WO2025166259A1 - Mineral binding mrna lipid nanoparticles - Google Patents

Abstract

The present disclosure relates to compositions and methods for improving mineral binding nucleic acid lipid nanoparticles. In one embodiment, the lipid nanoparticle formulation is modified to include DOTAP. Inclusion of DOTAP in the lipid nanoparticles improves mineral binding and maintenance of transfection following long-term storage. In another embodiment the mRNA lipid nanoparticle is conjugated to a hydroxyapatite (HA) binding peptide to promote binding to the mineral HA and to bone in vivo. In some embodiments, the composition includes mineral-coated microparticles (MCMs).

Description

MINERAL BINDING MRNA LIPID NANOPARTICLES

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is based on, claims priority to, and incorporates herein by reference in its entirety for all purposes, US Provisional Application Serial No. 63/548,715, fded February 1, 2024.

GOVERNMENT SUPPORT

[0002] This invention was made with government support under NS109427 awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

[0003] A Sequence Listing accompanies this application and is submitted as an XML fde of the sequence listing named “960296_04684.xml” which is 4,533 bytes in size and was created on January 31, 2025. The sequence listing is electronically submitted via Patent Center with the application and is incorporated herein by reference in its entirety.

FIELD OF INVENTION

[0004] The present disclosure generally relates to mineral binding mRNA lipid nanoparticles including lipid nanoparticle formulations that incorporate l ,2-dioleoyl-3-trimethylammonium- propane (DOTAP) to improve binding to minerals and maintenance of transfection following long term storage and lyophilization. The present disclosure further relates to mRNA lipid nanoparticles conjugated to a hydroxyapatite binding peptide to promote binding to the mineral hydroxyapatite (HA) and to bone in vivo.

BACKGROUND

[0005] Tissue specific targeting of systemically delivered therapeutic compounds is a difficult challenge. For most therapeutics, the native flow of the circulatory system results in 'first-pass- metabolism' and processing in the liver that often limits therapeutics to hepatic delivery. Bone specific targeting ligands for mRNA lipid nanoparticles (LNP) could allow for minimally invasive therapeutics to target bone fractures that do not heal (non-unions) or more widespread bone pathologies like osteoporosis.

[0006] Thus, there remains a need for improved strategies for systemic delivery of therapeutic compounds. The present disclosure provides a technical solution to these needs.

SUMMARY

[0007] Disclosed herein are methods to promote binding of nucleic acid LNPs to minerals. The method may include a substitution of a positively charged lipid (DOTAP) to a nucleic acid LNP formulation. The method allows for incorporation of the nucleic acid LNPs into a mineral coated microparticle and maintains the activity of the nucleic acid LNP after freeze-drying and storage for at least one month.

[0008] Further disclosed herein is a click chemistry -based method to conjugate a hydroxyapatite (HA) targeting peptide to RNA LNPs to create HA targeting RNA LNPs. HA is the main inorganic component of bone. These hydroxyapatite (HA) nanoparticles bind cells in vitro with minimal activity when not bound to HA. In both instances binding with HA and HA-like materials is demonstrated, allowing for systemic and local applications respectively.

[0009] In one aspect of the present disclosure, a method of preparing a nucleic acid-based therapeutic composition is described. The method comprises incubating a nucleic acid with a lipid nanoparticle (LNP) to form nucleic acid complexes, wherein the LNP includes a lipid membrane at least partially comprising a l,2-Dioleoyl-3 -trimethylammonium propane (DOTAP) lipid, incubating the nucleic acid complexes with a mineral-coated substrate to bind the nucleic acid complexes to the mi neral -coated substrate, suspending the bound nucleic acid complexes in a solution containing a lyoprotectant to provide the nucleic acid-based therapeutic composition, and lyophilizing the nucleic acid-based therapeutic composition to a dry powder.

[00010] In one aspect of the present disclosure, a nucleic acid-based therapeutic composition is described. The composition comprises a mineral -coated substrate, one or more nucleic acid complexes bound to the mineral -coated substrate, wherein the nucleic acid complexes include nucleic acid complexed with a lipid nanoparticle (LNP), wherein the LNP includes a lipid membrane at least partially comprising a l,2-Dioleoyl-3-trimethylammonium propane (DOTAP) lipid, and a lyoprotectant, wherein the nucleic acid-based therapeutic composition is lyophilized to a dry powder.

[00011] In one aspect of the present disclosure, a method of preparing a nucleic acid-based therapeutic composition is described. The method comprises incubating nucleic acid with a hydroxyapatite binding peptide (HABP)-functionalized lipid nanoparticle (LNP) to form nucleic acid complexes, wherein the HABP-functionalized LNP includes a lipid membrane at least partially comprising a HABP-functionalized lipid, and incubating the nucleic acid complexes with a mineral -coated substrate to bind the nucleic acid complexes to the mineral-coated substrate.

[00012] In one aspect of the present disclosure, a method of preparing functionalized nucleic acid complexes is described. The method comprises incubating a nucleic acid with a lipid nanoparticle (LNP) to form nucleic acid complexes, wherein the LNP includes a lipid membrane at least partially comprising a maleimide-modified lipid to form the nucleic acid complexes, and conjugating a peptide onto the nucleic acid complexes to form functionalized nucleic acid complexes.

[00013] In one aspect of the present disclosure, a nucleic acid-based therapeutic composition is described. The composition comprises a mineral-coated substrate, one or more nucleic acid complexes bound to the mineral -coated substrate, wherein the nucleic acid complexes include nucleic acid complexed with a lipid nanoparticle (LNP), wherein the LNP includes a lipid membrane at least partially comprising a hydroxyapatite binding peptide (HABP)-functionalized lipid, and a lyoprotectant, wherein the nucleic acid-based therapeutic composition is lyophilized to a dry powder.

[00014] In another aspect of the present disclosure, a nucleic acid-based composition made by any one of the methods described herein is presented.

[00015] In another aspect of the present disclosure, a method of treating a subject in need of a nucleic acid-based therapeutic is described. The method comprises administering any one of the compositions described herein to the subject in need of the RNA-based therapeutic.

[00016] In another aspect of the present disclosure, a method of treating a subject in need of a nucleic acid-based therapeutic is described. The method comprises administering a composition made by any one of the methods described herein to the subject in need of the nucleic acid-based therapeutic.

[00017] These aspects are non-limiting. Other aspects and features of the system and methods described herein will be provided below

BRIEF DESCRIPTION OF THE DRAWINGS

[00018] FIG. l is a schematic of a nucleic acid LNP, as described herein.

[00019] FIG. 2 is a schematic of example processing of the nucleic acid LNP of FIG. 1, as described herein.

[00020] FIG. 3A is a chart of the binding between LNP and MCM (%) with varying molar ratios of DMG-PEG-2000.

[00021] FIG. 3B shows charts of the binding ratio effect of LNP and MCM based on the MCM concentration, binding pH, and PEG concentration in a standard LNP (left) and DOTAP - substituted LNP (right, 20% DOTAP).

[00022] FIG. 3C shows charts of the effect of the MCM concentration, binding pH, and PEG concentration in a standard LNP (left) and DOTAP-substituted LNP (right, 20% DOTAP) on transfection of firefly luciferase mRNA.

[00023] FIG. 3D is a chart of the interaction effects of MCM concentration, pH, 1,2-DSPC content, and binding time on the binding ratio effect.

[00024] FIG. 3E is a chart of the interaction effects of MCM concentration, LNP concentration, MCM calcium concentration, and 2-(A-morpholino)ethanesulfonic acid (MES) molarity on the binding ratio effect.

[00025] FIG. 4 is a flowchart for forming the nucleic acid based therapeutic composition, as described herein.

[00026] FIG. 5 is a schematic of another nucleic acid LNP conjugated with a peptide, as described herein. HABP shown is SEQ ID NO: 1 and sham peptide is shown as SEQ ID NO: 2. [00027] FIG. 6 is a schematic of example processing of the nucleic acid LNP of FIG. 5, as described herein.

[00028] FIG. 7A is a flowchart for forming the nucleic acid LNP of FIG. 5, as described herein.

[00029] FIG. 7B is another flowchart for forming the nucleic acid LNP of FIG. 5, as described herein.

[00030] FIG. 8A is a schematic of the components of an example LNP composition (not to scale). Ionizable lipid (SM-102) helper lipid (1,2-DSPC), cholesterol, and PEG lipid (DMG-PEG-2000) were used at a ratio of 50/10/38.5/1.5 as base composition. In order to make charged LNPs, 1,2- DSPC was substituted completely for DOTAP (10% total lipids). For compositions incorporating more or less DOTAP, equal molar percentages of cholesterol were adjusted accordingly.

