Compositions and use of dry powder lipid nanoparticles

Abstract

Provided herein are embodiments of composition that include one or more particulates. Several embodiments provide one or more particulates that include one or more lipid nanoparticles coated in an excipient matrix, and encapsulating at least one pharmaceutically active ingredient. Several embodiments provide for particulates that at least partially encapsulate the pharmaceutically active ingredient and the excipient matrix that at least partially cover the outside of the particulates.

Description

DTS Ref: 40574. RNH. P1 10PCCOMPOSITIONS AND USE OF DRY POWDER LIPID NANOPARTICLESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to German Patent Application No. 10 2024 127 978.8, filed September 26, 2024, the entire content of which is incorporated by reference herein.BACKGROUNDFIELD

[0002] The present disclosure relates to the field of particulate carrier systems for delivery of active substances. Dry powder lipid nanoparticle compositions encapsulating therapeutic nucleic acids are provided in several embodiments.DESCRIPTION OF RELATED ART

[0003] Delivery of active substances, including therapeutic nucleic acids can be used in a range of indications. Such delivery can be used with different carrier systems.SUMMARY

[0004] Without protection strategies, active pharmaceutical substances, especially biologies and RNAs, are susceptible to degradation by enzymes, and are susceptible to elimination via renal filtration. In addition, the larger the substance, the more difficult it is to passively diffuse across biological membranes into the cell’s cytosol, the primary site of action for many drugs. To overcome these hurdles, specialized drug delivery systems are provided herein in several embodiments.

[0005] Several embodiments provided herein relate to compositions and methods that modulate the interaction of drug carriers with the biological environment and improve the efficacy of drug carriers as delivery vehicles. Associating particulate drug carriers with excipients and thereby modifying their ability to traverse different biological barriers and directly deliver pharmaceutically active substances into cells are provided in several embodiments herein.

[0006] The innovation described here, according to several embodiments, have one or more of the following benefits: (I) targeted delivery of therapeutic nucleic acids, (ii) targeted delivery to the lungs, throat, airways, nasal cavity, orother region fore.g., treating respiratory tract disorders, such as pulmonary disorders, (ill) targeted delivery where 50-100 percent of the nucleic acid orother active agent is efficiently delivered to the target location, thus enhancing efficacy and reducing issues that may arise from contact with undesired tissue, (iv) lipid nanoparticle compositions that have increased ability to cross tissue barriers, e.g., lung barriers, (v) increasedDTS Ref: 40574. RNH. P1 10PC cellular update, and (vi) improved efficiency in delivery of therapeutic delivery. Additional advantages in some embodiments include conversion of lipid nanoparticles into dry powder formulations, while protecting membranes (e.g., lipid membranes) during drying, and retaining integrity of the membranes upon reconstitution within the target environment, for example, in the lungs, thereby retaining the function of the lipid nanoparticles in targeted delivery of the therapeutic.

[0007] Several embodiments provide lipid nanoparticle compositions that comprise lipid nanoparticles. The lipid nanoparticles may comprise a membrane (e.g., lipid membrane) that partially or fully encapsulate one or more nucleic acids (or other therapeutic). In some embodiments, a matrix covers the lipid nanoparticles and comprises one or more excipient or combination of excipients configured to preserve the lipid nanoparticles during drying. In one aspect, the compositions are configured to deliver the nucleic acid to a target cell, wherein the composition is formulated as a dry powder. Several embodiments provided herein relate to dry powder lipid nanoparticle compositions that include a lipid membrane comprising one or more of ionizable amino lipid (e.g., a cationic ionizable amino lipid, C12-200, DOTAP (1 ,2-dioleyl-3-trimethytammonium propane), DODAP (1 ,2-dioleyl-3-dimethylammonium propane), DOTMA (1 ,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA, DLin-KC2-DMA, HGT4003, CKK-E12, ICE, DLin-MC3, 7C1 , ALC-0315, SM-102, CL-1 , 3060110, OF- 02, 7C1 , L319, A9, 93017S, Lipid C24, 1014, Lipidl 5, Lipid AX4, CP-LC-0729, Lipid A6, BAMEA-O16B, 98N12- 5, 4A3-SC8, 5A2-SC8, orother ionizable amino lipid), cholesterol, DSPC, and PEG (e.g., PEG-DMG 2000), siRNA, and matrix comprising lactose, wherein the composition is formulated as a dry powder. The membrane may be a lipid membrane, has lipid portions or is a non-lipid membrane, and may be single or multi-layers.

[0008] In several embodiments, the lipid nanoparticles comprise a lipid portion (e.g., a membrane or other component) that partially or fully incorporates, embeds, or otherwise interacts with one or more nucleic acids (or other therapeutic).

[0009] Several embodiments provide dry powder compositions that comprise one or more lipid nanoparticles, one or more corona excipients, and one or more nucleic acids. The one or more lipid nanoparticles may at least partially encapsulate the one or more nucleic acids. The one or more corona excipients may form a matrix that may at least partially cover the outside of the one or more lipid nanoparticles. The one or more lipid nanoparticles and the one or more corona excipients may modulate the one or more lipid nanoparticles interaction with a biological environment for tunable delivery of the one or more nucleic acids. The one or more lipid nanoparticles may include an ionizable amino lipid (e.g., a (cationic) (ionizable) amino lipid, C12-200, DOTAP (1 ,2- dioleyl-3-trimethytammonium propane), DODAP (1 ,2-dioleyl-3-dimethylammonium propane), DOTMA (1 ,2-di-O- octadecenyl-3-trimethylammonium propane), DLinDMA, DLin-KC2-DMA, HGT4003, cKK-E12, ICE, DLin-MC3, 7C1 , ALC-0315, SM-102, CL-1 , 3060110, OF-02, 7C1 , L319, A9, 93O17S, Lipid C24, 1014, Lipid15, Lipid AX4, CP-LC-0729, Lipid A6, BAMEA-O16B, 98N12-5, 4A3-SC8, 5A2-SC8, orother ionizable amino lipid), a sterol (e.g., cholesterol or other sterol), a phospholipid (e.g., phosphatidylcholine, such as distearoylphosphatidylcholineDTS Ref: 40574. RNH. P1 10PC(DSPC) or other phospholipid), and / or a polyether (e.g., polyethylene glycol such as PEG-DMG 2000 or other polyether). The composition may have a residual moisture of 10% or less. The composition may be formulated for various routes of administration, e.g., inhalable administration, enteral administration, transdermal administration, or parenteral administration. The composition may be formulated for respiratory tract administration. The one or more corona excipients may: facilitate cellular internalization of the one or more lipid nanoparticles; increase transfection efficiency of the one or more lipid nanoparticles; and / or prevent or reduce degradation or loss of the therapeutic nucleic acid. The one or more nucleic acids may include mRNA, circRNA, saRNA, antisense oligonucleotide, miRNA, shRNA, IncRNA, dsRNA, tracrRNA, siRNA, aptamer, gRNA, piRNA, pDNA, or an RNA conjugate or a combination thereof. The one or more corona excipients may comprise sugars, monosaccharides, disaccharides, oligosaccharides, polysaccharides, sugar alcohols, alcohols, amino acids, dipeptides, oligopeptides, polypeptides, proteins, esters, ethers, amides, amines, sulphates, thiols, urethanes, phosphoesters, phosphazenes, surfactants, lipids, stearates, polymers, salts, buffer, citric acid (monohydrate), (anhydrous) trisodium citrate, ascorbic acid, calcium carbonate, calcium chloride, EDTATE disodium, potassium chloride, potassium carbonate, potassium bicarbonite, sodium bicarbonate, sodium carbonate, sodium acetate, acetic acid, sodium bisulfate, sodium chloride, sodium hydroxide, sodium metabisulfate, sodium sulfate (anhydrous), sodium phosphate, sulfuric acid, trisodium citrate dihydrate, disodium hydrogen phosphate, potassium dihydrogen phosphate, phosphoric acid, or a combination, derivative, conjugate, or polymer thereof. Also, as an additional disclosure, excipients may comprise derivatives of sugars, monosaccharides, disaccharides, oligosaccharides, polysaccharides. Further, as an additional disclosure, excipients may comprise derivatives and / or conjugates of sugar alcohols, alcohols, amino acids, dipeptides, oligopeptides, polypeptides, proteins or derivatives and / or conjugates of esters, ethers, amides, amines, sulphates, thiols, urethanes, phosphoesters, phosphazenes, surfactants, lipids, stearates, polymers. Still further, as an additional disclosure, excipients may comprise salts, buffer, citric acid (monohydrate), (anhydrous) trisodium citrate, ascorbic acid, calcium carbonate, calcium chloride, EDTATE disodium, potassium chloride, potassium carbonate, potassium bicarbonite, sodium bicarbonate, sodium carbonate, sodium acetate, acetic acid, sodium bisulfate, sodium chloride, sodium hydroxide, sodium metabisulfate, sodium sulfate (anhydrous), sodium phosphate, sulfuric acid, trisodium citrate dihydrate, disodium hydrogen phosphate, potassium dihydrogen phosphate, phosphoric acid, or a combination, derivative, conjugate, or polymer thereof. Furthermore, as an additional disclosure, the excipient may comprise sugars, polysaccharides, sugar alcohols, alcohols, polypeptides, proteins, esters, ethers, amides, amines, sulphates, thiols, urethanes, phosphoesters, phosphazenes, amino acids, surfactants, lipids, stearates, or polymers.

[0010] Several embodiments provide a dry powder lipid nanoparticle composition comprising one or more lipid nanoparticles, one or more nucleic acid, and a matrix comprising one or more excipients. The one or more nucleic acids may be encapsulated within the one or more lipid nanoparticles and / or the matrix may at least partially cover the one or more lipid nanoparticles. The matrix may be configured to preserve the one or moreDTS Ref: 40574. RNH. P1 10PC lipid nanoparticles during drying and to modulate biological identity of the one or more lipid nanoparticles. The composition may be formulated in a powderform. The one or more excipients may: facilitate cellular internalization of the one or more lipid nanoparticles; increase transfection efficiency of the one or more lipid nanoparticles; prevent or reduce degradation or loss of the one or more nucleic acids. The composition may be formulated for inhalable administration, enteral administration, transdermal administration, or parenteral administration. The composition may be configured for redispersion (e.g., reconstitution). The one or more excipients may be configured to remain at least partially associated with the one or more lipid nanoparticles following redispersion. The one or more nucleic acids may be mRNA, circRNA, saRNA, antisense oligonucleotide, miRNA, shRNA, IncRNA, dsRNA, tracrRNA, siRNA, aptamer, gRNA, piRNA, pDNA, or an RNA conjugate, or a combination thereof. The one or more excipients may include sugars, monosaccharides, disaccharides, oligosaccharides, polysaccharides, sugar alcohols, alcohols, amino acids, dipeptides, oligopeptides, polypeptides, proteins, esters, ethers, amides, amines, sulphates, thiols, urethanes, phosphoesters, phosphazenes, surfactants, lipids, stearates, or polymers, salts, buffer, citric acid (monohydrate), (anhydrous) trisodium citrate, ascorbic acid, calcium carbonate, calcium chloride, EDTATE disodium, potassium chloride, potassium carbonate, potassium bicarbonite, sodium bicarbonate, sodium carbonate, sodium acetate, acetic acid, sodium bisulfate, sodium chloride, sodium hydroxide, sodium metabisulfate, sodium sulfate (anhydrous), sodium phosphate, sulfuric acid, trisodium citrate dihydrate, disodium hydrogen phosphate, potassium dihydrogen phosphate, phosphoric acid or a combination, derivative, conjugate, or polymer thereof. The membrane may include one or more of the following: ionizable amino lipid (e.g., a cationic ionizable amino lipid, CP-LC-0729, or other ionizable amino lipid), a sterol (e.g., cholesterol or other sterol), a phospholipid (e.g., phosphatidylcholine, such as distearoylphosphatidylcholine (DSPC) or other phospholipid), and / or a polyether (e.g., polyethylene glycol such as PEG-DMG 2000 or other polyether). The membrane may be a lipid membrane, has lipid portions or is a non-lipid membrane, and may be single or multilayers. The composition may be configured for administration with an inhaler device, spray device, dropper, or other administration route. The composition may have a residual moisture of 20, 15%, 10%, 5% or less.

[0011] Several embodiments provide for a dry powder lipid nanoparticle composition that includes a membrane comprising an ionizable amino lipid (e.g., a cationic (ionizable) (amino) lipid, C12-200, DOTAP (1 ,2- dioleyl-3-trimethytammonium propane), DODAP (1 ,2-dioleyl-3-dimethylammonium propane), DOTMA (1 ,2-di-O- octadecenyl-3-trimethylammonium propane), DLinDMA, DLin-KC2-DMA, HGT4003, cKK-E12, ICE, DLin-MC3, 7C1 , ALC-0315, SM-102, CL-1 , 3060110, OF-02, 7C1 , L319, A9, 93O17S, Lipid C24, 1014, Lipid15, Lipid AX4, CP-LC-0729, Lipid A6, BAMEA-O16B, 98N12-5, 4A3-SC8, 5A2-SC8 or other ionizable amino lipid), a sterol (e.g., cholesterol or other sterol), a phospholipid (e.g., phosphatidylcholine, such as distearoylphosphatidylcholine (DSPC) or other phospholipid), and / or a polyether (e.g., polyethylene glycol such as PEG-DMG 2000 or other polyether), one or more nucleic acids comprising siRNA, and one or more excipients. The composition may be formulated as a dry powder. The one or more lipid nanoparticles may deliver the one or more nucleic acids to lungDTS Ref: 40574. RNH. P1 10PC cells and / or cells within the respiratory tract. The one or more lipid nanoparticles may include an ionizable amino lipid in an amount of about 50%, a sterol in an amount ranging from about 20% to about 40%, a phospholipid in an amount ranging from about 10% to about 25%, and / or a polyether in an amount ranging from about 0.5% to about 1 .5%. The one or more excipients may comprise lactose. The one or more nucleic acids may be an siRNA that targets an mRNA, such as an mRNA that encodes a therapeutic target, such as for the alarmin TSLP, a cytokine, or a viral protein.

[0012] Several embodiments provide a use of any of the composition described herein for the treatment of a respiratory tract disease, e.g., asthma, chronic obstructive pulmonary disease, a viral infection, and / or an idiopathic pulmonary fibrosis, or other respiratory tract disease.

[0013] Several embodiments provide methods of delivering a dry powder lipid nanoparticle composition to a respiratory tract of a subject in need by administering any of the compositions described herein to the subject. The one or more excipients may have one or more of the following advantages facilitates cellular uptake of the one or more lipid nanoparticles to a lung cell and / or a cell within the respiratory tract of the subject; facilitate transport through mucus layers of a lung of the subject; increase transfection efficiency of the nucleic acid into a lung cell and / or a cell within the respiratory tract of the subject; and / or prevents or reduces degradation or loss of the nucleic acid. The one or more nucleic acids may be delivered in an effective amount.

[0014] Several embodiments provide methods of treating a respiratory tract disease in a subject and may include administering to a lung of the subject any ofthe compositions described herein. The administering may be performed using an inhaler device, sprayer, dropper or other device. The respiratory tract disease may be asthma, chronic obstructive pulmonary disease (COPD), a viral infection, or idiopathic pulmonary fibrosis. The one or more nucleic acids may be an siRNA against a target. The target may be a therapeutic target to a cytokine, a viral gene, and / or a nucleic acid sequence associated with a respiratory tract disease, e.g., pulmonary inflammation, epithelial barrier dysfunction, and / or immune dysregulation.

[0015] Several embodiments provide methods of making any of the compositions described herein by mixing lipid components with one or more nucleic acids to form one or more lipid nanoparticles, mixing the one or more lipid nanoparticles with one or more excipients to form a particulate comprising one or more lipid nanoparticles and the one or more excipients, and spray drying the particulate to form a dry powder comprising one or more lipid nanoparticles at least partially covered in a matrix comprising the one or more excipients. The one or more lipid nanoparticles may encapsulate, partially or fully, the one or more nucleic acids. The one or more excipients prevent or reduce degradation of the one or more lipid nanoparticles following drying (such as spray- draying) of the particulate.

[0016] Several embodiments provide kits for any of the compositions described herein that include any of the compositions and packaging. The packaging may include a capsule, a blister, and / or a reservoir. The packaging may be multidose or single dose packaging. The kit may include a nebulizer or an inhaler device.DTS Ref: 40574. RNH. P110PC

[0017] Some embodiments provided herein relate to compositions that include one or more particulates. In some embodiments, the one or more particulates include an excipient around, on, and / or interpenetrating a surface of a nanoparticle, an encapsulation structure, and at least one pharmaceutically active ingredient. In some embodiments, the encapsulation structure at least partially encapsulates the pharmaceutically active ingredient. In some embodiments, the excipient at least partially covers an outside surface of the encapsulation structure.

