Nanoparticle compositions

Abstract

Bioactive nanoparticle compositions containing metal ions, phenolic ligands, bioactive agents, and seeding agents are provided, as are methods for their preparation. Through variation of one or more of the components of the compositions, the bioactive nanoparticles can be formulated to target specific sites in vivo, for example specific organs.

Description

Nanoparticle compositions Field of the disclosure

[0001] The present disclosure relates to bioactive nanoparticle compositions containing metal ions, phenolic ligands, bioactive agents, and seeding agents. Through variation of one or more of the components of the compositions, they may be formulated to target specific sites in vitro and in vivo. Related application

[0002] This application claims the benefit of priority from Australian provisional application no.2023902964, filed 14 September 2024, the disclosure of which is incorporated herein in its entirety. Background of the disclosure

[0003] Nanoparticles have gained wide attention in therapeutic delivery. However, most nanoparticles tend to accumulate in the liver (due to the physiology of the liver) after intravenous (IV) administration, presenting a critical bottleneck for the clinical translation of nanoparticle-mediated therapy (i.e., nanomedicine). Although enormous efforts have been made to target specific tissues by incorporating a ligand that can bind to a specific biomolecule to the delivery system, it remains challenging to achieve targeting in vivo as the surface properties of ligand-integrated nanoparticles can be readily altered by plasma proteins during blood circulation.

[0004] Messenger RNA (mRNA)-based therapeutics have emerged as powerful agents for disease prevention and treatment, as highlighted by mRNA-based vaccines against COVID-19 from Pfizer and Moderna. Formulating mRNA therapeutics into nanoparticles is needed because: 1). mRNA is inherently unstable and susceptible to degradation by enzymes and hydrolysis, particularly in the complex biological environment in vivo; and 2). mRNA is long, high molecular weight, negatively charged biomolecule, which impairs its cellular membrane penetrability. Nanoparticles can protect the labile cargo from degradation and allow intracellular delivery and release to facilitate protein production. Lipid nanoparticles (LNPs) are the gold standard for mRNA delivery. However, challenges with LNPs remain for non-vaccine applications, including inflammation, the capacity to deliver drugs other than nucleic acids, and delivery that is 1005496795restricted to blood, liver, and spleen. Versatile alternative delivery platforms are required that can be used for mRNA and other classes of drugs (e.g., proteins, peptides, small molecule drugs) and be programmed to direct drugs to tissues of interest.

[0005] A diverse range of carriers have been leveraged for the protection and delivery of mRNA. Among these carriers, those equipped with a positive charge have emerged as the primary choice due to their capacity to accommodate a high payload of mRNA and potential to trigger endosomal escape. Cationic compounds / polymers such as polyethyleneimine (PEI), histidine / arginine rich amphipathic peptides, dendrimers, or hybrid systems have been investigated, alongside lipid-based nanoparticles. The positive charge of cationic carriers, however, is a safety concern, as they can potentially activate the innate immune response and exhibit potent immune adjuvant activity.

[0006] A recent publication (Chen, J., Pan, S., Zhou, J., Lin, Z., Qu, Y., Glab, A., Han, Y., Richardson, J.J. and Caruso, F., 2022. Assembly of bioactive nanoparticles via metal–phenolic complexation. Advanced Materials, 34, p.2108624), discloses nanoparticle compositions consisting of bioactive metal ion-phenolic materials. However, the disclosure is silent in regard to biodistribution, and is limited to nanoparticle compositions in which the molar ratio of metal ion to phenolic ligand is 2.

[0007] In view of the above, it would be desirable to develop rational design of nanoparticle delivery platforms with predictable biodistribution.

[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] A technology is disclosed for directing nanoparticles to different organs simply via the selection of particular metal ions or other nanoparticle composition parameters. The technology allows systematic engineering of nanoparticles to deliver therapeutics to desirable sites, which holds promise to improve therapeutic efficacy at diverse organs. 1005496795

[0010] In one aspect the present disclosure provides a bioactive nanoparticle composition comprising a metal ion–phenolic assembly and one or more bioactive agents, wherein the metal ion–phenolic assembly comprises one or more metal ions, one or more phenolic ligands, and one or more seeding agents.

[0011] In any aspect or embodiment, the metal ion comprises one or more of Cu(II), Fe(III), Zr(IV), Ti(IV), Ir(IV), Ta(V), W(VI), Bi(III), Zn(II), Mn(II), Gd(III), Pt(IV), Nb(V), Co(II), Cr(III), Tm(III), Al(III), and Na(I).

[0012] In any aspect or embodiment, the bioactive nanoparticle composition comprises a single metal ion, for example Cu(II) or Fe(III).

[0013] In any aspect or embodiment, the one or more phenolic ligands has a molecular weight between 110 and 3,000 Daltons.

[0014] In any aspect or embodiment, the one or more phenolic ligands comprise three or more ring-bonded hydroxy groups.

[0015] In any aspect or embodiment, the one or more phenolic ligands comprise one or more galloyl groups, optionally with one or more catechol groups.

[0016] In any aspect or embodiment, the one or more phenolic ligands comprises one or more of tannic acid, (-)-epigallocatechin-3-gallate, catechin, gallocatechin, catechin- 3-gallate, epigallocatechin, epicatechin-3-gallate, quercetin, gallic acid, procyanidin, ellagic acid, myricetin, fisetin, luteolin, and dopamine.

[0017] In any aspect or embodiment, the seeding agent comprises one or more polyethylene glycol or chemically functionalised polyethylene glycol.

[0018] In any aspect or embodiment, the one or more chemically functionalised polyethylene glycol derivatives comprise one or more of amine, acid, and ester functional groups.

[0019] In any aspect or embodiment, the one or more polyethylene glycol or chemically functionalised polyethylene glycol has a molecular weight from 100 to 10,000,000 Daltons. 1005496795

[0020] In any aspect or embodiment, the one or more polyethylene glycol or chemically functionalised polyethylene glycol has a molecular weight from 100 to 50,000 Daltons.

[0021] In any aspect or embodiment, the one or more polyethylene glycol or chemically functionalised polyethylene glycol has a molecular weight from 5,000 to 50,000 Daltons.

[0022] In any aspect or embodiment, the one or more polyethylene glycol or chemically functionalised polyethylene glycol has a molecular weight from about 2,000 to about 6,000 Daltons.

[0023] In any aspect or embodiment, the one or more polyethylene glycol or chemically functionalised polyethylene glycol has a molecular weight from about 10,000 to about 20,000 Daltons.

[0024] In any aspect or embodiment, the one or more polyethylene glycol or chemically functionalised polyethylene glycol has a molecular weight from about 2,000 to less than 10,000 Daltons.

[0025] In any aspect or embodiment, the one or more polyethylene glycol or chemically functionalised polyethylene glycol has a molecular weight of less than 2,000.

[0026] In any aspect or embodiment, the one or more polyethylene glycol or chemically functionalised polyethylene glycol has a linear, branched, star or comb structure.

[0027] In some embodiments, the one or more bioactive agents comprise one or more small molecules or biomacromolecules.

[0028] In some embodiments, the biomacromolecule comprises one or more of proteins, peptides, nucleic acid molecules and enzymes.

[0029] In embodiments, the biomacromolecule comprises single stranded RNA.

[0030] In some embodiments, the biomacromolecule comprises mRNA.

[0031] In embodiments, the molar ratio of metal ion to phenolic ligand in the bioactive nanoparticle composition is from 0.001:1 to 100:1. 1005496795

[0032] In embodiments, the molar ratio of metal ion to phenolic ligand in the bioactive nanoparticle composition is from 0.001:1 to less than 2:1.

[0033] In embodiments, the molar ratio of metal ion to phenolic ligand in the bioactive nanoparticle composition is from greater than 2:1 to 100:1.

[0034] In any aspect or embodiment, the bioactive agent is selected from the group of proteins, peptides and enzymes and the phenolic ligand is catechin, preferably (+)- catechin hydrate.

[0035] In any aspect or embodiment, the bioactive agent is mRNA and the phenolic ligand is (-)-epigallocatechin-3-gallate.

[0036] In embodiments, the bioactive agent is mRNA, the phenolic ligand is (-)- epigallocatechin-3-gallate, and the seeding agent is polyethylene glycol having a molecular weight from 100 to 10,000,000 Daltons, or from 5,000 to 50,000 Daltons.

[0037] In any aspect or embodiment, the presently disclosed bioactive nanoparticle compositions are substantially free, or free of organic solvents.

[0038] In another aspect the present disclosure provides a method of preparing a bioactive nanoparticle composition according to any one of the herein disclosed embodiments, comprising combining, in any order, one or more sources of metal ions, one or more phenolic ligands, one or more bioactive agents, and one or more seeding agents.

[0039] In some embodiments of the method the metal ion source is added to a mixture of phenolic ligand, bioactive agent and seeding agent.

[0040] In other embodiments of the method the bioactive agent is added to a mixture of phenolic ligand, metal ion source, and seeding agent.

[0041] In another aspect the present disclosure provides a bioactive nanoparticle composition comprising a metal ion–phenolic assembly and one or more nucleic acid molecules, wherein the metal ion–phenolic assembly comprises one or more metal ions, one or more phenolic ligands, and one or more seeding agents.

[0042] In any aspect or embodiment, the nucleic acid molecule is a therapeutic agent. 1005496795

[0043] In any aspect or embodiment, 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, or guide RNA molecule, or any combination thereof.

[0044] In any aspect or embodiment, 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.

[0045] In any aspect or embodiment, the nucleic acid molecule comprises a promoter or regulatory sequence.

[0046] 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. The relative amounts of nucleic acid and other elements (e.g., polyethylene glycol or chemically functionalised polyethylene glycol, phenolic ligands, and metal ions) may also vary.

[0047] In any aspect or embodiment, the mass ratio of the metal ion to phenolic ligand in the bioactive nanoparticle composition is from about 0.5:40 to about 3:40. In certain embodiments, the mass ratio is from about 0.5:40 to about 2:40. In other embodiments, the mass ratio is about 0.5:40, about 1:40, about 2:40 or about 3:40. Preferably, the mass ratio is about 1:40.

[0048] In any aspect or embodiment, the mass ratio of the metal ion to phenolic ligand in the bioactive nanoparticles composition is less than 1:5, preferably less than 3:40.

[0049] In any aspect or embodiment, the polyethylene glycol or chemically functionalised polyethylene glycol has a molecular weight of from about 5,000 to about 50,000 Da, from about 5,000 to about 40,000 Da, from about 5,000 to about 30,000 Da, from about 5,000 to about 25,000 Da, from about 5,000 to about 20,000 Da. In other certain embodiments, the molecular weight is from about 5,000 Da, about 10,000 Da, 1005496795about 15,000 Da, about 20,000 Da, about 25,000 Da, about 30,000 Da, about 40,000 Da or about 50,000 Da. Preferably, the molecular weight is about 20,000 Da.

[0050] In any aspect or embodiment, the polyethylene glycol or chemically functionalised polyethylene glycol has a linear structure.

[0051] In any aspect or embodiment, the polyethylene glycol or chemically functionalised polyethylene glycol has a star structure, preferably a 4-arm structure.

[0052] In any aspect or embodiment, the one or more phenolic ligands comprise (-)- epigallocatechin-3-gallate.

[0053] In any aspect or embodiment, the one or more metal ions has an oxidation state of at least +4, preferably +4.

[0054] In any aspect or embodiment, the one or more metal ions comprise Zr(IV) or Ti(IV).

[0055] In any aspect or embodiment, the seeding agent : nucleic acid molecule : phenolic ligand : metal ion has a mass ratio of 100 : 1 : 100 : 2.5.

[0056] In any aspect or embodiment, the bioactive nanoparticle composition is non- cationic.

[0057] In any aspect or embodiment, the bioactive nanoparticle composition is further functionalised, preferably wherein the bioactive nanoparticle composition comprises antibodies on the surface of the nanoparticle. In some embodiments, the antibodies specifically bind to one or more antigens expressed in a target organ or target site, or by a target cell.

[0058] In another aspect the present disclosure provides a method for delivering one or more bioactive agents to a target cell or tissue comprising bringing a bioactive nanoparticle composition according to any one of the herein disclosed embodiments into contact with the cell or tissue.

[0059] In another aspect, the present disclosure provides a method for delivering one or more nucleic acid molecules to a target cell or tissue comprising bringing a bioactive nanoparticle composition according to any one of the herein disclosed embodiments into contact with the cell or tissue. 1005496795

[0060] In any aspect or embodiment, the target cell may be a 2D or 3D cultured cell.

[0061] In any aspect or embodiment, the target cell is a mammalian cell.

[0062] In another aspect, the present disclosure provides a method of delivering bioactive nanoparticles to one or more target sites in a subject comprising administering the bioactive nanoparticle composition according to any one of the herein disclosed embodiments to a subject.

[0063] In any aspect or embodiment, the bioactive nanoparticle composition comprises a small molecule or biomacromolecule selected from proteins, peptides, and enzymes and at least one of the following: (i) the metal ions comprise one or more of Co(II), Zr(IV), Ti(IV), Ir(IV), Ta(V) and W(VI); (ii) the metal ions comprise one or more of Cu(II), Tm(III) and Bi(III); (iii) the metal ions comprise one or more Zn(II), Mn(II), Fe(III) and Cr(III); (iv) the phenolic ligand comprises tannic acid; (v) the phenolic ligand comprises (-)-epigallocatechin-3-gallate; (vi) the phenolic ligands comprise catechin, preferably (+)- catechin hydrate; (vii) the phenolic ligands comprise gallic acid; and (viii) the seeding agent comprises polyethylene glycol or chemically functionalised polyethylene glycols having a molecular weight of about 2,000 Da to about 6,000 Da; and (ix) the seeding agent comprises polyethylene glycol or chemically functionalised polyethylene glycols having a molecular weight of about 10,000 Da to about 20,000 Da.

[0064] In any aspect or embodiment, the bioactive nanoparticle composition comprises a nucleic acid molecule and at least one of the following: (i) the metal ions comprise one or more of Zr(IV), Ti(IV), Ir(IV), Ta(V) and W(VI), preferably Zr(IV) and / or Ti(IV); 1005496795(ii) the metal ions comprise one or more of Co(II), Tm(III), and Bi(III), preferably Co(II); (iii) the metal ion to phenolic ligand has a mass ratio of about 1:40; (iv) the metal ion to phenolic ligand has a mass ratio of about 3:40; (v) the seeding agent comprises polyethylene glycol or chemically functionalised polyethylene glycols having a star structure, preferably a 4-arm star structure; and (vi) the seeding agent comprises polyethylene glycol or chemically functionalised polyethylene glycols having a molecular weight of about 20,000 Da, optionally having a linear structure.

[0065] In another aspect, the present disclosure provides a method of delivering bioactive nanoparticles to multiple target sites in a subject comprising administering the bioactive nanoparticle composition according to any one of the herein disclosed embodiments to a subject.

[0066] In in any aspect or embodiment, the bioactive nanoparticle composition comprises one or more of Gd(III), Pt(IV) and Nb(V).

[0067] In some embodiments on the above methods, the presence of one or more metal ions; one or more phenolic ligands; and / or one or more seeding agents provides a greater amount or activity of the bioactive nanoparticle to the target site compared to that achieved in the absence thereof.

[0068] In some embodiments of the above methods, the target site is one or more of liver, kidney, lung, brain, spleen, heart, pancreas, stomach, small intestine, large intestine, lymph node, bone, bone marrow, skeletal muscle, bladder, breast, testes, ovaries, uterus, thymus, brainstem, cerebellum, spinal cord, eye, ear, tongue, prostate and skin.

[0069] In another aspect, the present disclosure provides a bioactive nanoparticle composition according to any one of the herein disclosed embodiments in the manufacture of a medicament for delivering bioactive nanoparticles to one or more target sites in a subject. 1005496795

[0070] In another aspect, the present disclosure provides a bioactive nanoparticle composition according to any one of the herein disclosed embodiments for use in a method of delivering bioactive nanoparticles to one or more target sites in a subject.

[0071] In another aspect, the present disclosure provides a method for treating or preventing a disease or condition in a subject in need thereof, the method comprising administering to the subject a bioactive nanoparticle composition according to any one of the herein disclosed embodiments, thereby treating or preventing a disease or condition in a subject in need thereof.

[0072] In another aspect, the present disclosure provides a bioactive nanoparticle 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.

[0073] In another aspect, the present disclosure provides a bioactive nanoparticle 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.

[0074] In another aspect, the present disclosure provides a method of producing a polypeptides of interest in a cell, preferably a mammalian cell, the method comprising contacting the cell with a bioactive nanoparticle composition according to any one of the herein disclosed embodiments, wherein the active agent is an mRNA encoding the polypeptides of interest, and wherein the mRNA is capable of being translated in the cell to produce the polypeptides of interest.

[0075] In another aspect, the present disclosure provides a method for the in vivo delivery of one or more bioactive agents, the method comprising administering a bioactive nanoparticle composition according to any one of the herein disclosed embodiments to a subject in need thereof, thereby delivering the one or more bioactive agents to the subject.

[0076] In another aspect, the present disclosure provides a method of delivering one or more components of a gene editing complex into a cell, comprising contacting a cell with a bioactive nanoparticle composition according to any one of the herein disclosed embodiments, wherein the bioactive agent comprises one or more components of the gene editing complex. 1005496795

[0077] In another aspect, the present disclosure provides a method of altering gene expression, comprising contacting a cell with a bioactive nanoparticle composition according to any one of the herein disclosed embodiments, thereby altering gene expression.