[00031] FIG. 8B is an overview of experimental groups, mRNA LNPs alone (MCM-), mRNA co-delivered with MCMs (MCM+ Adjuvant), or mRNA LNPs bound to MCMs and resuspended to remove unbound mRNA LNPs in the supernatant (MCM+ bound).

[00032] FIG. 9A is a chart of firefly luciferase activity after mRNA LNP delivery alone (MCM- ) or bound to MCMs (MCM+). LNPs were functionalized with the cationic lipid DOTAP or anionic lipid 18PA. LNPs were formulated with 4 components completely substituting charged lipid for DSPC (DSPC-) or 5 components in addition to DSPC (DSPC+).

[00033] FIG. 9B is a chart of excipient screening with lyophilization using normal saline (NS), the excipient in the Moderna COVID- 19 vaccine (Mod Exc), Pfizer COVID-19 vaccine (Pfi Exc), maltose and sucrose (M/S) or trehalose (T).

[00034] FIG. 9C is a fold change in luciferase activity of mRNA LNPs with MCMs relative to mRNA LNPs alone after 1 month storage frozen at -80°C, lyophilized and stored at 4°C or 25°C.

[00035] FIG 9D is a chart of firefly luciferase activity normalized to cell viability after 3 months storage at 25°C comparing free mRNA LNPs (MCM-) or mRNA LNPs co-delivered with MCMs (MCM+). [00036] FIG. 10A shows images of fluorescence microscopy visualizing MCM bound DiO labeled MC3 LNPs without DOTAP (0% - left) and with DOTAP (10% - right).

[00037] FIG. 10B is a chart of the quantification of MCM binding of MC3 LNPs +/- 10% DOTAP.

[00038] FIG. 10C is a chart of firefly luciferase (FFLuc) activity on day 1 (dl) of transfection with FFluc mRNA MC3 LNPs alone, bound to MCMs (Bound) or co-delivered with MCMs (Adjuvant).

[00039] FIG. 10D is a chart of firefly luciferase activity of MC3 LNPs frozen and stored for 7 days (d7) at -80°C in trehalose with MCMs.

[00040] FIG. 10E is a chart of firefly luciferase activity of MC3 LNPs frozen and stored for 7 days (d7) at 25°C in trehalose with MCMs.

[00041] FIG. 11A shows images of fluorescence microscopy localizing DiO labeled SM-102 LNPs bound to MCMs.

[00042] FIG. 1 IB is a plot of SM-102 LNP binding to MCMs as a function of % DOTAP in the LNP.

[00043] FIG. 11C is a chart of firefly luciferase activity on day 1 (dl) after delivery of freshly prepared mRNA LNPs delivered alone (MCM-), bound to MCMs with free LNPs removed (MCM+ - Bound) or co-delivered with MCMs (adjuvant).

[00044] FIG. 1 ID is a chart of firefly luciferase activity of SM-102 LNPs frozen and stored for 7 days (d7) at -80°C in trehalose with MCMs.

[00045] FIG. 1 IE is a chart of firefly luciferase activity of SM-102 LNPs frozen and stored for 7 days (d7) at 25°C in trehalose with MCMs.

[00046] FIG. 12A is a chart of the encapsulation efficiency of mRNA

[00047] FIG. 12B is a plot of the 2-(p-toluidino) naphthalene-6-sulfonic acid (TNS) assay determination of pK_a of mRNA LNPs. [00048] FIG. 12C is a chart of the dynamic light scattering assessment of mRNA LNP size.

[00049] FIG. 12D is a chart of the charges determined by zeta potential of mRNA LNPs.

[00050] FIG. 13 A Overview of mRNA Delivery and Vaccination experiments using 3: 1 pg Ovalbumin (OVA) mRNA to Firefly luciferase mRNA.

[00051] FIG. 13B is a set of IVIS imaging on day one (dl) following delivery of freshly prepared firefly luciferase mRNA LNPs, dl following delivery of firefly luciferase mRNA LNPs after 5 weeks storage at -80°C, or dl following delivery of firefly luciferase mRNA LNPs lyophilized and stored at 25°C. SM-102 LNPs with 10% DOTAP were used.

[00052] FIG. 13C is a plot of the quantification of IVIS data in FIG. 13B There were no statistical differences between groups by one-way ANOVA.

[00053] FIG. 13D is a plot of in vitro firefly luciferase expression in hMSCs using matched doses from FIGS. 13B-13C.

[00054] FIG. 13E is a plot of mRNA content of LNPs bound to MCMs.

[00055] FIG. 13F is a plot of OVA-antigen specific CD8 T cells harvested from the spleen measured by flow cytometry using MHC 1 tetramer antibody against OVA. There were no specific differences in OVA-antigen specific CD8 T cells.

[00056] FIG. 14 is a schematic overview (not to scale) of a functionalized nucleic acid LNP. LNPs were prepared at a 50: 10:38.5: 1.5 molar ratio of ionizable lipid, helper lipid, cholesterol, and PEGylated lipid. PEGylated lipid was substituted with a maleimide functionalized PEG lipid (0.15-1.2% of total lipid). Hydroxyapatite targeting peptides (HABP; SEQ ID NO: 1) or sham peptides (SEQ ID NO: 2) were conjugated by click chemistry to the PEG-Maleimide lipid.

[00057] FIG. 15A is a schematic overview of the functionalized nucleic acid compositions (not to scale).

[00058] FIG. 15B is a chart of firefly luciferase mRNA activity of unfunctionalized LNP delivered after no binding or binding to hydroxyapatite (HA). [00059] FIG. 15C is a chart of firefly luciferase mRNA expression of free LNPs (HA-) or bound LNPs (HA+).

[00060] FIG. 15D is a chart of the cytotoxicity data of peptide functionalized mRNA LNPs.

[00061] FIG. 15E shows fluorescence images of the localization of DiD labeled LNPs to hydroxyapatite microparticles for Sham peptide LNPs (top row) and HABP LNPs (bottom row). Significance Level by ANOVA at ****p<0.0001, ***p<0.001, **p<0.01 *p<0.05.

[00062] FIG. 16A shows charts of TNS Assay determination of pKa for sham (top) and HABP conjugated (bottom) mRNA LNPs.

[00063] FIG. 16B is a chart of the encapsulation efficiency of LNP formulations with varying DSPE-PEG-Maleimide %.

[00064] FIG. 16C is a chart of the sizes as determined by dynamic light scattering for LNPs with varying DSPE-PEG-Maleimide %.

[00065] FIG. 16D is a chart of the charges as determined by zeta potential for LNPs with varying DSPE-PEG-Maleimide %.

[00066] FIG. 17A is a plot of the binding of DiO labeled LNPs to hydroxyapatite microparticles over 120 min as measured by fluorescence depletion.

[00067] FIG. 17B shows images of fluorescence microscopy visualization of DiO labeled LNPs bound to HA microparticle after binding.

[00068] FIG. 17C is a plot of firefly luciferase expression 24 hours after transfection following binding to hydroxyapatite microparticles.

[00069] FIG. 17D shows images of the fluorescence microscopy visualization of DiD labeled LNP binding to cancellous cortical bone chips in vitro.

[00070] FIG. 18 A is a schematic overview of in vivo experiments. 3 : 1 eGFP to Firefly Luciferase mRNA was injected via tail vein and imaged after 24 hours in vivo and ex vivo. [00071] FIG. 18B shows ex vivo luminescence (top) and fluorescence (bottom) imaging.

[00072] FIG. 18C is a chart of the total flux of the liver (left) and spleen (right) of the luminescence images of FIG. 18B representing activity of delivered firefly luciferase mRNA.

[00073] FIG. 18D is a chart of the DIR labeled LNP fluorescence of the liver (left) and spleen (right) of the fluorescence images of FIG. 18B.

[00074] FIG. 19A shows hematoxylin and eosin staining images of mouse femurs (top) for sham LNP treated mice and HABP LNP treated mice. Immunofluorescence staining for eGFP in the proximal femoral head and shaft (bottom).

[00075] FIG. 19B shows the mean fluorescence intensity of immunofluorescence images of FIG. 18A for the femoral head (left) and femoral shaft (right).

[00076] FIG. 20 shows the hematoxylin and eosin staining (top) and immunofluorescence staining for eGFP (bottom) of mouse livers for sham LNP treated mice (left) and HABP LNP treated mice (right).