[0018] In some embodiments, the compositions include one or more particulates. The particulate may include an excipient around a surface of a nanoparticle, an encapsulation structure, and at least one pharmaceutically active ingredient. The encapsulation structure may at least partially encapsulate the pharmaceutically active ingredient, and the excipient at least partially covers the outside of the encapsulation structure, which may be a lipid structure and / or polymer structure. In some embodiments, the particulate is a nanoparticle. In some embodiments, the at least one pharmaceutically active ingredient is a nucleic acid, which may be an RNA or DNA. In some embodiments, the nucleic acid is a therapeutic nucleic acid, including for example, an mRNA, circRNA, saRNA, antisense oligonucleotide, miRNA, shRNA, IncRNA, dsRNA, tracrRNA, siRNA, aptamer, gRNA, piRNA, pDNA, or an RNA conjugate. The particulate may be in a dried, partially dried, or hydrated state, or may have been in a dried state or partially dried state at least once during its lifetime. The lipids or polymers of the encapsulation structure may be selected to control the interaction with the excipient, and may be configured such that a colloid electron density is different when at least partially covered by one or more excipients when compared to a not-covered colloid. In some embodiments, the lipids are selected from at least one of a cationic (ionizable) lipid, one or more helper lipids, and / or a stealth lipid. In some embodiments, at least two helper lipids are used and one of the helper lipids is a phospholipid, ester lipid, lyso-phospholipid, or a glycerol lipid and the second is a sterol. The cationic (ionizable) lipid may be selected from the group consisting of C12- 200, DOTAP (1 ,2-dioleyl-3-trimethytammonium propane), DODAP (1 ,2-dioleyl-3-dimethylammonium propane), DOTMA (1 ,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA, DLin-KC2-DMA, HGT4003, cKK-E12, ICE, DLin-MC3, CP-LC-0729, 7C1 , ALC-0315, SM-102, CL-1 , 3060110, OF-02, 7C1 , L319, A9, 93017S, Lipid C24, 1014, Lipidl 5, Lipid AX4, Lipid A6, BAMEA-O16B, 98N12-5, 4A3-SC8, 5A2-SC8, and combinations thereof. With respect to encapsulation, the lipid nanoparticles may comprise a lipid portion (e.g., a membrane or other component) that partially or fully incorporates, embeds, or otherwise combines with one or more nucleic acids (or other therapeutic). One or more nucleic acids may be provided, which optionally may be combined with non-nucleic acid therapeutics.

[0019] The excipient may be a pharmaceutically acceptable excipient, and may be selected based on characteristics of having an effect on the efficiency of the cellular internalization of the particulate system. In some embodiments, the selection of excipients can be used to adapt the penetration of the particulate system through the natural barriers of the human body. The pharmaceutically acceptable excipient may be selected basedDTS Ref: 40574. RNH. P1 10PC on characteristics of protecting lipids during a drying process. The pharmaceutically acceptable excipient may be a mono-, di-, tri-, oligo-, or sugars, alcohols, polysaccharides, sugar alcohols, mono-, di, tri, oligo- or polypeptides, proteins, esters, ethers, amides, amines, sulphates, thiols, urethanes, phosphoesters, phosphazenes, amino acids, surfactants, lipids, stearates, polymers, salts, and combination thereof. The polymers may be selected from one or more of Chitosan, polyvinylpyrrolidone (PVP), PVP / VA, Methacrylic polymers, polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), hydroxypropyl methylcellulose (HPMC), polycaprolactone (PCL), PEG, Polyvinyl alcohol, Polyvinyl acetate, crospovidone, HPMCAS, HPMCP, Methacrylic acid-ethyl acrylate copolymer, Poloxamer, microcrystalline cellulose (MCC), starch, sodium starch glycolate, dextran, pullulan, lactose, dicalcium phosphate, hydroxypropyl cellulose, methylcellulose, carboxymethyl cellulose, hydroxyethyl cellulose, ethylcellulose, acrylic polymers, polyethylene oxide, polymethacrylates, carbomers, chitosan, alginate, agarose, guar gum, xanthan gum, gelatin, polycarbophil, or combinations, analogues, modifications, or derivatives thereof. The excipient may at least partially cover an outside surface of the encapsulation structure, and / or may form a shell structure for and / or around the encapsulation structure.

[0020] In some embodiments, the plurality of particles is present in the form of a dry powder. The dry powder may have a residual moisture of 10% or less, a residual moisture of 8% or less, or a residual moisture of 5% or less. In some embodiments, the dry powder can at least partially be redispersed. The particulates, after redispersion, may remain at least partially associated with the one or more excipients.

[0021] In some embodiments, the composition is formulated for use as a pharmaceutical dosage form, including, for example, for pulmonary delivery, transdermal delivery, parenteral delivery (including intravenous, intramuscular, intrathecal, subcutaneous, or intraperitoneal), or oral delivery. The composition may be formulated for use to adapt the penetration of the particulate system through the natural barriers of the lung. The composition may be formulated such that the pharmaceutical dosage form can be administered using dry powder inhaler devices, soft mist inhaler, and / or a nebulizer. In some embodiments, the plurality of particles is present in form of a colloidal suspension.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The following drawings are for illustrative purposes only and show non-limiting embodiments. Features from different figures may be combined in several embodiments.

[0023] FIG. 1 depicts embodiments of a particulate system that includes an excipient and carrying a pharmaceutically active substance allows for tunable delivery of an active molecule through different biological barriers into a cell.

[0024] FIGs. 2A-2C depict embodiments of lipid nanoparticles with a matrix. As shown in FIG. 2A, a lipid nanoparticle and excipient composition are processed into a dry powder, and the excipient forms a matrix (or corona excipient) that covers the lipid nanoparticles, which themselves encapsulate an active molecule. WhenDTS Ref: 40574. RNH. P1 10PC the dry powder formulation comes in contact with a liquid environment, for example through dry powder administration or by redispersion, parts of the excipient remain associated with the lipid nanoparticles (FIG. 2B). In a physiological environment, the lipid nanoparticle and excipient composition result in formation of a specific protein corona that is different from a protein corona that would form around a lipid nanoparticle in the absence of the excipient.

[0025] FIG. 3 shows embodiments of diffusion coefficients of fresh and spray dried lipid nanoparticles in phosphate buffered saline or mucus, measured using fluorescence correlation spectroscopy (FCS).

[0026] FIGs. 4A-4C show embodiments of dialysis results and measurements of embodiments of compositions described herein before and after spray drying. FIG. 4A shows a schematic of the spray dryingbased process for the manufacturing of dry powder lipid nanoparticles with RNA encapsulated therein (RNA- LNPs). FIG. 4B shows a graph depicting the hydrodynamic diameter of redispersed LNPs before (dark bars) and after (light bars) 24 hour dialysis against phosphate buffered saline (PBS), measured via nanoparticle tracking analysis (NTA). FIG. 4C shows violin plots representing the distribution of all individual particle sizes within a D90 diameter range.

[0027] FIGs. 5A-5E show embodiments of all-atom simulation data of LNP membranes at different hydration states and with different D-Lin-MC3-DMA concentrations in the membrane. FIG. 5A shows simulations of hydrated membrane with 5% D-Lin-MC3-DMA and lactose in solution (top), dehydrated membrane with lactose (middle), rehydrated membrane with lactose (bottom) all after 10 nanosecond (ns) simulation. FIG. 5B shows the symmetrized mass density relative to the center of the simulation box (membrane) of Lactose, Membrane, DSPC, PEG-DMG-2000 hydrated (top), dehydrated (middle), rehydrated (bottom) for systems with 5% D-Lin-MC3-DMA content. FIG. 5C shows violin plots depicting the area per lipid for the membranes with and without lactose from 1 ns to 10 ns, where every 2 femtosecond (fs) on measurement point was calculated. FIG. 5D shows violin plots depicting the number of hydrogen bonds between lactose and the different membranes from 1 ns to 10 ns calculated every 4 fs. FIG. 5E shows graphs depicting the mean of the normalized number of hydrogen bonds between lactose and the different membrane components.

[0028] FIGs. 6A-6E show embodiments of LNP characteristics before and after spray drying. FIG. 6A shows a graph depicting DLS measurements before (LNP) and after (SD-LNP) spray drying of all four formulations of LNP, including: 10% DSPC, 0.5% PEG; 25% DSPC, 0.5% PEG; 10% DSPC, 1 .5% PEG; and 25% DSPC, 1 .5% PEG. FIG. 6B shows a graph depicting the difference in Z-A verage before and after spray drying for each of the four LNP formulations from FIG. 6A. FIG. 6C shows a graph depicting the difference in Gp value determined via Laurdan assay before and after spray drying. FIG. 6D shows a graph depicting the difference in apparent pKa value of LNPs before and after spray drying, determined via TNS (6-(p-toluidino)-2- naphthalenesulfonic acid sodium salt) assay. FIG. 6E shows cryo-TEM (cryogenic transmission electronDTS Ref: 40574. RNH. P110PC microscopy) micrographs of LNPs after concentration up to 1000 ng / pl of LNP3 or LNP4. LNP3 before and after spray drying (top), LNP 4 before and after spray drying (bottom).

[0029] FIGs. 7A-7D show graphs and confocal scanning microscopy images depicting embodiments of cellular uptake (left) and eGFP protein downregulation (right) in H1299-eGFP cells after transfection with siRNA -LNPs. FIG. 7A shows cellular uptake and eGFP protein down regulation in cells transfected with fresh LNP 1 and spray-dried LNP 1. FIG. 7B shows cellular uptake and eGFP protein downregulation in cells transfected with fresh LNP 2 and spray-dried LNP 2. FIG. 7C shows cellular uptake and eGFP protein downregulation in cells transfected with fresh LNP 3 and spray-dried LNP 3. FIG. 7D shows cellular uptake and eGFP protein downregulation in cells transfected with fresh LNP 4 and spray-dried LNP 4.

[0030] FIGs. 8A-8D depict embodiments of LNP transport through artificial mucus and transfection efficiency in air-liquid interface (ALI). FIG. 8A shows a schematic representation of a transwell mucus penetration assay. FIG. 8B shows graphs depicting the mucus penetration of fresh and spray dried LNPs. FIG. 8C depicts a schematic representation of Calu 3 cells seeded into a transwell and grown at an air-liquid-interface. FIG. 8D shows a graph depicting the percentage of transfected cells 6 hours after cells were transfected with fresh and SD-LNPs 1 -4.

[0031] FIGs. 9A-9B depict proteomic analysis of protein corona formation around various types of LNPs, including fresh and spray-dried LNPs from human bronchoalveolar lavage fluid (BALF). FIG. 9A depicts principal component analysis showing differences in protein profiles for BALF and LNP. FIG. 9B depicts enrichment of 20 proteins per LNP formulation based on ratio between LNP and BALF.DETAILED DESCRIPTION

[0032] The foregoing and other aspects of the present disclosure are described in more detail with respect to the description and methodologies provided herein. This description is not intended to be a detailed catalogue of all the ways in which the embodiments of the present disclosure may be implemented, or of all the features that may be added to the present disclosure. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be absent from that embodiment. In addition, variations and additions to the various embodiments described herein, which do not depart from the instant disclosure, are encompassed herein. Hence, the following specification is intended to illustrate some particular embodiments, and not to exhaustively specify all permutations, combinations and variations thereof.

[0033] Several embodiments provided herein provide targeted delivery, as off-target effects in nontarget organs can pose substantial risks. Local administration directly to target tissues can mitigate many toxicological issues associated with systemic delivery. While RNA therapeutics are theoretically well-suited for treating pulmonary disorders via localized lung administration, this presents significant challenges in practice. ToDTS Ref: 40574. RNH. P1 10PC address these difficulties, dry powder formulations of RNA-loaded lipid nanoparticles (RNA-LNPs) are disclosed herein. These formulations can be delivered to a cell or tissue of interest, for example, the lungs, using dry powder inhalers, which offer a rapid, non-invasive method, and are associated with favorable storage stability. In some embodiments, the compositions are formulated as a dry powder for delivery via various routes, including inhalation (for example, using an inhaler device), oral (for example, as a capsule or tablet), or parenteral (for example as reconstituted suspension). Targeted tissue includes the respiratory tract, including the lungs, throat, airways, nasal cavity, etc.

[0034] Although spray drying can be challenging, several embodiments used herein overcome issues such as membrane fusion or nucleic acid degradation by a rational selection and combination of (I) formulation factors, including the type of matrix excipients, lipid composition, and the ratio between matrix excipients and lipids, (II) process parameters, such as inlet and outlet temperature, drying gas flow rate, atomization gas flow, feed rate, aspirator rate, and nozzle, and (ill) equipment and environmental factors, including drying chamber geometry, and electrostatic effects. Thus, in several embodiments, spray drying is used as an effective method to form the powder. In other embodiments, freeze drying, atmospheric spray freeze-draying, supercritical fluid drying, vacuum drying, electrospray drying, microwave or radiofrequency-assisted drying, electrospraying / sputtering, vacuum-assisted thin film drying, and heat are also used to as a method of drying. Combinations of drying methods may also be used.

[0035] Several embodiments described herein provide effective and efficient delivery of active pharmaceutical ingredients (APIs) to a target cell or target tissue. For example, several embodiments relate to compositions of particulates that include an excipient matrix, lipid nanoparticles (LNPs), and a pharmaceutically active ingredient, and methods of making and using the same.

[0036] Several embodiments have one or more of the following advantages: (I) targeted and tunable delivery of therapeutic nucleic acids (II) targeted delivery to the lungs, throat, airway, or nasal cavity, for e.g., treating respiratory tract disorder, such as pulmonary disorders, (ill) targeted delivery where 50-100 percent of the nucleic acid or other active agent is efficiently delivered to the target location, thus enhancing efficacy and reducing issues that may arise from contact with undesired tissue, (iv) lipid nanoparticle formulations that have increased ability to cross lung barriers, (v) increased cellular update, and (vi) improved efficiency in delivery of therapeutic delivery. Additional advantages in some embodiments include conversion of lipid nanoparticles into dry powder formulations, while protecting membranes (such as lipid membranes) during drying, and retaining integrity of the membranes upon reconstitution within the target environment, for example, in the lungs, thereby retaining the function of the lipid nanoparticles in targeted delivery of the therapeutic.

[0037] Although several embodiments describe delivery to the lung, other tissue may also be targeted. For example, tissue in the nasal and / or oral cavities may be well suited for delivery of the powders disclosed herein.DTS Ref: 40574. RNH. P1 10PC

[0038] Several embodiments provided herein relate to delivery vehicles configured to deliver a pharmaceutically active substance using a particulate system as a carrier for the active substance. Some embodiments provided herein relate to development of a particulate drug delivery systems based on a combination of pharmacokinetics, including the rate and extent of absorption, distribution, metabolism, and excretion (ADME) of the drug delivery system. In particular, some embodiments provided herein relate to development of a particulate drug delivery systems by identification of the pharmacological target and analysis of the biological route, starting from the desired point of administration to the site of action.

[0039] In some embodiments, the compositions described herein allow for effective local delivery of particles to a target tissue. For example, at least 50% may be delivered to the desired target tissue (e.g., greater than 70%, 85%, 90%, 95%), thereby allowing for greater efficacy, less waste and lower cost of therapeutics, more rapid therapeutic effect, reduced side effects from contact with non-targeted tissue, etc. In several embodiments, the compositions are formulated for delivery, via inhalation, to the lungs, by addressing and / or overcoming biological hurdles. Forexample, embodiments of the compositions described herein must traverse the mucus layer in the upper airways and / or the lung lining fluid in the alveoli. The mucus layer may include water, mucins, proteins, salt, lipids, DNA, cells, and / or cellular debris, stabilized by covalent and noncovalent interactions, including hydrophobic, electrostatic, and hydrogen bonds, leading to gel formation. The makeup of mucus varies across the upper airways, with the mucus primarily consisting of MUC5B in the distal bronchioles and MUC5AC in the distal bronchi. In the alveoli, the lung lining fluid contains up to 60% surfactant protein B (SPB) secreted by type 2 alveolar cells, which is crucial for gas exchange. Several embodiments of the compositions described herein traverse the mucus layer and / or the lung lining fluid. Mucin, a highly glycosylated protein that forms a mesh, and surfactant present significant challenges for delivery of active pharmaceutical ingredients (API) due to their complex interactions. In some embodiments, the targeted tissue is to particular regions of the respiratory and / or pulmonary system (e.g., trachea, lung, bronchial tree, larynx, pharynx, diaphragm, bronchiole, pulmonary alveolus, pleura), and the majority of the therapeutic is delivered to said particular region(s).