[0078] In some embodiments, the cell is in vitro.

[0079] In some embodiment, the cell is in vivo, preferably within an animal, more preferably a mammal, even more preferably a human.

[0080] In another aspect, the present disclosure provides a method of treating or preventing 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. In some embodiments, the cancer is a metastatic cancer, such as lung metastases. In some embodiments, the cancer is metastatic melanoma, optionally metastatic melanoma to the lung.

[0081] In another aspect, the present disclosure provides a method for delivering one or more bioactive agents across the blood brain barrier in a subject in need thereof, the method comprising administering a bioactive nanoparticle composition according to any one of the herein disclosed embodiments to a subject, thereby delivering the one or more bioactive agents across the blood brain barrier.

[0082] In any aspect or embodiment, the bioactive nanoparticle composition may be administered intravenously, subcutaneously, intramuscularly, intranasally, intratracheally, intraperitoneally, intratumorally, or intracerebrally. Other administration routes are contemplated.

[0083] In any aspect or embodiment of the methods the bioactive agent comprises single stranded RNA.

[0084] In any aspect or embodiment of the methods the bioactive agent comprises mRNA.

[0085] In another aspect, the present disclosure provides a method for targeted delivery of one or more bioactive molecules to the lung of a subject, the method comprising administering a bioactive nanoparticle composition according to any one of the herein disclosed embodiments to the subject, 1005496795wherein the presence of one or more metal ions; one or more phenolic ligands; and / or one or more seeding agents provides a greater amount or activity of the bioactive nanoparticle to the lung compared to that achieved in the absence thereof, preferably wherein the bioactive molecule is a small molecule or biomacromolecule selected from proteins, peptides, and enzymes.

[0086] In preferred embodiments of the above aspect, the metal ions; one or more phenolic ligands; and / or one or more seeding agents provide a greater amount or activity of the bioactive nanoparticle composition to the lung compared to that achieved in the absence thereof is selected from: (i) metal ions comprising one or more of Co(II), Zr(IV), Ti(IV), Ir(IV), Ta(V) and W(VI); (ii) tannic acid; and (iii) polyethylene glycol or chemically functionalised polyethylene glycols having a molecular weight of about 2,000 to less than 10,000 Da, preferably 2,000 Da,

[0087] In another aspect, the present disclosure provides a method for targeted delivery of one or more nucleic acid molecules, preferably an mRNA, to the lung of a subject, the method comprising administering a bioactive nanoparticle composition according to any one of the herein disclosed embodiments to the subject, wherein the presence of one or more metal ions; one or more phenolic ligands; and / or one or more seeding agents provides a greater amount or activity of the bioactive nanoparticle to the lung compared to that achieved in the absence thereof.

[0088] In preferred embodiment of the above aspect, the metal ions that provide a greater amount or activity of the bioactive nanoparticle composition to the lung compared to that achieved in the absence thereof comprise Tm(III) or Co(II), preferably Co(II).

[0089] In another aspect, the present disclosure provides a method for targeted delivery of one or more bioactive molecules to the brain of a subject, the method comprising administering a bioactive nanoparticle composition according to any one of the herein disclosed embodiments to the subject, 1005496795wherein the presence of one or more metal ions; one or more phenolic ligands; and / or one or more seeding agents provides a greater amount or activity of the bioactive nanoparticle composition to the brain compared to that achieved in the absence thereof, preferably wherein the bioactive molecule is a small molecule or biomacromolecule selected from proteins, peptides, and enzymes.

[0090] In preferred embodiment of the above aspect, the phenolic ligands that provide a greater amount or activity of the bioactive nanoparticle to the brain compared to that achieved in the absence thereof comprise catechin, and / or gallocatechin and / or (-)- epigallocatechin-3-gallate; preferably (-)-epigallocatechin-3-gallate.

[0091] In another aspect, the present disclosure provides a method for targeted delivery of one or more nucleic acid molecules, preferably mRNA molecules, to the brain of a subject, the method comprising administering a bioactive nanoparticle composition to the subject, wherein the bioactive nanoparticle composition comprises a phenolic ligand assembly and one or more nucleic acid molecules, wherein the phenolic ligand assembly comprises one or more phenolic ligands, and one or more seeding agents.

[0092] In another aspect, the present disclosure provides a method for targeted delivery of one or more nucleic acid molecules, preferably mRNA molecules, to the brain of a subject, the method comprising administering a bioactive nanoparticle composition according to any one of the herein disclosed embodiments to the subject, wherein the presence of one or more metal ions; one or more phenolic ligands; and / or one or more seeding agents provide a greater amount or activity of the bioactive nanoparticle composition to the brain compared to that achieved in the absence thereof.

[0093] In preferred embodiments of the above aspect, the metal ion that provides a greater amount or activity of the bioactive nanoparticle composition to the brain compared to that achieved in the absence thereof comprises Zr(IV).

[0094] In another aspect, the present disclosure provides a method for targeted delivery of one or more bioactive molecules to the spleen of a subject, the method comprising administering a bioactive nanoparticle composition according to any one of the herein disclosed embodiments to the subject, 1005496795wherein the presence of one or more metal ions; one or more phenolic ligands; and / or one or more seeding agents provide a greater amount or activity of the bioactive nanoparticle composition to the spleen compared to that achieved in the absence thereof, preferably wherein the bioactive molecule is a small molecule or biomacromolecule selected from proteins, peptides, and enzymes.

[0095] In a preferred embodiment of the above aspect, the metal ions that provide a greater amount or activity of the bioactive nanoparticle to the spleen compared to that achieved in the absence thereof comprise one or more of Cu(II), Tm(III) and Bi(III).

[0096] In another aspect, the present disclosure provides a method for targeted delivery of one or more bioactive molecules to the kidney of a subject, the method comprising administering a bioactive nanoparticle composition according to any one of the herein disclosed embodiments to the subject, wherein the presence of one or more metal ions; one or more phenolic ligands; and / or one or more seeding agents provide a greater amount or activity of the bioactive nanoparticle composition to the kidney compared to that achieved in the absence thereof, preferably wherein the bioactive molecule is a small molecule or biomacromolecule selected from proteins, peptides, and enzymes.

[0097] In preferred embodiment of the above aspect, the metal ions that provide a greater amount or activity of the bioactive nanoparticle composition to the kidney compared to that achieved in the absence thereof comprise one or more of Zn(II), Mn(II), Fe(III) and Cr(III).

[0098] In another aspect, the present disclosure provides a method for targeted delivery of one or more nucleic acid molecules, preferably mRNA molecules, to the kidney of a subject, the method comprising administering a bioactive nanoparticle composition according to any one of the herein disclosed embodiments to the subject, wherein the presence of one or more metal ions; one or more phenolic ligands; and / or one or more seeding agents provide a greater amount or activity of the bioactive nanoparticle to the kidney compared to that achieved in the absence thereof. 1005496795

[0099] In preferred embodiments of the above aspect, the metal ion that provides a greater amount or activity of the bioactive nanoparticle composition to the kidney compared to that achieved in the absence thereof comprises Ti(IV) or Zr(IV), Optionally, the mass ratio of the metal ion to nucleic acid molecule is from about 1:40 to about 3:40, even more preferably about 3:40,

[0100] In another aspect, the present disclosure provides a method for targeted delivery of one or more nucleic acid molecules, preferably mRNA molecules, to the spleen of a subject, the method comprising administering a bioactive nanoparticle composition according to any one of the herein disclosed embodiments to the subject, wherein the presence of one or more metal ions; one or more phenolic ligands; and / or one or more seeding agents provide a greater amount or activity of the bioactive nanoparticle composition to the spleen compared to that achieved in the absence thereof.

[0101] In preferred embodiment of the above aspect, the seeding agent that provides a greater amount or activity of the bioactive nanoparticle to the spleen is polyethylene glycol or chemically functionalised polyethylene glycol having a star structure, preferably a 4-arm star structure

[0102] In any aspect or embodiment of the methods, the mRNA transfection efficiency is greater than 25%, or greater than 50%, or greater than 75%, or greater than 85%, or greater than 95%.

[0103] Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.

[0104] 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.

[0105] 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. 1005496795Brief description of the drawings

[0106] Figure 1 shows the biodistribution of nanoparticle compositions assembled from PEG (2 kDa, linear), bovine serum albumin (BSA), tannic acid (TA), with and without different metal ions, 4 h after intravenous administration in C57BL / 6J mice.

[0107] Figure 2 shows the biodistribution of TA / Ti(IV)-based BSA nanoparticle compositions prepared with different Ti(IV)-to-TA molar ratios, 4 h after intravenous administration in C57BL / 6J mice.

[0108] Figure 3 shows the biodistribution of TA / Ti(IV)-based nanoparticle compositions assembled with or without different types of proteins (i.e., no cargo, BSA, ovalbumin (OVA), glucose oxidase (GOx), catalase (Cat), and lysozyme (LYZ)), 4 h after intravenous administration in C57BL / 6J mice.

[0109] Figure 4 shows the biodistribution of Ti(IV)-based BSA nanoparticle compositions prepared with different phenolic ligands (i.e., tannic acid (TA), (-)- epigallocatechin-3-gallate (EGCG), (+)-catechin hydrate (CAT), or gallic acid (GA)), 4 h after intravenous administration in C57BL / 6J mice.

[0110] Figure 5(a) illustrates phenolic ligands, PEGs, and metal ions utilised for the assembly of mRNA-nanoparticle compositions and the synthesis of mRNA-nanoparticle compositions prepared through metal-phenolic-mediated assembly. The number indicates the sequence of adding reagents; (b) illustrates the loading efficiency of mCherry-encoding mRNA in mRNA-nanoparticle compositions (assembled with 20k linear PEG, EGCG, and Zr(IV) at different phenolic ligand-to-mRNA mass ratios. The inset is the image of agarose gel bands showing uncomplexed mRNA in the system; (c) Histogram of mCherry fluorescence from HEK 293T cells transfected by mRNA- nanoparticle compositions prepared with different phenolic ligands (with formulation 20k linear PEG:mRNA:phenolic ligand:ZrIVat 100:1:100:2.5 mass ratio) after 24h; (d) corresponding mean mCherry fluorescence intensity; (e) histogram of mCherry fluorescence from HEK 293T cells transfected by mRNA-nanoparticle compositions prepared with different PEGs (with formulation PEG:mRNA:EGCG:ZrIVat 100:1:100:2.5 mass ratio) after 24h; (f) corresponding mean mCherry fluorescence intensity. The inset is the image of agarose bands showing loading efficiency of formulations with different PEGs; (g) histogram of mCherry fluorescence; (h) mean mCherry fluorescence intensity 1005496795of HEK 293T cells transfected by mRNA-nanoparticle compositions with different metal ion concentrations (fixed with 20k linear PEG, EGCG, and ZrIV) after 24h. The inset is the image of agarose bands showing loading efficiency of formulations with different metal ion-to-EGCG mass ratios; (i) transfection efficiency (%) and (j) mean mCherry fluorescence intensity of HEK 293T cells transfected by mRNA-nanoparticle compositions assembled with various metal ions (with formulation 20k linear PEG:mRNA:EGCG:metal ion at 100:1:100:2.5 mass ratio) after 24h.

[0111] Figure 6 (a) and (b) is a schematic illustration of two routes used to load mRNA (a) assembly route 1 Co-Assembly (denoted as Co-A) and (b) assembly route 2 Post Assembly (denoted as Post-A), with formulation 20k linear PEG:mRNA:EGCG:Zr(IV) at 100:1:100:2.5 mass ratio; c) illustrates the UV-vis spectra of free EGCG, EGCG-PEG complex, and mRNA-nanoparticle compositions. The green region highlights the ligand- to-metal charge transfer (LMCT) band in the spectrum of mRNA –nanoparticle compositions; d) illustrates the size distribution of different nanoparticle compositions (20k linear PEG:mRNA:EGCG:Zr(IV) at 100:1:100:2.5 mass ratio) measured by DLS. Insets are structured illumination microscopy (SIM) images of nanoparticle compositions assembled using the two routes. Red signal stems from Cy5-labelled mRNA. Scale bars are 200 nm; (e) mRNA loading efficiency of nanoparticle compositions (20k linear PEG:mRNA:EGCG:Zr(IV) at 100:1:100:2.5 mass ratio) assembled via Co-A or Post-A routes; (f), (g) 24 h release profile of Co-A (f) and Post-A (g) mRNA-nanoparticle compositions (20k linear PEG:mRNA:EGCG:Zr(IV) at 100:1:100:2.5 mass ratio) in RNase-free water, PBS, and PBS + 10% FBS at 37 ˚C; (h) percentage of mRNA dissociated from nanoparticle compositions (20k linear PEG:mRNA:EGCG:Zr(IV) at 100:1:100:2.5 mass ratio; Co-A preparation route) after incubation in different solutions for 24 h.

[0112] Figure 7 illustrates (a) Representative structured illumination microscopy (SIM) images of the intracellular colocalization of mRNA-nanoparticle compositions (20k linear PEG:mRNA:EGCG:Zr(IV) at 100:1:100:2.5 mass ratio, prepared via co-assembly) with endo / lysosomes. Endo / lysosomes (green) were stained with LysoTracker Green DND- 26. mRNA (red) was conjugated with Cy-5. Scale bars are 10 μm. Representative high- magnification images are on the top right (scale bars are 2 μm). Bottom right is the corresponding colour scatter plot of endo / lysosomes (green channel) vs mRNA (red channel). (b) Confocal laser scanning microscopy (CLSM) immuno-fluorescence 1005496795staining showing the transfection of firefly luciferase-encoding mRNA by mRNA- nanoparticle compositions (20k linear PEG:mRNA:EGCG:Zr(IV) at 100:1:100:2.5 mass ratio) in HEK 293T cells after 24 h. Cell membranes (green) were stained with Alexa Fluor 488-wheat germ agglutinin conjugate (AF488-WGA). Luciferase was detected with Alexa Fluor 647-labelled anti-luciferase antibodies (red). Scale bars are 20 μm; (c) SIM images showing the transfection of Nerve Growth Factor Receptor (NGFR)-encoding mRNA in HEK 293T cells after 24 h. NGFR was stained with PE-conjugated antibodies. Scale bars are 10 μm; (d) CLSM images, e) percentage transfection; and f) mean fluorescence intensity of adherent cells (HEK 293T) transfected by mCherry mRNA- nanoparticle compositions (20k linear PEG:mRNA:EGCG:Zr(IV) at 100:1:100:2.5 mass ratio) with different assembly routes after 24 h. Cell membranes were stained in green. Scale bars are 20 μm. (g) CLSM images; (h) percentage transfection, and i) mean fluorescence intensity of suspension cells (Jurkat) transfected by mCherry mRNA- nanoparticle compositions (20k linear PEG:mRNA:EGCG:Zr(IV) at 100:1:100:2.5 mass ratio) with different assembly routes after 24h. Cell membranes (green) were stained with AF488-WGA. Scale bars are 10 μm.

[0113] Figure 8 illustrates in vivo mFlame transfection efficiency in C57BL / 6J mice with mRNA-nanoparticle compositions by altering (a) assembly routes (20k linear PEG:mRNA:EGCG:Zr(IV) at 100:1:100:2.5 mass ratio); (b) metal ion-to-EGCG ratio (wt:wt) (fixed with 20 kDa linear PEG, EGCG, and Zr(IV), prepared via Co-A); (c) types of metal ions (fixed with 20 kDa linear PEG, EGCG, and the mass ratio among each composition; prepared via Co-A), and (d) PEG (fixed with EGCG, Zr(IV), and the mass ratio among each composition; prepared via Co-A). Both quantitative results (i.e., the bar diagram) and inset representative images were obtained by In Vivo Imaging System (IVIS). Five mice were included in each group, and the quantitative data were normalized to DPBS-treated mice (negative control) and presented as mean ± standard deviation (SD). “Li” denotes “Liver”, “K” denotes “Kidney”, “Lu” denotes “Lung”, “H” denotes “Heart”, “S” denotes “Spleen”, and “B” denotes “Brain”.

[0114] Figure 9 (a) Size distribution of mRNA nanoparticle compositions and Nanoparticle compositions measured by dynamic light scattering. Inset shows a Lattice- SIM image of mRNA nanoparticle compositions using Cy5-labeled mRNA. Scale bar is 200 nm. (b) Transmission electron microscopy (TEM) image and EDX mapping of mRNA nanoparticle compositions. (c) ζ-Potential of mRNA nanoparticle compositions 1005496795across pH 4 to 9, as measured on a Zetasizer. (d) Percentage of mRNA loading into Nanoparticle compositions, as determined by agarose gel electrophoresis and fluorescence spectroscopy. (e) Release profiles of mRNA in RNase-free water and DMEM+10% FBS at 37 °C. (f) Percentage of mRNA released from mRNA nanoparticle compositions after incubation in different solutions for 24 h. All experiments were performed in triplicates (n = 3), and data are presented as the mean ± SD. Statistical significance was analyzed by the two-tailed unpaired t-test: ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, P > 0.05. The lead mRNA nanoparticle compositions were assembled with 20k linear PEG, mRNA, EGCG, and ZrIVat a mass ratio of 100:1:100:2.5.