DETAILED DESCRIPTION

[00077] As used herein, “nucleic acids” refer to any nucleotide-containing biomolecule. Nucleic acids may include ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In a non-limiting example, the RNA may include, but is not limited to messenger RNA (mRNA), microRNA (miRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), small interfering RNA (siRNA), small hairpin (shRNA), informational RNA (iRNA), guide RNA (gRNA), or apatamers. In a nonlimiting example, DNA may include, but is not limited to double stranded DNA (dsDNA) or single stranded DNA (ssDNA).

[00078] As used herein, “mRNA complexes” refer to complexes of mRNA with a complexing agent such as a lipid nanoparticle complexing agent.

[00079] A “complexing agent”, as used herein, refers to a transfection agent that binds to a nucleic acid, such as mRNA, and promotes cell transfection efficiency. Complexing agents used herein include but are not limited to lipid nanoparticles (LNPs). [00080] A “microparticle”, as used herein, refers to a particle that has a particle size in the micrometer range (or a particle size between about 0.2 um and about 1000 um).

[00081] As used herein, a “mineral-coated substrate” is a substrate coated with a mineral coating layer containing at least calcium, phosphate, and carbonate.

[00082] “Complex formation” as used herein refers to the complex formation between mRNA and the complexing agent.

[00083] As used herein, “binding” refers to binding between mRNA complexes and a mineral- coated substrate (e.g., a mineral -coated microparticle or a mi neral -coated glass vial).

[00084] “Lyoprotection”, as used herein, refers to the protection of a substance undergoing lyophilization against damage.

[00085] A “lyoprotectant”, as used herein, refers to a substance that that has lyoprotective properties.

[00086] As used herein, “mSBF” refers to modified simulated body fluid. The modified simulated body fluid is a solution with ion concentrations approximately equal to that of human blood plasma. As used herein, an mSBF differs from a standard simulated body fluid (SBF) in its concentrations of one or more of calcium, phosphate, carbonate, and dopants such as fluoride, silicates, and citrates.

[00087] As used herein, a “subj ect” refers to a live human or animal subj ect undergoing treatment.

[00088] FIG. 1 shows an example complexing agent that forms a complex with a nucleic acid and improves transfection efficiency. Suitable complexing agents may include positively charged lipid-based complexes such as LNPs. Such an LNP may comprise DLin-MC3-DMA, DMG- PEG(2000), and 1,2-DSPC (LNP-MC3 Exploration Kit, Cayman Chemical), SM-102, DMG- PEG(2000), 1,2-DSPC (LNP- 102 Exploration Kit, Cayman Chemical), or any other suitable lipids. FIG. 1 specifically illustrates a nucleic acid LNP composition 100. The LNP lipid membrane 101 includes an ionizable lipid 102, helper lipid 104, polyethylene glycol (PEG)-conjugated lipid 106, and cholesterol 108. One or more nucleic acids 110 are encapsulated within the lipid membrane 101. In a non-limiting example, the nucleic acid may be selected from the group consisting of mRNA, microRNA, siRNA, shRNA, iRNA, gRNA, and an aptamer. In some embodiments, an mRNA-based therapeutic composition may be a vaccine against a particular infectious disease such as, but not limited to, coronavirus 2019 (COVID-19), human immunodeficiency virus (HIV), malaria, or other known or emerging infectious disease. Suitable mRNAs also include RNAs with chemical modifications including labels (e.g., fluorescent labels, protein labels, radioactive labels, metal labels, nanoparticle labels, etc.) and/or chemically modified bases such as, but not limited to, 5-methylcytidine, pseudouridine, 2-thiouridine, and Ni-methyl-pseudouridine. In this specific example, and as used in the remaining disclosure and example, the nucleic acid 110 is mRNA.

[00089] In a specific non-limiting example of the LNP lipid membrane 101, the ionizable lipid 102 may be 9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102). Furthermore, the ionizable lipid 102 may be present in a molar ratio of 0-60%, suitably 0 - 50 % of the LNP, or any ratio between 0-50%.

[00090] The helper lipid 104 may be one of 1,2-Di stearoyl -sn-glycero-3 -PC (1,2-DSPC) or 1,2- Dioleoyl-3 -trimethylammonium propane (DOTAP). 1,2-DSPC is a neutral lipid, while DOTAP is a charged lipid with a positive charge on its hydrophilic end. In a non-limiting example. The helper lipid 104 may be present in a molar ratio of 0 - 40% of the LNP. In a specific example, DOTAP may be present in a molar ratio of 10-25%, suitably 0 - 20% of the LNP. In another non-limiting example, the helper lipid 104 may be a mix of DOTAP and 1,2-DSPC. For example, the helper lipid may be present in a range of 0-40% in which 1,2-DSPC may be present in a range of 0-10% and DPTAP is added for a total of not more than 40% helper lipid 104 in the LNP. In a non-limiting example where the LNP is DOTAP-substituted, the total DOTAP is from about 1 - 20 % of the LNP. In a further example, where the molar ratio of DOTAP is above 10%, the molar ratio of cholesterol is consequently decreased in the LNP.

[00091] The PEG-conjugated lipid 106 may be l,2-Dimyristoyl-sn-glycero-3- methoxypolyethylene glycol (DMG-PEG 2000). In a non-limiting example, the PEG-conjugated lipid 106 may be present in a molar ratio of 0-3%, suitably 0 - 1.5% of the LNP.

[00092] In a non-limiting example, cholesterol 108 may be present in a molar ratio of 8.5 - 43.5%, suitably 10-40%, suitably 15-35%, suitably 20-30% of the LNP. [00093] In a non-limiting example, a LNP lipid membrane 101 has molar ratios of 50% SM-102, 20% DOTAP, 28.5% Cholesterol, and 1.5% DMG-PEG-2000.

[00094] Referring to FIG. 2, the nucleic acid LNP 100, or mRNA complex 200, may be further processed to form an mRNA-based therapeutic composition 210. Specifically, the mRNALNP 100 may be incubated with a mineral-coated substrate 202. In a non-limiting example, the mineral- coated substrate 202 may be that of U.S. Application No. US 18/708,518, which is herein referenced in its entirety.

[00095] The mineral-coated substrate 202 may include a substrate having at least one mineral coating layer applied to one or more surfaces thereof. The mineral coating layer may have different ratios of calcium, phosphate, and carbonate. The calciunrphosphate ratio in the mineral coating layer may vary from about 0.1 to about 10, or from about 2 to about 5. The carbonate concentration of the mineral coating layer may vary from about 1 mM to about 150 mM, or from about 3 mM to about 100 mM. Other ratios/concentrations of calcium, phosphate, and carbonate may be used in alternative embodiments. Different morphologies of the mineral coating layer (e.g., plate-like, spherulite-like, etc.) may be achieved by varying the amounts and ratios of calcium, phosphate, and carbonate. For example, a high carbonate concentration may result in a mineral coating layer having a plate-like structure, whereas a low carbonate concentration may result in a mineral coating layer having a spherulite-like structure.

[00096] In some embodiments, the mineral-coated substrate 202 may include a mineral-coated microparticle (MCM) as shown in FIG. 2. The MCM 202 may include a core material in the form of a microparticle coated with the mineral coating layer. Suitable core materials on which the mineral coating layer is formed may include polymers, ceramics, metals, glass, and combinations thereof in the form of microparticles. Non-limiting examples of suitable microparticles include ceramics (e.g., hydroxyapatite, beta-tricalcium phosphate (P-TCP), magnetite, neodymium), plastics (e.g., polystyrene, poly-caprolactone), hydrogels (e.g., polyethylene glycol, poly(lactic- co-glycolic acid) and the like, and combinations thereof. Particularly suitable core materials include those that are dissolved in vivo such as P-TCP and hydroxyapatite. The mRNA complexes 200 may be adsorbed to the mineral coating layer of the MCM 202. Upon cell transfection or administration, the mRNA complexes adsorbed to the mineral coating layer may be released as the mineral coating layer degrades. Upon introduction to the cytoplasm (via endocytosis, micropinocytosis or other mechanism), the mRNAs may proceed to translation for protein production. After translation and processing, the MCMs used for initial delivery of the mRNA complexes may bind and sequester the secreted protein. The protein may be released from the MCM over time back to the cell, prolonging the biological response.