[0040] In some embodiments, the nasal cavity is the target tissue. In other embodiments, the throat region is the target tissue.

[0041] Several embodiments of the compositions described herein are prepared based on considerations of surface hydrophobicity, mucus clearance, and / or turnover time. For example, with respect to surface hydrophobicity, highly positively or negatively charged particles tend to interact more with the mucus, whereas slightly negatively charged particles interact less. In some embodiments, the shape and rigidity of the formulations may affect mucus penetration as well, with penetration decreasing as particle elasticity increases.

[0042] In some embodiments, after arriving at the target tissue, the composition is formulated to cross the extracellular matrix to be taken up by the cells. In some embodiments, crossing the extracellular matrix is achieved by designing the composition to exhibit physicochemical properties that result in uptake by the targetDTS Ref: 40574. RNH. P110PC cell. In several embodiments, the composition is formulated to be tunable for uptake by a particular tissue type, or a particular cell type.

[0043] Several embodiments of the compositions described herein are characterized in having enhanced stability to resist degradation via the endo-lysosomal pathway. For example, embodiments of the compositions are able to escape the endosomal compartment and resist degradation within the degrading environment of the lysosome, thereby enhancing therapeutic activity of the compositions. In contrast, conventional delivery systems are incapable of endosomal escape, with low therapeutic efficacy due to only a few percent of the nanoparticles that accomplished uptake by the target cell are able to escape the endosome. Conventional delivery systems also fail due to insufficient release of the therapeutic cargo from the carrier matrix. Consequently, the accumulation of unproductive and non-degradable material in the cytoplasm may interfere with the metabolism of the cell and potentially lead to cell death. In certain conventional therapies, less than 14 out of one million gold and silica nanoparticles with a specific targeting ligand reached the designated target cells (that is, less than 0.0014%). By contrast, in several embodiments herein, over 10% of the therapeutic reaches the targeted cells (and in some embodiments, over 25%, 50%, 75%, etc.). Moreover, in several embodiments, the therapeutic is a non-degraded, active form when it reaches the target.

[0044] In several embodiments, one, two, three or more therapeutics can be delivered simultaneously (e.g., in a single inhaler or other delivery device) or sequentially.

[0045] In several embodiments, the compositions herein include a distinctive matrix that covers (for example, envelops, surrounds, interacts with, and / or interpenetrates) the lipid nanoparticles, and portions of excipients from the matrix remain associated with the lipid nanoparticle after contact with a liquid environment (for example, during or post administration or after redispersion / reconstitution), and that can interact with specific cell surface receptors, leading to formation of a specific protein corona, which results in specific internalization of the lipid nanoparticles. As used herein, the term “matrix” shall include a structure that forms a shell-like structure, a net-like structure, a coating, a covering, a mesh, or similar structure that at least partially surrounds, envelops, covers, coats, interacts with, or interpenetrates a molecule, such as a lipid nanoparticle. As used herein, the terms redispersion and reconstitution are used interchangeably, and may include the process of combining a dried composition with a liquid to form a suspension, solution, or dispersion suitable for use. In some embodiments, the formation of a protein corona is tunable (for example, is designed to specifically interact with a target cell) based on the composition of the lipid nanoparticle and matrix. Several embodiments of the compositions include lipid nanoparticles (LNPs) that include Onpattro™ or an Onpattro™-like composition, designed for uptake into hepatocytes without a distinct attached targeting ligand. Onpattro, or patisiran, is an siRNA active compound formulated into lipid nanoparticles, which protect the siRNA and facilitate delivery. Thus, for example, such embodiments exhibit an interplay between surface charge and lipid composition that results in the formation of a protein corona with a significant portion of ApoE protein upon administration. The ApoE within the protein coronaDTS Ref: 40574. RNH. P110PC can interact with the LDL receptor on surface of hepatocytes, leading to its internalization. In several embodiments, tunable delivery includes design of positively and negatively charged lipid nanoparticles. Positively charged nanoparticles are known to interact with the endothelium in the lungs, while negatively charged particles tend to accumulate in the spleen. For instance, it has been discovered that the receptor aV|33 integrin within the lung endothelium can facilitate the cellular uptake of positively charged LNPs due to an increased concentration of vitronectin protein in the protein corona. Similarly, an enhanced concentration of p2-glycoprotein in the protein corona may lead to the accumulation of negatively charged LNPs in the spleen. The protein corona layer may be tunably designed to interact with and / or target a particular tissue or cell of interest. The composition may be tunable based on the selection and design of the lipid nanoparticle in combination with specific excipients, which form a matrix that covers the lipid nanoparticle following spray drying, and which result in specific surface properties of the lipid nanoparticle that determines the formation of a specific protein corona.

[0046] In several embodiments, the LNPs traverse the mucus layer in the upper airways or the lung lining fluid in the alveoli, overcoming issues with mucus clearance and turnovertime. Upon reaching the target tissue, the LNPs cross the extracellular matrix for cellular uptake, and cellular internalization is significantly influenced by the proteins adsorbed onto their surface, forming a protein corona upon administration, according to several embodiments.

[0047] In several embodiments, the compositions and uses thereof do not optionally include one or more of trehalose, sucrose, mannitol, and / or reducing agents. In several embodiments, the compositions and uses thereof are not prepared by freeze-drying nor by spray freeze drying. In several embodiments, the compositions and uses thereof are not acidic (are not within a pH ranging from pH 3 to 5) prior to drying. In several embodiments, the lipids do not include DOTAP.

[0048] Some embodiments provided herein relate to compositions that include one or more particulates. In some embodiments, the one or more particulates include a matrix that includes an excipient that acts as a corona (referred to herein as “corona excipient” or an “excipient matrix”), an encapsulation structure (also described herein as an LNP), and at least one pharmaceutically active ingredient. In some embodiments, the encapsulation structure at least partially encapsulates the pharmaceutically active ingredient, and the corona excipient at least partially covers the outside of the encapsulation structure.

[0049] In some embodiments, the particulate is a microparticle. The particulate may be in a dried, partially dried, or hydrated state. In some embodiments, the particulate has been in a dried (e.g., partially or fully) state at least once. In some embodiments, the particulate has a moisture content of 20% or less, for example, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, 0.2%, 0.1 %, or less. In some embodiments, the moisture content is negligible, for example, is in insignificant amount, an indetectable amount, or an amount that would be considered to not materially affect the composition.DTS Ref: 40574. RNH. P1 10PC

[0050] In some embodiments, the corona excipient at least partially covers the outside of the encapsulation structure. In some embodiments, the corona excipient covers at least partially the outside of the encapsulation structure, forming a shell structure or matrix for and / or around the encapsulation structure.

[0051] In some embodiments, the excipient is a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient comprises mono-, di-, tri-, oligo-, or sugars, alcohols, polysaccharides, sugar alcohols, mono-, di, tri, oligo- or polypeptides, proteins, esters, ethers, amides, amines, sulphates, thiols, urethanes, phosphoesters, phosphazenes, amino acids, surfactants, lipids, stearates, polymers, salts, and combination thereof. The polymers may be selected from one or more of Chitosan, polyvinylpyrrolidone (PVP), PVPA / A, Methacrylic polymers, polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), hydroxypropyl methylcellulose (HPMC), polycaprolactone (PCL), PEG, Polyvinyl alcohol, Polyvinyl acetate, crospovidone, HPMCAS, HPMCP, Methacrylic acid-ethyl acrylate copolymer, Poloxamer, microcrystalline cellulose (MCC), starch, sodium starch glycolate, dextran, pullulan, lactose, dicalcium phosphate, hydroxypropyl cellulose, methylcellulose, carboxymethyl cellulose, hydroxyethyl cellulose, ethylcellulose, acrylic polymers, polyethylene oxide, polymethacrylates, carbomers, chitosan, alginate, agarose, guar gum, xanthan gum, gelatin, polycarbophil, or combinations, analogues, modifications, or derivatives thereof

[0052] In some embodiments, the selection of excipients has an effect on the efficiency of the cellular internalization of the particulate system. In some embodiments, the selection of excipients can be used to adapt the penetration of the particulate system through the natural barriers of the human body. FIG. 1 depicts embodiments of a particulate system (001 ) associated with an excipient (002) and carrying a pharmaceutically active substance (003) allows for tunable delivery of the active molecule (003) through different biological barriers into a cell (004).

[0053] FIGs. 2A-2C depict an embodiment of a lipid nanoparticle composition in dry form (FIG. 2A), in contact with liquid after dry processing (FIG. 2B), and in a physiological environment (FIG. 2C). In some embodiments, to form a dry powder, lipid nanoparticles (e.g., in solution) are mixed or otherwise combined with one or more excipients and one or more active molecules (e.g., nucleic acids, APIs), and dried (e.g., spray dried). Several embodiments of the dried composition include the lipid nanoparticles that include a lipid membrane (205), and that encapsulate an active molecule(s) (203), such as a nucleic acid(s). The excipient (202) forms a matrix that covers the lipid nanoparticles. When the dry powder composition is in contact with a liquid environment, for example, through dry powder administration, or by redispersion / reconstitution, the matrix that covers the lipid nanoparticle disperses and parts of the excipient remain associated with the lipid nanoparticles. In some embodiments, the dry powder composition is tunably designed, such that in physiological environment, the resulting lipid nanoparticle and excipient forms a specific corona (such as a protein corona) that is different from a corona that would have formed around the lipid nanoparticle in the absence of the associated excipient (FIG. 2C). In some embodiments, specific lipid nanoparticles and excipients can be prepared in a dry powder compositionDTS Ref: 40574. RNH. P1 10PC that results in a protein corona that is specific for a cell or tissue of interest, for example, liver cells or tissue, kidney cells or tissue, lung cells or tissue, etc., based on the association of lipid nanoparticles with various excipients. In several embodiments, one advantage of the “tunability” aspect is that it allows for more efficient and / or specific uptake in the target region (because for example the protein corona that forms in vivo is influenced by the type / concentration of the excipient and / or lipid membrane and thus can be “tuned” by selection of these components).

[0054] In some embodiments, the encapsulation structure at least partially encapsulates the pharmaceutically active ingredient. In some embodiments, the encapsulation structure is a lipid structure and / or polymer structure. In some embodiments, the encapsulation structure is a lipid structure. In some embodiments, the encapsulation structure is a polymer structure. In some embodiments, the encapsulation structure is a lipid structure and polymer structure.

[0055] In some embodiments, the lipids of the encapsulation structure are selected to control the interaction with the excipient. In some embodiments, the lipids are configured such that the colloids electron density is different when at least partially covered by one or more excipients when compared to the not-covered colloid.

[0056] In some embodiments, the lipids are selected from at least one of a cationic (ionizable) lipid, one or more helper lipids, and / or a stealth lipid.

[0057] In some embodiments, the at least one cationic (ionizable) lipid is comprises or is selected from the group consisting of C12-200, DOTAP (1 ,2-dioleyl-3-trimethytammonium propane), DODAP (1 ,2-dioleyl- 3-dimethylammonium propane), DOTMA (1 ,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA, DLin- KC2-DMA, HGT4003, cKK-E12, ICE, DLin-MC3, 7C1 , ALC-0315, SM-102, CL-1 , 3060110, OF-02, 7C1 , L319, A9, 93O17S, Lipid C24, 1014, Lipid15, Lipid AX4, Lipid A6, BAMEA-O16B, 98N12-5, 4A3-SC8, 5A2-SC8 and combinations thereof. In some embodiments, the at least one cationic (ionizable) lipid is C12-200, DOTAP (1 ,2- dioleyl-3-trimethytammonium propane), DODAP (1 ,2-dioleyl-3-dimethylammonium propane), DOTMA (1 ,2-di-O- octadecenyl-3-trimethylammonium propane), DLinDMA, DLin-KC2-DMA, HGT4003, cKK-E12, ICE, DLin-MC3, 7C1 , ALC-0315, SM-102, CL-1 , 3060110, OF-02, 7C1 , L319, A9, 93O17S, Lipid C24, 1014, Lipid15, Lipid AX4, Lipid A6, BAMEA-O16B, 98N12-5, 4A3-SC8, or 5A2-SC8.

[0058] In some embodiments, the one or more helper lipids include at least two helper lipids. In some embodiments, the at least two helper lipids include a phospholipid, ester lipid, lyso-phospholipid, a glycerol lipid; or sterol.

[0059] In some embodiments, the polymers of the encapsulation structure are selected to control the interaction with the excipient. In some embodiments, the polymers are configured such that the colloids electron density is different when at least partially covered by one or more excipients when compared to the not- covered colloid.DTS Ref: 40574. RNH. P110PC

[0060] In some embodiments, the at least one pharmaceutically active ingredient is a nucleic acid. In some embodiments, the nucleic acid is a therapeutic nucleic acid. In some embodiments, the nucleic acid is an RNA or DNA. In some embodiments, the nucleic acid is an RNA. In some embodiments, the nucleic acid is a mRNA, circRNA, saRNA, antisense oligonucleotide, miRNA, shRNA, IncRNA, dsRNA, tracrRNA, siRNA, aptamer, gRNA, piRNA, pDNA, or an RNA conjugate.

[0061] Some embodiments provided herein relate to formulations that include any of the compositions described herein. In some embodiments, the composition is formulated for use as a pharmaceutical dosage form. In some embodiments, the composition is formulated for use as a pharmaceutical dosage form for pulmonary delivery. In some embodiments, the composition is formulated for use to adapt the penetration of the particulate system through the natural barriers of the lung.

[0062] In some embodiments, the composition is formulated such that the pharmaceutical dosage form can be administered using dry powder inhaler devices. In some embodiments, the plurality of particles is present in form of a dry powder. In some embodiments, the dry powder has a residual moisture of or less than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9.5%, 9%, 8.5%, 8%, 7.5%, 7%, 6.5%, 6%, 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1 %, 0.75%, 0.5%, 0.25%, 0.2%, 0.15%, or 0.1 % or a range a range defined by any two of the preceding values, for example, 20%-0.25%, 20%-1 %, 20%-5%, 20%-8%, 20%-10%, 15%-1 %, 15%-5%, 10%-0.25%, 10%-1 %, 10%-5%, 5%-1 %, 1 %-0.1 %, or 0.5%-0.1 %. In some embodiments, the dry powder has a residual moisture of less than 10%. In some embodiments, the dry powder has a residual moisture of less than 8%. In some embodiments, the dry powder has a residual moisture of less than 5%. In some embodiments, the dry powder has a residual moisture of less than 0.1 %. In some embodiments, the dry powder can at least partially be redispersed. In some embodiments, the particulates, after redispersion, remain at least partially associated with the one or more excipients.

[0063] In some embodiments, the plurality of particles is present in form of a colloidal suspension. In some embodiments, the composition is formulated such that the pharmaceutical dosage form can be administered using a nebulizer or a soft mist inhaler.

[0064] The term “composition” as used herein has its plain and ordinary meaning and shall include any combination (e.g., mixture or other combination) of two or more products, substances, or compounds. Composition may be, for example, a solution, a suspension, liquid, a gel, powder, a paste, a lyocake, aqueous, non-aqueous, or any combination thereof.

[0065] The term “excipient” as used herein has its plain and ordinary meaning and shall include a substance or component other than the active ingredient of a pharmaceutical composition or medicine.

[0066] The term “corona” as used herein has its ordinary meaning and shall include a coating of substances, components, excipients, biomolecules, and / or proteins around the surface of nanoparticle. The coating may form around the surface and / or is embedded / attached onto the external surface of the nanoparticleDTS Ref: 40574. RNH. P1 10PC through chemical and / or physical interactions. The term “corona excipient” as used herein has its ordinary meaning and shall include a coating of an excipient around the surface of a nanoparticle forming a matrix. The excipient coating may form around the surface and / or is embedded / attached, partially or fully, onto the external surface of the nanoparticle through e.g., chemical interactions. The term “protein corona” as used herein has its ordinary meaning and shall include an interaction of biomolecules that form a dynamic layer around the surface of a nanoparticle including but not limited to what is referred to in the literature as a hard corona, a soft corona, or any combination thereof. A protein corona may form during or post administration of the composition, whereas the corona excipient is present pre administration. The protein corona may form around the surface and / or is embedded / attached, partially or fully, onto the external surface of the administered composition through e.g., permanent and / or transient chemical and / or physical interactions. In one embodiment, the corona that forms during or post administration (protein corona) may in addition to proteins comprise peptides, amino-acids, cell-debris, lipids, nucleic acids, biomolecules, or the like.

