Nanoparticles and methods of manufacture for encapsulation of nucleic acids - Patent Application 20070123333

Abstract

Coated nanoparticles having a desired in vitro or in vivo effect, compositions of the coated nanoparticles, methods of using the coated nanoparticles to achieve a therapeutic or prophylactic effect in a cell, organ or subject, and processes for forming inverted nanoparticles encapsulating nucleic acids such as RNA stabilized by amphiphilic copolymers and coating the inverted nanoparticles with additional agents to create water-dispersible coated nanoparticles are provided.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 293,497, filed December 23, 2021, U.S. Provisional Patent Application No. 63 / 314,364, filed February 25, 2022, and U.S. Provisional Patent Application No. 63 / 389,349, filed July 14, 2022, all of which are incorporated by reference in their entireties herein.

[0002] This invention was made with Government support under Award No. 2032023 from the National Science Foundation. The Government has certain rights in this invention.

[0003] Embodiments of the present invention provide hybrid polymer-lipid nanoparticle compositions that encapsulate nucleic acids, processes for making the nanoparticles, and methods of using these compositions for therapeutic or prophylactic effects in cells, organs, tissues, or subjects. [Background technology]

[0004] Nucleic acids, including RNA and DNA, have short half-lives, are rapidly degraded by RNase or DNase, respectively, and are rapidly excreted when administered as soluble species. Nucleic acids can also be recognized and excreted by the immune system. A further limitation of nucleic acids is their inability to penetrate cell membranes due to their high water solubility. Summary of the Invention

[0005] U.S. Patent Nos. 10,231,937 and 11,103,461, U.S. Patent Application Publication Nos. US20200206136, US20210259984 and US20200023332, and International Application Publication Nos. WO2020227350 and WO2021046078, which are incorporated herein in their entireties, present methods for encapsulating hydrophilic compounds into nanoparticles that do not involve charge complexation, but instead rely on solubility-driven precipitation of water-soluble therapeutics, including nucleic acids. This encapsulation method is collectively referred to herein as "inverse flash nanoprecipitation" (iFNP). In one embodiment of the iFNP process, a hydrophilic active agent, referred to as an encapsulating agent, such as ribonucleic acid (RNA), deoxyribonucleic acid (DNA), oligonucleotides or other complex polynucleotide structures, and a stabilizing agent, such as a block copolymer, are dissolved in at least one polar solvent stream, which is rapidly mixed with at least one non-polar anti-solvent stream. Upon mixing, the encapsulating agent precipitates and forms a nanoparticle core. The hydrophilic region(s) of the stabilizing agent attaches or adheres to and / or is within the nanoparticle core, and the hydrophobic region(s) of the stabilizing agent faces the external non-polar anti-solvent phase, e.g., the hydrophobic region(s) of the stabilizing agent forms a shell around the core, forming an inverted nanoparticle.

[0006] In one embodiment, the inverse nanoparticles are transferred to a water-miscible modified solvent and the second stabilizing agent is dissolved in a desired ratio. In a second mixing step, the second stabilizing agent attaches, adheres and / or contacts the collapsed hydrophobic regions of the first stabilizing agent. This forms coated nanoparticles or coated nanocarriers containing a hydrophilic active agent such as a nucleic acid in the hydrophilic core surrounded by a hydrophobic shell with a hydrophilic surface of the coating to prevent aggregation under aqueous conditions.

[0007] Markwalter et al. (Polymeric Nanocarrier Formulations of Biologics Using Inverse Flash NanoPrecipitation. The AAPS Journal (2020) Vol. 22) reported encapsulation efficiency of RNA using this process as 48%, while lipid nanoparticle methods can report encapsulation efficiencies of over 80%.

[0008] The methods of the present invention surprisingly and unexpectedly enhance the properties of iFNP formulations through the incorporation of lipids.

[0009] One embodiment of the present invention includes a coated nanoparticle composition comprising one or more encapsulating agents and a first stabilizing agent, with a surface formed by additive(s) and a second stabilizing agent(s). The composition includes encapsulating agent(s) selected from a nucleic acid class including, but not limited to, RNA, DNA, mRNA (messenger RNA), siRNA (small interfering RNA), microRNA, circular RNA, antisense oligonucleotides, tRNA (transfer RNA), aptamers, DNA origami, or chemically modified polynucleotides including nucleic acid species with natural (e.g., from methylation or acetylation) or artificial substitutions, which may be circular or linear, or complexed with other molecules. The composition may include a salt, e.g., a calcium salt, or a free acid form. The encapsulating agent may be one or more of more than one agent, e.g., a nucleic acid, a peptide, a protein, or a second nucleic acid or nucleic acids. The encapsulating agent may be a plurality of nucleic acid sequences. In this specification, the terms nanoparticle, nanocarrier, and NC are used interchangeably.

[0010] In one embodiment, the composition comprises a first stabilizing agent that is a diblock, triblock, or comb copolymer. For example, the hydrophilic block(s) of the first stabilizing agent can be selected from dextran or other polysaccharides, poly(aspartic acid), or poly(glutamic acid). For example, the hydrophobic block(s) can be selected from poly(lactic acid), poly(lactic-co-glycolic acid), or poly(caprolactone).

[0011] In one embodiment, the composition comprises an additive selected from a lipid, a phospholipid, ethyl oleate, POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DSPC (1,2-distearoyl-s-glycero-3-phosphocholine), hydrogenated soy phosphatidylcholine (HSPC), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), a cationic lipid or cationic ionizable lipid, cholesterol, β-sitosterol, a fatty acid methyl ester, a fatty acid ethyl ester, a hydrophobic polymer, or a cationic or ionizable polymer, or a combination thereof.

[0012] The composition comprises a second amphiphilic stabilizing agent which is a diblock, triblock, or comb copolymer, or a lipid complex, or a mixture. The hydrophilic region of the polymer or lipid complex second stabilizing agent can be selected from poly(ethylene glycol), poly(sarcosine), poly(aspartic acid), poly(glutamic acid), poly(lysine), or poly(arginine), or a combination thereof. The hydrophobic polymer block can be selected from poly(lactic acid), poly(lactic acid-co-glycolic acid), poly(caprolactone). The composition comprises a PEG (polyethylene glycol, polyoxyethylene, poly(ethylene oxide)) lipid, such as PEG-DMG (1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol) or PEG-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]), or PEG-b-PLGA (polyethylene glycol-block-poly(lactic acid-co-glycolic acid)) or PEG-b-PLA (polyethylene glycol-block-poly(lactic acid)), or a mixture.

[0013] The process of the present invention is for preparing a coated nanocarrier composition comprising forming an inverse nanocarrier using reverse flash nanoprecipitation, exchanging the inverse nanocarrier into a modified solvent along with an additive and a second stabilizer, and assembling a coating using flash nanoprecipitation. The coated nanocarriers can then be further processed into a pharmaceutical form.

[0014] Inverse nanocarriers are produced by reverse flash nanoprecipitation. One or more solvent streams containing dissolved encapsulating agent(s) and a first stabilizing agent are rapidly micromixed with anti-solvent stream(s) optionally containing divalent cations such as calcium. This mixing process promotes the precipitation of the encapsulating agent(s), which is then stabilized from further growth by the stabilizing agent. The micromixing process is carried out using a mixer such as a confined impinging jet mixer or a multi-inlet vortex mixer. A typical solvent would be dimethylsulfoxide (DMSO) and a typical anti-solvent would be dichloromethane (DCM).

[0015] The inverse nanocarriers are then combined with additive(s) and second stabilizing agent(s) of a specific or desired composition. A solvent exchange process, such as distillation, is used to remove the poor solvent and introduce the modified solvent, e.g., acetonitrile.

[0016] The reverse nanocarriers in the modified solvent, as well as the additive(s) and the second stabilizer(s) are then rapidly micromixed with the aqueous antisolvent using a mixer as described above. The resulting nanoparticles are coated nanocarriers. Further processing steps common in the art, such as buffer exchange by ultrafiltration or diafiltration, can be used to remove residual modified solvent and prepare the pharmaceutical composition.

[0017] In one embodiment of the present invention, nanoparticles overcome the obstacles of RNA and DNA delivery by encapsulating nucleic acids, assisting in endocytic internalization, and providing an endosomal escape mechanism for cytoplasmic delivery. Cationic ionizable lipids can enable both encapsulation facilitated by charge complexation with anionic nucleic acids under acidic conditions, and endosomal escape for delivery to the cytosol. An encapsulation process that does not require charge complexation of nucleic acids can allow flexibility in nanoparticle design.

[0018] The methods of the invention provide for the delivery of therapeutic and / or prophylactic agents to a cell or organ. The delivery of therapeutic and / or prophylactic agents to a cell comprises administering to a subject a coated nanocarrier pharmaceutical composition comprising an encapsulating agent, and the administration of the composition comprises contacting the cell with the composition. The coated nanocarrier composition and / or pharmaceutical compositions comprising one or more coated nanocarrier compositions can be administered to any patient or subject, including patients or subjects who can benefit from a therapeutic effect provided by delivery of an encapsulating agent(s) to one or more specific cells, tissues, organs or systems or groups thereof, such as the liver system.

[0019] The step of contacting cells, e.g., mammalian cells, with the coated nanocarriers can be performed in vivo, ex vivo, in culture, or in vitro. In certain embodiments, the mRNA as an encapsulating agent in the coated nanocarriers can code for a recombinant polypeptide that can replace one or more polypeptides that may be reduced or substantially absent in cells contacted with the nanoparticle composition. One or more substantially absent polypeptides may be absent due to genetic mutations in the encoding gene or its regulatory pathway. In certain embodiments, one or more nucleic acids including an interfering RNA sequence (e.g., siRNA) can be encapsulated in the coated nanocarriers to provide a method for treating mammalian diseases or disorders in vitro and in vivo by downregulating or silencing the transcription and / or translation of one or more target nucleic acid sequences or genes of interest. In certain embodiments, mRNA sequences encoding endonucleases and small guide RNAs can be encapsulating agents in the coated nanocarriers to provide a method for treating by gene editing in vitro and in vivo.

[0020] In an embodiment of the invention, the nanoparticle comprises a core comprising a more polar region of a first stabilizing amphiphilic copolymer and at least one water-soluble agent, and a shell comprising a less polar region of the first stabilizing amphiphilic copolymer, at least one lipid, and a second stabilizing amphiphilic agent. The shell surrounds the core. The shell has an interior and an exterior. The interior of the shell is in contact with the core. The second stabilizing amphiphilic agent comprises a more polar region and a less polar region. The more polar region of the second stabilizing amphiphilic agent is at the exterior surface of the shell. The shell comprises a less polar region of the second stabilizing amphiphilic agent. The at least one water-soluble agent is not present at the exterior surface of the shell, a corona surrounds the shell, the corona comprises a more polar region of the second stabilizing amphiphilic agent. The at least one water-soluble agent is not in contact with the more polar region of the second stabilizing amphiphilic agent, and the corona is free of the at least one water-soluble agent.

[0021] In one embodiment, the at least one water-soluble agent is selected from the group consisting of nucleic acids, polynucleic acids, ribonucleic acids (RNA), messenger ribonucleic acids (mRNA), self-amplifying messenger ribonucleic acids (saRNA), small interfering ribonucleic acids (siRNA), microribonucleic acids (microRNA), circular ribonucleic acids (circular RNA), transfer ribonucleic acids (tRNA), small guide ribonucleic acids (sgRNA), deoxyribonucleic acids (DNA), antisense oligonucleotides (ASO), plasmids, episomes, and combinations.

[0022] In one embodiment, the at least one water-soluble agent comprises an mRNA encoding the SARS-CoV-2 spike protein receptor binding domain (RBD), optionally including a pseudo-uridine (the C5-glycosidic isomer of uridine, 5-ribosyluracil) modification.

[0023] The first stabilizing amphiphilic copolymer can be poly(aspartic acid)-block-poly(lactic acid) (PAsp-b-PLA), poly(aspartic acid)-block-poly(lactic acid-co-glycolic acid) (PAsp-b-PLGA), dextran-poly(lactic acid) (Dex-PLA), or dextran-poly(lactic acid-co-glycolic acid) (Dex-PLGA). Dextran and poly(aspartic acid) may have a molecular weight within the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da, respectively, and poly(lactic acid) and poly(lactic-co-glycolic acid) may have a molecular weight within the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da, respectively.

[0024] The first stabilizing amphiphilic copolymer can be poly(glutamic acid)-block-poly(lactic acid) (PGlu-b-PLA), poly(glutamic acid)-block-poly(lactic acid-co-glycolic acid) (PGlu-b-PLGA), poly(glutamic acid)-block-poly(caprolactone) (PGlu-b-PCL), poly(aspartic acid)-block-poly(caprolactone) (PAsp-b-PCL), or dextran-poly(caprolactone) (Dex-PCL). Dextran, poly(aspartic acid), and poly(glutamic acid) may have a molecular weight within the ranges of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da, respectively, and poly(lactic acid), poly(lactic-co-glycolic acid), and poly(caprolactone) may have a molecular weight within the ranges of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da, respectively.

[0025] The at least one lipid can be a phospholipid, a cationic lipid, an anionic lipid, a sterol, a monoglyceride, a triglyceride, a fatty acid methyl ester, a fatty acid ethyl ester, or a combination.

[0026] At least one lipid may be selected from the group consisting of 1,2-distearoyl-s-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DU ... Di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OchemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (Cl6 lysoPC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarrachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl The phosphatidylcholine may be 1-stearoyl-2-oleoyl-phosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), or a combination.

[0027] The at least one lipid may include 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The at least one lipid may include a cationic lipid and / or an ionized cationic lipid.

[0028] The at least one lipid may be a blend of a cationic lipid, a phospholipid, and cholesterol or a sterol.

[0029] The second stabilizer can be 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), pegylated distearoyl-phosphatidyl-ethanolamine (PEG-DSPE), polyethylene glycol-block-poly(lactic acid) (PEG-b-PLA), polyethylene glycol-block-poly(lactic-co-glycolic acid) (PEG-b-PLGA), or polyethylene glycol-block-poly(caprolactone) (PEG-b-PCL). Poly(lactic acid), poly(lactic-co-glycolic acid), and poly(caprolactone) can have molecular weights in the ranges of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da, respectively.

[0030] The nanoparticles can include at least one hydrophobic polymer. The at least one hydrophobic polymer can be poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), or a combination. The poly(lactic acid), poly(lactic-co-glycolic acid), and poly(caprolactone) can each have a molecular weight in the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da.

[0031] In one embodiment, the at least one water-soluble agent comprises an mRNA encoding a SARS-CoV-2 spike protein receptor binding domain (RBD) and / or an mRNA encoding a SARS-CoV-2 spike protein receptor binding domain (RBD) containing a pseudo-uridine (C5-glycoside isomer of uridine, 5-ribosyluracil) modification, the first stabilizing amphiphilic copolymer comprises dextran-poly(lactic acid) (Dex-PLA), dextran-poly(lactic-co-glycolic acid) (Dex-PLGA) and / or dextran-poly(caprolactone) (Dex-PCL), the at least one lipid comprises a cationic lipid, and the second stabilizing amphiphilic agent comprises a polyethylene glycol (PEG) copolymer and / or a polyethylene glycol (PEG) lipid.

[0032] In one embodiment, the at least one water-soluble agent comprises an mRNA encoding a SARS-CoV-2 spike protein receptor binding domain (RBD) containing a pseudo-uridine (C5-glycosidic isomer of uridine, 5-ribosyluracil) modification, the first stabilizing amphiphilic copolymer comprises dextran-poly(lactic-co-glycolic acid) (Dex-PLGA), the at least one lipid comprises 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM-102), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), and cholesterol, and the second stabilizing amphiphilic agent comprises 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG).

[0033] In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of nanoparticles and a pharma- ceutically acceptable carrier or diluent.

[0034] The method of administering to a subject includes administering to the subject a therapeutically effective amount of nanoparticles.

[0035] The method of administering to a cell includes contacting the cell with a nanoparticle. The cell can be a mammalian cell or a human cell. The administration to the cell can be performed in vitro.

[0036] A method for preventing or treating an infectious disease comprises administering to a subject suffering from an infectious disease a therapeutically effective amount of nanoparticles, wherein at least one water-soluble agent induces production of an antigen associated with the infectious disease by cells of the subject, and the antigen induces an immune response by the subject against the infectious disease.

[0037] The nanoparticles can be used in the prevention or treatment of infectious diseases.The nanoparticles can be used in the manufacture of a medicament for the prevention or treatment of infectious diseases.

[0038] The infection may be viral. The infection may be adenovirus, herpes simplex type 1, herpes simplex type 2, encephalitis virus, papillomavirus, varicella zoster virus, Epstein-Barr virus, human cytomegalovirus, human herpes virus type 8, human papillomavirus, BK virus, JC virus, smallpox, poliovirus, hepatitis B virus, human bocavirus, parvovirus B19, human astrovirus, Norwalk virus, coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, rubella virus, hepatitis E virus, The infection may be a virus, coronavirus, Middle East Respiratory Syndrome Coronavirus, Severe Acute Respiratory Syndrome Virus, SARS-CoV-2, rabies virus, influenza, zika virus, cytomegalovirus, or chikungunya virus.

[0039] A method for preventing or treating cancer comprises administering to a subject suffering from cancer a therapeutically effective amount of nanoparticles, wherein at least one water-soluble agent induces production by cells of the subject of a tumor antigen associated with cancerous cells of the cancer, and the antigen induces an immune response against the cancer by the subject.

[0040] The nanoparticles of claim 1 can be used for the prevention or treatment of cancer. The nanoparticles can be used in the manufacture of a medicament for the prevention or treatment of cancer.

[0041] A method for preventing or treating a loss-of-function disease comprises administering to a subject suffering from the loss-of-function disease a therapeutically effective amount of nanoparticles, wherein at least one water-soluble agent induces the production of a protein that restores the lost function.

[0042] The nanoparticles can be used for the prevention or treatment of loss-of-function diseases.The nanoparticles can be used in the manufacture of a medicament for the prevention or treatment of loss-of-function diseases.

[0043] The loss-of-function disease may be a urea cycle disorder, N-acetylglutamate synthase (NAGS) deficiency, carbamoyl phosphate synthase (CPS) deficiency, ornithine transcarbamoylase (OTC) deficiency, citrullinemia type 1 (CTLN1), citrullinemia type 2 (CTLN2), argininosuccinic aciduria, argininemia, or hyperornithinemic-hyperammonemia-homocitrullinuria (HHH) syndrome. The loss-of-function disease may be a polygenic disease, a monogenic disease, a polygenic liver disorder, or a monogenic liver disorder.

[0044] A method for preventing or treating a disease associated with a premature stop codon comprises administering a therapeutically effective amount of nanoparticles to a subject suffering from a disease associated with a premature stop codon. At least one water-soluble agent comprises a tRNA, which enables translation past the premature stop codon.

[0045] The nanoparticles can be used for the prevention or treatment of diseases associated with premature stop codons.The nanoparticles can be used in the manufacture of a medicament for the prevention or treatment of diseases associated with premature stop codons.

[0046] The disease associated with the premature stop codon may be β-thalassemia or Charcot-Marie-Tooth disease.

[0047] The gene editing method includes contacting nanoparticles with cells to insert a DNA sequence into the genome of the cell. The at least one water-soluble agent includes an endonuclease and / or an mRNA encoding the endonuclease, a small guide RNA (sgRNA), and a DNA sequence. The nanoparticles can be contacted with the cells in vitro. The endonuclease can be selected from the group consisting of Cas protein, Cas9, and TALEN.

[0048] The nanoparticles can be used in gene editing. The nanoparticles can be used in the manufacture of medicines for gene editing.

[0049] A method of making nanoparticles includes dissolving at least one water soluble agent in a first polar process solvent to form a water soluble agent solution, dissolving a first stabilizing amphiphilic copolymer in a second polar process solvent to form a copolymer solution, continuously mixing the water soluble agent solution and the copolymer solution with an anti-solvent to form a mixed solution from which nanoparticles assemble to form an inverse nanoparticle dispersion, adding at least one lipid to the inverse nanoparticle dispersion, adding a second stabilizing amphiphilic agent to the inverse nanoparticle dispersion, combining the inverse nanoparticle dispersion with a modifying solvent to form a modified dispersion, and continuously mixing the modified dispersion with an aqueous solvent to form nanoparticles. The first stabilizing amphiphilic copolymer includes at least one more polar region and at least one less polar region, the second polar process solvent may be the same as or different from the first polar process solvent, the anti-solvent is less polar than the first polar process solvent, and the anti-solvent is less polar than the second polar process solvent. The nanoparticles comprise a core and a shell, the core comprising a more polar region of a first stabilizing amphiphilic copolymer and at least one water-soluble agent, and the shell comprising a less polar region of the first stabilizing amphiphilic copolymer.

[0050] The aqueous solvent may be an aqueous buffer solution. The second polar process solvent may be the same as the first polar process solvent. The water-soluble agent solution and the copolymer solution may be a single mixed solution.

[0051] The second polar process solvent can be different from the first polar process solvent.

[0052] In one embodiment, the shell comprises an interior surface and an exterior surface, the interior surface of the shell is in contact with the core, the second stabilizing amphiphilic agent comprises a more polar region and a less polar region, the more polar region of the second stabilizing amphiphilic agent is on the exterior surface of the shell. The shell comprises the less polar region of the second stabilizing amphiphilic agent. The at least one water-soluble agent is not present on the exterior surface of the shell, a corona surrounds the shell, the corona comprises the more polar region of the second stabilizing amphiphilic agent. The at least one water-soluble agent is not in contact with the more polar region of the second stabilizing amphiphilic agent, and the corona is free of the at least one water-soluble agent.

[0053] The modification solvent can be acetonitrile.

[0054] The at least one water-soluble agent can be a nucleic acid, polynucleic acid, ribonucleic acid (RNA), messenger ribonucleic acid (mRNA), self-amplifying messenger ribonucleic acid (saRNA), small interfering ribonucleic acid (siRNA), microribonucleic acid (microRNA), circular ribonucleic acid (circular RNA), transfer ribonucleic acid (tRNA), small guide ribonucleic acid (sgRNA), deoxyribonucleic acid (DNA), antisense oligonucleotide (ASO), plasmid, episome, or a combination.

[0055] The at least one water-soluble agent comprises an mRNA encoding the SARS-CoV-2 spike protein receptor binding domain (RBD) and may optionally contain pseudo-uridine (the C5-glycosidic isomer of uridine, 5-ribosyluracil) modifications.

[0056] The first stabilizing amphiphilic copolymer can be poly(aspartic acid)-block-poly(lactic acid) (PAsp-b-PLA), poly(aspartic acid)-block-poly(lactic acid-co-glycolic acid) (PAsp-b-PLGA), dextran-poly(lactic acid) (Dex-PLA), dextran-poly(lactic acid-co-glycolic acid) (Dex-PLGA), poly(glutamic acid)-block-poly(lactic acid) (PGlu-b-PLA), poly(glutamic acid)-block-poly(lactic acid-co-glycolic acid) (PGlu-b-PLGA), poly(glutamic acid)-block-poly(caprolactone) (PGlu-b-PCL), poly(aspartic acid)-block-poly(caprolactone) (PAsp-b-PCL), or dextran-poly(caprolactone) (Dex-PCL). Dextran, poly(aspartic acid), and poly(glutamic acid) may have a molecular weight within the ranges of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da, respectively, and poly(lactic acid), poly(lactic-co-glycolic acid), and poly(caprolactone) may have a molecular weight within the ranges of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da, respectively.

[0057] The first stabilizing amphiphilic copolymer can be poly(glutamic acid)-block-poly(lactic acid) (PGlu-b-PLA) or poly(glutamic acid)-block-poly(lactic acid-co-glycolic acid) (PGlu-b-PLGA). The poly(glutamic acid) can have a molecular weight in the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da, and the poly(lactic acid) and poly(lactic acid-co-glycolic acid) can have a molecular weight in the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da, respectively.

[0058] The at least one lipid may be a phospholipid, a cationic lipid, an anionic lipid, a sterol, a monoglyceride, a triglyceride, a fatty acid methyl ester, a fatty acid ethyl ester, or a combination. The at least one lipid may include a cationic lipid and / or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The at least one lipid may be a blend of a cationic lipid, a phospholipid, and cholesterol or a sterol.

[0059] The second stabilizing amphiphilic agent can be PEGylated 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated distearoyl-phosphatidyl-ethanolamine (PEG-DSPE), polyethylene glycol-block-poly(lactic acid) (PEG-b-PLA), polyethylene glycol-block-poly(lactic-co-glycolic acid) (PEG-b-PLGA), or polyethylene glycol-block-poly(caprolactone) (PEG-b-PCL). The poly(lactic acid), poly(lactic-co-glycolic acid), and poly(caprolactone) can each have a molecular weight in the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da.

[0060] In one embodiment, the at least one water-soluble agent comprises an mRNA encoding a SARS-CoV-2 spike protein receptor binding domain (RBD) and / or an mRNA encoding a SARS-CoV-2 spike protein receptor binding domain (RBD) containing a pseudo-uridine (C5-glycoside isomer of uridine, 5-ribosyluracil) modification, the first stabilizing amphiphilic copolymer comprises dextran-poly(lactic acid) (Dex-PLA), dextran-poly(lactic-co-glycolic acid) (Dex-PLGA) and / or dextran-poly(caprolactone) (Dex-PCL), the at least one lipid comprises a cationic lipid, and the second stabilizing amphiphilic agent comprises a polyethylene glycol (PEG) copolymer and / or a polyethylene glycol (PEG) lipid.

[0061] In one embodiment, the at least one water-soluble agent comprises an mRNA encoding a SARS-CoV-2 spike protein receptor binding domain (RBD) containing a pseudo-uridine (C5-glycosidic isomer of uridine, 5-ribosyluracil) modification, the first stabilizing amphiphilic copolymer comprises dextran-poly(lactic-co-glycolic acid) (Dex-PLGA), the at least one lipid comprises 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM-102), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), and cholesterol, and the second stabilizing amphiphilic agent comprises 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG).

[0062] In one embodiment, the at least one water-soluble agent comprises a ribonucleic acid (RNA) in which at least one uridine is replaced with a modified uridine. At least 50% of the uridines of the ribonucleic acid (RNA) may be replaced with modified uridines, and the modified uridines may be the same or different. All the uridines of the ribonucleic acid (RNA) may be replaced with modified uridines, and the modified uridines may be the same or different.

[0063] For example, each modified uridine is independently selected from the group consisting of pseudouridine, 5-methoxyuridine, and N1-methylpseudouridine. For example, each modified uridine is independently selected from the group consisting of pseudouridine and 5-methoxyuridine.

[0064] The ribonucleic acid (RNA) can be messenger ribonucleic acid (mRNA), self-amplifying messenger ribonucleic acid (saRNA), small interfering ribonucleic acid (siRNA), micro ribonucleic acid (microRNA), circular ribonucleic acid (circular RNA), transfer ribonucleic acid (tRNA), small guide ribonucleic acid (sgRNA), or antisense oligonucleotide ribonucleic acid (ASO RNA).

[0065] The at least one water-soluble agent may be a nucleic acid at least partially neutralized with a base. The nucleic acid may be 50% neutralized with a base. The nucleic acid may be fully neutralized with a base. The nucleic acid may be a ribonucleic acid (RNA). The nucleic acid may be a messenger ribonucleic acid (mRNA), a self-amplifying messenger ribonucleic acid (saRNA), a small interfering ribonucleic acid (siRNA), a microribonucleic acid (microRNA), a circular ribonucleic acid (circular RNA), a transfer ribonucleic acid (tRNA), a small guide ribonucleic acid (sgRNA), or an antisense oligonucleotide ribonucleic acid (ASO RNA). The base may be an amine. The base may be a guanidine. The base may be a guanidine derivative or a guanidinium salt. The base may be arginine. The base may be a tertiary amine. The base may be triethylamine or diphenhydramine.

[0066] In one method, the at least one water-soluble agent comprises a ribonucleic acid (RNA) in which at least one uridine is replaced with a modified uridine. At least 50% of the uridines of the ribonucleic acid (RNA) may be replaced with a modified uridine, and the modified uridines may be the same or different. All the uridines of the ribonucleic acid (RNA) may be replaced with a modified uridine, and the modified uridines may be the same or different. For example, each modified uridine is independently selected from the group consisting of pseudouridine, 5-methoxyuridine, and N1-methylpseudouridine. For example, each modified uridine is independently selected from the group consisting of pseudouridine and 5-methoxyuridine.

[0067] The ribonucleic acid (RNA) can be messenger ribonucleic acid (mRNA), self-amplifying messenger ribonucleic acid (saRNA), small interfering ribonucleic acid (siRNA), micro ribonucleic acid (microRNA), circular ribonucleic acid (circular RNA), transfer ribonucleic acid (tRNA), small guide ribonucleic acid (sgRNA), or antisense oligonucleotide ribonucleic acid (ASO RNA).

[0068] The at least one water-soluble agent may be a nucleic acid that has been at least partially neutralized with a base prior to dissolving the at least one water-soluble agent in the first polar process solvent. The nucleic acid may be 50% neutralized with a base. The nucleic acid may be fully neutralized with a base. The nucleic acid may be a ribonucleic acid (RNA). The nucleic acid may be a messenger ribonucleic acid (mRNA), a self-amplifying messenger ribonucleic acid (saRNA), a small interfering ribonucleic acid (siRNA), a micro ribonucleic acid (microRNA), a circular ribonucleic acid (circular RNA), a transfer ribonucleic acid (tRNA), a small guide ribonucleic acid (sgRNA), or an antisense oligonucleotide ribonucleic acid (ASO RNA). The base may be an amine, guanidine, a guanidine derivative, a guanidine salt, arginine, a tertiary amine, triethylamine, or diphenhydramine.

[0069] In one method, the at least one water-soluble agent is a salt of a plasmid. The at least one water-soluble agent can be a triethylamine (TEA) salt of a plasmid or a guanidine salt of a plasmid. The plasmid can be an episome.

[0070] In one method, the first stabilizing amphiphilic copolymer can be a polysaccharide copolymer. The first stabilizing amphiphilic copolymer can be dextran-poly(lactic-co-glycolic acid) (Dex-PLGA). The dextran can have a molecular weight in the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da, and the poly(lactic-co-glycolic acid) can have a molecular weight in the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da.

[0071] In one method, the first stabilizing amphiphilic copolymer can be a polypeptide copolymer. The first stabilizing amphiphilic copolymer can be poly(aspartic acid)-poly(lactic acid-co-glycolic acid) (PAsp-PLGA). The poly(aspartic acid) can have a molecular weight in the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da, and the poly(lactic acid-co-glycolic acid) can have a molecular weight in the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da.

[0072] In one method, the at least one lipid can be a cationic lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol, or a combination.

[0073] In one method, the second amphiphilic stabilizing copolymer can be a PEGylated copolymer. The second amphiphilic stabilizing copolymer can be 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-DMG).

[0074] In one method, the anti-solvent comprises a metal salt, which can be MgCl2, CaCl2, or ZnCl2. [Brief description of the drawings]

[0075] [Figure 1]Schematic of nanocarrier structure during processing (not to scale). [Diagram 2] Expression in 293T cell cultures at 24 hours as measured by fluorescence intensity from flow cytometry analysis. Formulation identification (ID) corresponds to the formulation details provided in the Examples section. Bar graphs are for each RNA dose applied to cell supernatant. [Diagram 3] Expression in 293T cell cultures at (A) 24 hours and (B) 60 hours as measured by fluorescence intensity from flow cytometry analysis. Formulation ID corresponds to the formulation details provided in the Examples section. The x-axis indicates the RNA dose applied to the cell supernatant. [Figure 4] Expression in 293T cell cultures at (A) 24 hours and (B) 60 hours as measured by fluorescence intensity from flow cytometry analysis. Formulation ID corresponds to the formulation details provided in the Examples section. The x-axis indicates the RNA dose applied to the cell supernatant. [Figure 5A] Flow cytometry histograms for the indicated formulations showing similar expression between iFNP and lipid nanoparticle (LNP) formulations at an RNA dose of 1 ug / mL. The control formulation without mRNA (mRNA-55) was significantly less fluorescent. [Figure 5B] Epifluorescence and brightfield imaging of the formulations after 24 h incubation with 293T cells. [Figure 5C] Expression of GFP (Green Fluorescent Protein) in 293T cell cultures at 24 h as measured by fluorescence intensity from flow cytometry analysis. The y-axis shows the MFI (median fluorescence intensity) of GFP. [Figure 6A] Expression in 293T cell cultures as measured by fluorescence intensity from flow cytometry analysis at 24 hours. Formulation ID corresponds to the formulation details provided in the Examples section, and the x-axis indicates the RNA dose applied to the cell supernatant. [Figure 6B] Expression in HepG2 cell cultures as measured by fluorescence intensity from flow cytometry analysis at 24 hours. Formulation ID corresponds to the formulation details provided in the Examples section, and the x-axis indicates the RNA dose applied to the cell supernatant. [Figure 7A] Expression in 293T cell cultures as measured by fluorescence intensity from flow cytometry analysis at 24 hours. Formulation ID corresponds to the formulation details provided in the Examples section, and the x-axis indicates the RNA dose applied to the cell supernatant. (C) HepG2 data presented as mean instead of median fluorescence intensity. [Figure 7B] Expression in HepG2 cell cultures as measured by fluorescence intensity from flow cytometry analysis at 24 hours. Formulation ID corresponds to the formulation details provided in the Examples section, and the x-axis indicates the RNA dose applied to the cell supernatant. [Figure 7C] HepG2 data presented as mean instead of median fluorescence intensity. [Figure 8A] Expression in 293T cell cultures as measured by fluorescence intensity from flow cytometry analysis at 24 hours. Formulation ID corresponds to the formulation details provided in the Examples section, and the x-axis indicates the RNA dose applied to the cell supernatant. [Figure 8B] HepG2 cell cultures as measured by fluorescence intensity from flow cytometry analysis over 24 hours. Formulation ID corresponds to the formulation details provided in the Examples section, and the x-axis indicates the RNA dose applied to the cell supernatant. [Figure 9A] Expression in 293T cell cultures as measured by fluorescence intensity from flow cytometry analysis at 24 hours. Formulation ID corresponds to the formulation details provided in the Examples section, and the x-axis indicates the RNA dose applied to the cell supernatant. [Figure 9B] HepG2 cell cultures as measured by fluorescence intensity from flow cytometry analysis over 24 hours. Formulation ID corresponds to the formulation details provided in the Examples section, and the x-axis indicates the RNA dose applied to the cell supernatant. [Figure 10A] Expression in 293T as measured by fluorescence intensity from flow cytometry analysis at 24 hours. Formulation ID corresponds to the formulation details provided in the Examples section, and the x-axis indicates the RNA dose applied to the cell supernatant. [Figure 10B]HepG2 cell cultures as measured by fluorescence intensity from flow cytometry analysis over 24 hours. Formulation ID corresponds to the formulation details provided in the Examples section, and the x-axis indicates the RNA dose applied to the cell supernatant. [Figure 11A] Expression in 293T cell cultures 24 hours prior to freezing of coated nanocarriers as measured by fluorescence intensity from flow cytometry analysis. Formulation ID corresponds to the formulation details provided in the Examples section and the bar graph indicates the RNA dose applied to the cell supernatant. [Figure 11B] Expression in 293T cell cultures 24 hours after freezing of coated nanocarriers as measured by fluorescence intensity from flow cytometry analysis. Formulation ID corresponds to the formulation details provided in the Examples section and the bar graph indicates the RNA dose applied to the cell supernatant. [Figure 11C] Expression in HepG2 cell cultures 24 hours prior to freezing of coated nanocarriers as measured by fluorescence intensity from flow cytometry analysis. Formulation ID corresponds to the formulation details provided in the Examples section and the bar graph indicates the RNA dose applied to the cell supernatant. [Figure 11D] Expression in HepG2 cell cultures 24 hours after freezing of coated nanocarriers as measured by fluorescence intensity from flow cytometry analysis. Formulation ID corresponds to the formulation details provided in the Examples section and the bar graph indicates the RNA dose applied to the cell supernatant. [Figure 12A] Expression in 293T cell cultures 24 hours after freezing of coated nanocarriers as measured by fluorescence intensity from flow cytometry analysis. Formulation ID corresponds to the formulation details provided in the Examples section and the bar graph indicates the RNA dose applied to the cell supernatant. [Figure 12B] Expression in HepG2 cell cultures 24 hours after freezing of coated nanocarriers as measured by fluorescence intensity from flow cytometry analysis. Formulation ID corresponds to the formulation details provided in the Examples section and the bar graph indicates the RNA dose applied to the cell supernatant. [Figure 13A]Geometric mean of SARS-CoV2 receptor binding domain detected by fluorescent antibody binding in flow cytometry analysis of 293T cells transfected for 16 hours with either iFNP formulations, LNP formulations, DNA plasmid + Lipofectamine 2000, or no treatment. Dose ranges for iFNP and LNP mRNA formulations are listed. Detection was as described herein. [Figure 13B] Flow cytometry distribution data. [Figure 14A] iFNP antibody titers against the SARS-CoV2 receptor binding domain in mouse serum at the indicated time points during the immunization protocol. Log(titer) is the endpoint titer determined by serial dilution, with the endpoint defined as the luminescence signal two standard deviations above the mean background of the plate. [Figure 14B] LNP antibody titers against the SARS-CoV2 receptor binding domain in mouse serum at the indicated time points during the immunization protocol. Log(titer) is the endpoint titer determined by serial dilution, with the endpoint defined as the luminescence signal two standard deviations above the mean background on the plate. [Figure 15A] Green fluorescent protein (GFP) signal measured by flow cytometry after 24 h of incubation in 293T cultures. Median values ​​are reported for two mRNA doses in each well. [Figure 15B] Green fluorescent protein (GFP) signal measured by flow cytometry after 24 h of incubation in HepG2 cultures. Median values ​​are reported for two mRNA doses in each well. [Figure 16A] Firefly luciferase signal after 24 hours of incubation of 293T cells with nanoparticles. Dose values ​​are for mRNA in culture medium and detection was performed using standard manufacturer recommendations as described herein. [Figure 16B]Firefly luciferase signal after 24 h incubation of HepG2 cells with nanoparticles. Dose values ​​are for mRNA in culture medium and detection was performed using standard manufacturer recommendations as described herein. [Figure 17] Firefly luciferase bioluminescence signal after intravenous (IV) injection (total whole animal luminescence, ventral position). [Figure 18] Firefly luciferase bioluminescence signal after IV injection of iFNP or LNP formulations (whole animal total luminescence, dorsal position). [Figure 19] Firefly luciferase bioluminescence signal after intramuscular (IM) injection in the hind limb of iFNP or LNP formulations (whole animal total luminescence, ventral position). [Figure 20] Firefly luciferase bioluminescence signal after IM injection in the hind limb of iFNP or LNP formulations (whole animal total luminescence, dorsal position). [Figure 21] Firefly luciferase bioluminescence images at imaging time points for iFNP (formulation-fL3) and LNP nanoparticles (IV-ventral, IM-dorsal). The grayscale images show dark / light gradient spots due to increasing luciferase signal. Darker areas generally correspond to greater signal from luciferase and more concentrated expression of luciferase. [Figure 22] Firefly luciferase bioluminescence images 9 hours post-injection for LNP and iFNP (fL2-fL7) formulations ("No Tx" indicates untreated mice as control). Grayscale images show dark / light gradient spots due to elevated luciferase signal. IV injection produces liver / spleen signal, while IM injection produces localized signal (with some liver signal occurring with LNP). Darker areas generally correspond to greater signal from luciferase and more concentrated expression of luciferase. [Figure 23A] GFP signal measured by flow cytometry after 24 h incubation in 293T cultures. Median values ​​are reported for two mRNA doses for each well. [Figure 23B] GFP signal measured by flow cytometry after 24 h incubation in HepG2 cultures. Median values ​​are reported for two mRNA doses in each well. [Figure 24] Percentage of GFP-positive 293T cells measured by flow cytometry after 96 and 140 hours of treatment with 3 μg / mL of iFNP DNA formulations AG1-AG6. GFP positivity from iFNP-formulated DNA is compared to the same time points after transfection with Lipofectamine 2000 positive control or mock-transfected negative control. The DNA delivered encodes the GFP protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0076] The embodiments of the present invention are described in detail below. In describing the embodiments, specific terms are employed for clarity. However, the present invention is not intended to be limited to the specific terms so selected. Those skilled in the relevant art will recognize that other equivalent parts can be used and other methods can be developed without departing from the spirit and scope of the present invention. All references cited herein are incorporated by reference as if each was individually incorporated.