[00097] Referring still to FIG. 2, the mRNA complex 200 may also be suspended in a solution containing a lyoprotectant 204 to protect the mRNA complexes 200 from damage that may occur during freeze drying/lyophilization 206 to form a powder 208 of the mRNA-based therapeutic composition 210. In a non-limiting example, the lyoprotectant 204 may be a disaccharide that interacts with the polar head groups of the complexing agent (LNP) to prevent damage during lyophilization and help with long term storage of the composition. Lyoprotectants include, but are not limited to, trehalose, sucrose, or maltose. In some embodiments, the therapeutic composition may contain from about 5 millimolar (mM) to about 1 molar (M), or about 30 mM to about 1 M of the disaccharide (prior to lyophilization). In one specific embodiment, the composition 10 may contain about 150 mM trehalose. In another embodiment, the composition 10 may contain about 250 mM or about 254 mM sucrose. Other types of lyoprotectants may also be used including, but not limited to, glucose, maltose, lactose, inositol, dextran, hydroxypropyl-B-cyclodextrin, polyethylene glycol, and combinations thereof. In a non-limiting example, the disaccharide is trehalose, which may be present in a percentage from 5-20 % of the suspension solution. Alternatively, the disaccharide is any combination of maltose and sucrose, which may be present in a percentage from 5-20% of the suspension solution.

[00098] In a non-limiting example, a range of 0.05 mg/mL to 0.1 mg/mL of nucleic acid DOTAP lipid nanoparticle complexes 200 may be added to a range of 1 mg/mL to 20 mg/mL of MCMs 202 to form the nucleic acid based therapeutic composition 210.

[00099] FIGS. 3A-3D provide non-limiting examples if which factors affect LNP to MCM binding and/or transfection. FIG. 3 A shows that the amount of PEG-conjugated lipid 106 affects LNP to MCM binding for both a standard LNP (50: 10:38.5: 1.5 molar ratio of SM-102:l,2- DSCP:cholesterol:DMG-PEG-2000) and a 20% DOTAP LNP (50:20:28.5: 1.5 molar ration of SM- 102:DOTAP:cholesterol:DMG-PEG-2000). FIGS. 3B-3C further indicate that PEG-conjugated lipid 106 concentration is an influential binding (low PEG) and transfection factor (high PEG).

[000100] FIG. 3D-3E show the interaction effects of several factors. FIG. 3D illustrates that the ratio of MCM to RNA (ug MCM: 1 ug RNA) affect LNP to MCM binding. FIG. 3E illustrates that the initial concentration of MCM and the calcium to phosphate ratio of the mineral coated substrate affect LNP to MCM binding.

[000101] Referring now to FIG. 4, a workflow of a non-limiting method 400 of preparing the nucleic acid-based therapeutic composition is described. It is noted that the nucleic acid may be any of the enumerated types of RNA listed previously. In a specific embodiment, the nucleic acid may be mRNA. At step 402, nucleic acid is incubated with a LNP, whereby the lipid membrane at least partially comprises a DOTAP lipd to form RNA complexes. In a non-limiting example, the lipid membrane includes the DOTAP lipid in a molar ratio from about 1 to 20%.

[000102] At step 404, the nucleic acid complexes are incubated with a mineral -coated substrate to bind nucleic acid complexes to the mineral-coated substrate. Next, at step 406, the bound nucleic acid complexes are suspended in a solution containing a lyoprotectant to provide the nucleic acidbased therapeutic composition as described above. At step 408, the nucleic acid -based therapeutic composition is lyophilized into a dry powder.

[000103] FIG. 5 shows a non-limiting, alternative nucleic acid LNP composition 500. In this example, the nucleic acid LNP composition lipid membrane 501 includes ionizable lipid 102, helper lipid 502, (PEG)-conjugated lipid 106, cholesterol 108, and a mal eimide substituted lipid 504. One or more nucleic acids 110 are encapsulated within the lipid membrane 501. As previously described, the nucleic acid may be selected from the group consisting of mRNA, microRNA, siRNA, shRNA, iRNA, gRNA, and an aptamer. In a non-limiting example, the ionizable lipid 102 may be SM-102 as described previously with reference to FIG. 1. Here, helper lipid 502 is 1,2- DSPC as previously described. Alternatively, helper lipid 502 may be DOTAP as previously described. The (PEG)-conjugated lipid 106 may be DMG-PEG 2000 as previously described. In a non-limiting example, maleimide substituted lipid 504 may be l,2-Distearoyl-sn-glycero-3- phosphoethanolamine (DSPE)-PEG-Maleimide, which includes a DSPE phospholipid and a thiol or cysteine reactive maleimide group. The maleimide substituted lipid 504 may be present in a molar ratio of 0.15 - 1.2% of the LNP composition.

[000104]Nucleic acid LNP 500 may be further reacted with a peptide 508 via a click chemistry reaction to form a functionalized nucleic acid complex 510. In a non-limiting example, the click chemistry reaction includes a Thiol Michael Addition between the peptide and the thiol reactive maleimide group on DSPE-PEG-Maleimide 504. In a non-limiting example, the peptide 408 includes hydroxyapatite binding peptide (HABP) 512. For example, the HABP 512 has the sequence yEPRRyEVAyEL-GGGS-C (SEQ ID NO: 1). As used herein, “yE” is gamma carboxyglutamic acid instead of glutamate, which possess calcium binding properties. Further, the used herein are spacers for notation only to separate out the active portions of the peptide, where the first section before the first spacer is the active section forming a secondary structure to allow binding to hydroxyapatite, the “GGGS” sequence between the first and second spacer is a linker, and final “C” is cysteine for enabling thiol Michael addition. Furthermore, the HABP is synthesized using N-terminal acetylation and C-terminal amidation. Alternatively, a sham peptide 514 is provided for illustrative purposes in the following Examples. The sham peptide 514 may have the sequence EPRREVAEL-GGGS-C (SEQ ID NO: 2).

[000105] Referring to FIG. 6, the functionalized nucleic acid complex 510 may be further processed as previously presented for mRNA complex 200 in FIG. 2. For simplicity, FIG. 6 only shows incubation of the functionalized nucleic acid complex 510 with MCM 112 to form nucleic acid-based therapeutic composition 512. As described previously, the functionalized nucleic acid complex 510 may also be suspended in a solution containing a lyoprotectant for protecting the composition during freeze drying/lyophilization where it is turned into a dry powder.

[000106] Referring now to FIG. 7A, a workflow of a non-limiting method 700 for preparing a nucleic acid-based therapeutic composition is described. It is noted that the nucleic acid may be any of the enumerated types of RNA listed previously. In a specific embodiment, the nucleic acid may be mRNA. At step 702 nucleic acid is incubated with a HABP-functionalized LNP to form nucleic acid complexes. At step 704, the nucleic acid complexes are incubated with a mineral- coated substrate to bind the nucleic acid complexes to the mineral-coated substrate. [000107] More specifically, the non-limiting method 706 for preparing a nucleic acid-based therapeutic provides step 708, where nucleic acid is incubated with a LNP, whereby the lipid membrane includes maleimide-modified lipids to form nucleic acid complexes. Next, at step 710, a peptide is conjugated onto the nucleic acid complexes to form functionalized nucleic acid complexes. In a non-limiting example, the peptide may be the HABP as previously described, whereby conjugation onto the nucleic acid complexes includes a click chemistry reaction. As previously described, the click chemistry reaction includes a Thiol Michael Addition between the nucleic acid complexes and the peptide.

[000108] In a non-limiting example of the present disclosure, any one of the compositions described herein may be administer to a subject in need of the nucleic acid-based therapeutic.

[000109] Alternatively, any one of the compositions made by any one of the methods described herein may be administered to a subject in need of the nucleic acid-based therapeutic. In a nonlimiting example, the administration of theses compositions is via infusing or injecting the composition in the subject.

[000110] The present disclosure has described one or more embodiments, and it should be appreciated that many equivalents, alternatives, variations, and modifications, aside from those expressly stated, are possible and within the scope of the invention.

[000111] The following example provide illustrative compositions and methods but should not be construed as limiting.

EXAMPLE

Example 1 : Methods for DOTAP Modification to LNP

1.1.1. Chemicals and Reagents for Lipid Nanoparticle Synthesis

[000112] 9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102, MedKoo, Morrisville, NC, USA), DLin-MC3-DMA (Tocris Bioscience, Bristol, United Kingdom), Cholesterol (Sigma Aldrich, Burlington, Massachusetts, USA), 1,2- Distearoyl-sn-glycero-3-PC (1,2-DSPC, Avanti Polar Lipids, Burmingham, AL, USA), 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG PEG-2000, Avanti Polar Lipids, Burmingham, AL, USA),l,2-dioleoyl-3-trimethylammonium-propane chloride salt (DOTAP, Avanti Polar Lipids, Burmingham, AL, USA), l,2-dioleoyl-sn-glycero-3 -phosphate sodium salt (18PA, Avanti Polar Lipids, Burmingham, AL, USA). Firefly Luciferase mRNA(Trilink Biotechnologies, San Diego, Ca USA).