[0067] The term “particulate” as used herein has its ordinary meaning and shall include a particle that includes one or more lipid nanoparticle (LNP, also referred to herein as an encapsulation structure) coated in an excipient matrix. As used herein, the term “coated” or “coating” can refer to full or partial coating. In some embodiments, an excipient matrix includes polysaccharides, sugar alcohols, polypeptides, proteins (for example, a protein corona), esters, ethers, amides, amines, sulphates, thiols, urethanes, phosphoesters, phosphazenes, amino acids, surfactants, lipids, stearates, or polymers, or combinations thereof.

[0068] The term “nanoparticle” or “nanocarrier” as used herein has its ordinary meaning and shall include a matter particle that is 1 to 1000 nanometers in diameter. Nanoparticles can be composed of multiple substances and can contain compositions, molecules, and / or nucleic acids within its core.

[0069] The term “lipid nanoparticle (LNP)” as used herein has its ordinary meaning and shall include a spherical particle composed of lipids that is 1 to 1000 nanometers in diameter. Lipid nanoparticles can be composed of phospholipids, ionizable lipids, polyethylene glycol-derived lipids (PEGylated lipids), sterols, cholesterols, or a combination thereof. Lipid nanoparticles can contain compositions, molecules, and / or nucleic acids within its core. A lipid nanoparticle may be referred to herein as an encapsulation structure.

[0070] The term “pharmaceutically active ingredient” or “active pharmaceutical ingredient (API)” as used herein has its ordinary meaning and shall include a component that provides an active (e.g., biologically active) or other direct effect on the treatment, mitigation, cure, prevention or diagnosis of a disease of a subject or an effect on a physiological function of a subject.

[0071] The term “lipids” as used herein has its ordinary meaning and shall include a group of hydrophobic or amphiphilic organic molecules. Lipids can include but are not limited to monoglycerides, diglycerides, fats (triglycerides), fatty acids, phospholipids, and sterols.DTS Ref: 40574. RNH. P110PC

[0072] The term “cationic lipid” as used herein has its ordinary meaning and shall include a lipid molecule that is positively charged in an acidic pH condition and neutral at physiological pH.

[0073] The term “helper lipids” as used herein has its ordinary meaning and shall include a class of lipid molecules that contribute to the stability and delivery efficacy of a lipid nanoparticle.

[0074] The term “stealth lipid” as used herein has its ordinary meaning and shall include a class of lipid molecules (PEGylated lipids) that are modified with polyethylene glycol (PEG), or PEG alternatives, including, for example, polysarcosine, poly(2-oxazoline), poly(glycerol), poly(N-vinylpyrrolidone), zwitterionic polymers such as poly(carboxybetaine) or poly(sulfobetaine), polysaccharides such as dextran or hyaluronic acid, or peptide- based hydrophilic coatings.

[0075] The term “polymer” as used herein has its ordinary meaning and shall include a molecule composed of multiple repeating subunits (monomers), or block copolymer.

[0076] The term “dried state”, “dried”, or “dry” (e.g., dry powder) as used herein has its ordinary meaning and shall include the condition of particulates and / or nanoparticles that have been altered / processed to remove water / liquid through, for example, spray drying and / or lyophilization. A dried state may include a partially dried state, including for example a composition that includes 20% or less of moisture content, including for example, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, or 0.5% or less moisture content, and thus a dried state does not necessarily refer to a state that is entirely devoid of moisture.

[0077] The term “hydrated state” as used herein has its ordinary meaning and shall include the condition of particulates and / or nanoparticles in a liquid solution, in suspension, or dispersion.

[0078] The term “dry powder” as used herein has its ordinary meaning and shall include particulates and / or nanoparticles in a dry state (e.g., fine granular solid state).

[0079] The term “colloids electron density” as used herein has its ordinary meaning and shall include a measure of the probability of an electron of a colloid being present in an element of space at a given point. Colloids electron density can be measured and visualized using techniques known to those of skill in the art, including, for example, cryogenic transmission electron density (cryo-TEM), small-angle X-ray scattering (SAXS), and / or small-angle neutron scattering (SANS), or other microscopy, scattering, diffraction, or spectroscopy methods or techniques.

[0080] The term “colloidal suspension” or “colloid” as used herein has its ordinary meaning and shall include a mixture in which microscopically dispersed insoluble particles of one substance are suspended through a different substance. Colloidal suspensions can include aerosols, liquids, and gels.

[0081] The term “inhaler” or “inhaler device” as used herein has its ordinary meaning and shall include a delivery device used to administer a substance such as a pharmaceutical composition or medicine to the lungs or other tissue of a subject through the subject’s breathing or through forced ventilation. Types of inhalers include but are not limited to meter-dosed inhalers (MDI), dry powder inhalers (DPI), soft mist inhalers, smartDTS Ref: 40574. RNH. P110PC inhalers, and nebulizers. The term “nebulizer” as used herein has its ordinary meaning and shall include a delivery device used to administer a pharmaceutical composition or medicine in the form of a inhalable mist to respiratory tract (for example, the nasal cavities, airways, and / or lungs) of a subject. A nebulizer may use ultrasonic power, vibrating meshes, oxygen, or compressed air to break solutions and suspensions into aerosol droplets that include a mixture of gas and solid and / or liquid particulate. Nebulizers can include a mouthpiece or face mask for the subject to breathe the inhalable mist over a period of time. The term “soft mist inhaler” as used herein has its ordinary meaning and shall include a device that provides a metered dose to the user and that imposes pressure on a composition, causing composition to spray out of a nozzle, thus forming a soft mist to be inhaled.

[0082] The term “dry powder inhaler” as used herein has its ordinary meaning and shall include a delivery device used to administer a pharmaceutical composition or medicine in the form of a dry powder to the respiratory tract (for example, nasal cavities, airways, and / or lungs) of a subject through the subject’s breathing. The dry powder can be delivered to lungs of the subject without the use of an aerosol and / or a propellant as the subject takes an inhalation through a mouthpiece of the dry powder inhaler. Tissue other than the lung can be targeted using a dry powder inhaler.

[0083] A “ nucleic acid” sequence has its ordinary meaning and shall include a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequence. The term captures sequences that include any of the known base analogues of DNA and RNA such as, but not limited to 4-acetylcytosine, 8-hydroxy-N6- methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxyl- methyl) uracil, 5-fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6- isopentenyladenine, 1 -methyladenine, 1 -methylpseudouracil, 1 -methylguanine, 1 -methylinosine, 2,2- dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxy- aminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-th iocytosine, 5- methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N- uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

[0084] The term “oligonucleotide,” as used herein has its ordinary meaning and shall include a nucleic acid sequence comprising from about 2 to about to about 100 nucleotides (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100 nucleotides, or a range defined by any of the foregoing values).

[0085] The term “polynucleotide” has its ordinary meaning and shall include a polymeric form of nucleotides of any length, including DNA, RNA, or analogs thereof. A nucleotide may include mono-, di-, tri-, oligo- , and poly- forms. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to theDTS Ref: 40574. RNH. P1 10PC nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0086] The terms “polypeptide” and “protein” have their ordinary meaning and shall include a polymer of amino acid residues and are not limited to a minimum length. A peptide may include mono-, di-, tri-, oligo-, and poly- forms. Polypeptides, including therapeutic proteins and other peptides, e.g., linkers, tags, capsid proteins, may include amino acid residues including natural and / or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some respects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, such as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

[0087] Amino acids generally can be grouped according to the following common side- chain properties: (1 ) hydrophobic: Norleucine, Met, Ala, Vai, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Amino acids may include proteinogetic or nonproteinogetic forms.

[0088] Amino acid substitutions may also refer to one or more changes in a polypeptide sequence. The changes may include replacement of one amino acid in a polypeptide with another amino acid, insertion of one or amino acids, and / or deletion of one or more amino acids, or any combination thereof. Non-conservative amino acid substitutions will involve exchanging a member of one of these classes for another class.

[0089] The term “recombinant,” has its ordinary meaning and shall include a polynucleotide that is a product of various steps of alteration including but not limited to restriction enzyme digestion followed by ligation steps, alterations introduced by procedures such as polymerase chain reaction (PCR) (for example, introduction of restriction enzyme sites and nucleotide substitutions), and / or a combination of polynucleotides and proteins that is not found in nature.

[0090] The term “gene” has its ordinary meaning and shall include a polynucleotide containing at least one open reading frame that is capable of encoding a particular gene product. Any of the polynucleotide sequences described herein may be used to identify larger fragments or full-length coding sequences of the genes with which they are associated.

[0091] The term “administering” as used herein has its plain and ordinary meaning and shall include one or more steps that may be taken to introduce a composition to a cell, cell population, tissue, respiratory tract, lungs, airway, or other organ or body part of a subject. Administering includes “instructing administration.”DTS Ref: 40574. RNH. P1 10PC

[0092] As used herein, “therapeutically effective” has its ordinary meaning and shall include an amount of a pharmaceutically active compound(s) that is sufficient to treat or ameliorate, or in some manner reduce the symptoms associated with diseases and medical conditions. When used with reference to a method, the method is sufficiently effective to treat or ameliorate, or in some manner reduce the symptoms associated with diseases or conditions. Forexample, an effective amount in reference to diseases is that amount which is sufficient to block or prevent onset; or if disease pathology has begun, to palliate, ameliorate, stabilize, reverse or slow progression of the disease, or otherwise reduce pathological consequences of the disease. In any case, an effective amount may be given in single or divided doses.

[0093] The terms “treating,” “treatment,” “therapeutic,” or “therapy”, as used herein interchangeably, have their plain and ordinary meanings and do not necessarily mean total cure or abolition of the disease or condition. Any alleviation of any undesired signs or symptoms of a disease or condition, to any extent can be considered treatment and / or therapy. To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0094] As used herein, and unless otherwise specified, the terms “prevent,” “preventing” and “prevention” have their ordinary meaning and shall include the prevention of the onset, recurrence or spread of a disease ordisorder, or of one or more symptoms thereof. In certain embodiments, the terms refer to the treatment with or administration of a compound or dosage form provided herein, with or without one or more other additional active agent(s), prior to the onset of symptoms, particularly to subjects at risk of disease or disorders provided herein. The terms encompass the inhibition or reduction of a symptom of the particular disease. In certain embodiments, subjects with familial history of a disease are potential candidates for preventive regimens. In certain embodiments, subjects who have a history of recurring symptoms are also potential candidates for prevention. In this regard, the term “prevention” may be interchangeably used with the term “prophylactic treatment.”

[0095] The term “subject’, “individual”, or“patient”, as used herein interchangeably, have their plain and ordinary meanings and shall include a vertebrate, optionally a mammal, such as a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.EXAMPLES

[0096] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0097] Several embodiments provided herein relate to compositions of inhalable dry powder LNP formulations and methods of making and using the compositions. Structural changes in LNPs after dry powder manufacturing were systematically assessed through molecular dynamics simulations, as well as cryo-TEM, membrane fluidity measurements and apparent pKa values. The effects on how different compositions influenceDTS Ref: 40574. RNH. P1 10PC mucus penetration and diffusion, cellular uptake kinetics, and knockdown efficiency were also examined. In some embodiments, the LNP membranes include lipids that include DSPC and 1 ,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (PEG-DMG 2000). In some embodiments, the lipids provide interactions that include hydrogen bonding and the incorporation of lactose into the membrane, which may result in morphological changes. Afterthe reconstitution of spray-dried LNPs, membrane fluidity remains altered towards a less fluid state. In some embodiments, the structural changes in the membrane are influenced by the PEG and DSPC concentrations in the LNPs. The interactions of lung mucosa and lining fluid components with altered LNP structures were examined. These interactions improve the ability of LNPs to cross lung barriers, achieve cellular uptake, and release cargo. Although the lung barrier crossing is described herein, other barriers may also be effectively crossed in accordance with several embodiments described herein.Example 1 : Production of LNPs

[0098] In this non-limiting example, methods of characterizing LNPs were developed / utilized / performed.

[0099] Small interfering RNA-loaded LNPs were formulated based on the Onpattro® (patisiran) formulation, with variations in helper lipid and PEG-lipid content as shown in Table 1 . Lipids were dissolved in absolute ethanol at a concentration of 5 mM and mixed according to the specified ratios (Table 1 ). siRNA was diluted in 25 mM sodium acetate buffer (pH 4) to achieve an N / P ratio of 3. The lipid and siRNA solutions were mixed using either impingement jet mixing (KNAUER Nanoscaler®, KNAUER, Berlin, Germany) at a total flow rate (TFR) of 3 ml / minute, or syringe pumps (Landgraf LXXX) connected to a T-junction mixer (Techlab, Braunschweig, Germany) at a TFR of 2 ml / minute to produce precursor LNPs. For both mixing methods, a lipid-to-siRNA flow rate ratio (FRR) of 1 :3 was used. Precursor LNPs were dialyzed overnight at room temperature against PBS (pH 7.4) using a 3.5 kDa MWCO dialysis kit, then sterile filtered using a 0.22 m syringe filter. Final LNPs were stored at 4°C for up to 30 days until further use.Table 1 : Percentages of different lipids in the four LNP compositionsDTS Ref: 40574. RNH. P1 10PC

[0100] Other methods for loading LNPs with therapeutic nucleic acids may be used, including different mixing techniques, different concentrations, and different temperatures. In addition to siRNA, other therapeutic nucleic acids may be loaded into LNPs using a similar process. In addition, other lipids may be used for the composition of LNPs.Spray Drying of siRNA LNPs

[0101] Feed solutions were prepared by adding lactose monohydrate to LNP suspensions to reach a solid content of 50 mg / ml. This lactose-LNP solution was spray-dried following the method by Friis et al. (J Controlled Rel, 2023). Spray drying was performed using a Mini Spray Dryer B-290 (Buehl Labortechnik, Flawil, Switzerland) with the following adjusted parameters: inlet temperature 100°C, outlet temperature ~64°C, pump rate 10%, atomizing gas flow 473 L / h, and aspirator set to 80%. The powder was collected using a high- performance cyclone. Two siRNA concentrations were processed, resulting in final concentrations of 40 and 16 pmol siRNA / mg lactose.Hydrodynamic diameters and zeta potential

[0102] Hydrodynamic diameter and polydispersity index (PDI) were measured by dynamic light scattering (DLS) using a Zetasizer Advance Ultra (Malvern Instruments, Malvern, UK) at 25°C in disposable cuvettes. Spray-dried powders were redispersed in purified water to obtain a lactose concentration of 50 mg / ml and diluted 1 :5 in PBS or measured directly. Measurements were performed in triplicate (n = 3), and data were analyzed using ZS Xplorer software (v 3.1.5.1). Results are reported as Z-average (nm) ± SD. Nanoparticle tracking analysis (NTA) was performed using the NanoSight NS300 system (Malvern Instruments, Malvern, UK). Samples were diluted to achieve 10-200 particles per frame and measured at 25°C. Five runs per sample were conducted.Molecular Dynamics Simulations

[0103] All molecular dynamics (MD) simulations were performed using GROMACS 2021 .4 patched with PLUMED. All-atom (AA) membrane systems were constructed with the CHARMM-GUI Multicomponent Assembler. Lactose, built using the CHARMM-GUI Glycan Reader, was positioned in close proximity to the membrane within the simulation box. The Multicomponent Assembler utilizes CHARMM GUIs Membrane builder which was extended in 2021 to support ionizable lipids relevant for LNP systems.

[0104] Membrane components were selected to align with the Onpattro® (patisiran) LNP formulation, though different concentrations of unprotonated (6Z,9Z,28Z,31 Z)-heptatriaconta-6,9,23,31 -tetraen- 19-yl 4-(dimethylamino)butanoate (D-Lin-MC3-DMA) were used to better mimic the membrane composition of aDTS Ref: 40574. RNH. P1 10PC nucleic acid-loaded LNP. The concentrations of other membrane lipids were adjusted as shown in Table 2. In Table 2, the membranes were simulated as bilayers, and the numbers were given for the total amount of lipids in the system. The simulation box was 7 nm x 7 nm x 17 nm, with the extended z-dimension accommodating PEG- DMG 2000 molecules.

[0105] For hydrated and rehydrated simulations, the dehydrated membrane system was hydrated using the TIP3P water model, as recommended by GROMACS. The system was neutralized and isotonized with sodium and chloride ions following CHARMM-GUI guidelines. Simulations were run using the GROMACS- compatible Charmm36 force field. Energy minimization for hydrated and rehydrated systems was conducted in a stepwise manner for 50,000 steps. Coulombic interactions were treated using Particle Mesh Ewald (PME) electrostatics with a cutoff distance of 1 .2 nm. Van der Waals interactions were handled using a cutoff algorithm with the same 1 .2 nm cutoff. Equations of motion were integrated using the leap-frog algorithm during equilibration and production runs. The system underwent a 0.125 ns NVT equilibration at 303.15 K (v-rescale thermostat), maintaining constant number of atoms and volume. This was followed by a 0.125 ns NPT equilibration using position restraints at 1 atm pressure (Berendsen barostat) and 303.15 K temperature (v-rescale thermostat). Production simulations were conducted for 10 ns under constant temperature (303.15 K, Nose-Hooverthermostat) and semi-isotropic pressure (1 atm, Parrinello-Rahman barostat).

