 2, wherein the one or more phenolic compounds comprise one or more of apigenin, catechin, curcumin, luteolin, quercetin, gossypol, kaempferol, genistein, daidzein, fisetin, myricetin, naringenin, hesperetin, delphinidin, cyanidin, procyanidin, chalconaringenin, phloretin, tannic acid, (-)-epigallocatechin-3-gallate, gallic acid, ellagic acid, gallocatechin, catechin-3-gallate, epigallocatechin, epicatechin-3-gallate, resveratrol, and dopamine.

7. The lipid-based nanoparticle according to any one of claims 1 to 6, wherein the one or more lipids are partially or fully soluble in an aqueous solution.

8. The lipid-based nanoparticle according to any one of claims 1 to 6, wherein the one or more lipids are partially or fully soluble non-aqueous solution, such as an alcohol, preferably ethanol.

9. The lipid-based nanoparticle according to any one of the preceding claims, wherein the one or more lipids comprise one or more pegylated lipids, phospholipids, structural lipids, and cationic or ionisable lipids.

10. The lipid-based nanoparticle according to any one of the preceding claims, wherein the one or more lipids does not comprise one or more cationic or ionisable lipids.

11. The lipid-based nanoparticle according to any one of the preceding claims, wherein the one or more lipids does not comprise monoolein or phytantriol.

12. The lipid-based nanoparticle according to any one of the preceding claims, wherein the one or more lipids have a molecular weight from about 100 to about 100,000 Daltons, preferably from about 100 to about 3,000 Daltons.

13. The lipid-based nanoparticle according to any one of the preceding claims, wherein the one or more lipids comprise one or more of 1,2-dimyristoyl-rac-glycero-3-methoxy poly(ethylene glycol) (DMG-PEG), polyethylene glycol 2000-ester-stearic acid (PEG-SA); polyethylene glycol 2000-ester-myristic acid (PEG-MA); 2-[(polyethylene glycol)-N,N-ditetradecylacetamide (ALC-0159), polyethylene glycol-carbamate-1,2-dimyristoyl-sn-glycerol (PEG-c-DMG); 1,2-dimyristoyl-sn-glycero-3-succinyl-N-polysarcosine-25 (pSar-DMG); 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (ammonium salt) (DOPE-PEG 5000); methoxy polyethylene glycol 20k-distearoyl phosphatidyl ethanolamine (DSPE-PEG 20k), cholesterol-polyethylene glycol 5000 (CLS-PEG 5000), and cholesterol-polyethylene glycol 20k (CLS-PEG 20k); 1,2-dioleoyl-s / ?-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-s / ?-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DOPE-PEG), A / -(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-s / ?-glycero-3-phosphoethanolamine (sodium salt). (DSPE-PEG), 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (chloride salt) (EPC).

14. The lipid-based nanoparticle according to any one of the preceding claims, comprising a single type of lipid.

15. The lipid-based nanoparticle according to any one of the preceding claims, wherein the molar ratio of the one or more phenolic compounds to the one or more lipids is from about 0.5:1 to about 200:1.

16. The lipid-based nanoparticle according to any one of the preceding claims, wherein the lipid-based nanoparticle has a mean particle size from about 5 nm to about 1 micron, or from about 10 nm to about 500 nm.

17. The lipid-based nanoparticle according to any one of the preceding claims, wherein the polydispersity index of the lipid-based nanoparticle is less than about 0.3, or less than about 0.2, or less than about 0.1, or less than about 0.05.

18. The lipid-based nanoparticle according to any one of the preceding claims, wherein the zeta potential of the lipid-based nanoparticle is less than about -5 mV, or less than about -30 mV.

19. The lipid-based nanoparticle according to any one of the preceding claims, wherein the lipid-based nanoparticle comprises a non-lamellar lyotropic liquid crystalline phase nanostructure.

20. The lipid-based nanoparticle according to claim 19, wherein the non-lamellar lyotropic liquid crystalline phase nanostructure is a cubosome, a hexosome, or a micellarsome.

21. The lipid-based nanoparticle according to any one of the preceding claims, further comprising one or more metal ions.

22. The lipid-based nanoparticle according to claim 21, wherein the one or more metal ions comprises one or more of Cu(ll), Fe(lll), Zr(IV), Zn (II), Ni (II), Co(ll), Na(l), Ca(ll), Ti(IV), Mn(ll), Ir(IV), Ta(V), W(VI), Bi(lll), Zn(ll), Mn(ll), Gd(lll), Pt(IV), Nb(V), and Ga(lll).

23. The lipid-based nanoparticle according to claim 21 or claim 22 comprising a single type of metal ion.

24. The lipid-based nanoparticle according to any one of claims 21 to 23, wherein the molar ratio of one or more phenolic compounds to one or more metal ions is from about 200:1 to about 1:5.