1.1.2. Lipid Nanoparticle (LNP) Synthesis

[000113] Glass vials were used to store lipids dissolved in ethanol at -20°C. 4-component formulations of lipid nanoparticles were prepared at a molar ratio of 50:38.5: 10: 1.5 of ionizable lipid: cholesterol: 1,2-DSPC:PEG lipid at 5mM in ethanol. LNPs were synthesized by ethanol dilution method with mRNA in 100 mM citrate pH 4 at a ratio of 1 :3 by volume. These LNPs were then dialyzed for 2 hours in PBS prior to use. For 4 component LNP synthesis 1,2-DSPC was completely substituted with the lipids 18PA and DOTAP. For 5-component LNP synthesis DOTAP or 18PA were added to the 4-component formulation listed above with reduced molar ratio of cholesterol. For DOTAP LNPs 0-40% DOTAP was used completely substituting 1,2-DSPC (10% MW) and the molar ratio of cholesterol was changed in direct proportion to DOTAP (FIG. 8A).

1.3 Lipid Nanoparticle Characterization

1.3.1. pKa Determination

[000114] pK_a of lipid nanoparticle was determined by 6-(p-Toluidino)-2-naphthalenesulfonic acid (TNS).[1] TNS assay buffer (20 mM sodium phosphate, 25 mM citrate, 20 mM ammonium acetate, and 150 mM NaC)l were prepared at various pHs. 6 pM TNS reagent and 25 pM LNPs were added to each buffer in a black multi-well plate and fluorescence was measured at excitation/emission of 325/435. Normalized fluorescence measurements were fit determining the pKa at the pH value that measured 50% of the maximum fluorescence.

1.3.2 Encapsulation Efficiency

[000115] Encapsulation efficiency of mRNA LNPs was determined using a nucleic acid fluorophore (Ribogreen Reagent, Thermofisher Scientific, Waltham, Massachusetts, USA). Calculations were determined comparing the input mRNA mass of mRNA LNPs against the measured concentration of free mRNA as determined with a standard curve of free mRNA. 1.3.3 Dynamic Light Scattering and Zeta Potential

[000116] Size and charge of mRNA LNPs were determined using dynamic light scattering and zeta potential analysis using a Zetasizer (Malvern Panalytical, Malvern, UK).

1.4 Mineral Coated Microparticle Synthesis

[000117] Mineral coated microparticles were created as previously described. [2] Briefly, beta tricalcium phosphate micropowder (P-TCP; Plasma Biotal Limited, Maharashtra, India) was incubated in modified simulated body fluid (mSBF) solutions at 3 mg/mL. mSBF in this study is made with 141 mM NaCl, 4 mM KC1, 0.5mM MgSO₄, 1 mM MgCh, 2.1 mM NaHCCh, 20mM MES, 5 mM CaCh and 2 mM KH2PO4 for 2 days. Then the MCMs were incubated in mSBF as above with 5 mM CaCh, 10 mM KH2PO4 and 5mM citric acid and 1 mM sodium fluoride as previously described for 3 days. [2] The resulting citrate and fluoride doped MCMs were then washed twice in deionized water, filtered through a 40 pm pore cell strainer, and lyophilized for 48 hrs. The lyophilized MCMs were then analyzed for nanotopography, calcium/phosphate, release as previously described. [2-5]

1.5 Lyophilization

[000118] mRNA LNPs were mixed with MCMs at a ratio of 125 pg MCM: 1 pg of mRNA In mRNA LNPs for 1 hour. mRNA LNPs were spun down at 2000g for 30s and the supernatant was removed to assess the effect of mRNA LNPs only bound to MCMs (referred to as Bound) or were co-delivered without resuspending MCMs (referred to as Adjuvant) as shown in FIG. 8B. mRNA LNPs +/- MCMs in freezing excipient and frozen at -80°C. Freezing excipients screened include normal saline, maltose and sucrose, and trehalose or excipients from the mRNA COVID-19 vaccines. 20% trehalose was used for additional studies. mRNA LNPs +/- MCM were then lyophilized (Labconco, Kansas City, KA, USA) overnight. Lyophilized mRNA LNPs +/- MCM were then sealed in mylar pouches under nitrogen and stored at 4°C or 25°C.

1.6 Cell Culture and Transfection [000119]Human mesenchymal stromal cells (Passage 3-6, Lonza, Basel, CH) were grown in 10% FBS/1% penicillin/ streptomycin alpha minimum essential medium (Corning, NY, USA). Media was changed every three days and cells were grown to 80-90% confluence prior to transfection. In order to determine expression of firefly luciferase mRNA cells were lysed after 24 hours transfection. Cells were washed with PBS prior to addition of Cell Culture Lysis Reagent (Promega, Madison, WI, USA). 20 pl of lysates were mixed with 100 pL of luciferase assay buffer (50mM Tris, 10 mM MgSCU, 6mM cysteine, 0.02 mM sodium pyrophosphate, 1 mM EDTA, and 2g/L BSA) with 150 pg/mL D-Luciferin and 1 mM ATP and read on a plate reader (Synergy HTX, Biotek, Winooski, VT, USA).

1.62 Animal Study

[000120] Animal protocols were approved by the University of Wisconsin-Madison Institutional Animal Care and Use Committee (IACUC). C57B1/6 mice (stock no. 000664) were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were housed under aseptic conditions in a local vivarium.

[000121] mRNA LNPs were either freshly prepared or frozen with 20% trehalose and/or lyophilized and stored for 5 weeks. The mRNA was a mix of 3 pg ovalbumin and 1 pg of firefly luciferase mRNA. For groups incorporating MCMs 250 pg/pg of MCM to mRNA were mixed for further experiments. LNP formulation included SM-102/DOTAP/Cholesterol/DMG-PEG- 2000 at molar ratios of 50/10/38.5/1.5. Frozen formulations were stored at -80°C and lyophilized formulations were packaged in mylar under nitrogen and kept at room 25°C. Lyophilized formulations were resuspended in PBS prior to injection. Mice were anesthetized under isoflurane and mRNALNPs+/- MCMs were injected intradermally into the ear pinna using a low dead-space insulin syringe. Two immunizations were performed 1 week apart. One day after the first injection firefly luciferase expression was determined by IVIS Spectrum (PerkinElmer, Waltham, MA). Briefly, mice were anesthetized and injected with 150 mg/kg of D-Luciferin into the peritoneum. Peak luminescence was measured within

1.6.1 Statistical Analysis [000122] All statistical analysis were run using Prism software (v.10, GraphPad, Boston, MA, USA). Data reported are mean and standard deviation unless otherwise specified. One-way analysis of variance (ANOVA) was used to compare differences between three or more groups with post-hoc testing done by Tukey’s Honest Significant Difference test to determine differences between individual groups. Annotations for figures is as follows ****p<0.0001, ***p<0.001, **p<0.01 *p<0.05; n.s: no significance, unless otherwise specified.

Example 2: Results for DOTAP Modification to LNP

2.1 Long term storage of 10% DOTAP Functionalized mRNA LNPs is Improved with Biomimetic Minerals

[000123] Biomimetic mineral coated microparticles (MCM) maintain the activity of lyophilized DOTAP functionalized mRNA LNPs stored at 25°C (FIG. 9D). Substitution of 1,2-DSPC with the cationic lipid DOTAP in SM-102 LNPs improves transfection when bound to MCMs (FIG. 9A). MCMs improve activity of mRNA LNPs when lyophilized in the presence of trehalose or maltose and sucrose (FIG. 9B). Comparing mRNA LNPs stored with MCMs compared to those without MCMs significantly improve storage of lyophilized mRNA LNPs stored at 4°C and 25°C compared to those frozen and stored at -80°C (FIG. 9C). Lyophilized mRNA LNPs stored with MCMs at 25°C maintain luminescent activity in vitro over 3 months (FIG. 9D).

