TW202543583A - Materials and methods for encapsulating therapeutics in lipid nanoparticles - Google Patents

Abstract

The present disclosure provides methods for preparing lipid nanoparticle formulations that encompass nucleic acid therapeutics (e.g., mRNA). The lipid nanoparticles described herein include ionizable lipids, neutral lipids, charged lipids, steroids (including for example, all sterols and/or their analogs), and/or polymer conjugated lipids. In some instances, the lipid nanoparticles are used to formulate and deliver messenger RNA. Kits and methods for using lipid nanoparticles following formulation for treatment of various diseases or conditions, such as those caused by infectious entities and/or insufficiency of a protein, are also provided.

Description

Materials and methods for encapsulating therapeutic agents in lipid nanoparticles

This disclosure relates generally to a novel method for preparing lipid nanoparticles (LNPs) coated with nucleic acid molecules (e.g., oligonucleotides or polynucleotides). The lipid nanoparticles can be prepared using lipid components, such as ionizable lipids, neutral lipids, cholesterol, and lipids bound to polymers. Lipid nanoparticles coated with nucleic acid molecules facilitate delivery both in vitro and intracellularly.

Numerous challenges exist related to the storage and final delivery of nucleic acids to influence desired reactions in biological systems. Nucleic acid-based therapies hold immense potential, but more efficient methods for their preparation, storage, and delivery are still needed to realize this potential. It has been observed that lipid nanoparticles containing nucleic acids may degrade over time during manufacturing and storage. Although methods exist for the formulation of nucleic acid and lipid nanoparticles, these methods involve complex and/or cumbersome techniques (e.g., sample heating, complex fluid mixing equipment and solutions, buffer exchange via dialysis or TFF, and various analytical requirements to ensure the formulation meets specifications), which necessitates long-term exposure of nucleic acids to adverse conditions and makes them unsuitable for use in typical processing environments.

Therefore, there is a need for simplified techniques and conditions to prepare nucleic acids encapsulated in lipid nanoparticles, suitable for bedside preparation of formulations or with less invasiveness when handling the effective nucleic acid load during manufacturing. This disclosure provides these and related advantages.

In summary, this disclosure provides a method for preparing lipid nanoparticle formulations encapsulating nucleic acid therapeutics (e.g., mRNA) under mild conditions. The lipid nanoparticles described herein include ionizable lipids, neutral lipids, charged lipids, steroids (including, for example, all steroids) and/or their analogues and/or bound polymers. In some cases, lipid nanoparticles are used to formulate and deliver nucleic acids such as antisense RNA and/or messenger RNA and/or guide RNA. Methods for treating various diseases or conditions using formulated lipid nanoparticles are also provided, such as those caused by infectious agents and/or protein deficiencies and/or those with a genetic basis.

We also offer pharmaceutical compositions (such as vaccines or gene-editing products) that contain formulated lipid nanoparticles encapsulating nucleic acid molecules. These compositions can be used to deliver nucleic acid molecules and treat a variety of diseases and conditions.

In other embodiments, this disclosure provides a method for administering to a patient in need a composition comprising nucleic acids encapsulated in lipid nanoparticles prepared according to the methods described herein, the method comprising delivering the composition to the patient. Such methods can be used to induce protein expression in an individual, for example, to express antigens for vaccination purposes or to express gene-editing proteins for the correction of genetic disorders.

These and other forms of this disclosure will become apparent upon reference to the following embodiments.

In the following description, certain specific details are set forth to provide a comprehensive understanding of the various embodiments of this disclosure. However, those skilled in the art will understand that the embodiments of this disclosure can be practiced without using these details.

This disclosure is partly based on the discovery that nucleic acid molecules can be loaded into lipid nanoparticles in aqueous solution without any auxiliary agents (such as disruptors, ethanol, heating, etc.). The applicant unexpectedly discovered that aqueous solutions containing "empty" lipid nanoparticles can mix with nucleic acid molecules (e.g., in solution or in a dry state), and the nucleic acid molecules are subsequently encapsulated in the lipid nanoparticles, resulting in particles with surprisingly desirable characteristics (e.g., particle size, polydispersity index (PDI), encapsulation efficiency, loading capacity (RNA/lipid [wt/µm]), etc.). Surprisingly, without any auxiliary methods, when the pH of the solution is adjusted to a pH slightly lower than the pKa of the ionizable lipids that constitute LNPs, a neutral pH "empty" LNP solution (e.g. in phosphate-buffered saline or "PBS") can achieve ideal coating efficiency and yield LNP products (i.e. LNPs effectively coated with nucleic acid-based payloads).

The methods and materials disclosed herein offer advantages in the preparation of lipid nanoparticles (LNPs) for the in vivo delivery of active agents or therapeutics, such as nucleic acids, into mammalian cells. Some embodiments disclosed herein provide methods for the preparation of nucleic acid-lipid nanoparticle compositions via simple procedures that can be performed under mild conditions with minimal equipment.

In some embodiments, this disclosure provides methods for preparing solutions capable of forming modified LNPs for delivering mRNA and/or other polynucleotides in vitro, in vivo, and in vivo. In some embodiments, these modified LNPs can be used to express proteins encoded by RNA and/or DNA. In other embodiments, these modified LNPs can upregulate endogenous protein expression by delivering miRNA inhibitors targeting a specific miRNA or a set of miRNAs that regulate a target mRNA or several mRNAs. In other embodiments, these LNPs can be used to downregulate (e.g., silence) the protein and/or mRNA levels of a target gene. In some other embodiments, LNPs can also be used to deliver mRNA and plasmids for expressing transfected genes. In other embodiments, LNPs can be used to induce pharmacological effects produced by protein expression, such as increasing red blood cell production by delivering suitable erythropoietin mRNA, or preventing infection by delivering mRNA encoding suitable antibodies.

In some embodiments, LNPs can be used for gene editing, epigenome editing, cancer vaccines, Cart-T, gene insertion, leader editing, or combinations thereof.

LNPs and combinations thereof containing the LNPs disclosed herein can be used for a variety of purposes, including the delivery of coated or associated (e.g., complexed) therapeutic agents, such as nucleic acids, into cells, either in vitro, in vivo, or intracellularly. Therefore, embodiments of this disclosure provide a method for treating or preventing diseases or conditions in an individual by bringing the individual into contact (e.g., via injection) with an LNP coated or associated with a suitable therapeutic agent or nucleic acid.

As described herein, embodiments of the LNPs disclosed herein are particularly useful for delivering nucleic acids, including, for example, mRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA repressors (antagomir/antimir), messenger RNA-interference complementary RNA (micRNA), DNA, multivalent RNA, Dicer acceptor RNA, siRNA, circular RNA, self-amplifying RNA, adaptors, terminally blocked DNA, complementary DNA (cDNA), etc. Therefore, the LNPs disclosed herein and compositions containing LNPs can be used to induce the expression of desired proteins in vitro, in vivo, and in vivo by contacting cells with the LNPs described herein, wherein the LNPs coat or are associated with nucleic acids (e.g., messenger RNA or plasmids encoding the desired protein) that have been expressed to produce the desired protein. Alternatively, the LNPs disclosed herein and combinations thereof can be used to reduce the expression of target genes and proteins in vitro and in vivo by bringing cells into contact with the LNPs described herein, wherein the LNPs coat or are associated with nucleic acids that reduce the expression of target genes (e.g., antisense oligonucleotides or small interfering RNAs (siRNAs)). The LNPs disclosed herein and combinations thereof can also be used, alone or in combination, to co-deliver different nucleic acids (e.g., mRNA and plassomal DNA), such as to provide the desired effect of co-localization of different nucleic acids (e.g., mRNA encoding suitable gene-modifying enzymes and DNA fragments for incorporation into the host genome).

The nucleic acids used in this disclosure can be prepared using any available technology. For mRNA, the main preparation method (but not limited to) is enzymatic synthesis (also known as in vivo extratranscription), which is currently the most efficient method for producing long sequence-specific mRNA. In vivo extratranscription describes a process for synthesizing RNA molecules from an engineered DNA template, which is formed by linking an upstream phage promoter sequence (e.g., including, but not limited to, promoter sequences from T7, T3, and SP6 E. coli phages) with a downstream sequence encoding the gene of interest. Template DNA can be prepared from multiple sources using appropriate techniques known in this field for in vitro transcription, including (but not limited to) plasmid DNA and polymerase-linked amplification (see Linpinsel, JL and Conn, GL, General protocols for preparation of plasmid DNA template, and Bowman, JC, Azizi, B., Lenz, TK, Ray, P. and Williams, LD RNA in vitro transcription and RNA purification by denaturing PAGE, Recombinant and in vitro RNA syntheses Methods v. 941 Conn GL (ed.), New York, NY Humana Press, 2012).

In the presence of appropriate RNA polymerase and adenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates (rNTPs), RNA is transcribed in vivo using a linearized DNA template. The transcription conditions support polymerase activity while minimizing the potential degradation of the resulting mRNA transcript. In vitro transcription can be performed using various commercially available kits, including (but not limited to) the RiboMax large-scale RNA production system (Promega), the MegaScript transcription kit (Life Technologies), and commercially available reagents, including RNA polymerase and rNTPs. Methods for in vitro mRNA transcription are well known in this technique. (See, for example, Losick, R., 1972, In vitro transcription, Ann Rev Biochem v.41 409-46; Kamakaka, RT and Kraus, WL 2001. In Vitro Transcription. Current Protocols in Cell Biology. 2:11.6:11.6.1-11.6.17; Beckert, B. and Masquida, B., (2010) Synthesis of RNA by In Vitro Transcription in RNA in Methods in Molecular Biology v. 703 (Neilson, H. ed.), New York, NY Humana Press, 2010; Brunelle, JL and Green, R., 2013, Chapter Five - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology v. 530, 101-114; all of which are incorporated herein by reference.)

The desired in vitro transcribed mRNA is then purified from non-desired components of transcription or related reactions (including unincorporated rNTPs, proteases, salts, short RNA oligonucleotides, etc.). Techniques for isolating mRNA transcripts are well known in this field. Well-known procedures include phenol/chloroform extraction or precipitation with alcohols (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride. Non-limiting examples of additional purification procedures include size exclusion chromatography (Lukavsky, PJ and Puglisi, JD, 2004, Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides, RNA v.10, 889-893), silica-based affinity chromatography, and polyacrylamide gel electrophoresis (Bowman, JC, Azizi, B., Lenz, TK, Ray, P. and Williams, LD RNA in vitro transcription and RNA purification by denaturing PAGE, Recombinant and in vitro RNA syntheses Methods v. 941 Conn GL (ed.), New York, NY Humana Press, 2012). Purification can be performed using various commercially available kits, including (but not limited to) the SV total separation system (Promega) and the in vivo extracellular transcription cleansing and concentration kit (Norgen Biotek).

Furthermore, while reverse transcription produces a large amount of mRNA, the product may contain several aberrant RNA impurities associated with unwanted polymerase activity, which may need to be removed from full-length mRNA preparations. These impurities include short RNAs resulting from failed transcription initiation; and double-stranded RNAs (dsRNAs) generated by RNA-dependent RNA polymerase activity; which undergo RNA-guided transcription from the RNA template and self-complementary 3' extension. It has been demonstrated that these contaminants with dsRNA structures can induce unwanted immunostimulatory activity by interacting with various innate immune sensors in eukaryotic cells, which function to recognize specific nucleic acid structures and induce strong immune responses. This, in turn, can significantly reduce mRNA translation due to decreased protein synthesis during innate cellular immune responses. Therefore, additional techniques for removing such dsRNA contaminants have been developed, and these techniques are known in this field, including (but not limited to) scalable HPLC purification (see, for example, Kariko, K., Muramatsu, H., Ludwig, J. and Weissman, D., 2011, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA, Nucl Acid Res, v. 39 e142; Weissman, D., Pardi, N., Muramatsu, H. and Kariko, K., HPLC Purification of in vitro transcribed long RNA. Synthetic Messenger RNA and Cell Metabolism Modulation. Methods in Molecular Biology v.969 (Rabinovich, PH ed.), 2013). Reports indicate that HPLC-purified mRNA exhibits significantly higher translation levels, especially in primary cells and in vivo environments.

Various modifications have been described in this technique to alter specific properties of in vivo transcribed mRNA and enhance its efficacy. These modifications include, but are not limited to, modifications to the 5' and 3' ends of mRNA. Endogenous eukaryotic mRNA typically contains a cap at the 5' end of mature molecules. This cap plays a crucial role in mediating the binding of mRNA cap-binding proteins (CBPs), which in turn enhance mRNA stability in cells and the efficiency of mRNA transcription. Therefore, capped mRNA transcripts achieve the highest levels of protein expression. The 5' cap contains a 5'-5'-triphosphate bond between the terminal nucleotide and the guanine nucleotide. The bound guanine nucleotide is methylated at the N7 position. Additional modifications include methylation of the 2'-hydroxyl group of the 5' terminal and penultimate nucleotide.

A variety of cap structures can be used to generate the 5' cap of in vitro transcribed mRNA. 5' capping of synthetic mRNA can be co-transcribed with chemical cap analogs (i.e., capping during in vitro transcription). For example, the anti-retroviral cap analog (ARCA) contains a 5'-5'-guanine-triphosphate bond, where one guanine molecule contains an N7 methyl group and a 3'-O-methyl group. However, during this co-transcription process, up to 20% of the transcripts fail to cap, and the 5' cap structure of synthetic cap analogs differs from that of real cellular mRNA, potentially reducing transcribeability and cellular stability. Alternatively, synthetic mRNA molecules can also be enzymatically capped post-transcriptional. These methods may produce more realistic 5' cap structures that are structurally or functionally closer to the endogenous 5' cap. These structures can enhance the binding of cap-binding proteins, increase half-life, reduce sensitivity to 5' endonucleases, and/or reduce 5' cap removal. Numerous synthetic 5' cap analogues have been developed, and these analogues are known in this technique to enhance mRNA stability and translatability (see, for example, Grudzien-Nogalska, E., Kowalska, J., Su, W., Kuhn, A.N., Slepenkov, S.V., Darynkiewicz, E., Sahin, U., Jemielity, J. and Rhoads, R.E., Synthetic mRNAs with superior translation and stability properties. Synthetic Messenger RNA and Cell Metabolism Modulation. Methods in Molecular Biology v.969 (Rabinovich, P.H. ed.), 2013).

At the 3' end, during RNA processing, a long string of adenine nucleotides (poly-A tail) is typically added to the mRNA molecule. Immediately after transcription is complete, the 3' end of the transcript cleaves to release the 3' hydroxyl group. Poly-A polymerase then adds a string of adenine nucleotides to the 3' hydroxyl group of the RNA in a process called polyadenylation. It has been well demonstrated that the poly-A tail can enhance the translation efficiency and stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly (A), poly (A) binding protein and the regulation of mRNA stability, Trends Bio Sci v. 14 373-377; Guhaniyogi, J. and Brewer, G., 2001, Regulation of mRNA stability in mammalian cells, Gene, v. 265, 11-23; Dreyfus, M. and Regnier, P., 2002, The poly (A) tail of mRNAs: Bodyguard in eukaryotes, scavenger in bacteria, Cell, v.111, 611-613).

Poly(A) tailing of in vitro transcribed mRNA can be achieved through various pathways, including (but not limited to) selecting a poly(T) segment into a DNA template or adding it after transcription using a poly(A) polymerase. The first approach allows for in vitro transcription of mRNA with a poly(A) tail of a defined length, depending on the size of the poly(T) segment, but requires additional manipulation of the template. The latter approach involves enzymatically adding the poly(A) tail to in vitro transcribed mRNA using a poly(A) polymerase, which catalyzes the incorporation of an adenine residue into the 3' end of the RNA. This approach does not require additional manipulation of the DNA template but results in non-uniform poly(A) tail lengths in the mRNA. 5' capping and 3' poly(A) tailing can be performed using a variety of commercially available kits, including (but not limited to) the Poly(A) polymerase tailing kit (EpiCenter), the mMESSAGE mMACHINE T7 Ultra kit, and the Poly(A) tailing kit (Life Technologies), as well as using commercially available reagents such as various ARCA caps and Poly(A) polymerases.

Besides 5' capping and 3' polyadenylation, other modifications to in vitro transcripts have been reported to provide benefits related to translational efficiency and stability. It is well known in this art that in eukaryotes, pathogenic DNA and RNA can be recognized by multiple sensors and trigger a strong innate immune response. It has been shown that the ability to distinguish between pathogenic and self-generated DNA and RNA is at least partially based on structural and nucleoside modifications, as most naturally derived nucleic acids contain modified nucleosides. In contrast, in vitro synthesized RNA lacks these modifications, thus making it immunostimulatory, which in turn can inhibit efficient mRNA translation as outlined above. Introducing modified nucleosides into in vitro transcribed mRNA can prevent RNA sensor recognition and activation, thereby reducing this unwanted immunostimulatory activity and improving transcriptional efficiency (see, for example, Kariko, K. and Weissman, D. 2007, Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development, Curr Opin Drug Discov Devel, v.10 523-532; Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides, Synthetic Messenger RNA and Cell Metabolism Modulation in Methods, Molecular Biology v.969 (Rabinovich, PH ed.), 2013); Kariko, K., Muramatsu, H., Welsh, FA, Ludwig, J., Kato, H., Akira, S., Weissman, D., 2008, Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability, Mol Ther v.16, 1833-1840. Modified nucleosides and nucleotides used in the synthesis of modified RNA can be prepared, monitored, and applied using general methods and procedures known in this art. A variety of nucleoside modifications are available, and to some extent, these modifications can be incorporated, alone or in combination with other modified nucleosides, into mRNA transcribed in vivo (see, for example, U.S. Patent Application Publication No. 2012/0251618). It has been reported that nucleoside-modified mRNA synthesized in vivo exhibits reduced ability to activate immune sensors while simultaneously enhanced translational capacity.