[0106] For dehydrated systems, due to pressure instabilities in low-solvation conditions, energy minimization and NVT equilibration were performed as described above. The production run was conducted under NVT conditions with temperature coupling via the Nose-Hoover thermostat at 303.15 K.

[0107] Analysis included solvent-accessible surface area (SASA), number of hydrogen bonds, mean square displacement (MSD) of lactose, radial distribution function (RDF) of lactose relative to membrane components, symmetrized mass density, and area per lipid (for NPT simulations). SASA and hydrogen bond analysis was performed from 1 ns to 10 ns.Table 2: Number of lipids and lactose molecules per systemDTS Ref: 40574. RNH. P1 10PCLaurdan Assay

[0108] LNPs were stained with 6-dodecanyl-2-dimethylaminonaphthalene (Laurdan) at a lipid-to- Laurdan molar ratio of 100: 1 in a 1 mg / ml Laurdan ethanol solution. Non -spray-dried LNPs were diluted 1 :2 with highly purified water (HPW), LNPs were also supplemented with a lactose solution (50 mg / ml) and spray-dried LNPs were reconstituted to 50 mg / ml lactose in HPW and incubated for 30 minutes resulting in the same lipid concentrations in all solutions. Samples (100 pl) were transferred to a 96-well plate and fluorescence was measured in triplicates using a Spark® microplate reader. Excitation was at 340 nm, and emission was measured at 490 nm and 440 nm. Laurdan general polarization (Gp) was calculated using the formula introduced by Parasassi et al:Determination of apparent pKa

[0109] A master buffer stock was prepared by mixing 10 mM sodium phosphate, 10 mM sodium borate, 10 mM sodium citrate, and 150 mM sodium chloride as described by Sabnis et al. The pH was adjusted to values from 3 to 9 with sodium hydroxide, resulting in 14 pH conditions. 6-(p-toluidino)-2-naphthalenesulfonic acid sodium salt (TNS) reagent (20.75 mM in dimethyl sulfoxide (DMSO)) was added to the LNPs, which were concentrated to 500 pM lipid using a Vivaspin 500 column at 3,000 g. For non-spray-dried LNPs, 90 pl of each pH buffer was added into a 96-well plate. For spray-dried LNPs, the pH range was adjusted from 3 to 8.5 pl of LNP sample and 5 pl of TNS reagent (84 pM) were added to each well, incubated for 20 min at room temperature, and measured using a Spark® microplate reader at 320 nm excitation and 450 nm emission. The pKa was determined using a four-parameter logistic regression fit, with the value where 50% of fluorescence intensity was measured.Cryo-TEM

[0110] For Cryo-TEM measurements fresh or spray dried LNPs were concentrated via Vivaspin 6 columns using 3500 G for up to 3 hours, to a siRNA concentration of 1000 ng / pl.In vitro uptake and eGFP knockdown kinetics in H1299-eGFP cells

[0111] Human non-small lung carcinoma cell line H1299 stably expressing enhanced green fluorescent protein (H1299-eGFP) cells were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS), 1 % penicillin-streptomycin and 0.4% G418. Cells were maintained, grown and incubated in aDTS Ref: 40574. RNH. P1 10PC humidified incubator at 37°C and 5% CO2. 30,000 cells were seeded in 24-well plates and incubated for 24 hours before transfection. LNPs containing eGFP-siRNA labeled with Alexa-Fluor 647 were diluted in sterile PBS (fresh LNPs) or reconstituted in RNase-free water (spray-dried LNPs) to a final siRNA concentration of 800 pmol / mL and 100 l added to each well. Blank samples were treated with 100 pl PBS. At 0.5, 1 , 2, 4, 8, 16, and 24 h post transfection, the medium was removed and cells were washed with PBS and trypsin ized. The reaction was stopped by adding 200 pl of growth medium. The cell suspension was collected and centrifuged at 400 g for 5 min. Afterwards, supernatant was aspirated, cells washed with PBS, centrifuged, and finally suspended in 400 pl PBS with 2 mM EDTA. Cellular uptake and eGFP knockdown were measured using flow cytometry (Attune NxT, Thermo Fisher Scientific, Waltham, MA, USA), with excitation at 638 nm and 488 nm, detected with RL-1 H and BL-1 H filters. Data were collected from 10,000 viable gated cells per sample. Cellular uptake is reported as mean fluorescence intensity (MFI), while knockdown efficiency is expressed as % eGFP expression relative to blank controls. The knockdown half-time was derived by fitting the eGFP expression data to a four-parameter logistic regression model, with the time point corresponding to 50% maximal expression defined as T1 / 2.Confocal microscopy

[0112] H1299-eGFP cells (10,000 cells) were seeded in 8-well p-slides and incubated for 24 hours at 37°C with 5% CO2. One hour prior to transfection, the medium was aspirated, cells washed with PBS and stained with 50 nM LysoTracker Red DND-99 in growth medium. Fresh LNPs were diluted in sterile PBS, spray- dried LNPs were reconstituted in sterile RNase-free water to a siRNA concentration of 800 pmol / ml and 40 pl added to each well. Blank samples were treated with 40 pl PBS. After 4 hours, the medium was removed and cells were washed with PBS. For nuclei staining, cells were incubated with 0.5 pg / ml 4',6-diamidino-2-phenylindole (DAPI) in PBS for 15 minutes in absence of light, followed by 3x PBS washes. Live-cell imaging was performed using an SP8 inverted confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany) and analysis was performed using Leica Application Suite X software.Preparation and characterization of artificial mucus

[0113] Artificial mucus following a protocol published by Conte et al. with slight modifications. In brief, a mixture containing 25 pL of sterile egg yolk emulsion, 25 mg of mucin from bovine submaxillary glands, 20 mg of calf thymus DNA (Type I), 30 pL of an aqueous diethylenetriaminepentaacetic acid (DTPA) solution (1 mg / mL), 25 mg of sodium chloride, 11 mg of potassium chloride, 100 pL of RPM1 1640 medium, 50 pL of penicillinstreptomycin, and RNase-free water to obtain a total volume of 5 ml was prepared. The mixture was vortexed, stirred for 1 hour, and degassed for 4 hours in a vacuum oven at 20°C. The mucus was allowed to rest at 4°C overnight. Rheological characterization was performed using a Physica MCR 100 rheometer (Anton Paar, Graz, Austria) equipped with a cone- plate geometry (CP50-1 , Anton Paar). Viscosities were measured across shear rates ranging from dy / dt = 1000 s“1to dy / dt = 10 s“1. A strain-controlled frequency sweep from f = 0.10 Hz to f =DTS Ref: 40574. RNH. P1 10PC10 Hz was conducted to assess frequency-dependent viscoelasticity. Eleven equidistant data points were recorded on a logarithmic scale at 20°C. A solvent trap was used to prevent sample dehydration during measurements. Fluorescence Correlation Spectroscopy

[0114] Diffusion coefficient of LNPs in PBS and mucus was measured using Fluorescence Correlation Spectroscopy (FCS). LNPs were labeled using Atto 647N DOPE (Sigma). 20 l of LNPs were mixed with 1 microliter of 1 mg / ml Atto 647N DOPE solution in DMSO. Furthermore, 10 pl of labelled LNPs were added to 100 microliters of PBS or 100 pl of mucus. A Zeiss LSM 980 microscope with 40 x 1 .2 NA water immersion objective was used for FCS. A 633 nm He-Ne laser was used for excitation. The laser power was set to 0.05% of the total laser power, corresponding to 1 W. The emission detection was done with GaAsP spectral detector in the range of 650-700 nm.

[0115] FCS fitting and diffusion coefficient calculation was performed using the Python-based program Py-Profiler. Curves were fitted with the following three-dimensional diffusion model:

[0116] Where is a correlation function, stands for delay time, - for diffusion time, - for aspect ratio of the focal volume and - for an average number of molecules within the focal volume.

[0117] Calibration was performed using 10 nM solution of Alexa467 (ThermoFisher) in pure water.Diffusion was calculated from the diffusion time using the following expression:

[0118] Where is D a diffusion coefficient, co stands for full width at half maximum of focus point spread function, TD- for diffusion time.In vitro penetration through artificial mucus

[0119] LNP formulations for the mucus penetration assay were prepared with 2 mol% Rhodamine- labeled lipid. A volume of 50 pl artificial mucus was transferred to the insert of a 24-wel I Transwell system, and 20 pl of either fresh or reconstituted spray-dried LNPs was added on top. The acceptor compartment contained 500 pl PBS. The plate was incubated at 37°C in a humidified incubator. At 0.5, 1 , 2, 4, 8, and 24 h, 100 pl of the acceptor medium was collected for analysis and replaced with fresh PBS. Diffusion control wells contained 50 pl PBS instead of mucus and were processed identically. Fluorescence intensity was measured using a Spark®DTS Ref: 40574. RNH. P1 10PC microplate reader with excitation at 540 nm and emission at 590 nm. Results are presented as cumulative LNP penetration through mucus relative to the diffusion control (FIG. 3).Mucus penetration and transfection efficiency in mucus-secreting Calu-3 cells grown at the air liquid interface (AU)

[0120] Calu-3 cells were cultured in EMEM medium supplemented with 10% FBS and maintained in a humidified incubator at 37°C and 5% CO2. A total of 250, 000 cells were seeded onto Corning Transwell inserts and placed into wells containing 700 l culture medium at the basolateral side. After three days, a dense monolayer had formed. For air-liquid interface culture, the apical medium was removed and the basolateral medium was exchanged for 200 pl PneumaCult ALI medium, refreshed every two days. After six days, a stable polarized epithelial layer had formed. The diffuse mucus layer was washed with 200 pl PBS, and 24 hours later, cells were transfected with 100 pl fresh or reconstituted Alexa-Fluor 647-labeled siRNA-LNPs at 1 .6 nmol / ml siRNA. Blank samples were treated with 100 pl PBS. After 6 hours of incubation at 37°C and 5% CO2, cells were gently scraped off the Transwell membrane, suspended in PBS, and centrifuged at 400 g for 5 minutes. The supernatant was removed, cells were washed with PBS, centrifuged again, and resuspended in 400 pl PBS containing 2 mM EDTA. Flow cytometry (Attune NxT) was used to measure cellular uptake with a 638 nm excitation laser and RL-1 H filter detection. The experiment was performed once in triplicates. Results are presented as mean ± SD of transfected cells (%) relative to blank samples.Statistical analysis

[0121] If not stated otherwise, experiments were performed in triplicates, and results are represented as mean ± SD. Violin plots were used, where every data point of the measurements or simulations was considered; the median with quartiles is shown. The normality distribution of experimental data was tested using the Anderson-Darling and Shapiro-Wilk tests. One-way ANOVA (with the Tukey post-hoc test) was performed in GraphPad Prism (GraphPad Software, La Jolla, USA, v. 10.4.1) to calculate the p-values with 95% confidence. Affinity Designer 218.2 (version 2.5.7, Serif Ltd., West Bridgford, UK), PyMOL (version 2.5.8, Schrodinger Inc., New York, NY, USA), and GraphPad Prism were used for visualization. Some graphics contain elements were created in BioRender.Example 2: Alterations in LNP Characteristics

[0122] In this non-limiting example, structural changes and alterations were observed in LNPs, after LNPs were spray dried.

[0123] To assess whether the structural changes observed in spray-dried (SD) RNA-LNPs upon rehydration result from reversible sugar-membrane interactions or more permanent stress-induced modifications, Onpattro-like (patisiran-like) LNPs were spray-dried in the presence of 5% lactose (FIG. 4A) and subsequently dialyzed against PBS at room temperature for up to 96 hours. Fresh LNPs stored and dialyzed in 5% lactoseDTS Ref: 40574. RNH. P1 10PC served as controls. FIG. 4A shows a schematic of the spray drying-based process for the manufacturing of dry powder RNA-LNPs.

[0124] FIG. 4B shows a graph depicting the hydrodynamic diameter of redispersed LNPs before and after 24 hours dialysis against PBS, measured via nanoparticle tracking analysis (NTA). Data points represent the mean ± standard deviation (n = 3). Statistical analysis was performed using one-way ANOVA: ns, p > 0.05; *p < 0.05; **p < 0.01 ; ***p < 0.001 ; ****p < 0.0001 . FIG. 4C shows D90 values from size measurements by NTA for fresh LNPs, LNPs in 5% lactose, and spray-dried LNPs before and after dialysis; violin plots represent the distribution of all individual particle sizes within the D90 range; the median and quartiles are indicated (n = 3).

[0125] No significant changes were observed in the control LNPs, indicating that incubation with lactose alone did not impact particle integrity. In contrast, spray drying in the presence of lactose led to a substantial increase in particle size. Nanoparticle tracking analysis (NTA) (FIGs. 4B-4C) confirmed a shift toward larger hydrodynamic diameters, revealing upward shifts in both mean size and D90 values. The size distribution also broadened, which indicated increased heterogeneity among particles. Detailed analysis of the D90 population (FIG. 4C) showed that while a subset of SD-LNPs underwent substantial enlargement, approximately three- quarters of the population remained within the original D90 range.

[0126] Importantly, dialysis for 24 hours did not reverse the size increase, implying that the changes were not due to loosely bound lactose but rather to irreversible structural modifications in membrane organization. This finding is consistent with previous reports of persistent sugar coatings on Comirnaty LNPs detectable by NMR (Porat Dahlerbruch et al.) and supported by desiccation studies showing that removal of sugars from lipid membranes required significant energy input (Viera et al., Biomembranes, 1993).

[0127] Collectively, these data demonstrated that spray drying induces lasting, irreversible modifications in RNA-LNPs, characterized by stress-induced size increases, membrane rigidification, and partial particle heterogeneity. These alterations were consistent with the water replacement hypothesis, whereby sugars substitute for water molecules at the lipid headgroups, promoting increased lipid spacing and potential membrane thinning.Example 3: Lactose interactions with components of the LNP membranes

[0128] In this non-limiting example, lactose-LNP membrane interactions were observed and analyzed.

[0129] To investigate the molecular basis of lactose-membrane interactions, all-atom molecular dynamics simulations (MDS) were performed using bilayer membrane models. This approach followed previous studies that explored the influence of disaccharides, such as trehalose, on LNP membranes under different hydration states. The focus of these experiments was specifically on the behavior of lactose after spray drying.DTS Ref: 40574. RNH. P1 10PCLNP membranes were simulated under three conditions: (i) fully hydrated, mimicking fresh LNPs in lactose solution; (ii) nearly dehydrated; and (iii) rehydrated after dehydration.

[0130] Modeling the LNP surface as a bilayer, however, necessitates consideration of surface composition. Recent studies employing Cryo-TEM, NMR, and MDS demonstrate that PEG-lipids and 1 ,2- distearoyl-sn-glycero-3-phosphocholine (DSPC) are enriched at the LNP periphery, while the core is densely packed with 3|3-hydroxy-5-cholestene (cholesterol) and ionizable lipids. Membranes containing varying D-Lin- MC3-DMA (MC3) concentrations (50%, 5%, 0%) were simulated to reflect the spectrum of LNP surface compositions.

[0131] FIGs. 5A-5E show all-atom simulation data of LNP membranes at different hydration states and with different D-Lin-MC3-DMA concentrations in the membrane. FIG. 5A shows simulations of hydrated membrane with 5% D-Lin-MC3-DMA and lactose in solution (top), dehydrated membrane with lactose (middle), rehydrated membrane with lactose (bottom) all after 10 ns simulation. FIG. 5B shows the symmetrized mass density relative to the center of the simulation box (membrane) of Lactose, Membrane, DSPC, 1 , 2-d i my ristoyl-rac- glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG-2000) hydrated (top), dehydrated (middle), rehydrated (bottom) for systems with 5% D-Lin-MC3-DMA content. FIG. 5C shows violin plots depicting the area per lipid for the membranes without and with lactose from 1 ns to 10 ns, where every 2 fs on measurement point was calculated. FIG. 5D shows violin plots depicting the number of hydrogen bonds between lactose and the different membranes from 1 ns to 10 ns calculated every 4 fs. FIG. 5E shows graphs depicting the mean of the normalized number of hydrogen bonds between lactose and the different membrane components. Data points indicate mean ± standard deviation for FIG. 5B and the median with quartiles for FIGs. 5C-5D.