[0077] Definitions of some terms follow:

[0078] As used herein, solvent or solvent stream are terms used interchangeably to refer to organic solvents, aqueous buffers, or mixtures thereof, often with certain organic or inorganic compounds dissolved in them. "Stream" is often, but not exclusively, used in contexts where a continuous mixing or flow process is or will be used. The properties of the solvent are determined by the process as defined below.

[0079] As used herein, "anti-solvent" or "anti-solvent stream" are terms used interchangeably to refer to an organic solvent, an aqueous buffer, or a mixture thereof, often in which a particular organic or inorganic compound is dissolved. "Stream" is often, but not exclusively, used in contexts where a continuous mixed or flow process is or will be used. The characteristics of the anti-solvent are determined by the process, as defined below. In patents and literature related to flash nanoprecipitation (FNP) or inverse flash nanoprecipitation (iFNP), the terms "non-solvent" or "non-process solvent" may be used with the same intended meaning.

[0080] All solvents are miscible to some degree with each other. As referred to herein, a "miscible" solvent is one that, when mixed in the ratios used in the process, produces a solution with 20% or less of the volume of a minor phase that is not dissolved in the major phase. As referred to herein, a "non-miscible" solvent is one that, when mixed in the volume ratios used in the process, produces a second phase with more than 20% of the minor phase that is not dissolved in the major phase.

[0081] As used herein, the term "soluble" refers to the state of being molecularly dissolved in a given solvent or aqueous system. This requires that the solvent solubilize all regions, portions, or groups of the agent, species, polymer, or compound. Species that have precipitated, micellized, or formed collapsed polymer globules are not molecularly soluble.

[0082] As used herein, the terms "species" and "agent" refer to compounds or molecules - organic or inorganic - that are involved in the process.

[0083] As used herein, the terms "nanoparticle" ("NP"), "particle" and "nanocarrier" are used interchangeably unless the context indicates otherwise. Particles in embodiments of the invention having a hydrophilic or highly polar core are sometimes referred to as "inverse nanocarriers" in contrast to nanocarriers having a hydrophobic or less polar core. However, for brevity, when the context indicates that particles having a hydrophilic or highly polar core according to an embodiment of the invention are being described, they may be referred to simply as "particles" or "nanoparticles". "Coated nanocarriers" refer to "inverse nanocarriers" that have been further processed to have a surface that allows them to be dispersed in water without aggregation. Nanoparticles or nanocarriers typically have a hydrodynamic mass mean diameter of 10 nm to 800 nm as measured by dynamic light scattering.

[0084] The term "water-dispersible" refers to the property of being colloidally stable in an aqueous environment, such as an aqueous buffer. "Colloidally stable" refers to nanoparticles or other small particles that do not aggregate in the solvent. Colloidal stability can be imparted via polymer brushes that are solvated by the dispersing liquid, e.g., water, and thus provide steric hindrance to nanoparticle aggregation.

[0085] The term "lipid" refers to a group of organic compounds, including but not limited to esters of fatty acids, characterized by being insoluble in water but soluble in many organic solvents. They are usually classified into at least three classes: (1) "simple lipids", including fats and oils, as well as waxes; (2) "compound lipids", including phospholipids and glycolipids; and (3) "derived lipids" or "structured lipids", such as steroids or sterols. A "phospholipid" is a lipid that contains a phosphoric acid moiety and one or more carbon chains, e.g., unsaturated fatty acid carbon chains. Phospholipids may contain one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturated bonds).

[0086] As used herein, "expression" of a nucleic acid sequence refers to the transcription of DNA into mRNA, the translation of mRNA into a polypeptide or protein, and / or the post-translational modification of a polypeptide or protein.

[0087] As used herein, the term "in vitro" refers to events that take place not in a living organism (e.g., an animal, plant, or microorganism) but in an artificial environment, e.g., in a test tube or reaction vessel, in a cell culture, in a Petri dish, etc.

[0088] As used herein, the term "in vivo" refers to events that take place within an organism. The term "ex vivo" refers to events that occur outside of an organism.

[0089] As used herein, the term "polypeptide" or "polypeptide of interest" refers to a polymer of amino acid residues typically joined by peptide bonds, which may be produced naturally (e.g., isolated or purified) or synthetically. Polypeptides include proteins and enzymes.

[0090] As used herein, "target cell" refers to any one or more cells of interest. The cells may be found in vitro, in vivo, in situ, or in a tissue or organ of an organism. The organism may be an animal, such as a mammal or a human.

[0091] As used herein, the term "treating" refers to partially or completely alleviating, ameliorating, ameliorating, mitigating, delaying the onset of, inhibiting progression of, reducing the severity of, and / or reducing the incidence of one or more symptoms or characteristics of a particular infection, disease, disorder, and / or condition. For example, "treating" cancer can refer to inhibiting tumor survival, growth, and / or metastasis. Treatment can be administered to subjects who do not show signs of the disease, disorder, and / or condition and / or to subjects who show only early signs of the disease, disorder, and / or condition, with the intent of reducing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0092] As used herein, the term "immune response" refers to an integrated body response to an antigen or a cell expressing an antigen, and may refer to a cellular immune response or a cellular and humoral immune response. The immune response may be protective / preventive / prophylactic and / or therapeutic.

[0093] "Cellular immune response", "cellular response", "cellular response to an antigen" or similar terms are meant to include a cellular response directed to a cell expressing an antigen and is characterized by presentation of the antigen by class I or class II MHC. The cellular response involves cells called T cells or T lymphocytes, which act as either "helpers" or "killers".

[0094] The term "antigen" includes any molecule, e.g., a peptide or protein, that contains at least one epitope that will elicit an immune response and / or against which an immune response is directed. An antigen in the context of an embodiment of the present invention may be a molecule that, optionally after processing, induces an immune response that may be specific to the antigen or to a cell expressing the antigen. In particular, "antigen" relates to a molecule that, optionally after processing, is presented by MHC (major histocompatibility complex) molecules and reacts specifically with T lymphocytes (T cells). An antigen may be a tumor antigen, i.e. a constituent of a cancer cell, such as a protein or peptide expressed in the cancer cell, that may originate from the cytoplasm, the cell surface or the cell nucleus, in particular occurring mainly as an intracellular or surface antigen of the cancer cell.

[0095] Lipid nanoparticles are a non-viral formulation for intracellular delivery of RNA therapeutics such as siRNA and mRNA (Cullis & Hope, Lipid Nanoparticle Systems for Enabling Gene Therapies. Molecular Therapy (2017) 25). Additional methods for RNA encapsulation include polyplexes and liposomes.

[0096] Flash Nanoprecipitation (FNP) Flash nanoprecipitation (FNP) is a process of rapid micromixing (combining) in a confined geometry of miscible solvent and antisolvent streams to induce high supersaturation of components that are soluble in the solvent and insoluble in the antisolvent. Micromixing can occur in a variety of geometries. In FNP, a high velocity inlet stream induces turbulent mixing that occurs in a central cavity. The time of solvent / antisolvent mixing is faster than the precipitation of the components. Although not intended to be limiting, two such geometries have been previously described and analyzed: the confined impinging jet mixer (CIJ) (Johnson, BK, Prud'homme, RK Chemical processing and micromixing in confined impinging jets. AIChE Journal 2003, 49, 2264-2282) and the multi-inlet vortex mixer (MIVM) (Liu, Y., Cheng, C., Liu, Y., Prud'homme, RK, Fox, ROMixing in a multi-inlet vortex mixer (MIVM) for flash nano-deposition. Chemical Engineering Science 2008, 63, 2829-2842). These examples are intended to be illustrative, not limiting or exhaustive.

[0097] The stabilizing agent is incorporated into the solvent or anti-solvent stream. The drug to be incorporated or encapsulated in the nanoparticles is dissolved in the solvent stream. Mixing of the solvent and anti-solvent streams produces a composition in which the encapsulated species is insoluble. The resulting high supersaturation leads to rapid precipitation and growth of nanoparticles. The stabilizing agent in the formulation accumulates on the surface of the nanoparticles and stops the growth at the desired size. This process is described in Process and apparatuses for preparing nanoparticle compositions with amphiphilic copolymers and their use, BK Johnson, RK Prud'homme, U.S. Pat. No. 8,137,699 (2012). It is further described in a review article by Saad and Prud'hommee (see Saad, WS & Prud'homme, RK, Principles of nanoparticle formation by flash nanoprecipitation. Nano Today 2016, 11(2), 212-227). These and other documents mentioned herein are incorporated in their entirety into this application. In flash nanoprecipitation, the encapsulating agent is hydrophobic and the antisolvent (eg, an aqueous antisolvent) is more polar than the solvent.

[0098] The flash nanoprecipitation process includes a confined mixing volume having one or more solvent streams entering the mixing volume, one or more anti-solvent streams entering the mixing volume, and an exit stream for the process (exiting the mixing volume). The velocity of the inlet streams into the confined mixing volume can be from about 0.01 m / s to 100 m / s, or from about 0.1 m / s to 50 m / s, or from about 0.1 m / s to 10 m / s. The velocities of the streams may be equal to one another or may have different velocities. If the velocities are not equal, the velocity of the fastest stream is the specified velocity.

[0099] Inverse Flash Nanoprecipitation (iFNP) Inverse Flash Nanoprecipitation (iFNP) combines rapid micromixing in a confined geometry of miscible solvent and antisolvent streams to produce high supersaturation of components that are soluble in the solvent and insoluble in the antisolvent. Micromixing can occur in a variety of geometries. In iFNP, a high velocity inlet stream induces turbulent mixing that occurs in a central cavity. The time of solvent / antisolvent mixing is faster than the precipitation of the components. In iFNP, the encapsulant is hydrophilic and the antisolvent is less polar than the solvent. The inverse flash nanoprecipitation process is described in U.S. Patent Nos. 10,231,937 and 11,103,461, and U.S. Patent Application Nos. US20200206136 and US20210259984 and US20200023332, as well as WO2020227350 and WO2021046078;Pagels & Prud'homme, Polymeric nanoparticles and microparticles for the delivery of peptides, biologics, and soluble therapeutics.J Control Release(2015)vol.219;Markwalter et al.,Polymeric Nanocarrier Formulations of Biologics Using Inverse Flash NanoPrecipitation.The AAPS Journal(2020)Vol.22;Markwalter et al.,Sustained release of peptides and proteins from polymeric nanocarriers produced by inverse Flash NanoPrecipitation. Journal of Controlled Release (2021) Vol. 334. These documents are incorporated in their entirety into this application. In some cases, the term "flash nanoprecipitation" is used to refer to reverse flash nanoprecipitation. However, whether flash nanoprecipitation or reverse flash nanoprecipitation is used should be clear from the encapsulated materials, process solvents and anti-solvents.

[0100] Using the inverse flash nanoprecipitation (iFNP) process, "inverse" nanocarriers can be created with encapsulated hydrophilic substances, such as water-soluble peptides, proteins, and nucleic acids, such as RNA, DNA, antisense oligonucleotides, miRNA, siRNA, tRNA, plasmids, and nucleotides (Figure 1). These inverse nanocarriers have a hydrophobic surface imparted by a first stabilizing agent. The iFNP method can also include a processing step to generate a water-dispersible "coated nanocarrier" with a hydrophilic coating imparted by a second stabilizing agent. Additional processing steps can include solvent exchange to remove the non-polar solvent and can introduce a water-miscible modified solvent (if the anti-solvent is not already water-miscible). The method of the present invention provides for the incorporation of stabilizers and lipid components during this modification process. The inverse nanocarriers in the modified solvent as well as additional components and stabilizers are then rapidly micromixed in a manner similar to FNP using a constrained mixing geometry to generate a water-dispersible "coated" nanocarrier. Thus, iFNP can refer to both the processing steps to produce inverse nanocarriers and the series of steps required to produce coated nanocarriers.

[0101] The manufacturing process of inverse nanocarriers by iFNP can utilize encapsulating agents, stabilizers, solvent streams, anti-solvent streams, and optionally, salts.

[0102] Mounting agent In some embodiments of the method, the encapsulating agent is, for example, a hydrophilic peptide, a protein, or a nucleic acid. In one embodiment of the present invention, the encapsulating agent is a polynucleic acid or a nucleic acid, such as an oligonucleotide, a dinucleotide, a ribonucleic acid (RNA), a deoxyribonucleic acid (DNA), a plasmid DNA, an mRNA, a hybrid thereof, an RNAi-inducing (RNA interference-inducing) agent, a small interfering RNA (siRNA), a microRNA (miRNA), a small hairpin RNA (shRNA), a dicer substrate RNA (dsRNA), a transfer RNA (tRNA), a long non-coding RNA (lncRNA), a ribozyme, a catalytic DNA, an aptamer, a vector, or an antisense oligonucleotide. The nucleic acid can be linear, looped, circular, double-stranded, or single-stranded. The mRNA can be a self-amplifying mRNA. The mRNA can have natural nucleotides or can be a modified mRNA containing non-natural nucleotides. The antisense oligonucleotide can have a modified backbone or can include a locked nucleic acid, or can be a gap molecule. Nucleic acids containing different sequences can be co-encapsulated in the process.

[0103] The DNA or mRNA may code for a polypeptide of interest, including any naturally occurring or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA may be of any size and may have any secondary structure or activity. In some embodiments, the polypeptide encoded by the DNA or mRNA may have a therapeutic effect when expressed in a cell. The DNA or mRNA may include coding and non-coding flanking regions, such as the 5-UTR and 3-UTR (untranslated region).

[0104] siRNA can selectively knock down or downregulate the expression of a gene of interest.For example, siRNA can be selected to silence a gene associated with a particular disease, disorder or condition when administered to a subject in need of a nanoparticle composition comprising siRNA. siRNA can include a sequence that is complementary to the mRNA sequence that codes for a gene or protein of interest.In some embodiments, siRNA can be an immunomodulatory siRNA.

[0105] In other embodiments, recombinant polypeptides generated by translation of mRNA can alter DNA of gene expression in cells by encoding various endonucleases and auxiliary factors. Polypeptides including CRISPR (clustered regularly interspaced short palindromic repeats) associated (Cas) proteins, transcription activator-like effector nucleases (TALENs), or other endonucleases may be desirable to modify DNA of a subject to treat disease. For example, sgRNA-Cas9 complexes can affect mRNA translation of cellular genes. In certain embodiments, the therapeutic and / or prophylactic agent is an sgRNA and / or Cas9 encoded by mRNA, or other Cas protein variants, fragments, or subunits.

[0106] Nucleic acids and polynucleotides may include one or more naturally occurring components (either ribonucleic acids or deoxyribonucleic acids) that include any of the standard nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine) or T (thymidine). In some embodiments, all or substantially all nucleotides, including (a) the 5'-UTR, (b) the open reading frame (ORF), (c) the 3'-UTR, (d) the polyA tail, and any combination of (a, b, c or d above) include the naturally occurring standard nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine) or T (thymidine). Polynucleotides and nucleic acids may include one or more modified (e.g., altered or substituted) nucleobases, nucleosides, nucleotides, or combinations thereof. Nucleic acids and polynucleotides useful in nanoparticle compositions can include any useful modification or alteration, such as nucleobase, sugar, or internucleoside linkage (e.g., to phosphate / phosphodiester linkage / phosphodiester backbone). In certain embodiments, each of the nucleobase, sugar, and internucleoside linkage has a modification (e.g., one or more modifications). The modification according to the present disclosure can be a modification of ribonucleic acid (RNA) to deoxyribonucleic acid (DNA), such as a replacement of 2'-OH of the ribofuranosyl ring with 2'-H, a modification to threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or a hybrid thereof. Additional modifications are described herein.

[0107] A polynucleotide may contain (with respect to total nucleotide content or with respect to one or more types of nucleotides, i.e., any one or more of A, G, U, or C) from about 1% to about 100% alternative nucleotides or any intervening percentage (e.g., 1%-20%, 1%-25%, 1%-50%, 1%-60%, 1%-70%, 1%-80%, 1%-90%, 1%-95%, 10%-20%, 10%-25%, 10%-50%, 10%-60%, 10%-70%, 10%-80%, 10%-90%, 10%-10 ... 0%-95%, 10%-100%, 20%-25%, 20%-50%, 20%-60%, 20%-70%, 20%-80%, 20%-90%, 20%-95%, 20%-100%, 50%-60%, 50%-70%, 50%-80%, 50%-90%, 50%-95%, 50%-100%, 70%-80%, 70%-90%, 70%-95%, 70%-100%, 80%-90%, 80%-95%, 80%-100%, 90%-95%, 90%-100%, and 95%-100%). Any remaining percentages will be understood to be accounted for by the presence of standard nucleotides (eg, A, G, U or C).

[0108] Non-standard or modified bases may include one or more substitutions or modifications, including, for example, but not limited to, alkyl, aryl, halo, oxo, hydroxyl, alkyloxy, and / or thio substitutions; one or more condensations or ring openings; oxidations; and / or reductions. In some embodiments, the nucleobase is a surrogate uracil. Exemplary nucleobases and nucleosides with surrogate uracils include pseudouridine (y), pyridin-4-one ribonucleosides, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, 2-thio-uracil (s 2 U), 4-thio-uracil (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uracil (ho 5 U), 5-aminoallyl-uracil, 5-halo-uracil (e.g., 5-iodo-uracil or 5-bromo-uracil), 3-methyl-uracil (m 3 U), 5-methoxy-uracil (mo5 U), uracil 5-oxyacetic acid (cmo 5 U), uracil 5-oxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uracil (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uracil (chm 5 U), 5-carboxyhydroxymethyl-uracil methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uracil (mcm 5 U), 5-methoxycarbonylmethyl-2-thio-uracil (mcm 5 s 2 U), 5-aminomethyl-2-thio-uracil (nm 5 s 2 U), 5-methylaminomethyl-uracil (mnm 5 U), 5-methylaminomethyl-2-thio-uracil (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uracil (mnm 5 se 2 U), 5-carbamoylmethyl-uracil (ncm 5 U), 5-carboxymethylaminomethyl-uracil (cmnm 5 U), 5-carboxymethylaminomethyl-2-thio-uracil (cmnm 5 s 2 U), 5-propynyl-uracil, 1-propynyl-pseudouracil, 5-taurinomethyl-uracil (xm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uracil (xm 5 s 2 U), 1-taurinomethyl-4-thio-ploid uridine, 5-methyl-uracil (m 5 U, i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (mV), 1-ethyl-pseudouridine (EtV), 5-methyl-2-thio-uracil (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m's 4'|i), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 \| / ), 2-thio-1-methyl-pseudouridine, 1-methyl-l-deazo-pseudouridine, 2-thio-l-methyl-l-deazo-pseudouridine, dihydrouracil (D), dihydropseudouridine, 5,6-dihydrouracil, 5-methyl-dihydrouracil (m 5 D), 2-thio-dihydrouracil, 2-thio-dihydroploid uridine, 2-methoxy-uracil, 2-methoxy-4-thio-uracil, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uracil (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 y), 5-(isopentenylaminomethyl)uracil (inm 5 U), 5-(isopentenylaminomethyl)-2-thio-uracil (inm 5 s 2 U), 5,2'-O-dimethyl-uridine (m 5 Um), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3 Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 Um), 1-thio-uracil, deoxythymidine, 5-(2-carbomethoxyvinyl)-uracil, 5-(carbamoylhydroxymethyl)-uracil, 5-carbamoylmethyl-2-thio-uracil, 5-carboxymethyl-2-thio-uracil, 5-cyanomethyl-uracil, 5-methoxy-2-thio-uracil, and 5-[3-(1-E-propenylamino)]uracil.

[0109] In some embodiments, the nucleobase is an alternative cytosine. Exemplary nucleobases and nucleosides having an alternative cytosine are 5-aza-cytosine, 6-aza-cytosine, pseudoisocytidine, 3-methyl-cytosine (m3C), N4-acetyl-cytosine (ac4C), 5-formyl-cytosine (f5C), N4-methyl-cytosine (m4C), 5-methyl-cytosine (m5C), 5-halo-cytosine (e.g., 5-iodo-cytosine), 5- Hydroxymethyl-cytosine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytosine, pyrrolo-pseudoisocytidine, 2-thio-cytosine (s2C), 2-thio-5-methyl-cytosine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine Idoisocytidine, Zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytosine, 2-methoxy-5-methyl-cytosine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, Lysidine (k2C), 5,2'-O-dimethyl-cytidine (m5Cm), N These include 4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1-thio-cytosine, 5-hydroxy-cytosine, 5-(3-azidopropyl)-cytosine, and 5-(2-azidoethyl)-cytosine.

[0110] In some embodiments, the nucleobase is an alternative adenine. Exemplary nucleobases and nucleosides having an alternative adenine include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino- Purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenine (mlA), 2-methyl-adenine (m2A), N6-methyl-adenine (m6A), 2-methylthio-N6-methyl-adenine (ms2m6A), N6-isopentenyl-adenine (i6A), 2-methylthio-N6-isopentenyl-adenine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenine N6-glycinylcarbamoyl-adenine (g6A), N6-threonylcarbamoyl-adenine (t6A), N6-methyl-N6-threonylcarbamoyl-adenine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenine (ms2g6A), N6,N6-dimethyl-adenine (m62A), N6-hydroxy These include hydroxynorvalylcarbamoyl-adenine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenine (ms2hn6A), N6-acetyl-adenine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxyadenine, N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (ml Am), 2-amino-N6-methyl-purine, 1-thio-adenine, 8-azido-adenine, N6-(19-amino-pentaoxanonadecyl)-adenine, 2,8-dimethyl-adenine, N6-formyl-adenine, and N6-hydroxymethyl-adenine.

[0111] In some embodiments, the nucleobase is an alternative guanine. Exemplary nucleobases and nucleosides having an alternative guanine are inosine (I), 1-methyl-inosine (mll), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-l4), isowyosine (imG2), wyosine (yW), peroxywyosine (o2yW), hydroxywyosine (OHyW), undermodified hydroxywyosine (OHyW*), 7-deazaguanine (OHyW), hydroxywyosine (OHyW* ... anine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanine (preQO), 7-aminomethyl-7-deaza-guanine (preQ1), archeosine (G+), 7-deaza-8-aza-guanine, 6-thio-guanine, 6-thio-7-deaza-guanine, 6-thio-7-deaza-8-guanine, 7-methyl-guanine (m7G), 6 -thio-7-methyl-guanine, 7-methyl-inosine, 6-methoxy-guanine, 1-methyl-guanine (mlG), N2-methyl-guanine (m2G), N2,N2-dimethyl-guanine (m22), N2,7-dimethyl-guanine (m2,7G), N2,N2,7-dimethyl-guanine (m2,2,7G), 8-oxo-guanine, 7-methyl-8-oxo-guanine, 1-methyl-6-thio-guanine, N2-methyl-6-thio-guanine, N2,N These include 2-dimethyl-6-thio-guanine, N2-methyl-2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine (mlGm), N2,7-dimethyl-2'-O-methyl-guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (mllm), 1-thio-guanine, and O-6-methyl-guanine.

[0112] The alternative nucleobases of a nucleotide can be independently a purine, a pyrimidine, or a purine or pyrimidine analog. For example, the nucleobases can be alternatives to adenine, cytosine, guanine, uracil, or hypoxanthine. In some embodiments, nucleobases may also be selected from the group consisting of, for example, pyrazolo[3,4-d]pyrimidine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy, and other 8-substituted adenines. The bases may include naturally occurring and synthetic derivatives of bases, including uracil and guanine, 5-halo, e.g., 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5 triazinone, 9-deazapurine, imidazo[4,5-d]pyrazine, thiazolo[4,5-d]pyrimidine, pyrazin-2-one, 1,2,4-triazine, pyridazine; or 1,3,5-triazine. When nucleotides are designated using the abbreviations A, G, C, T, or U, each letter refers to a representative base and / or its derivatives, e.g., A includes adenine or an adenine analog, e.g., 7-deazaadenine.

[0113] Generally, RNA contains the sugar group ribose, which is a five-membered ring with oxygen. Exemplary non-limiting alternative nucleotides include substitution of oxygen in ribose (e.g., with S (sulfur), Se (selenium), or alkylenes, such as methylene or ethylene), addition of double bonds (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form cyclobutane or oxetane four-membered rings); ring expansion of ribose (e.g., anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino (which also has a phosphoramidate backbone). to form 6- or 7-membered rings with additional carbon or heteroatoms, such as those having additional carbon or heteroatoms); polycyclic forms (e.g., tricyclic and "unlocked" forms such as glycol nucleic acids (GNAs) (e.g., in which the ribose is replaced with a glycol unit attached to a phosphodiester bond, R-GNA or S-GNA), threose nucleic acids (in which the ribose is replaced with aL-threofuranosyl-(3'2'), TNA), and peptide nucleic acids (in which a 2-amino-ethyl-glycine bond replaces the ribose and phosphodiester backbone, PNA).

[0114] Alternative nucleotides can be modified on the internucleoside linkage (e.g., the phosphate backbone). In the present specification, the terms "phosphate" and "phosphodiester" are used interchangeably with respect to the polynucleotide backbone. The backbone phosphate group can be modified by replacing one or more oxygen atoms with different substituents. Alternative nucleotides can include large-scale replacement of unmodified phosphate sites with alternative internucleoside linkages as described herein. Examples of alternative phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced with sulfur. Phosphate linkers can also be modified by replacing linking oxygens with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).

[0115] Substitution of one or more oxygen atoms at the alpha position of a phosphate moiety (e.g., alpha-thiophosphate) can be provided to confer stability (such as against exonucleases and endonucleases) to RNA and DNA with non-natural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA exhibit increased resistance to nucleases and subsequently a longer half-life in the cellular environment.

[0116] A polynucleotide may comprise an internal ribosome entry site (IRES). An IRES may act as the sole ribosome binding site for an mRNA or as one of multiple ribosome binding sites. A polynucleotide comprising multiple functional ribosome binding sites may encode several peptides or polypeptides that are translated independently by the ribosome (e.g., a multicistronic mRNA). When a polynucleotide comprises an IRES, a second translatable region is optionally further provided. Examples of IRES sequences that can be used according to the present disclosure include, but are not limited to, those derived from picomaviruses (e.g., FMDV), plague viruses (CFFV), polioviruses (PV), encephalomyocarditis viruses (ECMV), foot and mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia viruses (MLV), simian immunodeficiency viruses (SIV) and / or cricket paralysis viruses (CrPV).

[0117] A polynucleotide (e.g., mRNA) may contain a 5'-cap structure. The 5'-cap structure of a polynucleotide is involved in nuclear export and enhancing polynucleotide stability, binds to mRNA cap-binding protein (CBP), and is responsible for polynucleotide stability and translational fitness in cells through the association of CBP with polyA-binding protein to form mature circular mRNA species. The cap also aids in the removal of 5'-proximal introns during mRNA splicing.

[0118] A polynucleotide or nucleic acid (e.g., mRNA) may contain a polyA sequence and / or a polyadenylation signal. The polyA sequence may be formed entirely or predominantly from adenine nucleotides or analogs or derivatives thereof. The polyA sequence may be a tail located adjacent to the 3' untranslated region of the nucleic acid.

[0119] The encapsulating agent may be an interfering RNA. The term "interfering RNA" or "RNAi" or "interfering RNA sequence" refers to single-stranded RNA (e.g., mature miRNA) or double-stranded RNA (i.e., double-stranded RNA such as siRNA, aiRNA (asymmetric interfering RNA) or pre-miRNA), which can reduce or inhibit the expression of a target gene or sequence (e.g., by mediating the degradation of the mRNA complementary to the interfering RNA sequence or inhibiting translation) when the interfering RNA is in the same cell as the target gene or sequence. Thus, an interfering RNA refers to a single-stranded RNA that is complementary to a target mRNA sequence, or to a double-stranded RNA formed by two complementary strands or one self-complementary strand. An interfering RNA may have substantial or complete identity to a target gene or sequence, or may contain a region of mismatch (i.e., a mismatch motif). The sequence of an interfering RNA may correspond to the full length of a target gene or a subsequence thereof.

[0120] Interfering RNA includes "small interfering RNA" or "siRNA", e.g., an interfering RNA of about 15-60, 15-50, or 15-40 (double-stranded) nucleotides in length, e.g., about 15-30, 15-25, or 19-25 (double-stranded) nucleotides in length, e.g., about 20-24, 21-22, or 21-23 (double-stranded) nucleotides in length (e.g., each complementary sequence of a double-stranded siRNA). The strings are 15-60, 15-50, 15-40, 15-30, 15-25 or 19-25 nucleotides long, e.g., about 20-24, 21-22 or 21-23 nucleotides long, and the double-stranded siRNAs are about 15-60, 15-50, 15-40, 15-30, 15-25 or 19-25 base pairs long, e.g., about 18-22, 19-20 or 19-21 base pairs long.) The siRNA duplexes can include a 3' overhang of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides and a 5' phosphate terminus. Examples of siRNA include, but are not limited to, double-stranded polynucleotide molecules assembled from two separate strand molecules (one strand is the sense strand and the other strand is the complementary antisense strand), double-stranded polynucleotide molecules assembled from single-stranded molecules (the sense and antisense regions are joined by a nucleic acid or non-nucleic acid based linker), double-stranded polynucleotide molecules having a hairpin secondary structure with self-complementary sense and antisense regions, and circular single-stranded polynucleotide molecules having two or more loop structures and a stem with self-complementary sense and antisense regions (circular polynucleotides can be processed in vivo or in vitro to generate active double-stranded siRNA molecules).

[0121] In one embodiment, the nucleic acid is an antisense oligonucleotide directed to a target gene or sequence of interest. The term "antisense oligonucleotide" or "antisense" includes oligonucleotides, such as a single strand of DNA or RNA, that is complementary to a selected polynucleotide sequence. Antisense RNA oligonucleotides prevent translation of a complementary RNA strand by binding to the RNA. Antisense DNA oligonucleotides can be used to target a specific complementary (coding or non-coding) RNA. If binding occurs, the DNA / RNA hybrid can be degraded by the enzyme RNase H. In certain embodiments, the antisense oligonucleotide comprises about 10 to about 60 nucleotides, for example, about 15 to about 30 nucleotides. The term also encompasses antisense oligonucleotides that may not be exactly complementary to the desired target gene. Thus, one embodiment of the present invention can be utilized when non-target specific activity is observed with the antisense or when an antisense sequence containing one or more mismatches with the target sequence is of particular use.

[0122] As mentioned above, the encapsulating agent can be a polynucleic acid or a nucleic acid, such as an oligonucleotide, a dinucleotide, a ribonucleic acid (RNA), a deoxyribonucleic acid (DNA), a plasmid DNA, an mRNA, a hybrid thereof, an RNAi inducer, a small interfering RNA (siRNA), a microRNA (miRNA), a small hairpin RNA (shRNA), a dicer substrate RNA (dsRNA), a transfer RNA (tRNA), a ribozyme, a catalytic DNA, an aptamer, a vector, or an antisense oligonucleotide. The encapsulating agent can be a single species or a combination of multiple types of oligonucleotide(s) with multiple sequences. The encapsulating agent(s) can include non-nucleic acid species, including hydrophilic small molecules, peptides, or proteins. For example, a protein encapsulating agent can be a Cas endonuclease. The non-nucleic acid species co-encapsulated with the nucleotide-based encapsulating agent can be included because it has a therapeutic benefit or may be useful for the encapsulation, delivery, or expression of the nucleotide-based encapsulating agent.