Other components of mRNA that can be modified to provide benefits in terms of translatability and stability include the 5' and 3' untranslated regions (UTRs). Optimization of the UTRs (favorable 5' and 3' UTRs can be obtained from cellular or viral RNA), whether optimized simultaneously or individually, has been shown to increase the mRNA stability and transcription efficiency of in vitro transcribed mRNAs (see, for example, Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides. Synthetic Messenger RNA and Cell Metabolism Modulation. Methods in Molecular Biology v.969 (Rabinovich, PH ed.), 2013).

Besides mRNA, other nucleic acid payloads can also be used in this disclosure. For oligonucleotides, preparation methods include (but are not limited to) chemical synthesis and enzymatic, chemical cleavage, and in vivo extracellular transcription of longer precursors, as described above. Methods for synthesizing DNA and RNA nucleotides are widely used and well-known in this technique (see, for example, Gait, M. J. (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, D.C.: IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press, 2005, both of which are incorporated herein by reference).

For plasso DNA, the preparation method used in conjunction with this disclosure typically utilizes (but is not limited to) in vitro amplification and isolation of plasso DNA in a bacterial liquid culture containing the plasso of interest. The plasso of interest contains genes encoding resistance to specific antibiotics (such as penicillin and kanamycin), which allows bacteria containing the plasso of interest to selectively grow in antibiotic-containing cultures. Methods for isolating plasmid DNA are widely used and well-known in this technique (see, for example, Heilig, J., Elbing, K. L. and Brent, R (2001) Large-Scale Preparation of Plasmid DNA. Current Protocols in Molecular Biology. 41:II:1.7:1.7.1-1.7.16; Rozkov, A., Larsson, B., Gillström, S., Björnestedt, R. and Schmidt, S. R. (2008), Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture. Biotechnol. Bioeng., 99: 557-566; and U.S. Patent No. 6,197,553). Plasmid separation can be performed using a variety of commercially available kits, including (but not limited to) Plasmid Plus, GenJET plasmid extraction (Thermo), and PureYield plasmid extraction (Promega) kits, as well as commercially available reagents.

The following describes in further detail various exemplary embodiments of lipid nanoparticles and compositions comprising such lipid nanoparticles, and their use in delivering active agents or therapeutics (such as nucleic acids for regulating gene and protein expression).

As used herein, unless otherwise specified, the following terms have the meanings assigned to them.

Unless the context otherwise requires, throughout the specification and the scope of the patent application, the word "comprise" and its variations (such as "comprises/comprising") shall be interpreted in an open and inclusive sense, that is, "including (but not limited to)".

Throughout this specification, references to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of this disclosure. Therefore, the phrases "in one embodiment" or "in an embodiment" appearing in different places throughout the specification do not necessarily refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic may be combined in one or more embodiments in any suitable manner.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. As used in this specification and the scope of the claims, unless the context clearly requires otherwise, the singular forms "a/an" and "the" include plural references.

The phrase "inducing desired protein expression" refers to the ability of nucleic acids to increase the expression of desired proteins. To detect the degree of protein expression, a test sample (e.g., a sample of cells in culture containing the desired protein) or a test mammal (e.g., a human mammal, or a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) is brought into contact with nucleic acids (e.g., nucleic acids combined with the lipid composition disclosed herein). The performance of the desired protein in the test sample or test animal is compared with the performance of the desired protein in a control sample (e.g., a sample of cells containing the desired protein in culture) or a control mammal (e.g., a human mammal, or an animal model of a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) where the control sample or control mammal has not been in contact with or administered the nucleic acid. When the desired protein is present in the control sample or control mammal, the performance of the desired protein in the control sample or control mammal can be assigned a value of 1.0. In some embodiments, the induced expression of the desired protein is achieved when the ratio of the desired protein expression level in the test sample or test mammal to that in the control sample or control mammal is greater than 1, for example, about 1.1, 1.5, 2.0, 5.0, or 10.0. The induced expression of the desired protein is achieved when it is not present in the control sample or control mammal, or when any measurable level of the desired protein is detected in the test sample or test mammal. Those familiar with this technique should understand the appropriate analytical methods used to determine the protein expression levels in a sample, such as dot speckling, northern speckling, in situ hybridization, ELISA, immunoprecipitation, enzyme function analysis, and phenotypic analysis, or analytical methods based on reporter proteins that can produce fluorescence or luminescence under appropriate conditions.

The "effective amount" or "therapeutic effective amount" of an active agent or therapeutic agent (such as a therapeutic nucleic acid) is the amount sufficient to produce the desired effect, such as increasing or inhibiting the expression of a target sequence compared to the normal expression level detected in the absence of nucleic acid. In the absence of nucleic acid and therefore no expression product, an increase in the expression of the target sequence is achieved when any measurable level of expression product is detected. An increase in expression occurs when the expression product is present at a certain level before contact with nucleic acids, and the value of nucleic acids such as mRNA increases relative to the control by approximately 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000 or greater. When the values obtained by nucleic acids such as antisense oligonucleotides are approximately 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% relative to the control, the expression of the target gene or target sequence is suppressed. Suitable analytical methods for measuring the expression of the target gene or target sequence include, for example, techniques known to those skilled in the art, such as dot spectrophotometry, northern spectrophotometry, in situ hybridization, ELISA, immunoprecipitation, enzyme function analysis, and suitable fluorescent or luminescent analysis of reporter proteins, as well as phenotypic analysis methods known to those skilled in the art to detect protein or RNA levels.

As used herein, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides and/or ribonucleotides in single-stranded or double-stranded form, including DNA, RNA, and their hybrids. DNA may be in the form of antisense molecules, plasmid DNA, cDNA, PCR products, or vectors. RNA may be in the form of hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA, dicer-accepted RNA, or viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, which are synthetic, naturally occurring, or non-natural, and which have binding properties similar to reference nucleotides. Examples of such analogs include (but are not limited to) phosphate thioesters, aminophosphate esters, methyl phosphonate, methyl palmaridate, 2'-O-methylribonucleotides, and peptide nucleic acids (PNA). Unless otherwise specified, this term covers nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleotide. Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes its conserved modified variants (e.g., degenerate codon substitutions), paired genes, heterologous homologs, single nucleotide polymorphisms (SNPs), complementary sequences, and explicitly indicated sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more (or all) selected codons is substituted with a mixed base and/or deoxyinosine residue (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). A "nucleotide" contains a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together by phosphate groups. "Base" includes purines and pyrimidines, and further includes natural compounds such as adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including (but not limited to) modifications that place new reactive groups such as (but not limited to) amines, alcohols, thiols, carboxylic acid esters, and alkyl halides.

The term "lipid" refers to a group of organic compounds, including (but not limited to) esters of fatty acids, and is generally characterized by poor water solubility but soluble in many organic solvents.

"Steroids" are compounds that contain the following carbon skeleton: Non-limiting examples of steroids include cholesterol and its analogues.

"Ionizable lipid" refers to a lipid capable of carrying a charge. In some embodiments, ionizable lipids are cationic lipids. As used herein, "cationic lipid" refers to a lipid capable of carrying a positive charge. Exemplary cationic lipids include one or more amino groups that can carry or actually carry a positive charge. In some embodiments, cationic lipids are ionizable, allowing them to exist in a positively charged or neutral form depending on the pH. The ionization of cationic lipids affects the surface charge of lipid nanoparticles under different pH conditions. This charge state can affect plasma protein uptake, blood clearance and tissue distribution (Semple, S.C. et al., Adv. Drug Deliv Rev 32:3-17 (1998)), as well as the ability to form non-bilayer structures for cleavage of nucleosomal nuclei, which are crucial for the intracellular delivery of nucleic acids (Hafez, I.M. et al., Gene Ther 8:1188-1196 (2001)).

The term "lipid nanoparticle" or "LNP" refers to at least one particle with a nanometer dimension (e.g., 1 to 1,000 nm) comprising components selected from: ionizable lipids (e.g., cationic lipids), charged lipids, neutral lipids, steroids, and/or lipids bound to polymers. In some embodiments, the LNP is included in a formulation for delivering an active agent or therapeutic agent, such as nucleic acid (e.g., mRNA), to a target site of interest (e.g., cells, tissues, organs, tumors, and analogues). In some embodiments, the LNP disclosed herein comprises nucleic acids. In some embodiments, such as nucleic acid active agents or therapeutic agents, may be encapsulated in the lipid fraction of lipid nanoparticles or in an aqueous space encapsulated by some or all of the lipid fractions of lipid nanoparticles, thereby protecting them from enzymatic degradation or other unwanted effects induced by host organism or cell mechanisms, such as adverse immune responses.

In various embodiments, the average diameter of the LNP is approximately 30 nm to approximately 150 nm, approximately 40 nm to approximately 150 nm, approximately 50 nm to approximately 150 nm, approximately 60 nm to approximately 130 nm, approximately 70 nm to approximately 110 nm, approximately 70 nm to approximately 100 nm, approximately 80 nm to approximately 100 nm, approximately 90 nm to approximately 100 nm, approximately 70 nm to approximately 90 nm, approximately 80 nm to approximately 90 nm, approximately 70 nm to approximately 80 nm, or approximately 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. The nanoparticles are nanometers in size and are essentially non-toxic. In some embodiments, when nucleic acids are present in LNPs, they are resistant to degradation by nucleases in aqueous solutions. Lipid nanoparticles containing nucleic acids and methods for their preparation are disclosed in, for example, U.S. Patent Publications Nos. 2004/0142025 and 2007/0042031, and PCT Publications Nos. WO 2013/016058 and WO 2013/086373, the entire disclosure of which is incorporated herein by reference for all purposes.

As used herein, "encapsulation" refers to the complete, partial, or both encapsulation of active agents or therapeutics such as nucleic acids (e.g., mRNA) by lipid nanoparticles. In one embodiment, nucleic acid molecules (e.g., mRNA) are completely encapsulated in lipid nanoparticles. In some embodiments, greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the nucleic acid molecules in a sample are completely or partially encapsulated by lipid nanoparticles.

The term "polymer-bound liposome" refers to a molecule comprising both a liposome and a polymer moiety. An example of a polymer-bound liposome is a polyethylene glycol-modified liposome. The term "polyethylene glycol-modified liposome" refers to a molecule comprising both a liposome and a polyethylene glycol moiety. Polyethylene glycol-modified liposomes are known in this art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristylglycerol (PEG-DMG) and its analogues.

In some embodiments, the lipid of the bound polymer is functionalized to promote surface modification of the LNP. In some embodiments, the lipid of the bound polymer is modified prior to LNP formation. In other embodiments, the lipid of the bound polymer is modified after LNP formation. In some embodiments, modification involves the addition of targeting groups (e.g., antibodies). In some embodiments, modification involves the addition of components that reduce LNP clearance.

The term "neutral lipids" refers to any of a number of lipid species that exist as uncharged or neutral zwitterionic ions at a selected pH. At physiological pH, these lipids include (but are not limited to) phospholipids, such as 1,2-distearyl-sn-glycero-3-phosphate choline (DSPC), 1,2-dipalmitinyl-sn-glycero-3-phosphate choline (DPPC), 1,2-dimyristinyl-sn-glycero-3-phosphate choline (DMPC), and 1-palmitinyl... Neutral lipids include 2-oleyl-sn-glycero-3-phosphate choline (POPC), 1,2-dioleyl-sn-glycero-3-phosphate choline (DOPC), phosphatidylethanolamine, such as 1,2-dioleyl-sn-glycero-3-phosphate cholineamine (DOPE), sphingomyelin (SM), ceramides, steroids, such as sterols and their derivatives. Neutral lipids can be synthetic or of natural origin.

The term "charged lipid" refers to any of a variety of lipid species that exist independently of pH in a positively or negatively charged form within a useful physiological pH range (e.g., pH from approximately 3 to approximately 9). Charged lipids can be synthetic or naturally derived. Examples of charged lipids include phosphatidylinosine, phosphatidic acid, phosphatidylinositol, sterol hemibutyrate, dialkyltrimethylammonium propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethyl phosphate choline, and dimethylaminoethaneaminomethyl sterol (e.g., DC-Chol).

As used herein, the term "aqueous solution" refers to a composition containing water. In some embodiments, the aqueous solution is essentially composed of water, salt, acid, and alkali (e.g., buffer). In some embodiments, the aqueous solution is a phosphate-buffered brine. In some embodiments, the aqueous solution is an acetate-buffered solution.

"Alkyl" refers to a straight-chain or branched-chain hydrocarbon group composed solely of carbon and hydrogen atoms. It is saturated (i.e., does not contain double or reference bonds) and has one to thirty-six carbon atoms ( _C1 - _C36 alkyl), one to twenty-four carbon atoms ( _C1 - _C24 alkyl), one to sixteen carbon atoms ( _C1 - _C16 alkyl), one to twelve carbon atoms ( _C1 - _C12 alkyl), six to twenty-four carbon atoms ( _C6 - _C24 alkyl), one to eight carbon atoms ( _C1 - _C8 alkyl), or one to six carbon atoms (C1- _C24 alkyl). _6- alkyl groups, and linked by single bonds to the remainder of the molecule, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tributyl), 3-methylhexyl, 2-methylhexyl, and similar groups. Unless otherwise specifically stated in this specification, alkyl groups are substituted as appropriate.

"Alkenyl" refers to a straight-chain or branched-chain hydrocarbon group consisting only of carbon atoms and hydrogen atoms, containing at least one carbon-carbon double bond, having two to twenty-four carbon atoms ( _C2 - _C24 alkenyl), two to twelve carbon atoms ( _C2 - _C12 alkenyl), six to twenty-four carbon atoms ( _C6 - _C24 alkenyl), two to sixteen carbon atoms ( _C2 - _C16 alkenyl), four to twelve carbon atoms ( _C4 - _C12 alkenyl), two to eight carbon atoms ( _C2 - _C8 alkenyl), or two to six carbon atoms ( _C2 - _C6 alkenyl), and connected to the rest of the molecule by a single bond, such as vinyl, n-propenyl, 1-methylvinyl, n-butenyl, n-pentenyl, 1,1-dimethylvinyl, 3-methylhexenyl, 2-methylhexenyl, and similar groups. Unless otherwise specifically stated in this specification, the alkenyl group is substituted as appropriate.

"Alynyl" refers to a straight-chain or branched hydrocarbon group consisting only of carbon atoms and hydrogen atoms, containing at least one carbon-carbon reference bond, having two to twenty-four carbon atoms ( _C2 - _C24 ynyl), two to twelve carbon atoms ( _C2 - _C12 ynyl), two to eight carbon atoms ( _C2 - _C8 ynyl), or two to six carbon atoms ( _C2 - _C6 ynyl), and linked by a single bond to the rest of the molecule, such as ethynyl, n-propynyl, 1-methylethynyl, n-butynyl, n-pentynyl, 1,1-dimethylethynyl, 3-methylhexynyl, 2-methylhexynyl, and similar groups. Unless otherwise specifically stated in this specification, the ynyl group is substituted as appropriate.

"Lylene" or "alkylene chain" refers to a straight or branched divalent saturated hydrocarbon chain that links the remainder of a molecule to a group, consisting only of carbon and hydrogen. In some embodiments, the alkylene chain has one to twenty-four carbon atoms ( _C1 - _C24 alkylene), one to fifteen carbon atoms ( _C1 - _C15 alkylene), one to twelve carbon atoms ( _C1 - _C12 alkylene), one to eight carbon atoms ( _C1 - _C8 alkylene), one to six carbon atoms ( _C1 - _C6 alkylene), four to six carbon atoms ( _C4 - _C6 alkylene), two to four carbon atoms ( _C2 - _C4 alkylene), and one to two carbon atoms ( _C1 - _C2 alkylene), such as methylene, ethylene, propylene, n-butylene, and similar groups. The alkyl chain is linked to the remainder of the molecule and to a group via single bonds. The connection points between the alkyl chain and the remainder of the molecule and to the group can be via one or any two carbons within the chain. Unless otherwise specifically stated in this specification, the alkyl chain is substituted as appropriate.

"Halogen" refers to halogen substituents (i.e., F, Cl, Br or I).

As used herein, the term "substitution" means any of the above groups (e.g., alkyl, alkenyl, and/or alkynyl) in which at least one hydrogen atom is replaced by a non-hydrogen atom, such as (but not limited to): halogen atoms, such as F, Cl, Br and I, cyano, -OH or _-NH₂ . "Optional" (e.g., substituted as appropriate) means that the situation or condition described below may or may not occur, and the description includes both the occurrence and non-occurrence of the event or condition. For example, "substituted alkyl as appropriate" means that the alkyl group may or may not be substituted, and the description includes both substituted and unsubstituted alkyl groups. In some embodiments, “substituted as appropriate” means that a particular group is substituted by one or more substituents selected from halogens (e.g., F, Cl, Br and I).