[0132] Analysis of the simulation trajectories revealed free diffusion of lactose molecules in both hydrated and rehydrated systems after approximately 1 ns. Examination of representative endpoint snapshots (5% MC3 membranes, after 10 ns) supported these findings (FIG. 5A). In fully hydrated systems, lactose molecules remained primarily associated with PEG-DMG-2000 in the aqueous phase. In contrast, following dehydration and rehydration, lactose localized closer to DSPC-rich membrane regions, in some cases penetrating the bilayer interior. Mass density distributions (5% MC3 membranes: FIG. 5B) revealed distinct lactose peaks within membranes after dehydration, consistent with clustering, while rehydrated membranes showed broader, less defined distributions, which indicates partial dissolution of clusters. These results support a model in which dehydration enhances lactose-membrane interactions via water replacement mechanisms, with partial persistence upon rehydration.

[0133] Lactose integration into membranes was evident across all simulations, which aligns with previous findings. The area per lipid, calculated over 10 ns in NPT ensembles, was consistently higher in lactose- containing membranes, with the greatest differences observed after rehydration (FIG. 5C). Membranes without lactose exhibited higher post-rehydration densities (data not shown), reinforcing the notion that lactose integrationDTS Ref: 40574. RNH. P1 10PC leads to membrane thinning. These observations align with the water replacement theory, which proposes that sugars stabilize membranes by occupying the space between lipid headgroups. Literature reports using smallangle neutron scattering (SANS) demonstrated that sugars at low concentrations (<0.2 M) strongly bind to bilayers, inducing membrane expansion and thinning. Given that lactose concentrations during spray drying were below 0.2 M, similar mechanisms underlie the observations.

[0134] Hydrogen bonding represents another key molecular interaction underlying lactose- membrane association. Calculation of hydrogen bonds over the final 9 ns of simulations revealed an increase upon dehydration, followed by a slow and incomplete decrease after rehydration (FIG. 5D). Among lipid components, DSPC and PEG-DMG-2000 accounted for the majority of hydrogen bonding with lactose (FIG. 5E), consistent with their surface exposure. The high affinity of PEG-DMG-2000 is attributed to its multiple polyethylene glycol monomers, which serve as efficient hydrogen bond acceptors. The prominent interaction with DSPC indicates its high availability at the membrane surface, consistent with prior MDS and NMR studies of LNP structure. The slow dissociation and persistence of sugar-lipid hydrogen bonds following rehydration has been reported in previous studies combining MDS with Fourier-transform infrared (FTIR) and two- dimensional infrared (2D-IR) spectroscopy. These studies demonstrated that sugar-lipid hydrogen bonds are stronger and more durable than water-lipid hydrogen bonds. These findings, validated experimentally by Viera et al., support a model where sugars protect membranes during dehydration stress via water replacement, with bond dissociation being a slow, energetically demanding process.

[0135] Combined, these results demonstrate that dehydration promotes enhanced lactose- membrane interactions via hydrogen bonding and partial membrane integration. DSPC and PEG-DMG-2000, abundantly present on the surface of LNPs, were identified as principal sites of interaction.Example 4: Modulation of lactose interactions by varying PEG and DPSC concentrations in LNPs

[0136] In this non-limiting example, the effects of variations in LNP surface composition on structural changes and lactose interactions were observed.

[0137] Given the central role of DSPC and PEG-lipids in mediating lactose-membrane interactions in simulations, the effects of variations in LNP surface composition on structural changes upon spray drying were investigated. Four distinct LNP formulations were generated, differing in DSPC and PEG content (Table 1), and subsequently subjected to spray drying. To control for inherent compositional effects on particle size (particularly driven by the PEG content), membrane fluidity (e.g., influenced by the cholesterol content), and apparent pKa, differences between pre- and post-spray drying values were evaluated as relative changes.

[0138] FIGs. 6A-6E show LNP characteristics before and after spray drying. FIG. 6A shows a graph depicting DLS measurements before and after spray drying of all four formulations. FIG. 6B shows a graph depicting the difference in Z-A verage before and after spray drying. FIG. 6C shows a graph depicting the differenceDTS Ref: 40574. RNH. P1 10PC in Gp value determined via Laurdan assay before and after spray drying. FIG. 6D shows a graph depicting the difference in apparent pKa value of LNPs before and after spray drying, determined via TNS (6-(p-toluidino)-2- naphthalenesulfonic acid sodium salt) assay. FIG. 6E shows cryo-TEM micrographs of LNPs after concentration up to 1000 ng / pl of LNP3 or LNP4. LNP3 before and after spray drying (top), LNP 4 before and after spray drying (bottom). Data points indicate mean ± SD (n = 3). One-way ANOVA, ns, > 0.05, *, p < 0.05 **, p < 0.01 , ***, p < 0.001 , ****, p < 0.0001.

[0139] Across all formulations, spray drying induced an increase in hydrodynamic diameter and polydispersity index (PDI) (FIGs. 6A-6B). Notably, LNP 4, which is characterized by the highest DSPC and PEG content, exhibited the most pronounced size increase and variability. This is not only consistent with the simulation results revealing that both PEG and DSPC strongly influence lactose interactions at the outer LNP shell but also aligns with prior studies indicating that sugars preferentially associate with phospholipid headgroups through hydrogen bonding and water replacement mechanisms.

[0140] For all LNPs, GP values increased after spray drying (FIG. 6C), reflecting reduced membrane hydration and greater lipid ordering. Notably, LNPs with lower initial DSPC content (LNP 1 and LNP 3) displayed a more substantial relative increase in GP, indicating that membranes with initially lower order are more susceptible to spray drying-induced rigidification.

[0141] These experimental results corroborate the simulation data, where an increase in area per lipid was observed after dehydration and rehydration (FIG. 5C), indicative of membrane expansion. Symmetrized density analyses confirmed these trends, revealing greater localization of lactose within or directly atop the membrane after rehydration (FIG. 5B, bottom). Notably, an increased presence of DSPC near the membrane surface was detected in rehydrated systems, particularly in the 5% and 0% MC3 simulations. Consistently, the persistent elevation of GP after rehydration supports the notion that lactose partially integrates into the membrane, altering lipid packing and organization after drying.

[0142] To probe surface reorganization, apparent pKa values were determined by TNS assay. In LNPs, the pKa is influenced by both, the presence of protonated ionizable lipid in the outer layer, and by the environment in which the ionizable lipid is situated. Here, while shifts were less pronounced than changes in GP, a consistent trend towards lower apparent pKa values after spray drying was observed across all formulations (FIG. 6D), indicating modified surface environments, potentially due to altered ionizable lipid exposure or local dielectric properties.

[0143] The most pronounced structural alterations, observed in LNP 3 and LNP 4, were visualized by cryo-TEM. In non-dried Onpattro-like (patisiran-like) LNPs (LNP 3), monodisperse particles of ~50 nm with featureless interiors were observed. Post-SD, LNP 3 exhibited increased size heterogeneity, with a notable fraction of particles exceeding 200 nm (FIG. 6E, top right ), consistent with DLS (FIG. 6A) data. A dark, electron-dense ring surrounding the particles was detected, reminiscent of previously reported sugar-associated membraneDTS Ref: 40574. RNH. P1 10PC coatings SD (Friis et al.), further supporting membrane remodeling. Micrographs revealed partial lamellar organization for some non-dried LNP 4 particles, likely due to the elevated DSPC content. Most LNPs displayed the previously reported distinct unilamellar structure typical for siRNA-LNPs formulated at an N / P ratio of 3, consistent with the relatively higher baseline GP values of LNP 4 compared to LNP 3 (FIG. 6E, bottom panels). After SD, LNP 4 particles frequently appeared elliptical, occasionally exhibiting polymorphic, fused, or bleb-like structures (FIG. 6E, bottom right). This observation is consistent with prior reports linking dehydration of LNPs during freeze drying with bleb formation. In contrast to SD LNP 3, LNP 4 particles lacked a distinct, dark electron- dense outer layer (FIG. 6E). Although some membrane-associated material remained visible, it appeared less prominent than in LNP 3, consistent with the smaller GP and pKa shifts observed for LNP 4 after drying. This indicates that membrane reorganization was less extensive in DSPC-rich LNPs. Nevertheless, morphological changes, including increased particle size, fusion events, and structural heterogeneity, were more pronounced in LNP 4 than in LNP 3, reflecting reduced mechanical stability during dehydration and rehydration at higher DSPC content.

[0144] Collectively, these results demonstrate that the extent and nature of lactose-membrane interactions and the resulting structural consequences are strongly dependent on LNP surface composition, particularly DSPC and PEG-lipid content.Example 5: Alterations in cellular internalization and silencing kinetics in spray-dried LNPs

[0145] In this non-limiting example, spray-dried LNPs were evaluated in in vitro lung models and penetration studies in artificial mucus.

[0146] RNA-LNPs enter cells via endocytosis, which through receptor-mediated pathways (e.g., clathrin- or caveolin-mediated endocytosis, CME) or receptor-independent routes such as macropinocytosis and phagocytosis. The efficiency of cytosolic delivery depends on the entry mode, influenced by both particle properties and cell type. Receptor-mediated endocytosis, is predominant and facilitated by a protein corona formed around the LNPs upon contact with serum. Components of this protein corona may include ApoE, which binds to low- density lipoprotein receptors (LDLR), as well as high-density lipoprotein (HDL), or albumin. The formation of the protein corona is dynamic, with PEG-lipid dissociation, further promoting protein adsorption and thereby nanoparticle-cell interactions.

[0147] To assess the impact of lipid composition and spray-drying on LNP behavior, all LNP formulations were evaluated for cellular uptake and eGFP silencing in H1299-eGFP cells. Cellular uptake of fresh and reconstituted spray-dried LNPs and eGFP protein levels were monitored at various time points posttransfection. A high-resolution kinetic analysis was conducted to capture subtle differences in uptake. Notably, PEG-shedding and protein corona formation are known to occur within 30 minutes of serum exposure with gradual accumulation of siRNA- LNPs in endosomes reported within the first 4 - 6 hours post-uptake.DTS Ref: 40574. RNH. P1 10PC

[0148] For cellular uptake and eGFP protein downregulation assays, cells were treated with 80 pmol AF647 -labeled eGFP-siRNA of the fresh and reconstituted spray dried LNP 1 - 4 formulations and analyzed simultaneously by flow cytometry 0.5, 1 , 2, 4, 8, 16 and 24 hours post transfection. Data points indicate mean ± SD (n= 3). Cellular uptake of AF647-labled siRNA of the fresh and reconstituted spray dried LNP 1 - 4 formulations in H1299-eGFP cells was visualized by confocal scanning microscopy. Samples were treated with 32 pmol siRNA and were analyzed 4 hours after transfection.

[0149] FIGs. 7A-7D show graphs and confocal scanning microscopy images depicting cellular uptake (left) and eGFP protein downregulation (right) in H1299-eGFP cells after transfection with siRNA-LNPs. FIG. 7A shows cellular uptake and eGFP protein downregulation in cells transfected with fresh LNP 1 and spray- dried LNP 1 . FIG. 7B shows cellular uptake and eGFP protein downregulation in cells transfected with fresh LNP 2 and spray-dried LNP 2. FIG. 7C shows cellular uptake and eGFP protein down regulation in cells transfected with fresh LNP 3 and spray-dried LNP 3. FIG. 7D shows cellular uptake and eGFP protein downregulation in cells transfected with fresh LNP 4 and spray-dried LNP 4. Microscopy images are presented as merge pictures. Cells underwent staining with DAPI to represent the nuclei and endosomal staining with LysoTracker red DND-99.

[0150] LNP 1 , with a PEG-lipid content of 0.5%, exhibited rapid and high uptake, potentially attributed to fast PEG shedding and efficient protein corona formation. In contrast, LNP 2, despite having the same PEG-lipid content, achieved only 20% of the uptake observed for LNP 1 after 24 hours. LNP 3, with a higher PEG- lipid content (1 .5%), also demonstrated efficient uptake over 24 hours, though slightly delayed due to the longer PEG-shedding process. A reduction in mean fluorescence intensity (MFI) at 16 hours for LNP 1 and LNP 3 demonstrate the onset of exocytosis (FIG. 7A; FIG. 7C). Contrary to initial expectations that LNP 4 combining high PEG and DSPC content would perform worse, instead LNP4 exhibited uptake twice that of LNP 2, though still only one third of the uptake of LNP 1 . LNPs with 25% DSPC content consistently performed worse than those with 10% DSPC. For spray-dried formulations, uptake was significantly reduced across all groups. LNP 1 retained only -10% of its fresh formulation uptake efficiency, LNP 2 (28%), LNP 3 (27%), and LNP 4 (53%). This reduction can be attributed to membrane-associated lactose and membrane reorganization during the drying process which alters the formation of a protein corona on the LNP surface. Relative performance comparisons revealed that formulations with minimal changes in GP value before and after spray drying experienced lower relative uptake losses.

[0151] The cellular uptake performance for all formulations was further verified by confocal microscopy 4 hours after transfection as shown in FIGs. 7A-7D.

[0152] Gene silencing efficiency was assessed alongside cellular uptake. Effective silencing relies not only on cellular uptake but also on efficient endosomal escape of siRNA, regarded as a bottleneck within RNA delivery. Studies found, depending on the ionizable lipid, only 1 -3.5% of siRNA internalized by endocytosis reaches the cytosol, once released, siRNA can exert its effect within 10 minutes. An exponential decay in eGFP expressionDTS Ref: 40574. RNH. P110PC was detected for all fresh LNP formulations. LNP 1 and LNP 3 achieved the fastest knockdown over 24 hours, while LNP 2 and LNP 4 showed delayed knockdown, with a 90% reduction after 16 hours. This tailing effect is due to the slower and lower particle uptake observed for these formulations and can also be attributed to a significantly reduced fusogen ic efficiency for LN Ps with high DSPC content as reported by Aliakbarinodehi et al. After 24 hours, all formulations reached > 90 % eGFP downregulation.

[0153] For the spray-dried LNPs, formulations LNP 3 and LNP 4 exhibited knockdown kinetics that matched those of the fresh formulation. Despite a lower level of particle uptake, the colloidal and fusogenic properties of these LNPs appear to have been preserved, resulting in comparable knockdown efficiency. In contrast, LNP 1 and LNP 2 demonstrated substantially reduced knockdown efficiency throughout the entire observation period, reaching only 88% (LNP 1) and 80% (LNP 2) after 24 hours. The eGFP expression data was fitted to a four-parameter logistic regression model, with the time point corresponding to 50% maximal expression defined as T1 / 2. The analysis of the knockdown half-time revealed the pronounced change in gene silencing efficiency, within the time required to achieve a 50% reduction in eGFP expression increasing from 5.8 hours to 16.7 hours for LNP 1 , and from 8.2 hours to 19.8 hours for LNP 2 following spray drying. This reduced performance can be attributed in part to markedly lower cellular uptake. Additionally, it can be inferred that the thermal stress and shear forces associated with the drying process had a more pronounced effect on these two formulations, which contained only 0.5% PEG-lipid. Kim et al. reported that LNPs containing 1 % PEG displayed superior colloidal stability compared to those with 0.5% PEG, even at temperatures as high as 90 °C. Similarly, in the context of nebulization, higher PEG-lipid content has been shown to afford improved protection against shear stress.Example 6: LNP transport through artificial mucus and transfection efficiency in mucus-secreting Calu-3 cells grown at the air-liquid interface (ALI)

[0154] In this non-limiting example, LNP transport through artificial mucus and transfection efficiency in Calu-3 cells were assessed.

[0155] Following inhalation and deposition in the airways of the lung, and prior to cellular uptake by pulmonary cells, nanoparticles must traverse a key physiological barrier: the airway mucus layer. This mucus has been estimated to exhibit a thickness of approximately 10-20 pm, based on measurements from in vitro bronchial epithelial cultures and ex vivo human lung grafts. In healthy individuals, airway mucus is composed of approximately 97% water, along with mucin (~2%), DNA, proteins, lipids, and cellular debris. The mucins act as gel-forming components that establish a mesh-like structure with pore sizes ranging from 100 to 500 nm. Successful drug delivery requires that nanoparticles penetrate this barrier and be internalized by cells before being cleared via mucus turnover mechanisms. Depending on the airway area can take minutes to hours.

