Metal-organic framework nanoparticles and methods of making the same

Abstract

Provided herein are metal-organic framework nanoparticles including a nucleic acid-based therapeutic agent and methods of making the same. The method includes adding the nucleic acid-based therapeutic agent to an imidazole solution to form a therapeutic agent containing solution, adding a polymer solution to the therapeutic agent containing solution to form an electrostatic complex solution, adding a zinc solution to the electrostatic complex solution to form a metal-organic framework nanoparticle precursor solution, and incubating the metal-organic framework nanoparticle precursor solution to form the metal-organic framework nanoparticles comprising the nucleic-acid based therapeutic agent. Dosage forms and metal-organic framework nanoparticle solutions including the metal-organic framework nanoparticles including a nucleic acid-based therapeutic agent are also provided.

Description

METAL-ORGANIC FRAMEWORK NANOPARTICLES AND METHODS OFMAKING THE SAMECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Patent Application No. 63 / 631,632, filed April 9, 2024, and United States Provisional Patent Application No. 63 / 729,631, filed December 9, 2024, the disclosures of which are hereby incorporated by reference in their entireties.BACKGROUND OF THE INVENTIONField of the Invention

[0002] Provided herein are metal-organic framework nanoparticles including a nucleic-acid based therapeutic agent, as well as methods of making the metal-organic framework nanoparticles, dosage forms including the metal-organic framework nanoparticles, metalorganic framework nanoparticle solutions, and methods of administering the metal-organic framework nanoparticle solutions to a patient.Description of Related Art

[0003] Metal-organic frameworks (MOFs) are porous coordinated polymers formed from metal ions such as zirconium (IV), iron (III), and zinc (II) and organic ligands that feature multiple amine and carboxyl groups extended from alkyl backbones or ring-based structures. MOFs can be used in catalysis, gas absorption and separation, sensors, energy storage, water treatment, and in biomedical applications, such as drug delivery.

[0004] Specific to drug delivery, MOFs have high drug loading capacity, biodegradability, biocompatibility, and a unique ability to deliver a variety of therapeutic modalities, including small molecules, gaseous transmitter molecules, biomolecules, and whole cell therapies. MOF-based drug loading can be achieved through surface attachment, post-synthetic pore encapsulation, one -pot co-precipitation, and biomimetic mineralization.

[0005] However, messenger RNA (mRNA) is extremely fragile, readily susceptible to intramolecular catalysis of strand breakage, oxidation, or reactions with impurities such as aldehydes, metals, and peroxides, and degradation by ubiquitous RNases, rendering the mRNA inactive.

[0006] Therefore, it would be desirable to have a MOF-based delivery system that encapsulates and can deliver fragile mRNA both in vitro and in vivo.SUMMARY OF THE INVENTION

[0007] Provided herein is a method of making metal-organic framework nanoparticles comprising a nucleic acid-based therapeutic agent. The method comprises adding the nucleic acid-based therapeutic agent to an imidazole solution comprising an imidazole containing compound and a first alkyl alcohol solvent and mixing the nucleic acid-based therapeutic agent and the imidazole solution to form a therapeutic agent containing solution. The method comprises adding a polymer solution comprising a cationic polymer to the therapeutic agent containing solution and mixing the polymer solution and the therapeutic agent containing solution to form an electrostatic complex solution. The method comprises adding a zinc solution comprising a hydrated zinc salt and a second alkyl alcohol to the electrostatic complex solution and mixing the zinc solution and the electrostatic complex solution to form a metalorganic framework nanoparticle precursor solution. The method comprises incubating the metal-organic framework nanoparticle precursor solution to form the metal-organic framework nanoparticles comprising the nucleic-acid based therapeutic agent.

[0008] Also provided herein is a method of making metal-organic framework nanoparticles comprising a nucleic acid-based therapeutic agent. The method comprises preparing a zinc solution by dissolving a hydrated zinc salt in a first alkyl alcohol solvent. The method comprises preparing an imidazole solution by dissolving an imidazole containing compound in a second alkyl alcohol solvent. The method comprises adding the nucleic acid-based therapeutic agent to the imidazole solution and mixing the nucleic acid-based therapeutic agent and the imidazole precursor solution to form a therapeutic agent containing solution. The method comprises adding a polymer solution comprising a cationic polymer to the therapeutic agent containing solution and mixing the polymer solution and the therapeutic agent containing solution to form an electrostatic complex solution. The method comprises adding the zinc solution to the electrostatic complex solution and mixing the zinc solution and the electrostatic complex solution to form a metal-organic framework nanoparticle precursor solution. The method comprises incubating the metal-organic framework nanoparticle precursor solution to form the metal-organic framework nanoparticles comprising the nucleic-acid based therapeutic agent.

[0009] Also provided herein is a metal-organic framework nanoparticle comprising a nucleic acid-based therapeutic agent comprising a zeolitic imidazole framework-8 (ZIF-8), a cationic polymer, and the nucleic acid-based therapeutic agent. The cationic polymer and the nucleic acid-based therapeutic agent form an electrostatic complex. The ZIF-8 encapsulates the electrostatic complex.

[0010] Also provided herein is a metal-organic framework nanoparticle solution comprising metal-organic framework nanoparticles comprising a nucleic acid-based therapeutic agent, wherein the metal-organic framework nanoparticles comprising the nucleic acid-based therapeutic agent comprise a zeolitic imidazole framework-8 (ZIF-8), a cationic polymer and the nucleic acid-based therapeutic agent, wherein the cationic polymer and the nucleic acidbased therapeutic agent form an electrostatic complex, and wherein the ZIF-8 encapsulates the electrostatic complex; and a solvent.

[0011] Also provided herein is a method of delivering a nucleic acid-based therapeutic agent to a patient. The method comprises administering to the patient an effective amount of a metalorganic framework nanoparticle solution. The metal-organic framework nanoparticle solution comprises metal-organic framework nanoparticles comprising: a zeolitic imidazole framework-8 (ZIF-8); a cationic polymer; and the nucleic acid-based therapeutic agent, wherein the cationic polymer and the nucleic acid-based therapeutic agent form an electrostatic complex, and wherein the ZIF-8 encapsulates the electrostatic complex; and a pharmaceutically-acceptable carrier.

[0012] This disclosure is further described in the following numbered clauses:

[0013] Clause 1 : A method of making metal-organic framework nanoparticles comprising a nucleic acid-based therapeutic agent, the method comprising: adding the nucleic acid-based therapeutic agent to an imidazole solution comprising an imidazole containing compound and a first alkyl alcohol solvent and mixing the nucleic acid-based therapeutic agent and the imidazole solution to form a therapeutic agent containing solution; adding a polymer solution comprising a cationic polymer to the therapeutic agent containing solution and mixing the polymer solution and the therapeutic agent containing solution to form an electrostatic complex solution; adding a zinc solution comprising a hydrated zinc salt and a second alkyl alcohol to the electrostatic complex solution and mixing the zinc solution and the electrostatic complex solution to form a metal-organic framework nanoparticle precursor solution; and incubating the metal-organic framework nanoparticle precursor solution to form the metal-organic framework nanoparticles comprising the nucleic-acid based therapeutic agent.

[0014] Clause 2: The method of clause 1, wherein the first alkyl alcohol solvent and the second alkyl alcohol solvent are the same and each independently comprise ethanol, methanol, or isopropyl alcohol.

[0015] Clause 3: The method of clause 1 or 2, wherein the first alkyl alcohol solvent and the second alkyl alcohol solvent each independently comprise ethanol.

[0016] Clause 4: The method of any one of clauses 1 to 3, wherein the cationic polymer comprises a linear or branched polyethyleneimine.

[0017] Clause 5: The method of clause 4, wherein the linear or branched polyethyleneimine comprises a weight average molecular weight of at least 10 kiloDaltons (kDa).

[0018] Clause 6: The method of clause 4, wherein the cationic polymer comprises a linear polyethyleneimine comprising a weight average molecular weight of at least 20 kDa.

[0019] Clause 7: The method of any one of clauses 4 to 6, wherein the polymer solution comprises less or equal to 2 milligrams per milliliter (mg / mL) of the linear or branched polyethyleneimine .

[0020] Clause 8: The method of any one of clauses 4 to 6, wherein the polymer solution comprises less or equal to 1.5 mg / mL of the linear or branched polyethyleneimine.

[0021] Clause 9: The method of any one of clauses 4 to 8, wherein the polymer solution comprises an aqueous solvent and a pH of approximately 7.

[0022] Clause 10: The method of clause 9, wherein the aqueous solvent comprises nuclease- free water.

[0023] Clause 11: The method of any one of clauses 1 to 10, wherein the electrostatic complex solution comprises a nitrogen-to-phosphate (N / P) ratio of at least 6:1.

[0024] Clause 12: The method of any one of clauses 1 to 10, wherein the electrostatic complex solution comprises a N / P ratio in a range of from 6:1 to 20:1.

[0025] Clause 13: The method of any one of clauses 1 to 10, wherein the electrostatic complex solution comprises a N / P ratio in a range of from 8:1 to 17:1.

[0026] Clause 14: The method of any one of clauses 1 to 10, wherein the electrostatic complex solution comprises a N / P ratio in a range of from 9:1 to 12:1.

[0027] Clause 15: The method of any one of clauses 1 to 10, wherein the electrostatic complex solution comprises a N / P ratio in a range of from 9:1 to 10:1.

[0028] Clause 16: The method of any one of clauses 1 to 15, wherein the imidazole solution is prepared by dissolving the imidazole containing compound in the first alkyl alcohol solvent.

[0029] Clause 17: The method of any one of clauses 1 to 16, wherein the zinc solution is prepared by dissolving the hydrated zinc salt in the second alkyl alcohol solvent.

[0030] Clause 18: The method of any one of clauses 1 to 17, further comprising incubating the polymer solution and the therapeutic agent containing solution for a time of not greater than 15 minutes to form the electrostatic complex solution.

[0031] Clause 19: The method of any one of clauses 1 to 18, wherein the zinc solution and the electrostatic complex solution are incubated for a time of at least 2 hours.

[0032] Clause 20: The method of any one of clauses 1 to 19, wherein the nucleic acid-based therapeutic agent comprises messenger RNA (mRNA), small interfering RNA (siRNA), plasmid DNA (pDNA), small harpin RNA (shRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), micro RNA (miRNA), small nuclear RNA (snRNA), long non-coding RNA (IncRNA), double- stranded RNA (dsRNA), piwi-interacting RNA (piRNA), circular RNA (circRNA), short interfering RNA (siRNA), antisense oligonucleotides, or combinations thereof.

[0033] Clause 21: The method of clause 20, wherein the nucleic acid-based therapeutic agent comprises mRNA.

[0034] Clause 22: The method of clause 20, wherein the nucleic acid-based therapeutic agent comprises mRNA, wherein the mRNA comprises chemically-modified bases.

[0035] Clause 23: The method of any one of clauses 1 to 22, wherein the hydrated zinc salt comprises zinc nitrate hexahydrate and the imidazole containing compound comprises 2- methy limidazole .

[0036] Clause 24: The method of any one of clauses 1 to 23, wherein the metal-organic framework is a zeolitic imidazole framework-8 (ZIF-8).

[0037] Clause 25: The method of any one of clauses 1 to 24, wherein the hydrated zinc salt comprises zinc nitrate hexahydrate, the imidazole containing compound comprises 2- methylimidazole, the cationic polymer comprises linear polyethyleneimine comprising a weight average molecular weight of at least 20 kDa, the first alkyl alcohol solvent comprises ethanol, and the second alkyl alcohol solvent comprises ethanol.

[0038] Clause 26: A dosage form comprising the metal-organic framework nanoparticles comprising the nucleic acid-based therapeutic agent prepared according to any one of clauses 1 to 25; and a pharmaceutically-acceptable carrier.

[0039] Clause 27: The dosage form of clause 26, wherein the dosage form is formulated as a parenteral dosage form.

[0040] Clause 28: The dosage form of clause 26 or 27, wherein the dosage form is a vaccine.

[0041] Clause 29: A method of making metal-organic framework nanoparticles comprising a nucleic acid-based therapeutic agent, the method comprising: preparing a zinc solution by dissolving a hydrated zinc salt in a first alkyl alcohol solvent; preparing an imidazole solution by dissolving an imidazole containing compound in a second alkyl alcohol solvent; adding the nucleic acid-based therapeutic agent to the imidazole solution and mixing the nucleic acidbased therapeutic agent and the imidazole precursor solution to form a therapeutic agent containing solution; adding a polymer solution comprising a cationic polymer to the therapeuticagent containing solution and mixing the polymer solution and the therapeutic agent containing solution to form an electrostatic complex solution; adding the zinc solution to the electrostatic complex solution and mixing the zinc solution and the electrostatic complex solution to form a metal-organic framework nanoparticle precursor solution; and incubating the metal-organic framework nanoparticle precursor solution to form the metal-organic framework nanoparticles comprising the nucleic-acid based therapeutic agent.

[0042] Clause 30: The method of clause 29, wherein the first alkyl alcohol solvent and the second alkyl alcohol solvent are the same and each independently comprise ethanol, methanol, or isopropyl alcohol.

[0043] Clause 31 : The method of clause 29 or 30, wherein the first alkyl alcohol solvent and the second alkyl alcohol solvent each independently comprise ethanol.

[0044] Clause 32: The method of any one of clauses 29 to 31, wherein cationic polymer comprises a linear or branched polyethyleneimine.

[0045] Clause 33: The method of clause 32, wherein the linear or branched polyethyleneimine comprises a weight average molecular weight of at least 10 kDa.

[0046] Clause 34: The method of clause 32, wherein the cationic polymer comprises a linear polyethyleneimine comprising a weight average molecular weight of at least 20 kDa

[0047] Clause 35: The method of any one of clauses 32 to 34, wherein the polymer solution comprises less or equal to 2.0 mg / mL of the linear or branched polyethyleneimine.

[0048] Clause 36: The method of any one of clauses 32 to 34, wherein the polymer solution comprises less or equal to 1.5 mg / mL of the linear or branched polyethyleneimine.

[0049] Clause 37: The method of any one of clauses 32 to 36, wherein the polymer solution comprises: an aqueous solvent; and a pH of approximately 7.

[0050] Clause 38: The method of clause 37, wherein the aqueous solvent comprises nuclease-free water.

[0051] Clause 39: The method of any one of clauses 29 to 38, wherein the electrostatic complex solution comprises a N / P ratio of at least 6:1.

[0052] Clause 40: The method of any one of clauses 29 to 38, wherein the electrostatic complex solution comprises a N / P ratio in a range of from 6:1 to 20:1.

[0053] Clause 41: The method of any one of clauses 29 to 38, wherein the electrostatic complex solution comprises a N / P ratio in a range of from 8:1 to 17:1.

[0054] Clause 42: The method of any one of clauses 29 to 38, wherein the electrostatic complex solution comprises a N / P ratio in a range of from 9:1 to 12:1.

[0055] Clause 43: The method of any one of clauses 29 to 38, wherein the electrostatic complex solution comprises a N / P ratio in a range of from 9:1 to 10:1.

[0056] Clause 44: The method of any one of clauses 29 to 43, further comprising incubating the polymer solution and the therapeutic agent containing solution for a time of not greater than 15 minutes to form the electrostatic complex solution.

[0057] Clause 45: The method of any one of clauses 29 to 44, wherein the zinc solution and the electrostatic complex solution are incubated for a time of at least 2 hours.

[0058] Clause 46: The method of any one of clauses 29 to 45, wherein the nucleic acid-based therapeutic agent comprises mRNA, siRNA, plasmid DNA, shRNA, tRNA, rRNA, miRNA, snRNA, IncRNA, dsRNA, piRNA, circRNA, siRNA, antisense oligonucleotides, or combinations thereof.

[0059] Clause 47: The method of clause 46, wherein the nucleic acid-based therapeutic agent comprises mRNA.