This disclosure is also intended to cover all pharmaceutically acceptable compounds that are isotopically labeled by replacing one or more atoms with atoms of different atomic masses or mass numbers (e.g., ionizable lipids, charged lipids, neutral lipids, lipids in polymers, steroids, etc.). Examples of isotopes that may be included in the compounds disclosed herein include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as ^2H , ^3H , ^11C , ^13C , ^14C , ^13N , ^15N , ^{15O, 17O , 18O} , ^31P , ^32P , 35S, ^18F , ^36Cl , ^123I , and ^125I , ^respectively . These radiolabeled compounds can be used to help determine or measure the effectiveness of compounds by characterizing, for example, the site of action or mode of action, or the binding affinity to pharmacologically important sites of action. Certain isotope-labeled compounds (e.g., those containing radioactive isotopes) are suitable for drug and/or recipient tissue distribution studies. The radioactive isotopes tritium (i.e., ^³H ) and carbon-14 (i.e., ^¹⁴C ) are particularly suitable for this purpose due to their ease of incorporation and readily available detection methods.

Substitution with heavier isotopes such as deuterium (i.e., ^2H ) can provide certain therapeutic advantages resulting from greater metabolic stability, such as prolonged in vivo half-life or reduced dose requirements, and may therefore be superior in some cases.

^Positron emission tomography (PET) studies using positron emission tomography (PET) is used to examine receptor occupancy rates, replacing positron emission tomography (PET) with appropriate isotopically labeled reagents instead of previously used unlabeled ^reagents , ^using techniques known to those skilled in the ^art or by processes similar to those described in the preparation methods and examples below.

This disclosure is also intended to cover the in vivo metabolites of the disclosed compounds. Such products can be produced, for example, by oxidation, reduction, hydrolysis, amination, esterification, and similar processes of the administered compound, primarily due to enzymatic processes. Therefore, this disclosure includes compounds produced by methods comprising administering the disclosed compound to mammals for a duration sufficient to produce its metabolites. Such products are typically identified by administering a detectable dose of a radiolabeled disclosed compound to animals (such as rats, mice, guinea pigs, monkeys) or humans, allowing sufficient time for metabolism, and separating the metabolites from urine, blood, or other biological samples.

"Pharmaceutical composition" refers to formulations of the disclosed compounds with media generally accepted in this art for delivering bioactive compounds to mammals (e.g., humans). Such media include all pharmaceutically acceptable carriers, diluents, or excipients.

"Effective dose" or "therapeutic effective dose" refers to the amount of the disclosed compound that, when administered to mammals, preferably humans, is sufficient to achieve therapeutic effects in mammals, preferably humans. The amount of lipid nanoparticles of the disclosed compound constituting a "therapeutic effective dose" will depend on the compound, the condition and its severity, the method of administration, and the age of the mammal to be treated, but can be conventionally determined by those skilled in the art based on their own knowledge and the contents of this disclosure.

As used herein, “treating” encompasses treating mammals, preferably humans, with a disease or condition of concern, and includes: (i) preventing the occurrence of the disease or condition in mammals, especially when such mammals are susceptible to the condition but have not yet been diagnosed with it; (ii) suppressing the disease or condition, i.e., halting its development; (iii) alleviating the disease or condition, i.e., causing the disease or condition to subside; or (iv) relieving symptoms caused by the disease or condition, i.e., relieving pain without addressing the underlying disease or condition. As used in this article, the terms “disease” and “symptom” may be used interchangeably or may differ, because a particular disease or symptom may not have a known causative factor (and therefore its cause has not been studied), and therefore it is not yet recognized as a disease, but is only regarded as an undesirable symptom or syndrome in which a clinician has identified a more or less specific set of symptoms.

Compounds comprising lipid nanoparticles or nucleic acids, or their pharmaceutically acceptable salts, may contain one or more asymmetric centers, thus giving rise to mirror isomers, non-mirror isomers, and other stereoisomers, which, in absolute stereochemistry, may be defined as ( R )- or ( S )-, or, for amino acids, as (D)- or (L)-. This disclosure is intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), ( R )- and ( S )- or (D)- and (L)- isomers can be prepared using palmitic synthons or palmitic reagents, or resolved using known techniques (e.g., chromatography and stepwise crystallization). Conventional techniques for the preparation/separation of individual mirror isomers include palmar synthesis of adaptive optically pure precursors, or the use of, for example, palmar high-pressure liquid chromatography (HPLC) to resolve racemates (or racemates of salts or derivatives). When the compounds described herein contain alkene double bonds or other geometrically asymmetrical centers, and unless otherwise specified, the intended compounds include both E-type and Z-type geometric isomers.

"Stereoisomers" refer to compounds that are composed of identical atoms linked by identical bonds but have different three-dimensional structures and are not interchangeable. This disclosure covers various stereoisomers and mixtures thereof, including "mirror isomers," which are two stereoisomers whose molecules are mirror images of each other that cannot be superimposed.

The preparation method as used herein, the “familiar method” for preparing lipid nanoparticles (LNPs) refers to a process that typically includes the following steps: (i) mixing RNA (dissolved in an aqueous buffer) and LNP components (dissolved in ethanol) via a T-mixer; (ii) removing ethanol and exchanging the buffer system via dialysis or tangential flow filtration (TFF); (iii) concentrating the resulting LNP-containing RNA to approximately 1 mg/mL via TFF or centrifugation; and (iv) diluting with a buffer (e.g., PBS and sucrose) and freezing at -80°C.

Step (i) takes approximately 2 to 5 hours. Step (ii) takes approximately 1 to 2 days. Step (iii) takes approximately 2 to 3 hours. Step (iv) takes approximately 0.5 hours.

In one embodiment, this disclosure provides a novel method for preparing nucleic acids encapsulated in lipid nanoparticles. The lipid nanoparticles may comprise components such as ionizable lipids (e.g., cationic lipids), neutral lipids, charged lipids, steroids, and/or lipids bound to polymers to form the lipid nanoparticles. It is not intended to be theoretically construed as such lipid nanoparticles can protect oligonucleotides from degradation in serum and can efficiently deliver oligonucleotides to cells both in vitro and in vivo.

One embodiment provides a method for encapsulating a nucleic acid-containing payload within lipid nanoparticles (LNPs), comprising: i) providing a first aqueous solution containing a plurality of LNPs, the LNPs containing a plurality of ionizable lipids, wherein the first solution substantially contains no destabilizing agent and the difference between the pH of the first solution and the pKa of the ionizable lipids is less than 1.0; ii) mixing the first solution with a dried payload or a payload in a second aqueous solution, wherein the second aqueous solution substantially contains no destabilizing agent, thereby encapsulating at least a majority of the payload in the LNPs in the resulting solution.

Another embodiment provides a method for encapsulating a nucleic acid-containing payload within lipid nanoparticles (LNPs), comprising: i) providing a first dried composition comprising a plurality of LNPs, the LNPs containing a plurality of ionizable lipids; ii) mixing the first composition with the payload in a second aqueous solution, wherein the second aqueous solution substantially contains no destabilizing agent, thereby encapsulating at least a majority of the payload in the LNPs in the first solution.

One embodiment provides a method for encapsulating a nucleic acid-containing payload within lipid nanoparticles (LNPs), comprising: i) providing a first dried composition comprising a plurality of LNPs, the LNPs containing a plurality of ionizable lipids; ii) mixing the first composition with a second composition comprising the dried payload; iii) mixing the first composition and the second composition together with a first aqueous solution, wherein the first solution substantially contains no destabilizing agent and the difference between the pH of the first solution and the pKa of the ionizable lipids is less than 1.0, thereby encapsulating at least a majority of the payload within the LNPs.

Unexpectedly, it was found that LNPs containing RNA could be prepared in a much shorter time than previously known methods according to the method of this invention.

In some embodiments, a first process is performed: mixing LNP components (e.g., cationic lipids, neutral lipids, polyethylene glycol-modified lipids and/or cholesterol), followed by a second process: dialysis or tangential flow filtration, thereby forming an LNP solution containing lipid nanoparticles (LNPs) and free from any destabilizing agents (e.g., ethanol).

In some embodiments, the first process is carried out in an acetate (aqueous) buffer solution with a pH range of 5.5 to 6.5, 5.6 to 6.6, 5.7 to 6.7, 5.8 to 6.8, 5.9 to 6.9, 6.0 to 7.0, 5.4 to 6.4, 5.3 to 6.3, 5.2 to 6.2, 5.1 to 6.1, or 5.0 to 6.0. In some embodiments, the first process is carried out in a solution containing ethanol.

In some embodiments, the second process is carried out in an acetate (aqueous) buffer solution with a pH range of 5.5 to 6.5, 5.6 to 6.6, 5.7 to 6.7, 5.8 to 6.8, 5.9 to 6.9, 6.0 to 7.0, 5.4 to 6.4, 5.3 to 6.3, 5.2 to 6.2, 5.1 to 6.1, or 5.0 to 6.0.

In some embodiments, the total time for the first and second processes (from the start of measurement of the mixed LNP components to the commencement of the next process step (e.g., the third process) is less than 48 hours. In some embodiments, the total time for the first and second processes (total) is less than 36 hours, less than 24 hours, less than 12 hours, or less than 8 hours. In some embodiments, the total time for the first and second processes (total) exceeds 12 hours, exceeds 18 hours, exceeds 24 hours, exceeds 36 hours, or exceeds 48 hours.

In some embodiments, the third process includes mixing the LNP solution with an aqueous solution containing a payload (e.g., mRNA), thereby encapsulating at least a majority of the payload in the LNP in the payload-LNP solution.

In some embodiments, the third process takes less than 4 hours from the start of mixing until the next process step (e.g., the fourth process). In some embodiments, the third process takes less than 3 hours, less than 2 hours, less than 1.5 hours, less than 1 hour, less than 30 minutes, less than 15 minutes, or less than 5 minutes. In some embodiments, the third process takes more than 5 minutes, more than 15 minutes, more than 30 minutes, more than 1 hour, more than 1.5 hours, more than 2 hours, or more than 3 hours.

In some embodiments, the third process is carried out at ambient temperature. In some embodiments, the third process is carried out at temperatures ranging from 15°C to 30°C, 20°C to 30°C, 25°C to 30°C, 15°C to 25°C, or 15°C to 20°C.

In some embodiments, the fourth process involves diluting the effective load-LNP solution with an aqueous buffer (e.g., 2× phosphate-buffered saline (PBS)). In some embodiments, the fourth process further includes mixing after dilution.

In some embodiments, the fourth process takes less than 2 hours from the start of dilution measurement until the next process step (e.g., the fifth process). In some embodiments, the fourth process takes less than 1.5 hours, less than 1 hour, or less than 30 minutes. In some embodiments, the fourth process takes more than 30 minutes, more than 1 hour, more than 1.5 hours, or more than 2 hours.

In some embodiments, the fifth process includes adding a sucrose-containing solution (e.g., an aqueous solution) to the solution produced in the fourth process. In some embodiments, the fifth process further includes mixing after dilution. In some embodiments, the fifth process includes filtration after dilution. In some embodiments, the aqueous buffer from the fifth process is added to the effective load-LNP solution at a 1:1 ratio (e.g., vol:vol or wt:wt). In some embodiments, the sucrose-containing solution further contains PBS.

In some embodiments, the time taken for the fifth process, from the start of measurement until the solution produced in the fifth process is placed in a refrigerator (e.g., a refrigerator set to -80°C), is less than 30 minutes. In some embodiments, the time taken for the fifth process is less than 20 minutes, less than 15 minutes, less than 10 minutes, or less than 5 minutes. In some embodiments, the time taken for the fifth process is more than 5 minutes, more than 10 minutes, more than 15 minutes, more than 20 minutes, or more than 25 minutes.

In some embodiments, RNA (e.g., mRNA) is mixed with LNPs that have not yet been encapsulated with an effective payload. In some embodiments, RNA is mixed at concentrations ranging from 0.05 to 2.0 mg/mL. In some embodiments, RNA is mixed at concentrations ranging from 0.05 to 1.75, 0.05 to 1.5, 0.05 to 1.25, 0.05 to 1.15, 0.05 to 1.0, 0.05 to 0.75, 0.05 to 0.65, 0.05 to 0.5, 0.05 to 0.35, 0.05 to 0.25, 0.05 to 0.15, 0.05 to 0.10, 0.05 to 0.085, and 0.05 to 0.075 mg/mL. In some embodiments, RNA is mixed at concentrations ranging from 0.065 to 2.0, 0.075 to 2.0, 0.085 to 2.0, 1.0 to 2.0, 1.15 to 2.0, 1.25 to 2.0, 1.35 to 2.0, 1.5 to 2.0, 1.65 to 2.0, 1.75 to 2.0, 1.85 to 2.0, or 1.95 to 2.0 mg/mL. In some more specific embodiments, RNA is mixed at a maximum concentration of 1.05 mg/mL or 0.065 mg/mL. In some embodiments, RNA is mixed in an acetate buffer at a pH range of 5.25 to 5.75 (e.g., 5.4, 5.5, 5.6, etc.). In some embodiments, the RNA is mixed in a phosphate buffer at a pH range of 5.6 to 6.0 (e.g., 5.7, 5.8, 5.9, etc.).

In some embodiments, the LNP in the first solution or the first composition does not encapsulate the effective load. In some embodiments, the pH of the first solution is less than 7.0. In some embodiments, the pH of the first solution is less than 6.8. In some embodiments, the pH of the first solution is less than 6.5. In some embodiments, the pH of the first solution is less than 6.4, less than 6.3, less than 6.2, less than 6.1, less than 6.0, less than 5.9, less than 5.8, or less than 5.7. In some embodiments, the pH of the first solution is less than 5.0. In some embodiments, the pH of the first solution is less than 5.7. In some embodiments, the pH of the first solution is in the range of 4.5 to 7.4, 5.5 to 6.5, or 5.7 to 6.2.

In some embodiments, the difference between the pH of the first solution and the pKa of the ionizable lipid is in the range of 0.1 to 1.5, 0.1 to 1.0, 0.1 to 0.5, 0.2 to 0.5, 0.5 to 0.5, 0.3 to 0.6, 0.2 to 0.6, or 0.4 to 0.8. In some embodiments, the difference between the pH of the first solution and the pKa of the ionizable lipid is less than 1.0. In some embodiments, the difference between the pH of the first solution and the pKa of the ionizable lipid is less than 0.7. In some embodiments, the difference between the pH of the first solution and the pKa of the ionizable lipid is less than 0.5, less than 0.3, or less than 0.1. In some embodiments, the difference between the pH of the first solution and the pKa of the ionizable lipid is at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.2, or at least 1.5. In some embodiments, the difference between the pH of the first solution and the pKa of the ionizable lipid is in the range of 0.3 to 1.0, 0.2 to 0.9, 0.1 to 1.1, 0.1 to 0.8, or 0.2 to 0.7.

In some embodiments, the coverage efficiency is at least 70%. In some embodiments, the coverage efficiency is at least 80%. In some embodiments, the coverage efficiency is at least 85%. In some embodiments, the coverage efficiency is at least 88%. In some embodiments, the coverage efficiency is at least 90%. In some embodiments, the coverage efficiency is at least 95%.

In some embodiments, the pH of the resulting solution is in the range of 6.0 to 7.8. In some embodiments, the pH of the resulting solution is in the range of 6.5 to 7.8. In some embodiments, the pH of the resulting solution is in the range of 7.0 to 7.5.

In some embodiments, the particle size of the LNPs effectively loaded in the first solution or the resulting solution is in the range of 20 to 120 nm, or 30 to 90 nm, 40 to 120 nm, 50 to 120 nm, or 30 to 100 nm. In some embodiments, the particle size of the LNPs effectively loaded in the first solution or the resulting solution is in the range of 60 to 100 nm. In some embodiments, the particle size of the LNPs effectively loaded in the first solution or the resulting solution is in the range of 70 to 90 nm. In some embodiments, the particle size of the LNPs effectively loaded in the first solution or the resulting solution is less than 90 nm. In some embodiments, the particle size of the LNPs effectively loaded in the first solution or the resulting solution is less than 80 nm. In some embodiments, the particle size of the LNPs effectively loaded in the first solution or the resulting solution is less than 70 nm, less than 60 nm, or less than 50 nm.

In some embodiments, the destabilizing agent in the first or second solution is an organic solvent. In some embodiments, the destabilizing agent in the first or second solution is methanol, ethanol, isopropanol, tetrahydrofuran, dimethylformamide, dimethyl sulfoxide, acetonitrile, sodium dodecyl sulfate, or combinations thereof.

In some embodiments, the temperature of the first solution prior to mixing is below 27°C, below 25°C, or below 22°C. In some embodiments, the temperature of the second solution during mixing is below 27°C, below 25°C, or below 22°C. In some embodiments, the temperature of the resulting solution immediately after mixing is below 27°C, below 25°C, or below 22°C.

In some embodiments, the plurality of LNPs in the first solution or the resulting solution contain 20 to 90 mol% or 30 to 90 mol% of ionizable lipids. In some embodiments, the plurality of LNPs in the first solution or the resulting solution contain 40 to 55 mol% of ionizable lipids. In some embodiments, the plurality of LNPs in the first solution or the resulting solution contain 46 to 49 mol% of ionizable lipids. In some embodiments, the plurality of LNPs in the first solution or the resulting solution contain one or more components of lipids selected from neutral lipids, steroids, and bound polymers.

In some embodiments, the plurality of LNPs in the first solution or the resulting solution contain neutral lipids at a concentration in the range of about 5 to about 15 mol% of the lipid nanoparticles. In some embodiments, the plurality of LNPs in the first solution or the resulting solution contain steroids at a concentration in the range of about 30 to about 50 mol% of the lipid nanoparticles.