[0123] Stabilizers Stabilizing agents suitable for iFNP are described in U.S. Patent Nos. 10,231,937 and 11,103,461, U.S. Patent Application Nos. US20200206136, US20210259984 and US20200023332, and published International Applications Nos. WO2020227350 and WO2021046078. The stabilizing agent used in iFNP is also referred to herein as a first stabilizing agent or a first stabilizing polymer. The agent may be a copolymer in which a more polar block is coupled with a less polar (or less polar) block. The term "block" may be interpreted as a separate domain with a single molecular composition, or may refer to a region of the polymer chain that has mainly more polar regions and other less polar regions. Polarity may be imparted by monomers forming the polymer backbone, or by grafted pendant groups or chains attached to the main polymer backbone. For example, the copolymer can be amphiphilic (the more non-polar block is not water soluble), but this is not required; the copolymer can be completely water soluble or completely water insoluble, as long as the solubility of the blocks is significantly different in the antisolvent to allow for surface stabilization facilitated by the incorporation of a stabilizer at the nanoparticle surface. The copolymer should self-assemble in the antisolvent, with the more polar block precipitating and the less polar block remaining soluble. When used in the iFNP process to produce particles, the more polar block reaches the core of the particle and the less polar block forms a sterically protective shell or brush. The sterically protective shell prevents particle aggregation and prevents the release of encapsulated materials during subsequent processing steps.

[0124] The nanoparticles formed by the disclosed processes can be formed using graft, block or random copolymers, for example, these copolymers can have a molecular weight of about 1000 g / mol to about 1,000,000 g / mol, or about 3000 g / mol to about 25,000 g / mol, or at least about 2000 g / mol.

[0125] Copolymers are formed from repeating units or blocks with different solubility properties. For example, these repeating units can be included in at least two groups forming a block of a given property. Depending on the synthesis method, these blocks can be all the same repeating units or can contain different repeating units distributed throughout the block, but still produce blocks of copolymers with polar and non-polar moieties. These blocks can be arranged in a series of two blocks (diblock) or three blocks (triblock) or more blocks (multiblock) forming the backbone of the block copolymer. Furthermore, the polymer chains can have chemical moieties covalently bonded or grafted to the backbone. Such polymers are graft polymers. The block units that make up the copolymer can occur at regular intervals or randomly to create a random copolymer. Furthermore, the grafted side chains can occur at regular intervals along the polymer backbone or randomly to create a random grafted copolymer. In a graft polymer, a polar block can be grafted to a non-polar polymer. Alternatively, a non-polar block can be grafted to a more polar polymer chain. In graft copolymers, the length of the grafted sites can vary. For example, the grafted segments can correspond to 2 to 22 ethylene units in length. The grafted hydrophobic groups that create at least one low polar region of the copolymer can include tocopherol, tocopherol derivatives, lipids, alcohols containing 12 to 40 carbon atoms, cholesterol, other sterols, unsaturated and / or hydrogenated fatty acids, their salts, esters or amides, fatty acids, mono-, di- or triglycerides, waxes, ceramides, cholesterol derivatives, or combinations. Grafting of the polymer backbone can be useful to enhance the solvation or stabilization properties of the nanoparticles.

[0126] The copolymers used in the compositions and methods of the present invention may be formed from blocks of at least two repeat units and may have a minimum contour length equivalent to at least 25 ethylene units. The contour length is the linear sum of the polymer backbones and its molecular dimensions may be approximated using the Polymer Handbook, 4th Edition, eds. J. Brandrup, EH Immergut and EA Grulke, assoc. ed. A. Abe, DR Bloch, 1999, New York, John Wiley & Sons, which is incorporated herein by reference in its entirety.

[0127] Examples of suitable non-polar (or hydrophobic or low polar) blocks in the copolymer include, but are not limited to, the following: acrylates, including methyl acrylate, ethyl acrylate, propyl acrylate, n-butyl acrylate (BA), isobutyl acrylate, 2-ethyl acrylate, and t-butyl acrylate; methacrylates, including ethyl methacrylate, n-butyl methacrylate, and isobutyl methacrylate; acrylonitrile; methacrylonitrile; vinyls, including vinyl acetate, vinyl versatate, vinyl propionate, vinyl formamide, vinyl acetamide, vinyl pyridine, vinyl phenol, and vinyl imidazole; aminoalkyls, including aminoalkyl acrylates, aminoalkyl methacrylates, and aminoalkyl (meth)acrylamides; styrene; cellulose acetate phthalate, cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, poly(D,L-lactide), poly(D,L-lactide-co-glycolide). ), poly(glycolide), poly(hydroxybutyrate), poly(alkylcarbonates), and poly(orthoesters), polyesters, poly(hydroxyvaleric acid), polydioxanone, poly(ethylene terephthalate), poly(malic acid), poly(tartronic acid), polyanhydrides, polyphosphazenes, poly(amino acids), lactic acid, polylactic acid (PLA), caprolactone, poly(caprolactone) (PCL), glycolic acid, poly(glycolic acid), and copolymers thereof, such as poly(lactic acid-co-glycolic acid), ) (PLGA); hydrophobic peptide-based polymers and copolymers based on poly(L-amino acids), poly(ethylene-vinyl acetate) ("EVA") copolymers, silicone rubber, polyethylene, polypropylene, polydienes (polybutadiene, polyisoprene, and hydrogenated forms of these polymers), maleic anhydride copolymers of vinyl methyl ether and other vinyl ethers, polyamides (nylon 6,6), polyurethanes, poly(ester urethanes), poly(ether urethanes), and poly(ester ureas).Hydrophobically modified sugar polymers, such as HPMC (hydroxypropyl methylcellulose), acetalized dextran, or acetylated dextran, can be used. For example, the polymer block can include poly(ethylene vinyl acetate), poly(D,L-lactic acid) oligomers and polymers, poly(L-lactic acid) oligomers and polymers, poly(glycolic acid), copolymers of lactic acid and glycolic acid, poly(caprolactone), poly(valerolactone), polyanhydrides, copolymers of poly(caprolactone) or poly(lactic acid), or poly(propylene sulfide). For example, in non-bio-related applications, the polymer block can include, for example, polystyrene, polyacrylate, and butadiene.

[0128] Natural products having sufficient hydrophobicity to act as the non-polar portion of the polymer include: hydrophobic vitamins (e.g., vitamin E, vitamin K and vitamin A), carotenoids and retinols (e.g., beta-carotene, astaxanthin, trans and cis retinal, retinoic acid, folic acid, dihydrofolic acid, retinyl acetate, retinyl palmitate), cholecalciferol, calcitriol, hydroxycholecalciferol, ergocalciferol, α-tocopherol, α-tocopherol acetate, α-tocopherol nicotinic acid, estradiol, lipids, alcohols having 12 to 40 carbon atoms, cholesterol, other sterols, unsaturated and / or hydrogenated fatty acids, their salts, esters or amides, fatty acids, mono-, di- or triglycerides, waxes, ceramides, cholesterol derivatives, or mixtures thereof. For example, a natural product is Vitamin E, which is readily available as Vitamin E succinate, which facilitates functionalization to amines and hydroxyls in the active species.

[0129] Examples of suitable polar (or hydrophilic or highly polar) blocks in amphiphilic polymers that are block copolymers include, but are not limited to, carboxylic acids, including acrylic acid, methacrylic acid, itaconic acid, and maleic acid; polyoxyethylene or polyethylene oxide; polyacrylamide and copolymers with dimethyl-aminoethyl-methacrylate, diallyl-dimethyl-ammonium chloride, vinylbenzyltrimethylammonium chloride, acrylic acid, methacrylic acid, 2-acrylamido-2-methylpropanesulfonic acid, and styrenesulfonate, polyvinylpyrrolidone, starch and starch derivatives, dextran and dextran derivatives; polypeptides, such as polylysine, polyarginine, polyaspartic acid, polyglutamic acid; polyhyaluronic acid, alginic acid, polylactide, polyethyleneimine, polyionene, polyacrylic acid, and polyiminocarboxylates, gelatin, and unsaturated ethylenic mono- or dicarboxylic acids. To prepare anionic copolymers, acrylic acid, methacrylic acid, and / or polyaspartic acid or glutamic acid polymers can be used. To produce cationic copolymers, DMAEMA (dimethylaminoethyl methacrylate), polyvinylpyridine (PVP), and / or dimethylaminoethylacrylamide (DMAMAM) can be used. The polar block of the stabilizing polymer can be uncharged, cationic, cationizable, anionic or anionizable, or a combination thereof. A list of suitable polar water-soluble polymers can be found in Handbook of Water-Soluble Gums and Resins, R. Davidson, McGraw-Hill (1980), which is incorporated herein by reference in its entirety.

[0130] The non-polar and polar polymers may each be selected to have a molecular weight or range of molecular weights. For example, but not limited to, dextran, poly(aspartic acid) and poly(glutamic acid) polymers (alone or as components of a copolymer) may have a molecular weight within the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da. For example, but not limited to, PLA, PLGA and PCL polymers (alone or as components of a copolymer) may have a molecular weight within the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da.

[0131] The above list of non-polar and polar polymers should not be considered mutually exclusive. Copolymers of two polymers shown in a single list may have sufficient difference in solubility in a given anti-solvent used in this process. As an illustrative example, poly(ethylene oxide) and poly(acrylic acid) are both listed in the list of polar polymers. However, poly(ethylene oxide) dissolves in chloroform and acetone, but poly(acrylic acid) does not. Therefore, in this process, copolymers of poly(ethylene oxide) and poly(acrylic acid) can be used with chloroform or acetone as anti-solvents.

[0132] The stabilizer polymer may be a linear polymer comprising one or more polar blocks and one or more non-polar blocks. For example, the non-polar block(s) may be poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), polyester, poly(orthoester), poly[(carboxyphenoxy)propane-sebacic acid], polyphosphoester, polyesteramide, polyurethane, polyvinyl acrylate, poly(amino acid), or hydrophobically modified polysaccharide. For example, the polar block(s) may be poly(amino acid), such as poly(glutamic acid) (PGlu), poly(aspartic acid) (PAsp), poly(lysine), poly(arginine), poly(serine), poly(threonine), poly(glutamine), poly(asparagine), poly(cysteine), or combinations or modifications thereof. For example, the polar block(s) can be a polysaccharide, such as cellulose, dextran, maltodextrin, dextrin, dextran sulfate, dextrin sulfate, hyaluronic acid, pectin, amylopectin, amylose, pullulan, xylan, carrageenan, chitin, chitosan, starch, or combinations or modifications thereof. For example, the stabilizing polymer can be PAsp-b-PLA, PAsp-b-PLGA, PAsp-b-PCL, PAsp-b-PLA-b-PAsp, PAsp-b-PLGA-b-PAsp, poly(ethylene glycol)-b-PLA-b-PAsp, poly(ethylene glycol)-b-PLGA-b-PAsp, poly(ethylene glycol)-b-PCL-b-PAsp, PGlu-b-PLA, PGlu-b-PLGA, PGlu-b-PCL, PGlu-b-PLA-b-PGlu, PGlu-b-PLGA-b-PGlu, poly(ethylene glycol)-b-PLA-b-PGlu, poly(ethylene glycol)-b-PLGA-b-PGlu, poly(ethylene glycol)-b-PCL-b-PGlu, or other combinations thereof. For example, the stabilizing polymer can be polysaccharide-b-PLGA, polysaccharide-b-PLA, polysaccharide-b-PCL, or higher order block copolymers composed of polysaccharide and PLA, PCL and / or PLGA blocks.

[0133] Stabilizers that are block copolymers include poly(styrene)-b-poly(acrylic acid), poly(lactic acid)-b-poly(aspartic acid), poly(lactic acid-co-glycolic acid)-b-poly(aspartic acid), poly(ethylene glycol)-b-poly(lactic acid)-b-poly(aspartic acid), poly(aspartic acid)-b-poly(lactic acid-co-glycolic acid)-b-poly(aspartic acid), dextran-b-poly(lactic acid), and dextran-b-poly(lactic acid-co-glycolic acid).

[0134] In one embodiment of the present invention, the stabilizing polymer is or is a comb polymer comprising a hydrophilic backbone and hydrophobic branches or grafts. In one embodiment of the present invention, the backbone of the comb polymer may be branched, such as dextran or poly(aspartic acid) produced by condensation polymerization. In one embodiment of the present invention, the backbone of the comb polymer may be linear, such as cellulose. In one embodiment of the present invention, the hydrophilic backbone of the comb polymer is a polysaccharide. For example, the backbone may be cellulose, dextran, maltodextrin, dextrin, dextran sulfate, dextrin sulfate, hyaluronic acid, pectin, amylopectin, amylose, pullulan, xylan, carrageenan, chitin, chitosan, starch, or combinations or modifications thereof. In one embodiment of the invention, the hydrophilic backbone of the comb polymer can be a poly(amino acid), such as poly(glutamic acid), poly(aspartic acid), poly(lysine), poly(arginine), poly(serine), poly(threonine), poly(glutamine), poly(asparagine), poly(cysteine), or combinations or modifications thereof. In one embodiment of the invention, the backbone is polyvinyl alcohol. In one embodiment of the invention, the non-polar side chains are poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), polyesters, poly(orthoesters), poly[(carboxyphenoxy)propane-sebacic acid], polyphosphoesters, polyesteramides, polyurethanes, polyvinyl acrylates, poly(amino acids), or hydrophobically modified polysaccharides. The comb polymer may have 20%, 30%, 40%, 50%, 60%, 70% or 80% to 70%, 80%, 90% or 95% by weight of non-polar side chains. The backbone of the comb polymer may be 500 Da, 1000 Da, 2500 Da, 5000 Da, 10000 Da or 20000 Da to 20000 Da, 50000 Da, 100000 Da or 500000 Da. Each non-polar side chain or graft may be 100, 500, 1000, 5000 Da, 10000 Da or 20000 Da to 20000 Da, 50000 Da, 100000 Da or 500000 Da.The comb polymer may have 20%, 30%, 40%, 50%, 60%, 70% or 80% to 70%, 80%, 90% or 95% by weight of non-polar side chains. Those skilled in the art will recognize that the number of non-polar side chains per polar backbone that results in the appropriate weight percentage of non-polar groups will depend on the molecular weight of the various species. For example, the stabilizing polymer may be dextran-graft-PLGA, dextran-graft-PLA, dextran-graft-PCL, or another polysaccharide to which PLA, PCL or PLGA is grafted. For example, the stabilizing comb polymer may be PAsp-graft-PLA, PAsp-graft-PLGA, PAsp-graft-PCL, or another poly(amino acid) to which PLA, PLGA or PCL is grafted.

[0135] Random copolymers include hydroxypropyl cellulose, methyl cellulose, ethyl methyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, or combinations thereof. Cellulosic polymers may include hydroxypropyl, hydroxyethyl, hydroxymethyl, succinate, and / or acetate substitution(s). In another embodiment of the invention, poly(meth)acrylate-based random copolymers are used as stabilizers. Eudragit polymers manufactured by Evonik Industries are one of the commercialized versions of these. However, embodiments of the invention are not limited to the random copolymers listed above. Stabilizing random copolymers may have molecular weights ranging from about 0.1 kDa, 1 kDa, 10 kDa, 100 kDa, or 1000 kDa to about 5 kDa, 50 kDa, 100 kDa, 1000 kDa, or more.

[0136] The first stabilizing agent may be a blend of different stabilizing agents.

[0137] salt Other non-liquid compounds that aid in the flow of the solvent may be added and are also considered part of the solvent or anti-solvent. For example, surfactants, salts, or co-solvents may be added to the solvent and are also considered part of the solvent. These excipient compounds may or may not be present in the coated nanocarriers depending on the requirements of the final product.

[0138] Patent application WO2021046078 describes suitable salt forms of nucleic acids or polynucleic acids. Protonated (free acid) forms may be used, as well as partially (1%, 5%, 10%, 15%, 25%, 50%, 75%, 99% of the acid groups neutralized) or fully neutralized forms of nucleic acids with sodium hydroxide, lithium hydroxide, tris(tris(hydroxymethyl)aminomethane), triethylamine, ammonia, cesium hydroxide, potassium hydroxide, choline, thiamine, pyroxidine, urea, guanidine, diphenhydramine, primary amines, secondary amines, quaternary amines or tertiary amines.

[0139] Any method known in the art can be used to modify the salt form of nucleic acid.Nucleic acid can be neutralized in situ in a solvent stream.Nucleic acid can be neutralized by salt exchange process, and then purified and isolated before dissolving in solvent.Nucleic acid salt form can be modified by buffer selection after in vitro transcription.

[0140] Cross-linking of acidic residues in stabilizing copolymers, such as poly(aspartic acid) or poly(acrylic acid) or poly(glutamic acid), has been described (see U.S. Pat. Nos. 10,231,937 and 11,103,461, as well as Pagels, RF; Prud'homme, RK, Polymeric nanoparticles and microparticles for the delivery of peptides, biologics, and soluble therapeutics. J Control Release 2015, vol. 219, 519-535, which are incorporated herein by reference in their entirety). Suitable cross-linking agents include, but are not limited to, salt forms of calcium, iron, zinc, manganese, divalent cations, and trivalent cations. Polyvalent amines, such as spermine, tetraethylenepentamine, protamine, poly(lysine), or other organic amines may also be used.

[0141] For example, the nanoparticles can be crosslinked during nanoparticle assembly. For example, the nanoparticles can be crosslinked after nanoparticle assembly. The crosslinks can be covalent crosslinks. For example, the crosslinks can be disulfide crosslinks. For example, the crosslinks can be performed via "click chemistry". The crosslinks can include cleavable ester bonds of the type described in USP Application No. 13 / 969,449, Particulate Constructs for Release of Active Agents, Lawrence Mayer, et al. The crosslinks can be non-covalent. For example, the crosslinks can be ionic, chelating, acid-base, or hydrogen bond crosslinks.

[0142] Crosslinking agents can be added to crosslink the copolymer. For example, crosslinking agents can be added to crosslinking groups of the copolymer that have anionic functional groups or properties. For example, the crosslinking agent can be an alkaline earth halide, magnesium halide, magnesium chloride, calcium halide, calcium chloride, transition metal halide, iron halide, iron(III) chloride, spermine, or a combination thereof. For example, the crosslinking agent can be a metal acetate, an alkaline earth metal acetate, a transition metal acetate, calcium acetate, or a combination. For example, the crosslinking agent can be chromium(III) acetate, or another chromium(III) salt. For example, the crosslinking agent can be a metal nitrate, an alkaline earth nitrate, a transition metal nitrate, calcium nitrate, zinc nitrate, iron nitrate, or a combination. Other biocompatible polycationic water-soluble agents can be used as crosslinking agents, for example, to crosslink the anionic portion of the copolymer. For example, the water-soluble agent can include tobramycin, which can crosslink the copolymer. One example is tetraethylenepentamine. Ammonia or another chemical with basic properties can be added to promote ionic interactions between the cationic crosslinkers and the hydrophilic groups of the copolymer if they have anionic functional groups.

[0143] Alternatively, the shell of the nanoparticles can be crosslinked to increase the stability of the nanoparticles. For example, the end groups or other groups of hydrophobic (non-polar) polymer chains or blocks can be crosslinked. For example, the crosslinks can be ionic or covalent. For example, two or more hydrophobic polymer chains can be crosslinked directly or by the addition of a multifunctional crosslinker. For example, a hydrophobic polymer shell containing groups with a functionality "X" (on the chain end or throughout the chain) reactive to a functionality "Y" can be crosslinked using a crosslinker with two or more "Y" functional groups. For example, the X and Y functional groups can be thiol and acrylate, thiol and maleimide, azide and alkyne, amine and acid, or vice versa. The functional groups X and Y can be groups that react by "click chemistry". Added catalysts or other agents can be required or used to induce crosslinking of the shell.

[0144] Crosslinkers can also be used to neutralize the charge on the nucleic acid. If there are no acidic residues on the stabilizing copolymer, crosslinkers can still be included in the process.

[0145] solvent Suitable anti-solvents are described in U.S. Patents 10,231,937 and 11,103,461. Examples of polar process solvents include, but are not limited to, water, alcohol, acetone, acetonitrile, glycol ethers, dimethylsulfoxide (DMSO), dimethylformamide, N-methyl-2-pyrrolidone, dihydrolevoglucosenone, glycofurol, and mixtures thereof. In one embodiment, the encapsulant is dissolved in the process solvent or mixture. The stabilizer can be dissolved in the same process solvent or mixture, or in a different process solvent. The encapsulant and stabilizer solutions may be combined to form a single solution or may be kept separate until the mixing process. The polar process solvent may be heated or pressurized, or both, to facilitate dissolution of the encapsulant and stabilizer, depending on the solubility characteristics. The choice of polar process solvent is provided by the solubility characteristics of the encapsulant and stabilizer. The polar process solvent containing the stabilizer is selected such that the stabilizer is molecularly soluble. This means that the process solvent solubilizes all portions of the stabilizing agent. If present, the process solvent containing the encapsulating agent is also selected so that the material is molecularly soluble. These process solvents may be the same, but are not required. In some cases, both the stabilizing agent and the encapsulating agent can be dissolved in a single solution of the process solvent.

[0146] The concentration of the encapsulating agent can be within one order of magnitude of the concentration of the stabilizer. If the concentration of the encapsulating agent is much lower than the concentration of the stabilizer, the final drug loading can be low (small). If the concentration of the encapsulating active agent is much higher than the concentration of the stabilizer, the inverse nanoparticles cannot be stabilized sufficiently to prevent aggregation. In one embodiment, the encapsulating agent that is poorly soluble in the poor solvent is coated, encapsulated or restrained as a core component and sterically stabilized by the stabilizer.

[0147] The stabilizer can be dissolved in the polar process solvent at a concentration of at least 0.01 wt%, and the concentration of the stabilizer can be at least 0.1 wt% to form the first process solution. In one embodiment, the stabilizer can be dissolved in the polar process solvent at a concentration in the range of about 0.01 wt%, 0.1 wt%, 0.5 wt%, 1 wt%, 2 wt%, 5 wt%, 10 wt%, or 20 wt% to about 0.2 wt%, 0.5 wt%, 1 wt%, 2 wt%, 5 wt%, 10 wt%, 20 wt%, or 40 wt%. Those skilled in the art will appreciate that the lower concentration limit can be limited by factors such as process economics, and the upper concentration limit can be limited by factors such as the viscosity of the process solution or the solubility limit of the copolymer in the polar process solvent. For example, if the viscosity of the first process solution is much greater than the viscosity of the anti-solvent, mixing of the first process solution with the anti-solvent can be inhibited. One of ordinary skill in the art will appreciate that factors such as the molecular weight of the copolymer and the composition of the copolymer can affect the maximum concentration that can be achieved in a polymer solution before the viscosity becomes too high (too large).

[0148] In one embodiment, the encapsulant may be dissolved in a polar process solvent at a concentration ranging from about 0.01%, 0.1%, 0.5%, 1%, 2%, 5%, 10%, or 20% by weight to about 0.2%, 0.5%, 1%, 2%, 5%, 10%, 20%, or 40% by weight. One skilled in the art will appreciate that factors such as process economics may limit the lower concentration limit, and factors such as the viscosity of the process solution or solubility limits in the polar process solvent may limit the upper concentration limit.

[0149] In one embodiment of this process, the solvent is DMSO (dimethyl sulfoxide).In an alternative embodiment, DMSO is in a mixture with water.For example, the water content in the mixture of DMSO and water can be 0.5% by volume (volume %), 1% by volume, 2.5% by volume, 5% by volume, 7.5% by volume, 10% by volume, 15% by volume, 20% by volume or 25% by volume.

[0150] Antisolvent In one embodiment of the present invention, a suitable anti-solvent is less polar than the process solvent and is described in US Patent Nos. 10,231,937 and 11,103,461. Examples include chloroform, dichloromethane, alcohols, alkanes such as hexane, tetrahydrofuran (THF), toluene, ethyl acetate, methyl acetate, acetonitrile, acetone, or a combination. The choice of anti-solvent is determined by the solubility characteristics of the stabilizing agent and the encapsulating agent. The encapsulating agent becomes supersaturated under the mixing conditions and precipitates from the solution. Furthermore, the anti-solvent is selected so that the different solubility characteristics of the regions or parts of the stabilizing agent are revealed and the more polar parts of the copolymer cannot exist in a soluble state, so that the stabilizing agent associates with the encapsulating agent and precipitates, generating a stable "inverse" nanocarrier. It is important to note that a process solvent in one system may effectively function as an anti-solvent in another system, and therefore, the listing as a process solvent above the examples should not be considered to preclude its use as an anti-solvent, and the listing as a anti-solvent above the examples should not be considered to preclude its use as a process solvent.

[0151] The anti-solvent is selected so that the more polar parts of the stabilizer precipitate quickly, while the more non-polar components of the stabilizer remain solubilized. Thus, the stabilizer can self-assemble into the desired nanoparticle shape in the anti-solvent. The anti-solvent is selected so that the encapsulant precipitates quickly in the final mixture. In some cases, the process solvent and the anti-solvent can be completely miscible in the final composition. In some cases, 20% or less by volume of the process solvent can phase separate in the final composition.

[0152] In one embodiment, the crosslinker or salt can be incorporated into the anti-solvent. In some embodiments, the crosslinker can be first dissolved in a secondary anti-solvent. For example, a solution in methanol could be made and then added to the dichloromethane anti-solvent. The secondary anti-solvent can be 1%, 2%, 5%, 10%, 20% or more (more) by volume in the anti-solvent composition. The concentration of the crosslinker is selected to provide the desired charge equivalent relative to the total acid residue composition of the encapsulant and stabilizer. The charge ratio is the molar ratio of the positive charge from the crosslinker and the negative charge from the total acid residues. The crosslinking charge can be 0.1, or 0.2, or 0.25, or 0.3, or 0.5, or 0.6, or 0.7, or 0.75, or 0.9, or 1, or 1.2, or 1.5, or 2, or more (more) relative to the acid charge.

[0153] mixture Intense micromixing of the process solution and antisolvent can be achieved in a variety of geometries. A high velocity inlet flow causes turbulence and mixing to occur in the central cavity. The time of process solvent / antisolvent mixing is faster than the association time of the nanoparticles. Although not intended to be limiting, two such geometries have been previously described and analyzed: the confined impinging jet mixer (CIJ) (Johnson, BK, Prud'homme, RK Chemical processing and micromixing in confined impinging jets. AIChE Journal 2003, 49, 2264-2282) and the multi-inlet vortex mixer (MIVM) (Liu, Y., Cheng, C., Liu, Y., Prud'homme, RK, Fox, ROMixing in a multi-inlet vortex mixer (MIVM) for flash nano-deposition. Chemical Engineering Science 2008, 63, 2829-2842). These examples are intended to be illustrative, not limiting or exhaustive.

[0154] The fast mixing and high energy dissipation involved in the iFNP process provide mixing time scales shorter than the time scales of particle nucleation and growth, thereby forming nanoparticles with active agent loading content and size distribution not provided by other techniques. When forming nanoparticles via inverse flash nanoprecipitation (iFNP), mixing is performed fast enough to allow high supersaturation levels, such as reaching a supersaturation level of 10,000 for all components, before aggregation begins. The supersaturation level is the ratio of the actual concentration of a material, such as an encapsulant, in a solvent to the saturation concentration of that material in that solvent. For example, the supersaturation level can be at least about 1, 3, 10, 30, 100, 300, 1000 or 3000, and can be up to about 3, 10, 30, 100, 300, 1000, 3000, 10,000, 30,000 or 100,000. The aggregation time scales of the encapsulant active substance and the copolymer self-assembly can be balanced. Thus, hydrophilic active materials and polymers may be precipitated simultaneously, overcoming the limitations of low encapsulant incorporation and aggregation seen in other techniques based on slow solvent exchange (e.g., dialysis).The reverse-flash nanoprecipitation process is a broadly applicable nanoparticle formation technique because it may be insensitive to the chemical specificities of the components.

[0155] In one embodiment, the encapsulant and stabilizing agent are dissolved in a single process solvent stream or solution. This stream is then rapidly mixed with the anti-solvent in a mixer such that the time of solvent / anti-solvent mixing is faster than the association time of the nanoparticles, as described, for example, in WO2017 / 112828A1, WO2015 / 200054 and US Pat. No. 8,137,699. The stream is synonymous with the liquid phase used as input to the mixing process. The single process solvent stream can alternatively be mixed with two or more anti-solvent streams. The flow rates of the solvent and anti-solvent streams can be the same or different. The flow rates can be altered to further tailor the solvent / anti-solvent composition in the central cavity of the mixer. For example, the composition in the central cavity of the mixer can be 50% by volume or less of solvent, or 40% by volume or less of solvent, or 25% by volume or less of solvent, or 10% by volume or less of solvent. In one embodiment, if a cross-linker is included, the cross-linker can be dissolved in all anti-solvent inlet streams. In one embodiment, if a cross-linking agent is included, the cross-linking agent can be dissolved in a single anti-solvent stream or in multiple anti-solvent streams.

[0156] In one embodiment, the encapsulant and the stabilizer are dissolved in separate process solvent streams. The process solvent used to dissolve the stabilizer and the process solvent used to dissolve the encapsulant may be the same, but need not be. For example, the encapsulant can be dissolved in a first polar process solvent to form an encapsulant solution, and the stabilizer can be dissolved in a second polar process solvent to form a stabilizer solution. The encapsulant and stabilizer solutions are mixed, for example, simultaneously, with an anti-solvent to form a mixed solution. The first polar process solvent and the second polar process solvent can be miscible or completely miscible at the volume ratio in which they are mixed (i.e., no separate phases are formed). The first polar process solvent and the anti-solvent can be miscible or completely miscible at the volume ratio in which they are mixed (i.e., no separate phases are formed). The second polar process solvent and the anti-solvent can be miscible or completely miscible at the volume ratio in which they are mixed (i.e., no separate phases are formed).

[0157] Those skilled in the art will recognize that all solvents are miscible with each other to some degree. Miscible solvents are used in the initial nanoparticle precipitation method. As referred to herein, a "miscible" solvent is one that, when mixed in the ratio used in the process, produces a solution with 20% or less of the volume of a minor phase that is not dissolved in the major phase (e.g., a polar process solvent). As referred to herein, a fully miscible solvent is one that, when mixed in the ratio used in the nanoparticle formation process, microparticle process, or another process, produces a solution without phase separation. As referred to herein, a "non-miscible" solvent is one that, when mixed in the ratio used in the process, produces a solution with 20% or more of the volume of a minor phase that is not dissolved in the major phase.

[0158] The nanoparticles can be made from the copolymer dissolved in a process solvent to which no hydrophilic active material has been added.

[0159] Additional anti-solvent can be added after the micromixing process to adjust the final composition of the solvent mixture if desired. Additional anti-solvent can be added by in-line mixing or can already be present in the collection vessel for the outlet of the mixer. The final composition of the mixture can be adjusted to have a process solvent content of 25% by volume or less, or 20% by volume or less, or 15% by volume or less, or 10% by volume or less, or 5% by volume or less. This can be referred to as a quench or collection bath or dilution bath.

[0160] The size of the nanoparticles resulting from this process can be controlled by controlling the mixing speed used to create them, the total mass concentration of the encapsulating agent and stabilizer in the process solvent, the process solvent(s) and anti-solvent(s), the ratio of encapsulating agent to stabilizer, and the supersaturation of the encapsulating agent and stabilizer when mixed with the anti-solvent.

[0161] In one method of the present invention, particles having a size in the range of 15 nm to 10,500 nm, 20 nm to 6,000 nm, 20 nm to 1,000 nm, 35 nm to 400 nm, or 40 nm to 300 nm can be produced. The size can be determined by dynamic light scattering. For example, particles can be made with a size of at least about 15 nm, 20 nm, 35 nm, 40 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 600 nm, 900 nm, 1000 nm, 2000 nm, 4000 nm, or 6000 nm, and up to about 20 nm, 35 nm, 40 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 600 nm, 900 nm, 1000 nm, 2000 nm, 4000 nm, 6000 nm, or 10500 nm. The sizes reported and cited herein are reported intensity average values ​​determined by Malvern Nanosizer deconvolution program for particles less than 2000 nm and determined by scanning electron microscopy. Other intensity-weighted deconvolution methods can be used to determine the size of nanoparticles.

[0162] Extraction of reverse nanocarriers After inverse nanoparticle formation, additional processing steps can be performed to create the desired formulation. Residual DMSO or other process solvents can be removed using extraction if the nanoparticles are dispersed in a water-immiscible antisolvent. The composition of the aqueous solution can be modified to promote the stability of the polymeric stabilizer. For example, the aqueous stream can contain 150 mM sodium chloride to adjust the osmolality. Sugars, polyethylene glycols (PEGs), or other osmolytes may be used to achieve a similar effect. The pH can be adjusted to limit the solubility of the stabilizer. Organic acids such as acetic or citric acid may be used, as well as mineral acids such as hydrochloric acid. Similarly, bases such as ammonium hydroxide and sodium hydroxide may be added. Buffer systems such as borates or carboxylates or others may be used. Extraction can be performed for 15 minutes or more using standard protocols that are acceptable depending on the processing scale. In one embodiment, the aqueous phase is sent to waste with the interface, and the organic phase containing the inverse nanoparticles is retained for further processing. Standard extraction methods known in the art can be used in this process. This is described in Markwalter, C. E. et al., Polymeric Nanocarrier Formulations of Biologics Using Inverse Flash NanoPrecipitation. The AAPS Journal (2020) Vol. 22, No. 2, p. 18 (doi:10.1208 / s12248-019-0405-z), which is incorporated by reference in its entirety.

[0163] Incorporation of additives and secondary stabilizers In one embodiment of the present invention, additives are added to the inverse nanocarriers and then processed so that they assemble on the surface of the inverse nanocarriers. The additive assembly is solubility driven and not controlled by charge complexation as in the case of lipid nanoparticles. Without being bound by theory, additives (e.g., hydrophobic or amphiphilic species) can be assembled on the surface during a successive solvent exchange process - first into a modified solvent and then into an aqueous system. The exchange into the modified solvent can be achieved by distillation or dialysis or other techniques known in the art. The exchange into an aqueous system can be achieved subsequently using flash nanoprecipitation techniques to rapidly mix an organic stream containing the components to be assembled with an aqueous anti-solvent.

[0164] It is surprising that the incorporation of additives, such as lipids, had a positive effect on the encapsulation efficiency of the encapsulating agent. The following examples demonstrate improved encapsulation efficiency and therapeutic efficacy using the processes and compositions reported herein.

[0165] The additive and the second stabilizer can be added to the inverse nanocarrier dispersion in pure form or as a solution in which they are dissolved in a suitable organic solvent. A suitable organic solvent is one in which the additive is soluble (molecularly dissolvable) but does not solubilize the first stabilizer from the surface of the inverse nanoparticles. For example, organic solvents such as chloroform, dichloromethane, ethanol, tetrahydrofuran, acetonitrile or methanol can be suitable solvents for adding the drug. The suitable solvent can be the same as the solvent in which the inverse nanoparticles are dispersed. The above list is representative and not exhaustive. Those skilled in the art will recognize that the particular drug and inverse nanocarrier composition can guide or determine the choice of solvent. The additive can be dissolved directly in the inverse nanocarrier dispersion without first being dissolved in an organic solution.

[0166] In one embodiment of the invention, the agent(s) comprise fatty acids, lipids, phospholipids, fatty acid methyl esters, fatty acid ethyl esters, sterols, cholesterol, organic acids, oils, cationic ionizable lipids, cationic lipids, polyvalent cationic lipids, sphingosines, sphingolipids, branched tail lipids, lipidoids, anionic lipids, glycosylated lipids, pH sensitive lipids, lipids containing modified head groups, biotinylated lipids, ceramides, diacylglycerin lipids, adjuvants, sphingomyelins, prostaglandins, eicosanoids, glycerides, glycosylated diacylglycerols, triglycerides, monoglycerides, diglycerides, oxygenated fatty acids, prenol, cholic acid, bile acids, taurocholic acid, carnitine lipids, oxysterols, cholesteryl esters, glycosylated sterols, hydrophobic small molecules, and / or biologically active small molecules. The second stabilizing agent may comprise a PEG-modified lipid, a PEG-containing block copolymer, PEG-b-PLA, PEG-b-PLGA, PEG-b-PCL, a dextran-modified lipid, a dextran-containing block copolymer, a poly(sarcosine)-modified lipid, a poly(sarcosine)-containing block copolymer, poly(sarcosine)-b-PLA, poly(sarcosine)-b-PLGA, and / or poly(sarcosine)-b-PCL. The stabilizing agent has a water-soluble (or hydrophilic) region and a hydrophobic region.