[0060] Clause 48: The method of clause 46, wherein the nucleic acid-based therapeutic agent comprises mRNA, wherein the mRNA comprises chemically-modified bases.

[0061] Clause 49: The method of any one of clauses 29 to 48, wherein the hydrated zinc salt comprises zinc nitrate hexahydrate and the imidazole containing compound comprises 2- methy limidazole .

[0062] Clause 50: The method of any one of clauses 29 to 49, wherein the metal-organic framework is a zeolitic imidazole framework-8 (ZIF-8).

[0063] Clause 51: The method of any one of clauses 29 to 50, wherein the hydrated zinc salt comprises zinc nitrate hexahydrate, the imidazole containing compound comprises 2- methylimidazole, the cationic polymer comprises linear polyethyleneimine comprising a weight average molecular weight of at least 20 kDa, the first alkyl alcohol solvent comprises ethanol, and the second alkyl alcohol solvent comprises ethanol.

[0064] Clause 52: A dosage form comprising the metal-organic framework nanoparticles comprising the nucleic acid-based therapeutic agent prepared according to any one of clauses 29 to 51 ; and a pharmaceutically-acceptable carrier.

[0065] Clause 53: The dosage form of clause 52, wherein the dosage form is formulated as a parenteral dosage form.

[0066] Clause 54: The dosage form of clause 52 or 53, wherein the dosage form is a vaccine.

[0067] Clause 55: A metal-organic framework nanoparticle comprising a nucleic acid-based therapeutic agent prepared according to the method of any one of clauses 1 to 28.

[0068] Clause 56: A metal-organic framework nanoparticle comprising a nucleic acid-based therapeutic agent prepared according to the method of any one of clauses 29 to 51.

[0069] Clause 57 : A metal-organic framework nanoparticle comprising a nucleic acid-based therapeutic agent comprising: a zeolitic imidazole framework-8 (ZIF-8); a cationic polymer; and the nucleic acid-based therapeutic agent, wherein the cationic polymer and the nucleic acid-based therapeutic agent form an electrostatic complex, and wherein the ZIF-8 encapsulates the electrostatic complex.

[0070] Clause 58: The metal-organic framework nanoparticles of clause 57, wherein the cationic polymer comprises a linear or branched polyethyleneimine.

[0071] Clause 59: The metal-organic framework nanoparticles of clause 58, wherein the linear or branched polyethyleneimine comprises a weight average molecular weight of at least 10 kDa.

[0072] Clause 60: The metal-organic framework nanoparticles of clause 58, wherein the cationic polymer comprises linear polyethyleneimine comprising a weight average molecular weight of at least 20 kDa.

[0073] Clause 61: The metal-organic framework nanoparticles of any one of clauses 57 to 60, wherein the nucleic acid-based therapeutic agent comprises mRNA, siRNA, pDNA, shRNA, tRNA, rRNA, miRNA, snRNA, IncRNA, dsRNA, piRNA, circRNA, siRNA, antisense oligonucleotides, or combinations thereof.

[0074] Clause 62: The metal-organic framework nanoparticles of clause 61, wherein the nucleic acid-based therapeutic agent comprises mRNA.

[0075] Clause 63: The method of clause 61, wherein the nucleic acid-based therapeutic agent comprises mRNA, wherein the mRNA comprises chemically-modified bases.

[0076] Clause 64: The metal-organic framework nanoparticles of any one of clauses 57 to63, wherein the metal-organic framework nanoparticles comprise a diameter ranging from 70 nanometers (nm) to 200 nm.

[0077] Clause 65: The metal-organic framework nanoparticles of any one of clauses 57 to64, further comprising an alkyl alcohol solvent, wherein the ZIF-8 encapsulates the alkyl alcohol solvent.

[0078] Clause 66: The metal-organic framework nanoparticles of clause 65, wherein the alkyl alcohol solvent comprises ethanol, methanol, or isopropyl alcohol.

[0079] Clause 67: The metal-organic framework nanoparticles of clause 65 or 66, wherein the alkyl alcohol solvent comprises ethanol.

[0080] Clause 68: A metal-organic framework nanoparticle solution comprising: metalorganic framework nanoparticles comprising a nucleic acid-based therapeutic agent, wherein the metal-organic framework nanoparticles comprising the nucleic acid-based therapeutic agent comprise: a zeolitic imidazole framework-8 (ZIF-8); a cationic polymer; and the nucleic acid-based therapeutic agent, wherein the cationic polymer and the nucleic acid-based therapeutic agent form an electrostatic complex, and wherein the ZIF-8 encapsulates the electrostatic complex; and a solvent.

[0081] Clause 69: The metal-organic framework nanoparticle solution of clause 68, wherein the cationic polymer comprises a linear or branched polyethyleneimine.

[0082] Clause 70: The metal-organic framework nanoparticle solution of clause 69, wherein the linear or branched polyethyleneimine comprises a weight average molecular weight of at least 10 kDa.

[0083] Clause 71: The metal-organic framework nanoparticle solution of clause 69, wherein the cationic polymer comprises linear polyethyleneimine comprising a weight average molecular weight of at least 20 kDa.

[0084] Clause 72: The metal-organic framework nanoparticle solution of any one of clauses 68 to 71, wherein the nucleic acid-based therapeutic agent comprises mRNA, siRNA, pDNA, shRNA, tRNA, rRNA, miRNA, snRNA, IncRNA, dsRNA, piRNA, circRNA, siRNA, antisense oligonucleotides, or combinations thereof.

[0085] Clause 73: The metal-organic framework nanoparticle solution of clause 72, wherein the nucleic acid-based therapeutic agent comprises mRNA.

[0086] Clause 74: The method of clause 72, wherein the nucleic acid-based therapeutic agent comprises mRNA, wherein the mRNA comprises chemically-modified bases.

[0087] Clause 75: The metal-organic framework nanoparticle solution of any one of clauses 68 to 74, wherein the metal-organic framework nanoparticles comprise a diameter ranging from 70 nm to 200 nm.

[0088] Clause 76: The metal-organic framework nanoparticle solution of any one of clauses 68 to 75, wherein the metal-organic nanoparticles comprising the nucleic acid-based therapeutic agent further comprise an alkyl alcohol solvent, wherein the ZIF-8 encapsulates the alkyl alcohol solvent.

[0089] Clause 77: The metal-organic framework nanoparticle solution of clause 76, wherein the alkyl alcohol solvent comprises ethanol, methanol, or isopropyl alcohol.

[0090] Clause 78: The metal-organic framework nanoparticle solution of clause 76 or 77, wherein the alkyl alcohol solvent comprises ethanol.

[0091] Clause 79: The metal-organic framework nanoparticle solution of any one of clauses 68 to 78, wherein the solvent comprises a mixture of an aqueous solvent and an alkyl alcohol solvent.

[0092] Clause 80: The metal-organic framework nanoparticle solution of clause 79, wherein aqueous solvent comprises phosphate buffered saline and the alkyl alcohol solvent comprises ethanol.

[0093] Clause 81 : A method of delivering a nucleic acid-based therapeutic agent to a patient, comprising administering to the patient an effective amount of a metal-organic framework nanoparticle solution comprising: metal-organic framework nanoparticles comprising: a zeolitic imidazole framework-8 (ZIF-8); a cationic polymer; and the nucleic acid-based therapeutic agent, wherein the cationic polymer and the nucleic acid-based therapeutic agent form an electrostatic complex, and wherein the ZIF-8 encapsulates the electrostatic complex; and a pharmaceutically-acceptable carrier.

[0094] Clause 82: The method of clause 81, wherein the cationic polymer comprises a linear or branched polyethyleneimine.

[0095] Clause 83: The method of clause 82, wherein the linear or branched polyethyleneimine comprises a weight average molecular weight of at least 10 kDa.

[0096] Clause 84: The method of clause 82, wherein the cationic polymer comprises linear polyethyleneimine comprising a weight average molecular weight of at least 20 kDa.

[0097] Clause 85: The method of any one of clauses 81 to 84, wherein the nucleic acid-based therapeutic agent comprises mRNA, siRNA, pDNA, shRNA, tRNA, rRNA, miRNA, snRNA, IncRNA, dsRNA, piRNA, circRNA, siRNA, antisense oligonucleotides, or combinations thereof.

[0098] Clause 86: The method of clause 85, wherein the nucleic acid-based therapeutic agent comprises mRNA.

[0099] Clause 87: The method of clause 85, wherein the nucleic acid-based therapeutic agent comprises mRNA, wherein the mRNA comprises chemically-modified bases.

[0100] Clause 88: The method of any one of clauses 81 to 87, wherein the metal-organic framework nanoparticles comprise a diameter ranging from 70 nm to 200 nm.

[0101] Clause 89: The method of any one of clauses 81 to 88, further comprising an alkyl alcohol solvent, wherein the ZIF-8 encapsulates the alkyl alcohol solvent.

[0102] Clause 90: The method of clause 89, wherein the alkyl alcohol solvent comprises ethanol, methanol, or isopropyl alcohol.

[0103] Clause 91: The method of clause 89 or 90, wherein the alkyl alcohol solvent comprises ethanol.

[0104] Clause 92: The method of any one of clauses 81 to 91, wherein the metal-organic framework nanoparticle solution is administered to the patient parenterally.BRIEF DESCRIPTION OF THE DRAWINGS

[0105] For a more complete understanding of the description provided herein and the advantages thereof, reference is now made to the brief descriptions below, taken in connection with the accompanying drawings and detailed description, wherein like reference numerals represent like parts.

[0106] FIG. 1 is a schematic of the method according to the present invention.

[0107] FIG. 2 is a schematic (not to scale) of a metal-organic framework nanoparticle according to the present invention.

[0108] FIGS. 3A-3C are schematic illustrations according to the present invention. FIG. 3A is a schematic illustration of the forming of the electrostatic complex between mRNA and polyethyleneimine (PEI) and one-pot co-precipitation of mRNA complexes inside zeolitic imidazole framework-8 (ZIF-8) to form polymer core-MOF shell nanoparticles (mRNA- PEI@ZIF-8 (mRPZ)). FIG. 3B is a schematic illustration of mRNA delivery from mRPZ nanoparticles and expression of green fluorescence protein (eGFP) in vitro. FIG. 3C is a schematic illustration of mRNA delivery from mRPZ nanoparticles and expression of firefly luciferase in vivo.

[0109] FIGS. 4A-4D show the stability of mRNA exposed to different experimental conditions using native RNA gel electrophoresis (FIGS. 4A-4C) and expressibility of encapsulated Cy5-eGFP-mRNA transfected into in HEK293t cells (FIG. 4D).

[0110] FIGS. 5A-5D show the particle protection against RNase and stability in biological mediums using native RNA gel electrophoresis. FIG. 5A is native RNA gel of mRNA@ZIF-8 after exposure to RNase A, compared with controls of mRNA, mRNA treated with RNase, and mRNA@ZIF-8. mRNA@ZIF-8 samples were exfoliated using EDTA before loading onto the gel. FIG. 5B is a Native RNA gel of mRNA @ ZIF-8 incubated for 1, 4, and 24 hours with various biological media including DMEM culture medium with 10% FBS, Opti-MEM culture medium, and PBS at pH 7 and 5. FIG. 5C is a native RNA gel of mRNA-PEI@ZIF-8 after exposure RNase A, compared with controls of mRNA, mRNA treated with RNase and mRNA- PEI@ZIF-8. mRNA-PEI@ZIF-8 samples were exfoliated using EDTA and heparin before loading onto the gel. FIG. 5D is a native RNA gel of mRNA-PEI@ ZIF-8 incubated for 1, 4,and 24 hours with various biological media including DMEM culture medium with 10% FBS, Opti-MEM culture medium, and PBS at pH 7 and 5.

[0111] FIG. 6 includes SEM and TEM micrographs of ZIF-8, mRNA@ZIF-8, and mRPZ nanoparticles, where FIG. 6(A) is an SEM micrograph of ZIF-8, FIG. 6(B) is a SEM micrograph of mRNA@ZIF-8, FIG. 6(C) is a SEM micrograph of mRPZ, FIG. 6(D) is a TEM micrograph of ZIF-8, FIG. 6(E) is a TEM micrograph of mRNA@ZIF-8, and FIG. 6(F) is a TEM micrograph of mRPZ. The SEM micrographs (FIGS. 6(A)-6(C)) have a scale bar of 500 nm and the TEM micrographs (FIGS. 6(D)-6(F)) have a scale bar of 100 nm.

[0112] FIGS. 7A-7C depict the characterization of ZIF-8, mRNA@ZIF-8, and mRPZ nanoparticles using size distribution measured by SEM (FIG. 7A), hydrodynamic diameter measured by DES (FIG. 7B), and Powder X-ray Diffraction (FIG. 7C).

[0113] FIGS. 8A-8C depict the evaluation of mRNA loading efficiency and capacity of mRPZ as determined by image analysis of a native RNA gel (FIG. 8A), a fluorescent probe (FIG. 8B), and thermogravimetric analysis (TGA; FIG. 8C).

[0114] FIGS. 9A-9B are graphs depicting the incorporation of mRNA inside ZIF-8 using zeta potential differences (FIG. 9A) and EDS (FIG. 9B).

[0115] FIG. 10 is a graph depicting resulting mRNA release as measured by a fluorescent probe.

[0116] FIG. 11 is a graph depicting eGFP expression of mRPZ by HEK293t cells.

[0117] FIGS. 12A- 12H are graphs depicting cell viability (FIG. 12A), cellular uptake (FIG. 12B), cellular expression of mRNA in live HEK293t cells (FIG. 12C), HeEA cells (FIG. 12D), and CHO cells (FIG. 12E), and normalized median fluorescence index (MFI) in live HEK293t cells (FIG. 12F), HeEA cells (FIG. 12G), and CHO cells (FIG. 12H). Error bars in FIGS. 12A- 12H represent the standard deviation.

[0118] FIG. 13 is a graph depicting eGFP expression after transfecting HEK293t cells using mRPZ nanoparticles that were stored at -80°C, 4°C, and room temperature (RT) and for various times.

[0119] FIGS. 14A-14B are graphs depicting luciferase expression kinetics measured over 96 hours (FIG. 14A) and the integrated luciferase activity (FIG. 14B).

[0120] FIGS. 15A-15B are graphs depicting the biodistribution of the mRNA (FIG. 15 A) and the bioavailability of the luciferase mRNA quantified after 12 hours (FIG. 15B).

[0121] FIGS. 16A-16F are graphs of the measurement of Urea concentration (FIG. 16A), Creatine concentration (FIG. 16B), Alkaline phosphatase (AFP) activity (FIG. 16C), alaninetransaminase activity (ALT) activity (FIG. 16D), aspartate transaminase (AST) activity (FIG. 16E), and Zinc concentration (FIG. 16F) in mouse serum.

[0122] FIG. 17 is a graph depicting mRNA expression in mice after mRPZ was stored at - 80°C and RT stored under vacuum for one-month.DESCRIPTION OF THE INVENTION

[0123] The following description is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses. While the description is designed to permit one of ordinary skill in the art to make and use the invention, and specific examples are provided to that end, they should in no way be considered limiting. It will be apparent to one of ordinary skill in the art that various modifications to the following will fall within the scope of the appended claims. The present invention should not be considered limited to the presently disclosed embodiments, whether provided in the examples or elsewhere herein.

[0124] The use of numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges are both preceded by the word “about”. In this manner, slight variations above and below the stated ranges (e.g., ± 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5%) can be used to achieve substantially the same results as values within the ranges. Also, unless indicated otherwise, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values. For example, a stated range of “1 to 10” should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more and ending with a maximum value of 10 or less, e.g., 1 to 3.3, 4.7 to 7.5, 5.5 to 10, and the like.

[0125] As used herein “a” and “an” refer to one or more.