In some embodiments, the concentration of polyethylene glycol-modified lipids in the plurality of LNPs in the first solution or the resulting solution is in the range of 0.1 to 10 mol%, 0.1 to 5 mol%, 0.1 to 3 mol%, 0.1 to 2 mol%, 0.1 to 1 mol%, 0.5 to 10 mol%, 0.5 to 5 mol%, 0.5 to 3 mol%, 0.5 to 2 mol%, 0.5 to 1 mol%, 1.0 to 10 mol%, 1.0 to 5 mol%, 1.0 to 3 mol%, 1.0 to 2 mol%, 1.5 to 10 mol%, 1.5 to 5 mol%, 1.5 to 3 mol%, 1.5 to 2 mol%, 2.0 to 10 mol%, 2.0 to 5 mol%, 2.0 to 3 mol%, 2.5 to 10 mol%, 2.5 to 5 mol%, or 2.5 to 3 mol% of the lipid nanoparticles.

In some embodiments, ionizable lipids have the following structure: Or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein: ^L1 and ^L2 are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -SS-, -C(=O) _S- , -SC(=O)-, -NR a C(=O)-, -C(=O)NR ^a- , -NR ^a C(=O)NR ^a- , -OC(=O)NR ^a- , ^{-NR a} C(=O)O-, or a direct bond; ^G1 is a _C1 - _C2 alkylene group, -(C=O)-, -O(C=O)-, -SC(=O)-, -NR ^a C(=O)-, or a direct bond; G ² is -C(=O)-, -(C=O)O-, -C(=O)S-, -C(=O) ^NRa- or a direct bond; ^G3 is _C1 - _C6 alkyl; ^Ra is H or _C1 - _C12 alkyl; ^R1a and ^R1b are independently: (a) H or _C1 - _C12 alkyl; or (b) ^R1a is H or _C1 - _C12 alkyl, and ^R1b together with its bonded carbon atom forms a carbon-carbon double bond with the adjacent ^R1b and its bonded carbon atom; ^R2a and ^R2b are independently: (a) H or _C1 - _C12 alkyl; or (b) ^R2a is H or _C1 - _C12 alkyl, and R ^R2b, together with its bonded carbon atom, forms a carbon-carbon double bond with the adjacent ^R2b and its bonded carbon atom; ^R3a and ^R3b, in each occurrence, are independently: (a) H or _C1 - _C12 alkyl; or (b) ^R3a is H or _C1 - _C12 alkyl, and ^R3b , together with its bonded carbon atom, forms a carbon-carbon double bond with the adjacent ^R3b and its bonded carbon atom; ^R4a and ^R4b, in each occurrence, are independently: (a) H or _C1 - _C12 alkyl; or (b) ^R4a is H or _C1 - _C12 alkyl, and ^R4b , together with its bonded carbon atom, forms a carbon-carbon double bond with the adjacent ^R4b and its bonded carbon atom; ^R5 and R ⁶ is independently H or methyl; ^R7 is _C4 - _C20 alkyl; ^R8 and ^R9 are independently _C1 - _C12 alkyl; or ^R8 and ^R9 together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocycle; a, b, c, and d are independently integers from 1 to 24; and x is 0, 1, or 2.

In some embodiments, ionizable lipids have the following structure: Or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein: one of ^L1 or ^L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) _x- , -SS-, -C(=O)S-, -SC(=O)-, -NR ^a C(=O)-, -C(=O)NR ^a- , NR ^a C(=O)NR ^a- , -OC(=O)NR ^a- , or -NR ^a C(=O)O-, and the other of ^L1 or ^L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) _x- , -SS-, -C(=O)S-, SC(=O)-, -NR ^a C(=O)-, -C(=O)NR ^a- , NR ^a C(=O)NR ^a- , -OC(=O)NR ^a- , or -NR ^a C(=O)O- or direct bond; ^G1 and ^G2 are each independently unsubstituted _C1 - _C12 alkyl or _C1 - _C12 alkenyl; ^G3 is _C1 - _C24 alkyl, C1-C24 alkenyl, _{C3 - C8} cycloalkyl, or _C3 - _C8 cycloalkyl; ^Ra is H or _C1 - _{C12 alkyl} ; ^R1 and ^R2 are each independently _C6 - _C24 alkyl or _C6 - _C24 alkenyl; ^R3 is H, ^OR5 , CN, -C(=O) ^OR4 , -OC(=O) ^R4 , or ^-NR5 C(=O) ^R4 ; ^R4 is _C1 - _C12 alkyl; ^R5 is H or C ₁ -C ₆ alkyl; and x is 0, 1 or 2.

In some embodiments, ionizable lipids have the following structure: Or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein: ^R1 is, where appropriate, a substituted _C1 - _C24 alkyl or a substituted _C2 - _C24 alkenyl; ^R2 and ^R3 are each independently, where appropriate, a substituted _C1 - _C36 alkyl; ^R4 and ^R5 are each independently, where appropriate, a substituted _C1 - _C6 alkyl, or ^R4 and ^R5 are combined with their attached N to form a heterocyclic or heteroaryl group; ^L1 , ^L2 , and ^L3 are each independently, where appropriate, a substituted _C1 - _C18 alkyl; ^G1 is a direct bond, -( _CH2 ) _nO (C=O)-, -( _CH2 ) _n (C=O)O- or -(C=O)-; ^G2 and ^G3 are each independently -(C=O)O- or -O(C=O)-; and n is an integer greater than 0.

In some embodiments, the ionizable lipids have one of the structures in Table A or B. In some embodiments, the ionizable lipids have one of the structures in Table C.

In some embodiments, the plurality of LNPs in the first solution or the resulting solution contain one or more neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.

In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the lipid that binds the polymer is a polyethylene glycol-based lipid. In some embodiments, the polyethylene glycol-based lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, or PEG dialkoxypropylcarbamate.

In some embodiments, the polyethylene glycol-modified lipid has the following structure: Or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: R ¹⁰ and R ¹¹ are each independently a straight-chain or branched-chain alkyl, alkenyl or alkynyl group containing 10 to 30 carbon atoms, wherein each alkyl, alkenyl or alkynyl group is, where appropriate, substituted with at least one fluorine; and z is an integer in the range of 30 to 60.

In some embodiments, ^R10 and ^R11 are each independently a straight-chain alkyl group containing 12 to 16 carbon atoms. In some embodiments, z is in the range of 45 to 50.

In some embodiments, the effective payload is DNA, siRNA, microcircular RNA, PNA, adaptor, guide RNA, PE guide RNA, saRNA, circular RNA, antisense RNA, messenger RNA, Cas9 mRNA, ribonucleoprotein, or a combination thereof. In some embodiments, the effective payload is mRNA.

In some embodiments, the method further includes delivering an effective payload encapsulated in an LNP to a patient in need.

One embodiment provides a method for delivering an effective payload encapsulated in lipid nanoparticles (LNPs) to a patient in need, the method comprising delivering lipid nanoparticles prepared as described in any of the embodiments disclosed herein.

In some embodiments, the injection is carried out within 72 hours of mixing. In some embodiments, the injection is carried out within 24 hours of mixing. In some embodiments, the injection is carried out within 12 hours of mixing. In some embodiments, the injection is carried out within 4 hours of mixing.

In some embodiments, the effective load is less than 1.5 mg per kilogram of patient. In some embodiments, the effective load is less than 1.0 mg per kilogram of patient. In some embodiments, the effective load is less than 0.5 mg per kilogram of patient.

In some embodiments, the effective load is less than 150 micrograms. In some embodiments, the effective load is less than 100 micrograms or less than 75 micrograms. In some embodiments, the effective load is in the range of 1 to 30 micrograms.

One embodiment provides a pharmaceutical composition comprising an LNP prepared by the method disclosed herein and a pharmaceutically acceptable diluent or excipient.

Another embodiment provides a kit comprising: i) a first aqueous solution containing a plurality of LNPs, the LNPs containing a plurality of ionizable lipids, wherein the first solution substantially contains no destabilizing agent and the difference between the pH of the first solution and the pKa of the ionizable lipids is less than 1.0; and ii) a dried effective load containing nucleic acids or an effective load containing nucleic acids in a second aqueous solution substantially containing no destabilizing agent.

Another embodiment provides a kit comprising a first aqueous solution containing a plurality of LNPs, the LNPs containing a plurality of ionizable lipids, wherein the first solution substantially contains no destabilizing agent, and the difference between the pH of the first solution and the pKa of the ionizable lipids is less than 1.0.

One embodiment provides a kit comprising a first dried composition, comprising: i) a plurality of dried LNPs comprising a plurality of ionizable lipids; and ii) a first solution substantially free of any destabilizing agent, wherein the difference between the pH of the first solution and the pKa of the ionizable lipids is less than 1.0.

In some embodiments, the kit further includes: a dried effective load containing nucleic acids or an effective load containing nucleic acids in a second aqueous solution that is substantially free of any destabilizing agent. In some embodiments, the kit further includes instructions for mixing the first solution with the dried LNP, the dried effective load, the second solution, or a combination thereof.

In some embodiments, when the first solution is mixed with the dried effective load, the second solution, or a combination thereof, most of the effective load is coated with LNP.

In some embodiments, the kit includes one or more unit doses of an effective payload containing nucleic acids. In some embodiments, the unit dose is less than 1.5 mg per kilogram of patient. In some embodiments, the unit dose is less than 1.0 mg per kilogram of patient. In some embodiments, the unit dose is less than 0.5 mg per kilogram of patient. In some embodiments, the unit dose is less than 150 micrograms. In some embodiments, the unit dose is less than 100 micrograms. In some embodiments, the unit dose is less than 75 micrograms. In some embodiments, the unit dose is in the range of 1 to 30 micrograms.

In one embodiment, the ionizable lipid has the following structure: Or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein: ^L1 and ^L2 are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -SS-, -C(=O) _S- , -SC(=O)-, -NR a C(=O)-, -C(=O)NR ^a- , -NR ^a C(=O)NR ^a- , -OC(=O)NR ^a- , ^{-NR a} C(=O)O-, or a direct bond; ^G1 is a _C1 - _C2 alkylene group, -(C=O)-, -O(C=O)-, -SC(=O)-, -NR ^a C(=O)-, or a direct bond; G ² is -C(=O)-, -(C=O)O-, -C(=O)S-, -C(=O) ^NRa- or a direct bond; ^G3 is _C1 - _C6 alkyl; ^Ra is H or _C1 - _C12 alkyl; ^R1a and ^R1b are independently: (a) H or _C1 - _C12 alkyl; or (b) ^R1a is H or _C1 - _C12 alkyl, and ^R1b together with its bonded carbon atom forms a carbon-carbon double bond with the adjacent ^R1b and its bonded carbon atom; ^R2a and ^R2b are independently: (a) H or _C1 - _C12 alkyl; or (b) ^R2a is H or _C1 - _C12 alkyl, and R ^R2b, together with its bonded carbon atom, forms a carbon-carbon double bond with the adjacent ^R2b and its bonded carbon atom; ^R3a and ^R3b, in each occurrence, are independently: (a) H or _C1 - _C12 alkyl; or (b) ^R3a is H or _C1 - _C12 alkyl, and ^R3b , together with its bonded carbon atom, forms a carbon-carbon double bond with the adjacent ^R3b and its bonded carbon atom; ^R4a and ^R4b, in each occurrence, are independently: (a) H or _C1 - _C12 alkyl; or (b) ^R4a is H or _C1 - _C12 alkyl, and ^R4b , together with its bonded carbon atom, forms a carbon-carbon double bond with the adjacent ^R4b and its bonded carbon atom; ^R5 and R ⁶ is independently H or methyl; ^R7 is _C4 - _C20 alkyl; ^R8 and ^R9 are independently _C1 - _C12 alkyl; or ^R8 and ^R9 together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocycle; a, b, c, and d are independently integers from 1 to 24; and x is 0, 1, or 2.

In some embodiments, ^L1 and ^L2 are each independently -O(C=O)-, -(C=O)O-, or direct keys. In other embodiments, ^G1 and ^G2 are each independently -(C=O)- or direct keys. In some different embodiments, ^L1 and ^L2 are each independently -O(C=O)-, -(C=O)O-, or direct keys; and ^G1 and ^G2 are each independently -(C=O)- or direct keys.

In some different embodiments, ^L1 and ^L2 are independently -C(=O)-, -O-, -S(O) _x -, -SS-, -C(=O)S-, -SC(=O)-, -NR a -, -NR ^a C(=O)-, -C(=O)NR ^a -, -NR ^a C(=O)NR ^a , -OC(=O)NR ^{a -} , -NR ^a C(=O)O-, -NR ^a S(O) _x NR ^a -, -NR ^a S(O) _x ^- or -S(O) _x NR ^a -.

In other of the aforementioned embodiments, the ionizable lipids have one of the following structures: or

In any of the foregoing embodiments, one of ^L1 or ^L2 is -O (C=O)-. For example, in some embodiments, each of ^L1 and ^L2 is -O (C=O)-.

In some different embodiments of either of the foregoing, one of ^L1 or ^L2 is -(C=O)O-. For example, in some embodiments, each of ^L1 and ^L2 is -(C=O)O-.

In different embodiments, one of ^L1 or ^L2 is a direct key. As used herein, "direct key" means that a basis (e.g., ^L1 or ^L2 ) does not exist. For example, in some embodiments, each of ^L1 and ^L2 is a direct key.

In the other different embodiments mentioned above, when ^R1a and ^R1b occur at least once, ^R1a is H or _C1 - _C12 alkyl, and ^R1b together with the carbon atom it is bonded to forms a carbon-carbon double bond with the adjacent ^R1b and the carbon atom it is bonded to.

In other different embodiments, when ^R4a and ^R4b occur at least once, ^R4a is H or _C1 - _C12 alkyl, and ^R4b together with the carbon atom it is bonded to forms a carbon-carbon double bond with the adjacent ^R4b and the carbon atom it is bonded to.

In further embodiments, when ^R2a and ^R2b occur at least once, ^R2a is H or _C1 - _C12 alkyl, and ^R2b together with the carbon atom it is bonded to forms a carbon-carbon double bond with the adjacent ^R2b and the carbon atom it is bonded to.

In other different embodiments of any of the foregoing, when ^R3a and ^R3b occur at least once, ^R3a is H or _C1 - _C12 alkyl, and ^R3b together with the carbon atom it is bonded to forms a carbon-carbon double bond with the adjacent ^R3b and the carbon atom it is bonded to.

It should be understood that a "carbon-carbon" double bond refers to one of the following structures: or ^Rc and ^Rd are independently H or substituents in each occurrence. For example, in some embodiments, ^Rc and ^Rd are independently H, _C1 - _C12 alkyl or cycloalkyl in each occurrence, such as H or _C1 - _C12 alkyl.

In various other embodiments, ionizable lipids have one of the following structures: or , where e, f, g and h are each an integer from 1 to 12.

In various implementations, e, f, g, and h are each an integer from 4 to 10.

In some of the foregoing embodiments, a, b, c, and d are each independently an integer from 2 to 12 or from 4 to 12. In other embodiments, a, b, c, and d are each independently an integer from 8 to 12 or from 5 to 9. In some embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In more embodiments, a is 3. In still other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In more embodiments, a is 7. In still other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In more embodiments, a is 11. In other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In many embodiments, a is 15. In other embodiments, a is 16.

In some embodiments, b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In still other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In still other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In still other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In still other embodiments, b is 16.

In some embodiments, c is 1. In other embodiments, c is 2. In more embodiments, c is 3. In still other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In still other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In still other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In more embodiments, c is 15. In still other embodiments, c is 16.

In some embodiments, d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In more embodiments, d is 3. In still other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In more embodiments, d is 7. In still other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In more embodiments, d is 11. In still other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In more embodiments, d is 15. In still other embodiments, d is 16.

In some embodiments, e is 1. In other embodiments, e is 2. In more embodiments, e is 3. In still other embodiments, e is 4. In some embodiments, e is 5. In other embodiments, e is 6. In more embodiments, e is 7. In still other embodiments, e is 8. In some embodiments, e is 9. In other embodiments, e is 10. In more embodiments, e is 11. In still other embodiments, e is 12.

In some embodiments, f is 1. In other embodiments, f is 2. In more embodiments, f is 3. In still other embodiments, f is 4. In some embodiments, f is 5. In other embodiments, f is 6. In more embodiments, f is 7. In still other embodiments, f is 8. In some embodiments, f is 9. In other embodiments, f is 10. In more embodiments, f is 11. In still other embodiments, f is 12.

In some embodiments, g is 1. In other embodiments, g is 2. In more embodiments, g is 3. In still other embodiments, g is 4. In some embodiments, g is 5. In other embodiments, g is 6. In more embodiments, g is 7. In still other embodiments, g is 8. In some embodiments, g is 9. In other embodiments, g is 10. In more embodiments, g is 11. In still other embodiments, g is 12.

In some embodiments, h is 1. In other embodiments, e is 2. In more embodiments, h is 3. In still other embodiments, h is 4. In some embodiments, e is 5. In other embodiments, h is 6. In more embodiments, h is 7. In still other embodiments, h is 8. In some embodiments, h is 9. In other embodiments, h is 10. In more embodiments, h is 11. In still other embodiments, h is 12.

In some other embodiments, a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments, a and d are the same and b and c are the same.