2.2 Functionalization of MC3 mRNA LNPs with DOTAP Improves Interactions with MCMs and mRNA Activity after Lyophilization

[000124] MCMs and MC3 DOTAP -LNPs improves mRNA activity with lyophilization compared to control MC3 LNPs (FIGS. 10A-10E). Substitution of DOTAP for helper lipid in MC3 mRNA LNPs improves localization of DiO labeled mRNA LNPs to MCMs seen with fluorescence microscopy (FIG. 10A). Inclusion of DOTAP in MC3 mRNA LNPs significantly improves binding to MCMs from 4.7% in control LNP to 40% binding in DOTAP LNP (FIG. 10B). MC3 DOTAP mRNA LNPs improve transfection of firefly luciferase mRNA compared to control LNPs (FIG. 10C). Binding to MCMs or co-deliveiy with MCMs further enhances mRNA transfection (FIG. 10C). DOTAP functionalized MC3 LNPs enhance transfection after storage frozen at -80°C or lyophilized and stored at 25°C for 7 days (FIGS. 10D-10E). MCMs further improve the transfection of frozen or lyophilized MC3 DOTAP mRNA LNPs (FIGS. 10D-10E). 2.3 Optimization of DOTAP Substitution in SM-102 mRNA LNPs Improves mRNA Activity Following Lyophilization

[000125] 20% DOTAP best improves preservation of mRNA LNPs with MCMs. Increasing DOTAP % improves binding with MCM from 17.9% without DOTAP to 50.8% for 40% DOTAP LNPs (FIGS. 11 A-l IB). Co-delivery of MCMs with DOTAP LNPs improve transfection across from 10-40% DOTAP (FIG. 11C). DOTAP SM-102 LNPs co-delivered MCMs improves transfection after storage frozen at -80°C (FIG. HD) and 25°C (FIG. HE) for 7 days. 20% DOTAP LNPs best maintain mRNA activity after storage for 7 days at 25°C (FIG. 1 IE).

2.4 Physicochemical Characterization of SM-102 mRNA LNPs with DOTAP

[000126] Functionalization of SM-102 mRNALNPs with increasing molar percentages of DOTAP changes the physicochemical properties of the LNP (FIGS. 12A-12D). LNPs with DOTAP percentages over 15% encapsulated significantly more mRNA than the control formulation without DOTAP (FIG. 12A). pK_a determination by 2-(p-toluidino) naphthalene-6-sulfonic acid (TNS) assay demonstrated a pK_a of 6.97 for control LNP without DOTAP, 6.82 for 10% DOTAP LNP and 7.17 for 20% DOTAP LNP (FIG. 12B). pK_a peaked at 7.34 for 25% DOTAP LNPs and declined to 6.24 for 40% DOTAP LNPs. (FIG. 12B). Handmixing of LNPs resulted in large LNPs in the 500nm range for most LNPs formulated but LNPs with 20% DOTAP were significantly smaller at 210nm (FIG. 12C). Zeta potential increased from -7.15 mV for control LNPs without DOTAP to - 4.22 mV for 20% DOTAP and -1.19 mV for 40% DOTAP (FIG. 12D).

2.5 In Vivo Experiments

[000127] Firefly Luciferase Expression in vivo demonstrates that mRNA LNPs maintain their activity on MCMs in fresh, frozen, and lyophilized formulations (FIGS. 13A-C). There were no significant differences by ANOVA between all groups tested (FIG. 13C). However, fresh mRNA LNPs without MCMs (MCM-) trended towards the highest radiance and lyophilized mRNALNPs with MCMs (MCM+) had the lowest radiance. Fresh or frozen mRNALNPs with MCMs (MCM+) had significantly higher firefly luciferase expression for matched doses scaled down for in vitro testing (FIG. 13D). There were no significant differences between mRNA LNPs that were lyophilized regardless of the presence of MCMs. However, mRNA LNPs with MCMs (MCM+) maintained significantly more mRNA content when frozen or lyophilized compared to freshly prepared mRNA LNPs (FIG. 13E). Furthermore, there were no significant differences in OVA antigen specific CD8 T cells detected by flow cytometry (Figure 13F).

Example 3 : Methods for enhanced mineral binding

3.1 Chemicals and Reagents for Lipid Nanoparticle Synthesis

[000128] 9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102, MedKoo, Morrisville, NC, USA), Cholesterol (Sigma Aldrich, Burlington, Massachusetts, USA), l,2-Distearoyl-sn-glycero-3-PC (1,2-DSPC, Avanti Polar Lipids, Burmingham, AL, USA), l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG PEG-2000, , Avanti Polar Lipids, Burmingham, AL, USA), , l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG 2000 Maleimide Avanti Polar Lipids, Burmingham, AL, USA). Firefly Luciferase mRNA (Trilink Biotechnologies, San Diego, USA).

3.2 Mineral Binding Peptide Description

[000129] We used a hydroxyapatite binding peptide (yEPRRyEVAYEL-GGGS-C; SEQ ID NO: 1) based on the N-terminal alpha helix of osteocalcin and sham peptide (EPRREVAEL-GGGS-C; SEQ ID NO: 2) with glutamine residues instead of gamma-carboxyglutamic acid as previously described. [6-8] In order to conjugate the peptides to the lipid nanoparticles a linker sequence and C-terminal cysteine were added as noted above. Peptides were synthesized from a commercial partner with N-terminal acetylation and C-terminal amidation (Genscript, Piscataway, NJ, USA).

3.3 Lipid Nanoparticle (LNP Preparation)

[000130] Lipids were dissolved in ethanol and stored in glass vials at -20°C. Lipid nanoparticles were prepared by mixing a molar ratio of 50:38.5: 10:1.5 of ionizable lipid: cholesterol: 1,2- DSPCPEG lipid at 5mM in ethanol. Lipid nanoparticles were formed by rapidly mixing the lipid-ethanol formulation described above with mRNA in citric acid buffer (100 mM, pH 4) at a ratio of 1 :3 by volume. LNPs were dialyzed (10K MWCO, Pur-A-Lyzer™ Midi, Sigma Aldrich, Burlington, Massachusetts, USA) for 2 hours into PBS prior to further use. To visualize mineral binding or quantify LNP binding 0.1% by molar weight of lipid label (DiO, DiD, Thermofisher Scientific, Waltham, MA, USA) were added prior to mixing.

[000131] For peptide conjugation, LNPs were synthesized as described above with 0.15 - 1.2% of the total lipid consisting of DSPE-PEG-Mal eimide (FIG. 14). DSPE-PEG2000-Maleimide served as a substitute for DMG-PEG-2000. For peptide conjugation by thiol Michael addition, hydroxyapatite binding peptide or sham peptide were incubated with LNPs at 10-times molar excess of peptide:DSPE-PEG-Maleimide overnight at 4°C. Peptide functionalized LNPs were then washed and concentrated two times using ultracentrifugation (Amicon Ultra 10KDA MWCO, Sigma Aldrich, Burlington, Massachusetts, USA), to remove residual peptide.

3.4 p/G Determination

[000132] Lipid nanoparticle pU_a was determined by 6-(p-Toluidino)-2-naphthalenesulfonic acid (TNS) assay as previously described. [1] Base buffer consisted of 20 mM sodium phosphate, 25 mM citrate, 20 mM ammonium acetate, and 150 mM NaCl. Buffers were adjusted with 0.1N of NaOH or HC1 to different pH levels. 100 pl of buffer at each pH was added to a black 96 well plate and TNS (6 pM final) and LNPs (25 pM final) were added to the plate. Fluorescence measurements were obtained using a plate reader at ex/em of 325/435. These fluorescence measurements were normalized to the minimum value and fit to give a titration curve. pN_a was evaluated by determining the pH representing 50% of the maximum fluorescence.

3.5 Encapsulation Efficiency

[000133] mRNA encapsulation efficiency was determined by incubating mRNALNPs with a nucleic acid fluorophore (Ribogreen Reagent, Thermofisher Scientific, Waltham, Massachusetts, USA). mRNA encapsulation efficiency was determined comparing the input mRNA mass of mRNA LNPs against the measured concentration of free mRNA as determined with a standard curve of free mRNA.

3.6 Dynamic Light Scattering and Zeta Potential [000134] Size and charge of mRNA LNPs were determined using dynamic light scattering and zeta potential analysis using a Zetasizer (Malvern Panalytical, Malvern, UK). mRNA LNPs were prepared at a concentration of 0.1 mg/mL by mass of total lipid in PBS.

3.7 Cell Culture and Transfection

[000135] Human mesenchymal stromal cells (Passage 3-6, Lonza, Basel, CH) were grown in 10% FBS/1% penicillin/ streptomycin alpha minimum essential medium (Corning, NY, USA). Media was changed every three days and cells were grown to 80-90% confluence prior to transfection. In order to determine expression of firefly luciferase mRNA cells were lysed after 24 hours transfection. Cells were washed with PBS prior to addition of Cell Culture Lysis Reagent (Promega, Madison, WI, USA). 20 pl of lysates were mixed with 100 pL of luciferase assay buffer (50mM Tris, 10 mM MgSCU, 6mM cysteine, 0.02 mM sodium pyrophosphate, 1 mM EDTA, and 2g/L BSA) with 0.15 mg/mL D-Luciferin and 1 mM ATP and read on a plate reader (Synergy HTX, Biotek, Winooski, VT, USA).