[0126] As used herein, the terms “comprising”, “comprise”, or “comprised”, and variations thereof, are open ended and do not exclude the presence of other elements not identified. In contrast, the term “consisting of’ and variations thereof is intended to be closed, and excludes additional elements in anything but trace amounts.

[0127] As used herein, spatial or directional terms, such as “left”, “right”, “inner”, “outer”, “above”, “below”, “over”, “under”, and the like, relate to the invention, are provided solely for ease of description and illustration, and do not imply directionality, unless specifically required for operation of the described aspect of the invention. It is to be understood that the inventioncan assume various alternative orientations and, accordingly, such terms are not to be considered as limiting.

[0128] Provided herein is a method 10 of making metal-organic framework (MOF) nanoparticles comprising a nucleic acid-based therapeutic agent (FIG. 1). The method 10 comprises: adding the nucleic acid-based therapeutic agent 12 to an imidazole solution 14 comprising an imidazole containing compound and a first alkyl alcohol solvent and mixing the nucleic acid-based therapeutic agent 12 and the imidazole solution to form a therapeutic agent containing solution 16; adding a polymer solution 18 comprising a cationic polymer to the therapeutic agent containing solution 16 and mixing the polymer solution 18 and the therapeutic agent containing solution 16 to form an electrostatic complex solution 20; adding a zinc solution 22 comprising a hydrated zinc salt and a second alkyl alcohol to the electrostatic complex solution 20 and mixing the zinc solution 22 and the electrostatic complex solution 20 to form a metal-organic framework nanoparticle precursor solution 24; and incubating the metal-organic framework nanoparticle precursor solution 24 to form the metal-organic framework nanoparticles comprising the nucleic-acid based therapeutic agent 26.

[0129] As used herein a “metal-organic framework nanoparticles” are nanoparticles formed from metal ions or metal clusters that are connected by organic linkers to form a porous, crystalline structure.

[0130] As used herein, a “nucleic acid-based therapeutic agent” is an agent including a nucleic acid that produces some desired local or systemic effect in a patient at a reasonable benefit / risk ratio applicable to any treatment. The metal-organic framework nanoparticles comprising the nucleic acid-based therapeutic agent described herein may be administered in a sufficient amount to produce a reasonable benefit / risk ratio applicable to such treatment.

[0131] ‘ ‘Nucleic acids” include deoxyribonucleic acids (DNA) and ribonucleic acids (RNA) as is found naturally, and chemically-modified nucleic acids, as are broadly-known. Nucleic acids useful in the nanoparticles and methods described herein may be polyanionic nucleic acids, having an overall negative charge under neutral or physiological conditions, such as in a nuclease-free aqueous solution pH 6-8, e.g., in water, blood, serum, Ringer’s, or normal saline. The nucleic acid comprises at least one phosphorus-containing moiety, such as a phosphate and / or a phosphorothioate moiety, making the nucleic acid polyanionic.

[0132] The nucleic acid-based therapeutic agent 12 is poly anionic and may comprise messenger RNA (mRNA), small interfering RNA (siRNA), plasmid DNA (pDNA), small harpin RNA (shRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), micro RNA (miRNA), small nuclear RNA (snRNA), long non-coding RNA (IncRNA), double- stranded RNA(dsRNA), piwi-interacting RNA (piRNA), circular RNA (circRNA), short interfering RNA (siRNA), antisense oligonucleotides, or combinations thereof. In some non-limiting embodiments, the nucleic acid-based therapeutic agent comprises mRNA. In another nonlimiting embodiment, the nucleic acid-based therapeutic agent comprises mRNA, wherein the mRNA comprises chemically-modified bases.

[0133] The nucleic acid-based therapeutic agent 12 may be suspended in a nuclease-free aqueous solution having a pH ranging from 6 to 8. For example, the nucleic acid-based therapeutic agent 12 may be suspended in the nuclease-free aqueous solution in an amount ranging from 0.1 milligram per milliliter (mg / mL) to 2 mg / mL, such as from 0.5 mg / mL to 2 mg / mL, or such as from 1 mg / mL to 2 mg / mL. For example, the nucleic acid-based therapeutic agent 12 may be suspended in nuclease-free water at a concentration of at least 2 mg / mL. Alternatively, the nucleic acid-based therapeutic agent 12 may be suspended in nuclease-free water at a concentration of at least 1 mg / mL.

[0134] The method 10 comprises adding the nucleic acid-based therapeutic agent 12 to an imidazole solution 14. The imidazole solution 14 comprises an imidazole containing compound and a first alkyl alcohol solvent.

[0135] An imidazole containing compound is a compound comprising at least one imidazole group as follows:

[0136] For example, the imidazole containing compound may be imidazole.

[0137] For example, the imidazole containing compound may be a substituted imidazole. Examples of substituted imidazoles include, but are not limited to: 2-methylimidazole, 2- aminoimidazole, 2-ethylimidazole, 4-aminoimidazole, 2-chloroimidazole, 4-bromo-2- methylimidazole, 2-isopropylimidazole, 2-propylimidazole, 2-iodoimidazole, 2- butylimidazole, and 2-fluoroimidazole.

[0138] For example, the imidazole containing compound may be 2-methylimidazole having the following structure:

[0139] As used herein, an “alkyl alcohol solvent” refers to an organic compound having a hydroxyl (-OH) group attached to a straight or branched alkyl chain. The straight or branchedalkyl chain may include from 1 to about 10 carbon atoms, for example and without limitation Ci-io, Ci-8, Ci-6, CM, Ci-3, or Ci-2 groups. The straight or branched alkyl chain can be, for example, a Ci, C2, C3, C4, C5, Ce, C7, Cs, C9, or C10 group that is substituted or unsubstituted. For example, the straight alkyl chain includes methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, or the like. Branched alkyl groups comprise any straight alkyl group substituted with any number of alkyl groups. Non-limiting examples of branched alkyl groups include isopropyl, n-butyl, isobutyl, sec-butyl, and / -butyl.

[0140] For example, the first alcohol solvent may comprise methanol. For example, the first alcohol solvent may comprise ethanol. For example, the first alcohol solvent may comprise propanol. For example, the first alcohol solvent may comprise isopropyl alcohol.

[0141] The imidazole solution 14 may be prepared by dissolving the imidazole containing compound in the first alkyl alcohol solvent.

[0142] The imidazole solution 14 may comprise the imidazole containing compound in a concentration of ranging from 30 millimolar (mM) to not greater than 200 mM, such as from 60 mM to not greater than 190 mM, such as from 80 mM to not greater than 180 mM, such as from 100 mM to not greater than 170 mM. For example, the imidazole solution 14 may comprise the imidazole containing compound in a concentration ranging from 100 mM to 165 mM, such as from 110 mM to 165 mM, such as from 120 mM to 165 nM, or such as from 130 mM to 160 mM. For example, the imidazole solution 14 may comprise the imidazole containing compound in a concentration of less than or equal to 160 mM.

[0143] In some non-limiting embodiments, the imidazole solution 14 comprises 2- methylimidazole and ethanol, wherein the 2-methylimidazole is dissolved in the ethanol.

[0144] Following the addition of the nucleic acid-based therapeutic agent 12 to the imidazole solution 14, the method 10 comprises mixing the nucleic acid-based therapeutic agent 12 and the imidazole solution 14 to form a therapeutic agent containing solution 16. The nucleic acid-based therapeutic agent 12 and the imidazole solution 14 may be mixed for a time to sufficiently mix the nucleic acid-based therapeutic agent 12 and the imidazole solution 14. For example, the nucleic acid-based therapeutic agent 12 and the imidazole solution 14 may be mixed for less than or equal to 1 minute, or less than or equal to 30 seconds.

[0145] The method 10 comprises adding a polymer solution 18 to the therapeutic agent containing solution 16. The polymer solution 18 comprises a cationic polymer.

[0146] As a class, “polymers” include, without limitation, homopolymers, heteropolymers, copolymers, block polymers, block co-polymers and can be both natural and synthetic.Homopolymers contain one type of building block, or monomer, whereas copolymers contain more than one type of monomer.

[0147] A polymer “comprises” or is “derived from” a stated monomer if that monomer is incorporated into the polymer. Thus, the incorporated monomer that the polymer comprises is not the same as the monomer prior to incorporation into the polymer, in that at the very least, during incorporation of the monomer, certain groups, e.g., terminal groups, that are modified during polymerization are changed, removed, and / or relocated, and certain bonds may be added, removed, and / or modified. An incorporated monomer is referred to as a “residue” of that monomer. A polymer is said to comprise a specific type of linkage if that linkage is present in the polymer. Unless otherwise specified, molecular weight for the polymer refers to a weight average molecular weight (Mw).

[0148] As used herein, a “cationic polymer” is a positively charged polymer.

[0149] The cationic polymer of the polymer solution 18 may comprise polyethyleneimine, poly(L-lysine), poly(2-(dimethylamino)ethyl methacrylate), spermine, diethylaminoethyldextran (DEAE-dextran), protamine, poly(beta-amino ester), poly(amidoamine), polybrene, salts thereof, and combinations thereof.

[0150] For example, the cationic polymer of the polymer solution 18 comprises polyethyleneimine .

[0151] The polyethyleneimine may be a linear or branched polyethyleneimine. As used herein, “linear polyethyleneimine” means a polymer that includes repeating units of an amine group and two aliphatic carbons that are linked in a continuous chain. As used herein, “branched polyethyleneimine” means a polymer having a non-linear structure with repeating units of ethylene diamine groups and containing primary, secondary, and tertiary amino groups.

[0152] The linear or branched polyethyleneimine may comprise a weight average molecular weight of at least 10 kiloDaltons (kDa). For example, the linear or branched polyethyleneimine may comprise a weight average molecular weight ranging from at least 10 kiloDaltons (kDa) to least 100 kDa. For example, the linear or branched polyethyleneimine may comprise a weight average molecular weight ranging from at least 10 kDa to at least 70 kDa, such as from at least 10 kDa to at least 60 kDa, such as from at least 10 kDa to at least 40 kDa, or such as from at least 20 kDa to at least 40 kDa. For example, the linear or branched polyethyleneimine may comprise a weight average molecular weight of at least 20 kDa. For example, the linear or branched polyethyleneimine may comprise a weight average molecular weight of at least 25 kDa. For example, the linear or branched polyethyleneimine may comprise a weight average molecular weight of at least 40 kDa. For example, the linear orbranched polyethyleneimine may comprise a weight average molecular weight of at least 60 kDa. For example, the linear or branched polyethyleneimine may comprise a weight average molecular weight of at least 70 kDa. For example, the linear or branched polyethyleneimine may comprise a weight average molecular weight of at least 80 kDa. For example, the linear or branched polyethyleneimine may comprise a weight average molecular weight of at least 100 kDa. The weight average molecular weight may be determined by any suitable method, such as gel permeation chromatography.

[0153] For example, the polyethyleneimine may be a linear polyethyleneimine comprising a weight average molecular weight of at least 10 kDa. Alternatively, the polyethyleneimine may be a branched polyethyleneimine comprising a weight average molecular weight of at least 10 kDa.

[0154] For example, the polyethyleneimine may be a linear polyethyleneimine comprising a weight average molecular weight of at least 20 kDa.

[0155] For example, the polyethyleneimine may be a linear polyethyleneimine comprising a weight average molecular weight of at least 40 kDa.

[0156] For example, the polyethyleneimine may be a branched polyethyleneimine comprising a weight average molecular weight of at least 25 kDa.

[0157] When the cationic polymer comprises polyethyleneimine, such as a linear polyethyleneimine comprising a weight average molecular weight of at least 20 kDa, the polymer solution 18 may comprise the polyethyleneimine in an amount of less than or equal to 2.0 mg / mL. For example, when the cationic polymer comprises polyethyleneimine, such as polyethyleneimine comprising a weight average molecular weight of at least 20 kDa, the polymer solution 18 may comprise the polyethyleneimine in an amount of less than or equal to 1.5 mg / mL. For example, when the cationic polymer comprises polyethyleneimine, such as polyethyleneimine comprising a weight average molecular weight of at least 20 kDa, the polymer solution 18 may comprise the polyethyleneimine in an amount ranging from 0.5 mg / mL to 2.0 mg / mL, such as in an amount ranging from 0.5 mg / mL to 1.5 mg / mL, such as in an amount ranging from 0.75 mg / mL to 1.4 mg / mL, or such as in an amount ranging from 1 mg / mL to 1.2 mg / mL.

[0158] The cationic polymer, such as polyethyleneimine, may be dissolved in an aqueous solvent, such as nuclease-free water or a buffer solution (e.g., a citrate buffer), to form the polymer solution 18. After dissolving the cationic polymer in the aqueous solvent, the polymer solution 18 may be adjusted to a neutral pH, such as a pH of approximately 7. The pH of thepolymer solution 18 may be adjusted by titrating the polymer solution 18 using an acid (e.g., hydrochloric acid) and / or a base (e.g., sodium hydroxide).

[0159] Alternatively, the cationic polymer, such as polyethyleneimine, may be obtained from the manufacturer in solution form. If necessary, this solution may be diluted to comprise the polyethyleneimine in an amount of less than or equal to 2.0 mg / mL. For example, this solution may be diluted to comprise the polyethyleneimine in an amount of less than or equal to 1.5 mg / mL. For example, this solution may be diluted to comprise the polyethyleneimine in an amount ranging from 0.5 mg / mL to 2.0 mg / mL, such as in an amount ranging from 0.5 mg / mL to 1.5 mg / mL, such as in an amount ranging from 0.75 mg / mL to 1.4 mg / mL, or such as in an amount ranging from 1 mg / mL to 1.2 mg / mL. This solution may be diluted in an aqueous solvent, such as nuclease-free water or a buffer solution (e.g., a citrate buffer). The pH of this solution may be adjusted to a neutral pH, such as a pH of 7, if necessary.

[0160] Following the addition of the polymer solution 18 to the therapeutic agent containing solution 16, the polymer solution 18 and the therapeutic agent containing solution 16 are mixed to form an electrostatic complex solution 20. The polymer solution 18 and the therapeutic agent containing solution 16 may be mixed for a time to sufficiently mix the polymer solution 18 and therapeutic agent containing solution 16. For example, the polymer solution and the therapeutic agent containing solution 16 may be mixed for less than or equal to 1 minute, or less than or equal to 30 seconds.

[0161] The addition of the polymer solution 18 to the therapeutic agent containing solution 16 forms an electrostatic complex, which forms between the nucleic acid-based therapeutic agent 12, having negative charges, and the cationic polymer, having positive charges.

[0162] The polymer solution 18 and the therapeutic agent containing solution 16 may be added together to provide the electrostatic complex solution 20 with a nitrogen-to-phosphate (or phosphorus) (N / P) ratio of at least 6:1, wherein the N / P ratio is determined by dividing the number of nitrogen atoms of the polymer in the polymer solution 18 by the number of phosphorus atoms in the nucleic acid-based therapeutic agent 12. For example, a N / P ratio of 6:1 corresponds to a weight ratio of nucleic acid-based therapeutic agent to cationic polymer of approximately 1:0.8.

[0163] For example, the polymer solution 18 and the therapeutic agent containing solution 16 may be added together to provide the electrostatic complex solution 20 with a N / P ratio in a range of from 6:1 to 20:1. For example, the polymer solution 18 and the therapeutic agent containing solution 16 may be added together to provide the electrostatic complex solution 20 with a N / P ratio in a range of from 7:1 to 19:1. For example, the polymer solution 18 and thetherapeutic agent containing solution 16 may be added together to provide the electrostatic complex solution 20 with a N / P ratio ranging from 8:1 to 17:1. For example, the polymer solution 18 and the therapeutic agent containing solution 16 may be added together to provide the electrostatic complex solution 20 with a N / P ratio ranging from 9:1 to 12:1. For example, the polymer solution 18 and the therapeutic agent containing solution 16 may be added together to provide the electrostatic complex solution 20 with a N / P ratio ranging from 9:1 to 10:1.