The sum of a and b, and the sum of c and d, are factors that can be varied to obtain lipids with the desired properties. In one embodiment, a and b are chosen such that their sum is an integer in the range of 14 to 24. In other embodiments, c and d are chosen such that their sum is an integer in the range of 14 to 24. In yet another embodiment, the sum of a and b, and the sum of c and d, are the same. For example, in some embodiments, the sum of a and b, and the sum of c and d, are all the same integer in the range of 14 to 24. In still more embodiments, a, b, c, and d are chosen such that the sum of a and b, and the sum of c and d, is 12 or greater.

The substituents at ^R1a , ^R2a , ^R3a , and ^R4a are not specifically limited. In some embodiments, at least one of ^R1a , ^R2a , ^R3a , and ^R4a is H. In some embodiments, ^R1a , ^R2a , ^R3a , and ^R4a are H in each occurrence. In some other embodiments, at least one of ^R1a , ^R2a , ^R3a , and ^R4a is a _C1 - _C12 alkyl group. In some other embodiments, at least one of ^R1a , ^R2a , ^R3a , and ^R4a is a _C1 - _C8 alkyl group. In some other embodiments, at least one of ^R1a , ^R2a , ^R3a , and ^R4a is a _C1 - _C6 alkyl group. In some of the aforementioned embodiments, the _C1 - _C8 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tributyl, n-hexyl, or n-octyl.

In some of the aforementioned embodiments, ^R1a , ^R1b , ^R4a and ^R4b are _C1 - _C12 alkyl groups in each occurrence.

In the other embodiments mentioned above, at least one of ^R1b , ^R2b , ^R3b and ^R4b is H, or ^R1b , ^R2b , ^R3b and ^R4b are H each time they occur.

In some of the foregoing embodiments, R ^1b, together with the carbon atom it bonds, forms a carbon-carbon double bond with adjacent R ^1b and its bonded carbon atom. In other of the foregoing embodiments, R ^4b , together with the carbon atom it bonds, forms a carbon-carbon double bond with adjacent R ^4b and its bonded carbon atom.

In the foregoing embodiments, the substituents at ^R5 and ^R6 are not specifically limited. In some embodiments, one of ^R5 or ^R6 is methyl. In other embodiments, each of ^R5 or ^R6 is methyl.

In the foregoing embodiments, the substituent at ^R7 is not specifically limited. In some embodiments, ^R7 is a _C6 - _C16 alkyl group. In some other embodiments, ^R7 is a _C6 - _C9 alkyl group. In some of these embodiments, R7 is ^substituted with -(C=O) ^ORb , -O(C=O) ^Rb , -C(=O) ^Rb , ^-ORb , -S(O) _xRb , ^-S - ^SRb , -C(=O) ^SRb , -SC(=O) ^Rb , ^-NRaRb , ^-NRaC (=O) ^{Rb ,} -C(=O ^{) NRaRb ,} -NRaC(=O) ^NRaRb , ^-OC (=O) ^NRaRb , ^{-NRaC (} =O) ^ORb , ^-NRaS (O ^)xNRaRb _, ^-NRaS (O) ^xRb or ^-S (O ⁾ _xNRaRb , wherein: ^Ra is ^H or _C1 - _C12 alkyl; ^Rb is _C1 - _C15 alkyl; and x is 0 _, 1 or 2. For example, in some embodiments, ^R7 is replaced by -(C=O)OR ^b or -O(C=O)R ^b .

In all the foregoing embodiments, ^Rb is a branched _C1 - _C15 alkyl group. For example, in some embodiments, ^Rb has one of the following structures: ; ; ; ; or .

In some other of the foregoing embodiments, one of ^R8 or ^R9 is methyl. In other embodiments, both ^R8 and ^R9 are methyl.

In some different embodiments, ^R8 and ^R9, together with the nitrogen atom to which they are attached, form a 5-membered, 6-membered, or 7-membered heterocycle. In some of the aforementioned embodiments, ^R8 and ^R9, together with the nitrogen atom to which they are attached, form a 5-membered heterocycle, such as a pyrrolidone ring. In some of the aforementioned different embodiments, ^R8 and ^R9, together with the nitrogen atom to which they are attached, form a 6-membered heterocycle, such as a piperidine ring.

In other embodiments of the aforementioned ionizable lipids, ^G3 is a _C2 - _C4 alkyl group, such as a _C3 alkyl group.

In various embodiments, ionizable lipids possess one of the structures described in Table A below. Table A. Representative ionizable lipids

In one embodiment, the ionizable lipid has the following structure: Or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein: one of ^L1 or ^L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) _x- , -SS-, -C(=O)S-, SC(=O)-, -NR ^a C(=O)-, -C(=O)NR ^a- , NR ^a C(=O)NR ^a- , -OC(=O)NR ^a- , or -NR ^a C(=O)O-, and the other of ^L1 or ^L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) _x- , -SS-, -C(=O)S-, SC(=O)-, -NR ^a C(=O)-, -C(=O)NR ^a- , NR ^a C(=O)NR ^a- , -OC(=O)NR ^a- , or -NR ^a C(=O)O- or direct bond; ^G1 and ^G2 are each independently unsubstituted _C1 - _C12 alkyl or _C1 - _C12 alkenyl; ^G3 is _C1 - _C24 alkyl, C1-C24 alkenyl, _{C3 - C8} cycloalkyl, or _C3 - _C8 cycloalkyl; ^Ra is H or _C1 - _{C12 alkyl} ; ^R1 and ^R2 are each independently _C6 - _C24 alkyl or _C6 - _C24 alkenyl; ^R3 is H, ^OR5 , CN, -C(=O) ^OR4 , -OC(=O) ^R4 , or ^-NR5 C(=O) ^R4 ; ^R4 is _C1 - _C12 alkyl; ^R5 is H or C ₁ -C ₆ alkyl; and x is 0, 1 or 2.

In some of the aforementioned embodiments, the ionizable lipids have one of the following structures: or Wherein: A is a 3 to 8-membered cycloalkyl or extended cycloalkyl ring; ^R6 is independently H, OH or _C1 - _C24 alkyl in each occurrence; and n is an integer in the range of 1 to 15.

In the other embodiments mentioned above, the ionizable lipids have one of the following structures: or y and z are each an integer in the range of 1 to 12.

In any of the foregoing embodiments, one of ^L1 or ^L2 is -O(C=O)-. For example, in some embodiments, each of ^L1 and ^L2 is -O(C=O)-. In some different embodiments of any of the foregoing embodiments, ^L1 and ^L2 are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments, each of ^L1 and ^L2 is -(C=O)O-.

In some of the aforementioned different embodiments, the ionizable lipids have one of the following structures: or .

In some of the aforementioned embodiments, the ionizable lipids have one of the following structures: ; ; or .

In some of the aforementioned embodiments, n is 2 to 12, for example, an integer in the range of 2 to 8 or 2 to 4. For instance, in some embodiments, n is 3, 4, 5, or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

In some other of the aforementioned embodiments, y and z are each independently an integer in the range of 2 to 10. For example, in some embodiments, y and z are each independently an integer in the range of 4 to 9 or 4 to 6.

In some of the foregoing embodiments, ^R6 is H. In other foregoing embodiments, ^R6 is a _C1 - _C24 alkyl group. In other embodiments, ^R6 is OH.

In some embodiments, ^G3 is unsubstituted. In other embodiments, ^G3 is substituted. In various embodiments, ^G3 is a straight-chain _C1 - _C24 alkyl or a straight-chain _C1 - _C24 alkenyl.

In some other of the foregoing embodiments, ^R1 or ^R2 , or both, are _C6 - _C24 alkenyl groups. For example, in some embodiments, ^R1 and ^R2 each independently have the following structures: In this embodiment, ^R7a and ^R7b are independently H or _C1 - _C12 alkyl groups in each occurrence; and a is an integer from 2 to 12, wherein ^R7a , ^R7b and a are each chosen such that ^R1 and ^R2 each independently contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer in the range of 5 to 9 or 8 to 12.

In some of the foregoing embodiments, ^R7a is H when it appears at least once. For example, in some embodiments, ^R7a is H when it appears in all of its occurrences. In other different foregoing embodiments, ^R7b is a _C1 - _C8 alkyl group when it appears at least once. For example, in some embodiments, the _C1 - _C8 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tributyl, n-hexyl, or n-octyl.

In different embodiments, ^R1 or ^R2 or both have one of the following structures: ; ; ; ; ; ; ; ; ; .

In some of the foregoing embodiments, ^R3 is OH, CN, -C(=O) ^OR4 , -OC(=O) ^R4 , or -NHC(=O) ^R4 . In some embodiments, ^R4 is methyl or ethyl.

In various embodiments, ionizable lipids possess one of the structures described in Table B below. Table B. Representative Ionizable Lipids

In one embodiment, the ionizable lipid has the following structure: Or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein: ^R1 is, where appropriate, a substituted _C1 - _C24 alkyl or a substituted _C2 - _C24 alkenyl; ^R2 and ^R3 are each independently, where appropriate, a substituted _C1 - _C36 alkyl; ^R4 and ^R5 are each independently, where appropriate, a substituted _C1 - _C6 alkyl, or ^R4 and ^R5 are combined with their attached N to form a heterocyclic or heteroaryl group; ^L1 , ^L2 , and ^L3 are each independently, where appropriate, a substituted _C1 - _C18 alkyl; ^G1 is a direct bond, -( _CH2 ) _nO (C=O)-, -( _CH2 ) _n (C=O)O- or -(C=O)-; ^G2 and ^G3 are each independently -(C=O)O- or -O(C=O)-; and n is an integer greater than 0.

In some embodiments, ionizable lipids have the following structure: .

In some embodiments, ionizable lipids have the following structure: .

In some embodiments, ^R1 is a substituted _C6 - _C18 alkyl or _C14 - _C18 alkenyl group, as appropriate. In some embodiments, ^R1 is a _C8 alkyl, _C9 alkyl, _C10 alkyl, _C12 alkyl, _C14 alkyl, or _C16 alkyl group. In some more specific embodiments, ^R1 is a _C16 alkenyl group. In some more specific embodiments, ^R1 is unbranched. In some embodiments, ^R1 is branched. In some embodiments, ^R1 is unsubstituted.

In some embodiments, ^G1 is a direct key -( _CH2 ) _nO (C=O)- or -( _CH2 ) _n (C=O)O-. In some embodiments, ^G1 is a direct key. In some more specific embodiments, ^G1 is -( _CH2 ) _n (C=O)O- and n is greater than 1. In some embodiments, n is 1 to 20. In some embodiments, n is 1 to 10. In some embodiments, n is 5 to 11. In some embodiments, n is 6 to 10. In some more specific embodiments, n is 5, 6, 7, 8, 9, or 10. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10.

In some embodiments, ^L1 is a _C1 - _C6 alkyl group. In some embodiments, ^L1 is a _C2 , _C3 , or _C4 alkyl group. In some more specific embodiments, ^L1 is unbranched. In some more specific embodiments, ^L1 is unsubstituted.

In some embodiments, ^R2 is a _C8 - _C24 alkyl. In some embodiments, ^R3 is a _C8 - _C24 alkyl. In some more specific embodiments, ^{both R2} and ^R3 are _C8 - _C24 alkyl. In some embodiments, ^R2 and ^R3 are each independently a _C11 alkyl, _C12 alkyl, _C13 alkyl, _C14 alkyl, _C15 alkyl, _C16 alkyl, _C18 alkyl, or _C20 alkyl. In some embodiments, ^R2 is branched. In more specific embodiments, ^R3 is branched. In some more specific embodiments, ^R2 and ^R3 each independently have one of the following structures: or Wherein: ^R6 and ^R7 are each independently _C2 - _C12 alkyl.

In some implementations, ^R2 and ^R3 each independently have one of the following structures: ; ; ; ; or .

In some embodiments, ^L2 and ^L3 are each independently _C4 - _C10 alkyl groups. In some embodiments, ^{both L2} and ^L3 are _C5 alkyl groups. In some more specific embodiments, ^{both L2} and ^L3 are _C6 alkyl groups. In some embodiments, ^{both L2} and ^L3 are _C8 alkyl groups. In some more specific embodiments, ^{both L2} and ^L3 are _C9 alkyl groups. In some embodiments, L2 is unbranched. In some embodiments, _L3 is unbranched. In more ^specific embodiments, ^L2 is unsubstituted. In some embodiments, _L2 is unsubstituted.

In some embodiments, ^R4 and ^R5 are each independently a _C1 - _C6 alkyl group. In more specific embodiments, both ^R4 and ^R5 are methyl. In some embodiments, both ^R4 and ^R5 are ethyl. In some embodiments, ^R4 is methyl and ^R5 is n-butyl. In some embodiments, both ^R4 and ^R5 are n-butyl. In various embodiments, ^R4 is methyl and ^R5 is n-hexyl.

In some embodiments, ^R4 and ^R5 , together with the N they are attached to, form a heterocyclic group. In some embodiments, the heterocyclic group is a 5-membered heterocyclic group. In some embodiments, the heterocyclic group has the following structure: .

In various embodiments, ionizable lipids possess one of the structures described in Table C below. Table C. Representative Ionizable Lipids

One embodiment provides an ionizable lipid having the following structure: Or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein: ^L1a and ^L1b are each independently a substituted _C3 - _C12 alkyl group, as appropriate; ^R1a is -C(=O) ^OR4a or -O(C=O) ^R4a ; ^R1b is -C(=O) ^OR4b or -O(C=O) ^R4b ; ^R2 is ^-NR6 (C=O) ^R5 , -(C=O)N( ^R6 ) ^R5 , or -(C=O) ^OR7 ; ^R3 and ^R6 are each independently hydrogen or a substituted _C1 - _C12 alkyl group, as appropriate; ^R4a , ^R4b , and ^R5 are each independently a substituted alkyl group, as appropriate; ^R7 is a substituted _C1 alkyl group, as appropriate. -C ₆ alkyl or, where applicable, substituted aryl alkyl; n1 is 2, 3, 4, 5 or 6; and X is C ₂ -C ₆ alkyl or C ₄ -C ₂₀ alkylene oxide.

In some embodiments, X is: or Where: n2 is 2, 3, 4, 5 or 6; n3 is 0, 1, 2, 3 or 4; n4 is 2, 3 or 4; and n5 is 2, 3, 4 or 5.

In some embodiments, ^L1a is a _C5 - _C9 alkyl. In some embodiments, ^L1b is a _C5 - _C9 alkyl. In some embodiments, ^L1a is a _C5 , _{C6 , C7} , or _C9 alkyl. In some embodiments, ^L1b is a _C5 alkyl. In some embodiments, ^L1a is _{a C6} alkyl. In some embodiments, ^L1a is a _C7 alkyl. In some embodiments, ^L1a is _{a C9} alkyl. In some embodiments, ^L1b is _{a C5} alkyl. In some embodiments, ^L1b is a _C6 alkyl. In some embodiments, ^L1b is a _C7 alkyl. In some embodiments, ^{L1b is} a _C9 alkyl. In some embodiments, ^L1a is not substituted. In some embodiments, ^L1b is not substituted. In some embodiments, ^L1a is a non-branched chain. In some embodiments, ^L1b is a non-branched chain.

In some embodiments, one of ^R1a is -O(C=O) ^R4a . In some embodiments, one of ^R1a is -(C=O) ^OR4a . In some embodiments, ^R1b is -O(C=O) ^R4b . In some embodiments, one of ^R1b is -(C=O) ^OR4b .

In some embodiments, ^R4a is a _C8 - _C24 alkyl. In some embodiments, ^R4a is a _C10 - _C18 alkyl. In some embodiments, ^R4a is a _C11 - _C16 alkyl. In some embodiments, ^R4a is a _C11 alkyl. In some embodiments, ^R4a is a _C15 alkyl. In some embodiments, ^R4a is a _C16 alkyl. In some embodiments, ^R4b is a _C8 - _C24 alkyl. In some embodiments, ^R4b is a _C10 - _C18 alkyl. In some embodiments, ^R4b is a _C11 - _C16 alkyl. In some embodiments, ^R4b is a _C11 alkyl. In some embodiments, ^R4b is a _C15 alkyl. In some embodiments, ^R4b is a _C16 alkyl.

In some embodiments, R ^4a is a branched chain. In some embodiments, R ^4b is a branched chain. In some embodiments, R ^4a is not substituted. In some embodiments, R ^4b is not substituted. In some embodiments, R ^4a has one of the following structures: ; ; ; ; or .

In some embodiments, R ^4b has one of the following structures: ; ; ; ; or .

In some embodiments, ^R2 is ^-NR6 (C=O) ^R5 . In some embodiments, ^R2 is -(C=O)N( ^R6 ) ^R5 . In some embodiments, ^R5 is _C2 - _C16 alkyl. In some embodiments, ^R5 is _C4 - _C13 alkyl. In some embodiments, ^R5 is _C4- , _C7- , _C8- , _C10- , or _C13 -alkyl. In some embodiments, ^R5 is unsubstituted. In some embodiments, ^R5 is hydroxylated. In some embodiments, ^R5 is branched. In some embodiments, ^R5 is unbranched. In some embodiments, ^R5 has one of the following structures: ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; or .

In some embodiments, ^R5 is a non-branched chain. In some embodiments, ^R5 has one of the following structures: ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; or .