3.8 Mineral Binding Experiments

[000136] To assess binding and transfection of mRNA LNPs in the presence of mineral sham or HABP conjugated mRNA LNPs were mixed with hydroxyapatite (HA) mineral micropowder powder (size 3-5 pm, Plasma Biotal, Buxton, UK) at a ratio of 25 pg HA : pg of mRNA for 30 minutes unless otherwise specified. For quantification of binding DiO labeled mRNA LNPs were incubated with HA as described above. At each time point the mixture was centrifuged for 30s at 2000g and the supernatant was taken off. Binding percentages were determined by comparing the fluorescence of binding supernatant to unbound control mRNA LNPs in a black multi -well plate using a plate reader (Synergy HTX, Biotek, Winooski, VT, USA). To determine binding to bone, DiD-peptide LNPs (5 pg) were incubated with cancellous cortical chips (4-6 mm, Regeneration Technologies inc., Alachua, FL, USA) for 1 hour then imaged. All imaging was performed using a fluorescent microscope with FITC, Cy5 filter cubes (Nikon Ti-Eclipse, Nikon, Toyko, Japan).

3.9 In Vivo Experiments [000137]DiR labeled mRNA LNPs were prepared as described above. C57BL/6 mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA). Mice were housed and monitored in the Wisconsin Institute for Medical Research vivarium and experiments were conducted under a University of Wisconsin-Madison lACUC-approved protocol, were injected with HABP or sham mRNA LNPs at 0.5 mg/kg mRNA with 3: 1 eGFP:Firefly Luciferase mRNA. 24 hours after transfection mice were injected with 150 mg/kg of D-Luciferin and imaged after 15 minutes. Mice were then sacrificed, organs were harvested and imaged for fluorescence and luminescence. Organs were kept moist in PBS in multi-well plates. Additional ATP (ImM) was added to bones for luminescence imaging.

3.10 Statistical Analysis

[000138] All statistical analysis were run using PRISM software (v.10, GraphPad, Boston, MA, USA). Reported data in figures are mean and standard deviation unless otherwise specified. Oneway analysis of variance (ANOVA) was used to compare differences between three or more groups with post-hoc testing done by Tukey’s Honest Significant Difference test to determine differences between individual groups. Annotations for figures is as follows ****p<0.0001, ***p<0.001, **p<0.01 *p<0.05; n.s: no significance, unless otherwise specified.

3.11 Histology and Immunofluorescence

[000139] Liver and femur specimens were fixed in 4% paraformaldehyde. Mouse femurs were decalcified with 20% EDTA for 48 hours, washed and placed in 70% EtOH prior to paraffin embedding and sectioning. Liver and femur specimens were stained using hematoxylin and eosin (H&E). Immunofluorescence for performed using anti-GFP primary antibody (Ab290, Abeam, Cambridge, UK) and Alexafluor 488 goat anti-rabbit secondary antibody (Al 1034, Invitrogen, Waithalm, MA, USA). Embedding, sectioning and staining was performed by the Translational Research Initiatives in Pathology laboratory at the University of Wisconsin - Madison.

Example 4: Results for enhanced mineral binding

4.1 Hydroxyapatite Binding Peptide Promotes mRNA LNP Transfection with HA Microparticles [000140] Hydroxyapatite binding peptide (HABP) conjugated mRNA lipid nanoparticles (LNP) promote transfection when bound to hydroxyapatite (HA). Fluorescently labeled peptide (HABP or sham) conjugated mRNA LNPs were incubated with HA microparticles, resuspended to remove free LNPs and added to cells (FIG. 15 A). Firefly luciferase mRNA is expressed after delivery of soluble unfunctionalized mRNA LNPs alone but do not express when bound to HA (FIG. 15B). HABP LNPs bound to HA improve transfection significantly over free HABP LNPs and free sham LNPs or HA bound sham LNPs. The most effective formulation of HABP LNPs bound to HA included 0.3% PEG-Maleimide. Soluble sham LNPs expressed minimal transgene while no appreciable signal was detected in sham LNPs bound to HA (FIG. 15C). HABP LNPs bound to HA microparticles reduced metabolic activity compared to free sham LNPs but did not significantly reduce metabolic activity compared to soluble HABP LNPs with 0.3 or 0.6% PEG- Maleimide (FIG. 15D). DiD labeled HABP mRNA LNPs were visualized bound to HA with fluorescence microscopy while sham LNPs were not detected (FIG. 15E).

4.2 Physiochemical Characteristics of Peptide Functionalized LNPs

[000141] Increasing the amount of DSPE-PEG-Maleimide lipid and, in turn, the amount of peptide decorating the LNPs changed the pKa and size of mRNA LNPs. HABP-LNPs formed with 0.3% DSPE-PEG-Maleimide had a significantly higher pKa of 6.16 (95% CI [5.93,6.42]) when compared to the 5.06 pKa of Sham-LNPs (p=0.010, 95% CI [5.06 [4.61,6.03]). LNPs with no peptide had a pKa of 5.89, which was not significantly different from the HABP-LNP pKa (p=0.31, 95% CI [4.97,6.40]) (FIG.16A, Table 1). HABP-LNPs tended to have higher pKa than Sham-LNPs for compositions created with between 0-0.9% DSPE-PEG-Maleimide (6.15 95% CI [5.90,6.42]; FIG.16A, Table 1). HABP-LNPs tended to be more negatively charged than Sham-LNPs (FIG. 16D). For example, HABP-LNPs formed with 0.3% DSPE-PEG-Maleimide had significantly less charge (-7.10 ± 0.50 ,p<0.001) than the comparable Sham-LNPs (-4.00 ± 0.80; FIG. 16D). LNPs without peptide were smaller (148.0 ± 17.1 nm) and less negatively charged (-2.90 ± 4.10 mV) than all peptide-functionalized LNPs (Figure 16D). Increasing the percentage of DSPE-PEG-Maleimide in the LNP formulation tended to increase the size of LNPs (FIG. 16D). HABP-LNPs formed with 0.3% DSPE-PEG- Maleimide were significantly smaller (134.50 ± 0.96 nm, p<0.001) than the comparable Sham- LNPs (195.50 ± 4.00 nm; FIG. 16D). [000142] There were no significant differences in mRNA encapsulation efficiency between Sham-LNPs and HABP-LNPs at DSPE-Peg-Maleimide of 0.15-0.9%, and efficiencies were in the 97-99% range (FIG. 16D). For example, the 0.3% DSPE-PEG-Maleimide compositions encapsulated mRNA at 99.82 ± 0.14% for Sham-LNPs and 99.52 ± 0.97 % for HABP-LNPs (FIG. 16D). However, there were significant decreases in encapsulation efficiency at the 1.2% level of DSPE-PEG-Mal eimide inclusion for both the Sham-LNPs (90.70% ± 0.51%) and HABP-LNPs (87.40% ± 1.50%, p<0.0001; FIG. 16D).

4.3 Hydroxyapatite Binding Peptide Improves mRNA LNP Binding to Mineral in vitro

[000143] HABP LNPs bind to hydroxyapatite microparticles and cancellous cortical chips in vitro. Binding of HABP LNPs to HA increases over 120 minutes while sham LNPs do not significantly change in binding over the same period (FIGS. 17A-17B). Increased binding time significantly increases transfection of HABP LNPs loaded on HA while sham LNPs do not significantly transfect (FIG. 17C). DiD labeled HABP LNPs bind to cancellous cortical chips in vitro as assessed by fluorescence while sham LNPs do not bind (FIG. 17D).

4.4 Hydroxyapatite Binding Peptide Improves Bone Marrow Transfection and Promotes Liver Escape in vivo

[000144] Intraosseous transfection is promoted by HABP LNPs.

[000145] HABP LNPs reduce liver transfection compared to sham LNPs in vivo. Ex vivo sham LNPs had significantly more luminescent and fluorescent signal in the liver compared to the HABP LNPs (FIGS. 18B-18D). There were no significant differences in splenic luminescence or fluorescence between groups (FIGS. 18B-18D). There was no abnormal infiltration of cells in the liver on hematoxylin and eosin staining in the liver (FIGS. 19A-19B and FIGS. 20).

[000146] Table 1. pKa Measurements of mRNA LNPs. p ?_a was evaluated by determining the pH representing 50% of the maximum fluorescence using a 6-(p-Toluidino)-2- naphthalenesulfonic acid (TNS) asay. p/L was evaluated for sham peptide and hydroxyapatite binding peptide (HABP) conjugated mRNA LNPs. p/L for control LNP without peptide was 5.89 95% confidence interval (CI) [4.97,6.40], n=3 replicates.