[0164] Following mixing, the polymer solution 18 and the therapeutic agent containing solution 20 may be incubated for a time of not greater than 20 minutes, such as a time of not greater than 15 minutes, to form the electrostatic complex solution 20. This incubation time promotes the formation of the electrostatic complex between the nucleic acid-based therapeutic agent 12 and the cationic polymer.

[0165] The method 10 comprises adding a zinc solution 22 to the electrostatic complex solution 20. The zinc solution 22 comprises a hydrated zinc salt and a second alkyl alcohol solvent.

[0166] The hydrated zinc salt may be any zinc salt that provides the zinc solution with zinc (II) ions (i.e., Zn2+ions). For example, the hydrated zinc salt may comprise zinc nitrate hexahydrate, zinc acetate dihydrate, zinc sulfate hydrate, zinc perchlorate hydrate, zinc chloride hydrate, zinc iodide hydrate, zinc acetylacetonate hydrate, or combinations thereof.

[0167] The second alkyl alcohol solvent may be any of the alkyl alcohol solvents described herein with respect to the first alkyl alcohol solvent. For example, the second alkyl alcohol solvent may comprise methanol. For example, the second alcohol solvent may comprise ethanol. For example, the second alcohol solvent may comprise propanol. For example, the second alcohol solvent may comprise isopropyl alcohol.

[0168] The second alkyl alcohol solvent may be the same as the first alkyl alcohol solvent. For example, the first alkyl alcohol solvent and second alkyl alcohol solvent may each independently comprise ethanol.

[0169] Alternatively, the second alkyl alcohol solvent may be different from the first alkyl alcohol solvent. For example, the first alkyl alcohol solvent may comprise methanol and second alkyl alcohol solvent may comprise ethanol. Alternatively, the first alkyl alcohol solvent may comprise ethanol and second alkyl alcohol solvent may comprise methanol.

[0170] The zinc solution 22 may be prepared by dissolving the hydrated zinc salt in the second alkyl alcohol solvent.

[0171] The zinc solution 14 may comprise the hydrated zinc salt in a concentration of not greater than 80 mM. For example, the zinc solution 22 may comprise the hydrated zinc salt ina concentration of not greater than 70 mM. For example, the zinc solution 22 may comprise the hydrated zinc salt in a concentration of not greater than 60 mM. For example, the zinc solution 22 may comprise the hydrated zinc salt in a concentration of less than or equal to 50 mM. For example, the zinc solution 22 may comprise the hydrated zinc salt in a concentration of less than or equal to 40 mM.

[0172] In some non-limiting embodiments, the zinc solution 22 comprises zinc nitrate hexahydrate and ethanol, wherein the zinc nitrate hexahydrate is dissolved in the ethanol.

[0173] Following the addition of the zinc solution 22 to the electrostatic complex solution 20, the method 10 comprises mixing the zinc solution 22 and electrostatic complex solution 20 to form a metal-organic framework nanoparticle precursor solution 24. The zinc solution and the electrostatic complex solution 20 may be mixed for a time to sufficiently mix the zinc solution 22 and the electrostatic complex solution 22. For example, the zinc solution 22 and the electrostatic complex solution 20 may be mixed for less than or equal to 1 minute, or less than or equal to 30 seconds.

[0174] The method 10 comprises incubating the metal-organic framework nanoparticle precursor solution 24 to form the metal-organic framework nanoparticles comprising the nucleic-acid based therapeutic agent 26. For example, the zinc solution 22 and the electrostatic complex solution 24 may be incubated for a time of at least 4 hours. The zinc solution 22 and the electrostatic complex solution 24 may be incubated for a time ranging from 1 hour to 4 hours, such as a time ranging from 1.5 hours to 3 hours, or such as a time ranging from 1.5 hours to 2.5 hours. For example, the zinc solution 22 and the electrostatic complex solution 24 may be incubated for a time of at least 2 hours.

[0175] The addition of the zinc solution 22 to the electrostatic complex solution 20 to form the metal-organic framework nanoparticle precursor solution 24 and the incubation of the metal-organic framework nanoparticle precursor solution to form the metal-organic framework nanoparticles comprising the nucleic-acid based therapeutic agent 26 is a one-pot coprecipitation method. The electrostatic complex formed between the nucleic acid-based therapeutic agent 12 and the cationic polymer is encapsulated by the metal-organic framework to generate polymer core-metal-organic framework shell nanoparticles.

[0176] When the zinc solution 22 comprises zinc nitrate hexahydrate in ethanol and the imidazole solution comprises 2-methylimidazole in ethanol, the metal-organic framework of the metal-organic framework nanoparticles comprising the nucleic acid-based therapeutic agent 26 may be zeolitic imidazole framework-8 (ZIF-8).

[0177] The method 10 may further comprise isolating the metal-organic framework nanoparticles comprising the nucleic-acid based therapeutic agent 26 by centrifugation.

[0178] The method 10 may further comprise washing the isolated metal-organic framework nanoparticles comprising the nucleic-acid based therapeutic agent 26 with an alkyl alcohol solvent, such as methanol or ethanol, to remove any unbound components. The isolated metalorganic framework nanoparticles comprising the nucleic-acid based therapeutic agent 26 may be washed one time with an alkyl alcohol solvent or may be washed two times with an alkyl alcohol solvent.

[0179] The method 10 may further comprise storing the metal-organic framework nanoparticles comprising the nucleic-acid based therapeutic agent 26 at a temperature of at least -10°C, such as at least -20°C, such as at least 30°C, such as at least -40°C, such as at least -50°C, such as at least -60°C, such as at least -70°C, or such as at least -80°C.

[0180] Metal-organic framework nanoparticles 30 comprising the nucleic acid-based therapeutic agent are shown in FIG. 2. The metal-organic framework nanoparticles 30 comprise a metal-organic framework 32, such as zeolitic imidazole framework-8 (ZIF-8), a cationic polymer, and the nucleic acid-based therapeutic agent. The cationic polymer and the nucleic acid-based therapeutic agent form an electrostatic complex 34, wherein the metalorganic framework 32, such as ZIF-8, encapsulates the electrostatic complex 34. The cationic polymer may be any of the cationic polymers described herein. The nucleic-based therapeutic agent may be any of the nucleic-acid based therapeutic agents described herein.

[0181] The metal-organic framework nanoparticles 30 comprising the nucleic acid-based therapeutic agent may be formed according to any of the methods described herein. For example, the ZIF-8 metal-organic framework may be formed through the combination zinc nitrate hexahydrate in the zinc solution 22 and 2-methylimidazole in the imidazole solution 14, wherein both the zinc solution 22 and the imidazole solution include ethanol.

[0182] The metal-organic framework 32, such as ZIF-8, of the metal-organic framework nanoparticles 30 may further encapsulate an alkyl alcohol solvent, such as ethanol, with the electrostatic complex 34. For example, the metal-organic framework 32, such as ZIF-8, of the metal-organic framework nanoparticles 30, may encapsulate the first alkyl alcohol solvent and the second alkyl alcohol solvent as described herein. For example, when the zinc solution 22 and the imidazole solution 14 both comprise ethanol as the alkyl alcohol solvent, the metalorganic framework nanoparticles 30 comprising the nucleic acid-based therapeutic agent maycomprise ethanol and the electrostatic complex 34 encapsulated within the metal-organic framework 32, such as ZIF-8.

[0183] The metal-organic framework nanoparticles 30 comprising the nucleic acid-based therapeutic agent may comprise a diameter in a range of from 40 nm to 400 nm, such as from 50 nm to 300 nm, such as from 60 nm to 250 nm, or such as from 70 nm to 200 nm.

[0184] The metal-organic framework nanoparticles 30 comprising the nucleic acid-based therapeutic agent may be suspended in a solvent to form a metal-organic framework nanoparticle solution. The metal-organic framework nanoparticles 30 comprising the nucleic acid-based therapeutic agent of the metal-organic framework nanoparticles comprises a metalorganic framework, such as zeolitic imidazole framework-8 (ZIF-8), a cationic polymer, and the nucleic acid-based therapeutic agent, wherein the cationic polymer and the nucleic acidbased therapeutic agent form an electrostatic complex, and wherein the metal-organic framework, such as ZIF-8, encapsulates the electrostatic complex. The cationic polymer may be any of the cationic polymers described herein. The nucleic acid-based therapeutic agent may be any of nucleic acid-based therapeutic agent described herein.

[0185] The metal-organic framework nanoparticles 30 comprising the nucleic acid-based therapeutic agent of the metal-organic framework nanoparticle solution may be formed according to any of the methods described herein. For example, the ZIF-8 metal-organic framework may be formed through the combination zinc nitrate hexahydrate in the zinc solution 22 and 2-methylimidazole in the imidazole solution 14, wherein both the zinc solution 22 and the imidazole solution include ethanol.

[0186] The solvent of the metal-organic framework nanoparticle solution may be any solvent that does not dissolve the metal-organic framework nanoparticles comprising the nucleic acid-based therapeutic agent. For example, the solvent may be an alkyl alcohol solvent, such as ethanol. Alternatively, the solvent may be an aqueous solvent, such as nuclease-free water or a buffer solution, such as phosphate buffered saline. Alternatively, the solvent may be a mixture of an alkyl alcohol solvent and an aqueous solvent. For example, the solvent may be a mixture of ethanol and phosphate buffered saline, such as a mixture of 5 volume percent (vol. %) and ethanol and 95 vol. % phosphate buffered saline.

[0187] The metal-organic framework nanoparticles comprising the nucleic acid-based therapeutic agent may be incorporated into a dosage form. The dosage form comprises the metal-organic framework nanoparticles comprising the nucleic acid-based therapeutic agent and a pharmaceutically-acceptable carrier.

[0188] The metal-organic framework nanoparticles comprising the nucleic acid-based therapeutic agent of the dosage form may be any of the metal-organic framework nanoparticles comprising the nucleic acid-based therapeutic agent described herein and may be prepared according to any of the methods described herein.

[0189] As used herein, a “dosage form” is a physical form of a dose of a composition used as a drug or medication intended for administration or consumption. A dosage form corresponds to a unit dosage form, even when dispensed from a drug product that includes multiple doses.

[0190] The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates, poly anhydrides, and / or vinyl polymers, such as polyethylene, polypropylene, polystyrene, polyvinylpyrrolidone, and polyvinyl chloride; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, Pa., 21st Edition (2005), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compositions, such as a chimeric virus, and additional pharmaceutical agents.

[0191] The metal-organic framework nanoparticles comprising the nucleic acid-based therapeutic agent may be compounded or otherwise manufactured into a suitable composition for use, such as a pharmaceutical dosage form or drug product in which the metal-organic framework nanoparticles comprising the nucleic acid-based therapeutic agent is the active ingredient.

[0192] Depending on the delivery route, the dosage form may comprise additional excipients. An “excipient” is an inactive substance used as a carrier for the active ingredients of a medication. Although “inactive”, excipients may facilitate and aid in increasing the delivery, stability or bioavailability of an active ingredient in a drug product. Non-limiting examples of useful excipients include: anti- adherents, binders, rheology modifiers, coatings, disintegrants, emulsifiers, oils, buffers, salts, acids, bases, fillers, diluents, solvents, flavors, colorants, glidants, lubricants, preservatives, antioxidants, sorbents, vitamins, sweeteners, etc., as are available in the pharmaceutical / compounding arts.

[0193] The dosage form may be formulated as a parenteral dosage form. The dosage form be suitable for use as an intravenous formulation. Alternatively, the dosage form may be suitable for use as a subcutaneous, intramuscular, intratumoral, or other suitable formulation.

[0194] The dosage form may be provided as a unit-dose or multi-dose containers, for example in sealed ampoules and / or vials. The unit-dose or multi-dose containers may comprise frozen metal-organic framework nanoparticles comprising the nucleic acid-based therapeutic agent and the pharmaceutically-acceptable carrier may be added immediately prior to use. Alternatively, the unit-dose or multi-dose containers may comprise a frozen metal-organic framework nanoparticle solution, wherein the solvent of the metal-organic framework nanoparticle solution is a pharmaceutically-acceptable carrier.

[0195] Also provided herein is a method of delivering a nucleic acid-based therapeutic agent to a patient. The method comprises administering to the patient an effective amount of a metal-organic framework nanoparticle solution. The metal-organic framework nanoparticle solution comprises metal-organic framework nanoparticles and a pharmaceutically-acceptable carrier. The metal-organic framework nanoparticles comprise a metal-organic framework, such as zeolitic imidazole framework-8 (ZIF-8), a cationic polymer, and the nucleic acid-based therapeutic agent, wherein the cationic polymer and the nucleic acid-based therapeutic agent form an electrostatic complex, and wherein the ZIF-8 encapsulates the electrostatic complex; and a pharmaceutically-acceptable carrier. The cationic polymer may be any of the cationic polymers described herein. The nucleic acid-based therapeutic agent may be any of the nucleicacid-based therapeutic agents described herein. The pharmaceutically-acceptable carrier may be any of the pharmaceutically-acceptable carriers described herein.

[0196] The metal-organic framework nanoparticles comprising the nucleic acid-based therapeutic agent may be any of the metal-organic framework nanoparticles comprising the nucleic acid-based therapeutic agent described herein and may be prepared according to any of the methods described herein. The metal-organic framework nanoparticle solution may be any of the metal-organic framework nanoparticle solutions described herein.

[0197] As used herein, the term “patient” or “subject” refer to members of the animal kingdom including but not limited to human beings and “mammal” refers to all mammals, including, but not limited to human beings.

[0198] The metal-organic framework nanoparticle solution may prevent, treat, or ameliorate a disease. As used herein, “preventing” a disease refers to inhibiting the full development of a disease or infection, from a subsequent exposure, “treating” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop, and “ameliorating” refers to the reduction in the number or severity of one or more signs or symptoms of a disease or infection.

[0199] The metal-organic framework nanoparticle solutions described herein may be formulated as a vaccine. A “vaccine” refers to a preparation of immunogenic material capable of stimulating an immune response, administered for the prevention, inhibition, amelioration, or treatment of infections, such as infections caused by a virus, such as a coronavirus, e.g. SARS-CoV-2, infections, or other types of disease. The immunogenic material may include attenuated or inactivated (killed) microorganisms (such as bacteria or viruses), or antigenic proteins, peptides, or DNA derived from them. In non-limiting embodiments, the immunogenic material is a nucleic acid-based therapeutic agent as described herein. An attenuated virus is a virulent organism that has been modified to produce a less virulent form, but nevertheless retains the ability to elicit antibodies and cell-mediated immunity against the virulent form. An inactivated (killed) virus is a previously virulent organism that has been inactivated with chemicals, heat, or other treatment, but elicits antibodies against the organism. Vaccines may elicit both prophylactic (preventative or protective) and therapeutic responses. Methods of administration vary according to the vaccine, but may include inoculation, ingestion, inhalation or other forms of administration. Vaccines may be administered with an adjuvant to boost the immune response.

[0200] As used herein, administering a composition (e.g., an immunogenic composition, such as a vaccine) to a subject means to give, apply or bring the composition into contact withthe subject. Administration can be accomplished by any of a number of routes, such as, for example, topical, oral, subcutaneous, intradermal intramuscular, transdermal, mucosal, intraperitoneal, intravenous, intrathecal, and intramuscular.

[0201] A therapeutically effective amount of vaccine is an amount sufficient to increase resistance to, prevent, ameliorate, and / or treat infection caused by a virus in a subject without causing a substantial cytotoxic effect in the subject. The effective amount of a vaccine useful for increasing resistance to, preventing, ameliorating, and / or treating infection in a subject will be dependent on, for example, the subject being treated, the manner of administration of the metal-organic framework nanoparticles, and other factors. Based on the teachings provided herein, one of ordinary skill can readily ascertain effective amounts of the elements of the described dosage form and produce a safe and effective dosage form and drug product.