[0156] An artificial mucus was prepared based on the formulation described by Conte et al. and slightly adopted to exhibiting a viscosity of approximately 250 mPa's at a shear rate of 10 Hz, consistent withDTS Ref: 40574. RNH. P1 10PC values reported in the literature for lung mucus. Mucus penetration was assessed using a transwell setup with a 6.5 mm diameter insert, into which 50 piL of artificial mucus was applied, corresponding to an estimated mucus layer thickness of 1.51 mm (FIG. 8A). FIGs. 8A-8D depict LNP transport through artificial mucus and transfection efficiency in air-liquid interface (ALI). FIG. 8A shows a schematic representation of a transwell mucus penetration assay. FIG. 8B shows graphs depicting the mucus penetration of fresh and spray dried LNPs. FIG. 8C depicts a schematic representation of Calu 3 cells seeded into a transwell. After three days confluence was reached and air lift was performed. After one week polarized and differentiated epithelium was transfected with 160 pmol fresh and spray-dried AF647 -labeled siRNA LNPs 1 . FIG. 8D shows a graph depicting the percentage of transfected cells 6 hours after cells were transfected with fresh and SD LNPs 1 - 4. Blank samples were treated with PBS. Data points indicate mean ± standard deviation (n = 3). One-way ANOVA, ns, > 0.05, *, p < 0.05 **, p < 0.01 , ***, p < 0.001 , ****, p < 0.0001.

[0157] Despite the artificial mucus layer being approximately 100 times thicker than physiological airway mucus, more than 85% of the freshly prepared LNPs were detected in the acceptor compartment within 2 hours (FIG. 8B). The effect of PEG content, which is generally considered to enhance mucus penetration due to its amphiphilic nature and neutral surface charge in nanoparticles <100-200 nm, was not assessed.

[0158] When comparing the mucus penetration of spray-dried and resuspended LNPs, all formulations except LNP 1 (100%) showed markedly reduced penetration after 24 hours, with values of 85% (LNP 2), 72% (LNP 3), and 73% (LNP 4), respectively (FIG. 8B). This decreased penetration can be attributed to the previously described increase in particle size following spray drying, which inhibits larger particles to diffuse through the dense and narrow mucin mesh.

[0159] Conventional two-dimensional (2D) cell cultures often fail to accurately predict in vivo performance. To better mimic the airway epithelial environment, air-liquid interface (ALI) cultures were established using human Calu-3 cells, a lung adenocarcinoma cell line. Under ALI conditions, Calu-3 cells differentiate into a functional epithelial monolayer that, despite being composed of a single cell type, replicates essential characteristics of the human bronchial epithelium, including tight junction formation, mucus secretion, receptor expression, and cytokine production (FIG. 8C). ALI cultures are difficult to transfect. In the case of nanoparticles, transfection challenges can be attributed to the physiologically relevant barrier properties of the model secreting mucus and the reduced availability of proteins required for forming a protein corona that facilitates receptor- mediated endocytosis.

[0160] ALI cultures were transfected for 6 hours with both fresh and spray-dried LNP formulations, and the percentage of transfected cells was subsequently quantified (FIG. 8D). Interestingly, the transfection profile closely mirrored the uptake performance observed in submerged H1299 cells. Among the fresh formulations, LNP 1 (34%) and LNP 3 (32%) demonstrated the highest transfection efficiency, whereas LNP 2 (12%) and LNP 4 (19%) achieved approximately half the efficiency. A similar trend was observed for the spray-DTS Ref: 40574. RNH. P1 10PC dried formulations, with LNP 3 performing best, followed by LNP 2, LNP 4, and LNP 1 , which showed the lowest efficiency. Notably, the decline in transfection efficiency post-spray-drying was less pronounced in ALI cultures than in H1299 cells, with LNP 1 maintaining 17%, LNP 2 69%, LNP 3 48%, and LNP 4 60% efficiency relative to their fresh counterparts. This indicates that cellular uptake in ALI cultures relies less on protein corona-mediated internalization and more on nonspecific uptake mechanisms.

[0161] Furthermore, overall transfection efficiency again correlated with the change in GP-value before and after spray-drying (FIG. 6C), demonstrating that formulations with minimal GP-value alterations exhibit reduced loss in cellular uptake because they exhibited a smaller change in lipid order.

[0162] During spray drying, patisiran-like LNPs undergo membrane surface reorganization, as indicated by an increase in Gp values, a decrease in apparent pKa, visual changes in electron density around the particles and impaired cellular uptake and changed mucus diffusion. These changes closely resemble the absolute values observed in LNPs formulated with 25% DSPC before spray drying indicating similar surface membrane organization.

[0163] These structural changes are driven by excipient interactions intensified during dehydration. Atomistic molecular dynamics simulations revealed the formation of stable hydrogen bond networks between DSPC, PEG, and lactose, which persist upon rehydration and likely hinder membrane fluidity. Importantly, despite retaining overall functionality after spray drying, the behavior of dried LNPs in vitro was strongly influenced by PEG and DSPC content, with reduced DSPC levels leading to more pronounced changes in membrane architecture.Example 7: Protein Corona Analysis in Bronchoalveolar Lavage Fluid

[0164] In this non-limiting example, protein corona formation around lipid nanoparticles is assessed and characterized.

[0165] Changes in lipid packing and surface chemistry can modulate nanoparticle interactions with biological fluids. Given the strong correlation between membrane order, post-drying stability, and uptake efficiency observed in ALI cultures, we next assessed whether such alterations affect LNP identity at the pulmonary interface. To this end, we profiled protein coronas formed in human bronchoalveolar lavage fluid (BALF). Well-known work on SORT LNPs demonstrated that differences in lipid composition can indirectly govern organ-specific delivery by modulating protein corona formation in plasma, where adsorbed proteins act as endogenous targeting ligands. Unlike plasma, BALF presents a distinct biochemical environment that contains not only soluble proteins, but also surfactants, vesicles, nucleic acids, and cellular debris, yet it remains largely unexplored in the context of inhaled nanocarriers.

[0166] To address this, we profiled the protein coronas of fresh and spray-dried LNPs (SD-LNPs) incubated in human BALF, freshly collected from a post-transplant donor under immunosuppressive therapy, and corona-associated proteins were quantified by mass spectrometry.DTS Ref: 40574. RNH. P1 10PC

[0167] Human BALF samples from post-lung transplantation (post-LTx) patients were obtained and stored at -80°C until analyzed. Fresh LNP samples (50 pl, 1 :2 with HPW) and reconstituted SD-LNPs (5 mg / ml lactose) were incubated with human BALF for 15 min at 37°C at a volumetric ratio of 1 : 1 . After incubation, samples were loaded onto 300 pl sucrose cushion (0.7 M) and centrifuged at 15,300 G at 4°C for 1.5 hours. Afterwards supernatant was removed and washed with 1 x PBS and centrifuged at 15,300 G at 4°C for 5 min. This washing step was performed twice more. LNPs and corona were kept dry at -20°C upon MS analysis. A positive control of PBS with BALF was treated similarly.

[0168] To each sample was added 25 pl of 50 mM HEPES pH 8 containing 0.04% n-dodecyl-|3-D- maltoside, followed by incubation for 1 h at 60°C with shaking. After brief centrifugation, 25 pl of enzyme solution was added (50 mM Hepes pH 8, 6 ng / pl trypsin, 4 ng / pl LysC, 2 mM CaCL) followed by overnight incubation at 37°C. Samples were acidified by addition of 5 pl of 1 % trifluoroacetic acid and stored at -20°C.

[0169] Samples were measured on a TimsTOF Ultra2 mass spectrometer (Bruker) online coupled to an EvoSep One HPLC (EvoSep). 20% of each sample was loaded on a EvoTip Pure (EvoSep) using the manufacturer’s instructions, followed by automatic injection of the sample and separation on a PepSep C18 column (15cmx75pm, 1.9pm; Bruker) using the 40SPD whisper zoom method at 50°C column oven temperature. The DIA-Pasef method covered a mass range from 300 to 1 ,250 m / z and a mobility range from 0.64 to 1 .30 1 / ko, with a ramp and accumulation time of each 100ms. ICC 2.0 was switched on with 95% target TIC. Precursor peptides were isolated using 27 variable MS / MS windows and 1 1 MS / MS ramps, resulting in a cycle time of 1 .28 sec. Collision energy for 0.6 1 / ko was set to 20 and for 1 .6 1 / ko to 59.

[0170] Generated measurement files were quantitatively analyzed in the Spectronaut 19 software (Biognosis) in directDIA using BGS settings, disabling carbamidomethylation as fixed modification, disabling crossrun normalization, allowing for quantification on only proteotypic peptides and applying the QUANT2.0 label-free quantification method allowing for a mean TOP3 protein group quantity calculation. Searches were performed using the canonical Swissprot Human (20432 sequences) database. TOP3 protein group quantities were imported in Perseus 2.0.1 1 software (MPI Martinsried). Abundances were Iog2 transformed and the data was filtered for at least 5 valid values per protein in all samples. Missing values were imputed from normal distribution using default settings. The average quantities per protein per group were calculated and used for ratio calculations and group comparisons applying a Student’s t-test.

[0171] Principal component analysis (PCA) revealed clear clustering by both LNP composition (LNP 3 vs. LNP 4) and processing status (fresh vs. spray dried, FIG. 9A), indicating that spray drying induces distinct shifts in corona profiles. These changes aligned with formulation-dependent differences in surface order (Gp-value), pKa, and cellular uptake.

[0172] Across all conditions, over 6,200 proteins were identified. To isolate corona-specific enrichments, protein abundances were benchmarked against a BALF-only control processed in parallel. The topDTS Ref: 40574. RNH. P1 10PC20 enriched proteins (FIG. 9B) exhibited divergent patterns across formulations and processing states. For example, NADH-ubiquinone oxidoreductase chain 6 appeared prominently on fresh LNPs, whereas TLC domaincontaining protein 4, a membrane-associated factor linked to lipid homeostasis, was enriched only on spray dried samples. Protein kinase C alpha, a DAG-sensitive signaling enzyme implicated in inflammation and proliferation, showed selective recruitment to DSPC-rich LNPs regardless of processing. While several proteins followed clear formulation-dependent trends, others, e.g., DNA repair protein XRCC1 or transcription factor IIH subunit XPD, exhibited variable, nonspecific enrichment.

[0173] To identify statistically significant shifts, pairwise comparisons were performed. SD-LNPs recruited broader and more distinct protein subsets relative to their fresh counterparts. This effect was more pronounced for LNP 3 than LNP 4, mirroring their differential changes in membrane order and cellular uptake. Interestingly, while fresh LNP 4 displayed the most diverse protein enrichment, its spray dried counterpart showed reduced recruitment, whereas SD-LNP 3 gained new protein interactions. These findings indicate that both lipid composition and physical processing shape the molecular fingerprint of LNPs at the lung interface.

[0174] All measurements were normalized to a shared BALF control, eliminating artifacts arising from nonspecific background binding. The observation that corona composition tracks with physicochemical parameters, particularly membrane order, supports the broader hypothesis that post-processing stability governs biological performance.

[0175] Any titles or subheadings used herein are for organizational purposes and should not be used to limit the scope of embodiments disclosed herein. All literature and similar materials cited in this application, including but not limited to, patents, patent applications, articles, books, treatises, and internet web pages are expressly incorporated by reference in their entirety for any purpose, including the disclosures specifically referenced herein. When definitions of terms in incorporated references appear to differ from the definitions provided in the present teachings, the definition provided in the present teachings shall control. It will be appreciated that there is an implied “about” prior to the temperatures, concentrations, times, etc. discussed in the present teachings, such that slight and insubstantial deviations are within the scope of the present teachings herein.

[0176] Although embodiments described herein have been disclosed in the context of certain embodiments and examples, the present disclosure extends beyond the specifically disclosed embodiments to other alternative embodiments and / or uses of the disclosure and obvious modifications and equivalents thereof. It is also contemplated that various combinations or sub-combinations of the specific features and aspects of the embodiments may be made and still fall within the scope of the disclosure. It should be understood that various features and aspects of the disclosed embodiments can be combined with, or substituted for, one another in order to form varying modes or embodiments. Thus, it is intended that the scope of the present disclosure should not be limited by the particular disclosed embodiments described above.DTS Ref: 40574. RNH. P1 10PC

[0177] The terminology used in the description presented herein is not intended to be interpreted in any limited or restrictive manner. Rather, the terminology is simply being utilized in conjunction with a detailed description of embodiments of the systems, methods, and related components. Furthermore, embodiments may include several novel features, no single one of which is solely responsible for its desirable attributes or is believed to be essential to practicing the embodiments herein described.

[0178] Changes and modifications in the embodiments described herein can be carried out without departing from the principles of the present disclosure. Each of the disclosed aspects and examples of the present disclosure may be considered individually or in combination with other aspects, examples, and variations of the disclosure. In addition, unless otherwise specified, none of the steps of the methods of the present disclosure are confined to any particular order of performance.

[0179] While the compositions and methods described herein may be susceptible to various modifications and alternative forms, specific examples thereof have been shown in the drawings and are herein described in detail. Embodiments are not to be limited to the particular forms or methods disclosed, but rather intended is to cover modifications, equivalents, and alternatives falling within the spirit and scope of the various examples and embodiments described herein and / or in the appended claims. Further, the disclosure herein of any particular feature, aspect, method, property, characteristic, quality, attribute, element, or the like in connection with an example can be used in all other examples set forth herein. Any methods disclosed herein need not be performed in the order recited. The use of sequential, or time-ordered language, such as “then,” “next,” “after,” “subsequently,” and the like, unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to facilitate the flow of the text and is not intended to limit the sequence of operations performed. Thus, some examples may be performed using the sequence of operations described herein, while other examples may be performed following a different sequence of operations.

[0180] Conditional language used herein, such as, among others, “can,” “might,” “may,” “e.g.,” and the like, unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that some examples include, while other examples do not include, certain features, elements, and / or states. Thus, such conditional language is not generally intended to imply that features, elements, blocks, and / or states are in any way required for one or more examples or that one or more examples necessarily include logic for deciding, with or without author input or prompting, whether these features, elements and / or states are included or are to be performed in any particular example.

[0181] Where compositions or methods “comprise” or “include” (the two being interchangeable) certain features or steps, such compositions or methods may also “consist essentially of such features or steps if identified as such in the claims. Where compositions or methods “comprise” or “include” (the two being interchangeable) certain features or steps, such compositions or methods may also “consist” of such features or steps if identified as such in the claims.DTS Ref: 40574. RNH. P1 10PC

[0182] The methods disclosed herein may include certain actions taken by a practitioner; however, the methods can also include any user (which may not be a practitioner) or third-party instruction of those actions, either expressly or by implication. For example, actions such as “administering a composition” include “instructing administration of a composition.”

[0183] The ranges disclosed herein also encompass any and all overlap, sub-ranges, and combinations thereof. Language such as “up to,” “at least,” “greater than,” “less than,” “between,” and the like includes the number recited. Numbers preceded by a term such as “about” or “approximately” include the recited numbers and should be interpreted based on the circumstances (e.g., as accurate as reasonable under the circumstances, for example ±5%, ±10%, ±15%, etc.). For example, “about 4 inches” includes “4 inches.” Phrases preceded by a term such as “substantially” include the recited phrase and should be interpreted based on the circumstances (e.g., as much as reasonably possible under the circumstances). Forexample, “substantially linear” includes “linear.” Unless stated otherwise, all measurements are at standard conditions including temperature and pressure. The phrase “at least one of” is intended to require at least one item from the subsequent listing, not one type of each item from each item in the subsequent listing. For example, “at least one of A, B, and C” can include A; B; C; A and B; A and C; B and C; or A, B, and C.

[0184] All numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification are to be understood as being modified in all instances by the term ‘about.’ Accordingly, unless indicated to the contrary, the numerical parameters set forth herein are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of any claims in any application claiming priority to the present application, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0185] Unless otherwise defined, all terms (including technical and scientific terms) are to be given their ordinary and customary meaning, and are not to be limited to a special or customized meaning unless expressly so defined herein. It should be noted that the use of particular terminology when describing certain features or aspects of the disclosure should not be taken to imply that the terminology is being re-defined herein to be restricted to include any specific characteristics of the features or aspects of the disclosure with which that terminology is associated. Terms and phrases used in this application, and variations thereof, especially in the appended claims, unless otherwise expressly stated, should be construed as open ended as opposed to limiting. As examples of the foregoing, the term ‘including’ should be read to mean ‘including, without limitation,’ ‘including but not limited to,’ or the like; the term ‘comprising’ as used herein is synonymous with ‘including,’ ‘containing,’ or ‘characterized by,’ and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; the term ‘having’ should be interpreted as ‘having at least;’ the term ‘includes’ should be interpreted as ‘includes but is not limited to;’ the term ‘example’ is used to provide example instances of the item in discussion,DTS Ref: 40574. RNH. P1 10PC not an exhaustive or limiting list thereof; adjectives such as ‘known’, ‘normal’, ‘standard’, and terms of similar meaning should not be construed as limiting the item described to a given time period or to an item available as of a given time, but instead should be read to encompass known, normal, or standard technologies that may be available or known now or at any time in the future; and use of terms like ‘preferably,’ ‘preferred,’ ‘desired,’ or ‘desirable,’ and words of similar meaning should not be understood as implying that certain features are critical, essential, oreven important to the structure or function of the invention, but instead as merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment of the invention. Likewise, a group of items linked with the conjunction ‘and’ should not be read as requiring that each and every one of those items be present in the grouping, but rather should be read as 'and / or' unless expressly stated otherwise. Similarly, a group of items linked with the conjunction ‘or1should not be read as requiring mutual exclusivity among that group, but rather should be read as ‘and / or1unless expressly stated otherwise.