REFERENCES

[1] M. Kim, M. Jeong, S. Hur, Y. Cho, J. Park, H. Jung, Y. Seo, H.A. Woo, K.T. Nam, K. Lee, H. Lee, Engineered ionizable lipid nanoparticles for targeted delivery of RNA therapeutics into different types of cells in the liver, Sci. Adv. 7 (2021) eabf4398. https://doi.org/10.1126/sciadv.abf4398.

[2] J.A. Choe, H.M. Brinkman, J.S. Lee, W.L. Murphy, Optimized biomimetic minerals maintain activity of mRNA complexes after long term storage, Acta Biomater. 174 (2024) 428- 436. https://doi.Org/10.1016/j.actbio.2023. ll.044.

[3] A S. Khalil, X. Yu, J.M. Umhoefer, C.S. Chamberlain, L A. Wildenauer, G.M. Diarra, T.A. Hacker, W.L. Murphy, Single-dose mRNA therapy via biomaterial-mediated sequestration of overexpressed proteins, Sci. Adv. 6 (2020) eaba2422. https://doi.org/10.1126/sciadv.aba2422.

[4] J.S. Lee, D. Suarez-Gonzalez, W.L. Murphy, Mineral coatings for temporally controlled delivery of multiple proteins, Adv. Mater. 23 (2011) 4279-4284. https://doi.org/10.1002/adma.201100060.

[5] A S. Khalil, X. Yu, A.W. Xie, G. Fontana, J.M. Umhoefer, H.J. Johnson, T.A. Hookway,

T.C. McDevitt, W.L. Murphy, Functionalization of microparticles with mineral coatings enhances non-viral transfection of primary human cells, Sci. Rep. 7 (2017) 14211. https://doi.org/10.1038/s41598-017-14153-x.

[6] J.S. Lee, J.S. Lee, W.L. Murphy, Modular peptides promote human mesenchymal stem cell differentiation on biomaterial surfaces, Acta Biomater. 6 (2010) 21-28. https://doi.Org/10.1016/j.actbio.2009.08.003. [7] S.H. Brounts, J.S. Lee, S. Weinberg, S.K. Lan Levengood, E L. Smith, W.L. Murphy, High Affinity Binding of an Engineered, Modular Peptide to Bone Tissue, Mol. Pharm. 10 (2013) 2086-2090. https://doi.org/10.1021/mp300662r.

[8] J.S. Lee, J.S. Lee, A. Wagoner-Johnson, W.L. Murphy, Modular peptide growth factors for substrate-mediated stem cell differentiation, Angew. Chem. Int. Ed Engl. 48 (2009) 6266-6269. https://doi.org/10.1002/anie.200901618.

Claims

CLAIMS What is claimed:

1. A method of preparing a nucleic acid-based therapeutic composition, comprising: incubating a nucleic acid with a lipid nanoparticle (LNP) to form nucleic acid complexes, wherein the LNP includes a lipid membrane at least partially comprising a 1,2- Dioleoyl-3 -trimethylammonium propane (DOTAP) lipid; incubating the nucleic acid complexes with a mineral -coated substrate to bind the nucleic acid complexes to the mineral -coated substrate; suspending the bound nucleic acid complexes in a solution containing a lyoprotectant to provide the nucleic acid-based therapeutic composition; and lyophilizing the nucleic acid-based therapeutic composition to a dry powder.

2. The method of claim 1, wherein the nucleic acid includes RNA.

3. The method of claim 2, wherein the RNA includes messenger RNA (mRNA).

4. The method of claim 2, wherein the RNA is selected from the group consisting of mRNA, microRNA, small interfering RNA (siRNA), small hairpin RNA (shRNA), informational RNA (iRNA), guide RNA (gRNA), and an aptamer.

5. The method of claim 1 or 2, wherein the lipid membrane includes the DOTAP lipid in a molar ratio from about 1 to 20%.

6. A nucleic acid-based therapeutic composition, comprising: a mineral-coated substrate; one or more nucleic acid complexes bound to the mineral -coated substrate, wherein the nucleic acid complexes include nucleic acid complexed with a lipid nanoparticle (LNP), wherein the LNP includes a lipid membrane at least partially comprising a l,2-Dioleoyl-3- trimethylammonium propane (DOTAP) lipid; and a lyoprotectant, wherein the nucleic acid-based therapeutic composition is lyophilized to a dry powder.

7. The composition of claim 6, wherein the nucleic acid includes RNA.

8. The composition of claim 7, wherein the RNA includes messenger RNA (mRNA).

9. The composition of claim 7, wherein the RNA is selected from the group consisting of mRNA, microRNA, small interfering RNA (siRNA), small hairpin RNA (shRNA), informational RNA (iRNA), guide RNA (gRNA), and an aptamer.

10. The composition of claims 6 or 7, wherein the lipid membrane includes the DOTAP lipid in a molar ratio from about 1 to 20%.

11. A method of preparing a nucleic acid-based therapeutic composition, comprising: incubating nucleic acid with a hydroxyapatite binding peptide (HABP)- functionalized lipid nanoparticle (LNP) to form nucleic acid complexes, wherein the HABP- functionalized LNP includes a lipid membrane at least partially comprising a HABP- functionalized lipid; and incubating the nucleic acid complexes with a mineral -coated substrate to bind the nucleic acid complexes to the mineral -coated substrate.

12. The method of claim 11, wherein the HABP has the sequence yEPRRyEVAyEL- GGGS-C (SEQ ID NO: 1).

13. The method of claim 11 or 12, wherein the HABP is synthesized using N-terminal acetylation and C-terminal amidation.

14. The method of claim 11 or 12, wherein the nucleic acid includes RNA.

15. The method of claim 14, wherein the RNA includes messenger RNA (mRNA).

16. The method of claim 14, wherein the RNA is selected from the group consisting of mRNA, microRNA, small interfering RNA (siRNA), small hairpin RNA (shRNA), informational RNA (iRNA), guide RNA (gRNA), and an aptamer

17. A method of preparing functionalized nucleic acid complexes, comprising: incubating a nucleic acid with a lipid nanoparticle (LNP) to form nucleic acid complexes, wherein the LNP includes a lipid membrane at least partially comprising a maleimide-modified lipid to form the nucleic acid complexes; and conjugating a peptide onto the nucleic acid complexes to form functionalized nucleic acid complexes.

18. The method of claim 17, wherein conjugating the peptide onto the nucleic acid complexes includes a click chemistry reaction between the nucleic acid complexes and the peptide.

19. The method of claim 18, wherein the click chemistry reaction includes a Thiol Michael Addition.

20. The method of claim 17 or 18, wherein the peptide includes hydroxyapatite binding peptide (HAPB).

21. The method of any one of claims 17-20, wherein the nucleic acid includes RNA.

22. The method of claim 21, wherein the RNA includes messenger RNA (mRNA).

23. The method of claim 21, wherein the RNA is selected from the group consisting of mRNA, microRNA, small interfering RNA (siRNA), small hairpin RNA (shRNA), informational RNA (iRNA), guide RNA (gRNA), and an aptamer.

24. A nucleic acid-based therapeutic composition, comprising: a mineral-coated substrate; one or more nucleic acid complexes bound to the mineral -coated substrate, wherein the nucleic acid complexes include nucleic acid complexed with a lipid nanoparticle (LNP), wherein the LNP includes a lipid membrane at least partially comprising a hydroxyapatite binding peptide (HABP)-functionalized lipid; and a lyoprotectant, wherein the nucleic acid-based therapeutic composition is lyophilized to a dry powder.

25. The composition of claim 24, wherein the HABP has the sequence YEPRRYEVAYEL-GGGS-C (SEQ ID NO: 1).

26. The composition of claim 25, wherein the HABP is synthesized using N-terminal acetylation and C-terminal amidation.

27. The composition of claim 24 or 25, wherein the nucleic acid includes RNA.

28. The composition of claim 27, wherein the RNA includes messenger RNA (mRNA).

29. The composition of claim 27, wherein the RNA is selected from the group consisting of mRNA, microRNA, small interfering RNA (siRNA), small hairpin RNA (shRNA), informational RNA (iRNA), guide RNA (gRNA), and an aptamer.

30. A nucleic acid-based composition made by a method of any one of claims 1-5 or 11-23.

31. A method of treating a subject in need of a nucleic acid-based therapeutic comprising administering the composition of any one of claims 6-10 or 24-30 to the subject in need of the nucleic acid-based therapeutic.

32. A method of treating a subject in need of nucleic acid-based therapeutic comprising administering a composition made by any one of the methods of claims 1-5 or 11-23 to the subject in need of the RNA-based therapeutic.

33. The method of claim 31 or 32, wherein the administration is via infusing or injecting the composition in the subject.