25. A bioactive lipid-based nanoparticle comprising the lipid-based nanoparticle according to any one of the preceding claims and one or more active agents.

26. The bioactive lipid-based nanoparticle according to claim 25, wherein the one or more active agents comprise on or more small molecules or biomacromolecules.

27. The bioactive lipid-based nanoparticle according to claim 26, wherein the biomacromolecule comprises one or more of proteins, peptides, nucleic acid molecules and enzymes.

28. The bioactive lipid-based nanoparticle according to any one of claims 25 to 27, wherein the one or more active agents are at least partly encapsulated within the lipid-based nanoparticle.

29. The bioactive lipid-based nanoparticle according to any one of claims 25 to 28, wherein the biomacromolecule comprises an RNA molecule.

30. The bioactive lipid-based nanoparticle according to any one of claims 25 to 29, wherein the biomacromolecule comprises an mRNA molecule or an siRNA molecule.

31. A pharmaceutical composition comprising the bioactive lipid-based nanoparticle according to any one of claims 25 to 30, and a pharmaceutically acceptable carrier, diluent, and / or excipient.

32. A method for producing a lipid-based nanoparticle according to any one of claims 1 to 20 comprising contacting one or more lipids and one or more phenolic compounds.

33. A method for producing a bioactive lipid-based nanoparticle according to any one of claims 25 to 30 comprising contacting one or more lipids, one or more phenolic compounds, and one or more active agents.

34. The method according to claim 32 or 33, wherein the lipid-based nanoparticle is produced in one-step by contacting a solution comprising the one or more phenolic compounds, optionally with one or more metal ions, with a solution comprising one or more lipids.

35. A method of delivering an active agent to a cell or tissue, the method comprising contacting the bioactive lipid-based nanoparticle according to any one ofclaims 25 to 30 or pharmaceutical composition according to claim 31 with the cell or tissue, thereby introducing the active agent into the cell or tissue.

36. The method according to claim 35, wherein the target cell is a 2D or 3D cultured cell.

37. The method according to claim 35 or claim 36, wherein the active agent is an RNA, such as an mRNA or siRNA.

38. The method according to claim 37, wherein an RNA transfection efficiency is greater than 25%, or greater than 50%, or greater than 75%, or greater than 85%, or greater than 95%.

39. A method of in vivo delivery of a bioactive agent, the method comprising administering the bioactive lipid-based nanoparticle according to any one of claims 25 to 30 or pharmaceutical composition according to claim 31 to a subject, thereby delivering the active agent to the subject.

40. A method of preventing, treating or ameliorating an infection, disease, disorder, or condition in a subject in need thereof, the method comprising administering to the subject the bioactive lipid-based nanoparticle according to any one of claims 25 to 30 or pharmaceutical composition according to claim 31, thereby treating or preventing an infection, disease, disorder or condition in a subject in need thereof.

41. A method of treating or preventing a cancer in a subject, comprising administering to the subject a bioactive nanoparticle composition according to any one of claims 25 to 30, or pharmaceutical composition according to claim 31, thereby treating or preventing cancer.

42. Use of a bioactive lipid-based nanoparticle according to any one of claims 25 to 30 or pharmaceutical composition according to claim 31 in the manufacture of a medicament for treating or preventing a disease or condition in a subject in need thereof.

43. Use of a bioactive lipid-based nanoparticle according to any one of claims 25 to 30 or pharmaceutical composition according to claim 31 in the manufacture of a medicament for treating or preventing a cancer in a subject.

44. A bioactive lipid-based nanoparticle according to any one of claims 25 to 30 or a pharmaceutical composition according to claim 31 for use in the treatment or prevention of a disease or condition in a subject in need thereof.

45. A bioactive lipid-based nanoparticle according to any one of claims 25 to 30 or a pharmaceutical composition according to claim 31 for use in the treatment or prevention of a cancer in a subject.

46. A method of delivering an RNA into a cell, preferably a mammalian cell, the method comprising administering to a subject a bioactive lipid-based nanoparticle according to any one of claims 25 to 30 or a pharmaceutical composition according to claim 31, wherein an active agent is an RNA, thereby delivering an RNA into a cell.

47. The method according to claim 46, wherein the RNA is an mRNA or siRNA.

48. A method of producing a polypeptide of interest in a cell, preferably amammalian cell, the method comprising contacting the bioactive lipid-based nanoparticle according to any one of claims 25 to 30 or pharmaceutical composition according to claim 31, with the cell or tissue, wherein the active agent is an mRNA encoding the polypeptide of interest, and wherein the mRNA is capable of being translated in the cell to produce the polypeptide of interest.

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