[0115] Figure 10 (a) i)–iii) are representative SIM images of the intracellular colocalization of mRNA nanoparticle compositions with endo / lysosomes 24 h post incubation. Endo / lysosomes (green) were stained with LysoTracker Green DND-26. mRNA (red) was conjugated with Cy5. Scale bars are 10 μm. iv)–vi) Representative high-magnification images (scale bars are 2 μm). vii) Corresponding color scatter plot of endo / lysosomes (green channel) vs mRNA (red channel). (b) CLSM immunofluorescence staining showing the transfection of FLuc-encoding mRNA by mRNA nanoparticle compositions in HEK 293T cells after 24 h. Cell membranes (green) were stained with AF488-wheat germ agglutinin conjugate (AF488-WGA). Luciferase was detected with AF647-labeled anti-luciferase antibodies (red). Scale bars are 20 μm. (c) Representative CLSM images showing the transfection of NGFR-encoding mRNA in HEK 293T cells after 24 h. NGFR was stained with phycoerythrin-conjugated anti- NGFR. Nuclei were stained with Hoechst 33342. Scale bars are 20 μm. (d–f), Percentage of transfection (d), MFI (e), and representative CLSM images (f) of adherent cells (HEK 293T) transfected by RNAiMax or mRNA (mCherry)-Nanoparticle compositions after 24 h. Cell membranes were stained in green. Scale bars are 20 μm. (g–i), Percentage of transfection (g), MFI (h), and representative CLSM images (i) of suspension cells (Jurkat) transfected by RNAiMax or mRNA (mCherry)-Nanoparticle compositions after 24 h. Cell membranes were stained in green. Scale bars are 20 μm. All experiments were performed in triplicates (n = 3) and data are presented as the mean ± SD. Statisticalwas analyzed using one-way ANOVA with Tukey’s multiple comparisons test: ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, P > 0.05. The lead mRNA nanoparticle compositions were assembled with 20k linear PEG, mRNA, EGCG, and ZrIVat a mass ratio of 100:1:100:2.5. 1005496795

[0116] Figure 11 (a) is a schematic illustration of IV administration of mRNA nanoparticle compositions and subsequent IVIS imaging. Created with BioRender.com. (b) mFlame expression in different organs of C57BL / 6J mice analyzed by IVIS 24 h post IV injection of mRNA nanoparticle compositions (lead formulation; mRNA at a dose of 0.25 mg kg−1). (c,d) Comparison of mFlame expression using mRNA nanoparticle compositions and mRNA-SM-102 LNPs: both quantitative (c) and representative images (d) were obtained from IVIS. Five mice were included in each group, and the quantitative data were normalized to DPBS-treated mice (negative control) and presented as mean ± SD. The fluorescence signal observed in DPBS-treated mice may be due to tissue autofluorescence, which is often observed at the excitation and emission wavelengths of 561 and 594 nm, respectively. Statistical significance was analyzed using the two-tailed unpaired t-test:***P < 0.001; **P < 0.01; *P < 0.05; ns, P > 0.05. e–g, CLSM images of sectioned liver (e), kidney (f), and brain (g) tissues post IVIS imaging. Cell nuclei were stained with 4′,6-diamidino-2- phenylindole (DAPI). Red fluorescence indicates expressed emiRFP670. h, Schematic illustration of an Ai14 Cre reporter model that could express tdTom by translating Cre- recombinase mRNA to Cre protein to delete the loxP-flanked STO cassette. i,j, CLSM images of sectioned liver and kidney (i) and brain (j) tissues 48 h post IV injection of Cre-Nanoparticle compositions (mRNA dose at 0.25 mg kg−1). Red fluorescence indicates expressed tdTom. Cell nuclei were stained with DAPI. Blood vessels were stained with Lectin-Dylight 488, which was injected intravenously 5 min before euthanizing the mice.

[0117] Figure 12 illustrates biocompatibility of using the presently disclosed nanoparticle compositions for mRNA transfection in vivo; (a) cytokine (IL-6 and TNF-ɑ) released in the plasma collected from mice treated by DPBS (negative control) and mRNA-nanoparticle compositions; (b) hematoxylin and eosin staining of organs harvested from mice treated with DPBS and mRNA-nanoparticle compositions after 24 hours. Statistical significance was analyzed using the two-tailed unpaired t-test. (c) Zr excretion profiles from harvested organs and body fluids (i.e., urine and blood) post treatment with DPBS or mRNA nanoparticle compositions, as measured by ICP-MS elemental analysis. Zr excretion is presented as the amount of Zr per gram of organ or body fluid collected from mice. Statistical significance was analyzed using two-way ANOVA: ns, P > 0.05. 1005496795

[0118] Figure 13 (a–i) mFlame expression in harvested organs using Nanoparticle compositions under varying conditions: treatment time (a,b); ZrIV-to-EGCG mass ratio (c–e); metal ions (f–h); and Mw and structure of PEG (i). Both quantitative results (i.e., bar diagrams) and representative images of the biodistribution were obtained by IVIS. Five biologically independent mice were included in each group (n = 5), and the quantitative data were normalized to DPBS (negative control) and presented as mean ± SD. Li, liver; K, kidney; Lu, lung; H, heart; S, spleen; B, brain. Statistical significance was analyzed using one-way ANOVA with Tukey’s multiple comparisons test: ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; NS, P > 0.05.

[0119] Figure 14 shows the biodistribution of TA / Ti(IV)-based BSA nanoparticle compositions prepared with PEG of different molecular weights, 4 h after intravenous administration in C57BL / 6J mice.

[0120] Figure 15 shows the biodistribution and colocalisation of TA / Ti(IV)-based BSA nanoparticle composition containing either Rh800 (for tracking nanoparticles) or Cyanine5 (Cy5)-labelled BSA (for tracking proteins), 4 h after intravenous administration in C57BL / 6J mice.

[0121] Figure 16 shows the average radiance efficiency of TA / Ti(IV)-based BSA nanoparticle, 1, 4, 10, 24, 48, 72, 120 and 168 hrs after intravenous administration in C57BL / 6J mice.

[0122] Figure 17 (a) shows a schematic illustration of an Ai14 Cre reporter model that could express tdTom by deletion of the loxP-flanked STO cassette when in the presence of Cre protein. TA / Ti(IV) nanoparticle compositions were loaded with 40 μg Cre-recombinase proteins with BSA with a mass ratio of 1:9 (Cre: BSA). Fluorescence indicates expression of tdTom by deletion of the loxP-flank STO cassette and was detected in whole harvested organs (b) using IVIS imaging; and sectioned organs for the lung (c) and spleen, liver and kidney (d) using confocal microscopy.

[0123] Figure 18 shows a B16-OVA lung metastatic mouse model being treated using DOX-loaded TA / Ti(IV)-based nanoparticle compositions, DOX (100 μL, 400 μg mL-1). B16-OVA melanoma cells were intravenously inoculated to C57BL / 6J mice (~4×105cells per mouse) on day 0 to develop a lung metastatic melanoma tumor model. The mice were treated by intravenously administrating DOX-loaded TA / Ti(IV) nanoparticle 1005496795compositions, DPBS (negative control), free DOX (free drug control), and DOX-loaded TA / Tm(III) nanoparticle compositions (non-lung targeting nanoparticle control) on day 4 and 11. The lungs were finally harvested 18 days post tumor inoculation, and the inhibition efficacy was evaluated by counting the metastatic nodules on the lung. Number of lung nodules, mouse weight and representative H&E staining of the mouse lung is shown.

[0124] Figure 19 shows representative H&E sections from mouse organs following administration of organ-targeted TA-BSA nanoparticle compositions, ie with varying metal ions, to C57BL / 6J mice. Two primary metabolic organs (i.e., liver and kidney) and targeted organs for each representative nanoparticle composition were harvested 7 days post injection. The levels of IL-6 and TNF-α were quantified using enzyme-linked immunosorbent assay kits (Invitrogen, USA).

[0125] Figure 20 shows the Zr excretion profile corresponding to single and double administration of mRNA nanoparticle compositions. Zr content in harvested organs, urine, and blood is presented as % ID g–1. Detailed description of the embodiments

[0126] 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

[0127] For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0128] 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.

[0129] "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% 1005496795from the specified value, as such variations are appropriate to perform the disclosed methods.

[0130] Ranges: throughout this disclosure, various aspects of the disclosure 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 disclosure. 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.

[0131] As used herein the term “phenolic ligand” refers to a molecule having two or more hydroxyl groups bonded to ring carbon atoms. It also includes molecules having, in addition to the hydroxyl groups, other functional groups, such as, for example, carboxylic acid and ester groups.

[0132] As used herein, the term "targeted delivery," "targeted deliver," or "targeted delivering" means delivery of more (e.g., at least 1.5 fold more, at least 2-fold more, at least 3-fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold more, at least 10-fold more) of a therapeutic and / or prophylactic by a nanoparticle to a target tissue of interest (e.g., mammalian lung) compared to an off-target tissue (e.g., mammalian liver). The level of delivery of a nanoparticle to a particular tissue may be measured, for example, by comparing the amount of protein produced in a tissue to the weight of said tissue, comparing the amount of therapeutic and / or prophylactic in a tissue to the weight of said tissue, comparing the amount of protein produced in a tissue to the amount of total protein in said tissue, or comparing the amount of therapeutic and / or prophylactic in a tissue to the amount of total therapeutic and / or prophylactic in said tissue.The present disclosure describes nanoparticle compositions comprising phenolic ligands, including natural phenolic ligands, which may form specific interactions with diverse bioactive molecules, including protein cargo. Many phenolic ligands are naturally abundant molecules that are characterized by multiple hydroxyl groups on ring carbons. This unique structure imparts phenolic ligands high binding affinity through diverse 1005496795interactive forces endowing an ability to complex with other molecules. When phenolic ligands coordinate with metal ions, they may form a stable, negatively charged network structure (metal-phenolic ligand network) under physiological conditions.

[0133] As used herein, “gene editing” refers to any methods by which a genomic sequence is modified by insertion, deletion or replacement at a selected locus by using at least an enzyme that cleaves phosphodiester bond within a polynucleotide chain. A “gene editing complex” refers to one or more therapeutically applicable genome editing molecules that together result in targeted gene editing.

[0134] The present invention is based on the discovery of a modular and noncationic metal–organic nanoparticle (NP) platform, ie bioactive nanoparticle compositions, for both in vitro and in vivo delivery of bioactive agents. Herein, nanoparticle compositions have been developed for bioactive agent delivery, including, for example, protein and mRNA delivery. In embodiments, non-cationic bioactive nanoparticle compositions are prepared using poly(ethylene glycol) (PEG) as a seeding agent which may enhance the local concentration and drive the self-assembly of bioactive agent, phenolic ligands, metal ions, and PEG into robust, negatively charged nanoparticles. The synthesis does not involve any organic solvents and may occur rapidly (less than or equal to 30 min).

[0135] When packaged with mRNA, these bioactive nanoparticle compositions, are approximately 200 nm in size and negatively charged. Surprisingly, the mRNA bioactive nanoparticle compositions have been demonstrated to have superior transfection efficiency in vitro compared to existing commercial transfection agents and a comparable in vivo transfection level to lipid-based nanoparticles without causing significant toxicity or inflammation.

[0136] Surprisingly, through variation of the composition of the bioactive nanoparticles, the nanoparticles may be targeted to specific sites. In embodiments, the nanoparticle compositions demonstrated effective triggering of mRNA endosomal escape and transfection of mRNA of various nucleotide lengths and function. Transfection efficiency of over 90% in the HEK 293T cell line and approximately 50% in the hard-to-transfect suspension cell line, Jurkat, was observed with negligible cytotoxicity. In embodiments, mRNA-nanoparticle compositions comprising EGCG, Zr(IV), and 20 k linear PEG, achieved high in vitro transfection efficiency (close to 100%) and showed transfection in the liver, kidneys, heart, lung, spleen, and brain of 1005496795mice. The mRNA-nanoparticle compositions are therefore an attractive alternative to current mRNA delivery platforms and demonstrates the inherent versatility of the system to deliver mRNA in addition to protein and small molecule drug cargoes.

[0137] The present disclosure provides bioactive nanoparticle compositions comprising a metal ion-phenolic ligand assembly and one or more bioactive agents, wherein the metal ion-phenolic ligand assembly comprises one or more metal ions, one or more phenolic ligands, and one or more seeding agents. Metal ions

[0138] The presently disclosed nanoparticle compositions may comprise a single metal ion, or multiple metal ions. Examples include, but are not limited to, one or more of Cu(II), Fe(III), Zr(IV), Ti(IV), Ir(IV), Ta(V), W(VI), Bi(III), Zn(II), Mn(II), Gd(III), Pt(IV), Nb(V), Co(II), Cr(III), Tm(III), Al(III), and Na(I).

[0139] In some embodiments, the nanoparticle compositions comprise a single metal ion, such as Fe(III). In other embodiments, the nanoparticle compositions comprises more than one metal ion, for example, both Fe(III) and Zr(IV).

[0140] Numerous simple metal salts or complexes are suitable for use in preparing the nanoparticle compositions of the present disclosure. Phenolic ligands

[0141] In embodiments, the one or more phenolic ligands has a molecular weight between 110 and 3,000 Daltons.

[0142] In preferred embodiments where the bioactive agent is a nucleic acid, the one or more phenolic ligands has a molecular weight greater than 2,000 Daltons, preferably about 20,000 Daltons.

[0143] In some embodiments, the one or more phenolic ligands comprise three or more ring-bonded hydroxy groups.

[0144] In some embodiments, the one or more phenolic ligands comprise one or more galloyl groups and catechol groups. 1005496795

[0145] In embodiments, the one or more phenolic ligands comprise one or more naturally occurring phenolic ligands.

[0146] In some embodiments, the one or more phenolic ligands comprises one or more of tannic acid, (-)-epigallocatechin-3-gallate, catechin, gallocatechin, catechin-3- gallate, epigallocatechin, epicatechin-3-gallate, quercetin, gallic acid, procyanidin, ellagic acid, myricetin, fisetin, luteolin, and dopamine. Other suitable phenolic ligands are contemplated.

[0147] In some embodiments, the one or more phenolic ligands comprises catechin, preferably (+)-catechin hydrate. Seeding agents

[0148] In embodiments, the seeding agent comprises one or more polyethylene glycol or chemically functionalised polyethylene glycol. Other seeding agents are contemplated.

[0149] In some embodiments, the one or more chemically functionalised polyethylene glycol derivatives comprise one or more of amine, acid and ester functional groups. Other functional groups are contemplated.

[0150] In embodiments, the one or more polyethylene glycol or chemically functionalised polyethylene glycol has a molecular weight from 100 to 10,000,000 Daltons, or from 100 to 50,000 Daltons, or from 1,000 to 50,000 Daltons, or from 5,000 to 50,000 Daltons. Higher molecular weight polyethylene glycols or chemically functionalised polyethylene glycols may also be utilised.

[0151] In embodiments, the one or more polyethylene glycol or chemically functionalised polyethylene glycol has a linear, branched, star or comb structure. Bioactive agents

[0152] The bioactive nanoparticle compositions as described herein comprise one or more bioactive agents. The disclosure features methods of delivering bioactive agents to a mammalian cell or organ, optionally producing a polypeptide of interest in a mammalian cell, and treating a disease or disorder in a mammal in need thereof 1005496795comprising administering to a mammal and / or contacting a mammalian cell with a bioactive nanoparticle composition including a bioactive agent.

[0153] 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.

[0154] The present disclosure provides novel 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).

[0155] Advantageously, the bioactive nanoparticle compositions as described herein have been shown to be substantially non-toxic to a mammal. 1005496795

[0156] 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 single- stranded or double-stranded DNA, RNA, or a DNA / RNA hybrid.

[0157] 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. The relative amounts of nucleic acid molecules and other elements (e.g., e.g., polyethylene glycol or chemically functionalised polyethylene glycol, phenolic ligands, and metal ions) may also vary.

[0158] In any aspect or embodiment, the mass ratio of seeding agent to nucleic acid molecules in the bioactive nanoparticle composition is from about 10:1 to about 200:1. In certain embodiments, the mass ratio is from about 20:1 to about 100:1. In certain embodiments, the mass ratio is about 10:1, 20:1, 50:1, 100:1 or 200:1. Preferably the mass ratio is about 100:1.

[0159] In any aspect or embodiment, the mass ratio of phenolic ligand to nucleic acid in the bioactive nanoparticle composition is from about 10:1 to about 200:1. In certain embodiments, the mass ratio is from about 20:1 to about 100:1. In certain embodiments, the mass ratio is about 10:1, 20:1, 50:1, 100:1 or 200:1. Preferably the mass ratio is about 100:1.

[0160] 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.

[0161] In some embodiments, the bioactive agent is a therapeutic agent, or a salt or derivative thereof. Therapeutic agent derivatives may be therapeutically active 1005496795themselves 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.

[0162] Bioactive agents include small molecules, and biomacromolecules such as proteins, peptides, nucleic acids and enzymes. In some embodiments, the bioactive agent comprises single stranded RNA, such as, but not limited to, mRNA.

[0163] In some embodiments, the nanoparticle compositions of the present disclosure may be loaded with diagnostic radionuclide, fluorescent materials or other materials that are detectable in both in vitro and in vivo applications. For example, suitable reporter materials for use in the present disclosure may include firefly luciferase mRNA, mCherry mRNA, and mFlame mRNA. Composition of nanoparticles

[0164] The ratio of components in the presently disclosed nanoparticle compositions may vary widely, depending on the nature of the components.