[0202] For example, an effective amount of a virus vaccine is an amount useful for eliciting an immune response in a subject and / or for preventing infection by the virus.

[0203] A non-limiting range for an effective amount of the disclosed metal-organic framework nanoparticle solution comprising the nucleic acid-based therapeutic agent prepared according to the methods described herein is about 0.0001 mg / kg body weight to about 10 mg / kg body weight, such as about 0.01 mg / kg, about 0.02 mg / kg, about 0.03 mg / kg, about 0.04 mg / kg, about 0.05 mg / kg, about 0.06 mg / kg, about 0.07 mg / kg, about 0.08 mg / kg, about 0.09 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, or about 10 mg / kg, for example, 0.01 mg / kg to about 1 mg / kg body weight, about 0.05 mg / kg to about 5 mg / kg body weight, about 0.2 mg / kg to about 2 mg / kg body weight, or about 1.0 mg / kg to about 10 mg / kg body weight. In some embodiments, the dosage includes a set amount of a disclosed metal-organic framework nanoparticle solution such as from about 1 pg to about 300 pg, for example, a dosage of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or about 300 pg.

[0204] The actual dosage of the metal-organic framework nanoparticle solution comprising the metal-organic framework nanoparticles comprising the nucleic acid-based therapeutic agent prepared according to the methods described herein will vary according to factors such as the disease indication and particular status of the subject (for example, the subject's age, size, fitness, extent of symptoms, susceptibility factors, and the like), time and route of administration, other drugs or treatments being administered concurrently, as well as the specific pharmacology of the composition for eliciting the desired activity or biological 1response in the subject. Dosage regimens can be adjusted to provide an optimum prophylactic or therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental side effects of the disclosed metal-organic framework nanoparticle solution and / or other biologically active agent is outweighed in clinical terms by therapeutically beneficial effects.

[0205] The metal-organic framework nanoparticle solution may be formulated as primeboost vaccines and / or booster vaccines. A prime-boost vaccination refers to an immunotherapy including administration of a first immunogenic composition (the primer vaccine) followed by administration of a second immunogenic composition (the booster vaccine) to a subject to elicit an immune response. The primer vaccine and / or the booster vaccine includes the metal-organic framework nanoparticle comprising the nucleic acid-based therapeutic agent, wherein the nucleic acid-based therapeutic agent is selected to express the antigen to which the immune response is directed. The booster vaccine is administered to the subject after the prime-boost vaccine; the skilled artisan will understand a suitable time interval between administration of the prime-boost vaccine and the booster vaccine, and examples of such timeframes are disclosed herein. In some embodiments, the prime-boost vaccine, the booster vaccine, or both prime-boost vaccine and the booster vaccine additionally include an adjuvant.

[0206] The prime-boost and booster immunizations may deliver the same nucleic acidbased therapeutic agent by the same route, as is commonly performed.

[0207] The following Examples illustrate various embodiments of the invention. However, it is to be understood that the invention is not limited to these specific embodiments.EXAMPLESExample 1

[0208] The encapsulation synthesis development, integration, and delivery of mRNA with ZIF-8 was investigated. A one-pot co-precipitation method was employed. The stability of the mRNA and its expression in vitro was systematically evaluated, considering the impact of ZIF- 8 synthesis conditions, ZIF-8 incubation, and encapsulation within ZIF-8.

[0209] A ZIF-8-based polymer composite mRNA delivery system, which involves initially complexing mRNA with polyethyleneimine (PEI) in a 2-methyl imidazole (2-MeIM) precursor solution, followed by zinc addition and encapsulation within ZIF-8 to form mRNA-PEI@ZIF- 8 (mRPZ) nanoparticles was completed, as shown in FIG. 3A. Characterization of the mRPZ nanoparticles confirmed the encapsulation of complexed mRNA within ZIF-8. The polymer complex core-MOF shell particle retained and delivered mRNA, producing eGFP expression in vitro, as shown in FIG. 3B, and producing firefly luciferase expression in vivo, as shown inFIG. 3C. Comparative analyses were performed against commercial transfection reagents and mRNA-PEI alone. Improved performance over mRNA-PEI, delayed expression kinetics, and the stabilization of mRNA complexes within mRPZ nanoparticles, with viable transfection maintained even after 3 months of room-temperature storage in vitro and 1 month in vivo, were observed.Materials and. Methods

[0210] Preparation ofZEF-8: Solutions of zinc nitrate hexahydrate (Zn(NO3)2-6H2O; Alfa Aesar, Haverhill MA) and 2-methylimidazole (2-MeIM; Sigma Aldrich, St. Louis MO) were prepared separately by dissolving 0.3 grams (g) and 0.66 g, respectively, in 14.3 milliliters (mL) methanol (Sigma Aldrich, St. Louis MO). The two solutions were mixed and stirred vigorously for 60 minutes. The particles were isolated by centrifugation at 10,000 x g (relative centrifugal force) for 10 minutes. The particles were washed with methanol two times. The resultant ZIF-8 particles were vacuum dried at 75 degrees Celsius (°C) overnight.

[0211] Preparation ofmRNA@ZEF-8: Precursor solutions of zinc nitrate hexahydrate (11.9 milligrams per milliliter (mg / mL)) and 2-methylimidazole (13.15 mg / mL) were separately prepared in methanol, ethanol (200 Proof; Pharmco, Toronto Ontario), or nuclease-free water (Invitrogen, Waltham MA). mRNA (1 mg / mL), 1 microgram (pg), was aliquoted into a centrifuge tube and mixed with 10 microliters (pL) of the 2-methylimidazole solution. 10 pL of the zinc nitrate hexahydrate solution was added and mixed for 30 seconds. The solution was incubated at room temperature for 2 hours. Particles were isolated by centrifugation at 16,000 x g for 5 minutes and washed with ethanol two times. Particles were stored at -80°C until use.

[0212] Preparation of mRNA-PEI@ZIF-8 (mRPZ): Precursor solutions of zinc nitrate hexahydrate (11.9 mg / mL) and 2-methylimidazole (13.15 mg / mL) were separately prepared in ethanol. mRNA (1 mg / mL), 1 pg, was aliquoted into a centrifuge tube and mixed with 10 pL of the 2-methylimidazole solution. After thoroughly mixing the mRNA, 1 pL of linear 20 kDa polyethyleneimine (PEI) (1-2 mg / mL in nuclease-free water; Sigma Aldrich (St. Louis, MO)) was added and mixed thoroughly. This solution was allowed to incubate for 15 minutes. After incubation, 10 pL of the zinc nitrate hexahydrate solution was added and mixed for 30 seconds. The solution was incubated at room temperature for 2 hours. Particles were isolated by centrifugation at 16,000 x g for 5 min and washed with ethanol two times. Particles were stored at -80°C until use. This procedure was also scaled using 24 pg of mRNA, maintaining the same weight ratios and precursor volumes.

[0213] Gel Electrophoresis: Equal volumes of samples were mixed with RNA loading dye (New England Biolab (Ipswich, MA)). The samples containing PEI were additionally treatedwith 4 pL of heparin (25 mg / mL of heparin sulfate, Sigma Aldrich (St. Louis, MO)). Samples were heated at 60°C for 10 min. A native 1% agarose (Fisher Scientific (Pittsburgh, PA)) gel was prepared in lx Tris-Borate Buffer (Invitrogen (Waltham, MA)) with a 1:10,000 dilution of SyberSafe (Invitrogen (Waltham, MA)). Samples were loaded into the gel and a potential of 80 Volts (V) was applied for 45 minutes. Gels were imaged using a BioRad ChemiDock imaging system.

[0214] RNA Loading Efficiency: Samples were run using gel electrophoresis. ssRNA ladders were purchased from New England Biolab (Ipswich, MA). The resulting image provided by the ChemiDock imaging system was analyzed using ImageJ software. The RNA band intensity, which was correlated with the amount of RNA, was quantified with ImageJ. A standard curve was used to determine the amount of RNA loaded. Loading efficiency was also determined using a Quantifluor RNA assay kit (Promega (Madison, WI)). Samples were first exfoliated with 100 mM ethylenediaminetetraacetic acid (EDTA; Ambeed (Arlington, IL)) (pH = 4.5) to prevent mRNA degradation and then treated with 100 pg of heparin. Samples were heated at 60°C for 10 minutes. The Quantifluor assay solution was prepared per the provided protocol. Samples (20 pL) were added to the assay solution and evaluated with a plate reader (SpectraMax i3) with 492 nanometer (nm) excitation and 540 nm emission.

[0215] RNase Treatment: Samples (approximately 0.5 pg RNA) were mixed with 0.85 pg of RNase A (New England Biolab (Ipswich, MA)) diluted in PBS and incubated at 37°C for 2 hours. Samples were centrifuged at 16,000 x g for 5 minutes and the supernatant was removed and then washed with lx phosphate buffered saline (PBS). One sample at a time, 10 pL of RNA loading dye was added to each MOF-containing sample and then 10 pL of 100 mM EDTA was immediately added with 4 pL of heparin sulfate (25 mg / mL). Samples were heated to 60°C for 10 min. Samples were run on a native, Sybersafe, 1% agarose gel at 80V for 40 minutes. The gel image was obtained using a Bio-Rad ChemiDock imager.

[0216] Scanning Electron Microscopy (SEM) and Energy-Dispersive X-ray Spectroscopy (EDS): Samples were suspended in ethanol, drop cast onto a silicon wafer, and mounted on an SEM stub using carbon tape. Samples were allowed to dry at room temperature. The samples were sputter-coated with 2 nm of platinum. Sample images were taken at 30 kilovolts (kV) and a working distance of 10 mm or less on a Tescan Mira3. EDS was performed at 10 kV, 3.2 nanoamperes (nA) current, and a working distance of 12 millimeters (mm) using a Thermofisher FEI Apreo equipped with APEX EDS.

[0217] Transmission Electron Microscopy (TEM), SEM, and EDS: Samples suspended in ethanol were drop-cast onto a mesh carbon support film with copper and dried. TEM sampleimages were taken using an FEI Tecnai F20 TEM / STEM or Thermo Fisher Themis 200 G3 Aberration Correction STEM at 80 kV with a spot size of 6. EDS / STEM was performed using the Thermo Fisher Themis with Super-X EDS detector at 200 kV and spot size 6. Element K- lines were used.

[0218] Fourier Transform Infrared (FTIR) Spectroscopy: FTIR was used to confirm the formation of MOF nanoparticles and determine functional groups of final components using a Perkin Elmer Frontier vibrational spectrophotometer (Spectrum 100, PerkinElmer Inc., USA). The particles were first lyophilized and then measured at room temperature in a wavenumber range of 4000-600 cm'1with a resolution of 2 cm-1against a potassium bromide (KBr) background.

[0219] Dynamic Light Scattering and Nanoparticle Tracking Analysis (NTA): Samples were resuspended in 1 mL of ethanol and sonicated. The zeta potential and particle size were determined using a Malvern Zetasizer. Samples for NTA were suspended in 10 pL ethanol and diluted in PBS and run on a ZetaView instrument by Particle Metrix.

[0220] Powder X-ray Diffraction: Samples were suspended in ethanol and drop-cast onto a silicon wafer. Samples were allowed to dry at room temperature. The crystalline structure of the particles was obtained using Malvern Panalytical Empyrean XRD with Cu anode K-alpha 1 = 1.540 at 45 mV and 40 mA.

[0221] Thermo gravimetric Analysis: Samples were resuspended in 100 pL of ethanol and sonicated to ensure that most of the sample was removed from the tube. The suspended sample was added to a crucible and run using a Perkin Elmer TGA 4000 under a nitrogen atmosphere. Samples were heated to 50°C and held at 50°C for 30 minutes to remove solvent. The sample was then ramp scanned from 50°C to 800°C at 10°C per minute (°C / min).

[0222] RNA Molecular Probing: Quantifluor dye was diluted in lx PBS 1:400 and 180 pL were aliquoted on a 96-well plate. Samples were resuspended in 5 pL ethanol and sonicated. Samples were treated accordingly: EDTA treated: 5 pL of 100 mM EDTA + 95 pL PBS; Heparin treated: 5 pL of heparin (25 mg / mL) + 95 pL PBS; and Dual treated: 5 pL of EDTA + 5 pL heparin + 90 pL PBS. Any samples treated with heparin were heated at 60°C for 10 min. After EDTA / heparin treatment, 20 pL were collected as the suspension samples, then the rest of the sample was centrifuged at 16,100 x g for 5 minutes. 20 pL of the supernatant was collected. 20 pL of the sample was added to a well with 180 pL of the Quantifluor dye solution, mixed well, and incubated on a shaker for 5 min. Resultant fluorescence was evaluated with a plate reader (SpectraMax i3) using 492 nm excitation and 540 nm emission.

[0223] Cell Culture: Human embryonic kidney (HEK-293t) cells and Chinese Hamster Ovary (CHO) cells were provided by the Ren Lab at Carnegie Mellon University. Human Cervical Cancer (HeLa) cells were provided by the Wayne Lab at Carnegie Mellon University. A549, MDA-MB-231, and RAW264.7 cells were obtained from the American Type Culture Collection (ATCC). All cell lines, except CHO, were cultured with DMEM (Cytiva (Marlborough, MA)) supplemented with 10% fetal bovine serum (LBS; VWR (Radnor, PA)) and Antibiotic- Antimycotic (Gibco (Grand Island, NY)). CHO cells were cultured in E12 / DMEM (1:1) (Gibco (Grand Island, NY)) supplemented with 10% BBS and Antibiotic- Antimycotic. The cells were kept in a 100% humified atmosphere containing 5% CO2 at 37°C. ATCC protocols were followed for culturing and handling.

[0224] Transfection: The particles were resuspended in 10 pL of ethanol and sonicated for 5 min then resuspended in Opti-MEM (Gibco (Grand Island, NY)) to a final concentration of 10 ng mRNA / pL. The particles were added to cells in a 96-well plate using 100 nanograms (ng) per well. Lipofectamine 2000 (Invitrogen (Waltham, MA)) and Lipofectamine Messenger Max (Lipo MM; Invitrogen (Waltham, MA)) were prepared per the manufacturer's protocol.

[0225] mRNA-PEI complexes were prepared by suspending mRNA and PEI separately in Opti-MEM then mixing both solutions and incubating for 15 mins.

[0226] Cells were incubated with particles for 48 hours, fixed using 4% paraformaldehyde (Thermo Scientific (Waltham, MA)) in PBS, permeabilized with a 0.1% triton X-100 solution (Sigma Aldrich (St. Louis, MO)), and stained with DAPI (Sigma Aldrich (St. Louis, MO)). A lipofectamine-positive control was used. The fluorescence intensity was measured using a SpectraMax i3 plate reader at 475 / 515 nm for eGFP and 350 / 460 nm for DAPI. Imaging was performed using a Thermofisher EVOS M7000 with a DAPI, Cy5, and GFP filter cube. Due to Cy5 interrupting the expression of eGFP, EZ CAP™ Cy5-eGFP-mRNA (5mo UTP) (APExBIO (Huston, TX) was mixed with untagged eGFP mRNA (5moU; Tri-Link (San Diego CA)) in a 1:10 ratio.

[0227] Flow cytometry: Two days post-transfection, media was removed. Cells were washed with PBS and then incubated in trypsin (Gibco (Grand Island, NY)) for 20-30 min. After incubation, PBS was added to each well, and cells were gently resuspended. Cells were moved to a 96-well u-bottom plate and centrifuged at 300 x g for 5 min. The supernatant was removed, and the pellet was resuspended in PBS containing a 1 : 10,000 dilution of Live / Dead™ Fixable Blue (Invitrogen (Waltham, MA)) and incubated for 15 min on ice in the dark. The plate was centrifuged again at 300 x g for 5 minutes and the staining buffer was removed. Cells were fixed with 4% paraformaldehyde in PBS on ice for 10 min. The cells were centrifuged at300 x g for 5 min and washed 2x with PBS. Cells were resuspended in flow buffer and run on a NovoCyte Flow Cytometer.