[0167] The drug(s) may be a mixture of components and is not limited to the addition of one component. The ratios of these drugs to each other and to the inverse nanocarrier are defined below. The additive mixture may be between phospholipids and cholesterol, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), a second lipid, a cationic lipid, a cationic ionizable lipid, anionic lipid, ethyl oleate, β-sitosterol, dioleoylphosphatidylethanolamine (DOPE), a fatty acid, a fatty acid ester, a polymer, a polyester, poly(lactic-co-glycolic acid), or a hydrophobic organic compound with known medicinal properties. This list is not intended to be exhaustive, and other additives with suitable properties may be selected as well.

[0168] One embodiment of the present invention uses an additive that is a lipid blend of cationic ionizable lipids, phospholipids, and structural lipids such as sterols.The cationic ionizable lipids can be known in the art.The cationic ionizable lipids can be permanently ionized, i.e., quaternary amines with a permanent charge to form cationic lipids.Such lipid blends are widely described in publications and patents such as U.S. Patent Nos. 8,058,069, 9,364,435, 10,221,127, WO2018170306A1, and WO2020061367A1.This list of patents and applications is not intended to be exhaustive, but rather to provide representative examples of suitable lipid blends.

[0169] Phospholipids useful in the compositions and methods include 1,2-distearoyl-s-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleo ... Lysero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (Cl6 lysoPC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarrachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl The nanoparticle composition may be selected from the non-limiting group consisting of 1-stearoyl-2-oleoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and combinations thereof. In some embodiments, the nanoparticle composition comprises POPC. In certain embodiments, the nanoparticle composition comprises DOPE. In some embodiments, the nanoparticle composition comprises DOPC. In some embodiments, the nanoparticle composition comprises both POPC and DOPE.

[0170] The additive(s) can be selected from anionic lipids. The term "anionic lipid" refers to lipids that are negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG) and other anionic modification groups attached to neutral lipids.

[0171] The cationic lipid or cationic ionizable lipid can be selected from any list known in the art. See, e.g., U.S. Pat. No. 10,221,127, International Application Publication Nos. WO2018170306A1 and WO2020061367A1, U.S. Patent Publication Nos. 20060083780, 20060240554 and 20210101875, U.S. Pat. Nos. 5,208,036, 5,264,618, 5,279,833, 5,283,185, 5,753,613 and 5,785,992, WO2019246203A1, and U.S. Pat. Nos. 10,562,849, 10,888,626 and 5,885,613. The term "cationic lipid" refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH (e.g., a pH of about 7.0). In some cases, the cationic lipid includes a protonated tertiary amine (e.g., pH-titratable) head group, an alkyl chain, an ether or ester bond between the head group and the alkyl chain, and 0-3 double bonds. In other cases, the cationic lipid includes a branched lipid tail formed from a saturated alkyl chain, as reported, for example, in WO2020061367A1. Other cationic lipid variants include thiourea and squaramide moieties at the head group. Commonly used cationic lipids are summarized in the literature (Hou, Zaks, Langer, and Dong. Nature Reviews Materials, 6, 1078-1094, 2021; Kauffman, Webber, and Anderson. Journal of Controlled Release, 240, 227-234, 2016) and include Dlin-MC3-DMA, SM-102, ALC-0315, ePC, C12-200, cKK-E12, OF-Deg-Lin, A2-iso5-2DC18, 306O i10 , 503O 13 , BAME-O16B, TT3, and FTT5. The cationic lipid may be multivalent, such as MVL5 and GL67.

[0172] Additional cationic lipids include 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (Dlin-KC2-DMA; "XTC2"), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (Dlin-KC3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (Dlin-KC4-DMA), and 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (Dlin-KC5-DMA). (Dlin-KC4-DMA), 2,2-Dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (Dlin-K6-DMA), 2,2-Dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane (Dlin-K-MPZ), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (Dlin-K-DMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (Dlin-C-DAP), 1,2-Dilinoleyloxy-3-(dimethylamino)acetoxypropane (Dlin-DAC ), 1,2-Dilinoleyloxy-3-morpholinopropane (Dlin-MA), 1,2-Dilinoleyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (Dlin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (Dlin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (Dlin-TMA), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (Dlin-TAP), 1,2-Dilinoleyloxy ... Dlin-MPZ), 3-(N,N-Dilinoleylamino)-1,2-propanediol (DlinAP), 3-(N,N-Dioleylamino)-1,2-propanediol (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (Dlin-EG-DMA), N,N-Dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-Dioleyloxy-N,N-dimethylaminopropane (DODMA), 1,2-Distearyloxy-N,N-Dimethylaminopropane (DSDMA), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOTAP), SPA), dioctadecylamidoglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-β-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-β-oxy)-3'-oxapentoxy]-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), or mixtures thereof. In certain embodiments, the cationic lipid is DLinDMA, Dlin-KC2-DMA ("XTC2"), 4-(dimethylamino)butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester (Dlin-MC3-DMA), or a mixture thereof. In addition, the cationic lipid may also be a lipid containing a cyclic amine group.

[0173] The additive(s) may include a polymer, such as poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(L-lactic-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone ... Poly(D,L-lactide-co-PEO-co-D,L-lactide), Poly(D,L-lactide-co-PPO-co-D,L-lactide), Polyalkylcyanoacrylate, Polyurethane, Poly-L-lysine (PLL), Hydroxypropyl methacrylate (HPMA), Polyethylene glycol, Poly-L-glutamic acid, Poly(hydroxy acid), Polyanhydride, Polyorthoester, Poly(ester amide), Polyamide, U.S. Pat. No. 9,801,636.No. 944, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohol (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone (PVP), polysiloxanes, polystyrene (PS), polyurethanes, derivative celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocellulose, hydroxypropyl cellulose, carboxymethyl cellulose, polymers of acrylic acid such as poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl cellulose), poly(ethyl ... butyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and copolymers and mixtures thereof, polydioxanone and copolymers thereof, polyhydroxyalkanoates, polypropylene fumarates, polyoxymethylenes, poloxamers, polyoxamines, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, poly(7V-acryloylmorpholine) (PacM), poly(2-methyl-2-oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), and polyglycerol.

[0174] The additive(s) may include structured lipids selected from, but not limited to, sterols, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, α-tocopherol, and mixtures thereof. Cholesterol derivatives may be selected, such as cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, and / or cholesteryl-4'-hydroxybutyl ether. In some embodiments, the structured lipid is cholesterol. In some embodiments, the structured lipid includes cholesterol and corticosteroids (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone), or combinations thereof.

[0175] In some embodiments, the nanoparticle compositions comprising one or more lipids described herein may also include one or more lipidated adjuvants, such as glucopyranosyl lipid adjuvants (GLA) and Pam3CSK4.

[0176] The second stabilizing agent can be a copolymer of a hydrophilic block bound to a hydrophobic block, for example, the second stabilizing agent can be a second stabilizing amphiphilic agent or a second stabilizing amphiphilic copolymer. The inverse nanocarriers coated by the disclosed process can be coated with graft, block or random amphiphilic copolymers. These amphiphilic copolymers can have a molecular weight of about 1000 g / mol to about 50,000 g / mol, about 3000 g / mol to about 25,000 g / mol, or at least about 2000 g / mol.

[0177] Examples of suitable hydrophobic blocks in amphiphilic copolymers that are block copolymers include, but are not limited to, the following: acrylates, including methyl acrylate, ethyl acrylate, propyl acrylate, n-butyl acrylate (BA), isobutyl acrylate, 2-ethyl acrylate, and t-butyl acrylate; methacrylates, including ethyl methacrylate, n-butyl methacrylate, and isobutyl methacrylate; acrylonitrile; methacrylonitrile; vinyls, including vinyl acetate, vinyl versatate, vinyl propionate, vinyl formamide, vinyl acetamide, vinyl pyridine, vinyl phenol, and vinyl imidazole; aminoalkyls, including aminoalkyl acrylates, aminoalkyl methacrylates, and aminoalkyl (meth)acrylamides; styrene; cellulose acetate phthalate, cellulose acetate succinate, hydroxypropyl methylcellulose, and cellulose acetate phthalate. poly(hydroxybutyrate), poly(alkylcarbonate), and poly(orthoesters), polyesters, poly(hydroxyvaleric acid), polydioxanones, poly(ethylene terephthalate), poly(malic acid), poly(tartronic acid), polyanhydrides, polyphosphazenes, poly(amino acids), and copolymers thereof; hydrophobic peptide-based polymers and copolymers based on poly(L-amino acids), poly(ethylene-vinyl acetate) ("EVA") copolymers, silicone rubber, polyethylene, polypropylene, polydienes (polybutadiene, polyisoprene, and hydrogenated forms of these polymers), maleic anhydride copolymers of vinyl methyl ether and other vinyl ethers, polyamides (nylon 6,6), polyurethanes, poly(ester urethanes), poly(ether urethanes), and poly(ester ureas).For example, polymer blocks include poly(ethylene vinyl acetate), poly(D,L-lactic acid) oligomers and polymers, poly(L-lactic acid) oligomers and polymers, poly(glycolic acid), copolymers of lactic acid and glycolic acid, poly(caprolactone), poly(valerolactone), polyanhydrides, copolymers of poly(caprolactone) or poly(lactic acid).

[0178] Natural products that are sufficiently hydrophobic to act as the hydrophobic portion of an amphiphilic polymer include, for example, hydrophobic vitamins (e.g., vitamin E, vitamin K, and vitamin A), carotenoids, sterols, cholesterol, and retinol (e.g., β-carotene, astaxanthin, trans- and cis-retinal, retinoic acid, folic acid, dihydrofolic acid, retinyl acetate, retinol palmitate), cholecalciferol, calcitriol, hydroxycholecalciferol, ergocalciferol, α-tocopherol, α-tocopherol acetate, α-tocopherol nicotinate, and estradiol.

[0179] Examples of suitable hydrophilic blocks in amphiphilic polymers include, but are not limited to, carboxylic acids, including acrylic acid, methacrylic acid, itaconic acid and maleic acid; polyoxyethylene or polyethylene oxide; polyacrylamide and its copolymers with dimethylaminoethyl methacrylate, diallyldimethylammonium chloride, vinylbenzyltrimethylammonium chloride, acrylic acid, methacrylic acid, 2-acrylamido-2-methylpropanesulfonic acid and styrenesulfonate, polyvinylpyrrolidone, starch and starch derivatives, dextran and dextran derivatives; polypeptides, such as poly(lysine), poly(arginine), poly(glutamic acid), poly(sarcosine); polyhyaluronic acid, alginic acid, polylactide, polyethyleneimine, polyionene, polyacrylic acid, and polyiminocarboxylate, poly(ethylene glycol), gelatin, and unsaturated ethylenic mono- or dicarboxylic acids. For example, the hydrophilic block can be poly(ethylene glycol). To prepare cationic amphiphilic polymers, DMAEMA (dimethylaminoethyl methacrylate), polyvinylpyridine (PVP), chitosan, poly(lysine), poly(ethyleneimine), or dimethylaminoethylacrylamide (DMAMAM) can be used. A representative list of suitable cationic polymers can be found in the literature, for example, Kauffman, Webber, and Anderson. Journal of Controlled Release, 240, 227-234 (2016), and a further list of suitable hydrophilic polymers can be found in Handbook of Water-Soluble Gums and Resins, R. Davidson, McGraw-Hill (1980), which are incorporated herein by reference in their entirety.

[0180] The polymer block can be a diblock, triblock or multiblock repeat. For example, the amphiphilic polymer can be polystyrene-block-poly(ethylene glycol) (PS-b-PEG), poly(lactic acid)-block-poly(ethylene glycol) (PLA-b-PEG), poly(caprolactone)-block-poly(ethylene glycol) (PCL-b-PEG), poly(lactic acid-co-glycolic acid)-block-poly(ethylene glycol) (PLGA-b-PEG), or a triblock form of the above diblock copolymer. In addition, triblock copolymers such as poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (PEO-b-PPO-b-PEO), or poly(ethylene oxide)-block-poly(butylene oxide)-block-poly(ethylene oxide) (PEO-b-PBO-b-PEO) can be used.

[0181] In amphiphilic polymers that are graft copolymers, the length of the grafted moieties can vary. For example, the grafted segments can be alkyl chains of 4 to 22 carbons in length, or alkyl chains corresponding to 2 to 11 ethylene units. The grafted groups can also include lipids, phospholipids, or cholesterol. Grafting of the polymer backbone can be useful to enhance the solvation or stabilization properties of the nanoparticles. Suitable chemical moieties grafted to the block units of the copolymer include alkyl chains containing species such as amide, imide, phenyl, carboxy, aldehyde, or alcohol groups.

[0182] In some embodiments, the second stabilizer is a complex lipid.Examples of suitable complex lipid include, but are not limited to, PEG-lipid complexes, dextran-lipid complexes, cationic polymer-lipid complexes (CPLs), sarcosine-lipid complexes, and mixtures thereof.In certain embodiments, the particle comprises any of the PEG-lipid complexes together with CPLs.In certain embodiments, the PEG lipid is selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and / or PEG-modified dialkylglycerol. For example, some embodiments include PEGylated diacylglycerols (PEG-DAG) such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerols (PEG-S-DAG) such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DAG). AG), pegylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamates such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate, or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (PEG-DPSE).

[0183] The second stabilizer can be a blend of different stabilizers. For example, two block copolymers can be used in a desired ratio. For example, PEG-lipid and block copolymer can be used in a desired ratio.

[0184] The additive(s) are added at a desired mass or molar ratio relative to the encapsulating agent mass (e.g., mRNA mass). For purposes herein, the encapsulating agent mass is the amount of encapsulating agent in the inverse nanocarrier after the iFNP association step. (This takes into account the process hold-up in the mixer). If multiple additives are added, they are also added to each other at a defined mass or molar ratio. Simple screening experiments can be used to identify the appropriate composition of encapsulating agent, first stabilizing agent, additive(s), and second stabilizing agent(s). For a given inverse nanocarrier composition, the additive(s) can be added at a mass ratio selected from the range of 1:0.01 to 1:100 (encapsulating agent to additive(s)). The mass ratio can be selected from the range of 1:0.25 to 1:25. The mass ratio can be selected from the range of 1:0.5 to 1:15.

[0185] When two additives are added, they can be in a defined ratio to each other. The ratio can be selected from the range of 1:0.01 to 1:100. The ratio can be selected from the range of 1:0.1 to 1:10. For example, the additive can be a lipid and a polyester. In some cases, the additive can be two lipids, or a lipid and a sterol. The ratio can be selected based on known mechanisms or synergistic ratios. For example, cholesterol and lipid can be used in a ratio similar to the mammalian cell membrane composition. On a molar basis, the ratio of cholesterol to lipid can be 20-50 mol% or 25-40 mol% or 30-40 mol%. These examples are intended to be illustrative and not limiting.

[0186] Similarly, multiple additives may be added. The total mass of the additives may follow the ranges outlined above. The additives may be added in a mass ratio of 1:0.1 to 1:100 relative to the encapsulating agent. The ratio of the multiple additives may be empirically selected by straightforward screening experiments. The ratio may be selected based on known mechanisms or synergistic ratios. For example, the multiple additives may include cationic ionizable lipids, phospholipids, and structural lipids such as cholesterol. Considering the molar composition of these three additives, the cationic ionizable lipids may be included in a molar ratio of 0% to 75%, the phospholipids may be included in a ratio of 0% to 50%, and the structural lipids may be included in a ratio of 0% to 50%. The cationic ionizable lipids may be included in a molar ratio of 20% to 60%, the phospholipids may be included in a ratio of 0% to 40%, and the structural lipids may be included in a ratio of 10% to 50%. In certain cases where the second stabilizing agent is a PEG-lipid, the molar ratio of the PEG-lipid may also be considered. For example, cationic ionizable lipids can be included at a molar ratio of 0% to 75%, phospholipids can be included at a ratio of 0% to 50%, structural lipids can be included at a ratio of 0% to 50%, and PEG-lipids can be included at a ratio of 0.5% to 10%. Cationic ionizable lipids can be included at a molar ratio of 20% to 60%, phospholipids can be included at a ratio of 5% to 25%, structural lipids can be included at a ratio of 15% to 50%, and PEG-lipids can be included at a ratio of 1% to 8%. For example, cationic ionizable lipids can be included at a ratio of 45% to 60%, phospholipids can be included at a ratio of 5% to 10%, structural lipids can be included at a ratio of 30% to 40%, and PEG-lipids can be included at a ratio of 2% to 8%. In one embodiment, a PEG block copolymer such as PEG-b-PLA can be used instead of the PEG-lipid.

[0187] In embodiments where a cationic agent, such as a cationic ionizable lipid, is co-formulated with a nucleic acid species, an "N:P ratio" can be reported, which is the molar ratio of the nitrogen groups of the cationic species to the phosphate groups of the nucleic acid species. In the disclosed process, when a cationic ionizable lipid species is included, the cationic species can be included at an N:P ratio of 0.1:1 to 10:1. The N:P ratio can be selected from the range of 1:1 to 6:1. This ratio is a further refinement of the full composition range defined by mass basis above. The particular value can be empirically selected by activity-based screening assays appropriate for the particular application.

[0188] After the drug(s) are added, the dispersion of reverse nanocarrier and drug(s) can be aged for a specified period of time. The aging time can be 5 minutes or more. The aging time can be 15 minutes or more. The aging time can be 1 hour or more. The aging time can be 24 hours or more. One skilled in the art will recognize that the aging conditions can be determined by consideration of stability, including degradation profile, of the nucleic acid. The aging process can be carried out under controlled temperature conditions. The dispersion of reverse nanocarrier and drug(s) can be aged at 37°C or more. The dispersion of reverse nanocarrier and drug(s) can be aged at 20°C or more. The dispersion of reverse nanocarrier and drug(s) can be aged at 2-8°C or more. The dispersion of reverse nanocarrier and drug(s) can be aged at -20°C or more. The dispersion of inverse nanocarriers and drug(s) may be stirred or agitated (using any technique known in the art, including magnetic stir bars, stirred tanks using impellers, overhead impellers of any suitable design, including swept-blade impellers or chevron impellers), or may be stored unstirred.

[0189] Generally, the second stabilizing agent(s) are added to the modified solvent prior to solvent exchange. Drugs that are fully soluble in the modified solvent (not partially or fully insoluble and therefore do not aggregate on the inverse nanocarriers) can be added after solvent exchange but prior to aqueous mixing to form coated nanocarriers.

[0190] The second stabilizing agent(s) are added at a desired mass or molar ratio relative to the encapsulating agent mass (e.g., mRNA mass). For purposes herein, the encapsulating agent mass is the amount of encapsulating agent in the inverse nanocarrier after the iFNP association step. (This takes into account the process hold-up in the mixer). If multiple second stabilizing agents are added, they are also added to each other at a defined mass or molar ratio. Simple screening experiments can be used to identify the appropriate composition of encapsulating agent, first stabilizing agent, additive(s), and second stabilizing agent(s). For a given inverse nanocarrier composition, the second stabilizing agent(s) can be added at a mass ratio selected from the range of 1:0.01 to 1:100 (encapsulating agent to second stabilizing agent(s)). In one embodiment, the mass ratio can be selected from the range of 1:0.25 to 1:25. In one embodiment, the mass ratio can be selected from the range of 1:0.5 to 1:15.

[0191] When two stabilizers are added, they can be in a defined ratio to each other. The ratio can be selected from the range of 1:0.01 to 1:100. The ratio can be selected from the range of 1:0.1 to 1:10. The ratio can be selected based on known mechanisms or synergistic ratios, or based on standard screening experiments.

[0192] Solvent exchange Solvent exchange for iFNPs can serve to transfer the inverse nanocarriers to a water-miscible organic solvent, referred to as the modified solvent, in preparation for the coating process. In the disclosed process, the solvent exchange serves the additional purpose of facilitating the association of additives to the inverse nanocarrier surface prior to the coating step. In the examples provided below using POPC or POPC blends as the additive(s), the process performance (defined by high encapsulation efficiency and low burst release) was adversely affected when the additive(s) were added after the modified solvent was added. In general, the second stabilizer may be molecularly dissolved (both the hydrophobic and hydrophilic portions remain soluble in the modified solvent) or it may associate with the modified solvent similar to the additive during the solvent exchange. This role of the modified solvent was unexpected.

[0193] The modification solvent should be selected so that the hydrophobic block or region of the first stabilizer remains soluble during the solvent exchange. The modification solvent should be selected so that the additive does not exhibit bulk precipitation when the modification solvent is added to the inverse nanocarrier dispersion in the iFNP poor solvent, but bulk precipitation of the additive when the modification solvent is added may occur even in the absence of the inverse nanocarrier dispersion. For additives with amphiphilic characteristics, the modification solvent can be selected so that the hydrophilic characteristics are poorly soluble but the hydrophobic characteristics are soluble. When the additive(s) include phospholipids, lipids or cationic lipids, representative but non-limiting modification solvents include acetonitrile, propionitrile, acetone, or tetrahydrofuran.

[0194] Solvent exchange from the anti-solvent to the modified solvent can be accomplished by any method known in the art. For example, distillation or dialysis can be used. When distillation is used, the distillation can be performed using standard techniques known in the art. When the solvent exchange is achieved by distillation, the modified solvent can have a higher boiling point than the anti-solvent or an azeotrope composition that allows removal of the anti-solvent by evaporation. The distillation can be completed in a put-and-take manner (concentrate and then add more modified solvent before repeating the process). The distillation can also be performed as a constant volume distillation (concentrate to the desired volume and then continuously add modified solvent at a rate that approximately matches the evaporation rate of the solvent mixture).

[0195] In one embodiment of the present invention, the dispersion of the inverse nanocarrier, additive(s) and second stabilizer in the antisolvent is first concentrated to the desired concentration, and then the first portion of the modified solvent required by the selected distillation method is added. As a non-limiting example, given an RNA batch size of about 1 mg in the antisolvent dichloromethane (DCM), the volume was reduced from about 4.5 mL to about 2 mL by distillation. Next, 8 mL of the modified solvent acetonitrile was added. The mixture was then concentrated to about 1.5 mL, and 8 mL of acetonitrile was added again. This was repeated until acetonitrile was added three times. The dispersion in acetonitrile was then concentrated to the target range. Those skilled in the art will recognize that the amounts and associated concentration ranges listed herein can be adjusted to meet process constraints, such as minimum or maximum amount constraints.

[0196] Distillation should be carried out until the residual anti-solvent content is reduced to such an extent that the solution can be homogeneously mixed with the aqueous anti-solvent during the coating process, i.e., the distillation should be carried out so that the residual anti-solvent does not form a second liquid phase.

[0197] Those skilled in the art will recognize that distillation unit operations can be performed at a range of operating temperatures, pressures, and total mass concentrations of species in the solvent. Considerations such as process throughput, economics, vessel volume, time constraints, cooling and heating capabilities, and encapsulant stability can guide these decisions. Generally speaking, temperatures and pressures should be selected to obtain distillation conditions identifiable from vapor-liquid equilibrium data for the contemplated solvent mixture. Temperatures can be selected in the range of 5°C to 50°C, or 15°C to 40°C, or 25°C to 35°C. Pressures can be selected in the range of 10 Torr to 400 Torr, or 50 Torr to 250 Torr. As the distillation progresses and the solvent composition changes, temperatures and pressures can also be adjusted as necessary. The total mass concentrations of the encapsulant, first stabilizing agent, additive(s), and second stabilizing agent can range from 0.1 mg / mL to 200 mg / mL, or 0.5 mg / mL to 40 mg / mL.

[0198] Formation of coated nanocarriers - Coating step mixing process In one embodiment of the present invention, the inverse nanocarriers containing the additive and the second stabilizer are then processed in an aqueous environment. This process is referred to as the "coating step" because it promotes the assembly of the second stabilizer on the nanocarrier surface and ensures the formation of a hydrophobic layer around the encapsulant. The method of coating the nanocarriers is as previously described by Johnson et al., termed "Flash NanoPrecipitation" (FNP), Johnson, BK, et al., AIChE Journal (2003) 49: 2264-2282, and U.S. Patent No. 8,137,699, which are incorporated herein by reference in their entirety. This mixing technique was also used to form the inverse nanocarriers. The quality of the solvent is rapidly degraded by micromixing with water or aqueous buffer or mixture, and the time of mixing is faster than the aggregation of nanoparticles and commensurate with the time scale of the self-assembly of the stabilizer.

[0199] Although not intended to be limiting, two such configurations are the confined impinging jet mixer (CIJ) and the multi-inlet vortex mixer (MIVM), examples of which are intended to be illustrative rather than limiting or exhaustive, and are set forth above.

[0200] The vortex mixer consists of a closed volume chamber in which one solvent stream containing the modified solvent with inverse nanocarriers, additives and stabilizers is mixed at high speed with one or more solvent streams containing water or aqueous buffers. When coating nanoparticles by flash nanoprecipitation, the mixing is fast enough to allow high supersaturation levels of all components to be achieved before the onset of inverse nanocarrier aggregation. The flash nanoprecipitation process is a widely applicable nanoparticle coating technique because it may be less sensitive to the chemical specificity of the components. The confined impinging jet mixer is a similar mixing geometry to the vortex mixer, but has only one inlet stream for the modified solvent and one for the aqueous stream.

[0201] In one embodiment, the coating formed by the second stabilizing agent can have an inner region and an outer region, The inner region can include the hydrophobic or less polar region(s) of the second stabilizing agent, and the outer region can include the hydrophilic or more polar region(s) of the second stabilizing agent.

[0202] In one embodiment, the coated nanocarrier comprises a nanoparticle having a plurality of regions. The core comprises an encapsulating agent, e.g., a water-soluble agent, e.g., a nucleic acid, and a region(s) comprising a hydrophilic or more polar region(s) of a first stabilizing agent, e.g., a first stabilizing amphiphilic copolymer, e.g., dextran. Around this core, i.e., surrounding the core, is a shell comprising a region(s) of a less hydrophobic or less polar region(s) of a first stabilizing agent, e.g., a first stabilizing amphiphilic copolymer, e.g., a region(s) comprising PLGA, an additive(s) (e.g., lipid, phospholipid, or lipid blend), and a region(s) of a more hydrophobic or more polar region(s) of a second stabilizing agent, e.g., a second stabilizing amphiphilic agent, e.g., a second stabilizing amphiphilic copolymer (also referred to as a coating stabilizer or coating polymer). The shell can have an inner surface and an outer surface, and the inner surface can be in contact with the core. The second stabilizing amphiphilic agent may include a hydrophilic or more polar region(s) and a hydrophobic or less polar region(s). The hydrophilic or more polar region(s) of the second stabilizing amphiphilic agent may be present on the outer surface of the shell, e.g., as a corona of the outer surface of the shell, extending beyond the outer surface of the shell away from the center of the coated nanocarrier or nanoparticle, e.g., toward the environment surrounding the coated nanocarrier or nanoparticle. The corona may be around the shell, i.e., the corona may surround the shell. That is, the hydrophilic or more polar region(s) of the second stabilizing amphiphilic agent may be in contact with the environment outside the shell and outside the nanoparticle as a whole. The hydrophobic or less polar region(s) of the second stabilizing amphiphilic agent may be within the shell. For example, the hydrophobic or less polar region(s) of the first stabilizing amphiphilic copolymer and the hydrophobic or less polar region(s) of the second stabilizing amphiphilic agent may be in contact with each other. For example, the surface of the coated nanocarriers may include hydrophilic or less polar region(s) of a second stabilizing agent, such as poly(ethylene glycol) (PEG).

[0203] For example, the shell can include regions of a non-polar, less polar, or hydrophobic agent or drug, including a first stabilizer, an additive, and a second stabilizer.

[0204] In one method of the present invention, nanoparticles having a size in the range of 15 nm to 10,500 nm, 20 nm to 6,000 nm, 20 nm to 1,000 nm, 35 nm to 400 nm, or 40 nm to 300 nm can be produced. The size can be determined by dynamic light scattering. For example, particles can be produced having a size of at least about 15 nm, 20 nm, 35 nm, 40 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 600 nm, 900 nm, 1000 nm, 2000 nm, 4000 nm or 6000 nm, and up to about 20 nm, 35 nm, 40 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 600 nm, 900 nm, 1000 nm, 2000 nm, 4000 nm, 6000 nm or 10500 nm. The sizes reported and cited herein are determined by dynamic light scattering using a deconvolution program installed by the manufacturer.

[0205] The nanoparticle composition may be relatively uniform. The polydispersity index may be used to indicate the homogeneity of the nanoparticle composition, for example, the particle size distribution of the nanoparticle composition. A small polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. The nanoparticle composition may have a polydispersity index of about 0 to about 0.3, for example, 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, 0.25, 0.29, or 0.30, or within a range between two of these values. In some embodiments, the polydispersity index of a nanoparticle composition can be from about 0.10 to about 0.20.

[0206] The zeta potential can represent the surface charge of a nanoparticle. The composition can be selected to produce different zeta potentials of nanocarriers. In some embodiments, the zeta potential of the nanoparticle composition can be about -30 mV to about +30 mV, about -20 mV to about +20 mV, about -12 mV to about +10 mV, about -12 mV to about +5 mV, about -12 mV to about 0 mV, about -12 mV to about -5 mV, about -10 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

[0207] Nanoparticles that are mixtures of polymers and lipids are reviewed in Hadinoto, Sundaresan, Chewo, European Journal of Pharmaceutics and Biopharmaceutics (2013) 85, 427-443, the entire contents of which are incorporated herein by reference. All existing methods rely on charge complexation to encapsulate RNA as well as lipid nanoparticles and utilize different emulsification or nanoprecipitation techniques to form a polymer core. For example, the process described herein differs in that it uses successive nanoprecipitation steps to apply successive layers to the nanoparticles.

[0208] Formation of coated nanocarriers – flow composition The concentrations, stabilizers, modified solvents and aqueous buffers (anti-solvents) used in the coating process can be optimized for each individual inverse nanocarrier to be coated. The number of mixer inlet streams can be controlled by the mixer geometry. Multi-inlet vortex mixers typically contain four streams, whereas confined impinging jet mixers typically contain two streams. The volume ratio of these streams is selected as described above for inverse flash nanoprecipitation (iFNP) and flash nanoprecipitation (FNP) to ensure adequate mixing speed and intensity. The water content after mixing can be 50% by volume or more with the modified solvent. The water content after mixing can be 75% by volume or more, or 90% by volume or more. Furthermore, additional aqueous dilution after mixing can be used to adjust the residual modified solvent content to generate a stable nanocarrier dispersion.

[0209] The aqueous composition may be deionized water, or a suitable buffer, such as phosphate buffered saline, or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, acetate buffer, citrate buffer, or other organic or inorganic buffer that controls the pH in the range of 2-8 or 4-7.5, such as those listed in the Good's List of Buffers. These representative buffers are not intended to be limiting. The aqueous composition in the mixer may be deionized water, but additional aqueous dilution after mixing may be an appropriate buffer. The aqueous stream may be unbuffered, but may contain species to adjust the osmolality of the aqueous system. The osmolyte may be selected from sugars, PEG oligomers, salts, or amino acids, etc.

[0210] The concentration of the species in the modifying solvent may be selected to achieve the desired result. Those skilled in the art will recognize that a concentration may be selected that is too dilute and therefore uneconomical. Alternatively, a concentration may be selected that is too high and may lead to inverse nanocarrier aggregation, for example, because the concentration approaches an area during coating where the collision of the inverse nanocarriers occurs faster than the second stabilizer can reach the surface, or where the stabilizer becomes a polymer overlap area. Typical concentration ranges suitable for coating include 0.5 mg / mL to 50 mg / mL, or 1 mg / mL to 20 mg / mL, or 5 mg / mL to 15 mg / mL.

[0211] When using a vortex mixer with four inlet streams, compositions of the additional inlet streams are possible. For example, one stream may be the modified solvent, two streams may be aqueous, and one stream may contain a second modified solvent or solvent mixture. For example, the second modified solvent may contain a second stabilizing agent if the agent is not added directly to the inverse nanocarrier dispersion at another point in the process. This method allows for the use of stabilizers that do not have an appropriate solubility profile for the desired modified solvent.

[0212] Post-processing and preparation of pharmaceutical forms - Buffer exchange The coated nanocarriers can be further processed into pharmaceutical forms useful for treating a patient or subject in need of treatment. These methods of preparing pharmaceutical forms are known in the art. For example, the coated nanocarriers can be prepared in a pharma- ceutically acceptable carrier (e.g., saline or phosphate buffer) selected according to the route of administration and standard pharmaceutical practice. As a pharma-ceutically acceptable carrier, normal buffered saline (e.g., 135-150 mM NaCl) can be used. Other suitable carriers include, for example, water, buffered water, 0.4% saline, 0.3% glycine, and the like, including glycoproteins for improving stability such as albumin, lipoproteins, globulins, and the like. These are examples and are not intended to be limiting of those that can be used. As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coating agents, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier liquids, and the like. Carriers are distinct from the term "nanocarriers." For example, the phrase "pharmaceutical acceptable" refers to molecular entities and compositions that do not produce unwanted allergic responses or similar unacceptable responses when administered to humans.

[0213] The pharma- ceutically acceptable carrier is generally added after the formation of the coated nanocarrier. Residual modified solvent can be removed to an acceptable level as determined according to the ICH (International Council for Harmoisation of Technical Requirements for Pharmaceutical for Human Use) guidelines. Any suitable method known in the art can be used for removing modified solvent and introducing carrier. For example, suitable methods include, but are not limited to, dialysis, ultrafiltration, tangential flow filtration, diafiltration, and centrifugal ultrafiltration.

[0214] Post-processing and preparation of pharmaceutical forms - excipients The coated nanocarriers can include any material useful in pharmaceutical compositions. For example, the composition can include one or more pharma- ceutically acceptable excipients or auxiliary ingredients, such as, but not limited to, one or more solvents, dispersion media, diluents, dispersing aids, suspending aids, granulating aids, disintegrants, fillers, lubricants, liquid vehicles, binders, surfactants, isotonicity agents, thickening or emulsifying agents, buffers, lubricants, oils, preservatives, and other species. Excipients such as waxes, butters, colorants, coating agents, flavors, and perfumes can also be included. Many pharma-ceutically acceptable excipients are known in the art.

[0215] Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and / or combinations thereof. The granulating and dispersing agents may be selected from the non-limiting list consisting of potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked polyvinylpyrrolidone (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, and / or combinations thereof.

[0216] Surfactants and / or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyisobutene ... sorbitan monolaurate [TWEEN® 20], polyoxyethylene sorbitan [TWEEN® 60], polyoxyethylene sorbitan monooleate [TWEEN® 80], sorbitan monopalmitate [SPAN® 40], sorbitan monostearate [SPAN® 60], sorbitan tristearate [SPAN® 65], glyceryl monooleate, sorbitan monooleate [SPAN® 80]), polyoxyethylene esters (e.g., polyoxyethylene ...), polyoxyethylene esters (e.g., polyoxyethylene sorbitan monolaurate [TWEEN® 20], polyoxyethylene sorbitan monolaurate [TWEEN® 60], polyoxyethylene sorbitan monooleate [TWEEN® 80]), polyoxyethylene sorbitan monolaurate [TWEEN® 80], polyoxyethylene sorbitan monolaurate [TWEEN® 80], polyoxyethylene sorbitan monolaurate [TWEEN® 80], polyoxyethylene sorbitan monolaurate [TWEEN® 80], polyoxyethylene sorbitan monolaurate [TWEEN® 8 Ethylene monostearate [MYRJ® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether [BRIJ® 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate,These may include, but are not limited to, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLURONIC® F68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or combinations thereof.