[0165] In embodiments, the molar ratio of metal ion to phenolic ligand in the bioactive nanoparticle composition is from 0.001:1 to 100:1.

[0166] In embodiments, the molar ratio of metal ion to phenolic ligand in the bioactive nanoparticle composition is from 0.001:1 to less than 2:1.

[0167] In embodiments, the molar ratio of metal ion to phenolic ligand in the bioactive nanoparticle composition is from greater than 2:1 to 100:1.

[0168] In embodiments, the molar ratio of metal ion to phenolic ligand in the bioactive nanoparticle composition is from 0.002:1 to 1:1, or from 0.005:1 to 1:1, or from 0.01:1 to 1:1, or from 0.02:1 to 1:1, or from 0.03:1 to 1:1. The molar ratio of metal ion to phenolic ligand may be, for example, 0.01:1, 0.02:1, 0.03:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.10:1, 0.11:1, 0.12:1, 0.13:1, 0.14:1, 0.15:1, 0.16:1, 0.17:1, 0.18:1, 0.19:1, or 0.20:1. Such ratios are particularly useful when the bioactive agent is mRNA. However, other molar ratios are contemplated. 1005496795

[0169] In a preferred embodiment, the bioactive nanoparticle composition comprises a linear polyethylene glycol seeding agent having a molecular weight of 20,000 Dalton, (-)- epigallocatechin-3-gallate, mRNA, and one or more metal ions.

[0170] In a preferred embodiment, the mass ratio of the components is 100 PEG:1 mRNA:100 (-)-epigallocatechin-3-gallate. Other mass ratios are contemplated. Bioactive nanoparticles

[0171] The use of metal-phenolic networks 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 agent associated with a phenolic ligand and / or metal ion may be encapsulated in the interior of a nanoparticle, attached to a 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.

[0172] In any aspect or embodiment, the bioactive nanoparticle compositions may have a composition or property as described herein including the Examples. For example, the bioactive nanoparticle composition may be a broad or targeted bioactive nanoparticle as described in the Examples.

[0173] The mean size of a nanoparticle with the nanoparticle composition of the present disclosure may be greater than, or equal to about 40nm or 100nm e.g., measured by dynamic light scattering (DLS). For example, the mean size may be greater than, or equal to about, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm, 150 nm, 155 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205nm, 210nm, 215 nm, 220nm, 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, 350 nm, 355 nm, 360 nm, 365 nm, 370 nm, 375 nm, 380 nm, 385 nm, 390 nm, 395 nm, or 400 nm. 1005496795

[0174] In some embodiments, the mean size of a nanoparticle of the present disclosure may be from about 50nm to about 400nm, from about 60nm to about 400nm, from about 70nm to about 400nm, from about 80nm to about 400nm, from about 90nm to about 400nm, from about 100nm to about 400nm, from about 110nm to about 400nm, from about 120nm to about 400nm from, about 130nm to about 400nm from, about 140nm to about 400nm, from about 150nm to about 400nm, from about 160nm to about 400nm, from about 170nm to about 400nm, from about 180nm to about 400nm, from about 190nm to about 400nm, from about 200nm to about 400nm, from about 210nm to about 400nm, from about 220nm to about 400nm, from about 230nm to about 400nm, from about 240nm to about 400nm, from about 250nm to about 400nm, from about 260nm to about 400nm, from about 270nm to about 400nm, from about 280nm to about 400nm, from about 300nm to about 400nm, from about 310nm to about 400nm, from about 320nm to about 400nm, from about 330nm to about 400nm, from about 340nm to about 400nm, from about 350nm to about 400nm, from about 360nm to about 400nm, from about 370nm to about 400nm, from about 380nm to about 400nm, from about 390nm to about 400nm, from about 410nm to about 400nm, from about 420nm to about 400nm, from about 430nm to about 400nm, from about 440nm to about 400nm, from about 450nm to about 400nm, from about 460nm to about 400nm, from about 470nm to about 400nm, from about 480nm to about 400nm, or from about 490nm to about 400nm.

[0175] In some embodiments, the mean size of a nanoparticle of the present disclosure may be from about 150nm to about 400nm, from about 150nm to about 390nm, from about 150nm to about 380nm, from about 150nm to about 370nm, from about 150nm to about 360nm, from about 150nm to about 350nm, from about 150nm to about 340nm, from about 150nm to about 330nm, from about 150nm to about 320nm, from about 150nm to about 310nm, from about 150nm to about 300nm, from about 150nm to about 290nm, from about 150nm to about 280nm, from about 150nm to about 270nm, from about 150nm to about 260nm, from about 150nm to about 250nm, from about 150nm to about 240nm, from about 150nm to about 230nm, from about 150nm to about 220nm, from about 150nm to about 210nm, or from about 150nm to about 200nm.

[0176] A nanoparticle may be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size 1005496795distribution of the nanoparticle compositions. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A nanoparticle composition may have a polydispersity index from about 0 to about 0.25, such as 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. In some embodiments, the polydispersity index of a nanoparticle composition may be from about 0.10 to about 0.20.

[0177] As used herein, the "zeta potential" is the electrokinetic potential of a nanoparticle.

[0178] 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.

[0179] In some embodiments, the zeta potential of a nanoparticle may be from about -65 mV to about 0 mV, preferably about -65mV to about -5mV. In some embodiments, the zeta potential of a nanoparticle may be from -65 mV to +0 mV, preferably -30mV to - 5mV, or -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 -10MV, 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 across a broad pH range. For example the bioactive nanoparticles may have a zeta potential as described herein at a pH of about 1 to about 14, about 2 to 14, about 3 to about 14, about 4 to about 14, about 5 to about 14, about 6 to about 14, about 7 to about 14, about 8 to about 14, about 9 to about 14, or about 10 to about 14. Alternatively, the bioactive nanoparticles may have a zeta potential as described herein at a pH of about 2 to about 13, about 2 to about 12, about 2 to about 11, about 2 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, or about 2 to about 6. Alternatively again, the bioactive 1005496795nanoparticles may have a zeta potential as described herein at a pH of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. Preferably, the bioactive nanoparticles have a zeta potential as described herein at a pH of about 3 to about 12, about 3 to about 11, about 3 to about 10, about 4 to about 12, about 4 to about 11, or about 4 to about 10. Even more, preferably about 4 to about 9.

[0180] The bioactive nanoparticle compositions may be any one described herein including those listed in the Examples, e.g. Example 29. Preparation of nanoparticle compositions

[0181] The nanoparticle compositions may be prepared by combining, in any order, one or more sources of metal ions, one or more phenolic ligands, one or more bioactive agents, and one or more seeding agents.

[0182] In some embodiments of the method the metal ion source is added to a mixture of phenolic ligand, bioactive agent and seeding agent. In this method (referred to as co-assembly (Co-A)) the metal ion source is added in the presence of bioactive agent.

[0183] In other embodiments of the method the bioactive agent is added to a mixture of phenolic ligand, metal ion source, and seeding agent. In this method (referred to as post-assembly or Post-A) the bioactive agent is added to a preformed nanoparticle composition. Pharmaceutical Compositions

[0184] Bioactive nanoparticle compositions may be formulated in whole or in part as pharmaceutical compositions. Pharmaceutical compositions may include one or more bioactive nanoparticles as described herein. 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 such as those described herein. 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, 21stEdition, A. R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Conventional excipients and 1005496795accessory 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 a bioactive nanoparticle. An excipient or accessory ingredient may be incompatible with a component of a bioactive nanoparticle if its combination with the component may result in any undesirable biological effect or otherwise deleterious effect.

[0185] 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 bioactive nanoparticle. For example, the one or more excipients or accessory ingredients may make up 50%, 60%, 70%, 80%, 90%, or more of a pharmaceutical convention. 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.

[0186] Relative amounts of the one or more bioactive nanoparticles, the 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 bioactive nanoparticles.

[0187] Bioactive nanoparticles as described herein, including pharmaceutical compositions including bioactive nanoparticles may be administered to any patient or subject, including those patients or subjects that may benefit from a therapeutic or prophylactic effect provided by the delivery of a bioactive agent to one or more particular cells, tissues, organs, or systems or groups thereof, such as the lung and brain. Although the descriptions provided herein of bioactive nanoparticles and pharmaceutical compositions including bioactive nanoparticles are principally directed to compositions which are suitable for administration to humans, it will be understood by 1005496795the 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.

[0188] A pharmaceutical composition including one or more bioactive 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.

[0189] 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., bioactive nanoparticle). 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.

[0190] 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. 1005496795Application and Administration

[0191] 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.

[0192] The present disclosure provides a method for delivering one or more bioactive agents to a target cell or tissue comprising bringing a bioactive nanoparticle composition according to any one of the herein disclosed embodiments into contact with the cell or tissue.

[0193] In embodiments, the target cell may be a 2D or 3D cultured cell.

[0194] The present disclosure further provides a method of delivering bioactive nanoparticles to one or more target sites in a subject comprising administering the bioactive nanoparticle composition according to any one of the herein disclosed embodiments to a subject.

[0195] In embodiments, the bioactive nanoparticle composition may be administered intravenously, subcutaneously, intramuscularly, intranasally, intratracheally, intraperitoneally, intratumorally, or intracerebrally.

[0196] In embodiments of the methods the bioactive agent comprises single stranded RNA.

[0197] In embodiments of the methods the bioactive agent comprises mRNA.

[0198] In embodiments of the methods, the target site is one or more of liver, kidney, lung, brain, spleen, heart, pancreas, stomach, small intestine, large intestine, lymph node, bone, bone marrow, skeletal muscle, bladder, breast, testes, ovaries, uterus, thymus, brainstem, cerebellum, spinal cord, eye, ear, tongue, prostate and skin.

[0199] In embodiments of the methods, the mRNA transfection efficiency is greater than 25%, or greater than 50%, or greater than 75%, or greater than 85%, or greater than 95%.

[0200] The compositions and methods of the present disclosure provide for the delivery of mRNA to treat a number of disorders. In embodiments, the compositions and methods of the present disclosure are suitable for the treatment of diseases or disorders 1005496795relating to the deficiency of proteins and / or enzymes that are excreted or secreted by the target cell into the surrounding extracellular fluid (e.g., mRNA encoding hormones and neurotransmitters).

[0201] In embodiments, the compositions of the present disclosure exhibit high mRNA transfection efficiency. Transfection efficiencies of almost 100% may be realised.

[0202] The bioactive nanoparticle compositions as disclosed herein can be used to deliver one or more components of a gene editing complex in vivo and in vitro. In particular, these gene editing complexes can be used to mutate any gene of interest, by delivering a gene editing system to target cells. The bioactive nanoparticle compositions as described herein may be used for the delivery of DNA or RNA molecules for gene targeted insertion of a polynucleotide sequence encoding a polypeptide of interest at a desired locus by Non-homologous end joining or homologous recombination.

[0203] In one embodiment, a subject in need thereof can be administered one or more bioactive nanoparticle compositions as described herein containing different components of a gene editing complex.

[0204] The bioactive nanoparticle compositions of the present invention may be useful for treating a disease, disorder, or condition. In particular, such compositions may be useful in treating a disease, disorder, or condition characterized by missing or aberrant protein or polypeptide activity. For example, a bioactive nanoparticle composition comprising a bioactive agent, such as mRNA encoding a missing or aberrant polypeptide, may be administered or delivered to a cell. Subsequent translation of the mRNA may produce the polypeptide, thereby reducing or eliminating an issue caused by the absence of or aberrant activity caused by the polypeptide. A bioactive agent included in a nanoparticle composition may also be capable of altering the rate of transcription of a given species, thereby affecting gene expression.

[0205] Diseases, disorders, and / or conditions characterized by dysfunctional or aberrant protein or polypeptide activity 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 1005496795conditions 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.

[0206] The disclosure provides methods involving administering bioactive nanoparticles including one or more bioactive 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 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.

[0207] A bioactive nanoparticle including one or more bioactive agents may be administered by any route. In some embodiments, compositions, including prophylactic, diagnostic, or imaging compositions including one or more bioactive 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, 1005496795intraocular, 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. Examples Materials

[0208] Dulbecco’s phosphate-buffered saline (DPBS), Ultrapure DNase / RNase free distilled water, Dulbecco’s modified Eagle medium (DMEM), Reduced-Serum Medium (Opti-MEM) and Roswell Park Memorial Institute (RPMI)-1640 medium were purchased from Life Technologies (USA). (-)-epigallocatechin-3-O-gallate (EGCG), tannic acid (TA), (+)-gallocatechin (CAT), gallic acid monohydrate (GA), sodium chloride (NaCl), copper (II) chloride tetrahydrate (CuCl2·2H2O), cobalt (II) chloride (CoCl2), thulium (III) chloride hexahydrate (TmCl3·6H2O), chromium (III) chloride hexahydrate (CrCl3·6H2O), titanium (IV) bis(ammonium lactato)dihydroxide, zirconium (IV) chloride (ZrCl4), iron(III) chloride hexahydrate (FeCl3·6H2O), zinc (II) chloride (ZnCl2), Manganese (II) chloride (MnCl2), bismuth (III) chloride (BiCl3), Gadolinium (III) chloride hexahydrate (GdCl3·6H2O), Iridium (IV) chloride (IrCl4), platinum (IV) chloride (PtCl4), niobium (V) chloride (NbCl5), tantalum (V) chloride (TaCl5), tungsten (VI) chloride (WCl6), poly(ethylene glycol) (PEG; 2k linear, 20k linear), agarose (low EEO), ethylenediaminetetraacetic acid (EDTA), urea, Rhodamine 800 (Rh800),3 (N- morpholino)propanesulfonic acid (MOPS), bovine serum albumin (BSA), ovalbumin (OVA), and lysozyme (LYZ), glucose oxidase (Gox), catalase (Cat), and TWEEN 20 were purchased from Sigma-Aldrich (USA). PEG (2k 4-arm) was purchased from JenKem (USA). Heparin sodium was purchased from Jomar Life Research (Australia). mCherry mRNA, firefly luciferase mRNA, NGFR mRNA, mFlame mRNA (also referred 1005496795to as “mScarlet3” herein), Cre-recombinase (both mRNA and proteins), and Cy5- labelled mRNA were purchased from Messenger Bio (Australia). All chemicals were used as received without further purification. All aqueous solutions were filtered with 220 nm diameter membranes before the in-vivo experiments.

[0209] A number of bioactive nanoparticle compositions according to the present disclosure were prepared and studied. The nature of the metal ion, the nature of the phenolic ligand, the nature of the protein cargo, and the phenolic ligand to metal ion ratio, were examined in formulations using a linear 2k MW PEG as seeding agent. The following examples 1 to 4 detail the procedures and results. Example 1: Fabrication of TA-based BSA nanoparticle compositions with different metal ions

[0210] TA-based BSA nanoparticle compositions were prepared through a one-pot assembly protocol. Briefly, PEG (200 μL, MW 2 kDa, 20 mg mL−1), BSA (100 μL, 10 mg mL−1), Rh800 (200 μL, 23.8 μg mL−1), and FeCl3·6H2O (64 μL, 5 mg mL−1) were successively added to a vial containing Milli-Q water (2 mL). The assembly was initiated by introducing TA (200 μL, 5 mg mL−1) into the vial (Note: the Fe(III)-to-TA molar ratio was 2:1). The nanoparticle composition was then obtained by stirring the mixture for 10 min at room temperature. High-speed centrifugation (8000 g, 10 min) was performed to remove excess materials, and the resultant TA / Fe(III)-based BSA nanoparticle composition was finally dispersed in 2 mM of MOPS (pH7) for future use.

[0211] To replace Fe(III) with the other metal ions (except Ti(IV), Ir(IV), and W(VI)): the method and other components remained the same, while simply replacing FeCl3·6H2O with CoCl2 (30.8 μL, 5 mg mL−1), CuCl2·2H2O (40 μL, 5 mg mL−1), CrCl3·6H2O (63 μL, 5 mg mL−1), TmCl3.6H2O (63.4 μL, 7.1 mg mL−1), or ZrCl4 (55 μL, 5 mg mL−1), ZnCl2 (32 μL, 5 mg mL−1), MnCl2 (46.2 μL, 5 mg mL−1), NbCl5(45 μL, 7 mg mL−1in ethanol), TaCl5 (26.5 μL, 16 mg mL−1in ethanol) (Note: the molar ratio of metal ions to TA was 2:1). Nanoparticle compositions assembled from Cu(II), Mn(II), Zn(II), and Cr(III) were dispersed in 2 mM of MOPS (pH7), while those assembled from other metal ions were dispersed in Milli-Q water for future use.

[0212] To replace Fe(III) with Ti(IV), Ir(IV), or W(VI): PEG (200 μL, 2 kDa, 20 mg mL−1), BSA (100 μL, 10 mg mL−1), Rh800 (200 μL, 23.8 μg mL−1), TA, and titanium(IV) 1005496795bis(ammonium lactato)dihydroxide (i.e, Ti(IV)) (69.1 μL, 5 mg mL−1), Ir(IV) (79 μL, 5 mg mL−1), or W(VI)(232.5 μL, 1 mg mL−1in methanol) were successively added to a vial containing 2 mL of Milli-Q water and 500 μL of DPBS. Note, the metal ion-to-TA molar ratio was 2:1. Then, TA / Ti(IV), TA / Ir(IV), or TA / W(VI)-based BSA-nanoparticle compositions were obtained by stirring the mixture for 10 min at room temperature. The purification method was the same as for Fe(III), and the resultant nanoparticle composition suspension was re-dispersed in Milli-Q water for future use.