[0228] Cell Metabolic Activity: HEK293t cells were plated at a seed density of 30,000 cells per well in a black-walled, clear bottom 96-well plate, phenol-red free media was used to reduce background, and one column of wells was left without cells. Resazurin was prepared for a final working concentration of 44 micromolar (pM). Cells were treated with mRPZ, ZIF- 8, linear 20 kDa PEI, and lipid particles and allowed to incubate for 4-6 hours. One column of wells was treated with 70% ethanol to kill all the cells. After incubation, resazurin solution was added to the well, and the fluorescence (excitation 560 nm, emission 590 nm) was measured between 4-24 hours after adding resazurin on the SpectraMax i3 plate reader.

[0229] Time to Expression Assay: The particles were resuspended in 10 pL of ethanol and sonicated for 5 min then resuspended in Opti-MEM to a final concentration of 10 ng mRNA / pL. The particles were added to cells in a 96-well plate using 100 ng per well. Lipofectamine 2000 (Invitrogen (Waltham, MA)) and Lipofectamine Messenger Max (Invitrogen (Waltham, MA)) were prepared per the manufacturer's protocol.

[0230] mRNA-PEI complexes were prepared by suspending mRNA and PEI separately in Opti-MEM then mixing both solutions and incubating for 15 mins.

[0231] Cells were maintained in phenol red-free medired-free 0% FBS. Fluorescence was measured at different time points using a SpectraMax i3 plate reader at 475 / 515 nm.

[0232] Thermal Storage: Samples were prepared as noted above. Samples were dried using a DNA drier at room temperature for 1 hour and stored closed under a vacuum in descant. After a set amount of time, samples were resuspended and used to transfect cells. RNA and exfoliated (Ex) RNA from ZIF-8 were transfected using lipofectamine Messenger Max. Lipofectamine 2000 and lipofectamine Messenger Max were prepared with RNA stored at -80°C as a control. Gel electrophoresis was performed on stored samples as previously described.

[0233] In vivo mRNA delivery and efficiency: Six- to 8-week-old Balb / c mice were purchased from Charles River and allowed to acclimate to vivarium conditions for at least 3 days before experiments were performed. Mice were anesthetized and injected in the retro- orbital vein with mRPZ particles generated with Firefly Luciferase expressing mRNA (5moU; Tri-Link (San Diego CA). Before injection, particles were suspended in 5 pL of 200 proof ethanol and sonicated, then DPBS was added stepwise, with repeated sonication, until a final volume of 170 pL was achieved.

[0234] The PEI control was prepared by complexing mRNA and linear 20 kDa PEI in a 5% sterile glucose solution for 15 min followed by retro-orbital injections. In vivo, JetRNA+ (Polyplus (Illkirch-Graffenstaden, France)) was prepared per the manufacturer's protocol.

[0235] Luciferase expression kinetics were collected starting at 3 hours post-injection. Mice were injected with 110 pL of D-Luciferin (30 mg / mL in DPBS; Gold Biotechnology (St. Louis, MO)) and luminescence was measured 10 to 15 minutes post-injection using IVIS® (PerkinElmer, Waltham, MA). Particle bioavailability and biodistribution were assessed by sacrificing the mice after 12 and 6 hours, respectively, and harvesting the heart, lungs, spleen, pancreas, kidneys, and liver. The organs’ fluorescence and luminescence were measured using the IVIS® system. Blood was collected from mice, then serum was isolated and assessed for nitrogen urea, creatine, alkaline phosphatase, aspartate transaminase, alanine transaminase, and zinc concentration. Blood assays for creatine (ECRT- 100), alkaline phosphatase (QFAP-100), aspartate transaminase (EASTR-100), alanine transaminase (EALT- 100), and zinc concentration (DIZN-250) were obtained from BioAssay Systems (Hayward, CA). A Urea Nitrogen (Bun) assay detection kit was obtained from Arbor Assays (Ann Arbor, MI).

[0236] Graphing and Statistical Analysis: Graphing and statistical analyses were performed using Prism GraphPad vlO. Statistical analysis was performed by first removing outliers, testing for data normality, and using appropriate comparison tests as applicable (i.e., one-way ANOVA, Dunn’s multiple comparisons, and two-way ANOVA). Bars shown on graphs are standard error mean unless noted.Results and Discussion

[0237] Recognizing the delicate nature of mRNA, synthetic conditions for ZIF-8 encapsulation were identified that preserve mRNA stability, considering mRNA length and expressibility in vitro. mRNA length using a native agarose gel was assessed and expressibility was evaluated with treated mRNA and Lipofectamine Messenger Max (Lipo MM) in HEK293t cells.

[0238] For encapsulation, biomimetic concentrations of 160 mM 2-methylimidazole (2- MelM) and 40 mM Zn(NO3)2-6H2O were employed, along with common solvents for ZIF-8 synthesis, namely nuclease-free water, ethanol, and methanol, in a coprecipitation method. Individual samples of mRNA underwent the following experimental conditions: a 2-hour incubation in synthesis conditions for encapsulation, a 2-hour co-incubation of mRNA with synthesized ZIF-8 (previously reported in Venna et al. (“Structural Evolution of Zeolitic hnidazolate Framework-8”, J Am Chem Soc, 2010, 132(51): 18030-18033)), or an encapsulation within ZIF-8 (mRNA@ZIF-8) using nuclease-free water, ethanol, and methanol.

[0239] Generated ZIF-8 and mRNA@ZIF-8 particles were characterized using powder x- ray diffraction (PXRD) and SEM. After isolating or exfoliating mRNA from ZIF-8 using EDTA, the samples were analyzed on a native RNA gel. The EDTA was used to strip the MOF coating from the mRNA before gel preparation and was found to prevent mRNA degradation during the process. Exposure to water alone was determined to lead to mRNA degradation in the presence of ZIF-8. From FIG. 4A the native RNA gel revealed evident mRNA degradation, indicated by arrows, when incubated with ZIF-8 in methanol and subjected to 40 mM Zn(NO3)2-6H2O in methanol. Similarly, mRNA incubation with ZIF-8 in nuclease-free water exhibited visible degradation (FIG. 4B), whereas incubation with precursor solutions and encapsulated mRNA showed no degradation.

[0240] The water- synthesized samples lacked matching PXRD patterns to simulated ZIF- 8 and featured a variety of morphologies. Ethanol demonstrated minimal to no mRNA degradation under all tested conditions (FIG. 4C), and mRNA@ZIF-8 generated in ethanol produced PXRD patterns agreeing with simulated ZIF-8 and monodispersed particles. Specifically, mRNA@ZIF-8 formed in methanol and ethanol showed the distinct dodecahedron shape of literature-synthesized ZIF-8 and had matching diffraction patterns as compared to simulated and literature- synthesized ZIF-8.

[0241] After evaluating mRNA length, in vitro transfections to assess mRNA expressibility were completed. Cy5-eGFP-mRNA samples encapsulated within ZIF-8 were prepared with nuclease-free water, methanol, and ethanol and isolated with centrifugation. The particles were then introduced to HEK293t cells. As controls, mRNA samples exfoliated from mRNA@ZIF- 8 (Exf. mRNA@ZIF-8), prepared with all mentioned solvents, were applied to transfect the same cells using Lipo MM. Following a 24-hour incubation, cells were fixed, and fluorescence was observed under a microscope, with quantification performed using a plate reader (FIG. 4D). The presence of Cy5 (red) indicated mRNA uptake, while eGFP (green) indicated expressed mRNA. mRNA@ZIF-8 synthesized with any of the mentioned solvents did not induce eGFP expression and no Cy5 signal was visible. The lack of Cy5 signal suggested a delivery issue with mRNA@ZIF-8, as exfoliated mRNA (Exf. mRNA@ZIF-8) delivered with Lipo MM exhibited eGFP expression. Additionally, eGFP expression levels were compared among exfoliated mRNA samples. While particles synthesized with water and ethanol showed statistically similar eGFP expression, samples synthesized with methanol exhibited significantly lower eGFP expression, indicating that mRNA is incompatible with the synthetic conditions in methanol.

[0242] mRNA@ZIF-8 synthesized with any solvent maintained mRNA length and expressibility, but significant mRNA degradation occurred with methanol-based synthesis conditions and when mRNA was incubated with ZIF-8 in deionized water, nuclease-free water, or methanol. When heparin (50 pg) was added to nuclease-free water, the mRNA showed slightly less degradation, possibly due to the heparin blocking the mRNA from interacting with ZIF-8. ZIF-8-mRNA co-incubation caused degradation in all solvents tested except PBS and ethanol for up to 4 hours.

[0243] The deviation of mRNA@ZIF-8 particles synthesized in water from simulated ZIF- 8 PXRD patterns and morphology indicated that these biomimetic mineralization concentrations were insufficient for uniform ZIF-8 nanoparticle formation. Ethanol-based coprecipitation maintained mRNA length better than the other tested solvents when dried and stored for up to 7 days.

[0244] mRNA@ZIF-8 samples were exposed to nuclease-free water, deionized water, PBS, methanol, and ethanol and were then exfoliated with EDTA. The mRNA inside of mRNA@ZIF-8 was stable in PBS and ethanol post-synthesis, but was not stable in unbuffered aqueous solutions and methanol.

[0245] As no Cy5-mRNA uptake was visible with mRNA@ZIF-8, the particle’s protection against RNase and stability in biological media were tested. After exposure to RNase A for 2 hours or biological media for over 24 hours, particles were isolated with centrifugation. ZIF-8 was exfoliated off of the mRNA, and the mRNA was run on a native RNA gel (FIGS. 5A-5B). When exposed to RNase A for 2 hours suspended in PBS, mRNA@ZIF-8 demonstrated protection of the mRNA, as the isolated mRNA matches the control band, and no degradation is visible when compared to mRNA treated with RNase A (FIG. 5A). However, when mRNA@ZIF-8 was exposed to biological media, the isolated mRNA@ZIF-8 pellet yielded no mRNA bands in the 10% FBS- supplemented media after 1 hour of incubation (FIG. 5B). When mRNA@ZIF-8 was exposed to other biological media, such as Opti-MEM and PBS (pH 7 and pH 5), mRNA isolated from the mRNA@ZIF-8 pellet showed retention of mRNA for at least 1 hour. Opti-MEM and PBS pH 5 treatments showed significant mRNA leakage / degradation over 24 hours. The medium supernatants were also tested and it was confirmed that mRNA@ZIF-8 incubation with 10% FBS DMEM resulted in mRNA leakage. The mRNA@ZIF-8 supernatants showed a weak, but distinct band at the control mRNA’s length. PBS at pH 7 showed the weakest band intensities.

[0246] To address the issue of mRNA leakage, linear 20 kDa, polycationic polyethyleneimine (PEI) was introduced into the synthetic process. Additional PEI isoforms,specifically linear 10 kDa PEI (Sigma Aldrich (St. Louis, MO)), linear 40 kDa PEI (Polysciences (Warrington, PA)), and branched 25 kDa PEI (Sigma Aldrich (St. Louis, MO)), were also tested in the synthetic process.

[0247] HEK293t cells were transfected in serum media with mRNA complexed with varying amounts of the PEI isoforms, ranging from 0 to 12.5 pg of PEI per 1 pg of mRNA), which were complexed in Opti-MEM for 10-15 min. The mRNA-PEI was then applied to cells in a 96-well plate with 100 ng mRNA per well. The cells were fixed and GFP expression was measured at 48 hours post-transfection and normalized to the DAPI signal. The linear 20 kDa PEI and the linear 40 kDa PEI each performed better than the linear 10 kDa PEI and the branched 25 kDa PEI in terms of GFP expression, with GFP expression maximized in an amount of 2.26 and 3.76 pg of PEI per pg mRNA. The linear 20 kDa PEI isoform was chosen for further studies.

[0248] The minimum amount of PEI needed to bind all mRNA was determined through a retardation assay, where PEI and mRNA were combined at various weight ratios. mRNA- PEI@ZIF-8, having different weight ratios of RNA:PEI, and therefore different nitrogen-to- phosphate (N / P) ratios, were suspended in Opti-MEM and delivered to HEK293t cells plated in a 96-well plate with 100 ng of mRNA per well. Encapsulation within ZIF-8 improved mRNA expression significantly over the best-performing mRNA-PEI complex (N / P ratio of 28, or 3.76 pg of PEI per pg mRNA), at a lower N / P ratio of 9.3 (or 1.25 pg PEI per pg mRNA). Additionally, it was shown that the complexation of mRNA-PEI in ZIF-8 2-MeIM precursor did not elicit a high GFP expression.

[0249] The minimum binding ratio of mRNA and PEI occurred at a weight ratio of (RNA:PEI) 1:0.8, which represents a nitrogen-to-phosphate (N / P) ratio of about 6.

[0250] With the addition of PEI, the synthetic protocol was carried out by first mixing mRNA with 2-MeIM, then adding an optimized amount of PEI allowing a 15 -minute incubation, mixing in zinc nitrate hexahydrate, and incubating for 2 hours. The PEI is electrostatically complexed with mRNA before ZIF-8 encapsulation, creating a ZIF-8 shell around the mRNA-PEI complex (mRNA-PEI@ZIF-8), as shown in FIG. 3A. By creating an mRNA-PEI complex core, the mRNA is retained in the core, while PEI aids in the stability of the ZIF-8 coating, possibly due to its strong binding of Zn ions with uncomplexed or partially complexed PEI.

[0251] The generated composite particles, mRNA-PEI@ZIF-8 (mRPZ), underwent treatment with RNase A and exposure to biological media (FIGS. 5C-5D). When comparing mRPZ to RNase-treated mRNA, minimal to no degradation of the mRNA was observed, as theband remained approximately the same length as the RNA control. This validated that mRPZ shields the mRNA from RNase, as well as mRNA@ZIF-8. Following exposure to biological media, the isolated mRPZ pellet was analyzed on a native RNA gel, revealing a distinct band of mRNA present for all biological media for up to 4 hours. This confirmed that mRPZ is stable longer than mRNA@ZIF-8 in biological media.

[0252] Synthesized mRPZ was imaged with SEM and TEM, along with literature- synthesized ZIF-8 and mRNA@ZIF-8 (FIG. 6). SEM and TEM images of ZIF-8 are shown in FIGS. 5(A) and 5(D), respectively. SEM and TEM images of mRNA@ZIF-8 are shown in FIGS. 5(B) and 5(E), respectively. SEM and TEM images of mRPZ are shown in FIGS. 5(C) and 5(F), respectively. The TEM micrographs of the synthesized mRPZ (FIG. 5(F)) showed the distinct dodecahedron morphology that is present in the ZIF-8 literature, as well as a uniform inner atomic weight distribution (based on intensity). Furthermore, the inclusion of PEI appeared to improve the stability of ZIF-8 under the TEM beam. Using imaging analysis on acquired SEM images, mRPZ was about 107 nm, which is larger than literature- synthesized ZIF-8 and mRNA@ZIF-8, 74 nm and 80 nm, respectively (FIG. 7A). Some variation in size occurred from batch to batch. The mean hydrodynamic diameter, as determined by dynamic light scattering (DLS), of mRPZ was 146 nm (FIG. 7B) which may be explained by uncomplexed PEI. The hydrodynamic diameter of mRNA@ZIF-8 was larger than mRPZ and literature-synthesized ZIF-8, contrary to the size obtained with SEM (Ad = 80 nm larger). The difference in diameter may indicate that mRNA is loosely associated with the surface of the mRNA@ZIF-8 particle, which increases its hydrodynamic diameter. PXRD confirmed the synthesis of the ZIF-8 particles. The diffraction patterns of mRPZ and mRNA@ZIF-8 match well with simulated ZIF-8 and literature-synthetized ZIF-8 peaks (FIG. 7C).