[0217] The binders may be starches (e.g., corn starch and starch paste); gelatin; sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic gums (e.g., acacia, sodium alginate, Irish moss extract, panwar gum, ghatti gum, psyllium mucilage, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGEUM®), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol, and combinations thereof, or any other suitable binders.

[0218] Examples of preservatives may include, but are not limited to, antioxidants, chelating agents, antibacterial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Examples of antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, butyl hydroxyanisole, butyl hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Examples of antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Examples of antifungal preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and / or phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and / or phytic acid.Other preservatives include, but are not limited to, tocopherol, tocopheryl acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN® II, NEOLONE®, KATHON®, and / or EUXYL®.

[0219] Examples of buffering agents include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levuvinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixture, tromethamine, aminosulfonic acid buffer (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and / or combinations thereof. The lubricant may be selected from the non-limiting group consisting of magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

[0220] Examples of oils include almond oil, apricot kernel oil, avocado oil, babassu oil, bergamot oil, blackcurrant seed oil, borage oil, cade oil, chamomile oil, canola oil, caraway oil, camauba oil, castor oil, cinnamon oil, cocoa butter, coconut oil, cod liver oil, coffee, corn oil, cottonseed oil, emu oil, eucalyptus oil, evening primrose oil, fish oil, linseed oil, geraniol oil, gourd oil, grapeseed oil, hazelnut oil, hyssop oil, isopropyl myristate, jojoba oil, kukuni nut oil, lavandin oil, lavender oil, lemon oil, macadamia nut oil, mallow oil, mango seed oil, meadowfoam seed oil, mink oil, nutmeg oil, olive oil, orange ... Oils that may be used include, but are not limited to, oleic acid, stearyl alcohol ... oil, stearyl oil, stearyl oil, stearyl oil, stearyl oil, stearyl oil, stearyl oil, stearyl oil, stearyl oil, stearyl oil, stearyl oil, stearyl oil, stearyl oil, stearyl oil, stearyl oil, stearyl oil, stearyl oil, stearyl oil, stearyl oil, stearyl oil, stearyl oil, stearyl oil, stearyl oil, stearyl oil, stearyl oil, ste

[0221] Excipients may be included as cryoprotectants or cryoprotectants. Examples of excipients are glucose, sucrose, trehalose, lactose, mannitol, sorbitol, aerosil, maltose, fructose, dextran, glycerol, poly(vinylpyrrolidone), poly(vinyl alcohol), glycine, cyclodextrin, hydroxypropyl-β-cyclodextrin, gelatin, poly(ethylene glycol), alanine, sodium chloride, citric acid, histidine, and starch derivatives.

[0222] Post-processing and preparation of pharmaceutical forms - freezing or lyophilization The pharmaceutical form may be prepared in liquid or solid form. The liquid form may be stored at an appropriate temperature. For example, the liquid form may be stored at room temperature or at 2-8°C. The dispersion may be stored as a frozen liquid. For example, the coated nanocarrier dispersion may be stored at -12°C or below, or -20°C or below, or -80°C or below. The coated nanocarrier pharmaceutical form may be prepared as a lyophilized or spray-dried powder. To lyophilize or spray-dry the coated nanocarrier pharmaceutical form, methods known in the art can be used, including the addition of cryoprotectants or cryoprotective excipients as described above.

[0223] composition The composition may include an encapsulating agent(s) selected from a nucleic acid class, including, but not limited to, RNA, DNA, mRNA, siRNA, microRNA, circular RNA, antisense oligonucleotide, tRNA, or plasmid. In one embodiment, the composition may include a salt, for example, a calcium salt. In one embodiment, the encapsulating agent(s) may be a nucleic acid and a peptide, a protein, or a second nucleic acid, or a combination thereof. The encapsulating agent may be a plurality of nucleic acid sequences.

[0224] The composition may include a first stabilizing agent that is a diblock, triblock, or comb copolymer. The hydrophilic block may be selected from dextran, poly(aspartic acid), and poly(glutamic acid). The hydrophobic block is selected from poly(lactic acid), poly(lactic-co-glycolic acid), and poly(caprolactone).

[0225] The composition can include an additive selected from a lipid, a phospholipid, ethyl oleate, POPC, DSPC, HSPC, DOPC, DOPE, a cationic lipid or a cationic ionizable lipid, cholesterol, β-sitosterol, a fatty acid methyl ester, a fatty acid ethyl ester, a hydrophobic polymer, a cationic or ionizable polymer, and a combination.

[0226] The composition comprises a second stabilizing agent which is a diblock, triblock or comb copolymer, or a lipid complex, or a mixture. The hydrophilic polymer block or hydrophilic lipid complex can be selected from poly(ethylene glycol), poly(sarcosine), poly(aspartic acid), poly(glutamic acid), poly(lysine), or poly(arginine), and combinations thereof. The hydrophobic polymer block can be selected from poly(lactic acid), poly(lactic-co-glycolic acid), and poly(caprolactone). The composition comprises a PEG lipid, such as PEG-DMG or PEG-DSPE, or PEG-b-PLGA or PEG-b-PLA, or a mixture.

[0227] How to use The present application provides compositions and methods for delivering therapeutic and / or prophylactic agents to cells, tissues, or organs by administering a coated nanocarrier pharmaceutical composition of a drug to a subject in need of the nanocarrier pharmaceutical composition. Delivering the therapeutic and / or prophylactic agent to the subject comprises administering a coated nanocarrier pharmaceutical composition comprising an encapsulating agent, and administering the composition comprises contacting a cell membrane with the composition. The coated nanocarrier composition, and / or pharmaceutical compositions comprising one or more coated nanocarrier compositions, can be administered to any patient or subject, including patients or subjects who can benefit from a therapeutic effect provided by delivery of a therapeutic and / or prophylactic agent to one or more specific cells, tissues, organs, or systems or groups thereof, such as the liver system. Although the description provided herein includes those of compositions suitable for administration to humans or human-derived materials, one of skill in the art will appreciate that such compositions may be suitable for administration to other biological systems, including animals or animal-derived materials. Modifications of compositions suitable for administration to humans may be made to make the compositions suitable for administration to various animals or animal-derived materials, and an ordinarily skilled veterinary pharmacologist may be able to design and / or perform such modifications, if any, by routine experimentation. Subjects to which the compositions are contemplated for administration include, but are not limited to, humans, other primates, and other animals, including commercially relevant animals such as, for example, cows, pigs, horses, sheep, cats, dogs, rodents, mice, chickens, and / or rats. Subjects to which the compositions are contemplated for administration also include materials derived from humans or other animals, such as cells, tissues, and organs.

[0228] The coated nanocarrier pharmaceutical form may be prepared for administration by routes including, but not limited to, oral, topical, transdermal, inhalation, parenteral, intraocular, subretinal, sublingual, mucosal, buccal, rectal, vaginal, and nasal. As used herein, the term parenteral includes subcutaneous injection, intravenous, intramuscular or other direct injection into tissues or organs, intradermal, intrathecal, intraperitoneal, intraarterial, intracisternal, intracerebral, or intratumoral injection or infusion techniques. The pharmaceutical composition of the present invention embodiment is formulated so that the active ingredient contained therein is bioavailable upon administration of the composition to a patient. The composition for administration to a subject or patient may be in the form of one or more dosage units, for example, a tablet, or other dosage form may be a single dosage unit, and a container of the compound of the present invention embodiment in aerosol form may hold multiple dosage units. Actual methods of preparing such dosage forms will be known or apparent to those skilled in the art.

[0229] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated according to known techniques using suitable dispersing agents, wetting agents, and / or suspending agents. Compositions for rectal or vaginal administration are usually suppositories. They can be prepared by mixing the composition with suitable non-irritating excipients, such as cocoa butter, polyethylene glycol, and / or suppository wax. They are solid at ambient temperature, but liquid at body temperature, so that they melt in the rectum or vaginal cavity and release the active ingredient. Solid dosage forms for oral administration include capsules, tablets, pills, films, powders, and granules. Dosage forms for topical and / or transdermal administration of the composition can include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and / or patches. In addition, the present disclosure contemplates the use of transdermal patches, which often have the added advantage of providing a controlled delivery of the compound to the body. Such dosage forms can be prepared, for example, by dissolving and / or dispensing the compound in a suitable medium. Suitable devices for use in delivering the intradermal pharmaceutical compositions described herein include the short-needle devices described in U.S. Patent Nos. 4,886,499, 5,190,521, 5,328,483, 5,527,288, 4,270,537, 5,015,235, 5,141,496, and 5,417,662. Pharmaceutical compositions formulated for pulmonary or nasal delivery can provide the active ingredient in the form of droplets of solution and / or suspension, or as a dry powder.

[0230] Generally, contacting cells with the coated nanocarriers can occur in vivo, ex vivo, in culture, or in vitro. The amount of coated nanocarriers contacted with cells, and / or the amount of encapsulating agent therein, can depend on the type of cell or tissue being contacted, the means of administration, physiochemical properties, and other factors.

[0231] In certain embodiments, the mRNA contained in the coated nanocarrier can code for a natural or recombinant polypeptide, e.g., a protein, that can replace one or more polypeptides that may be reduced or substantially absent in a cell contacted with the nanoparticle composition. One or more substantially absent polypeptides may be absent due to genetic mutations in the encoding gene or its regulatory pathway. One or more substantially absent polypeptides may be enzymes whose activity is partially or completely absent. Alternatively, the recombinant polypeptides produced by translation of the mRNA may antagonize the activity of endogenous proteins present within the cell, present on the cell surface, or secreted from the cell. Antagonistic recombinant polypeptides may be desirable to overcome deleterious effects caused by the activity of endogenous proteins, such as altered activity or localization caused by mutations. In other embodiments, the recombinant polypeptides produced by translation of the mRNA can alter the DNA of gene expression in the cell by encoding various endonucleases and cofactors. Polypeptides including Cas proteins, TALENs, and other endonucleases may be desirable to alter the DNA of a subject to treat disease. In other embodiments, the recombinant polypeptides produced by translation of the mRNA in a cell can encode polypeptides that are not normally found in that cell. Such polypeptides may include, but are not limited to, transcription factors, insulin, antibodies, hormones, cytokines, complement factors, clotting factors and growth factors, and may be used to treat a variety of diseases.

[0232] In certain embodiments, coated nanocarrier compositions having an encapsulating agent comprising one or more nucleic acids forming an interfering RNA sequence (e.g., siRNA) or an antisense oligonucleotide sequence provide a method of treating a disease or disorder in a mammal or animal subject in vitro and / or in vivo by downregulating or silencing the transcription and / or translation of one or more target nucleic acid sequences or genes of interest.

[0233] In certain embodiments, coated nanocarrier compositions having encapsulating agents comprising one or more nucleic acids forming non-coding ribonucleic acid (RNA) sequences (e.g., transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), Piwi-interacting RNA (piRNA), small nuclear RNA (snoRNA), messenger RNA (mRNA), or long non-coding RNA (lncRNA)) provide a method of treating a disease or disorder in an animal in vitro and / or in vivo by modulating the transcription and / or translation of one or more target nucleic acid sequences or genes of interest.

[0234] In certain embodiments, coated nanocarrier compositions having encapsulants containing one or more nucleic acid forming DNA sequences (e.g., DNA aptamers or donor sequences for homologous repair) provide a method of treating a disease or disorder in an animal in vitro and / or in vivo by controlling the sequence, transcription and / or translation of one or more target nucleic acid sequences or genes of interest.

[0235] In certain embodiments, the DNA contained in the coated nanocarrier may code for a natural or recombinant RNA sequence, for example, mRNA, siRNA, or another RNA species, whether natural or non-natural. In some embodiments, the utility of the DNA contained in the coated nanocarrier is encompassed by the same utility as RNA. In other embodiments, the DNA contained in the coated nanocarrier may contain or code for additional elements that facilitate retention in the cell as an episomal complex, or may facilitate integration into chromosomal DNA. In some embodiments, the coated nanocarrier-DNA complex may be used to treat a variety of diseases with both acute and widespread expression. In some embodiments, the DNA contained in the nanocarrier may be used as a non-viral gene therapy with applications to a variety of diseases.

[0236] Methods of Use: Treatment of Diseases and Disorders The coated nanocarrier composition may be useful for treating a disease, disorder, or condition. For example, such a composition may be useful for treating a disease, disorder, or condition characterized by a lack or abnormality in the activity of a protein or polypeptide. For example, a nanoparticle composition containing an mRNA encoding a missing or abnormal polypeptide may be administered or delivered to a cell. Subsequent translation of the mRNA may produce the polypeptide, thereby reducing or eliminating the problem caused by the lack of activity or abnormal activity caused by the polypeptide. Because translation may occur rapidly, the method and composition may also be useful for treating acute diseases, disorders, or conditions, such as sepsis, stroke, hyperammonemia, and myocardial infarction, or chronic diseases. Diseases, disorders and / or conditions characterized by dysfunctional or abnormal protein or polypeptide activity for which the compositions may be administered include, but are not limited to, rare diseases, infectious diseases (both as vaccines and therapeutics), cancer, proliferative and hyperproliferative diseases, fibrotic diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, neurological diseases, cardiac and renal vascular diseases, endocrine diseases, neuroendocrine diseases, hepatobiliary diseases, ophthalmological diseases, musculoskeletal diseases, gastrointestinal diseases, reproductive diseases, genitourinary diseases, lymphatic system diseases, skin diseases, blood diseases, and metabolic diseases.

[0237] For example, nanoparticle compositions comprising DNA or mRNA can treat hepatic single gene disorders such as urea cycle disorders by replacing substantially reduced or absent proteins. Examples of such inborn errors of metabolism include NAGS (N-acetylglutamate synthase) deficiency, CPS (carbamoyl phosphate synthase) deficiency, OTC (ornithine transcarbamoylase) deficiency, citrullinemia type 1, citrullinemia type 2, argininosuccinic aciduria, argininemia, or HHH (hyperornithinemic-hyperammonemia-homocitrullinuria) syndrome. Administration of nanoparticle compositions comprising DNA or mRNA of natural or recombinant polypeptides encoding NAGS, CPS1, OTC1, ASS1, SLC25A13, ASL1, ARG1, SLC25A15, or ORNT1 can treat urea cycle disorders in subjects in need of treatment of urea cycle disorders.

[0238] Nanoparticle compositions containing DNA or mRNA can treat other single or multiple diseases, including, but not limited to, lysosomal storage diseases such as Fabry disease, acidurias such as methylmalonic aciduria, and Wilson's disease (diseases in which liver enzymes are severely reduced or deficient). Additionally, nanoparticle compositions containing translated DNA or mRNA in the liver can produce polypeptides or proteins that are not present in other tissues. This method can treat diseases by increasing plasma levels of therapeutic proteins, including, but not limited to, insulin in type 1 or type 2 diabetes, clotting factors in hemophilia, or protease inhibitors in alpha-1 antitrypsin deficiency.

[0239] Nanoparticle compositions comprising DNA or mRNA can also treat diseases including, but not limited to, neurological diseases, musculoskeletal diseases, blood diseases, methylmalonic acidemia, tyrosinemia, Wilson's disease, hemophilia A, hemophilia B, hereditary fructose intolerance, glutaric aciduria, chronic granulomatous disease, fatty acid oxidation disorders, MTP (mitochondrial triglyceride) deficiency, propionic acidemia, maple syrup urine disease, alpha 1 antitrypsin inhibitor deficiency, intermittent porphyria, galactosemia, hyperoxaluria, Fabry's disease, ADA (adenosine deaminase) deficiency, cystic fibrosis, familial hypercholesterolemia, hemophilia, Duchenne muscular dystrophy, Becker muscular dystrophy, Rett syndrome, fragile X syndrome, sarcosinemia, ataxia, dystrophies, Fanconi anemia, sickle cell anemia, Gaucher disease, Hunter syndrome, X-linked SCID (severe combined immunodeficiency), and the like. These provided diseases are intended to be exemplary and non-limiting.

[0240] Nanoparticle compositions containing DNA or mRNA can treat cancer or proliferative diseases by encoding growth-suppressing or apoptotic proteins. Delivery of native or recombinant TP53, p21INK4a, Rb, APC, SMAD2, SMAD3, BAX or other DNA / mRNA can be therapeutic in various cancers or fibrotic diseases, such as colorectal cancer or hepatocellular carcinoma.

[0241] Nanoparticle compositions comprising DNA or mRNA can treat autoimmune or immune-mediated inflammatory diseases by encoding autoantigens or other peptides or proteins involved in the development of autoimmune or adverse immune responses. Such autoantigens include, but are not limited to, any of those known to play a role in any of a variety of autoimmune or immune-mediated inflammatory diseases, such as, for example, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, psoriasis, dermatomyositis, pemphigus vulgaris, inflammatory bowel disease, and interstitial lung disease. In another embodiment, the nanoparticle composition comprises DNA or mRNA co-encapsulated with other compounds capable of modulating immune responses, including, but not limited to, immunomodulatory drugs, biologics, or other molecules.

[0242] In another embodiment, the disorder being treated involves one of the following polypeptides or proteins, which are exemplary and not intended as limiting choices: ABCA4; ABCD3; ACADM; AGL; AGT; ALDH4A1; ALPL; AMPD1; APOA2; AVSD1; BRCD2; C1QA; C1QB; C1QG; C8A; C8B; CACNA1S; CCV; CD3Z; CDC2L1; CHML; CHS1; CIAS1; CLCNKB; CMD1A; CMH2; CMM; COL11A1; COL8A2; COL9A2; CPT2; CRB1; CSE; CSF3R; CTPA; CTSK; DBT; DIO 1;DISC1;DPYD;EKV;ENO1;ENO1P;EPB41;EPHX1;F13B;F5;FCGR2A;FCGR2B;FCGR3A;FCHL;FH;FMO3;FMO4;FUCA1;FY;GALE;GBA;GFND;GJA8;GJB 3;GLC3B;HF1;HMGCL;HPC1;HRD;HRPT2;HSD3B2;HSPG2;KCNQ4;KCS;KIF1B;LAMB3;LAMC2;LGMD1B;LMNA;LOR;MCKD1;MCL1;MPZ;MTHFR;MTR;MUTY H;MYOC;NB;NCF2;NEM1;NPHS2;NPPA;NRAS;NTRK1;OPTA2;PBX1;PCHC;PGD;PHA2A;PHGDH;PKLR;PKP1;PLA2G2A;PLOD;PPOX;PPTO;PRCC;PRG4;P SEN2;PTOS1;REN;RFX5;RHD;RMD1;RPE65;SCCD;SERPINC1;SJS1;SLC19A2;SLC2A1;SPG23;SPTA1;TAL1;TNFSF6;TNNT2;TPM3;TSHB;UMPK;UOX;U ROD;USH2A;VMGLOM;VWS;WS2B;ABCB11;ABCG5;ABCG8;ACADL;ACP1;AGXT;AHHR;ALMS1;ALPP;ALS2;APOB;BDE;BDMR;BJS;BMPR2;CHRNA1;CMCWTD ;CNGA3;COL3A1;COLAA3;COL4A4;COL6A3;CPS1;CRYGA;CRYGEP1;CYP1B1;CYP27A1;DBI;DES;DYSF;EDAR;EFEMP1;EIF2AK3;ERCC3;FSHR;GINGF;<h2 style=";text-align:left;direction:ltr">GLC1B; GPD2; GYPC; HADHA; HADHB; HOXD13; HPE2; IGKC; IHH; IRS1; ITGA6; KHK; KYNU; LCT; LHCGR; LSFC; MSH2; MSH6; NEB; NMTC; NPHP1; PAFAH1P1; PAX3; PAX8; PMS1; PNKD; PPH1; PROC; REG1A; SAG; SFTPB; SLC11A1; SLC3A1; SOS1; SPG4; SRD5A2; TCL4; TGFA; TMD; TPO; UGT1A@; UV24; WSS; XDH; ZAP70; ZFHX1B; AC AA1;AGS1;AGTR1;AHSG;AMT;ARMET;BBS3;BCHE;BCPM;BTD;CASR;CCR2;CCR5;CDL1;CMT2B;COL7A1;CP;CPO;CRV;CTNNB1;DEM;ETM1;FANCD2;FIH;FOXL2 GBE1 OU1F1;PPARG;PROS1;PTHR1;RCA1;RHO;SCA7;SCLC1;SCN5A;SI;SLC25A20;SLC2A2;TF;TGFBR2;THPO;THRB;TKT;TM4SF1;TRH;UMPS;UQCRC1;USH3A;VHL;WS2A;XPC;ZNF35;ADH1B;ADH1C;AFP;AGA;AIH2;ALB;ASMD;BFHD;CNGA1;CRBM;DCK;DSPP;DTDP2;ELONG;ENAM;ETFDH;EVC;F11;FABP2;FGA;FGB;FGFR 3;FGG;FSHMD1A;GC;GNPTA;GNRHR;GYPA;HCA;HCL2;HD;HTN3;HVBS6;IDUA;IF;JPD;KIT;KLKB1;LQT4;MANBA;MLLT2;MSX1;MTP;NR3C2;PBT;PDE6B;PEE1;PITX2;PKD2;QDPR;SGCB;SLC25A4;SNCA;SOD3;STATH;TAPVR1;TYS;WBS2;WFS1;WHCR;ADAMTS2;ADRB2;AMCN;AP3B1;APC;ARSB;B4GALT7;BHR1;C6;C7;<h2 style=";text-align:left;direction:ltr">CCAL2;CKN1;CMDJ;CRHBP;CSF1R;DHFR;DIAPH1;DTR;EOS;EPD;ERVR;F12;FBN2;GDNF;GHR;GLRA1;GM2A;HEXB;HSD17B4;ITGA2;KFS;LGMDLA;LOX;LTC4S;MAN2A1;MCC;MCCC2;MSH3;MSX2;NR3C1;PCSK1;PDE6A;PFBI;RASA1;SCZD1;SDHA;SGCD;SLC22A5;SLC26A2;SLC6A3;SM1;SMA@;SMN1;SMN2;SPINK5;TCO F1;TELAB1;TGFBI;ALDH5A1;ARG1;AS;ASSP2;BCKDHB;BF;C2;C4A;CDKN1A;COL10A1;COL11A2;CYP21A2;DYX2;EJM1;ELOVL4;EPM2A;ESR1;EYA4;F13A1; FANCE;GCLC;GJA1;GLYS1;GMPR;GSE;HCR;HFE;HLA-A;HLA-DPB1;HLA-DRA;HPFH;ICS1;IDDM1;IFNGR1;IGAD1;IGF2R;ISCW;LAMA2;LAP;LCA5;LPA;MCDR 1;MOCS1;MUT;MYB;NEU1;NKS1;NYS2;OA3;ODDD;OFC0;PARK2;PBCA;PBCRA1;PDB1;PEX3;PEX6;PEX7;PKHD1;PLA2G7;PLG;POLH;PPAC;PSORS1;PUJO;RCD 1;RDS;RHAG;RP14;RUNX2;RWS;SCA1;SCZD3;SIASD;SOD2;ST8;TAP1;TAP2;TFAP2B;TNDM;TNF;TPBG;TPMT;TULP1;WISP3;AASS;ABCB1;ABCB4;ACHE;AQP 1;ASL;ASNS;AUTS1;BPGM;BRAF;C7orf2;CACNA2D1;CCM1;CD36;CFTR;CHORDOMA;CLCN1;CMH6;CMT2D;COL1A2;CRS;CYMD;DFNA5;DLD;DYT11;EEC1;ELN; ETV1;FKBP6;GCK;GHRHR;GHS;GLI3;GPDS1;GUSB;HLXB9;HOXA13;HPFH2;HRX;IAB;IMMP2L;KCNH2;LAMBI;LEP;MET;NCF1;NM;OGDH;OPN1SW;PEX1;PGAM2;<h2 style=";text-align:left;direction:ltr">PMS2;PON1;PPP1R3A;PRSS1;PTC;PTPN12;RP10;RP9;SERPINE1;SGCE;SHFM1;SHH;SLC26A3;SLC26A4;SLOS;SMAD1;TBXAS1;TWIST;ZWS1;ACHM3;ADRB3 ;ANK1;CA1;CA2;CCAL1;CLN8;CMT4A;CNGB3;COH1;CPP;CRH;CYP11B1;CYP11B2;DECR1;DPYS;DURS1;EBS1;ECA1;EGI;EXT1;EYA1;FGFR1;GNRH1;GSR;GU LOP;HR;KCNQ3;KFM;KWE;LGCR;LPL;MCPH1;MOS;MYC;NAT1;NAT2;NBS1;PLAT;PLEC1;PRKDC;PXMP3;RP1;SCZD6;SFTPC;SGM1;SPG5A;STAR;TG;TRPS1;T TPA;VMD1;WRN;ABCA1;ABL1;ABO;ADAMTS13;AK1;ALAD;ALDH1A1;ALDOB;AMBP;AMCD1;ASS;BDMF;BSCL;C5;CDKN2A;CHAC;CLA1;CMD1B;COL5A1;CRAT;DB H;DNAI1;DYS;DYT1;ENG;FANCC;FBP1;FCMD;FRDA;GALT;GLDC;GNE;GSM1;GSN;HSD17B3;HSN1;IBM2;INVS;JBTS1;LALL;LCCS1;LCCS;LGMD2H;LMX1B;MLLT3;MROS;MSSE;NOTCH1;ORM1;PAPPA;PIP5K1B;PTCH;PTGS1;RLN1;RLN2;RMRP;ROR2;RPD1;SARDH;SPTLC1;STOM;TDFA;TEK;TMC1;TRIM32;TSC1;TYRP 1; ODAL;OAT;OATL3;PAX2;PCBD;PEO1;PHYH;PNLIP;PSAP;PTEN;RBP4;RDPA;RET;SFTPA1;SFTPD;SHFM3;SIAL;THC2;TLX1;TNFRSF6;UFS;UROS;AA;ABCC8;<h2 style=";text-align:left;direction:ltr">ACAT1; ALX4; AMPD3; ANC; APOAL; APOA4; APOC3; ATM; BSCL2; BWS; CALCA; CAT; CCND1; CD3E; CD3G; CD59; CDKNLC; CLN2; CNTF; CPT1A; CTSC; DDB1; DDB2; DHCR7; DLAT; DRD4; ECB2; ED4; EVR1; EXT2; F2; FSHB; FTH1; G6PT1; G6PT2; GIF; HBB; HBBP1; HBD; HBE1; HBG1; HBG2; HMBS; HND; HOMG2; HRAS; HVBS1; IDDM2; IG ER;INS;JBS;KCNJ11;KCNJ1;KCNQ1;LDHA;LRP5;MEN1;MLL;MYBPC3;MYO7A;NNO1;OPPG;OPTB1;PAX6;PC;PDX1;PGL2;PGR;PORC;PTH;PTS;PVRL1;PYGM; RAG1;RAG2;ROM1;RRAS2;SAA1;SCA5;SCZD2;SDHD;SERPING1;SMPD1;TCIRG1;TCL2;TECTA;TH;TREH;TSG101;TYR;USH1C;VMD2;VRNI;WT1;WT2;ZNF145; A2M; AAAS; ACADS; ACLS; ACVRL1; ALDH2; AMHR2; AOM; AQP2; ATD; ATP2A2; BDC; CIR; CD4; CDK4; CNA1; COL2A1; CYP27B1; DRPLA; ENUR2; FEOM1; FGF23; FPF; GNB3; GNS; HAL; HBP1; HMGA2; HMN2; HPD; IGF1; KCNA1; KERA; KRAS2; KRT1; KRT2A; KRT3; KRT4; KRT5; KRT6A; KRT6B; KRTHB6; LDHB; LYZ; MGCT; MPE; MVK; MYL 2;OAP;PAH;PPKB;PRB3;PTPN11;PXR1;RLS;RSN;SAS;SAX1;SCA2;SCNN1A;SMAL;SPPM;SPSMA;TBX3;TBX5;TCF1;TPI1;TSC3;ULR;VDR;VWF;ATP7B;BRCA2;BRCD1;CLN5;CPB2;ED2;EDNRB;ENUR1;ERCC5;F10;F7;GJB2;GJB6;IPF1;MBS1;MCOR;NYS4;PCCA;RB1;RHOK;SCZD7;SGCG;SLC10A2;SLC25A15;STARP1;ZNF198; ACHM1; ARVD1; BCH; CTAA1; DAD1; DFNB5; EML1; GALC; GCH1; IBGC1; IGH@; IGHC group; IGHG1; IGHM; IGHR; IV; LTBP2; MCOP; MJD; MNG1; MPD1; MPS3C; MYH6; MYH; <h2 style=";text-align:left;direction:ltr">7;NP;NPC2;PABPN1;PSEN1;PYGL;RPGRIP1;SERPINA1;SERPINA3;SERPINA6;SLC7A7;SPG3A;SPTB;TCL1A;TGM1;TITF1;TMIP;TRA@;TSHR;USHLA;VP;ACCPN;AHO2;ANCR;B2M;BBS4;BLM;CAPN3;CDAN1;CDAN3;CLN6;CMH3;CYP19;CYP1A1;CYP1A2;DYX1;EPB42;ETFA;EYCL3;FAH;FBN1;FES;HCVS;HEXA;IVD;LC S1;LIPC;MYO5A;OCA2;OTSC1;PWCR;RLBP1;SLC12A1;SPG6;TPM1;UBE3A;WMS;ABCC6;ALDOA;APRT;ATP2A1;BBS2;CARD15;CATM;CDH1;CETP;CHST6;CLN3 ;CREBBP;CTH;CTM;CYBA;CYLD;DHS;DNASE1;DPEP1;ERCC4;FANCA;GALNS;GAN;HAGH;HBA1;HBA2;HBHR;HBQ1;HBZ;HBZP;HP;HSD11B2;IL4R;LIPB;MC1R; MEFV; MHC2TA; MLYCD; MMVP1; PHKB; PHKG2; PKD1; PKDTS; PMM2; PXE; SALL1; SCA4; SCNN1B; SCNN1G; SLC12A3; TAT; TSC2; VDI; WT3; ABR; ACACA; ACADVL; ACE; ALDH3A2; APOH; ASPA; AXIN2; BCL5; BHD; BLMH; BRCA1; CACD; CCA1; CCZS; CHRNB1; CHRNE; CMT1A; COL1A1; CORD5; CTNS; EPX; ERBB2; G6PC; GAA; GALK1; GCG R;GFAP;GH1;GH2;GP1BA;GPSC;GUCY2D;ITGA2B;ITGB3;ITGB4;KRT10;KRT12;KRT13;KRT14;KRT14L1;KRT14L2;KRT14L3;KRT16;KRT16L1;KRT16L2;KRT 17;KRT9;MAPT;MDB;MDCR;MGI;MHS2;MKS1;MPO;MYO15A;NAGLU;NAPB;NF1;NME1;P4HB;PAFAH1B1;PECAM1;PEX12;PHB;PMP22;PRKAR1A;PRKCA;PRKWNK4;<h2 style=";text-align:left;direction:ltr">PRP8; PRPF8; PTLAH; RARA; RCV1; RMSA1; RP17; RSS; SCN4A; SERPINF2; SGCA; SGSH; SHBG; SLC2A4; SLC4A1; SLC6A4; SMCR; SOST; SOX9; SSTR2; SYM1; SYNS1; TCF2; THRA; TIMP2; TOC; TOP2A; TP53; TRIM37; VBCH; ATP8B1; BCL2; CNSN; CORD1; CYB5; DCC; F5F8D; FECH; FEO; LAMA3; LCFS2; MADH4; MAFD1; MC2R; MCL; M YP2;NPC1;SPPK;TGFBRE;TGIF;TTR;AD2;AMH;APOC2;APOE;ATHS;BAX;BCKDHA;BCL3;BFIC;C3;CACNA1A;CCO;CEACAM5;COMP;CRX;DBA;DDU;DFNA4;DLL3;DM1;DMWD;E11S;ELA2;EPOR;ERCC2;ETFB;EXT3;EYCL1;FTL;FUT1;FUT2;FUT6;GAMT;GCDH;GPI;GUSM;HB1;HCL1;HHC2;HHC3;ICAM3;INSR;JAK3;KLK3; 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[0243] <h2 style=";text-align:left;direction:ltr"> In other examples, nanoparticle compositions comprising mRNA encoding an endonuclease, a DNA-binding or RNA-binding factor, and further comprising one or more small guide RNAs (sgRNAs) can be used for various diseases or conditions. For example, mRNA and guide RNA encoding Cas9 can be used to treat various genetic diseases such as sickle cell anemia or β-thalassemia. Alternatively, mRNA and guide RNA encoding Cas13d can be useful for treating RNA repeat disorders such as Huntington's disease or ALS (amyotrophic lateral sclerosis).

[0244] In other examples, nanoparticle compositions comprising endonucleases, DNA-binding or RNA-binding agents, and further comprising one or more small guide RNAs, can be used to treat various diseases or conditions. For example, Cas9 and guide RNAs (also called ribonucleoproteins) can be used to treat various genetic diseases, such as sickle cell anemia or beta thalassemia.

[0245] Nanoparticle compositions containing siRNA or miRNA encapsulating agent(s) can be used to downregulate or silence the translation (i.e., expression) of genes of interest, including, but not limited to, genes associated with viral infection and survival, genes associated with metabolic diseases and disorders (e.g., liver diseases and disorders), genes associated with tumorigenesis and cell transformation (e.g., cancer), angiogenesis genes, immunomodulatory genes, such as genes associated with inflammation and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative diseases.

[0246] Nanoparticle compositions comprising non-coding RNA such as tRNA can be used for a variety of diseases resulting from dysregulation or loss of tRNA synthesis, including, but not limited to, diseases associated with premature stop codons such as Charcot-Marie-Tooth disease, or β-thalassemia. Additionally, the tRNA constructs can be engineered with unnatural amino acids to treat various diseases such as cancer.

[0247] In another embodiment, the coated nanocarrier composition can be used to induce an immune response directed against one or more tumor-associated antigens or cells, such as cancer cells, expressing one or more tumor-associated antigens. One embodiment of the present invention contemplates the use of coatings or non-coding DNA or RNA that result in expression of tumor-associated antigens (also referred to herein as "antigens") as an encapsulating agent in the coated nanocarrier. These antigens may include sequences that essentially correspond to or are identical to the sequences of the tumor-associated antigens or one or more fragments thereof. In the methods of the present invention, the coated nanocarrier composition encapsulating an mRNA encoding an antigen can elicit an antigen-specific immune response in a subject, comprising administering to the subject an effective amount to generate an antigen-specific immune response. In some embodiments, the antigen-specific immune response comprises a T cell response. In some embodiments, the antigen-specific immune response comprises a B cell response. In some embodiments, the method of generating an antigen-specific immune response comprises a single administration of a vaccine. In some embodiments, the method further comprises administering one or more booster doses of the vaccine. In some embodiments, the vaccine is administered to the subject by intradermal or intramuscular injection.

[0248] In some embodiments, the disease-associated antigen is a tumor antigen. In this embodiment, the coated nanocarrier compositions described herein may be useful in treating cancer or cancer metastasis. Examples of tumor antigens that may be useful in embodiments of the present invention include p53, ART-4, BAGE, β-catenin / m, Bcr-abL, and the like. CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, cell surface proteins of the claudin family such as claudin-6, claudin-18.2 and claudin-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-B10, MAGE-B20, MAGE-B30, MAGE-B40, MAGE-B50, MAGE-B60, MAGE-B70, MAGE-B80, MAGE-B90, MAGE-B100, MAGE-B110, MAGE-B120, MAGE-B130, MAGE-B140, MAGE-B150, MAGE-B160, MAGE-B170, MAGE-B180, MAGE-B190, MAGE-B210, MAGE-B220, MAGE-B230, MAGE-B240, MAGE-B250, MAGE-B310, MAGE-B320, MAGE-B430, MAGE-B440, MAGE-B450, MAGE-B50, MAGE-B60, MAGE-B70, MAGE-B80, MAGE-B90, MAGE-B150, MAGE-B160, MAGE-B170, MAGE-B180, A9, MAGE-A10, MAGE-A11 or MAGE-A12, MAGE-B, MAGE-C, MART-1 / Melan-A, MCIR, Myosin / m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190 minor BCR-abL, Pm1 / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, as well as WT, e.g., WT-1.