[0213] The nanoparticle compositions were injected into C57BL / 6J mice (female, 8–10 weeks of age) via intravenous administration at the dosage of 10 mg kG−1, while the same volume of DPBS was injected as a negative control. After 4 h, major organs, including the spleen, liver, kidney, lung, heart, and brain, were harvested, followed by IVIS imaging. The biodistribution of nanoparticle compositions is presented (Figure 1) as the colocalized image of organ and fluorescence signal from Rh800.

[0214] Table 1 summarises the formulations prepared and the primary organ deposition observed. In the absence of metal ion the compositions were primarily deposited in the liver. However, compositions comprising metal ions were deposited in different organs and variation of the metal ion influenced the particular location. Table 1 Component Component Component Component Component Fabrication Primary organ 1: 2: 3: 4: mass ratio method deposition PEG Model Metal ion Phenolic protein ligand Linear, 2k BSA None TA 4:1:0:1 Sequentially add Liver components 1, 2, 3, and 4 Linear, 2k BSA Cu(II) TA 4:1:0.2:1 Sequentially add Even distribution components 1, in the spleen, 2, 3, and 4 liver, and kidney Linear, 2k BSA Fe(III) TA 4:1:0.32:1 Sequentially add Kidney components 1, 2, 3, and 4 Linear, 2k BSA Zr(IV) TA 4:1:0.275:1 Sequentially add Lung components 1, 2, 3, and 4 Linear, 2k BSA Ti(IV) TA 4:1:0.345:1 Sequentially add Not tested components 1, 2, 3, and 4 1005496795Table 1 Component Component Component Component Component Fabrication Primary organ 1: 2: 3: 4: mass ratio method deposition PEG Model Metal ion Phenolic protein ligand Linear, 2k BSA Co(II) TA 4:1:0.154:1 Sequentially add Lung components 1, 2, 3, and 4 Linear, 2k BSA Cr(III) TA 4:1:0.32:1 Sequentially add Kidney components 1, 2, 3, and 4 Linear, 2k BSA Tm(III) TA 4:1:0.45:1 Sequentially add Even distribution components 1, in spleen, liver 2, 3, and 4 and kidney Linear, 2k BSA Ti(IV) TA 4:1:0.345:1 Sequentially add Lung components 1, 2, and 4, and then add component 3 to stabilise NPs Linear, 2k BSA Bi(III) TA 4:1:0.37:1 Sequentially add Even distribution components 1, in spleen, liver, 2, 3, and 4 and kidney Linear, 2k BSA Ir(IV) TA 4:1:0.395:1 Sequentially add Lung components 1, 2, 4, and 3 to stabilize NPs Linear, 2k BSA Ta(V) TA 4:1:0.424:1 Sequentially add Lung components 1, 2, 3, and 4 Linear, 2k BSA W(VI) TA 4:1:0.233:1 Sequentially add Lung components 1, 2, 4, and 3 to stabilize NPs Linear, 2k BSA Zn(II) TA 4:1:0.16:1 Sequentially add Kidney (and components 1, small portion in 2, 3, and 4 the brain) Linear, 2k BSA Mn(II) TA 4:1:0.231:1 Sequentially add Kidney (and a components 1, small portion in 2, 3, and 4 the brain) Linear, 2k BSA Gd(III) TA 4:1:0.44:1 Sequentially add Broad components 1, distribution in 2, 3, and 4 spleen, liver, kidney, lung, and heart Linear, 2k BSA Pt(IV) TA 4:1:0.313:1 Sequentially add Broad components 1, distribution in 1005496795Table 1 Component Component Component Component Component Fabrication Primary organ 1: 2: 3: 4: mass ratio method deposition PEG Model Metal ion Phenolic protein ligand 2, 4, and 3 to spleen, liver, stabilize NPs kidney, lung, and heart Linear, 2k BSA Nb(V) TA 4:1:0.315:1 Sequentially add Broad components 1, distribution in 2, 3, and 4 spleen, liver, kidney, lung, and heart Example 2: Fabrication of TA / Ti(IV)-based nanoparticle compositions with different TA-to-Ti(IV) molar ratio

[0215] The fabrication protocol was the same as per described for TA / Ti(IV)-based BSA nanoparticle compositions in Example 1. The molar amounts of other components were fixed, while only altering the Ti(IV) amount to change the Ti(IV)-to-TA molar ratio (i.e., 0:1, 0.1:1, 0.5:1, 1:1, and 2:1). The nanoparticle compositions were purified with Milli-Q water via centrifugation (8000 g, 10 min), and the resultant suspension was re- dispersed in Milli-Q water for future use. Organ deposition studies were performed as in Example 1. Table 2 collects the results and Figure 2 illustrates the biodistribution. Variation of the metal ion to phenolic ligand ratio over a wide range did not influence primary organ deposition location, but an increase in metal ion to phenolic ligand ratio did indicate stronger targeting capability. Table 2 Component Component Component Component TiIVto TA Fabrication Primary organ 1: 2: 3: 4: molar ratio method deposition PEG Cargo Metal ion Phenolic ligand Linear, 2k BSA none TA 0:1 Sequentially add Liver components 1, 2, and 4 Linear, 2k BSA Ti(IV) TA 0.1:1 Sequentially add Lung components 1, 2, and 4 and then add component 3 Linear, 2k BSA Ti(IV) TA 0.5:1 Sequentially add Lung components 1, 2, and 4 and then add component 3 1005496795Linear, 2k BSA Ti(IV) TA 1:1 Sequentially add Lung components 1, 2, and 4 and then add component 3 Linear, 2k BSA Ti(IV) TA 2:1 Sequentially add Lung components 1, 2, and 4 and then add component 3 Example 3: Fabrication of TA / Ti(IV)-based nanoparticle compositions with or without different proteins

[0216] The fabrication protocol was the same as per described for TA / TiIV-based nanoparticle compositions in Example 1, except either removing BSA or replacing BSA with LYZ (100 μL, 2.1 mg mL−1) or OVA (100 μL, 6.8 mg mL−1), GOx (50 μL, 1.28 mg mL−1in PBS), or Cat (50 μL, 2 mg mL−1in PBS). Further, the order of addition of the components was sequentially mixing PEG, protein and phenolic ligand, followed by addition of the metal ion (Co-A protocol). The nanoparticle compositions were purified with Milli-Q water via centrifugation (8000 g, 10 min), and the resultant suspension was re-dispersed in Milli-Q water for future use. Organ deposition studies were performed as in Example 1. Table 3 collects the results and Figure 3 illustrates the biodistribution. Variation of the protein cargo did not affect primary organ deposition location. Table 3 Component Component Component Component Component Fabrication Primary 1: 2: 3: 4: mass ratio method organ PEG Cargo Metal ion Phenolic deposition ligand Linear, 2k BSA Ti(IV) TA 4:1:0.345:1 Sequentially mix Lung components 1, 2, 4 to form NPs, and then add component 3 to stabilize NPs Linear, 2k No protein Ti(IV) TA 4:0:0.345:1 Sequentially mix Lung cargo components 1, 2, 4 to form NPs, and then add component 3 to stabilize NPs Linear, 2k OVA Ti(IV) TA 4:0.68:0.345:1 Sequentially mix Lung components 1, 2, 4 to form NPs, and then add component 3 to stabilize NPs Linear, 2k LYZ Ti(IV) TA 4:0.2:0.345:1 Sequentially mix Lung components 1, 2, 4 to form NPs, 1005496795and then add component 3 to stabilize NPs Linear, 2k Luc Ti(IV) TA 4:0.05:0.345:1 Sequentially mix Lung components 1, 2, 4 to form NPs, and then add component 3 to stabilize NPs Linear, 2k Gox Ti(IV) TA 4:0.064:0.345: Sequentially add Lung 1 components 1, 2, 4, and 3 to stabilize NPs Linear, 2k Cat Ti(IV) TA 4:0.1:0.345:1 Sequentially add Lung components 1, 2, 4, and 3 to stabilize NPs Example 4: Fabrication of Ti-based BSA-nanoparticle compositions with different phenolic ligands

[0217] The fabrication protocol was the same as per described in Example 3, except either replacing TA with EGCG (180 μL, 5 mg mL−1), CAT (200 μL, 5 mg mL−1; Note: CAT should be dissolved in ethanol first to prepare 50 mg mL−1stock solution and diluted to 5 mg mL−1in Milli-Q water) or GA (250 μL, 4 mg mL−1). The nanoparticle compositions were purified with Milli-Q water via centrifugation (8000 g, 10 min), and the resultant nanoparticle suspension was re-dispersed in Milli-Q water for future use. Organ deposition studies were performed as in Example 1. Table 4 collects the results and Figure 4 illustrates the biodistribution. The selection of phenolic ligand influenced the primary organ deposition location. 1005496795Table 4 Component Component Component Component Component Fabrication Primary organ 1: 2: 3: 4: mass ratio method deposition PEG Cargo Metal ion Phenolic ligand Linear, 2k BSA Ti(IV) TA 4:1:0.345:1 Sequentially mix Lung components 1, 2, 4 to form NPs, and then add component 3 to stabilize NPs Linear, 2k BSA Ti(IV) EGCG 4:1:0.345:0.9 Sequentially mix Kidney and components 1, 2, heart 4 to form NPs, and then add component 3 to stabilize NPs Linear, 2k BSA Ti(IV) GA 4:1:0.345:1 Sequentially mix Kidney and components 1, 2, heart 4 to form NPs, (including a and then add small portion in component 3 to the brain) stabilize NPs Linear, 2k BSA Ti(IV) CAT 4:1:0.345:1 Sequentially add Broad components 1, 2, distribution 4, and 3 to (including a stabilize NPs small portion in the brain) Example 5: Fabrication of mRNA-Metal ion–Phenolic Nanoparticle compositions

[0218] 20k MW linear PEG (20 μL, 10 mg mL−1in RNase-free water) and mRNA (2 μL, 1 mg mL−1in citric buffer) were mixed in a 1.7 mL Eppendorf tube. EGCG (40 μL, 5 mg mL−1in RNase-free water) was introduced to initiate the assembly, followed by addition of ZrCl4 (10 μL, 0.5 mg mL−1in RNase-free water). The mixture was then kept on ice for 30 min. For in vitro mRNA transfection during the formulation screen, the mixture was directly added to cells without purification. For mRNA-nanoparticle composition characterization and in vivo studies, particles were purified by centrifugation (8000 rcf, 5 min). The resulting pellet was dispersed in 100 μL RNase-free water, followed by 10 s sonication to fully resuspend the nanoparticles. The mass ratio of each component was fixed PEG:mRNA:metal ion:phenolic ligand at 100:1:2.5:100 unless otherwise specified. Figure 5(a) illustrates the phenolic ligands, seeding agents (PEGs), and metal ions utilised for the assembly of the nanoparticle compositions, and the synthesis of mRNA- nanoparticle compositions through metal-phenolic ligand-mediated assembly. Table 5 collects the results and Figure 8(a) illustrates the mRNA expression in different organs. 1005496795mRNA-nanoparticle compositions were injected into C57BL / 6J mice (female, 8–10 weeks of age) via intravenous administration, while the same volume of DPBS was injected as a negative control. After 24 h, major organs, including the liver (Li), kidney (K), lung (Lu), heart (H), spleen (S), and brain (B) were harvested, followed by IVIS imaging (Perkin Elmer). Unless otherwise stated, the dosage was 5 µg of mFlame per mouse.

[0219] In these studies, fluorescence comes from the fluorescence protein encoded by the delivered mRNA. The fluorescence can only be detected if the mRNA transfection is successful (i.e., the fluorescence intensity indicates the level of mRNA expression). Table 5 Component Component Component Component Component Fabrication mRNA transfection 1: 2: 3: 4: mass ratio method PEG mRNA Metal ion Phenolic ligand Linear, 20k mFlame ZrIVEGCG 100:1:2.5:100 Co-A Both in vitro and in vivo Primary organs showing mRNA expression: Liver, kidney, (including a small portion in the brain) Linear, 20k mFlame ZrIVEGCG 100:1:2.5:100 Post-A Both in vitro and in vivo Primary organs showing mRNA expression: Liver, kidney Linear, 20k mFlame TiIVEGCG 100:1:2.5:100 Co-A Both in vitro and in vivo Primary organs showing mRNA expression: Liver, kidney 4-arm, 2k mFlame ZrIVEGCG 100:1:2.5:100 Co-A Both in vitro and in vivo Primary organs showing mRNA expression: spleen Linear, 20k mFlame TmIIIEGCG 100:1:2.5:100 Co-A Both in vitro and in vivo Primary organs showing mRNA expression: Liver and lung 1005496795Table 5 Component Component Component Component Component Fabrication mRNA transfection 1: 2: 3: 4: mass ratio method PEG mRNA Metal ion Phenolic ligand Linear, 20k mFlame CoIIEGCG 100:1:2.5:100 Co-A Both in vitro and in vivo Primary organs showing mRNA expression: Lung Linear, 20k mFlame none EGCG 100:1:0:100 Co-A Both in vitro and in vivo Overall low level of mRNA expression in vivo. It showed a small portion in the brain Linear, 20k mFlame ZrIVEGCG 100:1:7.5:100 Co-A Both in vitro and in vivo Primary organs showing mRNA expression: Kidney

[0220] Note: All formulations showed a small portion of mRNA expression in the brain, but one preferred formulation (i.e., Linear 20 k PEG, mFlame, Zr(IV), EGCG, mass ratio 100:1:2.5:100, Fabrication method: Co-A ) demonstrated the highest level of expression. The formulation absent metal ion showed a low level of mRNA expression in vivo. Example 6: Effect of metal ion to EGCG ratio

[0221] A series of nanoparticle formulations were prepared using the Co-A protocol with 20 kDa linear PEG, mFlame, EGCG, and Zr(IV), and varying the metal ion to EGCG ratio (mass ratios 0:1, 0.025:1, and 0.075:1, corresponding to molar ratios 0:1, 0.05:1, and 0.15:1). The results of mRNA expression in harvested organs are illustrated in Figure 8(b) and Figure 13(c). 1005496795Example 7: Effect of metal ions

[0222] A series of nanoparticle formulations were prepared using the Co-A protocol with 20 kDa linear PEG, mFlame, EGCG, and varying the metal ion. The results of mRNA expression in harvested organs are illustrated in Figure 8(c) and Figure 13(f-h). Example 8: Effect of PEG seeding agent

[0223] A series of nanoparticle formulations were prepared using the Co-A protocol with mFlame, EGCG, Zr(IV), and varying the PEG seeding agent. The results of mRNA expression in harvested organs are illustrated in Figure 8(d) and Figure 13(i). Example 9: Characterization of mRNA-nanoparticle compositions

[0224] To determine the mRNA loading capacity of mRNA nanoparticle compositions, the mixture of PEG, mRNA, phenolic ligand, and metal ion was diluted with RNase-free water and loaded on 1% agarose gels with a concentration of 200 ng mRNA per well. A Zetasizer Nano-ZS instrument (Malvern Instrument, UK) was used to measure the hydrodynamic diameter (number mean), polydispersity index (PDI) and ζ potential of mRNA-nanoparticle compositions at different buffered pH buffers (10mM). UV–Vis absorption spectra were recorded on a Specord 250 Plus spectrophotometer (Analytik Jena AG, Germany). Fourier transform infrared (FTIR) spectroscopy analysis was performed on a Tensor II FTIR spectrometer (Bruker Optics, US). Super-resolution fluorescence images of the particles in the aqueous phase were obtained on a structured illumination microscope (Elyra 7 Lattice SIM with Lattice SIM2processing function, Zeiss).

[0225] Figure 5(b) illustrates the loading efficiency of mCherry-encoding mRNA in nanoparticle compositions (assembled with 20k linear PEG, EGCG, and Zr(IV)) at different phenolic ligand to mRNA mass ratios. The inset is the image of agarose gel bands showing uncomplexed mRNA in the system. At a phenolic ligand to mRNA mass ratio of 100:1, the loading efficiency was close to 100%.

[0226] Figure 5(c) is a histogram of mCherry fluorescence of HEK 293T cells transfected by mRNA-nanoparticle compositions prepared with different phenolic ligands (assembled with 20k linear PEG and Zr(IV)) after 24h. Figure 5(d) illustrates mCherry mean fluorescence intensity. 1005496795

[0227] Figure 5(e) is a histogram of mCherry fluorescence from HEK 293T cells transfected by mRNA nanoparticle compositions prepared with different PEGs of different molecular weight and architecture (with formulation PEG:mRNA:EGCG: ZrIVat 100:1:100:2.5 mass ratio) after 24h and 5(f) corresponding mean mCherry fluorescence intensity. The inset is the image of agarose bands showing loading efficiency of formulations with different PEGs.

[0228] Figure 5(g) is a histogram of mCherry fluorescence and 5(h) mean mCherry fluorescence intensity of HEK 293T cells transfected by mRNA nanoparticle compositions with different metal ion concentrations (fixed with 20k linear PEG, EGCG, and ZrIV) after 24h. The inset is the image of agarose bands showing loading efficiency of formulations with different metal ion-to-EGCG mass ratios.

[0229] Figure 5(i) illustrate transfection efficiency (%) and 5(j) mean mCherry fluorescence intensity of HEK 293T cells transfected by mRNA nanoparticle compositions assembled with various metal ions (with formulation 20k linear PEG:mRNA:EGCG:metal ion at 100:1:100:2.5 mass ratio) after 24h.