In some embodiments, ^R6 is a _C1 - _C6 alkyl. In some embodiments, ^R6 is a _C1 - _C10 alkyl. In some embodiments, ^R6 is a _C1 - _C4 alkyl. In some embodiments, ^R6 is a _C1- , _C2- , _C3- , _C6- , _C8- , or _C10 -alkyl. In some embodiments, ^R6 is methyl, ethyl, n-butyl, n-hexyl, n-octyl, or n-decyl. In some embodiments, ^R6 is unbranched. In some embodiments, R6 is methyl or n-butyl. In some embodiments, ^R6 is unsubstituted. In some embodiments, ^R6 is substituted. In some embodiments, ^R6 is ^a _C1 - _C6 alkyl group substituted with one or more hydroxyl groups. In some embodiments, ^R6 is a _C2 , _C3 , _C4 , or _C6 alkyl group substituted with one or more hydroxyl groups. In some embodiments, ^R6 is hydrogen. In some embodiments, ^R2 is -(C=O) ^OR7 .

In some embodiments, ^R7 is a _C1 - _C3 alkyl or a _C7 - _C16 arylalkyl. In some embodiments, ^R7 is a _C7 - _C16 arylalkyl. In some embodiments, ^R7 is a _C1 - _C3 alkyl. In some embodiments, ^R7 is unsubstituted.

In some embodiments, R ⁷ is -CH ₃ or has the following structure: .

In some implementations, R ⁷ has the following structure: .

In some embodiments, ^R3 is a substituted _C1 - _C6 alkyl group, as appropriate. In some embodiments, ^R3 is a substituted methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tributyl, or n-hexyl group, as appropriate. In some embodiments, ^R3 is a substituted methyl group, as appropriate. In some embodiments, ^R3 is a _C1 - _C6 alkyl group substituted with one or more hydroxyl groups. In some embodiments, ^R3 is a _C2 or _C4 alkyl group substituted with one or more hydroxyl groups. In some embodiments, ^R3 is unsubstituted. In some embodiments, ^R3 is hydrogen.

In some embodiments, X is For example, in some states, ionizable lipids have the following structure: or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. For example, in some of these embodiments, n2 is 3, 4, or 5.

In other embodiments, X is In some of these embodiments, ionizable lipids have the following structure: Or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. In different embodiments, n3 is 0 or 1. In other embodiments, n4 is 2 or 3. In some other different embodiments, n5 is 3.

In some embodiments, n1 is 3, 4, or 5. In some embodiments, n1 is 2.

In some embodiments, the ionizable lipids have one of the structures described in Table D below, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof . Table D. Representative ionizable lipids Number Structure D-1 D-2 D-3 D-4 D-5 D-6 D-7 D-8 D-9 D-10 D-11 D-12 D-13 D-14 D-15 D-16 D-17 D-18 D-19 D-20 D-21 D-22 D-23 D-24 D-25 D-26 D-27 D-28 D-29 D-30 D-31 D-32 D-33 D-34 D-35 D-36 D-37 D-38 D-39 D-40 D-41 D-42 D-43 D-44 D-45 D-46 D-47 D-48 D-49 D-50 D-51 D-52 D-53

The LNP prepared according to this disclosure can be used to deliver therapeutic agents, such as nucleic acids. The components of the LNP are present in an amount that can effectively form the LNP, encapsulate the therapeutic agent (e.g., nucleic acid) under mild conditions, and deliver the therapeutic agent. In some embodiments, delivery is helpful for treating specific diseases or symptoms of concern. Appropriate concentrations and dosages can be easily determined by those skilled in the art.

One embodiment provides a composition comprising lipid nanoparticles, a therapeutic agent, and, optionally, additional lipid adjuvants. In some embodiments, the composition further comprises one or more components selected from ionizable lipids, neutral lipids, steroids, and bound polymers.

In some embodiments, the treatment contains nucleic acids. In some embodiments, the nucleic acids are selected from antisense RNA and messenger RNA. In some embodiments, the nucleic acids are mRNA. In some embodiments, the combination is a vaccine (e.g., a COVID-19 vaccine).

Exemplary ionizable lipids (e.g., cationic lipids) and their synthesis can be found in the following publications: US Patent No. 9,738,593; US Patent No. 10,221,127; US Patent No. 10,166,298; US Patent No. 11,357,856; US Patent No. 11,712,481; US Patent No. 11,453,639; US Patent Publication No. 2018/0185516; US Patent No. 2022/0106257; PCT Publication No. WO 2017/117528; WO 2016/176330; WO 2018/191719; WO 2018/200943; WO The contents of the following WOs are hereby incorporated by reference: WO 2019/036000; WO 2019/036028; WO 2019/036030; WO 2019/036008; WO 2019/089828; WO 2020/061426; WO 2020/081938; WO 2021/030701; WO 2023/114944; WO 2023/114939; WO 2023/114943.

Lipids (e.g., ionizable lipids) and methods for their preparation are disclosed in, for example, U.S. Patent Nos. 8,569,256 and 5,965,542; and U.S. Patent Publications Nos. 2016/0199485, 2016/0009637, 2015/0273068, 2015/0265708, 2015/0203446, 2015/0005363, 2014/0308304, 2014/0200257, 2013/086373, 2013/0338210, 2013/0323269, and others. US No. 2013/0245107, US No. 2013/0195920, US No. 2013/0123338, US No. 2013/0022649, US No. 2013/0017223, US No. 2012/0295832, US No. 2012/0183581, US No. 2012/0172411, US No. 2012/0027803, US No. 2012/0058188, US No. 2011/0311583, US No. 2011/0311582, US No. 2011/0262527, US No. 2011/0216622, US No. US No. 2011/0117125, US No. 2011/0091525, US No. 2011/0076335, US No. 2011/0060032, US No. 2010/0130588, US No. 2007/0042031, US No. 2006/0240093, US No. 2006/0083780, US No. 2006/0008910, US No. 2005/0175682, US No. 2005/017054, US No. 2005/0118253, US No. 2005/0064595, US No. 2004/0142025, US No. The entire contents of the following disclosures are incorporated herein by reference in their entirety for all purposes: No. 2007/0042031, No. 1999/009076; and PCT disclosures No. 99/39741, No. 2017/004143, No. 2017/075531, No. 2015/199952, No. 2014/008334, No. 2013/086373, No. 2013/086322, No. 2013/016058, No. 2013/086373, No. 2011/141705 and No. 2001/07548.

Other illustrative lipids and their manufacture are described in this art, for example, in the following publications: U.S. Patent Application Publication No. US 2012/0276209, Semple et al., 2010, Nat Biotechnol., 28(2):172-176; Akinc et al., 2010, Mol Ther., 18(7): 1357-1364; Basha et al., 2011, Mol Ther., 19(12): 2186-2200; Leung et al., 2012, J Phys Chem C Nanomater Interfaces, 116(34): 18440-18450; Lee et al., 2012, Int J Cancer., 131(5): E781-90; Belliveau et al., 2012, Mol Ther nucleic acid Acids, 1: e37; Jayaraman et al., 2012, Angew Chem Int Ed Engl., 51(34): 8529-8533; Mui et al., 2013, Mol Ther Nucleic Acids. 2, e139; Maier et al., 2013, Mol Ther., 21(8): 1570-1578; and Tam et al., 2013, Nanomedicine, 9(5): 665-74, each incorporated herein by reference in its entirety. Lipids and their manufacture can be found, for example, in U.S. Patent Application Publications 2015/0376115 and 2016/0376224, both of which are incorporated herein by reference.

In some embodiments, the lipid nanoparticles comprise one or more neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the neutral lipid is DSPC. In some embodiments, the molar ratio of ionizable lipids to neutral lipids is in the range of about 2:1 to about 8:1. In some embodiments, the steroid is cholesterol. In some embodiments, the molar ratio of ionizable lipids to cholesterol is in the range of about 2:1 to about 1:1. In some embodiments, the molar ratio of ionizable lipids to cholesterol is in the range of about 5:1 to about 1:1 or from about 2:1 to about 1:1.

In some embodiments, the lipid in the polymer is a polyethylene glycol-modified lipid. In various embodiments, the lipid in the polymer is a polyethylene glycol-modified lipid. For example, some embodiments include polyethylene glycol-modified diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristylglycerol (PEG-DMG); polyethylene glycol-modified phosphatidylethanolamine (PEG-PE); PEG-S-DAG, such as 4-O-(2',3'-bis(tetradecyloxy)propyl-1-O-( ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG); polyethylene glycol-modified ceramide (PEG-cer); or PEG dialkoxypropyl aminocarbamate, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecoxy)propyl)aminocarbamate or 2,3-di(tetradecoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)aminocarbamate.

In some embodiments, the molar ratio of the ionizable lipid to the polyethylene glycol-modified lipid is in the range of about 100:1 to about 10:1 or about 100:1 to about 25:1. In some embodiments, the molar ratio of the ionizable lipid to the polyethylene glycol-modified lipid is in the range of about 100:1 to about 20:1 or about 100:1 to about 10:1. In some embodiments, the polyethylene glycol-modified lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, or PEG dialkoxypropylcarbamate.

In some embodiments, the lipid nanoparticles comprise at least one polyethylene glycol-modified lipid having the following structure: Or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: ^R10 and ^R11 are each independently a straight-chain or branched-chain alkyl, alkenyl or ynyl group containing 10 to 30 carbon atoms, wherein each alkyl, alkenyl or ynyl group is, where appropriate, substituted with at least one fluorine atom; and z is an integer in the range of 30 to 60.

In some embodiments, ^R10 and ^R11 are each independently a linear alkyl group containing 12 to 16 carbon atoms, wherein each alkyl group is, where appropriate, substituted with at least one fluorine atom. In some embodiments, ^R10 and ^R11 are each independently a linear alkyl group containing 12 to 16 carbon atoms.

In some implementations, ^R4 and ^R5 are independent: , ,or .

In some embodiments, z is an integer in the range of 45 to 50. In some embodiments, z is an integer in the range of 42 to 48. In some embodiments, at least one polyethylene glycol ester has the following structure ("PEG ester 1"): "PEG lipid 1" or its pharmaceutically acceptable salt or stereoisomer.

In some embodiments, the lipid nanoparticles comprise multiple polyethylene glycol-modified lipids. In some embodiments, the z-mean value of the multiple polyethylene glycol-modified lipids is in the range of 40 to 55. In some embodiments, the z-mean value of the multiple polyethylene glycol-modified lipids is in the range of 40 to 50 or 42 to 48. In some embodiments, the z-mean value of the multiple polyethylene glycol-modified lipids is in the range of 30 to 55, 30 to 50, 30 to 45, 30 to 40, or 30 to 35. In some embodiments, the z-mean value of the multiple lipids is in the range of 35 to 55, 40 to 55, 42 to 55, 45 to 55, or 48 to 55.

The synthesis of polyethylene glycol-modified lipids can be found in U.S. Patent No. 9,738,593, the disclosure of which is incorporated herein by reference.

As used herein, “mol percentage”, “mol%” or “mol%” refers to the percentage of a component relative to the total number of moles of all components in the lipid nanoparticles other than the therapeutic agent (e.g., the total number of moles of ionizable lipids, neutral lipids, steroids and/or lipids bound to polymers).

In some embodiments, the ionizable lipids are present at concentrations ranging from about 20 to about 70 mol% of lipid nanoparticles. In some embodiments, the ionizable lipids are present at concentrations ranging from about 35 to about 70 mol%, about 40 to about 60 mol%, about 45 to about 50 mol%, about 45 to about 49 mol%, about 40 to about 55 mol%, or about 46 to about 48 mol% of lipid nanoparticles.

In some embodiments, the neutral lipids are present at a concentration of about 5 to about 15 mol% of lipid nanoparticles. In some embodiments, the neutral lipids are present at a concentration of about 7 to about 12 mol%, about 6 to about 11 mol%, or about 8 to about 13 mol% of lipid nanoparticles.

In some embodiments, the steroid is present at a concentration of about 30 to about 60 mol% of lipid nanoparticles. In some embodiments, the steroid is present at a concentration of about 40 to about 50 mol%, about 41 to about 49 mol%, or about 46 to about 44 mol%.

In some embodiments, the concentration of the polyethylene glycol-modified lipid is in the range of about 3.5 to about 5.5 mol% of the lipid nanoparticles. In some embodiments, the concentration of the polyethylene glycol-modified lipid is in the range of about 1.0 to about 3.0 mol% of the lipid nanoparticles. In some embodiments, the concentration of the polyethylene glycol-modified lipid is in the range of about 1.0 to about 2.5 mol% of the lipid nanoparticles.

The administration of the lipid nanoparticles disclosed herein can be carried out via any recognized administration method used to provide similar efficacy. The pharmaceutical compositions disclosed herein can be formulated into solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalers, gels, microspheres, and aerosols. Typical routes of administration for such pharmaceutical compositions include (but are not limited to) oral, topical, percutaneous, inhalation, non-enteric, sublingual, buccal, rectal, vaginal, and intranasal administration. As used herein, non-enteric administration includes subcutaneous, intravenous, intramuscular, intradermal, and intrasternal injection or infusion techniques. The pharmaceutical compositions disclosed herein are formulated to allow for bioavailability of the active ingredient contained therein when administered to an individual. The compositions administered to an individual or patient are in the form of one or more dosage units, wherein, for example, tablets may be a single dosage unit, and containers for the disclosed compounds in aerosol form may hold multiple dosage units. The actual methods used to prepare such dosage forms are known to those skilled in the art, or will be obvious to those skilled in the art; see, for example, Remington : The Science and Practice of Pharmacy , 20th edition (Philadelphia College of Pharmacy and Science, 2000). In any event, the composition to be administered will contain a therapeutically effective amount of the therapeutic agent of this disclosure (e.g., nucleic acid) or its medically acceptable salt, for the treatment of the disease or condition of concern in accordance with the teachings of this disclosure.

In some implementations, the administration is localized (e.g., injected directly into the eye or brain).

Some embodiments provide pharmaceutical compositions comprising lipid nanoparticles (e.g., dried or in aqueous solution) as defined herein. Some embodiments provide pharmaceutical compositions comprising lipid nanoparticles (e.g., dried or in aqueous solution) and an effective load (e.g., dried or in aqueous solution) as defined herein. Some other embodiments provide pharmaceutical compositions comprising an effective load (e.g., dried or in aqueous solution) as defined herein. The composition may be prepared, packaged, or marketed in bulk, in a single unit dose, or in multiple single unit doses. As used herein, a “unit dose” is a dispersed amount of a pharmaceutical composition containing a pre-quantitative amount of active ingredient (e.g., mRNA). The amount of active ingredient is generally equal to the dose of active ingredient to be administered to an individual, or a suitable portion of such doses, such as one-half or one-third of such doses. Administration can be performed by syringe injection or intravenous infusion, and the unit dose can be contained in a suitable container (e.g., bottle, vial, ampoule, bag, etc.). The unit dose can be administered over a short period of time (e.g., less than 5 seconds or less than 30 seconds) or over a longer period of time (e.g., over several hours or days). In some embodiments, the unit dose contains an effective mRNA and is administered as a single injection. In some embodiments, the unit dose is less than 100 micrograms. In some embodiments, the unit dose is less than 100 mg/kg (patient weight). In some embodiments, the unit dose is contained in a syringe.

In some embodiments, the disclosed pharmaceutical composition is in the form of an aqueous solution. In some embodiments, the aqueous solution contains one or more carriers, which may be liquid or dissolved in a liquid. In some embodiments, the carrier facilitates aerosol delivery, which can be used, for example, for inhalation administration. In some embodiments, administration is performed by inhalation and the composition is aerosolized. In some embodiments, the composition is administered to treat surfaces with mucous membranes.

In some embodiments, administration targets extrahepatic tissue by prolonging circulation. In some embodiments, administration targets extrahepatic tissue, and the LNP includes a targeting component (e.g., an antibody).

In some embodiments, the pharmaceutical composition is in liquid form (e.g., aqueous solution), such as an elixir, syrup, solution, emulsion, or suspension. As two examples, the liquid can be delivered by intravenous or intramuscular injection. The composition intended to be administered by injection may include one or more surfactants, preservatives, wetting agents, dispersants, suspensions, buffers, stabilizers, isotonic agents, or combinations thereof.

The liquid pharmaceutical compositions disclosed herein, whether in solution, suspension or similar form, may include one or more of the following adjuvants: sterile diluents, such as water for injection, saline solution (preferably physiological saline), Ringer's solution. Solutions), isotonic sodium chloride; nonvolatile oils, such as synthetic monoglycerides or diglycerides, polyethylene glycol, glycerol, propylene glycol or other solvents that can be used as solvents or suspension media; antibacterial agents, such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates; and reagents for adjusting tension, such as sodium chloride or dextrose; reagents used as cryoprotectants, such as sucrose or trehalose. Non-enteric preparations can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. Injectable pharmaceutical compounds are preferably sterile.

In some embodiments, the liquid pharmaceutical composition of this disclosure intended for non-enteral administration should contain a certain amount of the therapeutic agent of this disclosure to obtain an appropriate dosage.

The pharmaceutical compositions disclosed herein are intended for topical administration, in which case the loading agent may suitably comprise a solution, emulsion, ointment, or gel matrix. For example, the matrix may comprise one or more of the following: paraffin, lanolin, polyethylene glycol, beeswax, mineral oil, thinners (such as water and alcohol), emulsifiers, and stabilizers. Thickeners may be present in pharmaceutical compositions intended for topical administration. If intended for transdermal administration, the composition may comprise a transdermal patch or iontophoresis device.