[0186] As used in the claims below and throughout this disclosure, by the phrase “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0187] With respect to the use of substantially any plural and / or singular terms herein, these shall be translated from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / pl u ral permutations may be expressly set forth herein for sake of clarity. The indefinite article “a” or “an” does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0188] Furthermore, although the foregoing has been described in some detail by way of illustrations and examples for purposes of clarity and understanding, changes and modifications may be practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention to the specific embodiments and examples described herein, but rather to also cover modifications and alternatives coming with the true scope and spirit of the inventions described herein.

Claims

DTS Ref: 40574. RNH. P110PCWHAT IS CLAIMED IS:1 . A dry powder composition comprising: a lipid nanoparticle; a corona excipient; and a nucleic acid, wherein the lipid nanoparticle at least partially encapsulates the nucleic acid and wherein the corona excipient forms a matrix that at least partially covers the outside of the lipid nanoparticle, wherein the lipid nanoparticle and the corona excipient are configured to modulate the lipid nanoparticle interaction with a biological environment for tunable delivery of the nucleic acid.

2. The composition of claim 1 , wherein lipid nanoparticle comprises an ionizable amino lipid, cholesterol, DSPC, and PEG-DMG 2000.

3. The composition of any one of claims 1 -2, wherein the composition has a residual moisture of 10% or less.

4. The composition of any one of claims 1 -3, wherein the composition is formulated for inhalable administration, enteral administration, transdermal administration, or parenteral administration.

5. The composition of any one of claims 1 -4, wherein the composition is formulated for respiratory tract administration.

6. The composition of any one of claims 1 -5, wherein the corona excipient facilitates cellular internalization of the lipid nanoparticle.

7. The composition of any one of claims 1 -6, wherein the corona excipient increases transfection efficiency of the lipid nanoparticle.

8. The composition of any one of claims 1 -7, wherein the corona excipient prevents or reduces degradation or loss of the therapeutic nucleic acid.

9. The composition of any one of claims 1 -8, wherein the nucleic acid is mRNA, circRNA, saRNA, antisense oligonucleotide, miRNA, shRNA, IncRNA, dsRNA, tracrRNA, siRNA, aptamer, gRNA, piRNA, pDNA, or an RNA conjugate or a combination thereof.

10. The composition of any one of claims 1 -9, wherein the corona excipient comprises sugars, monosaccharides, disaccharides, oligosaccharides, polysaccharides, sugar alcohols, alcohols, amino acids, dipeptides, oligopeptides, polypeptides, proteins, esters, ethers, amides, amines, sulphates, thiols, urethanes, phosphoesters, phosphazenes, surfactants, lipids, stearates, polymers, , salts, buffer, citric acid (monohydrate), (anhydrous) trisodium citrate, ascorbic acid, calcium carbonate, calcium chloride, EDTATE disodium, potassium chloride, potassium carbonate, potassium bicarbonite, sodium bicarbonate, sodium carbonate, sodium acetate, acetic acid, sodium bisulfate, sodium chloride, sodium hydroxide, sodium metabisulfate, sodium sulfateDTS Ref: 40574. RNH. P110PC(anhydrous), sodium phosphate, sulfuric acid, trisodium citrate dihydrate, disodium hydrogen phosphate, potassium dihydrogen phosphate, phosphoric acid, or a combination, derivative, conjugate, or polymer thereof.1 1. A dry powder lipid nanoparticle composition, comprising: lipid nanoparticles comprising a membrane; nucleic acid, wherein the nucleic acid is encapsulated within the one or more lipid nanoparticles; and a matrix comprising a excipient, wherein the matrix at least partially covers the lipid nanoparticles; wherein the matrix is configured to preserve the lipid nanoparticles during drying and configured to modulate biological identity of the lipid nanoparticle, wherein the composition is in a powder form configured for administration.

12. The composition of claim 11 , wherein the excipient facilitates cellular internalization of the lipid nanoparticles.

13. The composition of any one of claims 1 1 -12, wherein the excipient increases transfection efficiency of the lipid nanoparticles.

14. The composition of any one of claims 1 1 -13, wherein the excipient prevents or reduces degradation or loss of the nucleic acid.

15. The composition of any one of claims 1 1 -14, wherein the powder form is formulated for inhalable administration, enteral administration, transdermal administration, or parenteral administration.

16. The composition of any one of claims 1 1 -15, wherein the powder is configured for redispersion.

17. The composition of claim 16, wherein the excipient is configured to remain at least partially associated with the lipid nanoparticles following redispersion.

18. The composition of any one of claims 1 1 -17, wherein the nucleic acid is mRNA, circRNA, saRNA, antisense oligonucleotide, miRNA, shRNA, IncRNA, dsRNA, tracrRNA, siRNA, aptamer, gRNA, piRNA, pDNA, or an RNA conjugate, or a combination thereof.

19. The composition of any one of claims 1 1 -18, wherein the excipient(s) comprises sugars, monosaccharides, disaccharides, oligosaccharides, polysaccharides, , sugar alcohols, alcohols, amino acids, dipeptides, oligopeptides, polypeptides, proteins, esters, ethers, amides, amines, sulphates, thiols, urethanes, phosphoesters, phosphazenes, surfactants, lipids, stearates, or polymers, salts, buffer, citric acid (monohydrate), (anhydrous) trisodium citrate, ascorbic acid, calcium carbonate, calcium chloride, EDTATE disodium, potassium chloride, potassium carbonate, potassium bicarbonite, sodium bicarbonate, sodium carbonate, sodium acetate, acetic acid, sodium bisulfate, sodium chloride, sodium hydroxide, sodium metabisulfate, sodium sulfate (anhydrous), sodium phosphate, sulfuric acid, trisodium citrate dihydrate, disodium hydrogen phosphate, potassium dihydrogen phosphate, phosphoric acid or a combination, derivative, conjugate, or polymer thereof.DTS Ref: 40574. RNH. P110PC20. The composition of any one of claims 1 1 -19, wherein the membrane comprises an ionizable amino lipid, cholesterol, DSPC, and PEG-DMG 2000.21 . The composition of any one of claims 1 1 -20, wherein the powder is configured for administration with an inhaler device.

22. The composition of claim 11 , wherein the composition has a residual moisture of 10% or less.

23. A dry powder lipid nanoparticle composition, comprising: a membrane comprising an ionizable amino lipidT, cholesterol, DSPC, and PEG-DMG 2000; a nucleic acid comprising siRNA; and an excipient; wherein the composition is formulated as a dry powder.

24. The composition of any one of claims 1 1 -23, wherein the lipid nanoparticles deliver the nucleic acid to lung cells and / or cells within the respiratory tract.

25. The composition of any one of claims 1 1 -24, wherein the lipid nanoparticles comprise: an ionizable amino lipid in an amount of about 50%; cholesterol in an amount ranging from about 20% to about 40%;DSPC in an amount ranging from about 10% to about 25%; andPEG-DMG 2000 in an amount ranging from about 0.5% to about 1 .5%.

26. The composition of any one of claims 1 1 -25, wherein the comprises lactose.

27. The composition of any one of claims 11 -26, wherein the nucleic acid is an siRNA that targets the mRNA encoding for the alarmin TSLP, a cytokine or a viral protein.

28. A method of delivering a dry powder lipid nanoparticle composition to a respiratory tract of a subject, the method comprising: administering the composition of any one of claims 1 -27 to the subject.

29. The method of claim 28, wherein the excipient facilitates cellular uptake of the lipid nanoparticle to a lung cell and / or a cell within the respiratory tract of the subject.

30. The method of any one of claims 28-29, wherein the excipient facilitates transport through mucus layers of a lung of the subject.31 . The method of any one of claims 28-30, wherein the excipient increases transfection efficiency of the nucleic acid into a lung cell and / or a cell within the respiratory tract of the subject.

32. The method of any one of claims 28-31 , wherein the excipient prevents or reduces degradation or loss of the nucleic acid.

33. The method of any one of claims 28-32, wherein the nucleic acid is delivered in an effective amount.

34. A method of treating a respiratory tract disease in a subject, the method comprising administering to a lung of the subject an inhalable dry powder lipid nanoparticle composition of any one of claims 1 -27.DTS Ref: 40574. RNH. P110PC35. The method of claim 34, wherein the administering is performed using an inhaler device.

36. The method of any one of claims 34-35, wherein the respiratory tract disease is asthma, chronic obstructive pulmonary disease (COPD), a viral infection, or idiopathic pulmonary fibrosis.

37. The method of any one of claims 34-36, wherein the nucleic acid is an siRNA against a target.

38. The method of claim 37, wherein the target is a cytokine or a viral gene or a nucleic acid sequence associated with pulmonary inflammation, epithelial barrier dysfunction, or immune dysregulation.

39. Use of the composition of any one of claims 1 -27 for treatment of a respiratory tract disease.

40. The use of claim 39, wherein the respiratory tract disease comprises asthma, chronic obstructive pulmonary disease (COPD), a viral infection, or idiopathic pulmonary fibrosis.41 . The use of any one of claims 39-40, wherein the nucleic acid is an siRNA against a target.

42. The use of claim 41 , wherein the target is a mRNA of TSLP, a cytokine or a viral gene.

43. A method of making the composition of any one of claims 1 -27, comprising: mixing lipid components with nucleic acid to form lipid nanoparticles, the lipid nanoparticles encapsulating the nucleic acid; mixing the lipid nanoparticles with the excipient to form a particulate comprising lipid nanoparticles and the excipient; and spray drying the particulate to form a dry powder comprising a lipid nanoparticle at least partially covered in a matrix comprising an excipient.

44. The method of claim 43, wherein the excipient prevents or reduces degradation of the lipid nanoparticles following spray-draying of the particulate.

45. A kit comprising an inhalable dry powder lipid nanoparticle composition of any one of claims 1 -27 and packaging.

46. The kit of claim 45, wherein the packaging comprises a capsule, a blister, or a reservoir.

47. The kit of any one of claims 45-46, wherein the packaging is multidose or single dose packaging.

48. The kit of any one of claims 45-47, further comprising a nebulizer or an inhaler device.

49. A composition comprising one or more particulates, wherein the particulate comprises one or more corona excipients, an encapsulation structure, and at least one pharmaceutically active ingredient, wherein the encapsulation structure at least partially encapsulates the pharmaceutically active ingredient and wherein the one or more corona excipients at least partially covers the outside of the encapsulation structure.

50. The composition according to claim 49, wherein the particulate is a nanoparticle.

51. The composition according to claim 49 or claim 50, wherein the encapsulation structure is a lipid structure and / or polymer structure.DTS Ref: 40574. RNH. P110PC52. The composition according to any one of claims 49-51 , wherein the at least one pharmaceutically active ingredient is a nucleic acid.

53. The composition according to claim 52, wherein the nucleic acid is an RNA or DNA.

54. The composition according to claim 53, wherein the nucleic acid is a mRNA, circRNA, saRNA, antisense oligonucleotide, miRNA, shRNA, IncRNA, dsRNA, tracrRNA, siRNA, aptamer, gRNA, piRNA, pDNA, or an RNA conjugate, or a combination thereof.

55. The composition according to any one of claims 49-54, wherein the particulate is in a dried, partially dried, or hydrated state.

56. The composition according to any one of claims 49-55, wherein the particulate was at least once during its lifetime in a dried or partially dried state.

57. The composition according to claim 51 , wherein the lipids or polymers of the encapsulation structure are selected to control the interaction with the one or more corona excipients.

58. The composition according to claim 57, wherein the lipids or polymers are configured such that the colloids electron density is different when at least partially covered by one or more excipients when compared to the not-covered colloid.

59. The composition according to claim 57 or 58, wherein the lipids are selected from at least one of a cationic (ionizable) lipid, one or more helper lipids, and / or a stealth lipid.

60. The composition according to claim 59, wherein at least two helper lipids are used and one of the helper lipids is a phospholipid, ester lipid, lyso-phospholipid, or a glycerol lipid and the second is a sterol.61 . The composition according to claim 59 or claim 60, wherein the cationic (ionizable) lipid is selected from the group consisting of C12-200, DOTAP (1 ,2-dioleyl-3-trimethytammonium propane), DODAP (1 ,2-dioleyl- 3-dimethylammonium propane), DOTMA (1 ,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA, DLin- KC2-DMA, HGT4003, cKK-E12, ICE, DLin-MC3, 7C1 , ALC-0315, SM-102, CL-1 , 3060110, OF-02, 7C1 , L319, A9, 93O17S, Lipid C24, 1014, Lipid15, Lipid AX4, Lipid A6, BAMEA-O16B, 98N12-5, 4A3-SC8, 5A2-SC8 and combinations thereof.

62. The composition according to any one of claims 49-61 , wherein the one or more corona excipients is a pharmaceutically acceptable excipient.

63. The composition according to claim 62, wherein the selection of excipients has an effect on the efficiency of the cellular internalization of the particulate system.

64. The composition according to claim 62 or claim 63, wherein the selection of excipients can be used to adapt the penetration of the particulate system through the natural barriers of the human body.

65. The composition according to one of claims 62 to 64, wherein the at least one pharmaceutically acceptable excipient is selected from the group consisting of sugars, monosaccharides, disaccharides, oligosaccharides, polysaccharides, sugar alcohols, alcohols, amino acids, dipeptides, oligopeptides, polypeptides,DTS Ref: 40574. RNH. P1 10PC proteins, esters, ethers, amides, amines, sulphates, thiols, urethanes, phosphoesters, phosphazenes, surfactants, lipids, stearates, salts, buffer, citric acid (monohydrate), (anhydrous) trisodium citrate, ascorbic acid, calcium carbonate, calcium chloride, EDTATE disodium, potassium chloride, potassium carbonate, potassium bicarbonite, sodium bicarbonate, sodium carbonate, sodium acetate, acetic acid, sodium bisulfate, sodium chloride, sodium hydroxide, sodium metabisulfate, sodium sulfate (anhydrous), sodium phosphate, sulfuric acid, trisodium citrate dihydrate, disodium hydrogen phosphate, potassium dihydrogen phosphate, phosphoric acid, or a combination, derivative, polymer, or conjugate thereof.

66. The composition according to claim 65, wherein the polymers are selected from Chitosan, PVP, PVP / VA, Methacrylic polymers, PLA, PLGA, HPMC, PCL, PEG, Polyvinyl alcohol, Polyvinyl acetate, HPMCAS, HPMCP, Methacrylic acid-ethyl acrylate copolymer, Poloxamer, or a derivative thereof.

67. The composition according to any one of claims 49-66, wherein the one or more corona excipients covers at least partially the outside of the encapsulation structure, preferably forming a shell structure for and / or around the encapsulation structure.

68. The composition according to any one of claims 49-67, wherein the plurality of particles is present in form of a dry powder.

69. The composition according to claim 68, wherein the dry powder has a residual moisture of less than 10%, preferably less than 8%, most preferably less than 5%.

70. The composition according to claim 68 or claim 69, wherein the dry powder can at least partially be redispersed.71 . The composition according to claim 70, wherein the particulates, after redispersion, remain at least partially associated with the one or more excipients.

72. The composition according to one of claims 68 to 71 , wherein the composition is formulated for use as a pharmaceutical dosage form, especially for pulmonary delivery.

73. The composition according to one of claims 68 to 72, wherein the composition is formulated for use to adapt the penetration of the particulate system through the natural barriers of the lung.

74. The composition according to one of claims 68 to 73, wherein the composition is formulated such that the pharmaceutical dosage form can be administered using dry powder inhaler devices.

75. The composition according to any one of claims 49-74, wherein the plurality of particles is present in form of a colloidal suspension.

76. The composition according to any one of claims 49-75, wherein the composition is formulated for use as a pharmaceutical dosage form, especially for pulmonary delivery.

77. The composition according to one of claims 75 or claim 76, wherein the composition is formulated for use to adapt the penetration of the particulate system through the natural barriers of the lung.DTS Ref: 40574. RNH. P1 10PC78. The composition according to claim 77, wherein the composition is formulated such that the pharmaceutical dosage form can be administered using a nebulizer.

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