[0230] Figure 6(c) shows the UV-vis spectra of free EGCG, EGCG-PEG complex, and mRNA-nanoparticle composition (20k linear PEG:mRNA:EGCG:Zr(IV) at 100:1:100:2.5 mass ratio). The green region highlights the ligand-to-metal charge transfer (LMCT) band in the spectrum of mRNA-nanoparticle composition. Figure 6(d) shows the size distribution of nanoparticle compositions measured by DLS. Insets are structured illumination microscopy (SIM) images of nanoparticles assembled using the two routes. Red signal stems from Cy5-labelled mRNA. Scale bars are 200 nm. Figure 6(e) illustrates mRNA loading efficiency of these mRNA-nanoparticle compositions assembled via Co-A or Post-A. Example 10: In Vitro Release Profile

[0231] To study the kinetics of mRNA release, mRNA-nanoparticle compositions with Cy5-labelled mRNA were incubated in different media (RNase-free water, DPBS, and DPBS with 10% FBS) at 37 ˚C. At each different time point, the particles were centrifuged at 8000 rcf for 5 min. The supernatant was removed for fluorescence measurements and replaced with the same amount of fresh medium for the next time point. The fluorescence of the collected supernatant was measured using an Infinite 1005496795M200 microplate reader (Tecan, Switzerland), and the concentration of Cy5-labelled mRNA in the supernatant was calculated using a standard curve. Figure 6 (f and g) illustrate the results. Example 11: Disassembly Assay

[0232] To determine the dominant interactive forces for mRNA-nanoparticle composition assembly, the particles were incubated in NaCl, EDTA, Tween 20, and urea (100 mM). After 24 h, the mRNA-nanoparticle compositions in different solutions were then loaded into 1% agarose gels. The gels were run at 90V for 45 min. Bands of naked mRNA and untreated mRNA-nanoparticle compositions were normalized to 100% and 0% disassembly respectively. Band intensity was obtained and analysed by Image J software. Figure 6(h) illustrates the results. Example 12: Cell culture

[0233] HEK 293T cells with low passage numbers (passages: 14-25) were cultured in DMEM medium supplemented with 10% (v / v) fetal bovine serum (FBS,) and 1% (v / v) penicillin / streptomycin (VWR). Jurkat cells (passages: 11-15) were cultured in supplemented RPMI 1640. Cells were grown in 37 °C, 5% CO2, and 95% humidity. Example 13: In Vitro Transfection

[0234] Two reporter genes including Firefly luciferase and mCherry, and one functional gene NGFR were used to evaluate the mRNA transfection efficiency of mRNA- nanoparticle compositions. Before transfection, HEK 293T cells were seeded at a density of 6×104cells per well in 48-well plates and Jurkat cells were seeded at a density of 3×104cells per well in 96-well plates. After overnight incubation, 80%-90% cell confluency was reached. The cell media was aspirated and 300 µL of mRNA- nanoparticle compositions in serum free media (Opti-MEM) was added for a final mRNA concentration of 1 µg / mL. Equivalent concentration of mRNA was mixed with the commercial transfection reagent RNAiMax according to the product manual as positive control. Naked mRNA (equivalent concentration) was also set as control. After 6 h of incubation, serum-free media were aspirated and replaced with complete DMEM (RPMI-1640 for Jurkat). Transfection was then continued for another 18 h, and then the cells were washed with PBS for 3 times, trypsinized, and transferred into a 96-well plate for washing by centrifugation and FACS analysis. 1005496795

[0235] For CLSM imaging following mCherry mRNA transfection, cells were seeded in 8-well Lab-Tek chamber slides with the other conditions kept the same. After transfection finished, cells were then fixed with 4% paraformaldehyde (in PBS) for 10 min, followed by staining the cell membrane with 5 µg / mL WGA-488 (Invitrogen, USA) and cell nuclei with 5 µg / mL Hoechst 33342 (Invitrogen, USA). Figure 7(d) contains CLSM images, 7(e) percentage transfection, and 7(f) mean fluorescence intensity of adherent cells (HEK 293T) transfected by mCherry mRNA-nanoparticle compositions (20k linear PEG:mRNA:EGCG:Zr(IV) at 100:1:100:2.5 mass ratio), with different assembly routes after 24 h. Cell membranes were stained in green. Scale bars are 20 μm. Figure 7(g) contains CLSM images, 7(h) percentage transfection, and 7(i) mean fluorescence intensity of suspension cells (Jurkat) transfected by mCherry mRNA- nanoparticle compositions (20k linear PEG:mRNA:EGCG:Zr(IV) at 100:1:100:2.5 mass ratio) with different assembly routes after 24h. Cell membranes (green) were stained with AF488-WGA. Scale bars are 10 μm.

[0236] For CLSM imaging following firefly luciferase mRNA transfection, cells were seeded in 8-well Lab-Tek chamber slides with the other conditions kept the same. After transfection, cells were fixed with 4% paraformaldehyde for 10 min. Then, the cell membranes were permeabilized using 0.2% Triton-X for 10 min, followed by 2% BSA blocking for 1 h at room temperature (RT). The cells were incubated with a primary antibody against luciferase (1 / 500 dilution, host species: rabbit, ABcam) at 4 °C overnight. After washing, the secondary antibody (goat anti-rabbit, AF-647 labelled) was added for 1 h incubation at RT, followed by 3 rounds of washing with DPBS. Figure 7(b) illustrates CLSM immuno-fluorescence staining showing the transfection of firefly luciferase-encoding mRNA by mRNA-nanoparticle composition (20k linear PEG:mRNA:EGCG:Zr(IV) at 100:1:100:2.5 mass ratio, prepared via co-assembly) in HEK 293T cells after 24 h. Cell membranes (green) were stained with Alexa Fluor 488- wheat germ agglutinin conjugate (AF488-WGA). Luciferase was detected with Alexa Fluor 647-labelled anti-luciferase antibodies (red). Scale bars are 20 μm.

[0237] For SIM imaging following NGFR mRNA transfection, cells were seeded in 8- well Lab-Tek chamber slides with the other conditions kept the same. After transfection, cells were fixed with 4% paraformaldehyde for 10 min.2% BSA was then added to block non-specific binding, followed by treatment with anti-NGFR (PE conjugated, Invitrogen, USA) for 3 h at room temperature and 3 rounds of washing with DPBS. 1005496795Figure 7(c) contains SIM images showing the transfection of Nerve Growth Factor Receptor (NGFR)-encoding mRNA in HEK 293T cells after 24 h. NGFR was stained with PE-conjugated antibodies. Scale bars are 10 μm. Example 14: Endo / lysosomal Colocalization.

[0238] HEK 293T cells were seeded at a density of 6 × 104cells per well in 8-well Lab- Tek chamber slides and cultured overnight to allow for cellular adhesion to the substrate. The following day, the cell media was aspirated and 300 µL of mRNA- nanoparticle compositions prepared with Cy5-labelled mRNA in Opti-MEM was added for a final mRNA concentration of 1 µg / mL. After 6 h, Opti-MEM was aspirated and replaced with complete DMEM. After a further 18 h, 75 nM LysoTracker Green DND-26 (Invitrogen, US), dissolved in complete DMEM, was added in the medium and incubated at 37 °C for 1 h to stain endo / lysosomes, followed by washing with PBS (three times). The colocalization of endo / lysosomes with mRNA was observed via SIM with a 63 × oil immersion objective. The images were further processed by Image J software to obtain PCC values. Figure 7(a) shows representative structured illumination microscopy (SIM) images of the intracellular colocalization of mRNA-nanoparticle compositions (20k linear PEG:mRNA:EGCG:Zr(IV) at 100:1:100:2.5 mass ratio, prepared via co-assembly) with endo / lysosomes. Endo / lysosomes (green) were stained with LysoTracker Green DND- 26. mRNA (red) was conjugated with Cy-5. Scale bars are 10 μm. Representative high- magnification images are on the top right (scale bars are 2 μm). Bottom right is the corresponding colour scatter plot of endo / lysosomes (green channel) vs mRNA (red channel). Example 15: Live / Dead Staining

[0239] HEK 293T cells were seeded in 96-well plates at a density of 2×104cells / well and cultured overnight. mRNA-nanoparticle compositions in Opti-MEM with a gradient of mRNA concentrations (10, 50, 100, 200, 300, 500 ng / well) were added for incubation. After 6 h, Opti-MEM was aspirated and replaced with complete DMEM. The aspirated media of each well with floating cells were collected and incubated in a new plate.18 h after, complete DMEM was aspirated and the media with floating cells were collected. All the removed cells were combined back to their original well before being washed with PBS for 3 times and incubated with live / dead fixable far-red stain (Invitrogen, US) at 37 °C for 30 min. The viability of cells was then analysed with FACS. 1005496795Example 16: In vivo safety and biocompatibility of mRNA nanoparticle composition

[0240] Comprehensive toxicity and biocompatibility evaluations post IV administration were conducted. Cytokine (interleukin-6 (IL-6) and TNFs) release assays in the blood (Figure 12a) and tissue histological analyses (i.e., hematoxylin and eosin (H&E) staining, Figure 12b) revealed no significant inflammatory response, cellular swelling, or histological alterations, denoting minimal in vivo toxicity of the NPs within the 24 h time period examined. Furthermore, mice exhibited negligible weight loss within 10 days post IV administration (data not shown). To assess any potential toxicity of the metal ions in the NPs, the biodistribution of Zr in organs was assessed up to 10 days post administration using inductively coupled plasma mass spectrometry (ICP-MS) on digested organ and body fluid (i.e., urine and blood) preparations. The mRNA nanoparticle compositions contained a Zr load of 64%, implying that for every 0.25 mg kg−1of mRNA delivered to mice, approximately 0.8 mg kg−1of metal will be introduced. However, under experimental conditions, NPs resulted in only nanogram-level changes in Zr content per gram of organ or body fluid (Figure 12c). At 4 h post injection, approximately 60 ng of Zr was detected per gram of blood, which was the highest level among all tested time points within 10 days (Figure 12c), suggesting the presence of Zr in circulation within 4 h. The gradual reduction of Zr content in the blood after 4 h indicates gradual organ deposition or excretion. In the urine samples, ~70 ng of Zr was detected per gram of urine at 4 h, which decreased rapidly thereafter (Figure 12c), suggesting efficient metal excretion from mice. Although Zr signals in harvested organs peaked on either day 3 (e.g., liver, kidney) or day 6 (e.g., brain, lung), they reduced to the baseline level (i.e., Zr content in DPBS-treated mice), implying that the overall Zr accumulation owing to the NPs was negligible within the 10-day study. The dose- dependent effect on Zr accumulation was also evaluated by applying a second injection on day 5. The Zr content slightly increased in the urine, blood, and most harvested organs on day 6 and was then excreted by day 10 (data not shown). Overall, the percentage of the injected dose of Zr per gram tissue (%ID g−1) in harvested organs and body fluids was low (i.e., <2%) regardless of single or double injections, further implying minimal in vivo metal accumulation. Together these data highlight the safety and biocompatibility of the noncationic mRNA nanoparticle compositions. 1005496795Example 17: Fabrication of TA / Ti(IV)-based BSA nanoparticle compositions with different PEG

[0241] The fabrication protocol was the same as per described for TA / Ti(IV)-based BSA nanoparticle compositions, except replacing PEG (2k) with PEG having different molecular weights (i.e., 6k, 10k, 20k, 35k). The nanoparticle compositions were purified with Milli-Q water via centrifugation (8000 g, 10 min), and the resultant nanoparticle suspension was re-dispersed in Milli-Q water for future use. Organ deposition studies were performed as in Example 1.

[0242] In these studies, selection of phenolic ligand influenced the primary organ deposition location (Table 5 and Figure 14). Table 5 Component Component Component Component Component Fabrication Primary 1: 2: 3: 4: mass ratio method Organ PEG mRNA Metal ion Phenolic Deposition ligand Linear, 2k BSA Ti(IV) TA 4:1:0.345:1 Sequentially add Lung components 1, 2, 4, and 3 to stabilize NPs Linear, 6k BSA Ti(IV) TA 4:1:0.345:1 Sequentially add Lung, spleen, components 1, 2, liver, and 4, and 3 to kidney stabilize NPs Linear, 10k BSA Ti(IV) TA 4:1:0.345:1 Sequentially add Spleen, liver, components 1, 2, and kidney 4, and 3 to stabilize NPs Linear, 20k BSA Ti(IV) TA 4:1:0.345:1 Sequentially add Spleen, liver, components 1, 2, and kidney 4, and 3 to stabilize NPs Linear, 35k BSA Ti(IV) TA 4:1:0.345:1 Sequentially add Spleen, liver, components 1, 2, and kidney 4, and 3 to stabilize NPs Example 18: Colocalization of encapsulated protein and TA / Ti(IV)-based BSA nanoparticle compositions

[0243] The colocalization of TA / Ti(IV)-based BSA nanoparticle compositions and encapsulated BSA was evaluated through both IVIS imaging and microscopy. The TA / Ti(IV)-based BSA nanoparticle compositions containing either Rh800 (for tracking nanoparticles) or Cyanine5 (Cy5)-labelled BSA (for tracking proteins) were prepared. 1005496795The organ deposition studies were performed as in Example 1. The colocalization of protein and nanoparticle compositions at the organ level can be illustrated by tracking Cy5 and Rh800 through IVIS imaging.

[0244] The microscopy allows for exploring the colocalization of BSA and nanoparticle compositions at the cellular level. In this experiment, TA / Ti(IV)-based BSA nanoparticle compositions were prepared with both Rh800 and commercial Fluorescein isothiocyanate-labelled BSA (FITC-BSA) and intravenously administrated into C57BL / 6J mice. The harvested organs were fixed with 4% PFA (4 °C, overnight), dehydrated with 15% and 30% sucrose solution (prepared with 1x PBS) until the organ sediment, followed by embedding in frozen optimal cutting temperature medium. The entire tissue block was frozen in dry ice and cut into sections using a Leica Biosystems CM1950 Cryostat (Leica Biosystems, AU) with a thickness of 10 μm. The sections were placed on glass slides with DAPI fluoromount (to stain the cell nucleus) for subsequent confocal microscopy.

[0245] In these studies, nanoparticles and encapsulated proteins accumulated in the same organ and were primarily associated with the same cells (according to microscopy results) (Figure 15). Example 19: Time-dependent lung-retention of TA / Ti(IV)-based BSA nanoparticle compositions

[0246] The TA / Ti(IV)-based BSA nanoparticle compositions were fabricated and intravenously administrated to C57BL / 6J mice. The major organs, including the spleen, liver, kidney, lung, heart, and brain, were harvested after 1, 4, 10, 24, 48, 72, 120, and 168 h. The fluorescence intensity (i.e., average radiance efficiency) of the lung indicates the lung retention corresponding to different time points (Figure 16). Example 20: Microscopy of sectioned tissues for TA-based BSA nanoparticle compositions

[0247] Representative organ-targeted TA-based nanoparticle compositions were selected for microscopy analysis. The target organs were harvested 4 h post intravenous administration. These organs were fixed with 4% PFA (4 °C, overnight), dehydrated with 15% and 30% sucrose solution (prepared with 1x PBS) until the organ sediment, followed by embedding in frozen optimal cutting temperature medium. The 1005496795entire tissue block was frozen in dry ice and cut into sections using a Leica Biosystems CM1950 Cryostat (Leica Biosystems, AU) with a thickness of 10 μm. The sections were placed on glass slides with DAPI fluoromount (to stain the cell nucleus) for subsequent confocal microscopy.

[0248] Microscopy results were consistent with IVIS observations, and most nanoparticles were associated with cells upon organ deposition (data not shown). Example 21: Delivery of Cre-recombinase protein for targeted gene editing

[0249] To enable gene editing in the lung, Cre-recombinase proteins (i.e., Cre, 40 μg) were premixed with BSA with a mass ratio of 1:9 (Cre: BSA) to shield the cationic charge of Cre-recombinase. Then, the Cre-BSA complexes were assembled into TA / Ti(IV)-nanoparticle compositions as described in Example 1, except replacing BSA with Cre-BSA complexes. The Cre-loaded TA / Ti(IV) nanoparticle compositions were intravenously administrated into B6.Cg-Gt(ROSA)26Sortm14 (CAG-tdTomato)Hze / J mice (Common name: Ai14 ) at a dosage of 40 μg of Cre per mouse. The mice were euthanized after 1, 3, and 5 days post injection, with the major organs harvested (Figure 17a). The TdTom activation, indicative of successful gene editing, was investigated through IVIS imaging (Figure 17b). As TA / Ti(IV)-based nanoparticle compositions feature lung targeting, the lung was collected, sectioned, and prepared for confocal microscopy.

[0250] Cre-loaded TA / Ti(IV)-based nanoparticle compositions enabled gene editing in the lung, consistent with the organ tropism of this composition (Figure 17c).

[0251] To enable gene editing in the spleen, liver, and kidney, TA / Tm(III)-based nanoparticle compositions (i.e., typically distributed across the spleen, liver, and kidney) were used to encapsulate the Cre-BSA complexes (mass ratio of Cre and BSA =1:9). Then, the Cre-loaded TA / Tm(III) nanoparticle compositions were intravenously administrated into Ai 14 mice at a dosage of 80 μg of Cre per mouse. As TA / Tm(III)- based nanoparticle compositions distribute across the spleen, liver, and kidney, these organs were collected, sectioned, and prepared for confocal microscopy.