The pharmaceutical compositions disclosed herein are intended for rectal administration, for example in suppository form, which dissolves in the rectum and releases the drug. Compositions intended for rectal administration may contain an oily matrix as a suitable, non-irritating excipient. Such matrices include (but are not limited to) lanolin, cocoa butter, and polyethylene glycol.

The pharmaceutical compositions disclosed herein may also include agents that bind to the surface of the lipid nanoparticles disclosed herein and thereby facilitate the delivery of the therapeutic agent. Suitable agents that can exert this capability include monoclonal or polyclonal antibodies or proteins.

The pharmaceutical compositions disclosed herein can be composed of dosage units that can be administered in aerosol form. The term aerosol is used to refer to a variety of systems ranging from colloidal properties to systems consisting of pressurized encapsulation. Delivery can be carried out by liquefying or compressing gas or by a suitable pump system for dispensing the active ingredient. The aerosols of the disclosed compounds can be delivered in single-phase, two-phase, or three-phase systems to deliver the active ingredient. Aerosol delivery includes the necessary containers, starters, valves, sub-containers, and the like, which can be assembled together. Those skilled in the art can determine the optimal aerosol without excessive experimentation.

The pharmaceutical compositions disclosed herein can be prepared using methods well known in pharmaceutical technology. For example, pharmaceutical compositions intended for injection can be prepared by combining the disclosed lipid nanoparticles with sterile distilled water or other carriers to form a solution. Surfactants may be added to promote the formation of a homogeneous solution or suspension. The surfactant is a compound that non-covalently interacts with the disclosed compound to promote the dissolution or homogeneous suspension of the compound in an aqueous delivery system.

The therapeutically effective dose of the disclosed combination or its pharmaceutically acceptable salt will vary depending on a number of factors, including the activity of the particular treatment used; the metabolic stability and duration of action of the treatment; the patient's age, weight, general health, sex and diet; the administration method and time; the rate of excretion; the combination of drugs; the severity of the particular disease or symptom; and the individual undergoing the treatment.

The disclosed combination may also be administered concurrently with, before, or after one or more other therapeutic agents. Such combination therapies include single pharmaceutical dosage forms administering the disclosed combination and one or more additional active agents, as well as separate pharmaceutical dosage forms administering the disclosed combination and each active agent individually. For example, the disclosed combination and other active agents may be administered to the patient together in a single dose combination (e.g., injection), or each agent may be administered in a separate dosage form. When using separate dosage forms, the disclosed compound and one or more additional active agents may be administered at substantially the same time, i.e., simultaneously, or at staggered times, i.e., sequentially; combination therapies should be understood to include all such options.

The methods for preparing the above compounds and compositions are described herein and/or are known in the art.

Those skilled in the art will understand that in the methods described or incorporated herein, the functional groups of intermediate compounds may require protection by suitable protecting groups. These functional groups include hydroxyl , amino, tert-hydroxyl, and carboxylic acids. Suitable protecting groups for hydroxyl groups include trialkylsilyl or diarylalkylsilyl (e.g., tributyldimethylsilyl, tributyldiphenylsilyl, or trimethylsilyl), tetrahydropiperanyl, benzyl, and similar groups. Suitable protecting groups for amino, formamidinyl, and guanidine groups include tributoxycarbonyl, benzoxycarbonyl, and similar groups. Suitable protecting groups for hydroxyl groups include -C(O)-R'' (where R" is alkyl, aryl, or arylalkyl), p-methoxybenzyl, triphenylmethyl, and similar groups. Suitable protecting groups for carboxylic acids include alkyl, aryl, or arylalkyl esters. Protecting groups may be added or removed according to standard techniques known to those skilled in the art and described herein. The use of protecting groups is described in detail in Green, TW, and PGM Wutz, Protective Groups in Organic Synthesis (1999), 3rd edition, Wiley. As will be known to those skilled in the art, protecting groups may also be polymer resins, such as Wang resin, Rink resin, or 2-chlorotriphenylmethyl chlororesin.

Furthermore, all the disclosed compounds existing in free base or acid form can be processed by methods known to those skilled in the art, using appropriate inorganic or organic bases or acids, to convert them into their pharmaceutically acceptable salts. The salts of the lipid components disclosed herein can be converted into their free base or acid forms using standard techniques.

It should be understood that those skilled in the art may be able to prepare the ionizable lipids disclosed or incorporated herein by using the methods mentioned or by combining the methods mentioned with methods known to those skilled in the art. Generally, the starting components used to prepare the ionizable lipids may be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Maybridge, Matrix Scientific, TCI, and Fluorochem USA, or synthesized from sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Edition (Wiley, December 2000)), or prepared as described in this disclosure.

Example 1: Formation of Lipid Nanoparticles The following parameters and procedures are used to test the nucleic acid loading of lipid nanoparticles after their formation (i.e., not during or concurrent with the lipid nanoparticle formation process, hence the term "post-formulation loading," "post-formation loading," or "PFL"). More specifically, for the PFL method, LNPs are formed, the lipid solubilizer (e.g., ethanol) required in the formation step is removed, and then the resulting LNPs are combined with the effective payload (e.g., mRNA) under specific conditions to encapsulate the effective payload. Different loading methods and procedures, as well as different post-loading neutralization methods, are used for these experiments.

The LNP sample for reference was prepared using a standard method with specific variations described herein. Such general methods are as described elsewhere (e.g., U.S. Patent No. 11,453,639) and with the variations specified herein.

A) Preparation of empty LNP: Individual stock solutions of each lipid were prepared by weighing powders of distearate phosphatidylcholine (DSPC), cholesterol, and PEG lipid 1 and dissolving them in ethanol to achieve a lipid concentration of approximately 10 mg/mL. Using these stock solutions, a mixture containing lipids was prepared by adding an appropriate amount of the stock solution to 27.6 mg of pure ionizable lipid C-18 oil, with a molar ratio of ionizable lipid C-18/DSPC/cholesterol/PEG lipid 1 of 47.5:10:40.7:1.8. The final lipid mixture was diluted with ethanol to achieve a final total lipid concentration of 11.83 mM.

B) Preparation of effective mRNA payload: The mRNA encoding hAAT was thawed at room temperature, and the RNA concentration was verified by measuring its absorbance at 260 nm, assuming a conversion factor of 40 µg/OD260. The measured concentration was 1.08 mg/mL. The RNA was then diluted in 10 mM phosphate buffer at pH 7.4 to achieve a final concentration of 0.100 mg/mL.

C) Preparation of LNPs with RNA Storage Solution at Neutral pH: LNPs are prepared by mixing a lipid solution with an aqueous RNA solution diluted in 10 mM phosphate buffer at pH 7.4, as commonly described elsewhere (e.g., U.S. Patent No. 11,453,639). In short, a T-type mixing system is used to combine solutions at a 3:1 ratio of aqueous to organic phase (i.e., RNA solution to lipid solution) with an aqueous flow rate of 30 mL/min and an organic flow rate of 10 mL/min, respectively. As shown in Table 1 below, when LNPs are formed with RNA storage solution at pH 7.4 as described, the RNA is not coated during the mixing process, and the resulting LNP suspension consists of empty LNPs and RNA in the external solution of the LNPs. The mixture of empty LNP and external RNA was divided into two aliquots and dialyzed in 1.5 L of 10 mM phosphate buffer (pH 7.4) for 1.5 hours. The first aliquot (amplitude 1 in Table 1) was transferred to 6 L of Durbecco's phosphate-buffered saline (DPBS) (pH 7.4) and dialyzed until the next day. The second aliquot (amplitude 2 in Table 1) was transferred to 2 L of 25 mM acetate buffer (pH 5.9) and dialyzed for 11.5 hours, then transferred to 1 L of DPBS and dialyzed for 6 hours. After the dialysis procedure of aliquot 2 was completed, the two LNP formulations were removed from the dialysis chamber and filtered (0.2 µm), followed by analysis to determine particle size (by dynamic light scattering) and RNA coating efficiency (by Ribogreen). The results are shown in Table 1 below. Table 1: Effect of intermediate dialysis at acetate pH 5.9 on post-load formation Sample Description Z- mean (nm) PDI Coating efficiency (%) Aliquot 1: LNP was prepared using DPBS pH 7.4 and then dialyzed using PBS pH 7.4. 62 0.059 0 Aliquot 2: LNPs were prepared using DPBS pH 7.4, and dialyzed with acetate pH 5.9 before final dialysis with DPBS pH 7.4. 117 0.096 95

This data indicates that under neutral pH conditions, the merging of lipids and RNA does not lead to encapsulation during the mixing process, but exposing the resulting mixture to lower pH conditions leads to encapsulation after the initial formation of LNPs. Furthermore, this method of RNA encapsulation does not require any additional auxiliary means, such as solvents, surfactants, or temperature increases.

Example 2A) Preparation of Empty LNP: Individual stock solutions of each lipid were prepared by weighing powders of distearate phospholipid choline (DSPC), cholesterol, and PEG lipid 1 and dissolving them in ethanol to achieve a lipid concentration of approximately 10 mg/mL. Using these stock solutions, a mixture containing lipids was prepared by adding an appropriate amount of the stock solution to 30.3 mg of pure ionizable lipid C-18 oil, with a molar ratio of ionizable lipid C-18/DSPC/cholesterol/PEG lipid 1 of 47.5:10:40.7:1.8. The final lipid mixture was diluted with ethanol to achieve a final total lipid concentration of 11.83 mM.

B) Preparation of effective mRNA payload: The mRNA encoding firefly luciferase was thawed at room temperature, and the RNA concentration was verified by measuring its absorbance at 260 nm, assuming a conversion factor of 40 µg/OD260. The measured concentration was 1.04 mg/mL. Subsequently, the RNA was diluted in 10 mM phosphate buffer at pH 7.4 to achieve a final concentration of 0.100 mg/mL.

C) LNP preparation is performed by mixing a lipid solution with an aqueous RNA solution diluted in 10 mM phosphate buffer at pH 7.4, as commonly described elsewhere (e.g., U.S. Patent No. 11,453,639). In short, a T-type mixing system is used to combine solutions at a 3:1 ratio of aqueous to organic phase (i.e., RNA solution to lipid solution) with an aqueous flow rate of 30 mL/min and an organic flow rate of 10 mL/min, respectively. As demonstrated in Preparation Example 1 and in the first column of Table 2 below, when LNPs are formed with the RNA storage solution at pH 7.4 as described, the RNA is not coated during the mixing process, and the resulting LNP suspension consists of an empty LNP and RNA in the external solution of the LNP. The mixture of empty LNPs and external RNA was divided into five aliquots and dialyzed for 2 hours in 1.4 L of 10 mM phosphate-buffered saline (pH 7.4) using 12–14 kDa regenerated cellulose dialysis tubes. After 2 hours of dialyzing in 10 mM phosphate-buffered saline (pH 7.4), one aliquot was transferred to 5 L of DPBS, and the remaining four aliquots were transferred to 2 L of 25 mM acetate-buffered saline (pH 5.9). At each time point shown in Table 2, 200 µL aliquots of one of the LNP samples dialyzed in 25 mM acetate-buffered saline were used for in-process analysis of particle size, coating efficiency, and pH. The remaining LNPs were transferred to DPBS (pH 7.4) and dialyzed overnight. Approximately 16 hours later (i.e., overnight dialysis), all LNP formulations were collected from dialysis and filtered (0.2 µm) for subsequent analysis to determine particle size (by dynamic light scattering) and RNA coating efficiency (by Ribogreen). The results of the intermediate dialysis phase after dialysis in acetate at pH 5.9, and the corresponding final product analysis after dialysis in DPBS, are shown in Table 2. Table 2: Effect of intermediate dialysis time in acetate at pH 5.9 After intermediate dialysis in acetate at pH 5.9 After final dialysis in PBS pH 7.4 Acetate pH 5.9 Dialysis time ( min) Z- mean (nm) PDI Coating efficiency (%) Sample pH Z- mean (nm) PDI Coating efficiency (%) 0 n/a n/a n/a n/a 55 0.029 3 30 61 0.054 30 7.00 61 0.042 twenty four 60 62 0.052 51 6.78 69 0.036 53 120 70 0.048 93 6.16 71 0.002 93 180 70 0.060 95 6.00 71 0.021 93

This data suggests that nucleic acid coating can be achieved after LNP formation by exposing a mixture of empty LNPs and external RNA to an acidic pH. RNA coating occurs without any additional aids (such as surfactants, solvents, or increased temperature), or it may not occur simultaneously with the initial formation of LNPs.

Data shows that the pH of the sample decreased with dialysis time in acetate at pH 6. This was related to a significant increase in coating and a slight increase in particle size, but had no effect on polydispersity, which remained at a uniformly low level.

Formulation Example 3 : Formation of Lipid Nanoparticles A) Preparation of Empty LNPs A lipid mixture was prepared by appropriately adding individual lipid reserve solutions (10 mg/mL) to ionizable lipids. The final lipid mixture contained lipids in a molar ratio of 47.5:10:40.7:1.8 (ionizable lipid C-18, DSPC, cholesterol, PEG lipid 1), with a total lipid concentration of 23.65 mM. To prepare empty LNPs, a 25 mM acetate buffered aqueous solution at pH 5.9 (i.e., RNA-free) and a lipid solution were combined at a 3:1 ratio of aqueous solution to organic solution (i.e., buffered aqueous solution to organic lipid solution) using a T-type mixing system, with an aqueous solution flow rate of 30 mL/min and an organic solution flow rate of 10 mL/min. The LNP suspension was collected and allowed to stand at room temperature for approximately 15 minutes. Ethanol was then removed by overnight dialyzing of empty LNPs in 25 mM acetate buffer (pH 5.9), 200 times the volume of the LNP suspension. After dialyzing, the empty LNPs were filtered (0.2 µm) and analyzed to determine their physical properties. The empty LNP samples were stored at 2°C to 8°C for two days, and particle size and lipid concentration were subsequently analyzed. Total lipid concentration was calculated as follows: cholesterol concentration was first determined using cholesterol E enzyme assay, and then calculated using the theoretical molar ratio of cholesterol to all lipid components. Total lipid concentration is used to calculate the amount of lipid required to prepare RNA-carrying LNPs at an amino lipid nitrogen to nucleic acid phosphate (N:P) ratio of 6.

B) Loading LNPs with mRNA: The mRNA encoding firefly luciferase was thawed at room temperature, and the absorbance at 260 nm showed a concentration of 1.09 mg/mL, assuming a conversion factor of 40 µg/OD260 nm. The empty LNP solution was diluted in 25 mM acetate buffer (pH 5.9) to achieve a total lipid concentration of 3.17 mM. RNA (0.686 mL, 1.09 mg/mL) was added to the LNP solution by pipetting, followed by gently inverting the tube several times. This produced an RNA-LNP mixture with a total lipid concentration of 2.96 mM and an RNA concentration of 0.075 mg/mL. The RNA-LNP mixture was incubated at room temperature, and aliquots (1 mL) were obtained at 10, 30, 50, 70, and 90 minutes. At each time point, the RNA-LNP aliquots were added to 19 mL of 1×DPBS. The PBS-diluted RNA-LNPs were slowly mixed by inverting the container, and the particle size and RNA coating of the samples were then analyzed (Table 3). In addition to this dilution process, at time points of 10, 30, 70, and 90 minutes, other RNA-LNP aliquots (1 mL) were transferred to dialysis bags and dialyzed in DPBS (200-fold volume). The remaining empty LNPs were also dialyzed in PBS from time 0. The dialysis process was performed overnight at room temperature with stirring. The empty LNP formulation was then filtered (0.2 µm) and analyzed to determine particle size and RNA coating. Table 3: Physical properties of LNPs after 20-fold direct dilution of PFL RNA-LNPs with DPBS. After direct dilution with PBS at pH 7.4 After dialysis overnight in PBS pH 7.4 Acetate incubation time at room temperature and pH 5.9 Z- mean (nm) PDI Coating efficiency % Z- mean (nm) PDI Coating efficiency % N/A; Empty LNP 51 0.188 N/A 50 0.063 N/A 10 72 0.126 96.4 63 0.050 99.2 30 71 0.087 97.3 64 0.034 98.8 50 69 0.110 98.4 - - - 70 69 0.145 98.3 64 0.035 98.9 90 69 0.103 99.5 64 0.054 99.0

This data suggests that by introducing RNA into an LNP suspension maintained at a weakly acidic pH of 5.9, and then adjusting the mixture back to a neutral pH suitable for in vivo drug administration, nucleic acid coating can be achieved after LNPs have formed. RNA coating occurs without any additional auxiliary means (such as surfactants, solvents, or temperature), or may not need to occur during LNP formation.

RNA-LNPs were prepared by directly adding RNA to an empty LNP suspension, followed by direct dilution in DPBS to neutralize the pH, resulting in RNA-LNPs with reasonable particle size and polydispersity and high RNA coating efficiency. Data indicated that the coating process was effectively completed within 10 minutes (e.g., coating efficiency greater than 95%), although some minor improvements were observed with longer incubation times before direct dilution. Prior to direct dilution, there was a trend towards smaller particle size and increased coating efficiency with increasing incubation time.

The corresponding samples neutralized by dialysis showed no trend in terms of incubation time at pH 5.9, which again indicates that the coating process was completed within the shortest incubation time (10 minutes) studied in this paper. Overall, the results show smaller particle size, lower polydispersity, and virtually the highest coating efficiency across all incubation times studied compared to the corresponding directly diluted samples.

Formulation Example 4 : Formation of Lipid Nanoparticles The following examples demonstrate the results of the PFL method using different sample neutralization methods after loading a series of formulations based on different ionizable cationic lipids.