[0249] In some embodiments, the coated nanoparticle pharmaceutical compositions described herein may also include one or more adjuvants, including, but not limited to, glucopyranosyl lipid adjuvant (GLA), CpG oligodeoxynucleotide (e.g., class A or B), poly(I:C), or aluminum hydroxide.

[0250] In some embodiments, the encapsulating agent is DNA or mRNA encoding one or more antigens or other proteins from an infectious agent. Such infectious agents include any microorganism capable of causing disease in humans or other mammals. In one embodiment, the infectious agent is an adenovirus, herpes simplex type 1, herpes simplex type 2, encephalitis virus, papillomavirus, varicella zoster virus, Epstein-Barr virus, human cytomegalovirus, human herpes virus type 8, human papillomavirus, BK virus, JC virus, smallpox, poliovirus, hepatitis B virus, human bocavirus, parvovirus B19, human astrovirus, Norwalk virus, coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, severe acute respiratory syndrome virus, SARS-CoV-2, hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, rubella virus, hepatitis E virus, human immunodeficiency virus (HIV), influenza virus, ... The strain of virus is selected from influenza virus, Guanarito virus, Junin virus, Lassa virus, Machupo virus, Sabia virus, Crimean-Congo hemorrhagic fever virus, Ebola virus, Marburg virus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus, Hendra virus, Nipah virus, rabies virus, hepatitis D virus, rotavirus, orbivirus, Coltivirus, Banna virus, human enterovirus, Hantavirus, West Nile virus, Middle East respiratory syndrome coronavirus, coronavirus, Japanese encephalitis virus, swine vesicular ecchymosis virus, Eastern equine encephalitis, Zika virus, and / or Chikungunya virus.

[0251] In some embodiments, the virus is a strain of influenza A or influenza B, or a combination thereof. In some embodiments, the influenza A or influenza B strain is associated with an avian, porcine, equine, canine, human, or non-human primate. In some embodiments, the antigenic polypeptide encodes a hemagglutinin protein or a fragment thereof.

[0252] In some embodiments, the infectious agent is a bacterial strain such as Mycobacterium tuberculosis, clindamycin-resistant Clostridium difficile, fluoroquinolone-resistant Clostridium difficile, methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Enterococcus faecalis, multidrug-resistant Enterococcus faecium, multidrug-resistant Pseudomonas aeruginosa, multidrug-resistant Acinetobacter baumannii, and vancomycin-resistant Staphylococcus aureus (VRSA). In some embodiments, the bacterium is Clostridium difficile.

[0253] In some embodiments, the encapsulating agent is multivalent. In some embodiments, the open reading frame (e.g., mRNA sequence) of one or more encapsulating agents encodes two or more antigenic polypeptides. In some embodiments, the open reading frame (e.g., mRNA sequence) of one or more encapsulating agents encodes at least 10, 15, 20 or 50 antigenic polypeptides.

[0254] Mammalian cells contacted by the compositions include, but are not limited to, hepatic cells, epithelial cells, hematopoietic cells, monocytic cells, endothelial cells, lung cells, alveolar cells, type I pneumocytes, type II pneumocytes, bone cells, stem cells, mesenchymal cells, neuronal cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiac muscle cells, skeletal muscle cells, lymphoid cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor or cancer cells. Target cells may be antigen-presenting cells, dendritic cells, macrophages, spleen cells, lung cells, hepatic sinusoidal wall cells, or Claudius cells, Hensen cells, Merkel cells, Müller cells, Paneth cells, Purkinje cells, Schwann cells, Sertoli cells, eosinophil cells, acinar cells, lipoblasts, adipocytes, brown or white alpha cells, amacrine cells, beta cells, thecal cells, cement cells, chief cells, chondroblasts, chondrocytes, chromaffin cells, chromophobe cells, corticotropin-secreting cells, delta cells, Langerhans cells, follicular dendritic cells, enterochromaffin cells, ependymal cells, epithelial cells, basal cells, squamous cells, endothelial cells, transitional cells, erythroblasts, red blood cells, fibroblasts, Follicle, fibrocyte, follicular cell, germ cell, gamete, egg, sperm, oocyte, primary oocyte, secondary oocyte, spermatid, spermatocyte, primary spermatocyte, secondary spermatocyte, germinal epithelium, giant cell, glial cell, astroblast, astrocyte, oligodendrocyte, oligodendrocyte, glioblast, goblet cell, gonadotropic cell, granulosa cell, hemocyte cell, hair cell, hepatoblast, hepatocyte, vitreous cell, stromal cell, juxtaglomerular cell, keratinocyte, keratocyte, leukocyte, granulocyte, basophil, eosinophil, neutrophil, lymphoblast, B lymphoblast, T lymphoblast, lymphocyte, B lymphocyte, T lymphocyte, helper-inducing T lymphocyte, Th1 T lymphocyte, Th2T lymphocyte, natural killer cell, thymocyte, macrophage, Kupffer cell, alveolar macrophage, foam cell, histiocyte, luteal cell, lymphoid stem cell, lymphoid cell, lymphoid stem cell, astroglia, mammotroph, mast cell, medulloblast, megakaryoblast, megakaryocyte, melanoblast, melanocyte, mesangial cell, mesothelial cell, metamyelocyte, monoblast, monocyte, mucosal cervical cell, muscle cell, cardiac muscle cell, skeletal muscle cell, smooth muscle cell, myelocyte, myeloid cell, bone marrow stem cell, myoblast, myoepithelial cell, myeloid cell The cell may be a fibroblast, neuroblast, neuroepithelial cell, neuron, odontoblast, osteoblast, osteoclast, osteocyte, oxyntic cell, parafollicular cell, paraluteal cell, digestive cell, pericyte, peripheral blood mononuclear cell, brown cell, supporting cell, pineal cell, pituitary cell, plasma cell, platelet, podocyte, proerythroblast, promonocyte, promyeloblast, promyelocyte, pronormoblast, reticulocyte, retinal pigment epithelial cell, retinoblast, small cell, growth hormone secreting cell, stem cell, supporting cell, teloglial cell, zymogen cell, kidney cell, or glandular cell.

[0255] One of skill in the art can use standard methods to determine appropriate dosage ranges and dosage levels for coated nanocarrier pharmaceutical compositions. For example, nanocarrier compositions coated according to the present disclosure may be administered in doses of about 0.0001 mg / kg to about 100 mg / kg, about 0.001 mg / kg to about 50 mg / kg, about 0.005 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.05 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg / kg, about 2 ... The composition may be administered in vivo at dosage levels sufficient to deliver about 10 mg / kg, about 5 mg / kg to about 10 mg / kg, about 0.0001 mg / kg to about 5 mg / kg, about 0.001 mg / kg to about 5 mg / kg, about 0.005 mg / kg to about 5 mg / kg, about 0.01 mg / kg to about 5 mg / kg, about 0.05 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 5 mg / kg, or about 1 mg / kg to about 5 mg / kg, with a dose of 1 mg / kg (mpk) providing 1 mg of encapsulating agent per kg of subject body weight.

[0256] The coated nanocarrier compositions containing one or more encapsulating agents can be used in combination with one or more other therapeutic, prophylactic, diagnostic or imaging agents. The term "in combination with" is not intended to imply that the agents must be formulated for simultaneous administration and / or delivery together, although these delivery methods are within the scope of this application. For example, one or more nanoparticle compositions containing one or more different therapeutic and / or prophylactic agents can be administered in combination. The compositions can be administered simultaneously with, prior to, or following one or more other desired therapeutic agents or medical procedures. For example, each agent may be administered at a dose and / or time schedule determined for that agent. EXAMPLES

[0257] Note on the naming of the first stabilizer used in the examples As described above, the iFNP process employs a first stabilizing agent (e.g., a first stabilizing amphiphilic copolymer). In the examples that follow, the stabilizing agent used in the iFNP process is composed of either poly(aspartic acid) (PAsp) or dextran (Dex) hydrophilic (more polar) region(s) and poly(lactic acid) (PLA) or poly(lactic-co-glycolic acid) (PLGA) hydrophobic (less polar) region(s). PAsp-b-PLA or PAsp-b-PLGA polymers are diblock copolymers in which the PAsp block is polymerized from a PLA or PLGA macroinitiator. Dextran-PLGA (or Dex-PLGA) polymers are comb-shaped polymers in which PLGA is grafted to a dextran backbone such that each dextran chain has one or more PLGA chains grafted onto it. The abbreviations used for these stabilizing polymers, and a description of each polymer, are provided in Table 1 below. The polymers shown in this table are not intended to reflect the full range of stabilizer compositions that can be used in this process. For example, poly(glutamic acid) (PGlu) can be used in place of or together with PAsp and / or dextran to form the hydrophilic (more polar) regions of the first stabilized amphiphilic copolymer. For example, polycaprolactone (PCL) can be used in place of or together with PLA and / or PLGA to form the hydrophobic (less polar) regions of the first stabilized amphiphilic copolymer.

[0258] In the examples below, encapsulation efficiency (EE) is the amount of RNA inside the nanoparticles divided by the total amount in solution. Alternatively, EE can be calculated as 100% minus the amount of free (unencapsulated) RNA in solution divided by the total amount of RNA in solution.

[0259] Burst release is the percentage of RNA (not encapsulated within nanocarriers) that is released within 15 minutes. The released RNA is separated from the nanocarriers using an ultrafilter. (Burst release = [RNA outside nanocarriers] / [total RNA inside and outside the nanocarriers]).

[0260] RNA loading is the mass fraction of RNA within the nanocarriers (RNA loading=[RNA mass] / [mass of RNA+first stabilizing agent+additive+second stabilizing agent]).

[0261] The terms RNAse protection and protection are used interchangeably. Protection is the percentage of total RNA in the nanocarriers that is not degraded by the RNAse enzyme added to the dispersion, as measured by the RiboGreen assay protocol. (Protection = [non-degraded RNA] / [total RNA]). [Table 1]

[0262] Example 1: Unexpected effects of lipid and solvent choice on RNA encapsulation RNA in triethylamine salt form (size approx. 5 kDa with unmodified bases) was dissolved in 0.5 mL of DMSO at 2.5 mg / mL (based on triethylamine (TEA) salt) with block copolymer stabilizer (2.5 mg / mL) as shown in Table 2. The naming system of the stabilizing polymer is shown at the beginning of the Examples section in Table 1. The DMSO contained 5% by volume of deionized water. This solution was rapidly mixed with an anti-solvent stream of DCM in a multi-inlet vortex mixer. DCM (dichloromethane) was placed in the mixer in three separate streams of 0.5 mL volume. One charge equivalent of CaCl2 relative to the acid groups in the formulation was included in one DCM stream. This mixing process formed the inverse nanocarriers with RNA encapsulated. The RNA content, taking into account mixer hold-up and losses, was approximately 0.75 mg (based on free acid). The inverted nanocarriers were then coated with PLA-b-PEG (2.5 mg) after brine extraction and solvent exchange into a water-miscible modified solvent (THF (tetrahydrofuran)). With the inclusion of 5 mg of bulk polymer (PLGA or PLA) before solvent exchange, salt changes of the block copolymer stabilizer, and additional crosslinker, EE values ​​below 45% were obtained (Formulation IDs 1-3).

[0263] RNA release from PEG-coated nanocarriers was measured by separating the nanocarriers from soluble RNA using a 300 kDa molecular weight cutoff ultrafilter. The RNA content of the crude nanoparticles and ultrafilter flow-through was then measured spectrophotometrically by absorbance at 260 nm. As a screening standard, PEG-coated nanocarriers were diluted into 150 mM NaCl ("brine"). Burst release from the nanocarriers was measured after 15 minutes of incubation in this medium. Formulations IDs 1, 2 and 3 all had a burst release of >50% (greater than 50%).

[0264] Unexpectedly, the addition of lipid POPC in the antisolvent stream during the iFNP process resulted in EE values ​​ranging from 57% to 71% with burst release values ​​less than 32% (formulations 4-7). Continued improvement in EE was seen with increasing the POPC amount from 1.95 mg to 3.9 mg (2x POPC). The following examples demonstrate that POPC can be included after the iFNP process with similar effects.

[0265] Surprisingly, a specific and enhanced effect on process performance was observed when acetonitrile (MeCN) was replaced with THF as the modification solvent. Formulations 8 and 9 showed EE values ​​>75% and burst release <20% when this substitution was used. However, when POPC was added after the solvent exchange to MeCN, the EE and burst release values ​​were comparable to the THF results (53% EE and 37% burst, not included in Table 2). [Table 2]

[0266] To confirm that the composition alone did not provide the process index enhancement, the same formulation composition was prepared using a modified process. RNA in triethylamine salt form (size approx. 5 kDa for unmodified bases) was dissolved at 2.5 mg / mL in 0.5 mL of DMSO with block copolymer stabilizer (5 mg / mL) as shown in Table 3. The DMSO contained 5% by volume of deionized water. This solution was rapidly mixed with an anti-solvent stream of either THF or MeCN in a confined impinging jet (CIJ) mixer. The anti-solvent stream contained one charge equivalent of CaCl2 and, optionally, two different amounts of POPC (1.95 mg or 3.9 mg). After mixing the solvent and anti-solvent streams, 2.5 mg of PLA-b-PEG and 5 mg of PLGA homopolymer were added to the inverse nanoparticles. The mixed stream was not further diluted before the coating step, which was achieved by CIJ mixing with an equal volume of water. The coated nanocarrier dispersion was then diluted 10-fold with additional water.

[0267] The EE and burst release values ​​are reported in Table 3 and show that composition alone was not responsible for the high EE and low burst values ​​achieved by the use of POPC and acetonitrile in the iFNP process. [Table 3]

[0268] Example 2: Variation of the modified iFNP process using POPC and acetonitrile A variation of the process was performed using a POPC additive and the modified solvent acetonitrile (MeCN). RNA in triethylamine salt form (size approx. 5 kDa for unmodified bases) was dissolved at 2.5 mg / mL in 0.5 mL of DMSO with block copolymer stabilizer (5 mg / mL) as shown in Table 4. The DMSO contained the indicated volume percent of deionized water. This solution was rapidly mixed with an anti-solvent stream of DCM in a multi-inlet vortex mixer (MIVM) or confined impinging jet (CIJ) mixer. For MIVM, DCM was entered into the mixer in three separate streams of 0.5 mL volume. For CIJ, a single stream of 0.5 mL of DCM was used. One charge equivalent of CaCl2 or Ca(NO3)2 (except where noted) was included in one DCM stream. Additional DCM was added to the mixer outlet to bring the DMSO content to 10% by volume or less. This produces an inverse nanocarrier dispersion in DCM containing approximately 0.75 mg of RNA (free acid basis, taking into account hold-up and losses in the mixer apparatus).

[0269] The samples were then aged for approximately 18 hours, except where noted in Table 4, and then extracted with a volume of 150 mM NaCl in water (collectively, approximately 25-75% of the total inverse nanocarrier dispersion volume) for approximately 30 minutes. The DCM phase was isolated and lipid(s), hydrophobic additive, and PEG-based coating stabilizer (PLA-b-PEG or Vitamin E TPGS (tocopheryl polyethylene glycol succinate)) were added (this coating stabilizer is also referred to as the second stabilizing amphiphilic agent).

[0270] A solvent exchange to the modification solvent, acetonitrile (MeCN), was performed as follows: MeCN was added to DCM at an equal volume, and the dispersion was then concentrated by rotary evaporation. A put-and-take distillation was performed involving two additions of MeCN. For a DMSO input of 0.5 mL, these additions totaled 8 mL, with a final concentration volume of approximately 1 mL. The temperature used was 35° C., and the vacuum setting was altered as necessary to achieve the distillation.

[0271] The inverse nanocarrier dispersion in acetonitrile was then rapidly mixed in a CIJ mixer with an equal volume of deionized water (except as noted in Table 4) and an additional deionized water collection bath was used to reduce the MeCN volume content to less than 10% by volume. This process produced PEG-coated nanocarriers. Encapsulation efficiency (EE) was determined by isolating the free RNA from the nanocarriers using a 300 kDa molecular weight cutoff ultrafilter. The coated nanocarriers were further diluted approximately 4-fold in water prior to analysis. The RNA content of filtered and unfiltered samples was then measured spectroscopically by absorbance at 260 nm. As a screening standard, 600 mM aqueous NaCl was added to the PEG-coated nanocarriers dispersed in water, resulting in the PEG-coated nanocarriers dispersed in 150 mM aqueous NaCl ("brine"). The burst release from the nanocarriers was measured after 15 minutes of incubation in this medium in the same manner as EE was measured.

[0272] Table 5 reports the EE and burst release data for process variations. Table 6 reports the size determined by dynamic light scattering (DLS) analysis for selected formulations. These data demonstrate that process variations can be used to produce formulations with high EE (greater than 80%) and low burst (less than 10%). In contrast to previous observations by iFNP, the inclusion of suitable lipids, such as POPC, allows for high EE, which is further enhanced by using acetonitrile (MeCN) as a modification solvent. In the compositions tested, DSPC and DOPE do not provide as good process indicators as POPC. [Table 4] TIFF2025501764000006.tif225159 [Table 5] [Table 6]

[0273] Example 3: Removal of the extraction step in a modified iFNP process RNA in triethylamine salt form (approximately 5 kDa in size for unmodified bases) was dissolved at 2.5 mg / mL in 0.5 mL of DMSO with block copolymer stabilizers (5 mg / mL) as shown in Table 7. The nomenclature of the stabilizers is consistent with Table 1. The DMSO solution contained the indicated volume % of deionized water. This solution was rapidly mixed with an anti-solvent stream of DCM in a multi-inlet vortex mixer (MIVM) or confined impinging jet (CIJ) mixer. For MIVM, DCM was placed in the mixer in three separate streams of 0.5 mL volume. For CIJ, a single stream of 0.5 mL of DCM was used. One charge equivalent of CaCl2 relative to the total acid content of the formulation was included in one DCM stream. No additional DCM was included in the collection vessel. Thus, the MIVM formulation produces an inverse nanocarrier dispersion in DCM with 25% DMSO by volume, and the CIJ formulation produces an inverse nanocarrier in 50% DCM / 50% DMSO. Surprisingly, the resulting inverse nanocarriers were stable in this environment and towards further processing. The RNA content was approximately 0.75 mg (free acid basis) after taking into account the mixer hold-up and losses.

[0274] Samples were aged for 45 min to 18 h as shown in Table 7, and samples were not subjected to extraction to remove DMSO. After inverse nanocarrier formation, lipid(s), hydrophobic additive, and PEG-b-PLA coating stabilizer (unless otherwise noted) were added directly to the dispersion.

[0275] Solvent exchange to the modification solvent, acetonitrile, was performed as follows: Acetonitrile (MeCN) was added to DCM at an equal volume, and the dispersion was then concentrated by rotary evaporation. A put-and-take distillation was performed involving two additions of MeCN. For a DMSO input of 0.5 mL, these additions totaled 8 mL, resulting in a concentration volume of approximately 1 mL. The temperature used was 35°C, and the vacuum set point was modified as necessary to achieve distillation. Surprisingly, the inverse nanocarrier (NC) dispersion was stable during the solvent exchange, even in the presence of relatively high DMSO concentrations.

[0276] The inverse NC dispersion in acetonitrile was then rapidly mixed in a CIJ mixer with an equal volume of deionized water (except as noted in Table 7) and an additional deionized water collection bath was used to reduce the MeCN volume content to less than 10% by volume. This process produced PEG-coated nanocarriers. Encapsulation efficiency (EE) was determined by isolating the free RNA from the nanocarriers using a 300 kDa molecular weight cutoff ultrafilter. The nanocarriers were further diluted approximately 4-fold in water prior to analysis. The RNA content of filtered and unfiltered samples was then measured spectroscopically by absorbance at 260 nm. As a screening standard, 600 mM aqueous NaCl was added to the PEG-coated nanocarriers dispersed in water, resulting in the PEG-coated nanocarriers dispersed in 150 mM aqueous NaCl ("brine"). The burst release from the nanocarriers was measured after 15 minutes of incubation in this medium in the same manner as EE was measured. These data are summarized in Table 7. [Table 7] TIFF2025501764000010.tif92159

[0277] These data demonstrate that it is possible to eliminate the extraction process, optimize the formulation for high EE, and include alternative hydrophobic additives other than polymers and a PEG coating.

[0278] Example 4: Use of lipid blends and ionizable lipids in a modified iFNP process RNA in triethylamine salt form (size approximately 5 kDa with unmodified bases) was dissolved at 2.5 mg / mL in 0.5 mL of DMSO with Dex-PLGA polymer (503-80) stabilizer (2.5 mg / mL). The DMSO contained 10% by volume of deionized water. This solution was rapidly mixed with an anti-solvent stream of DCM in a multi-inlet vortex mixer (MIVM). DCM was entered into the mixer in three separate streams of 0.5 mL volume. Ca(NO3)2 with the charge equivalents (relative to the total acid content of the formulation) listed in Table 8 was included in one DCM stream from a concentrated solution in methanol. The outlet stream from the mixer was collected in a vessel containing additional DCM to bring the DMSO content to 10% by volume or less. The RNA content was 0.75 mg (based on free acid) after accounting for the hold-up losses of the mixer.

[0279] After inverse nanocarrier formation, 2.85 mg of a cationic ionized lipid called Dlin-DMA (CAS number: 871258-12-7) was added to the dispersion and aged for the time indicated in Table 8. Next, 0.84 mg of POPC and 1.46 mg of cholesterol were added to the dispersion and aged for an additional 15 minutes. Finally, PEG stabilizer was added as indicated in Table 8. Either 2 mg of the lipid system PEG-DMG (Cas number: 1397695-86-1) or 1.5 mg of the polymer PEG-b-PLA was added as indicated in Table 8.

[0280] Solvent exchange into the modified solvent, acetonitrile (MeCN), was performed as follows: The DCM dispersion was concentrated to about 1 mL, and then 4 mL of MeCN was added with stirring. The MeCN dispersion was then concentrated to about 0.75 mL, and an additional 4 mL of MeCN was added. The dispersion was concentrated again to 0.5 mL. The reversed NC dispersion in acetonitrile was then mixed with an equal volume of aqueous buffer (deionized (DI) water, phosphate buffered saline (PBS) or citrate buffer (citric acid)) as shown in Table 8 using a CIJ mixer. An additional buffer collection bath was used to reduce the MeCN volume content to less than 10% by volume. This process produced PEG-coated nanocarriers. The encapsulation efficiency (EE) was determined by separating the free RNA from the nanocarriers using a 300 kDa molecular weight cutoff ultrafilter. The coated nanocarriers were further diluted, typically about 4-fold, in water before analysis. The RNA content in the filtered and unfiltered samples was then measured using standard methods for RiboGreen. Table 8: Variation of parameters for Dlin-containing formulations. Aging time is the delay before lipid addition or extraction. Ca equivalent is the calcium equivalent to the acid residues (RNA phosphates) in the formulation. Coating stabilizer is that used in the FNP coating process to create a stabilized surface on the coated nanoparticles. EE is the encapsulation efficiency measured spectroscopically. Size was measured by DLS using a cumulant fit. (ND = not determined) [Table 8]

[0281] These data demonstrate that ionizable lipids and lipid blends can be successfully incorporated into the process. Significant process improvements were observed when using blends containing EE values ​​>95%, independent of process variations. Size control was demonstrated with coated nanocarrier sizes suitable for sterile filtration. Additionally, the process demonstrates the use of PEG anchored to PLA rather than lipids. The high polydispersity index (PDI) observed in DLS reflects the presence of a small micelle population in addition to the coated nanocarrier population.

[0282] Example 5: Formulation screening with model mRNA using a modified iFNP process RNA in triethylamine salt form (>200 base pair size with unmodified bases, Qiagen Carrier RNA) was dissolved at 2.5 mg / mL in 0.5 mL of DMSO. The block copolymer stabilizer 753-Asp was included to obtain the RNA loadings shown in Table 9. The DMSO stream contained 10% by volume of deionized (DI) water unless otherwise noted. This solution was rapidly mixed with an anti-solvent stream of DCM in a multi-inlet vortex mixer (MIVM). Table 9 shows some examples using different anti-solvent or CIJ mixers. For MIVM, DCM was placed in the mixer in three separate streams of 0.5 mL volume. For CIJ, a single stream of 0.5 mL of DCM was used. One charge equivalent of Ca(NO3)2 relative to the total acid content of the formulation was included in one DCM stream unless otherwise noted. No additional DCM was included in the collection container except as indicated in the table. This additional DCM is referred to as the "iFNP quench." Thus, the MIVM formulation produces an inverse nanocarrier dispersion in DCM with 25% DMSO by volume.

[0283] The samples were aged and extracted with 150 mM brine according to the data in Table 9. Extraction was performed as described in Example 2. After inverse nanocarrier formation, lipid(s), hydrophobic additives and PEG-b-PLA coating stabilizer (except where noted) were added directly to the dispersion. Solvent exchange to the modification solvent, acetonitrile, was performed as described in Example 4.

[0284] The inverse NC dispersion in acetonitrile was then mixed with an equal volume of deionized water in a CIJ mixer, and an additional deionized water collection bath was used to reduce the MeCN volume content to less than 10% by volume. Where the use of a separate PEG-b-PLA block copolymer (BCP) coating stream is indicated in Table 9, a MIVM was used with one inlet containing the inverse nanocarrier dispersion, one inlet containing the coating stabilizer, and two inlets containing antisolvent (deionized water or 10 mM HEPES buffer, pH 7). This process produced PEG-coated nanocarriers. Encapsulation efficiency (EE) was determined by separating the free RNA from the nanocarriers using a 300 kDa molecular weight cutoff ultrafilter. Nanocarriers were typically diluted approximately 4-fold in water prior to this analysis. The RNA content of filtered and unfiltered samples was then measured spectroscopically by absorbance at 260 nm. In some cases, DLS size data was obtained using standard techniques. These data are summarized in Table 9. [Table 9] TIFF2025501764000013.tif241159TIFF2025501764000014.tif236159

[0285] These results indicate broadly applicable conditions using different lipids, different coatings, and different iFNP compositions.

[0286] Example 6: Screening using an RNAse digestion-based enzyme protection assay RNA in triethylamine salt form (>200 base pair size with unmodified bases, Qiagen Carrier RNA) was dissolved at 2.5 mg / mL in 0.5 mL of DMSO. The inclusion of Dex-PLGA stabilizing polymer (503-80) resulted in the RNA loadings shown in Table 10. This also describes some formulations using alternative block copolymer stabilizers. The DMSO stream contained 10% deionized water by volume. This solution was rapidly mixed with an anti-solvent stream of DCM in a multi-inlet vortex mixer (MIVM). DCM was placed in the mixer in three separate streams of 0.5 mL volume. Unless otherwise stated, one charge equivalent of Ca(NO3)2 relative to the total acid content of the formulation was included in one DCM stream. An additional DCM, referred to as "iFNP quench", was included in the collection vial to ensure that the DMSO content after collection was less than 10% by volume. The RNA content, after taking into account mixer hold-up and losses, was approximately 0.75 mg (free acid basis).

[0287] Samples were aged for 1-2 hours and were not extracted prior to lipid addition and solvent exchange unless noted in Table 10. The lipid(s), hydrophobic additive(s) and coating stabilizer (listed in Table 10) were added directly to the dispersion. Generally, any ionizable components were added first, followed by POPC and cholesterol, followed by coating stabilizer. The coating stabilizer was generally 2-3 mg at the input concentration used in this example. Solvent exchange to the modified solvent, acetonitrile, was performed as described in Example 4.

[0288] The inverse NC dispersion in acetonitrile was then mixed with an equal volume of deionized water in a CIJ mixer, and the MeCN volume content was reduced to less than 10% by volume using a collection bath of additional deionized water. This process produced PEG-coated nanocarriers. The nanocarriers were then buffer-exchanged by ultrafiltration into deionized water (to remove residual acetonitrile) or PBS, as described in Table 10.

[0289] DLS size data was obtained using standard techniques. The nanocarriers were also evaluated for RNAse A protection of the encapsulated RNA. This assay was performed as follows: 50 μL of nanocarriers in PBS (approximately 200 ug / mL of RNA) were mixed with 100 μL of RNAse A in deionized water (20 μg / mL). After 1 hour of incubation at 37°C, 100 μL of proteinase K (1 mg / mL) + DTT (dithiothreitol) (5 mM) was added. This digestion continued for 30 minutes at 55°C. A negative control was performed using deionized water without enzyme. RNA content was then measured by dissolving the nanoparticles in Triton X-100 (a non-ionic surfactant with polyethylene oxide chains) (1% in Tris-EDTA, pH 8) for 15-30 minutes at 55°C. Exposed RNA is degraded, resulting in no signal in the RiboGreen concentration measurement, while protected RNA is detected by RiboGreen. Each assay included a positive control of soluble RNA. Further controls confirmed that the proteinase K degradative conditions were sufficient to abolish RNAse activity. The RiboGreen assay does not detect single-strand breaks, but an average degree of degradation. Protection values ​​are calculated as [RNA signal with RNAse] / [RNA signal without RNAse]*100. These data are summarized in Table 10.

[0290] As a positive control, standard lipid nanoparticles were produced using methods known in the art. A 5-fold or 6-fold charge ratio (lipid:RNA) was used. Lipid nanoparticles were assembled in MIVM using one ethanol stream containing lipid components and three aqueous streams (citrate buffer, pH 4) containing RNA. Lipid nanoparticles were then dialyzed overnight in phosphate buffer saline (pH 7.3) and characterized for size and RNAse protection. Known lipid formulations for DODAP (1,2-dioleoyl-3-dimethylammonium propane), DC-Cholesterol (DC-Chol) (3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride), and Dlin-DMA (1,2-dilinoleyloxy-n,n-dimethyl-3-aminopropane) were used. However, the charge ratio (lipid:RNA) of lipid nanoparticles was significantly higher than that used for iFNP. The lipid nanoparticles generally had sizes ranging from 70 to 100 nm, and RNAse protection ranged from 90 to 100%.

[0291] To confirm that the enhanced RNAse protection seen for certain lipid compositions was due to iFNP structure and not charge complexation of the RNA, control experiments characterized the RNAse protection when RNA was added to the aqueous phase during the coating step. In these experiments, the iFNP / coating process was performed with all the same components, but RNA was not included in the formation of the inverted nanocarriers. The inverted nanocarriers behaved as if they were the same throughout the process. An amount of RNA, adjusted for hold-up losses in the mixer during inverted nanocarrier assembly, was dissolved in the aqueous stream used as the antisolvent in the coating process. The nanocarriers were visually similar to those produced with encapsulated RNA. The nanocarriers were then analyzed using the RNAse protection assay described above after buffer exchange into PBS. Ionized lipids (Dlin-DMA, DODAP) showed equal protection rates (approximately 5-10% protection) to controls where soluble RNA was added to the digestion assay. This indicates that the enhanced protection seen (>60%) is dependent on the iFNP process and not charge complexation. In the case of DC-Chol, which has a permanent charge, the control showed 100% protection, indicating that soluble RNA that was not encapsulated within the iFNP nanocarriers was complexed by DC-Chol. [Table 10] TIFF2025501764000016.tif239159TIFF2025501764000017.tif47159

[0292] Example 7: Screening of formulations using SM-102 cationic lipid RNA in triethylamine salt form (>200 base pair size with unmodified bases, Qiagen Carrier RNA) was dissolved in 0.5 mL of DMSO at 2.5 mg / mL. (In one case, as noted in Table 11, the triethylamine salt was recovered from the solution by standard LiCl / ethanol precipitation and then used in the process). Dex-PLGA stabilizing polymer (503-80 unless otherwise noted in Table 11) was added to the DMSO stream at 2.5 mg / mL. The DMSO stream contained 10% by volume of deionized water. This solution was rapidly mixed with an anti-solvent stream of DCM in a multi-inlet vortex mixer (MIVM). DCM was placed in the mixer in three separate streams of 0.5 mL volume. Ca(NO3)2 was included in one DCM stream at a specific charge ratio (Table 11) relative to the total acid content of the formulation. Additional DCM, referred to as "iFNP quench," was included in the collection vial to ensure that the DMSO content after collection was less than 10% by volume. This resulted in RNA encapsulated in inverse nanocarriers with a total content of approximately 0.75 mg (based on free acid, taking into account hold-up and losses in the mixer apparatus).

[0293] Samples were not extracted prior to lipid addition and solvent exchange. Dispersions were aged for 10 minutes unless otherwise noted in Table 11. Cationic lipid SM-102 (heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate) (CAS number 2089251-47-6) was added to the dispersion. POPC, cholesterol and PEG-DMG were then added. Stock solutions were in chloroform or ethanol (no differences were observed). SM-102 was included at a charge ratio of approximately 1.9. The molar ratio of lipid components was approximately 47% SM-102, 10% POPC, 37% cholesterol and 6% PEG-DMG. Solvent exchange to the modified solvent acetonitrile was performed as described in Example 4, but with three acetonitrile additions instead of two.

[0294] The inverse NC dispersion in acetonitrile was then mixed with an equal volume of deionized water in a CIJ mixer, and an additional deionized water collection bath was used to reduce the MeCN volume content to less than 10% by volume. This process produced PEG-coated nanocarriers. The nanocarriers were then buffer exchanged by ultrafiltration into PBS. Where noted, some formulations were diluted with the same volume of buffer prior to ultrafiltration. The same buffer was then used for the buffer exchange step of ultrafiltration. The buffers tested were 150 mM NaCl, PBS, and 25 mM acetate buffer (pH 4).

[0295] DLS size data was measured using standard techniques. Nanocarriers were also evaluated for RNAse A protection of encapsulated RNA. The assay was performed as follows: 50 μL of nanocarriers in PBS (approximately 200ug / mL RNA) were mixed with 100 μL of RNAse A in deionized water (20 μg / mL). After 1 hour of incubation at 37°C, 100 μL of proteinase K (1mg / mL) + DTT (5mM) was added. This digestion continued for 30 minutes at 55°C. A negative control was performed using deionized water without enzyme. RNA content was then measured by dissolving the nanoparticles in Triton X-100 (1% in Tris-EDTA, pH 8) for 15-30 minutes at 55°C. Exposed RNA is degraded, resulting in no signal in the RiboGreen concentration measurement, and protected RNA is detected by RiboGreen. Each assay included a positive control of soluble RNA. Further controls confirmed that the proteinase K degradative conditions were sufficient to abolish RNAse activity. The RiboGreen assay does not detect single strand breaks, but an average degree of degradation. Protection values ​​are calculated as [RNA signal with RNAse] / [RNA signal without RNAse]*100. These data are summarized in Table 11. [Table 11] TIFF2025501764000019.tif69159

[0296] Example 8: Cell transfection assay The ability of these formulations to transduce cells was evaluated using an mRNA sequence encoding green fluorescent protein (GFP) (Dasher GFP mRNA from Aldevron). Blends of mRNA encoding GFP in TEA salt form and carrier RNA were prepared (ratios as shown in Table 12). RNA was mixed with block copolymer in DMSO containing 10% by volume of deionized water, unless otherwise noted in Table 12. The stabilizing polymer was Dex-PLGA(503-80), unless otherwise noted in the Process Notes column of Table 12. RNA was added to the DMSO stream at 2.5 mg / mL, and polymeric stabilizer was added at the concentration required to produce the loading amount listed in Table 12. This solution was rapidly mixed with an anti-solvent stream of DCM in a multi-inlet vortex mixer (MIVM). DCM was placed in the mixer in three separate streams of 0.5 mL volume each. Ca(NO3)2 at one charge equivalent ratio to the total acid content of the formulation was included in one DCM stream (except as noted in Table 12). Additional DCM, referred to as "iFNP quench", was included in the collection vial to ensure that the DMSO content after collection was less than 10% by volume. The resulting RNA content in the inverse nanocarriers was 0.75 mg, taking into account hold-up losses up to the mixer device.