[0252] Cre-loaded TA / Tm(III)-based nanoparticle compositions enabled gene editing in the spleen, liver, and kidney, consistent with the organ tropism of this composition (Figure 17d). 1005496795Example 22: Delivery of doxorubicin (DOX) using TA / Ti(IV)-based nanoparticle compositions for lung-metastasis inhibition

[0253] The DOX-loaded TA / Ti(IV)-based nanoparticle compositions were prepared as described in Example 1, except replacing BSA with DOX (100 μL, 400 μg mL-1).

[0254] B16-OVA melanoma cells were intravenously inoculated to C57BL / 6J mice (~4×105cells per mouse) on day 0 to develop a lung metastatic melanoma tumor model. The mice were treated by intravenously administrating DOX-loaded TA / Ti(IV) nanoparticle compositions, DPBS (negative control), free DOX (free drug control), and DOX-loaded TA / Tm(III) nanoparticle compositions (non-lung targeting nanoparticle control) on day 4 and 11. The lungs were finally harvested 18 days post tumor inoculation, and the inhibition efficacy was evaluated by counting the metastatic nodules on the lung.

[0255] DOX-loaded TA / Ti(IV)-based nanoparticle compositions reduced metastatic nodules in the lung, indicating their potential for melanoma lung metastasis inhibition (Figure 18). Example 23: Biocompatibility analysis through Hematoxylin and Eosin (H&E) staining and cytokine assays

[0256] The representative organ-targeted TA-based BSA nanoparticle compositions were intravenously administrated into C57BL / 6J mice. Two primary metabolic organs (i.e., liver and kidney) and targeted organs for each representative nanoparticle composition were harvested 7 days post injection. These harvested organs were fixed with 4% PFA (4 °C, overnight), dehydrated with 15% and 30% sucrose solution until the organs settled down, followed by embedding in a frozen optimal cutting temperature medium. The entire tissue block was frozen in dry ice and cut into sections using a Leica Biosystems CM1950 Cryostat (Leica Biosystems, AU) with a thickness of 10 μm. The sections were placed on glass slides for subsequent H&E staining (manufacturer’s protocol). For cytokine assays, the mouse blood was collected 7 days post injection via cardiac puncture, and plasma was then obtained via centrifugation (900g 15 mins). The levels of IL-6 and TNF-α were quantified using enzyme-linked immunosorbent assay kits (Invitrogen, USA). 1005496795

[0257] No histological alterations, negligible inflammatory responses, and negligible weight loss indicate the minimal toxicity of these nanoparticle compositions within 7 days (Figure 19). Example 24: Physicochemical properties of mRNA nanoparticle compositions

[0258] The physicochemical properties of mRNA nanoparticle compositions are key to their function. The bioactive mRNA nanoparticle formulation of PEG:mRNA:EGCG:Zr(IV) with a mass ratio of 100:1:100:2.5, displayed a spherical morphology with an average diameter of ~220 nm and a narrow size distribution post purification (via centrifugation), as deduced by a polydispersity index (PDI) of 0.19 (Figure 9a) and monodisperse NPs observed by lattice-structured illumination microscopy (Lattice-SIM; data not shown). Notably, the nanoparticle compositions were smaller (by ~120 nm) than the NP formulation without mRNA (i.e., Nanoparticle compositions), indicating that mRNA incorporation may enhance the overall cross- linking density within the NPs. The uniform distribution of elements O, N, P, and Zr within the NPs, as revealed by energy-dispersive X-ray spectroscopy (EDX) mapping (Figure 9b), along with the stretching vibration of –NH2 group in the Fourier transform infrared (FTIR) spectrum of the lead mRNA nanoparticle compositions (data not shown), indicate the successful incorporation of mRNA and the metal–organic nature of the NPs. Furthermore, the thickness of the mRNA nanoparticle compositions in the dried state was ~0.8 nm, indicating the collapsible nature of the NPs (data not shown). The ζ- potential of the mRNA nanoparticle compositions was –20 mV at physiological pH (i.e., pH 7), which is consistent with those observed for other metal ion-phenolic ligand assembly materials. Moreover, the mRNA nanoparticle compositions remained negatively charged (i.e., –25 to –10 mV) over a wide pH range (i.e., pH 4–9) (Figure 9c). This noncationic feature sets the current mRNA nanoparticle composition delivery platform apart from most existing mRNA delivery platforms that contain cationic components, which have raised safety concerns regarding inflammation and toxicity.

[0259] To elucidate the assembly and stability of mRNA within the Nanoparticle compositions, mRNA labeled with cyanine 5 (Cy5) was incorporated during NP assembly. Fluorescence spectroscopy showed ~50% loading efficiency, confirming the observations on agarose gels (Figure 9d). The stability of the mRNA nanoparticle compositions and the kinetics of mRNA release were examined in both Milli-Q water 1005496795and Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). As indicated in Figure 9e, mRNA release from the mRNA nanoparticle compositions in water was minimal (<12% over 24 h) and without significant change in NP size (data not shown), confirming the stability of the NPs in water. In FBS- supplemented DMEM, the size of the NPs initially increased (by 30%) within the first 30 min of incubation before gradually decreasing with time. These changes in particle size were accompanied with an mRNA release of 60% in the first 2 h and ~90% by 24 h. The size change in the biological medium suggests that the mRNA nanoparticle compositions readily interact with proteins present in the environment. This interaction was further supported by incubating the mRNA nanoparticle compositions in Alexa Fluor (AF) 488-conjugated immunoglobulin G (IgG) for 10 min. The Lattice-SIM results revealed an IgG envelope surrounding the NPs (data not shown), with a computational estimation of 89 antibodies adsorbed per NP. These findings highlight the potential and amenability of the mRNA nanoparticle compositions toward surface modification and for active delivery via ligand–receptor interactions.

[0260] To understand the dominant interactive forces that stabilize mRNA within Nanoparticle compositions, the mRNA nanoparticle compositions were incubated in different solutions for 24 h and the release of mRNA quantified. Upon exposure of the mRNA nanoparticle compositions to ethylenediaminetetraacetic acid (EDTA; metal chelator) and Tween 20 (hydrophobic competitor), ~70 and ~50% of mRNA was released from the NPs, respectively (Figure 9f). Incubation with urea, a hydrogen bond competitor, triggered the release of ~30% of mRNA, underlying the role of hydrogen bonds in the stabilization of the NP structure. However, the presence of sodium chloride (NaCl), serving as an ionic competitor, led to an mRNA release of <10%, indicating a minimal contribution of electrostatic forces to the overall stability of the mRNA within the NPs (Figure 9f). Example 25: mRNA delivery from mRNA nanoparticle compositions in vitro

[0261] The efficient delivery of biomacromolecular therapeutics, including mRNA, to the intracellular environment is often impeded by endosomal entrapment. To address this, the capability of mRNA nanoparticle compositions to facilitate endosomal escape was investigated via super-resolution microscopy (Lattice-SIM). Endo / lysosomes were stained with LysoTracker Green, while mRNA was labeled with Cy5. Following incubation of the NPs with HEK 293T cells for 2 h, a notable colocalization of mRNA 1005496795was observed within endo / lysosomal compartments. The obtained Pearson’s correlation coefficient (PCC) of 0.62 suggests a positive correlation between the mRNA and endo / lysosomal markers (data not shown). At 6 h post incubation, the extent of colocalization decreased, with an accompanying reduction in PCC to 0.45 (data not shown). At 24 h post incubation, most of the red and green fluorescence signals appeared as separate signals in the merged SIM image and the color scatter plot, and a corresponding low PCC value (i.e., 0.27) was observed (Figure 10a). These results collectively suggest a low degree of colocalization of mRNA and endo / lysosomes, indicating endosomal escape of mRNA. The endosomal escape ability of mRNA nanoparticle compositions is likely attributed to the buffering capacity imparted by metal ion – phenolic ligand compositions. Although incorporating polyphenols into NPs can facilitate endosomal escape of mRNA (data not shown), metal–phenolic coordination exerts synergistic buffering effects, enabling superior endosomal escape capability via enhanced proton-sponge effects (data not shown).

[0262] The versatility of mRNA delivery by Nanoparticle compositions was further assessed across different mRNA sequences and cell lines in vitro. Given the negligible cytotoxicity of the NPs at concentrations up to 500 ng mRNA per 100 μL media in HEK 293T cells (data not shown), an intermediate concentration of 166 ng mRNA per 100 μL media was selected for subsequent in vitro transfection studies. The mRNA nanoparticle compositions were benchmarked against RNAiMax, a commercial cationic lipid-based transfection agent. Transfection was conducted using mRNA encoding firefly luciferase (FLuc). To analyze FLuc transfection, immunofluorescence staining was conducted instead of a typical bioluminescence assay (i.e., based on the reaction between luciferase and luciferin substrate to produce a luminescent product) as EGCG interferes with the assay (data not shown). The presence of luciferase detected by anti- luciferase antibodies confirmed effective transfection using mRNA nanoparticle compositions, as shown in Figure 10b. Expanding beyond reporter proteins, the delivery system was assessed with mRNA encoding a functional protein (i.e., nerve growth factor receptor (NGFR), a member of the tumor necrosis factor (TNF) transmembrane receptor family). The mRNA nanoparticle compositions mediated surface expression of NGFR with a greater transfection efficiency (by 17.4%) than RNAiMax (Figure 10c).

[0263] A comparative analysis between adherent and suspension cell lines highlighted the efficacy of Nanoparticle compositions. In adherent HEK 293T cells, mRNA 1005496795nanoparticle compositions enabled nearly 100% transfection efficiency, surpassing RNAiMax by approximately 40%, although with a lower level of protein expression in each cell, as indicated by cell MFI (22000 for mRNA nanoparticle compositions vs 40000 for RNAiMax; Figure 10d, e). Suspension cells are typically difficult to transfect owing to the limited membrane binding of the NPs44. Nevertheless, the mRNA nanoparticle compositions exhibited superior performance in transfecting Jurkat cells (a suspension T cell line), with both higher transfection efficiency (by 2.5-fold) and average level of protein expression in each cell (i.e., MFI; by ~3-fold) than RNAiMax (Figure 10g–i). Collectively, these results indicate some degree of structural integrity of the mRNA released from mRNA nanoparticle compositions and demonstrate the broad applicability of these NPs as an mRNA delivery platform. Example 26: mRNA delivery from mRNA nanoparticle compositions in vivo

[0264] Prior to conducting the in vivo studies, a hemolysis assay was performed to analyze the safety of mRNA nanoparticle compositions for IV injection. The mRNA nanoparticle compositions induced <1% cell lysis across a range of NP concentrations from 10–100 µg mL−1(data not shown), showing the nonhemolytic nature and suitability of the NPs for IV injection. The NPs were then injected intravenously into C57BL / 6J mice at an mRNA dosage of 0.25 mg kg−1. After 24 h post injection, major organs, i.e., liver, kidney, lung, heart, spleen, and brain, were harvested for analysis using an In Vivo Imaging System (IVIS) (Figure 11a).

[0265] For the biodistribution studies, rhodamine 800 (Rh800)-labeled mRNA nanoparticle compositions were examined. The fluorescently labeled NPs displayed no significant difference in size or ζ-potential (data not shown) from the nonlabeled NPs. The NPs were broadly distributed in the harvested organs at 4 h, as well as 24 h post injection (data not shown). To explore the in vivo mRNA transfection efficacy, an engineered mRNA encoding a brighter red fluorescent protein (mFlame) was used, which revealed expression across all harvested organs 24 h after NP injection. Among all organs analyzed, the liver exhibited the highest mFlame expression (~30% of total intensity from all harvested organs; ~13-fold higher radiance efficiency than mice treated with Dulbecco’s phosphate-buffered saline (DPBS)) (Figure 11b). mFlame expression was also predominant in the kidney, and notably, the brain (~20%; 8-fold higher radiance efficiency than in DPBS-treated mice). As the liver exhibited the most prominent level of mRNA expression for mRNA nanoparticle compositions, the in vivo 1005496795transfection efficacy of the mRNA nanoparticle compositions was compared against LNPs formulated with SM-102 ionizable lipid (used in Moderna’s SARS-CoV-2 vaccine vector, which show predominant liver expression). The results showed that the mRNA nanoparticle compositions enabled 2-fold higher expression in the liver, as well as overall higher expression across all harvested organs relative to the mRNA-SM-102 LNPs at the same mRNA dosage (Figure 11c, d,).

[0266] To further validate mRNA expression in the organs using mRNA nanoparticle compositions, sectioned organ analysis was performed. In this analysis, mRNA encoding a far-red fluorescent protein (i.e., emiRFP670) was employed to alleviate the interference from autofluorescence in tissues (emission occurs at a longer wavelength where there is less tissue autofluorescence). The emiRFP670-encoding mRNA (1232 nucleotides) had a similar nucleotide number to mFlame-encoding mRNA (1004 nucleotides) (data not shown). The mRNA (emiRFP670)-Nanoparticle compositions and mRNA (mFlame)-Nanoparticle compositions displayed comparable particle size (data not shown) and comparable in vivo transfection (data not shown). CLSM images revealed the presence of emiRFP670-positive cells in the liver, kidney, and brain tissues (Figure 11 e–g), consistent with the organ-level mRNA expression.

[0267] Next, the ability of the mRNA nanoparticle compositions to mediate gene editing was examined by employing genetically engineered tdTomato (tdTom) reporter mice and Cre recombinase (Cre) mRNA. In these reporter mice, tdTom expression is silenced by a loxP-flanked stop cassette, which can be removed by the Cre enzyme to activate fluorescence (Figure 11h). Successful gene editing across the liver, kidney, and brain was indicated by the presence of tdTom signals (Figure 11i,j). Upon staining the vasculature with lectin, some of the tdTom signals did not colocalize with the cerebral vascular structures, indicating transfection beyond the blood–brain barrier. This brain deposition may primarily be mediated by the composition of the mRNA nanoparticle compositions. Based on the compositional mass ratio of the mRNA nanoparticle formulation (i.e., PEG:mRNA:EGCG:Zr(IV) = 100:1:100:2.5), EGCG is one of the most predominant constituents in mRNA nanoparticle compositions. Example 27: Organ tropism of mRNA expression modulated by NP composition 1005496795

[0268] The effects of mRNA dosage, treatment time, and NP composition on in vivo mRNA transfection were examined. Unlike a dosage of 5 µg of mRNA per mouse (~0.25 mg kg−1) that led to prominent expression in the liver, kidney, and brain, using a lower dosage of 2 µg per mouse (~0.1 mg kg−1) was insufficient to trigger expression that could be detected via IVIS. In contrast, increasing the dosage to 0.5 mg kg−1only resulted in a comparable level of mRNA expression to that observed using 0.25 mg kg−1(data not shown). The expression of mFlame mRNA was negligible at 12 h post IV administration and peaked at 24 h (Figure 13a, b, with the intensity of mRNA expression illustrated as normalized radiance efficiency). Overall, mRNA expression level decreased at 48 h, although mRNA expression remained strong in the spleen, likely owing to the predominant splenic retention of the NPs after 48 h (data not shown).

[0269] Leveraging the high degree of modularity of Nanoparticle compositions, the inventors investigated the effect of varying mRNA nanoparticle composition constituents on in vivo mRNA expression. Altering the ZrIV-to-EGCG mass ratio influenced both mRNA expression levels and the percentage of mRNA expression in the harvested organs (data not shown). For example, using mRNA nanoparticle compositions without metal ions (i.e., ZrIV-to-EGCG mass ratio = 0:40) led to a negligible mFlame fluorescence signal or expression in most harvested organs, suggesting the vital role of metal ions in improving the overall level of mRNA transfection. However, this ratio resulted in a higher proportion of mFlame expression in the brain (a 4.5-fold increase in radiance efficiency compared to the DPBS-treated controls) than 1:40 and 3:40 ratios (Figure 13e). Assembling mRNA nanoparticle compositions at a ZrIV-to-EGCG mass ratio 1:40 ratio led to the most pronounced mFlame expression in the liver (Figure 13c) among the different ZrIV-to-EGCG mass ratios examined. Further increasing the ZrIV-to- EGCG mass ratio improved the proportion of mFlame expression in the kidney, while reducing the proportion of the expression in the brain (Figure 13d, e).

[0270] The modularity and versatility of the mRNA nanoparticle compositions were further demonstrated by varying the metal ion component. Similar to using ZrIV, using CoII, TmIII, or TiIVenabled successful mRNA transfection in vivo (data not shown). The type of metal ions influenced the organ tropism of expressed mRNA (Figure 6f–h). Replacing ZrIVwith CoIIresulted in <5% mRNA expression in the liver and kidney but ~50% expression in the lung (i.e., ~50%, 13-fold higher radiance efficiency than DPBS- treated mice). In contrast, substituting ZrIVby TiIVled to a comparable expression profile 1005496795in the liver (i.e., ~30%, ~10-fold higher radiance efficiency than DPBS-treated mice), kidney (i.e., ~20%, ~9-fold higher radiance efficiency than DPBS-treated mice), and lung (i.e., ~11%, 4.5-fold higher radiance efficiency than DPBS-treated mice). Without being bound by theory, the inventors believe that varying the type of metal ions leads to compositional changes on mRNA nanoparticle compositions, potentially altering the composition of the biomolecular corona formed on the NP surface. This variability in corona composition may influence the organ tropism of mRNA expression, which was also observed with LNPs.