[0253] The loading efficiency of mRPZ was measured as the amount of RNA released from nanoparticles after exfoliation of ZIF-8 with EDTA and dissociation from PEI with heparin sulfate divided by the mRNA used in synthesis. Gel electrophoresis and Image J analysis were utilized to compare band intensities of released RNA to standards. The intensity of the resultant mRNA standard was used to create standard curves. The linear regression equations of the standard curves were used to calculate the amount of mRNA successfully encapsulated in mRPZ and mRNA@ZIF-8 and the amount encapsulated was used to estimate a loading efficiency. The loading efficiency of mRPZ and mRNA@ZIF-8 was found to be 91.1% + / - 7.7% and 91.5% + / - 9.5%, respectively (FIG. 8A). The loading efficiency of mRPZ was further validated using a fluorescent probe assay, confirming a similar loading capacity of 92.3% + / - 2.5% (FIG. 8B).

[0254] The loading capacity of mRPZ was determined through thermogravimetric analysis (TGA), where the organic components of mRPZ exhibit lower burning temperatures than ZIF- 8. Differential mass loss over a temperature range helped identify the weight composition of each particle component. Weight compositions of mRNA@ZIF-8, PEI-ZIF-8, and ZIF-8 were employed to confirm the decomposition of each component (FIG. 8C). From the literature, RNA, PEI, and ZIF-8, decompose between 100-300°C (M.A., G “Thermal and Chemical Stability of the Solid State RNA Samples: Could It Survive at the Extreme Conditions of the Early Earth?” Viva Origino, 2020, 48(1): 1) 300-400°C (Zhu et al. “Effect of Polyethylenimine on Hydrolysis and Dispersion Properties of Aqueous ShN4 Suspensions”, Journal of the American Ceramic Society, 2007, 90(3):797-804) and 400°C-600°C (James et al. “Kinetics of ZIF-8 Thermal Decomposition in Inert, Oxidizing, and Reducing Environments”, Journal of Physical Chemistry C, 2016, 120(26): 14015-14026) under nitrogen, respectively. The mRNA loading capacity of mRPZ was calculated to be 5.3%. The loading capacity for mRNA@ZIF- 8 was slightly higher at 6.0%, possibly due to the absence of PEI contributing to the overall weight. The incorporation of mRNA and PEI within ZIF-8 reduced the decomposition temperature, evidenced by shifts in the composite ZIF-8 materials.

[0255] FTIR was also performed to ensure chemical consistency between tested samples and ZIF-8. Peaks observed in the PEI-ZIF-8 fingerprint region were absent in mRPZ, which may indicate the loss of bond stretching due to the binding of mRNA to PEI.

[0256] To validate the integration of mRNA with ZIF-8, an analysis involving zeta potential measurements, EDS, and a molecular probe assay was conducted. Zeta potential assessments were carried out for individual components, as well as the mRPZ particles and mRNA-PEI nanoparticles (FIG. 9A). The binding of PEI with mRNA to form the mRNA-PEI complex (27.3 mV) resulted in a discernible reduction in zeta potential compared to PEI alone (43.1 mV). Subsequent coating with ZIF-8 led to a significant increase in the zeta potential of the mRPZ particles, reaching 59.5 mV, which is greater than ZIF-8 alone (45.63 mV). This observation provided evidence of the incorporation of mRNA-PEI within a ZIF-8 shell.

[0257] EDS was employed to affirm the presence of PEI and mRNA within ZIF-8, manifested by an increase in the nitrogen: zinc atomic weight percent ratio, as the composite particles contain PEI and mRNA, which add to the nitrogen content of the entire particle. The atomic weight compositions were determined at various locations within the bulk sample. A distinct rise in nitrogen atomic percent, when comparing ZIF-8 with mRPZ, was indicated, signifying the incorporation of mRNA and PEI into the bulk material (FIG. 9B). Other tested composite materials, namely PEI-ZIF-8 and mRNA@ZIF-8, also showed increased nitrogen:zinc weight percent ratio compared to ZIF-8. Qualitative element mapping of mRPZ, as compared to ZIF-8, mRNA@ZIF-8, and PEI-ZIF-8, was performed using an EDS-equipped STEM / TEM, where carbon, nitrogen, zinc, phosphorus, and oxygen were analyzed. Elemental mapping with STEM / TEM demonstrated that mRNA was encapsulated within mRPZ. Due to the sensitive nature of ZIF-8 to the electron beam damage, the measured atomic percentages obtained with TEM were not consistent with SEM or literature. However, there was a notable increase in the zinc nitrogen ratio of mRPZ compared to ZIF-8 as well as visible signatures of phosphorous in mRPZ.

[0258] To further establish the incorporation of mRNA-PEI within ZIF-8, a molecular probe assay utilizing an intercalating dye was employed, emitting fluorescence upon contact with free mRNA or bound mRNA which is not blocked from the intercalating dye. By treating mRPZ, mRNA-PEI, and mRNA@ZIF-8 with EDTA and heparin, followed by probing with the intercalating dye, how mRNA is incorporated into mRPZ could be elucidated. EDTA served to exfoliate ZIF-8, while heparin displaced or released mRNA from electrostatically binding surfaces or molecules, the surface of ZIF-8 (+45.6 mV) or PEI. Particles were suspended in PBS and treated with EDTA and heparin, separately or together, or received no treatment. The suspension was probed with dye and then centrifuged to pellet any suspended particles. After centrifugation, the supernatant fluid, which could contain free mRNA, was probed with dye. By probing both the suspension and the supernatant after centrifugation, it was possible to determine if mRNA was released from the particle or bound to the particle. The data (FIG. 10) indicated that mRNA was encapsulated within mRPZ, as treatment with heparin alone did not fully release RNA into the supernatant or suspension, akin to mRNA- PEI. Conversely, treatment with EDTA alone did not cause the release of mRNA from mRPZ, as observed with mRNA@ZIF-8. Only the combined treatment of mRPZ with EDTA and heparin resulted in the unbinding and release of mRNA into the supernatant. mRNA @ ZIF-8 did have a signal without treatment and with heparin treatment, alluding to some mRNA loosely associating with the surface of mRNA@ZIF-8, explaining its larger hydrodynamic diameter. Additionally, this loosely associated mRNA appeared to dislodge from mRNA@ZIF-8 when treated with heparin, explaining the signal increase in the heparin-treated suspension and supernatant fractions. Controls confirmed that no fluorescence is caused by interactions with ZIF-8, PEI, EDTA, and heparin.

[0259] mRPZ was applied for in vitro cell transfection. Briefly, Cy5-tagged, eGFP- encoding mRNA-loaded mRPZ nanoparticles were suspended in Opti-MEM and administered to HEK293t in serum-containing media. After 48 hours, cells were fixed and stained with anuclear dye (DAPI), and the resulting fluorescence was imaged with a microscope and quantified with a plate reader (FIG. 11). The presence of Cy5 located within the fixed cells, coupled with the expression of eGFP, indicated the successful delivery of mRNA by mRPZ and the subsequent expression of its corresponding protein. For comparison, cells treated with mRNA@ZIF-8 were absent of Cy5 and eGFP signals. mRNA-PEI transfection was also performed as a comparison, utilizing the same nitrogen-to-phosphate ratio of 9.3 as mRPZ. mRPZ was found to perform five to ten times better than mRNA-PEI (N / P=9.3) in HEK293t, HeLa, and CHO cells. Specifically in HEK cells, mRPZ utilizing an N / P ratio of 9.3 outperformed mRNA-PEI complexes with an optimized N / P ratio of 28-30, which is consistent across the PEI isoforms tested (linear 10 kDa PEI, linear 20 kDa PEI, linear 40 kDa PEI, and branched 25 kDa PEI. Additionally, across all three cell lines, mRPZ outperforms Lipofectamine 2000 (Lipo 2k), and mRPZ performs better than or similar to Lipo MM, except in CHO cells.

[0260] Cy5-labeled, eGFP-encoding mRPZ nanoparticles and Lipofectamine were incubated with HEK293t, HeLa, and CHO cells for 48 hours, and the resultant cells were evaluated by flow cytometry and imaged. The same gating strategy was used across cell lines. Cells were gated by first identifying the cell population among the debris, then singlet cells were selected. Of those remaining, singlet cells were gated by comparing viable cells to an untreated group. The live, single-cell populations were then measured for GFP and Cy5 intensity. The HEK293t, HeLa, and CHO cells treated with Cy5-Tagged, eGFP expressing mRNA carried by mRPZ had successful uptake (Cy5) and expression (GFP) of the mRNA in cells stained with a nuclear stain (DAPI).

[0261] Cell viability was assessed using an amine-reactive dye, LIVE / DEAD™ Fixable Blue, and cell uptake was assessed using the Cy5 mRNA tag. As shown in FIGS. 12A-12B, the cellular viability and uptake of all cell lines were above 92% (except Lipo MM) and 88%, respectively. This demonstrates that mRPZ was efficiently taken up by cells and caused minimal toxicity. Comparing the relative eGFP expression in all cell lines, mRPZ induces eGFP expression in 82.2%, 76.1%, and 49.5% of live cells for HEK293t, HeLa, and CHO cells, respectively (FIGS. 12C-12E). In comparison to positive Lipofectamine controls, mRPZ performs equally well or slightly worse depending on the cell line and the specific Lipofectamine control. The Median Fluorescence Index (MFI), normalized with the percentage of GFP-positive cells, shows mRPZ performed similarly to Lipofectamine controls on a singlecell basis, except in comparison to Lipo MM in CHO cells (FIG. 12F-12H). mRPZ also outperformed mRNA-PEI at an N / P ratio of 9.3 and outperformed or equivalently performedto mRNA-PEI at an N / P ratio of 28 in terms of normalized MFI and percentage of GFP positive cells. The normalized MFI measurement captures the intensity of eGFP expression on a singlecell basis. The increased MFI caused by mRPZ may allude to more mRNA strands being delivered into a single cell. Using nanoparticle tracking to obtain particle concentrations, the number of mRNA strands per nanoparticle was back-calculated to be about 33.

[0262] Additional cell lines, RAW264.7, A549, and 231, were tested using mRPZ. The RAW264.7, A549, and 231 cells treated with Cy5-Tagged, eGFP expressing mRNA carried by mRPZ had successful uptake (Cy5) and expression (GFP) of the mRNA in cells stained with a nuclear stain (DAPI). In RAW 264.7 mouse macrophages, it was found that mRPZ performed equivalently to Lipo 2k.

[0263] In comparison to mRNA-PEI, mRPZ outperforms or equivalently performs mRNA- PEI at both N / P ratios of 9.3 and 28.

[0264] The toxicity of mRPZ, components, and commercial transfection reagents were assessed using a resazurin assay which measures cell metabolic activity. HEK293t cells were treated with mRPZ, components, and transfection reagents. After a 4-6-hour exposure, the metabolic activity of the cells was measured by resazurin conversion for 8 hours. The viability of the cells after transfection with Lipo 2K, Lipo MM, mRNA-PEI (N / P=28), and mRPZ were not statically different from the untreated cells. Cells treated with Linear 20 kDa PEI alone showed no signs of toxicity up to a concentration of 3.6 pg / mL, which corresponds to transfecting with PEI at an N / P ratio of about 28. At higher concentrations, such as concentrations exceeding 12 pg / mL, PEI significantly reduced the metabolic activity of the cells. Cells treated with 3.6 pg / mL of PEI and transfected with mRNA-PEI at N / P ratios of about 28 showed visible signs of reduced growth, as final cell confluency was lower compared to other treatments. Cells treated with ZIF-8 alone showed no significant metabolic change up to 50 pg / mL, where mRPZ used to transfect one 96-well plate well would correspond to 16.62 pg / mL of ZIF-8. Higher concentrations of ZIF-8 caused a significant drop in metabolic activity and caused the cell morphology to become round. The combination of PEI and ZIF-8 shows better transfection over PEI alone and reduces the amount of PEI needed.

[0265] The kinetics of eGFP expression after transfection by treating HEK293t cells with mRPZ was studied and the eGFP fluorescence in phenol red-free media for 96 hours was monitored with a plate reader. mRPZ reached a maximum eGFP signal at 72 hours and reached 80% of its maximum expression at 44 hours. Lipo 2k and Lipo MM reached maximum expression around 48 hours and reached 80% of their maximum expression around 24 hours. mRNA-PEI (PEI N / P=9.3) reached its maximum expression at 96 hours and 80% of itsmaximum expression at around 21 hours. This delayed expression may be explained by the slow dissociation of the ZIF-8 coating from mRPZ.

[0266] The stability of the encapsulated mRNA over 12 weeks at room temperature, 4°C, and -80°C was investigated by taking mRPZ, drying it for 1 hour, and storing it. Room temperature storage condition was under vacuum as particles stored without vacuum had little transfection viability and formed cavities visible under TEM. After one month, it was found that the encapsulated mRNA transfected well across all temperatures, producing eGFP signals that were several folds above an untreated background sample (FIG. 13). At 8 and 12 weeks, a significant drop in the eGFP signal was observed in mRPZ stored at 4°C and room temperature. However, mRPZ maintained its ability to successfully transfect cells even after 12 weeks at room temperature.

[0267] A clear degradation of mRNA in mRNA-PEI was evident by 8 weeks, rendering it unable to transfect cells regardless of storage time or temperature. The material stability of mRPZ was recorded over 3 months and compared to mRNA @ ZIF-8. mRPZ and mRNA@ZIF- 8 demonstrate a similar size and morphology at 1 month, but more round morphologies at 3 months. The crystallinity was assessed using PXRD and mRPZ and mRNA@ZIF-8 demonstrated retention of the ZIF-8 diffraction pattern.

[0268] When comparing mRNA stored under the same thermal conditions and delivered with Eipo MM with mRPZ, it was observed that mRNA was stable and able to transfect regardless of the time or temperatures tested. Therefore, it was determined that the ZIF-8 coating on mRPZ improves the stability of the mRNA-PEI complex but does not aid the stability of the mRNA for cell transfection. mRNA from mRNA@ZIF-8 stored at room temperature, exfoliated, and transfected using Eipo MM did illicit eGFP expression but was much lower than mRPZ or other Eipofectamine controls with fresh mRNA.

[0269] mRPZ was scaled and translated into an in vivo gene delivery model using Balb / C mice. Instead of eGFP expressing mRNA, utilizing the same synthetic encapsulation process, 12 pg of firefly luciferase mRNA (1920 nt) was encapsulated. Firefly luciferase generates bioluminescence in the presence of luciferin, which can be injected into mice to monitor luciferase expression.

[0270] Balb / C mice were injected intravenously through the retro-orbital vein with mRPZ, mRNA-PEI utilizing an N / P ratio of 9.3 (PEI N / P=9.3), in vivo JetRNA+ (JetRNA, a commercial lipid reagent), or PBS. The expression of luciferase was monitored over 96 hours by injecting mice in their intraperitoneal cavity with a luciferin DPBS solution and measuring the luminescence with an IVIS® live imaging system. The images were analyzed using IVIS®software and the mean radiance was collected measuring the entire body of each mouse. The kinetics of luciferase expression were tracked using the image data over 96 hours (FIG. 14A). mRPZ showed the same delayed maximum expression of mRNA in vivo as it does in vitro when compared to a commercial lipid reagent. mRPZ reached a maximum expression between 12 to 18 hours post- injection, while JetRNA reached a maximum expression at 3 hours postinjection, with both gradually tapering off over 96 hours. mRNA-PEI (N / P=9.3) showed some expression in vivo but not statically above background (PBS), even when controlling for sex.