[0297] Samples were not extracted prior to lipid addition, except as noted in Table 12. A cationic lipid, typically SM-102 (CAS number 2089251-47-6), or sometimes DODAP (CAS number 127512-29-2) or Dlin-MC3-DMA ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) (CAS number 1224606-06-7), was added to the dispersion prior to the solvent exchange process. The other components were then added as shown in Table 12. Stock solutions were in chloroform or ethanol. Solvent exchange to the modified solvent, acetonitrile, was performed as described in Example 7.

[0298] The inverse NC dispersion in acetonitrile was then mixed with an equal volume of deionized water in a CIJ mixer (except where noted) and an additional deionized water collection bath was used to reduce the MeCN volume content to less than 10% by volume. This process produced PEG-coated nanocarriers. The nanocarriers were then buffer exchanged by ultrafiltration into deionized water or PBS. Where noted, some formulations were diluted with the same volume of buffer prior to ultrafiltration. The same buffer was then used for the buffer exchange step of ultrafiltration. The buffers used were 150 mM NaCl, PBS and 25 mM acetate buffer (pH 4), as well as isotonic HEPES / NaCl.

[0299] DLS size data was obtained using standard techniques. The nanocarriers were also evaluated for RNAse A protection of the encapsulated RNA. The assay was performed as follows: 50 μL of nanocarriers in PBS (approximately 200 ug / mL RNA) were mixed with 100 μL of RNAse A in deionized water (20 μg / mL). After 1 hour of incubation at 37°C, 100 μL of proteinase K (1 mg / mL) + dithiothreitol (DTT) (5 mM) was added. This digestion continued for 30 minutes at 55°C. A negative control was performed using deionized water without enzyme. RNA content was then measured by dissolving the nanoparticles in Triton X-100 (1% in Tris-EDTA, pH 8) for 15-30 minutes at 55°C. Exposed RNA is degraded, resulting in no signal in the RiboGreen concentration measurement, and protected RNA is detected by RiboGreen. Each assay included a positive control of soluble RNA. Further controls confirmed that the proteinase K degradative conditions were sufficient to abolish RNAse activity. The RiboGreen assay does not detect single strand breaks, but an average degree of degradation. Protection values ​​are calculated as [RNA signal with RNAse] / [RNA signal without RNAse]*100. These data are summarized in Table 13.

[0300] As a positive control, standard lipid nanoparticles were produced using methods known in the art. A 5-fold or 6-fold charge ratio (lipid:RNA) was used. Lipid nanoparticles were assembled in MIVM using one ethanol stream containing lipid components and three aqueous streams (citrate buffer, pH 4) containing RNA. Lipid nanoparticles were then dialyzed overnight in phosphate buffered saline (PBS, pH 7.3) and characterized for size and RNAse protection. The lipid formulations of DODAP, DC-Cholesterol (DC-Chol), Dlin-DMA, and SM-102 used standard compositions. The charge ratio (lipid:RNA) of lipid nanoparticles was significantly higher than that used for iFNP. Lipid nanoparticles are characterized in Table 13. [Table 12] TIFF2025501764000021.tif241159TIFF2025501764000022.tif237159TIFF2025501764000023.t if241159TIFF2025501764000024.tif236159TIFF2025501764000025.tif237159TIFF20255017640 00026.tif236159TIFF2025501764000027.tif235159TIFF2025501764000028.tif233159TIFF202 5501764000029.tif236159TIFF2025501764000030.tif240159TIFF2025501764000031.tif199159

[0301] Nanoparticles were added to adherent cell cultures with a starting confluence ranging from 30-50% and incubated for 24 h in standard cell culture conditions. Cells were washed and resuspended in PBS + 500 ng / mL DAPI (2-(4-carbamimidoylphenyl)-1H-indole-6-carboxyimidamide) and then analyzed using an Attune NxT flow cytometer (ThermoFisher). Singlet cells were gated using SSC-A × SSC-H and live cells were gated from the DAPI negative population. Fluorescence intensity of GFP was captured in the BL-1 channel without compensation. Cell cultures were HEK-293T and HepG2.

[0302] The results of the characterization of each formulation are summarized in Table 13. As a summary value, the GFP signal is reported in Table 13. It is calculated as [nanoparticle signal-negative control]-[positive control-negative control] at mRNA doses of 100-200 ng / mL in the 293T cell line. The positive control is the standard lipid nanoparticle run in that cohort. The negative control is an iFNP formulation containing only carrier RNA (0% mRNA). Dose curves were generated for most formulations, which are summarized in Figure 2, Figure 3A, Figure 3B, Figure 4A, Figure 4B, Figure 6A, Figure 6B, Figure 7A, Figure 7B, Figure 7C, Figure 8A, Figure 8B, Figure 9A, Figure 9B, Figure 10A, and Figure 10B. Figure 5A shows flow cytometry histograms for the indicated formulations showing equivalent and similar expression between the iFNP and lipid nanoparticle (LNP) formulations at an RNA dose of 1 ug / mL. A control formulation without mRNA (mRNA-55) was significantly less fluorescent; a control formulation, mRNA-55, was produced with the same ingredients and steps and conditions as mRNA-54, but without the mRNA. Figure 5B shows epifluorescence imaging and brightfield images of the formulations after 24 hours of incubation with 293T cells. Figure 5C shows the expression of GFP in 293T cell cultures at 24 hours, as measured by fluorescence intensity from flow cytometry analysis. [Table 13] TIFF2025501764000033.tif241159TIFF2025501764000034.tif28159

[0303] Example 9: Alternative buffer exchange treatment of GFP mRNA formulations The mRNA encoding GFP was mixed with Dex-PLGA(503-80) in DMSO containing 10% by volume of deionized water. The RNA in the TEA salt form was present at 2.5 mg / mL (mass including TEA counterion) and the polymeric stabilizer was present at 5 mg / mL. This solution (0.5 mL) was rapidly mixed with an antisolvent stream of DCM in a multi-inlet vortex mixer (MIVM). DCM was placed in the mixer in three separate streams of 0.5 mL volume. CaCl2 at a 0.75 charge equivalent ratio to the total acid content of the formulation was included in one DCM stream. Additional DCM, referred to as "iFNP quench", was included in the collection vial such that the DMSO content after collection was less than 10% by volume. Taking into account the hold-up in the mixer apparatus, the mRNA mass was 0.75 mg (free acid basis) and the Dex-PLGA mass was 1.875 mg.

[0304] Prior to the solvent exchange process, cationic lipid SM-102 (CAS number 2089251-47-6) was added to the dispersion (3 mg). After an aging time of 5 min, POPC (0.7 mg) and cholesterol (1.3 mg) were added. After an additional aging time of 5 min, PEG-DMG (1.5 mg) was added. All these components were added in ethanol. They were in a molar ratio of 47:10:37:6 (SM-102:POPC:cholesterol:PEG-DMG) relative to each other. SM-102 was added at approximately 1.9 charge equivalents relative to the RNA acid groups. The DCM solution was concentrated approximately 4-fold by rotary evaporation to 1-1.5 mL. Acetonitrile (8 mL) was then slowly added to the vial. The dispersion was concentrated to 1-1.5 mL at 70 Torr with a heat bath set at 35 °C. Acetonitrile (8 mL) was then added to the vial. The dispersion was concentrated to 1-1.5 mL at 65 Torr with a heat bath set at 35 °C. Acetonitrile (8 mL) was then added to the vial. The dispersion was concentrated to 1 mL at 60 Torr with a heat bath set at 35 °C.

[0305] The inverse NC dispersion in acetonitrile was then rapidly mixed with an equal volume of deionized water in a CIJ mixer, and the MeCN volume content was reduced to below 10 vol.% using a collection bath of additional deionized water (sample A) or phosphate-buffered saline (sample B). This process produced PEG-coated nanocarriers.

[0306] The formulations were processed into the final buffer as follows: For sample A1, the nanocarriers were dialyzed in a 500 volume equivalent excess of PBS at 2-8°C for 18 hours. The nanocarriers were then concentrated by ultrafiltration over a 50 kDa molecular weight cutoff filter to the desired RNA concentration. For sample A2, the nanocarriers were diluted with an equal volume of PBS and stored at 2-8°C for 18 hours. The nanocarriers were then concentrated by ultrafiltration over a 50 kDa molecular weight cutoff filter. A volume of PBS equal to the total nanocarrier input volume was added to the filter and the concentration step was repeated. A second addition of PBS and concentration to the desired RNA range yielded the nanocarriers in the final buffer. For A3, the nanocarriers were stored undiluted and without stirring at 2-8°C for 18 hours. The nanocarriers were then concentrated by ultrafiltration over a 50 kDa molecular weight cutoff filter. A volume of deionized water equal to the total nanocarrier input volume was added to the filter and the concentration step was repeated. A second water addition to the desired RNA range and concentration gave the nanocarriers in concentrated form. Sample B1, prepared with a collection bath that was PBS rather than deionized water, was dialyzed in a 500 volume equivalent excess of PBS at 2-8°C for 18 hours. The nanocarriers were then concentrated by ultrafiltration over a 50 kDa molecular weight cutoff filter to the desired RNA concentration. For sample B2, the nanocarriers were stored at 2-8°C for 18 hours. The nanocarriers were then concentrated by ultrafiltration over a 50 kDa molecular weight cutoff filter. Twice the total nanocarrier input volume of PBS was added to the filter and the concentration step was repeated. A second PBS addition to the desired RNA range and concentration gave the nanocarriers in the final buffer.

[0307] DLS size data was obtained using standard techniques. The nanocarriers were also evaluated for RNAse A protection of the encapsulated RNA. The assay was performed as follows: 50 μL of nanocarriers in PBS (approximately 200 ug / mL RNA) were mixed with 100 μL of RNAse A in deionized water (20 μg / mL). After 1 hour of incubation at 37°C, 100 μL of proteinase K (1 mg / mL) + DTT (5 mM) was added. This digestion continued for 30 minutes at 55°C. A negative control was performed using deionized water without enzyme. RNA content was then measured by dissolving the nanoparticles in Triton X-100 (1% in Tris-EDTA, pH 8) for 15-30 minutes at 55°C. Exposed RNA is degraded, resulting in no signal in the RiboGreen concentration measurement, and protected RNA is detected by RiboGreen. Each assay included a positive control of soluble RNA. Further controls confirmed that the proteinase K degradation conditions were sufficient to abolish RNAse activity. This RiboGreen assay does not detect single-strand breaks, but does detect an average degree of degradation. Protection values ​​are calculated as [RNA signal with RNAse] / [RNA signal without RNAse]*100. Encapsulation efficiency (EE) was measured with the standard RiboGreen protocol, rather than ultrafiltration, as was done in the previous examples. Briefly, nanocarrier dispersions were diluted in either Tris(hydroxymethyl)aminomethane (Tris) buffer or Tris / EDTA / Triton x-100 (1%) buffer and incubated at 37°C for 30 minutes. The nanocarrier mixture was added to Tris buffer containing a 200-fold dilution of RiboGreen dye stock, and the fluorescence signal was collected at 525 nm with excitation at 485 nm. Standard curves of mRNA prepared in Tris buffer or Tris / EDTA / Triton buffer were used to calculate the total RNA detected in each buffer. The Tris buffer allows the detection of free RNA, while the Tris / EDTA / Triton buffer measures total RNA content. These data are summarized in Table 14. [Table 14]

[0308] The stability of nanocarrier formulations against rapid freezing was evaluated using sucrose or trehalose cryoprotectants added at 5 wt% of the final composition. Rapid freezing was performed using a dry ice bath. Nanocarrier size was evaluated after the frozen compositions were thawed again. The results are summarized in Table 15. [Table 15]

[0309] Example 10: Co-encapsulation with small molecule co-cores The effect of incorporating a small molecule co-core was tested on the encapsulation of either a carrier RNA (>200 base pair size with unmodified bases, Qiagen Carrier RNA) in TEA salt form or an mRNA encoding GFP (Dasher GFP mRNA from Aldeveron) (Table 16). A solvent stream was prepared by dissolving RNA and Dex-PLGA stabilizer (503-80) in DMSO containing 10% by volume of deionized water. For formulations without a co-core, the solvent stream contained 2.5 mg / mL RNA in TEA salt form and 2.5 mg / mL or 5 mg / mL Dex-PLGA, as shown in Table 16. For formulations with a choline co-core, 1.65 mg / mL choline was included in the solvent stream. For formulations with a trehalose co-core, 5 mg / mL trehalose was included in the solvent stream. This solution (0.5 mL) was rapidly mixed with three anti-solvent streams of DCM in a multi-inlet vortex mixer (MIVM). DCM was fed into the mixer in three separate streams of 0.5 mL volume each. CaCl2 was included in one DCM stream at a 0.5 or 0.75 charge equivalence ratio to the acid content of the RNA. Additional DCM, referred to as "iFNP quench", was included in the collection vial to ensure that the DMSO content after collection was less than 10% by volume. Taking into account the hold-up losses of the mixer, the RNA content of the inverse nanocarrier was 0.75 mg (free acid basis).

[0310] Samples were not extracted before lipid addition.Before the solvent exchange process, 3.1mg (2 charge equivalents to the acid group in RNA) SM-102 (CAS number 2089251-47-6) was added to the dispersion, followed by 0.7mg POPC, 1.3mg cholesterol, and 1.5mg PEG-DMG.Solvent exchange to the modified solvent acetonitrile was carried out as described in Example 8.

[0311] The inverse nanocarrier dispersion in acetonitrile was then mixed in a CIJ mixer against an equal volume of deionized water and collected in a bath of either additional deionized water or PBS, as shown in Table 16, such that the final MeCN volume content was less than 10% by volume. This process produced PEG-coated nanocarriers. When the bath was deionized water, the nanocarrier dispersion was further diluted with an equal volume of PBS after coating. [Table 16]

[0312] The nanocarriers were then buffer exchanged by ultrafiltration into PBS. DLS size data was obtained using standard techniques both immediately after coating and after ultrafiltration. The ability of these nanoparticles to transduce cells was tested as shown in Example 7. The GFP signal in 293T cell cultures as defined in Example 7 is shown in Table 17. The dose variations are summarized in Figures 11A, 11B, 11C and 11D, and 12A and 12B. [Table 17]

[0313] Example 11: Effect of variations in lipid ratios on transfection mRNA encoding GFP was mixed with Dex-PLGA(503-80) in DMSO containing 10% by volume of deionized water. The RNA in the TEA salt form was present at 2.5 mg / mL, and the polymeric stabilizer was present at 5 mg / mL or 2.5 mg / mL as indicated. This solution (0.5 mL) was rapidly mixed with an antisolvent stream of DCM in a multi-inlet vortex mixer (MIVM). DCM was placed in the mixer in three separate streams of 0.5 mL volume each. CaCl2 at 0.75 or 0.5 charge equivalent ratio to the total acid content of the formulation was included in one DCM stream. Additional DCM, referred to as "iFNP quench", was included in the collection vial such that the DMSO content after collection was less than 10% by volume. Taking into account hold-up, the mRNA mass was 0.75 mg (free acid basis) and the Dex-PLGA mass was 1.875 mg.

[0314] Prior to the solvent exchange process, cationic lipid SM-102 (CAS number 2089251-47-6) was added to the dispersion (3.1 mg or 4.85 mg). After an aging time of 5 min, POPC (0.7 mg or 1.1 mg or 0.43 mg) and cholesterol (1.3 mg or 2.2 mg) were added. After another aging time of 5 min, PEG-DMG (1.5 mg) was added. All these components were added in ethanol. They were in a molar ratio of 47:10:39:4 or 60:5:30:5 (SM-102:POPC:cholesterol:PEG-DMG) relative to each other. SM-102 was added at approximately 2 or 3 charge equivalents relative to the RNA acid groups. The DCM solution was concentrated approximately 4-fold by rotary evaporation to 1-1.5 mL. Acetonitrile (8 mL) was then slowly added to the vial. The dispersion was concentrated to 1-1.5 mL at 70 Torr with a heat bath set at 35 °C. Acetonitrile (8 mL) was then added to the vial. The dispersion was concentrated to 1-1.5 mL at 65 Torr with a heat bath set at 35 °C. Acetonitrile (8 mL) was then added to the vial. The dispersion was concentrated to 1 mL at 60 Torr with a heat bath set at 35 °C.

[0315] The inverse nanocarrier dispersion in acetonitrile was then mixed with an equal volume of deionized water in a CIJ mixer, and the MeCN volume content was reduced to less than 10% by volume using a collection bath of additional deionized water or phosphate buffered saline. This process produced PEG-coated nanocarriers. These formulations are summarized in Table 18. [Table 18]

[0316] The formulation was processed into the final buffer as follows: Nanocarriers were concentrated by ultrafiltration at 10°C on a 50 or 100 kDa molecular weight cutoff filter. Twice the total nanocarrier input volume of PBS was added to the filter and the concentration process was repeated. A second addition of PBS and concentration to the desired RNA range yielded nanocarriers in the final buffer. The final form was then supplemented with 5 wt% trehalose dihydrate as a cryoprotectant and aliquots were flash frozen at -196°C. DLS size and zeta potential were measured using standard techniques throughout the process.

[0317] Nanoparticles were added to adherent cell cultures of HEK-293T or HepG2 with a starting confluence ranging from 30-50% and incubated for 24 h in standard cell culture conditions. Cells were washed and resuspended in PBS + 500 ng / mL 4',6-diamidino-2-phenylindole (DAPI) and then analyzed using an Attune NxT flow cytometer (ThermoFisher). Singlet cells were gated using SSC-A × SSC-H and live cells were gated from the DAPI negative population. GFP fluorescence intensity was captured in the BL-1 channel without compensation.

[0318] The results of the characterization of each formulation are summarized in Table 19. As a summary value, the GFP signal is reported in Table 19. It is calculated as [nanoparticle signal-negative control]-[positive control-negative control] at an mRNA dose of 200 ng / mL. The positive control is the standard lipid nanoparticles performed in that cohort. The negative control is the cell background signal (cell blank). The dose variations are summarized in Figures 11A, 11B, 11C and 11D, as well as Figures 12A and 12B. [Table 19]

[0319] Example 12: iFNP formulation of SARS RBD mRNA mRNA encoding the SARS-CoV-2 spike protein receptor binding domain (RBD) with pseudouridine (C5-glycosidic isomer of uridine, 5-ribosyluracil) modifications was mixed with Dex-PLGA (503-80) in DMSO containing 10% by volume deionized water. RNA in TEA salt form was present at 2 mg / mL and polymeric stabilizer was present at 2 mg / mL. This solution (0.5 mL) was rapidly mixed with an anti-solvent stream of DCM in a multi-inlet vortex mixer (MIVM). DCM was placed in the mixer in three separate streams of 0.5 mL volume each. CaCl2 at a 0.5 charge equivalent ratio to the total acid content of the formulation was included in one DCM stream. Additional DCM, referred to as "iFNP quench", was included in the collection vial to ensure that the DMSO content after collection was less than 10% by volume. Taking hold-up into account, the mRNA mass was 0.75 mg (free acid basis) and the Dex-PLGA mass was 1.875 mg.

[0320] Prior to the solvent exchange process, cationic lipid, SM-102 (CAS number 2089251-47-6, 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester) was added to the dispersion (4.85 mg). After an aging time of 5 minutes, POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine, 1.1 mg) and cholesterol (chol, 2.2 mg) were added. After an additional aging time of 5 minutes, PEG-DMG (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, 1.5 mg) was added. All these components were added in ethanol. They were in a molar ratio of 47:10:39:4 (SM-102:POPC:cholesterol:PEG-DMG) relative to each other. SM-102 was added at approximately 3 charge equivalents relative to the RNA acid groups. The DCM solution was concentrated approximately 4-fold by rotary evaporation to 1-1.5 mL. Acetonitrile (8 mL) was then slowly added to the vial. The dispersion was concentrated to 1-1.5 mL at 70 Torr with a heat bath set at 35 °C. Acetonitrile (8 mL) was then added to the vial. The dispersion was concentrated to 1-1.5 mL at 65 Torr with a heat bath set at 35 °C. Acetonitrile (8 mL) was then added to the vial. The dispersion was concentrated to 1 mL at 60 Torr with a heat bath set at 35 °C.

[0321] The inverse nanocarrier dispersion in acetonitrile was then mixed with an equal volume of deionized water in a CIJ mixer, and an additional 0.5x collection bath of phosphate-buffered saline (PBS) was used to reduce the MeCN volume content to less than 10% by volume. This process produced PEG-coated nanocarriers.

[0322] As a control, standard lipid nanoparticles (LNPs) were prepared with a charge ratio of 6:1 N:P (nitrogen:phosphorus) and a lipid composition of 50:10:38.5:1.5 (SM-102:DSPC:Chol:PEG-DMG) molar ratio. Ethanol streams containing these lipids at 15 mM were rapidly mixed in a MIVM with three citrate streams (pH 4, 50 mM) containing the mRNA. The LNPs were then dialyzed overnight against 0.5x PBS at 4°C. Samples were then concentrated to the desired range (400 μg / mL) and flash frozen in 10 wt% trehalose. They were stored at -80°C until thawed for use. These formulations are summarized in Table 20.

[0323] The particle size for each of the examples described herein was characterized by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern, Worcestershire, UK) at 25°C by diluting each sample 10 times in water. The particle size distribution was determined from the CONTIN analysis implemented by the Zetasizer software. The polydispersity index (PDI) is obtained from the Taylor series expansion of the autocorrelation function implemented by the Zetasizer software. The ratio of the second moment to the first moment is defined as the PDI. The zeta potential was measured in 0.1x PBS using a Zetasizer Nano ZS.

[0324] Testing of the formulations for transfection of 293T cells (an adherent human embryonic kidney 293 cell line containing SV40 T antigen) was performed in 6-well plates with mRNA doses of 3 μg, 1.5 μg, 0.75 μg and 0.375 μg per well. After 18 hours, staining for SARS-CoV2 RBD was performed using anti-RBD primary antibody and fluorescent secondary goat anti-human IgG Fcy fragment-APC (Jackson ImmunoResearch; Cat. No. 109-135-098). These results are summarized in Figure 13A and Figure 13B. As a further confirmation of expression, treated cells were fixed with 4% paraformaldehyde before and after staining with fluorescent antibody against SARS-CoV2 RBD. Epifluorescence images confirmed uniform expression across the cell population with significant signal at the cell membrane. [Table 20]

[0325] LNP and iFNP formulations were administered to BALB / c mice at 20 μg of mRNA via two 25 μL injections in each hind thigh muscle. A booster dose was administered on day 21 as well. Serum was collected before the first injection (prebleed), on day 7 (prime) and on day 28 (boost). Antibody titers were analyzed by ELISA (enzyme-linked immunosorbent assay) using standard techniques with plates coated with SARS-CoV2 RBD and detected using a standard chemiluminescence assay. Both iFNP (FIG. 14A) and LNP (FIG. 14B) produced antibody titer responses. In FIG. 14A and FIG. 14B, circles are data points, gray boxes indicate the inner interquartile range, the horizontal line within the gray box is the median, and the horizontal line above the gray box is 1.5 times the inner interquartile range.

[0326] These results demonstrate the suitability of iFNP for the encapsulation of mRNA sequences containing modified uridine residues (pseudouridine) and subsequent expression of said mRNA sequences as proteins in mammalian cells. These results also demonstrate the suitability of iFNP for the manufacture and administration to mammalian subjects of pharmaceutical compositions comprising iFNP nanoparticles encapsulating mRNA sequences encoding infectious disease antigens, and the use of iFNP to formulate vaccines that elicit an immune response against the antigens encoded by the mRNA sequences.

[0327] Example 13: In vitro testing of iFNP formulations with varying parameters mRNA encoding green fluorescent protein (GFP, DasherGFP, Aldevron, Cat. No. 3870) was incubated with Dex-PLGA(503-80) or PAsp in DMSO containing 10% by volume of deionized water. 2kDa -b-PLGA 15kDa RNA in TEA salt form was present at 2 mg / mL (PAsp formulation) or 2.5 mg / mL (Dex formulation). Polymeric stabilizer was present at 5 mg / mL (Dex) or 3 mg / mL (PAsp). This solution (0.5 mL) was rapidly mixed with an anti-solvent stream of DCM in a multi-inlet vortex mixer (MIVM). DCM was placed in the mixer in three separate streams of 0.5 mL volume. CaCl2 at 0.5 charge equivalent ratio to the total acid content of the formulation was included in one DCM stream. Additional DCM, referred to as "iFNP quench", was included in the collection vial to ensure that the DMSO content after collection was less than 10% by volume.

[0328] Prior to the solvent exchange process, cationic lipid SM-102 (CAS number 2089251-47-6) was added to the dispersion. After an aging time of 5 minutes, POPC and cholesterol were added. After an additional aging time of 5 minutes, PEG-DMG was added. All these components were added in ethanol. SM-102 was added at either 2 or 4 charge equivalents relative to the RNA acid groups. The lipid components were maintained at a molar ratio of 47:10:39:4 (SM-102:POPC:Chol:PEG-DMG) relative to each other. The DCM solution was concentrated approximately 4-fold by rotary evaporation to 1-1.5 mL. Acetonitrile (8 mL) was then slowly added to the vial. The dispersion was concentrated to 1-1.5 mL at 70 Torr with a heat bath set at 35 °C. Acetonitrile (8 mL) was then added to the vial. The dispersion was concentrated to 1-1.5 mL at 65 Torr with a heat bath set at 35 °C. Acetonitrile (8 mL) was then added to the vial. The dispersion was concentrated to 1 mL at 60 Torr with the heat bath set at 35° C.

[0329] The inverse nanocarrier dispersions in acetonitrile were then mixed in a CIJ mixer against an equal volume of deionized water. Collection baths containing various buffer compositions were used to reduce the acetonitrile (MeCN) volume content to less than 10% by volume. This process produced PEG-coated nanocarriers. Collection baths were selected from the following: deionized water, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 30 mM), HEPES+NaCl (135 mM), HEPES+trehalose (10 wt%), Tris / acetate (30 mM), Tris / acetate+NaCl (120 mM), or Tris / acetate+trehalose (10 wt%). Nanoparticles with PAsp cores were produced using deionized water for the collection bath. All buffering components were at pH 7.4. After 2–5 min, 1x PBS was added to each dispersion in equal volumes. The formulation was processed into the final buffer as follows: The nanocarriers were concentrated by ultrafiltration on a 50 or 100 kDa molecular weight cutoff filter at 10°C. PBS was added to the filter twice and the concentration step was repeated each time. The concentration was adjusted to 0.15 mg / mL RNA and tested in cell culture. DLS size and zeta potential were measured using standard techniques (Table 21).

[0330] Nanoparticles were added to adherent cell cultures with a starting confluence ranging from 30-50% and incubated for 24 h in standard cell culture conditions. Cells were washed and resuspended in PBS + 500 ng / mL 4',6-diamidino-2-phenylindole (DAPI) and then analyzed using an Attune NxT flow cytometer (ThermoFisher). Singlet cells were gated using SSC-A x SSC-H and live cells were gated from the DAPI negative population. Fluorescence intensity of GFP was captured in the BL-1 channel without compensation. Cell cultures were HEK-293T and HepG2. HepG2 is a hepatocellular carcinoma cell line derived from human liver. Figure 15A shows the green fluorescent protein (GFP) signal measured by flow cytometry after 24 h of culture in HEK-293T cultures. Median values ​​are reported for two mRNA doses in each well. Figure 15B shows green fluorescent protein (GFP) signals measured by flow cytometry after 24 h of incubation in HepG2 cultures. Median values ​​are reported for the two mRNA doses in each well. [Table 21]

[0331] For example, results for PAsp-PLGA NCs with either 2 or 4 equivalents of ionized lipid showed a large dose-dependent expression of GFP, demonstrating cell transfection.

[0332] These results demonstrate additional suitable iFNP nanoparticle compositions and process conditions for producing same. The process and composition produce nanoparticles with appropriate size, polydispersity and surface charge. The nanoparticles have been shown to effectively transfect mammalian cells in culture.

[0333] Example 14: In vitro evaluation of iFNP formulations of firefly luciferase mRNA The mRNA encoding firefly luciferase modified with 5-methoxyuridine (TriLink Biotech, Cat. No. L-7202) was incubated with Dex-PLGA(503-80) or PAsp in DMSO containing 10% by volume of deionized water. 2kDa -b-PLGA 15kDa RNA in TEA salt form was present at 2 mg / mL (PAsp formulations) or 2.5 mg / mL (Dex formulations). Polymeric stabilizer was present at 5 mg / mL (Dex) or 3 mg / mL (PAsp). This solution was rapidly mixed with an anti-solvent stream of DCM in a multi-inlet vortex mixer (MIVM). DCM was placed in the mixer in three separate streams of equal volume. CaCl2 at 0.5 charge equivalent ratio to the total acid content of the formulation was included in one DCM stream. Additional DCM, referred to as "iFNP quench", was included in the collection vial to ensure that the DMSO content after collection was less than 10% by volume.

[0334] Prior to the solvent exchange process, cationic lipid SM-102 (CAS number 2089251-47-6) was added to the dispersion. After an aging time of 5 minutes, POPC and cholesterol were added. After an additional aging time of 5 minutes, PEG-DMG was added. In one sample, PLA-PEG was added instead of PEG-DMG at 27% by weight relative to the total weight of the formulation. All these components were added in ethanol. SM-102 was added at either 2 or 3 charge equivalents relative to the RNA acid groups. The lipid components were maintained at a molar ratio of 47:10:39:4 (SM-102:POPC:Chol:PEG-DMG) relative to each other. The DCM solution was concentrated approximately 4-fold by rotary evaporation. Acetonitrile was then slowly added to the vial. The dispersion was concentrated at 70 Torr with a heat bath set at 35° C. Acetonitrile was then added to the vial. The dispersion was concentrated at 65 Torr with a heat bath set at 35° C. Acetonitrile was then added to the vial. The dispersion was concentrated at 60 Torr with the heat bath set at 35°C.

[0335] The inverse nanocarrier dispersions in acetonitrile were then mixed in a CIJ mixer against an equal volume of deionized water. Collection baths containing various buffer compositions were used to reduce the MeCN volume content to less than 10% by volume. This process produced PEG-coated nanocarriers. Collection baths were selected from the following: deionized water, Tris / acetate (30 mM), or Tris / acetate + NaCl (120 mM). All buffering components were at pH 7.4. After 2-5 min, 1x PBS was added to each dispersion in equal volumes. The formulations were processed into the final buffer as follows: Nanocarriers were concentrated by ultrafiltration at 10°C over 50 or 100 kDa molecular weight cutoff filters. PBS was added to the filters three times, and the concentration step was repeated each time. Sucrose was added at 5 wt%, the samples were filtered through 0.45 μm filters, and the concentration was adjusted to 0.25 mg / mL RNA. Samples were then flash frozen using liquid nitrogen until thawed for use. DLS size and zeta potential were measured using standard techniques throughout handling (Table 22).

[0336] As a control, two lipid nanoparticles were prepared with a 6:1 N:P ratio and lipid compositions of 50:10:38.5:1.5 (SM-102:DSPC:Chol:PEG-DMG) molar ratios for LNP1 and 50:10:39.6:0.4 (SM-102:DSPC:Chol:PEG-DMG) molar ratios for LNP2. The ethanol streams containing these lipids were rapidly mixed in a MIVM with the three citrate streams (pH 4, 50 mM) containing the mRNA. The LNPs were then dialyzed overnight against 1x PBS at 4°C. [Table 22]

[0337] Nanoparticles were added to adherent cell cultures with a starting confluence ranging from 30-50% and incubated for 12-24 hours in standard cell culture conditions with serial dilutions of nanoparticles compared to a PBS-only control. At the end point, cells were lysed and luminescence was quantified using the Pierce Firefly Luciferase Glow Assay Kit according to the manufacturer's instructions. Samples were tested for cell transfection immediately after thawing. Figure 16A shows the firefly luciferase signal after 24 hours of incubation of 293T cells with nanoparticles. Dose values ​​are for mRNA in culture medium and detection used standard manufacturer recommended values ​​as described herein. Figure 16B shows the firefly luciferase signal after 24 hours of incubation of HepG2 cells with nanoparticles. Dose values ​​are for mRNA in culture medium and detection used standard manufacturer recommended values ​​as described herein.

[0338] iFNP NCs induced a large and dose-dependent expression of encapsulated mRNA, demonstrating cell transfection.

[0339] These results demonstrate that iFNPs are suitable for encapsulation of mRNA sequences with 5-methoxyuridine modifications, Cap1 structures and polyadenylation. iFNP nanoparticles are suitable for expression of mRNA coding sequences in mammalian cell culture and show a well-defined dose-response curve. These results demonstrate expression in cell culture using iFNP nanoparticles with a stably attached PLA-PEG coating, unlike PEG-DMG, which is known to detach and expose the underlying lipid surface.

[0340] Example 15: In vivo screening of luciferase mRNA formulations by whole animal imaging The following samples from Example 14 (Table 22) were tested for expression kinetics and biodistribution in BALB / c mice (female 6-8 weeks): LNP1, fL2, fL3, fL5, fL6, fL7. Aliquots were prepared for intravenous (IV) injection (tail vein) and intramuscular (IM) injection (thigh) at 125 μg / mL and 250 μg / mL of RNA, respectively. Each mouse was administered 12.5 μg of mRNA. Bioluminescence imaging was performed 9, 24, 48, 96, and 144 hours after injection. A fresh stock solution of luciferin was prepared at 15 mg / ml in Dulbecco's phosphate buffered saline and filter sterilized through a 0.22 μm filter. Mice were injected with a dose of 6 mg (compound) / kg (body weight) of luciferin via intraperitoneal injection. Mice were anesthetized with isoflurane and imaged 15 min after luciferin injection for exposure times of 60 s (unless saturated) on the ventral and dorsal sides, respectively.

[0341] Whole animal luciferase bioluminescence (total luminescence) for time points is summarized in Figures 17-20. Representative imaging results can be seen in Figures 21 and 22 showing the differences in biodistribution of expression for the formulations.

[0342] These results demonstrate the suitability of iFNP to encapsulate mRNA sequences into nanoparticles and process them into pharmaceutical compositions. These results also demonstrate that the pharmaceutical compositions can be administered to mammalian subjects by various routes to achieve expression of the desired encoded enzyme (in this case, luciferase), which can be used to treat loss-of-function diseases such as inborn errors of metabolism. The results show that iFNP formulations can produce liver expression or spleen expression, or both, after IV administration. The results also show that iFNP formulations can produce local muscle / lymphatic expression after IM injection.

[0343] Example 16: Effect of RNA salt form on carrier RNA formulation Carrier RNA (size >200 base pairs with unmodified bases, Qiagen Carrier RNA, Cat# 1017647) was prepared in salt forms other than TEA. Guanidine and choline salt forms were prepared by adding 1.1 molar equivalents of guanidine*HCl or choline*HCl to carrier RNA (RNA in sodium salt form relative to the phosphate group) in water. The solution was incubated at 37°C for 10 minutes and then lyophilized. Diphenhydramine (Benadryl) salt forms were prepared by adding 10 equivalents of diphenhydramine*HCl to carrier RNA (RNA in sodium salt form relative to the phosphate group) in water, resulting in precipitation of the new salt form of RNA. The mixture was incubated at 37°C for 10 minutes and the RNA precipitate was removed from the dispersion by centrifugation at 20k rcf for 10 minutes. The supernatant was removed from the RNA pellet and fresh deionized water was added to the RNA, which was then lyophilized.

[0344] Carrier RNA in guanidine, choline or benadryl salt form was mixed with Dex-PLGA(503-80) in DMSO containing 10% by volume of deionized water. RNA was present at 1.6 mg / mL (based on free acid) and polymeric stabilizer was present at 4 mg / mL. This solution (0.5 mL) was rapidly mixed with an anti-solvent stream of DCM in a multi-inlet vortex mixer (MIVM). DCM was placed in the mixer in three separate streams of 0.5 mL volume each. CaCl2 at 0.5 charge equivalent ratio to the total acid content of the formulation was included in one DCM stream. Additional DCM, referred to as "iFNP quench", was included in the collection vial to ensure that the DMSO content after collection was less than 10% by volume.