[0271] The mRNA expression could also be adjusted by using different PEGs. Assembling mRNA nanoparticle compositions using 2k linear PEG led to a reduced level of overall mRNA expression compared to using 20k linear PEG but exhibited liver tropism. Using 2k 4-arm PEG also resulted in an overall diminished level of mRNA expression compared to using 20k linear PEG (data not shown), but the splenic expression increased from 10% to 50% with the normalized radiance efficiency increasing from 4-fold to 16-fold (normalized to DPBS-treated mice) (Figure 13i). The increment of splenic expression might be attributed to the change in ζ-potential. NPs prepared with 2k 4-arm PEG displayed more negative ζ-potential (–47 ± 5 mV) than those prepared with the linear PEG (–17 ± 4 mV; data not shown), which might increase the likelihood of splenic deposition of NPs. In addition, protein adsorption (i.e., the formation of a protein corona) on the NPs may play a role in driving organ tropism, as demonstrated with other NP systems. Example 28: In vivo biocompatibility of and metal excretion from mRNA- metal ion-phenolic nanoparticle compositions

[0272] mRNA nanoparticle compositions were injected into C57BL / 6J mice (mixed gender, 8–10 weeks old) at a dosage of 0.25 mg kg−1via the lateral tail vein on day 0. For single injection groups, the treated mice were sacrificed after 4 h, 1 day, 3 days, 6 days, and 10 days post-injection. For double injection groups, a second injection with the same dose was performed on day 5, and the mice were sacrificed on day 6 and day 10. Urine samples were collected before euthanasia. The mice were euthanized using a slow influx of CO2, after which cervical dislocation was carried out. After sacrificing the animals, blood was collected via cardiac puncture. Six organs, including the liver, brain, kidney, lung, spleen and heart, were resected surgically. 1005496795

[0273] The fresh organs and body fluids were then weighed and digested in 70% HNO3 (0.6 mL) at 90 °C for 30 min, then diluted to achieve a HNO3 concentration of 2%. Zr standards were prepared using ICP multi-element standard solution IV (Merck, Germany). Zr content in the digested samples and the standard was quantified on an ICP-MS Vitesse instrument (Nu Instruments, UK), which was tuned and optimized for detection of isotopes 781 around 100 amu with standard tuning solution (Nu Instruments, UK), and on a NexION 2000 ICP-MS instrument (PerkinElmer, USA). Zr amount in organs and body fluids was calculated as the mass of Zr as a fraction of the injected dose and normalized for organ weight (%ID g−1).

[0274] Overall, the percentage of the injected dose of Zr per gram tissue (%ID g−1) in harvested organs and body fluids was low (i.e., <2%) regardless of single or double injections, further implying minimal in vivo metal accumulation (Figure 20). Example 29: Characterisation of mRNA nanoparticle compositions Table 7: Characterisation of mRNA nanoparticle compositions with different mRNA sequencesTable 8: mRNA sequencesTable 9: Characterisation of mRNA nanoparticle compositions prepared using the PEG:mRNA: EGCG:Zr(IV) nanaoparticles at a mass ratio of 100:1:100:2.5 1005496795Table 10: Characterisation of mRNA nanoparticle compositions using different Zr(IV)-to-EGCG mass ratios and examined in in vivo studiesTable 11: Characterisation of mRNA nanoparticle compositions prepared with different metal ions and examined in in vivo studiesTable 12: Characterisation of mRNA nanoparticle compositions prepared by varying Mw or structure of PEG and examined in in vivo studies1005496795

Claims

CLAIMS 1. A bioactive nanoparticle composition comprising a metal ion-phenolic ligand assembly and one or more bioactive agents, wherein the metal ion-phenolic ligand assembly comprises one or more metal ions, one or more phenolic ligands, and one or more seeding agents.

2. The bioactive nanoparticle composition according to claim 1, wherein the metal ion comprises one or more of Cu(II), Fe(III), Zr(IV), Ti(IV), Ir(IV), Ta(V), W(VI), Bi(III), Zn(II), Mn(II), Gd(III), Pt(IV), Nb(V), Co(II), Cr(III), Tm(III), Al(III), and Na(I).

3. The bioactive nanoparticle composition according to claim 1 or claim 2 comprising a single metal ion.

4. The bioactive nanoparticle composition according to any one of claims 1 to 3, wherein the one or more phenolic ligands has a molecular weight from 110 to 3,000 Daltons.

5. The bioactive nanoparticle composition according to any one of claims 1 to 4, wherein the one or more phenolic ligands comprise three or more ring-bonded hydroxy groups.

6. The bioactive nanoparticle composition according to any one of claims 1 to 5, wherein the one or more phenolic ligands comprise one or more galloyl groups, optionally with one or more catechol groups.

7. The bioactive nanoparticle composition according to any one of claims 1 to 6, wherein the one or more phenolic ligands comprise one or more of tannic acid, (-)- epigallocatechin-3-gallate, catechin, gallocatechin, catechin-3-gallate, epigallocatechin, epicatechin-3-gallate, quercetin, gallic acid, procyanidin, ellagic acid, myricetin, fisetin, luteolin, and dopamine.

8. The bioactive nanoparticle composition according to any one of claims 1 to 7, wherein the seeding agent comprises one or more of polyethylene glycol or chemically functionalised polyethylene glycol. 10054967959. The bioactive nanoparticle composition according to claim 8, wherein the one or more chemically functionalised polyethylene glycol comprises one or more of amine, acid, and ester functional groups.

10. The bioactive nanoparticle composition according to claim 8 or claim 9, wherein the one or more polyethylene glycol or chemically functionalised polyethylene glycol has a molecular weight from 100 to 10,000,000 Daltons, or from 100 to 50,000 Daltons, or from 1000 to 50,000 Daltons, or from 5,000 to 50,000 Daltons.

11. The bioactive nanoparticle composition according to any one of claims 8 to 10, wherein the one or more polyethylene glycol or functionalised polyethylene glycol is linear, branched, star or comb.

12. The bioactive nanoparticle composition according to any one of claims 1 to 11, wherein the one or more bioactive agent comprises one or more small molecules and biomacromolecules.

13. The bioactive nanoparticle composition according to claim 12, wherein the biomacromolecule comprises one or more of proteins, peptides, nucleic acid molecules and enzymes.

14. The bioactive nanoparticle composition according to claim 13, wherein the biomacromolecule comprises one or more nucleic acid molecules.

15. The bioactive nanoparticle composition according to claim 14, wherein the nucleic acid molecule comprises single stranded RNA.

16. The bioactive nanoparticle composition according to claims 14 or 15, wherein the nucleic acid molecule comprises cDNA, mRNA, miRNA, siRNA, sgRNA, modified RNA, antagomir, antisense molecule, or guide RNA molecule, or any combination thereof.

17. The bioactive nanoparticle composition according to any one of claims 14 to 16, wherein the nucleic acid molecule comprises mRNA.

18. The bioactive nanoparticle composition according to claim 17, wherein the mRNA encodes one or more antigens. 100549679519. The bioactive nanoparticle composition according to claim 18, wherein 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.

20. The bioactive nanoparticle composition according to any one of claims 14 to 19, wherein the mass ratio of the metal ion to phenolic ligand in the bioactive nanoparticle composition is from about 0.5:40 to about 3:

40.

21. The bioactive nanoparticle composition according to any one of claims 14 to 20, wherein the nucleic acid molecule is less than 1:

5.

22. The bioactive nanoparticle composition according to any one of claims 14 to 21, wherein the seeding agent comprises polyethylene glycol or chemically functionalised polyethylene glycol having a molecular weight of from about 5,000 to about 50,000 Da.

23. The bioactive nanoparticle composition according to any one of claims 14 to 22, wherein the seeding agent comprises polyethylene glycol or chemically functionalised polyethylene glycol having a linear structure.

24. The bioactive nanoparticle composition according to any one of claims 14 to 22, wherein the seeding agent comprises polyethylene glycol or chemically functionalised polyethylene glycol having a star structure, preferably a 4-arm structure.

25. The bioactive nanoparticle composition according to any one of claims 14 to 24, wherein the one or more phenolic ligands comprise (-)-epigallocatechin-3-gallate.

26. The bioactive nanoparticle composition according to any one of claims 14 to 25, wherein the one or more metal ions has an oxidation state of at least +4, preferably +4.

27. The bioactive nanoparticle composition according to any one of claims 14 to 26, wherein the metal ions comprise one or more of Zr(IV), Ti(IV), Ir(IV), Ta(V) and W(VI).

28. The bioactive nanoparticle composition according to any one of claims 14 to 27, wherein the metal ions comprise Zr(IV).

29. The bioactive nanoparticle composition according to any one of claims 14 to 27, wherein the metal ions comprise Ti(IV). 100549679530. The bioactive nanoparticle composition according to any one of claims 14 to 25, wherein the metal ions comprise one or more of Gd(III), Pt(IV) and Nb(V).

31. The bioactive nanoparticle composition according to any one of claims 14 to 25, wherein the one or more metal ions Co(II), Tm(III), and Bi(III), preferably Co(II).

32. The bioactive nanoparticle composition according to any one of claims 14 to 31, wherein the mass ratio of seeding agent to nucleic acid molecules in the bioactive nanoparticle composition is from about 10:1 to about 200:

1.

33. The bioactive nanoparticle composition according to any one of claims 14 to 32, wherein the mass ratio of phenolic ligand to nucleic acid in the bioactive nanoparticle composition is from about 10:1 to about 200:

1.

34. The bioactive nanoparticle composition according to claim 33, wherein the biomacromolecule comprises one or more of proteins, peptides and enzymes.

35. The bioactive nanoparticle composition according to claim 34, wherein the seeding agent comprises one or more polyethylene glycol or chemically functionalised polyethylene glycol having a molecular weight from about 2,000 to less than 10,000 Daltons.

36. The bioactive nanoparticle composition according to claim 34, wherein the seeding agent comprises the one or more polyethylene glycol or chemically functionalised polyethylene glycol has a molecular weight from about 10,000 to about 20,000 Daltons.

37. The bioactive nanoparticle composition according to any one of claims 34 to 36, wherein the molar ratio of metal ion to phenolic ligand in the bioactive nanoparticle composition is from 0.001:1 to 100:

1.

38. The bioactive nanoparticle composition according to any one of claims 34 to 36, wherein the molar ratio of metal ion to phenolic ligand in the bioactive nanoparticle composition is from 0.001:1 to less than 2:

1.

39. The bioactive nanoparticle composition according to any one of claims 34 to 36, wherein the molar ratio of metal ion to phenolic ligand in the bioactive nanoparticle composition is from greater than 2:1 to 100:

1. 100549679540. The bioactive nanoparticle composition according to any one of claims 1 to 39, wherein the nanoparticle composition has a mean nanoparticle size of from about 50nm to about 400nm.

41. The bioactive nanoparticle composition according to any one of claims 1 to 40, wherein nanoparticle composition has a polydispersity index from about 0 to about 0.

25.

42. The bioactive nanoparticle composition according to any one of claims 1 to 41, wherein the zeta potential of the nanoparticle is about -65mV to about -5mV.

43. The bioactive nanoparticle composition according to any one of claims 1 to 42, wherein the zeta potential of the nanoparticle is about -65mV to about -5mV at a pH of about 2 to about 12.

44. A method of preparing the bioactive nanoparticle composition according to any one of claims 1 to 43 comprising combining, in any order, one or more sources of metal ions, one or more phenolic ligands, one or more bioactive agents, and one or more seeding agents.

45. The method according to claim 44, wherein the metal ion source is added to a mixture of phenolic ligand, bioactive agent and seeding agent.

46. The method according to claim 44, wherein the bioactive agent is added to a mixture of phenolic ligand, metal ion source, and seeding agent.

47. A method for delivering one or more bioactive agents to a target cell or tissue comprising bringing a bioactive nanoparticle composition of any one of claims 1 to 43 into contact with the cell or tissue.

48. The method according to claim 47, wherein the target cell is a 2D or 3D cultured cell.

49. The method according to claim 47 or 48, wherein the target cell is a mammalian cell.

50. A method of delivering bioactive nanoparticles to one or more target sites in a subject comprising administering a bioactive nanoparticle composition according to any one of claims 1 to 43 to a subject. 100549679551. The method according to claim 50, wherein the bioactive agent comprises single stranded RNA.

52. The method according to claim 50 or 51, wherein the bioactive agent comprises mRNA.

53. The method according to claim 52, wherein the mRNA has an mRNA transfection efficiency greater than 25%, or greater than 50%, or greater than 75%, or greater than 85%, or greater than 95%.

54. The method according to claim 50, wherein the bioactive nanoparticle composition comprises a small molecule or biomacromolecule selected from proteins, peptides, and enzymes and at least one of the following: (i) the metal ions comprise one or more of Co(II), Zr(IV), Ti(IV), Ir(IV), Ta(V) and W(VI); (ii) the metal ions comprise one or more of Cu(II), Tm(III) and Bi(III); (iii) the metal ions comprise one or more Zn(II), Mn(II), Fe(III) and Cr(III); (iv) the phenolic ligand comprises tannic acid; (v) the phenolic ligand comprises (-)-epigallocatechin-3-gallate; (vi) the phenolic ligands comprise catechin, preferably (+)- catechin hydrate; (vii) the phenolic ligands comprise gallic acid; and (viii) the seeding agent comprises polyethylene glycol or chemically functionalised polyethylene glycols having a molecular weight of about 2,000 Da to about 6,000 Da; and (ix) the seeding agent comprises polyethylene glycol or chemically functionalised polyethylene glycols having a molecular weight of about 10,000 Da to about 20,000 Da.

55. The method according to any one of claims 50 to 53, wherein the bioactive nanoparticle composition comprises a nucleic acid molecule and at least one of the following: 1005496795(i) the metal ions comprise one or more of Zr(IV), Ti(IV), Ir(IV), Ta(V) and W(VI), preferably Zr(IV) and / or Ti(IV); (ii) the metal ions comprise one or more of Co(II), Tm(III), and Bi(III), preferably Co(II); (iii) the metal ion to phenolic ligand has a mass ratio of about 1:40; (iv) the metal ion to phenolic ligand has a mass ratio of about 3:40; (v) the seeding agent comprises polyethylene glycol or chemically functionalised polyethylene glycols having a star structure, preferably a 4-arm star structure; and (vi) the seeding agent comprises polyethylene glycol or chemically functionalised polyethylene glycols having a molecular weight of about 20,000 Da, optionally having a linear structure.

56. A method of delivering bioactive nanoparticles to multiple target sites in a subject comprising administering the bioactive nanoparticle composition according to any one of claims 1 to 43 to a subject.

57. The method according to claim 56, wherein the bioactive nanoparticle composition comprises one or more of Gd(III), Pt(IV) and Nb(V).

58. The method according to any one of claims 50 to 57, wherein the target site is one or more of liver, kidney, lung, brain, spleen, heart, pancreas, stomach, small intestine, large intestine, lymph node, bone, bone marrow, skeletal muscle, bladder, breast, testes, ovaries, uterus, thymus, brainstem, cerebellum, spinal cord, eye, ear, tongue, prostate, and skin.

59. The method according to claim 58, wherein the presence of one or more metal ions; one or more phenolic ligands; and / or one or more seeding agents provides a greater amount or activity of the bioactive nanoparticle to the target site compared to that achieved in the absence thereof.

60. A method for treating or preventing a disease or condition in a subject in need thereof, the method comprising administering to the subject a bioactive nanoparticle composition according to any one of claims 1 to 43, thereby treating or preventing a disease or condition in a subject in need thereof. 100549679561. A method of producing a polypeptide of interest in a cell, preferably a mammalian cell, the method comprising contacting the cell with a bioactive nanoparticle composition according to any one of claims 1 to 43, 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.

62. A method for the in vivo delivery of one or more bioactive agents, the method comprising administering a bioactive nanoparticle composition according to any one of claims 1 to 43 to a subject in need thereof, thereby delivering the one or more bioactive agents to the subject.

63. A method of delivering one or more components of a gene editing complex into a cell, comprising contacting a cell with a bioactive nanoparticle composition according to any one of 1 to 43, wherein the bioactive agent comprises one or more components of the gene editing complex.

64. A method of altering gene expression, comprising contacting a cell with a bioactive nanoparticle composition according to any one of claims 1 to 43, thereby altering gene expression.

65. The method according to claim 63 or 64, wherein the cell is in vitro.

66. The method according to claim 63 or 64, wherein the cell is in vivo, preferably within an animal, more preferably a mammal, even more preferably a human.

67. A method of treating or preventing cancer in a subject, comprising administering to the subject a bioactive nanoparticle composition according to any one of claims 1 to 43, thereby treating or preventing cancer.

68. The method according to claim 67, wherein the cancer is a metastatic cancer, such as lung metastases.

69. A method for delivering one or more bioactive agents across the blood brain barrier in a subject in need thereof, the method comprising administering a bioactive nanoparticle composition according to any one of claims 1 to 43 to a subject, thereby delivering the one or more bioactive agents across the blood brain barrier. 100549679570. The method according to any one of claims 51 to 64 and 66 to 69, wherein the bioactive nanoparticle composition is administered intravenously, subcutaneously, intramuscularly, intranasally, intratracheally, intraperitoneally, intratumorally, or intracerebrally. 1005496795

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