[0271] Trapezoidal integration approximation was used to find the area under the curve for each mouse, and the log of the integrated mean radiance, which represents the total luciferase expression was used to compare each group’s luciferase expression (FIG. 14B). mRPZ, which contained the same amount of PEI, significantly outperformed mRNA-PEI (N / P=9.3) over tenfold. However, JetRNA significantly outperformed mRPZ by about three-fold in total expression. mRNA-PEI (N / P=9.3) did express luciferase, but luciferase expression was not statically different from the background of the PBS mice. This held even when controlling for sex. When controlling for sex, both mRPZ and JetRNA demonstrate significantly more expression in female mice compared to male mice. Smaller doses of mRPZ, at 6 pg per mouse, yielded total luciferase expression levels that were not statistically different from the 12-pg dose.

[0272] The biodistribution of mRPZ, compared to mRNA-PEI (N / P=9.3) and PBS, was determined by injecting particles encapsulating Cy5-tagged mRNA expressing luciferase into mice, waiting 6 hours, then collecting their organs (heart, lungs, pancreas, spleen, kidneys, and liver) and imaging them for Cy5 fluorescence. Biodistribution, as determined by Cy5 localization, represents the location of the mRNA, and hence the injected particle trafficked in mice. The mRPZ particles trafficked primarily to the lungs, spleen, and liver in the first 6 hours when comparing the log of the Cy5 fluorescence against the PBS control (FIG. 15A). This distribution pattern was consistent with the distribution of mRNA-PEI (N / P=9.3). mRPZ particles were observed to traffic significantly more to the lungs and significantly less to the liver as compared to mRNA-PEI (N / P=9.3) particles. This result was consistent across sex for the lungs, but not for the spleen and liver. When comparing the spleen, mRNA-PEI (N / P=9.3) particles trafficked significantly less to the spleen than mRPZ in male mice while female mice showed no significant difference. mRNA-PEI (N / P=9.3) particles trafficked significantly more to the liver as compared to mRPZ in female mice, while male mice showed no statistical difference.

[0273] mRNA expressing firefly luciferase was encapsulated in mRPZ or delivered with PEI (mRNA-PEI) to Balb / c mice intravenously. After 12 hours, mice were injected with luciferin and sacrificed, their organs harvested, and imaged (FIG. 15B). The luminescence of the organs was measured with the IVIS® imaging system, and the log mean radiance was compared. In comparison to mRNA-PEI (N / P=9.3), mRPZ resulted in significantly higher expression in all collected organs, well above PBS background. These findings were consistent across sex. mRNA-PEI (N / P = 9.3) mRNA was only expressed above background in the lungs and spleen.

[0274] Toxicity of the particles was assessed using the collected serum from the sacrificed mice 7 days after injection, measuring the Urea (FIG. 16A), Creatine (FIG. 16B), and Zinc (Zn2+) (FIG. 16C) concentrations as well as the alkaline phosphatase (ALP) (FIG. 16D), alanine transaminase (ALT) (FIG. 16E), and aspartate transaminase activity (AST) (FIG. 16F). mRPZ, mRNA-PEI (N / P=9.3), and JetRNA caused no statistical difference in the serum factors measured compared to a healthy control (PBS). The zinc concentration of mRPZ was lower than that of the healthy control, but it was also equivalent to mRNA-PEI (N / P=9.3), which contained no zinc. Overall, the mice showed minimal signs of toxicity when compared to a healthy control and are within healthy standards. High concentrations of mRPZ caused the death of 4 out of 6 mice at a 24-pg dose and mRPZ particles that were poorly resuspended caused the death of 2 mice at a 12-pg dose.

[0275] The long-term stability of the particles was assessed at room temperature for one month. Particles were synthesized and stored at room temperature under vacuum and injected intravenously in mice. The resulting luciferase activity was measured between 12 and 24 hours, capturing the maximum expression time range identified in FIG. 14A. The integrated mean radiance was used to find the luciferase expression at that time point and compared to mRPZ stored at -80°C for a month and mRPZ freshly synthesized. A PBS control was included to determine background radiance (FIG. 17). Freshly synthesized mRPZ performed equally to mRPZ stored at -80°C for one month. mRPZ stored at room temperature produced luciferase expression that was significantly less than -80°C stored mRPZ, but still significantly above background (PBS) over 5-fold.

[0276] A delay in luciferase expression compared to the commercial lipid reagent and improved luciferase expression compared to mRNA-PEI (N / P=9.3) was observed. mRPZ was taken up more and expressed more in the lungs compared to mRNA-PEI (N / P=9.3) alone. Many of the trends were found to be consistent across mouse sex. Based on the dosage response, less mRNA can be used to achieve a similar expression level. No toxicity was evidentbased on the blood chemistry, even zinc ion levels were within a healthy range. Additionally, the storage of mRPZ at room temperature displayed expression after a month of storage.Example 2

[0277] The synthetic methodology for mRNA encapsulation described in Example 1 was found to also work for pDNA. eGFP-pDNA (13031 pcDNA3-EGFP) (6.1 kbp) was purchased from Addgene (Watertown, MA). After encapsulation of eGFP-pDNA in ZIF-8 using the coprecipitation method described in Example 1, pDNA-PEI@ZIF-8 (pDPZ) was imaged using SEM and applied for the transfection of HEK293t cells. The eGFP expression in the cells was observed at 48 and 96 hours. Similar to mRPZ, the expression of the eGFP-pDNA was also delayed, this time by 48 hours.

[0278] It will be readily appreciated by those skilled in the art that modification may be made to the invention without departing from the concepts disclosed in the foregoing description. Accordingly, the particular embodiments described in detail herein are illustrative only and are not limiting to the scope of the invention, which is to be given the full breadth of the appended claims and any and all equivalents thereof.

Claims

What is Claimed Is:

1. A method of making metal-organic framework nanoparticles comprising a nucleic acid-based therapeutic agent, the method comprising: adding the nucleic acid-based therapeutic agent to an imidazole solution comprising an imidazole containing compound and a first alkyl alcohol solvent and mixing the nucleic acid-based therapeutic agent and the imidazole solution to form a therapeutic agent containing solution; adding a polymer solution comprising a cationic polymer to the therapeutic agent containing solution and mixing the polymer solution and the therapeutic agent containing solution to form an electrostatic complex solution; adding a zinc solution comprising a hydrated zinc salt and a second alkyl alcohol to the electrostatic complex solution and mixing the zinc solution and the electrostatic complex solution to form a metal-organic framework nanoparticle precursor solution; and incubating the metal-organic framework nanoparticle precursor solution to form the metal-organic framework nanoparticles comprising the nucleic-acid based therapeutic agent.

2. The method of claim 1, wherein the first alkyl alcohol solvent and the second alkyl alcohol solvent are the same and each independently comprise ethanol, methanol, or isopropyl alcohol.

3. The method of claim 2, wherein the first alkyl alcohol solvent and the second alkyl alcohol solvent each independently comprise ethanol.

4. The method of claim 1, wherein the cationic polymer comprises a linear or branched polyethyleneimine.

5. The method of claim 4, wherein the linear or branched polyethyleneimine comprises a weight average molecular weight of at least 10 kiloDaltons (kDa).

6. The method of claim 4, wherein the cationic polymer comprises a linear polyethyleneimine comprising a weight average molecular weight of at least 20 kDa.

7. The method of claim 4, wherein the polymer solution comprises less or equal to 2.0 milligrams per milliliter (mg / mL) of the linear or branched polyethyleneimine.

8. The method of claim 4, wherein the polymer solution comprises: an aqueous solvent; and a pH of approximately 7.

9. The method of claim 1, wherein the electrostatic complex solution comprises a nitrogen-to -phosphate (N / P) ratio of at least 6:1.

10. The method of claim 1, wherein the imidazole solution is prepared by dissolving the imidazole containing compound in the first alkyl alcohol solvent.

11. The method of claim 1, wherein the zinc solution is prepared by dissolving the hydrated zinc salt in the second alkyl alcohol solvent.

12. The method of claim 1, further comprising incubating the polymer solution and the therapeutic agent containing solution for a time of not greater than 15 minutes to form the electrostatic complex solution.

13. The method of claim 1, wherein the zinc solution and the electrostatic complex solution are incubated for a time of at least 2 hours.

14. The method of claim 1 , wherein the nucleic acid-based therapeutic agent comprises messenger RNA (mRNA), small interfering RNA (siRNA), plasmid DNA (pDNA), small harpin RNA (shRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), micro RNA (miRNA), small nuclear RNA (snRNA), long non-coding RNA (IncRNA), double-stranded RNA (dsRNA), piwi-interacting RNA (piRNA), circular RNA (circRNA), short interfering RNA (siRNA), antisense oligonucleotides, or combinations thereof.

15. The method of claim 14, wherein the nucleic acid-based therapeutic agent comprises mRNA.

16. The method of claim 14, wherein the nucleic acid-based therapeutic agent comprises mRNA, wherein the mRNA comprises chemically-modified bases.

17. The method of claim 1, wherein the hydrated zinc salt comprises zinc nitrate hexahydrate and the imidazole containing compound comprises 2-methylimidazole.

18. The method of claim 1, wherein the metal-organic framework is a zeolitic imidazole framework-8 (ZIF-8).

19. A dosage form comprising: the metal-organic framework nanoparticles comprising the nucleic acid-based therapeutic agent prepared according to claim 1; and a pharmaceutically-acceptable carrier.

20. The dosage form of claim 19, wherein the dosage form is formulated as a parenteral dosage form.

21. A method of making metal-organic framework nanoparticles comprising a nucleic acid-based therapeutic agent, the method comprising: preparing a zinc solution by dissolving a hydrated zinc salt in a first alkyl alcohol solvent; preparing an imidazole solution by dissolving an imidazole containing compound in a second alkyl alcohol solvent; adding the nucleic acid-based therapeutic agent to the imidazole solution and mixing the nucleic acid-based therapeutic agent and the imidazole precursor solution to form a therapeutic agent containing solution; adding a polymer solution comprising a cationic polymer to the therapeutic agent containing solution and mixing the polymer solution and the therapeutic agent containing solution to form an electrostatic complex solution; adding the zinc solution to the electrostatic complex solution and mixing the zinc solution and the electrostatic complex solution to form a metal-organic framework nanoparticle precursor solution; andincubating the metal-organic framework nanoparticle precursor solution to form the metal-organic framework nanoparticles comprising the nucleic-acid based therapeutic agent.

22. The method of claim 21, wherein the first alkyl alcohol solvent and the second alkyl alcohol solvent are the same and each independently comprise ethanol, methanol, or isopropyl alcohol.

23. The method of claim 22, wherein the first alkyl alcohol solvent and the second alkyl alcohol solvent each independently comprise ethanol.

24. The method of claim 21, wherein cationic polymer comprises a linear or branched polyethyleneimine.

25. The method of claim 24, wherein the linear or branched polyethyleneimine comprises a weight average molecular weight of at least 10 kDa.

26. The method of claim 24, wherein the cationic polymer comprises a linear polyethyleneimine comprising a weight average molecular weight of at least 20 kDa.

27. The method of claim 24, wherein the polymer solution comprises less or equal to 2.0 mg / mL of the linear or branched polyethyleneimine.

28. The method of claim 24, wherein the polymer solution comprises: an aqueous solvent; and a pH of approximately 7.

29. The method of claim 21, wherein the electrostatic complex solution comprises a N / P ratio of at least 6:1.

30. The method of claim 21, further comprising incubating the polymer solution and the therapeutic agent containing solution for a time of not greater than 15 minutes to form the electrostatic complex solution.

31. The method of claim 21, wherein the zinc solution and the electrostatic complex solution are incubated for a time of at least 2 hours.

32. The method of claim 21, wherein the nucleic acid-based therapeutic agent comprises mRNA, siRNA, plasmid DNA, shRNA, tRNA, rRNA, miRNA, snRNA, IncRNA, dsRNA, piRNA, circRNA, siRNA, antisense oligonucleotides, or combinations thereof.

33. The method of claim 32, wherein the nucleic acid-based therapeutic agent comprises mRNA.

34. The method of claim 32, wherein the nucleic acid-based therapeutic agent comprises mRNA, wherein the mRNA comprises chemically-modified bases.

35. The method of claim 21, wherein the hydrated zinc salt comprises zinc nitrate hexahydrate and the imidazole containing compound comprises 2-methylimidazole.

36. The method of claim 21, wherein the metal-organic framework is a zeolitic imidazole framework-8 (ZIF-8).

37. A metal-organic framework nanoparticle comprising a nucleic acidbased therapeutic agent comprising: a zeolitic imidazole framework- 8 (ZIF-8); a cationic polymer; and the nucleic acid-based therapeutic agent, wherein the cationic polymer and the nucleic acid-based therapeutic agent form an electrostatic complex, and wherein the ZIF-8 encapsulates the electrostatic complex.

38. The metal-organic framework nanoparticles of claim 37, wherein the cationic polymer comprises a linear or branched polyethyleneimine.

39. The metal-organic framework nanoparticles of claim 38, wherein the linear or branched polyethyleneimine comprises a weight average molecular weight of at least10 kDa.

40. The metal-organic framework nanoparticles of claim 38, wherein the cationic polymer comprises linear polyethyleneimine comprising a weight average molecular weight of at least 20 kDa.

41. The metal-organic framework nanoparticles of claim 37, wherein the nucleic acid-based therapeutic agent comprises mRNA, siRNA, pDNA, shRNA, tRNA, rRNA, miRNA, snRNA, IncRNA, dsRNA, piRNA, circRNA, siRNA, antisense oligonucleotides, or combinations thereof.

42. The metal-organic framework nanoparticles of claim 41, wherein the nucleic acid-based therapeutic agent comprises mRNA.

43. The method of claim 41, wherein the nucleic acid-based therapeutic agent comprises mRNA, wherein the mRNA comprises chemically-modified bases.

44. The metal-organic framework nanoparticles of claim 37, wherein the metal-organic framework nanoparticles comprise a diameter ranging from 70 nanometers (nm) to 200 nm.

45. The metal-organic framework nanoparticles of claim 37, further comprising an alkyl alcohol solvent, wherein the ZIF-8 encapsulates the alkyl alcohol solvent.

46. The metal-organic framework nanoparticles of claim 45, wherein the alkyl alcohol solvent comprises ethanol, methanol, or isopropyl alcohol.

47. The metal-organic framework nanoparticles of claim 46, wherein the alkyl alcohol solvent comprises ethanol.

48. A metal-organic framework nanoparticle solution comprising: metal-organic framework nanoparticles comprising a nucleic acid-based therapeutic agent, wherein the metal-organic framework nanoparticles comprising the nucleic acid-based therapeutic agent comprise: a zeolitic imidazole framework- 8 (ZIF-8); a cationic polymer; and the nucleic acid-based therapeutic agent, wherein the cationic polymer and the nucleic acid-based therapeutic agent form an electrostatic complex, and wherein the ZIF-8 encapsulates the electrostatic complex; and a solvent.

49. The metal-organic framework nanoparticle solution of claim 48, wherein the solvent comprises a mixture of an aqueous solvent and an alkyl alcohol solvent.

50. The metal-organic framework nanoparticle solution of claim 49, wherein aqueous solvent comprises phosphate buffered saline and the alkyl alcohol solvent comprises ethanol.

51. A method of delivering a nucleic acid-based therapeutic agent to a patient, comprising administering to the patient an effective amount of a metal-organic framework nanoparticle solution comprising: metal-organic framework nanoparticles comprising: a zeolitic imidazole framework- 8 (ZIF-8); a cationic polymer; and the nucleic acid-based therapeutic agent, wherein the cationic polymer and the nucleic acid-based therapeutic agent form an electrostatic complex, and wherein the ZIF-8 encapsulates the electrostatic complex; and a pharmaceutically-acceptable carrier.

52. The method of claim 51, wherein the metal-organic framework nanoparticle solution is administered to the patient parenterally.

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