[0345] Prior to the solvent exchange process, the cationic lipid SM-102 was added to the dispersion. After an aging time of 5 minutes, POPC and cholesterol were added. After an additional aging time of 5 minutes, PEG-DMG was added. All these components were added in ethanol. SM-102 was added at 2 charge equivalents relative to the RNA acid groups. The lipid components were maintained at a molar ratio of 47:10:39:4 (SM-102:POPC:Chol:PEG-DMG) relative to each other. The DCM solution was concentrated approximately 4-fold by rotary evaporation to 1-1.5 mL. Acetonitrile (8 mL) was then slowly added to the vial. The dispersion was concentrated to 1-1.5 mL at 70 Torr with a heat bath set at 35 °C. Acetonitrile (8 mL) was then added to the vial. The dispersion was concentrated to 1-1.5 mL at 65 Torr with a heat bath set at 35 °C. Acetonitrile (8 mL) was then added to the vial. The dispersion was concentrated to 1 mL at 60 Torr with the heat bath set at 35 °C.

[0346] The inverse nanocarrier dispersion in acetonitrile was then mixed with an equal volume of deionized water in a CIJ mixer. The MeCN volume content was reduced to less than 10% by volume using a collection bath containing 30 mM HEPES and 135 mM NaCl at pH 7.4. This process produced PEG-coated nanocarriers. DLS size and zeta potential were measured using standard techniques (Table 23). [Table 23]

[0347] These results demonstrate the formation of alternative RNA salt forms and their use in the iFNP process to produce nanoparticles with appropriate size, PDI values ​​indicating low polydispersity, and near-neutral zeta potential values.

[0348] Example 17: Effect of RNA salt forms on GFP mRNA formulations The mRNA encoding GFP (DasherGFP, Aldevron) was prepared in a salt form other than TEA. The guanidine salt form was prepared by adding 1.1 molar equivalents of guanidine*HCl to the mRNA in water (mRNA in sodium salt form relative to the phosphate group). This solution was incubated at 37°C for 10 minutes and then lyophilized. The diphenhydramine (Benadryl) salt form was prepared by adding 10 equivalents of diphenhydramine*HCl to the mRNA in water (mRNA in sodium salt form relative to the phosphate group), resulting in the precipitation of the new salt form of the mRNA. The mixture was incubated at 37°C for 10 minutes and the mRNA precipitate was removed from the dispersion by centrifugation at 20k rcf for 10 minutes. The supernatant was removed from the mRNA pellet and fresh deionized water was added to the mRNA and the material was then lyophilized.

[0349] The mRNA in Benadryl or guanidine salt form was mixed with Dex-PLGA(503-80) in DMSO containing 10% by volume of deionized water. The RNA was present at 1.6 mg / mL (based on free acid) and the polymeric stabilizer was present at 4 mg / mL. This solution (0.5 mL) was rapidly mixed with an anti-solvent stream of DCM in a multi-inlet vortex mixer (MIVM). DCM was placed in the mixer in three separate streams of 0.5 mL volume each. CaCl2 at 0.5 charge equivalent ratio to the total acid content of the formulation was included in one DCM stream. Additional DCM, referred to as "iFNP quench", was included in the collection vial to ensure that the DMSO content after collection was less than 10% by volume.

[0350] Cationic lipid SM-102 was added to the dispersion prior to the solvent exchange process. After an aging period of 5 minutes, POPC and cholesterol were added. For some formulations, PEG-DMG was added after an additional aging period of 5 minutes. All these components were added in ethanol. SM-102 was added at 2 charge equivalents each relative to the RNA acid groups. The lipid components were maintained at a molar ratio of 47:10:39:4 (SM-102:POPC:Chol:PEG-DMG) relative to each other. For formulations without PEG-DMG, poly(caprolactone) 5kDa -b-PEG 5kDa (PCL-PEG) was added as a coating material such that the final coated particles were 28% by mass of PCL-PEG. The DCM solution was concentrated approximately 4-fold by rotary evaporation to 1-1.5 mL. Acetonitrile (8 mL) was then slowly added to the vial. The dispersion was concentrated to 1-1.5 mL at 70 Torr with a heat bath set at 35 °C. Acetonitrile (8 mL) was then added to the vial. The dispersion was concentrated to 1-1.5 mL at 65 Torr with a heat bath set at 35 °C. Acetonitrile (8 mL) was then added to the vial. The dispersion was concentrated to 1 mL at 60 Torr with a heat bath set at 35 °C.

[0351] The inverse nanocarrier dispersions in acetonitrile were then mixed in a CIJ mixer against an equal volume of deionized water. The acetonitrile volume content was reduced to less than 10% by volume using a collection bath containing 30 mM Tris / acetate at pH 7.4 and 120 mM NaCl. This process produced PEG-coated nanocarriers. After 2-5 min, 1x PBS was added to each dispersion in an equal volume. The formulations were processed into the final buffer as follows: For the PEG-DMG-coated formulations, the nanocarriers were concentrated by ultrafiltration at 10°C over a 50 or 100 kDa molecular weight cut-off filter. PBS was added to the filter twice and the concentration step was repeated each time. For the PCL-PEG-coated formulations, the nanocarriers were dialyzed in excess PBS at 4°C for 12 hours. The PCL-PEG formulations were then concentrated by ultrafiltration once at 10°C over a 100 kDa molecular weight cut-off filter. The concentration of all formulations was adjusted to 0.15 mg / mL RNA and tested in cell culture. DLS size and zeta potential were measured using standard techniques (Table 24).

[0352] Nanoparticles were added to adherent cell cultures with a starting confluence ranging from 30-50% and incubated for 24 h in standard cell culture conditions. Cells were washed and resuspended in PBS + 500 ng / mL 4',6-diamidino-2-phenylindole (DAPI) and then analyzed using an Attune NxT flow cytometer (ThermoFisher). Singlet cells were gated using SSC-A x SSC-H and live cells were gated from the DAPI negative population. Fluorescence intensity of GFP was captured in the BL-1 channel without compensation. Cell cultures were HEK-293T and HepG2 (Figure 23). Figure 23A shows the GFP signal measured by flow cytometry after 24 h incubation in 293T culture. Median values ​​are reported for two mRNA doses for each well. Figure 23B shows the GFP signal measured by flow cytometry after 24 h incubation in HepG2 culture. Median values ​​are reported for duplicate mRNA doses for each well. [Table 24]

[0353] These results demonstrate that alternative salt forms of RNA retain the ability to transform mammalian cells after encapsulation in the iFNP process. These results demonstrate dose-dependent transfection of mammalian cells by iFNP nanocarriers with stably attached PCL-PEG surface coatings for both RNA salt forms tested (diphenhydramine and guanidine salt forms).

[0354] Example 18: Preparation of TEA and guanidine salts of plasmids To generate the TEA salt, the plasmid in water (pEPito-EF1-EGFPLuc from PlasmidFactory) was concentrated in a centrifugal filter. TEA carbonate buffer (0.5M) was added to the plasmid and concentrated. This process was repeated two more times. The plasmid was then washed five times with deionized water in a centrifugal filter to remove excess TEA, and then concentrated. DMSO was added to the plasmid to a final solution of 65% DMSO and 35% water by volume. The plasmid concentration was quantified by UV-Vis spectroscopy, and this stock solution was used for subsequent iFNP formulations.

[0355] To generate the guanine salt, the plasmid in water (pEPito-EF1-EGFPLuc from PlasmidFactory) was concentrated as much as possible with a centrifugal filter. Guanidine*HCl (20 mg / mL) in water was added to the plasmid and incubated at 37°C for 10 min. DMSO was added to the plasmid to a final solution of 65% DMSO and 35% water by volume. Plasmid concentration was quantified by UV-Vis spectroscopy, and this stock solution was used for subsequent iFNP formulations.

[0356] Example 19: iFNP formulation of plasmid DNA stabilized with Dex-PLGA The TEA salt form of the plasmid (pEPito-EF1-EGFPLuc from PlasmidFactory) was mixed with Dex-PLGA(503-80) in DMSO containing 10% by volume of deionized water. DNA-TEA was present at 2 mg / mL and polymeric stabilizer was present at 4 mg / mL. This solution (0.5 mL) was rapidly mixed with an anti-solvent stream of DCM in a multi-inlet vortex mixer (MIVM). DCM was placed in the mixer in three separate streams of 0.5 mL volume each. CaCl2 at 0.5 charge equivalent ratio to the total acid content of the formulation was included in one DCM stream. Additional DCM, referred to as "iFNP quench", was included in the collection vial to ensure that the DMSO content after collection was less than 10% by volume.

[0357] Prior to the solvent exchange process, cationic lipid, SM-102 (CAS number 2089251-47-6) or D-Lin-MC3-DMA (MC3, [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl]-4-(dimethylamino)butanoate), was added to the dispersion at either 2 or 3 equivalents relative to the phosphate groups in ...

Claims

1. A nanoparticle, the nanoparticle comprising: a core comprising a highly polar region of a first stabilizing amphiphilic copolymer and at least one water-soluble agent; a shell comprising a less polar region of the first stabilizing amphiphilic copolymer, at least one lipid, and a second stabilizing amphiphilic agent; The shell surrounds the core of the nanoparticle.

2. the shell includes an inner surface and an outer surface; the inner surface of the shell is in contact with the core; the second stabilizing amphiphilic agent comprises a more polar region and a less polar region; The nanoparticle of claim 1 , wherein the more polar regions of the second stabilizing amphiphilic agent are at the outer surface of the shell.

3. The nanoparticle of claim 2 , wherein the shell comprises the less polar region of the second stabilizing amphiphilic agent.

4. The nanoparticle of claim 3 , wherein the at least one water-soluble agent is not present on the exterior surface of the shell.

5. Including Corona, the corona surrounds the shell; The nanoparticle of claim 3 , wherein the corona comprises the more polar region of the second stabilizing amphiphilic agent.

6. the at least one water-soluble agent is not in contact with the more polar regions of the second stabilizing amphiphilic agent; The nanoparticle of claim 5 , wherein the corona is free of the at least one water-soluble agent.

7. 2. The nanoparticle of claim 1, wherein the at least one water-soluble agent is selected from the group consisting of nucleic acids, polynucleic acids, ribonucleic acids (RNA), messenger ribonucleic acids (mRNA), self-amplifying messenger ribonucleic acids (saRNA), small interfering ribonucleic acids (siRNA), micro ribonucleic acids (microRNA), circular ribonucleic acids (circular RNA), transfer ribonucleic acids (tRNA), small guide ribonucleic acids (sgRNA), deoxyribonucleic acids (DNA), antisense oligonucleotides (ASO), plasmids, episomes, and combinations.

8. The nanoparticle of claim 1 , wherein the at least one water-soluble agent is selected from the group consisting of DNA, mRNA and saRNA.

9. 2. The nanoparticle of claim 1, wherein the at least one water-soluble agent comprises an mRNA encoding the SARS-CoV-2 spike protein receptor binding domain (RBD).

10. 2. The nanoparticle of claim 1, wherein the at least one water-soluble agent comprises an mRNA encoding a SARS-CoV-2 spike protein receptor binding domain (RBD) with pseudouridine (the C5-glycosidic isomer of uridine, 5-ribosyluracil) modifications.

11. 2. The nanoparticle of claim 1, wherein the first stabilizing amphiphilic copolymer is selected from the group consisting of poly(aspartic acid)-block-poly(lactic acid) (PAsp-b-PLA), poly(aspartic acid)-block-poly(lactic acid-co-glycolic acid) (PAsp-b-PLGA), dextran-poly(lactic acid) (Dex-PLA), or dextran-poly(lactic acid-co-glycolic acid) (Dex-PLGA).

12. the dextran and the poly(aspartic acid) each have a molecular weight in the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da; 12. The nanoparticle of claim 11, wherein the poly(lactic acid) and the poly(lactic-co-glycolic acid) each have a molecular weight in the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da.

13. 2. The nanoparticle of claim 1, wherein the first stabilizing amphiphilic copolymer is selected from the group consisting of poly(glutamic acid)-block-poly(lactic acid) (Pglu-b-PLA), poly(glutamic acid)-block-poly(lactic acid-co-glycolic acid) (Pglu-b-PLGA), poly(glutamic acid)-block-poly(caprolactone) (Pglu-b-PCL), poly(aspartic acid)-block-poly(caprolactone) (PAsp-b-PCL), or dextran-poly(caprolactone) (Dex-PCL).

14. the dextran, the poly(aspartic acid), and the poly(glutamic acid) each have a molecular weight in the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da; 14. The nanoparticle of claim 13, wherein the poly(lactic acid), the poly(lactic-co-glycolic acid), and the poly(caprolactone) each have a molecular weight in the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da.

15. 2. The nanoparticle of claim 1, wherein the at least one lipid is selected from the group consisting of phospholipids, cationic lipids, anionic lipids, sterols, monoglycerides, triglycerides, fatty acid methyl esters, fatty acid ethyl esters, and combinations.

16. The at least one lipid may be 1,2-distearoyl-s-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3 ... phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (Ochems PC), 1-hexadecyl-sn-glycero-3-phosphocholine (Cl6 lysoPC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarrachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2. The nanoparticle of claim 1, wherein the nanoparticle is selected from the group consisting of 2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and combinations. ,

17. The nanoparticle of claim 1, wherein the at least one lipid comprises 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC).

18. The nanoparticle of claim 1 , wherein the at least one type of lipid comprises a cationic lipid and / or an ionizable cationic lipid.

19. The nanoparticle of claim 1 , wherein the at least one lipid is a blend of a cationic lipid, a phospholipid, and cholesterol or a sterol.

20. 2. The nanoparticle of claim 1, wherein the second stabilizing agent is selected from the group consisting of 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-DMG), pegylated distearoyl-phosphatidyl-ethanolamine (PEG-DSPE), polyethylene glycol-block-poly(lactic acid) (PEG-b-PLA), polyethylene glycol-block-poly(lactic-co-glycolic acid) (PEG-b-PLGA), and polyethylene glycol-block-poly(caprolactone) (PEG-b-PCL).

21. 21. The nanoparticle of claim 20, wherein the poly(lactic acid), the poly(lactic-co-glycolic acid), and the poly(caprolactone) each have a molecular weight in the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da.

22. The nanoparticle of claim 1 , further comprising at least one hydrophobic polymer.

23. 23. The nanoparticle of claim 22, wherein the at least one hydrophobic polymer is selected from the group consisting of poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), and combinations.

24. 24. The nanoparticle of claim 23, wherein the poly(lactic acid), the poly(lactic-co-glycolic acid), and the poly(caprolactone) each have a molecular weight in the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da.

25. the at least one water-soluble agent comprises an mRNA encoding the SARS-CoV-2 spike protein receptor binding domain (RBD) and / or an mRNA encoding the SARS-CoV-2 spike protein receptor binding domain (RBD) with pseudouridine (the C5-glycosidic isomer of uridine, 5-ribosyluracil) modifications; the first stabilizing amphiphilic copolymer comprises dextran-poly(lactic acid) (Dex-PLA), dextran-poly(lactic-co-glycolic acid) (Dex-PLGA) and / or dextran-poly(caprolactone) (Dex-PCL); the at least one lipid comprises a cationic lipid; The nanoparticle of claim 1 , wherein the second stabilizing amphiphilic agent comprises a polyethylene glycol (PEG) copolymer and / or a polyethylene glycol (PEG) lipid.

26. the at least one water-soluble agent comprises an mRNA encoding a SARS-CoV-2 spike protein receptor binding domain (RBD) with pseudouridine (the C5-glycosidic isomer of uridine, 5-ribosyluracil) modifications; the first stabilizing amphiphilic copolymer comprises dextran-poly(lactic-co-glycolic acid) (Dex-PLGA); the at least one lipid comprises 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM-102), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), and cholesterol; 26. The nanoparticle of claim 25, wherein the second stabilizing amphiphilic agent comprises 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol-2000 (PEG-DMG).

27. A pharmaceutical composition comprising a therapeutically effective amount of the nanoparticles of claim 1 and a pharma- ceutically acceptable carrier or diluent.

28. 13. A method of administering to a subject, comprising administering to the subject a therapeutically effective amount of the nanoparticles of claim 1.

29. A method of administering to a cell, the method comprising contacting the cell with the nanoparticle of claim 1.

30. 30. The method of claim 29, wherein the cell is selected from the group consisting of a mammalian cell and a human cell.

31. 30. The method of claim 29, wherein said administering to said cell is performed in vitro.

32. 13. A method for preventing or treating an infectious disease, comprising administering to a subject suffering from said infectious disease a therapeutically effective amount of the nanoparticles of claim 1, said at least one water-soluble agent induces the production of an antigen associated with said infection by cells of said subject; The method, wherein the antigen induces an immune response by the subject against the infectious disease.

33. 10. The nanoparticle of claim 1 for use in the prevention or treatment of an infectious disease.

34. 13. Use of the nanoparticles according to claim 1 in the manufacture of a medicament for the prevention or treatment of an infectious disease.

35. 33. The method of claim 32, wherein the infectious disease is a virus.

36. The infectious diseases include adenovirus, herpes simplex type 1, herpes simplex type 2, encephalitis virus, papilloma virus, varicella zoster virus, Epstein-Barr virus, human cytomegalovirus, human herpes virus type 8, human papilloma virus, BK virus, JC virus, smallpox, poliovirus, hepatitis B virus, human bocavirus, parvovirus B19, human astrovirus, Norwalk virus, coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, rubella virus, hepatitis E virus, human immunodeficiency virus, 33. The method of claim 32, wherein the virus is selected from the group consisting of HIV, Guanarito virus, Junin virus, Lassa virus, Machupo virus, Sabia virus, Crimean-Congo hemorrhagic fever virus, Ebola virus, Marburg virus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus, Hendra virus, Nipah virus, Hepatitis D virus, Rotavirus, Orbivirus, Coltivirus, Banna virus, Human enterovirus, Hantavirus, West Nile virus, Japanese encephalitis virus, Swine vesicular rash virus, and Eastern equine encephalitis.

37. 33. The method of claim 32, wherein the infectious disease is selected from the group consisting of a virus, coronavirus, Middle East Respiratory Syndrome Coronavirus, Severe Acute Respiratory Syndrome Virus, SARS-CoV-2, rabies virus, influenza, Zika virus, cytomegalovirus, and Chikungunya virus.

38. 13. A method for preventing or treating cancer, comprising administering to a subject suffering from said cancer a therapeutically effective amount of the nanoparticle of claim 1, the at least one water-soluble agent induces production by cells of the subject of a tumor antigen associated with cancerous cells of the cancer; The method, wherein the antigen induces an immune response by the subject against the cancer.

39. The nanoparticle of claim 1 for use in the prevention or treatment of cancer.

40. 13. Use of the nanoparticles according to claim 1 in the manufacture of a medicament for the prevention or treatment of cancer.

41. A method for preventing or treating a loss-of-function disease, comprising administering to a subject suffering from said loss-of-function disease a therapeutically effective amount of the nanoparticle of claim 1, The method, wherein the at least one water-soluble agent induces the production of a protein that restores the lost function.

42. The nanoparticle of claim 1 for use in the prevention or treatment of a loss-of-function disease.

43. 13. Use of the nanoparticles according to claim 1 in the manufacture of a medicament for the prevention or treatment of a loss-of-function disease.

44. 42. The method of claim 41, wherein the loss-of-function disease is selected from the group consisting of urea cycle disorders, N-acetylglutamate synthase (NAGS) deficiency, carbamoyl phosphate synthase (CPS) deficiency, ornithine transcarbamoylase (OTC) deficiency, citrullinemia type 1 (CTLN1), citrullinemia type 2 (CTLN2), argininosuccinic aciduria, argininemia, or hyperornithinemic-hyperammonemia-homocitrullinuria (HHH) syndrome.

45. 42. The method of claim 41, wherein the loss-of-function disease is selected from the group consisting of a polygenic disease, a monogenic disease, a polygenic liver disorder, and a monogenic liver disorder.

46. A method for preventing or treating a disease associated with a premature stop codon, comprising administering to a subject suffering from the disease associated with the premature stop codon a therapeutically effective amount of the nanoparticle of claim 1, the at least one water-soluble agent comprises a tRNA; The method, wherein the tRNA allows translation past a premature stop codon.

47. The nanoparticle of claim 1 for use in the prevention or treatment of a disease associated with a premature stop codon.

48. 2. Use of the nanoparticles according to claim 1 in the manufacture of a medicament for the prevention or treatment of a disease associated with a premature stop codon.

49. 47. The method of claim 46, wherein the disease associated with a premature stop codon is selected from the group consisting of β-thalassemia and Charcot-Marie-Tooth disease.

50. A gene editing method, comprising: contacting a nanoparticle according to claim 1 with a cell, thereby inserting a DNA sequence into the genome of said cell; The method, wherein the at least one water-soluble agent comprises an endonuclease and / or an mRNA encoding an endonuclease, a small guide RNA (sgRNA), and the DNA sequence.

51. 51. The method of claim 50, wherein the nanoparticles are contacted with the cells in vitro.

52. 51. The method of claim 50, wherein the endonuclease is selected from the group consisting of a Cas protein, Cas9, and a TALEN.

53. 10. The nanoparticle of claim 1 for use in gene editing.

54. 2. Use of the nanoparticles described in claim 1 in the manufacture of a medicament for gene editing.

55. 1. A method for producing nanoparticles, comprising: dissolving at least one water soluble agent in a first polar process solvent to form a water soluble agent solution; dissolving a first stabilizing amphiphilic copolymer in a second polar process solvent to form a copolymer solution; sequentially mixing the water-soluble agent solution and the copolymer solution with a poor solvent to form a mixed solution, and nanoparticles are aggregated from the mixed solution to form an inverse nanoparticle dispersion; adding at least one lipid to the inverse nanoparticle dispersion; adding a second stabilizing amphiphilic agent to the inverse nanoparticle dispersion; combining the inverse nanoparticle dispersion with a modified solvent to form a modified dispersion; and successively mixing the modified dispersion with an aqueous solvent to form the nanoparticles; the first stabilizing amphiphilic copolymer comprises at least one region of high polarity and at least one region of low polarity; the second polar process solvent may be the same as or different from the first polar process solvent; the anti-solvent is less polar than the first polar process solvent; the anti-solvent is less polar than the second polar process solvent; The nanoparticles comprise a core and a shell, the core comprises the more polar regions of the first stabilizing amphiphilic copolymer and the at least one water soluble agent; The method, wherein the shell comprises the less polar region of the first stabilizing amphiphilic copolymer.

56. 56. The method of claim 55, wherein the aqueous solvent is an aqueous buffer.

57. 56. The method of claim 55, wherein the second polar process solvent is the same as the first polar process solvent.

58. 56. The method of claim 55, wherein the water-soluble agent solution and the copolymer solution are a single mixed solution.

59. 56. The method of claim 55, wherein the second polar process solvent is different from the first polar process solvent.

60. the shell includes an inner surface and an outer surface; the inner surface of the shell is in contact with the core; the second stabilizing amphiphilic agent comprises a more polar region and a less polar region; 56. The method of claim 55, wherein the more polar regions of the second stabilizing amphiphilic agent are on the exterior surface of the shell.

61. 61. The method of claim 60, wherein the shell comprises the less polar region of the second stabilizing amphiphilic agent.

62. 62. The method of claim 61, wherein the at least one water soluble agent is not present on the exterior surface of the shell.

63. a corona surrounding the shell; 62. The method of claim 61 , wherein the corona comprises the more polar region of the second stabilizing amphiphilic agent.

64. the at least one water-soluble agent is not in contact with the more polar regions of the second stabilizing amphiphilic agent; 64. The method of claim 63, wherein the corona is free of the at least one water soluble agent.

65. 56. The method of claim 55, wherein the modification solvent is acetonitrile.

66. 56. The method of claim 55, wherein the at least one water-soluble agent is selected from the group consisting of nucleic acids, polynucleic acids, ribonucleic acids (RNA), messenger ribonucleic acids (mRNA), self-amplifying messenger ribonucleic acids (saRNA), small interfering ribonucleic acids (siRNA), micro ribonucleic acids (microRNA), circular ribonucleic acids (circular RNA), transfer ribonucleic acids (tRNA), small guide ribonucleic acids (sgRNA), deoxyribonucleic acids (DNA), antisense oligonucleotides (ASO), plasmids, episomes, and combinations.

67. 56. The method of claim 55, wherein the at least one water-soluble agent comprises an mRNA encoding the SARS-CoV-2 spike protein receptor binding domain (RBD).

68. 56. The method of claim 55, wherein the at least one water-soluble agent comprises an mRNA encoding a SARS-CoV-2 spike protein receptor binding domain (RBD) with pseudouridine (the C5-glycosidic isomer of uridine, 5-ribosyluracil) modifications.

69. The first stabilizing amphiphilic copolymer may be selected from the group consisting of poly(aspartic acid)-block-poly(lactic acid) (PAsp-b-PLA), poly(aspartic acid)-block-poly(lactic acid-co-glycolic acid) (PAsp-b-PLGA), dextran-poly(lactic acid) (Dex-PLA), dextran-poly(lactic acid-co-glycolic acid) (Dex-PLGA), poly(glutamic acid)-block-poly(lactic acid) (Pglu-b 56. The method of claim 55, wherein the polymer is selected from the group consisting of poly(glutamic acid)-block-poly(lactic acid-co-glycolic acid) (Pglu-b-PLGA), poly(glutamic acid)-block-poly(caprolactone) (Pglu-b-PCL), poly(aspartic acid)-block-poly(caprolactone) (PAsp-b-PCL), and dextran-poly(caprolactone) (Dex-PCL).

70. the dextran, the poly(aspartic acid), and the poly(glutamic acid) each have a molecular weight in the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da; 70. The method of claim 69, wherein the poly(lactic acid), the poly(lactic-co-glycolic acid), and the poly(caprolactone) each have a molecular weight in the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da.

71. 56. The method of claim 55, wherein the first stabilizing amphiphilic copolymer is selected from the group consisting of poly(glutamic acid)-block-poly(lactic acid) (Pglu-b-PLA) and poly(glutamic acid)-block-poly(lactic acid-co-glycolic acid) (Pglu-b-PLGA).

72. the poly(glutamic acid) has a molecular weight in the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da; 72. The method of claim 71, wherein the poly(lactic acid) and the poly(lactic-co-glycolic acid) each have a molecular weight in the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da.

73. 56. The method of claim 55, wherein the at least one lipid is selected from the group consisting of phospholipids, cationic lipids, anionic lipids, sterols, monoglycerides, triglycerides, fatty acid methyl esters, fatty acid ethyl esters, and combinations.

74. 56. The method of claim 55, wherein the at least one lipid comprises a cationic lipid and / or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC).

75. 56. The method of claim 55, wherein the at least one lipid is a blend of a cationic lipid, a phospholipid, and cholesterol or a sterol.

76. 56. The method of claim 55, wherein the second stabilizing amphiphilic agent is selected from the group consisting of pegylated 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-DMG), pegylated distearoyl-phosphatidyl-ethanolamine (PEG-DSPE), polyethylene glycol-block-poly(lactic acid) (PEG-b-PLA), polyethylene glycol-block-poly(lactic-co-glycolic acid) (PEG-b-PLGA), and polyethylene glycol-block-poly(caprolactone) (PEG-b-PCL).

77. 77. The method of claim 76, wherein the poly(lactic acid), the poly(lactic-co-glycolic acid), and the poly(caprolactone) each have a molecular weight in the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da.

78. the at least one water-soluble agent comprises an mRNA encoding the SARS-CoV-2 spike protein receptor binding domain (RBD) and / or an mRNA encoding the SARS-CoV-2 spike protein receptor binding domain (RBD) with pseudouridine (the C5-glycosidic isomer of uridine, 5-ribosyluracil) modifications; the first stabilizing amphiphilic copolymer comprises dextran-poly(lactic acid) (Dex-PLA), dextran-poly(lactic-co-glycolic acid) (Dex-PLGA) and / or dextran-poly(caprolactone) (Dex-PCL); the at least one lipid comprises a cationic lipid; 56. The method of claim 55, wherein the second stabilizing amphiphilic agent comprises a polyethylene glycol (PEG) copolymer and / or a polyethylene glycol (PEG) lipid.

79. the at least one water-soluble agent comprises an mRNA encoding a SARS-CoV-2 spike protein receptor binding domain (RBD) with pseudouridine (the C5-glycosidic isomer of uridine, 5-ribosyluracil) modifications; the first stabilizing amphiphilic copolymer comprises dextran-poly(lactic-co-glycolic acid) (Dex-PLGA); the at least one lipid comprises 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM-102), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), and cholesterol; 79. The method of claim 78, wherein the second stabilizing amphiphilic agent comprises 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG).

80. 2. The nanoparticle of claim 1, wherein the at least one water-soluble agent comprises a ribonucleic acid (RNA) in which at least one uridine has been replaced with a modified uridine.

81. at least 50% of the uridines in the ribonucleic acid (RNA) are each replaced with a modified uridine; 81. The nanoparticle of claim 80, wherein the modified uridines can be the same or different.

82. each of said uridines of said ribonucleic acid (RNA) is replaced with a modified uridine; 81. The nanoparticle of claim 80, wherein the modified uridines can be the same or different.

83. 81. The nanoparticle of claim 80, wherein each modified uridine is independently selected from the group consisting of pseudouridine, 5-methoxyuridine, and N1-methylpseudouridine.

84. 84. The nanoparticle of claim 83, wherein each modified uridine is independently selected from the group consisting of pseudouridine and 5-methoxyuridine.

85. 81. The nanoparticle of claim 80, wherein the ribonucleic acid (RNA) is selected from the group consisting of messenger ribonucleic acid (mRNA), self-amplifying messenger ribonucleic acid (saRNA), small interfering ribonucleic acid (siRNA), micro ribonucleic acid (microRNA), circular ribonucleic acid (circular RNA), transfer ribonucleic acid (tRNA), small guide ribonucleic acid (sgRNA), and antisense oligonucleotide ribonucleic acid (ASO RNA).

86. The nanoparticle of claim 1 , wherein the at least one water-soluble agent is a nucleic acid that is at least partially neutralized with a base.

87. 87. The nanoparticle of claim 86, wherein the nucleic acid is 50% neutralized with bases.

88. 87. The nanoparticle of claim 86, wherein the nucleic acid is fully neutralized with bases.

89. 87. The nanoparticle of claim 86, wherein the nucleic acid is ribonucleic acid (RNA).

90. 87. The nanoparticle of claim 86, wherein the nucleic acid is selected from the group consisting of messenger ribonucleic acid (mRNA), self-amplifying messenger ribonucleic acid (saRNA), small interfering ribonucleic acid (siRNA), micro ribonucleic acid (microRNA), circular ribonucleic acid (circular RNA), transfer ribonucleic acid (tRNA), small guide ribonucleic acid (sgRNA), and antisense oligonucleotide ribonucleic acid (ASO RNA).

91. 87. The nanoparticle of claim 86, wherein the base is an amine.

92. 87. The nanoparticle of claim 86, wherein the base is guanidine.

93. 87. The nanoparticle of claim 86, wherein the base is a guanidine derivative or a guanidinium salt.

94. 87. The nanoparticle of claim 86, wherein the base is arginine.

95. 87. The nanoparticle of claim 86, wherein the base is a tertiary amine.

96. 87. The nanoparticle of claim 86, wherein the base is selected from the group consisting of triethylamine and diphenhydramine.

97. 56. The method of claim 55, wherein the at least one water-soluble agent comprises a ribonucleic acid (RNA) in which at least one uridine has been substituted with a modified uridine.

98. at least 50% of the uridines in the ribonucleic acid (RNA) are each replaced with a modified uridine; 98. The method of claim 97, wherein the modified uridines can be the same or different.

99. each of said uridines of said ribonucleic acid (RNA) is replaced with a modified uridine; 98. The method of claim 97, wherein the modified uridines can be the same or different.

100. 98. The method of claim 97, wherein each modified uridine is independently selected from the group consisting of pseudouridine, 5-methoxyuridine, and N1-methylpseudouridine.

101. 101. The method of claim 100, wherein each modified uridine is independently selected from the group consisting of pseudouridine and 5-methoxyuridine.

102. 98. The method of claim 97, wherein the ribonucleic acid (RNA) is selected from the group consisting of messenger ribonucleic acid (mRNA), self-amplifying messenger ribonucleic acid (saRNA), small interfering ribonucleic acid (siRNA), micro ribonucleic acid (microRNA), circular ribonucleic acid (circular RNA), transfer ribonucleic acid (tRNA), small guide ribonucleic acid (sgRNA), and antisense oligonucleotide ribonucleic acid (ASO RNA).

103. 56. The method of claim 55, wherein the at least one water-soluble agent is a nucleic acid that is at least partially neutralized with a base prior to dissolving the at least one water-soluble agent in the first polar process solvent.

104. 104. The method of claim 103, wherein the nucleic acid is 50% neutralized with bases.

105. 104. The method of claim 103, wherein the nucleic acid is fully neutralized with bases.

106. 104. The method of claim 103, wherein the nucleic acid is ribonucleic acid (RNA).

107. 104. The method of claim 103, wherein the nucleic acid is selected from the group consisting of messenger ribonucleic acid (mRNA), self-amplifying messenger ribonucleic acid (saRNA), small interfering ribonucleic acid (siRNA), micro ribonucleic acid (microRNA), circular ribonucleic acid (circular RNA), transfer ribonucleic acid (tRNA), small guide ribonucleic acid (sgRNA), and antisense oligonucleotide ribonucleic acid (ASO RNA).

108. 104. The method of claim 103, wherein the base is an amine.

109. 104. The method of claim 103, wherein the base is guanidine.

110. 104. The method of claim 103, wherein the base is a guanidine derivative or a guanidinium salt.

111. 104. The method of claim 103, wherein the base is arginine.

112. 104. The method of claim 103, wherein the base is a tertiary amine.

113. 104. The method of claim 103, wherein the base is selected from the group consisting of triethylamine and diphenhydramine.

114. 56. The method of claim 55, wherein the at least one water solubility agent is a salt of a plasmid.

115. 115. The method of claim 114, wherein the at least one water solubility agent is a triethylamine (TEA) salt of the plasmid.

116. 115. The method of claim 114, wherein said at least one water solubility agent is a guanidine salt of said plasmid.

117. 115. The method of claim 114, wherein the plasmid is an episome.

118. 115. The method of claim 114, wherein the first stabilizing amphiphilic copolymer is a polysaccharide copolymer.

119. 115. The method of claim 114, wherein the first stabilizing amphiphilic copolymer is dextran-poly(lactic-co-glycolic acid) (Dex-PLGA).

120. the dextran has a molecular weight in the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da; 120. The method of claim 119, wherein the poly(lactic-co-glycolic acid) has a molecular weight in the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da.

121. 115. The method of claim 114, wherein the first stabilizing amphiphilic copolymer is a polypeptide copolymer.

122. 115. The method of claim 114, wherein the first stabilizing amphiphilic copolymer is poly(aspartic acid)-poly(lactic-co-glycolic acid) (PAsp-PLGA).

123. the poly(aspartic acid) has a molecular weight in the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da; 123. The method of claim 122, wherein the poly(lactic-co-glycolic acid) has a molecular weight in the range of about 500 to about 500,000 Da, about 500 to about 50,000 Da, or about 750 Da to about 20,000 Da.

124. 115. The method of claim 114, wherein said at least one lipid is selected from the group consisting of cationic lipids, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol, and combinations.

125. 115. The method of claim 114, wherein the second amphiphilic stabilizing copolymer is a PEGylated copolymer.

126. 115. The method of claim 114, wherein the second amphiphilic stabilizing copolymer is 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-DMG).

127. 56. The method of claim 55, wherein the anti-solvent comprises a metal salt.

128. The metal salt is MgCl ２ , CaCl ２ , and ZnCl ２ 128. The method of claim 127, selected from the group consisting of:

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