A) Preparation of empty LNPs: For each of the three ionizable cationic lipids (ionizable lipid A-15, ionizable lipid B-3, and ionizable lipid C-18), a lipid mixture was prepared by appropriately adding individual lipid reserve solutions (10 mg/mL) to separately weighed pure ionizable lipids. The final lipid mixture contained lipids (ionizable lipids, DSPC, cholesterol, and PEG lipid 1) in a molar ratio of 47.5:10:40.7:1.8, with a total lipid concentration of 23.65 mM. To prepare empty LNPs, a T-type mixing system was used, with the aqueous solution flowing at a rate of 30 mL/min and the organic solution flowing at a rate of 10 mL/min, and the 25 mM acetate buffered aqueous solution (pH 5.9, i.e., RNA-free) and lipid solution were combined at a 3:1 ratio of aqueous phase to organic phase (i.e., buffered aqueous solution and organic lipid solution). The LNP suspension was collected and allowed to stand at room temperature for approximately 15 minutes. Ethanol was then removed by overnight dialyzing of the empty LNPs in 25 mM acetate buffer (pH 5.9) at a volume 200 times that of the LNP suspension. After dialyzing, the empty LNPs were filtered (0.2 µm) and the particle size and lipid concentration were analyzed. Total lipid concentration was calculated as follows: cholesterol concentration was first determined using cholesterol E enzyme assay, and then calculated using the theoretical mortise ratio of cholesterol to all lipid components. Total lipid concentration was used to calculate the amount of lipid required to prepare RNA-loaded LNPs at an aminolipid nitrogen to nucleic acid phosphate (N:P) ratio of 6.

B) Loading LNPs with mRNA: A 1:1 molar mixture of two mRNAs encoding the heavy and light chains of the anti-influenza IgG antibody was thawed at room temperature and diluted in nuclease-free water to a working concentration of approximately 1 mg/mL. RNA concentration was verified by measuring absorbance at 260 nm, assuming a conversion factor of 40 µg/OD260. The measured RNA concentration was 1.12 mg/mL. Empty LNP solution was added to polypropylene cone tubes and diluted in 25 mM acetate buffer (pH 5.9) to achieve a total lipid concentration of 3.17 mM. Then, 0.806 mL of 1.12 mg/mL RNA stock solution was added to the LNP solution by pipetting, followed by gently inverting the tube several times. This produced an RNA-LNP mixture with a total lipid concentration of 2.96 mM and an RNA concentration of 0.075 mg/mL. The RNA-LNP mixture was incubated at room temperature for 90 minutes.

C) Processing and Analysis of RNA-LNP (Neutralization and Concentration): After incubation in acetate at pH 5.9 for a period of time, the RNA-LNP mixture was separated and diluted or dialyzed in a neutral pH buffer solution to restore the sample to neutral pH. In this experiment, three different methods were investigated for this neutralization step: 5× dilution with 1× DPBS: 1 volume of LNP mixture was added to 4 volumes of DPBS in a polypropylene cone tube. The sample was then concentrated to approximately 0.6 mg/mL RNA by ultracentrifugation and stored overnight at 2–8°C. 2× dilution with 2× DPBS: 1 volume of LNP mixture was added to an equal volume of 2× concentrated DPBS. The sample was then concentrated to approximately 1 mg/mL RNA by ultracentrifugation and stored overnight at 2–8°C. Dialysis in DPBS: The LNP mixture was placed in a dialysis bag and dialyzed overnight in 1× DPBS, which was 200 times the volume of the RNA-LNP mixture in acetate buffer. After dialysis, the sample was subsequently concentrated to approximately 1 mg/mL RNA by ultracentrifugation.

D) UPLC was used to analyze the particle size, RNA coating efficiency, and lipid and RNA content of all LNP samples.

E) Testing Freeze/Thaw Stability: After the treatments described above, all samples were diluted with DPBS, and an appropriate amount of 1.2 M sucrose cryoprotectant was added to achieve 300 mM sucrose and 0.8 mg/mL RNA in preparation for freezing at -80°C. However, samples diluted 5× in DPBS could not be adequately concentrated to the final target concentration of 0.8 mg/mL RNA by ultracentrifugation, and in these cases, the samples underwent the minimum dilution required to achieve the final solution of 300 mM sucrose with cryoprotectant. The total RNA concentrations of these samples are shown in the table below. After this dilution, the samples were passed through a 0.2 µm filter and aliquoted as needed. Subsequently, after a single freeze/thaw cycle, the particle size, RNA coating efficiency, and lipid and RNA concentrations of the RNA-LNP formulation were analyzed. Table 4a: Data generated using formulations based on ionizable lipid A-15 – particle size, polydispersity, coating efficiency, and drug-lipid ratio. Sample describe Z- mean (nm) PDI Coverage rate % Total mRNA / lipids (mg/µmol) 4a-1 Empty ionizable lipid A-15 LNP stock solution - after dialysis with acetate at pH 5.9 49 0.101 N/A N/A 4a-2 Empty LNP control - after DPBS dialysis and concentration (final) 56 0.093 N/A N/A 4a-3 Empty LNP control - final sample after freezing/thawing 62 0.066 N/A N/A 4a-4 Add the mRNA to 4a-1, incubate for 90 minutes, then dilute 5-fold in 1× DPBS. 80 0.093 - - 4a-5 After concentration (final) 77 0.046 - - 4a-6 After storing overnight at 2 to 8°C 78 0.032 97 0.028 4a-7 The final sample of 0.38 mg/mL after freezing/thawing. 78 0.031 98 0.026 4a-8 Add mRNA to 4a-1, incubate for 90 minutes, then dilute 2-fold in 2× DPBS. 78 0.060 - - 4a-9 After concentration (final) 80 0.035 - - 4a-10 After storing overnight at 2 to 8°C 76 0.044 97 0.029 4a-11 Final sample at 0.8 mg/mL after freezing/thawing 77 0.071 98 0.025 4a-12 Add mRNA to 4a-1, incubate for 90 minutes, dialyze and concentrate in DPBS (final). 75 0.018 99 0.029 4a-13 Final sample at 0.8 mg/mL after freezing/thawing 75 0.033 98 0.027 Table 4b: Data generated using formulations based on ionizable lipid B-3 – particle size, polydispersity, encapsulation efficiency, and drug-lipid ratio Sample describe Z- mean (nm) PDI Coverage rate % Total mRNA / lipids (mg/µmol) 4b-1 Empty ionizable lipid B-3 LNP stock solution - after dialysis with acetate at pH 5.9 52 0.085 N/A N/A 4b-2 Empty LNP control - after DPBS dialysis and concentration (final) 56 0.073 N/A N/A 4b-3 Empty LNP control - final sample after freezing/thawing 66 0.113 N/A N/A 4b-4 Add mRNA to 4b-1, incubate for 90 minutes, then dilute 5-fold in 1× DPBS. 76 0.064 - - 4b-5 After concentration (final) 73 0.026 - - 4b-6 After storing overnight at 2 to 8°C 73 0.017 88% 0.028 4b-7 The final sample at 0.38 mg/mL after freezing/thawing. 74 0.022 87% 0.025 4b-8 Add the mRNA to 4b-1, incubate for 90 minutes, then dilute 2-fold in 2× DPBS. 74 0.051 - - 4b-9 After concentration (final) 73 0.047 - - 4b-10 After storing overnight at 2 to 8°C 74 0.019 88% 0.027 4b-11 Final sample at 0.8 mg/mL after freezing/thawing 73 0.065 90% 0.025 4b-12 Add mRNA to 4b-1, incubate for 90 minutes, dialyze and concentrate in DPBS (final). 73 0.002 92% 0.028 4b-13 Final sample at 0.8 mg/mL after freezing/thawing 72 0.029 90% 0.025 Table 4c: Data generated using formulations based on ionizable lipid C-18 – particle size, polydispersity, encapsulation efficiency, and drug-lipid ratio Sample describe Z- mean (nm) PDI Coverage rate % Total mRNA / lipids (mg/µmol) 4c-1 Empty ionizable lipid C-18 LNP stock solution - after dialysis with acetate at pH 5.9 48 0.053 N/A N/A 4c-2 Empty LNP control - after DPBS dialysis and concentration (final) 55 0.048 N/A N/A 4c-3 Empty LNP control - final sample after freezing/thawing 66 0.027 N/A N/A 4c-4 Add mRNA to 4c-1, incubate for 90 minutes, then dilute 5-fold in 1× DPBS. 74 0.097 - - 4c-5 After concentration (final) 76 0.045 - - 4c-6 After storing overnight at 2 to 8°C 73 0.080 99% 0.028 4c-7 The final sample at 0.38 mg/mL after freezing/thawing. 75 0.067 99% 0.025 4c-8 Add the mRNA to 4c-1, incubate for 90 minutes, then dilute 2-fold in 2× DPBS. 74 0.079 - - 4c-9 After concentration (final) 75 0.051 - - 4c-10 After storing overnight at 2 to 8°C 73 0.068 99% 0.028 4c-11 Final sample at 0.8 mg/mL after freezing/thawing 76 0.086 99% 0.025 4c-12 Add mRNA to 4c-1, incubate for 90 minutes, dialyze and concentrate in DPBS (final). 70 0.051 100% 0.028 4c-13 Final sample at 0.8 mg/mL after freezing/thawing 78 0.017 99% 0.025

Tables 4a-c summarize that, regardless of the variations in the post-loading sample neutralization strategy employed in this study, the particle size/PDI, RNA coating, and drug-to-lipid ratio of formulations based on three chemically distinct classes of ionizable lipids generally remained within the desired parameter ranges. In all cases, the empty LNP stock solution exhibited a smaller average particle size, increasing by approximately 15 to 25 nm in the final state. All samples were freeze-thaw stable; that is, any increase in particle size or PDI after freeze-thaw was minimal and had no impact on coating efficiency.

It should be noted that the formulation based on ionizable lipid B-3 exhibited a coating efficiency of approximately 90%, lower than the approximately 97% or higher coating efficiency observed in other formulations in this study. The apparent pKa of ionizable lipid B-3 was 6.09, while that of ionizable lipid A-15 was 6.35, and that of ionizable lipid C-18 was 6.45.

Formulation Example 5 : Formation of Lipid Nanoparticles The following examples demonstrate alternative PFL conditions for improving the encapsulation efficiency of formulations based on ionizable lipid B-3, and demonstrate the PFL method for formulations based on the relevant ionizable lipid B-45 using the original conditions.

A) Preparation of empty LNPs: For each of the two ionizable cationic lipids (ionizable lipid B-3 and ionizable lipid B-45), a lipid mixture was prepared by appropriately adding individual lipid reserve solutions (10 mg/mL) to separately weighed pure ionizable lipids. The final lipid mixture contained lipids (ionizable lipids, DSPC, cholesterol, and PEG lipid 1) in a molar ratio of 47.5:10:40.7:1.8, with a total lipid concentration of 23.65 mM. This experiment used two acetate buffers with different pH values: a lower pH acetate buffer (pH 5.5) was used for the ionizable lipid B-3 LNP, while the same pH 5.9 buffer used in the previous example was used for the ionizable lipid B-45. To prepare empty LNPs, a T-type mixing system was used to combine 25 mM acetate buffered solution (i.e., RNA-free) and lipid solution at a 3:1 ratio (30 mL/min aqueous solution and 10 mL/min organic solution flow rate) corresponding to the ionizable lipids described above, along with the organic phase (i.e., buffered aqueous solution and organic lipid solution). After mixing, the suspension was allowed to stand at room temperature for approximately 15 minutes. Following this step, ethanol was removed by dialyzing empty LNPs overnight in 25 mM acetate buffer (pH 5.5 for ionizable lipid B-3; pH 5.9 for ionizable lipid B-45) at a volume of 200 × LNP formulation (using 12 to 14 kDa regenerated cellulose dialysis tubing). After dialysis, the empty LNP formulation was filtered using a 0.2 µm syringe filter and analyzed to determine particle size (by dynamic light scattering) and lipid concentration. Total lipid concentration was calculated as follows: cholesterol concentration was first determined using a cholesterol E enzyme assay, and then calculated using the theoretical molar ratio of cholesterol to all lipid components. Total lipid concentration is used to calculate the amount of lipid required to prepare RNA-carrying LNPs at an amino lipid nitrogen to nucleic acid phosphate (N:P) ratio of 6.

B) Loading LNPs with mRNA: A 1:1 molar mixture of two mRNAs encoding the heavy and light chains of anti-influenza IgG antibodies was thawed at room temperature and diluted in nuclease-free water to a working concentration of approximately 1 mg/mL. RNA concentration was verified by measuring absorbance at 260 nm, assuming a conversion factor of 40 µg/OD260. The measured RNA concentration was 1.12 mg/mL. Empty LNP solutions were added to individual test tubes and diluted with acetate buffer (25 mM) at pH 5.5 (for ionizable lipid B-3) or pH 5.9 (for ionizable lipid B-45) to achieve a total lipid concentration of 3.17 mM in both cases. The RNA preparation solution (0.806 mL, 1.12 mg/mL) was then pipetted into various LNP solutions, with the test tubes gently inverted several times after each addition. This produced an RNA-LNP solution with a total lipid concentration of 2.96 mM and an RNA concentration of 0.075 mg/mL. The RNA-LNP mixture was then incubated at room temperature for 90 minutes.

C) Processing and Analysis of RNA-LNP (Neutralization and Concentration): After incubation in the acetate buffer described above for a period of time, the RNA-LNP mixture was separated and diluted or dialyzed in a neutral pH buffer solution to restore the sample to neutral pH. In this experiment, two different methods were investigated for this neutralization step: dialysis dilution with 2× PBS; and after incubation for a period of time, the mixture was added to an equal volume of 2× DPBS in polypropylene cone tubes. The particle size, RNA coating efficiency, and lipid and RNA concentrations of the DPBS-diluted RNA-LNP were analyzed. Dialysis in DPBS: After incubation, the LNP mixture is placed in a dialysis bag and dialyzed in 1× DPBS solution (pH 7.4) with a volume of 200× LNP mixture in acetate buffer.

D) Analyze all LNPs as previously described, analyzing the particle size, RNA coating efficiency, and lipid and RNA content of all final LNP samples.

E) Testing Freeze/Thaw Stability: After the treatments described above, all samples were diluted with DPBS, and an appropriate amount of 1.2 M sucrose cryoprotectant was added to achieve 300 mM sucrose and approximately 0.75 mg/mL RNA, in preparation for freezing at -80°C. After this dilution, the samples were passed through a 0.2 µm filter and aliquoted as needed. Subsequently, after a single freeze/thaw cycle, the particle size, RNA coating efficiency, and lipid and RNA concentrations of the RNA-LNP formulation were analyzed. Table 5a: Data generated using cationic lipid B-3 (pH 5.5) – particle size, polydispersity, coating efficiency, and drug-lipid ratio. Sample describe Z- mean (nm) PDI Coverage rate % Total mRNA / lipids (mg/µmol) 5a-1 Empty ionizable lipid B-3 LNP stock solution - after dialysis with acetate at pH 5.5 57 0.125 N/A N/A 5a-2 Empty LNP control - after DPBS dialysis and concentration (final) 57 0.111 N/A N/A 5a-3 Empty LNP control - final sample after freezing/thawing 69 0.097 N/A N/A 5a-4 Add mRNA to 5a-1, incubate for 90 minutes, then dilute 2-fold in 2× DPBS. 81 0.140 - - 5a-5 After concentration (final) 78 0.078 - - 5a-6 After storing overnight at 2 to 8°C 77 0.076 99 0.023 5a-7 Final sample after freezing/thawing 82 0.118 97 0.028 5a-8 Add mRNA to 5a-1, incubate for 90 minutes, then dialyze overnight in DPBS. 75 0.077 - - 5a-9 After concentration (final) 76 0.077 98 0.023 5a-10 Final sample after freezing/thawing 80 0.092 97 0.026 Table 5b: Data generated using ionizable lipid B-45 (pH 5.9) – particle size, polydispersity, encapsulation efficiency, and drug-lipid ratio Sample describe Z- mean (nm) PDI Coverage rate % Total mRNA / lipids (mg/µmol) 5b-1 Empty ionizable lipid B-45 LNP stock solution - after dialyzing with acetate at pH 5.5 75 0.103 N/A N/A 5b-2 Empty LNP control - after DPBS dialysis and concentration (final) 76 0.132 N/A N/A 5b-3 Empty LNP control - final sample after freezing/thawing 76 0.077 N/A N/A 5b-4 Add mRNA to 5b-1, incubate for 90 minutes, then dilute 2-fold in 2× DPBS. 90 0.092 - - 5b-5 After concentration (final) 89 0.005 - - 5b-6 After storing overnight at 2 to 8°C 88 0.077 97 0.024 5b-7 Final sample after freezing/thawing 90 0.080 96 0.026 5b-8 Add mRNA to 5b-1, incubate for 90 minutes, then dialyze overnight in DPBS. 88 0.021 - - 5b-9 After concentration (final) 87 0.078 97 0.024 5b-10 Final sample after freezing/thawing 90 0.054 96 0.028

The data presented here indicate that for formulations based on ionizable lipid B-3, pH adjustment during the acetate incubation step leads to high coating efficiencies consistent with other formulations incubated at pH 5.9, such as those shown in earlier examples and, in this example, formulations based on ionizable lipid B-45, which has a similar chemical structure but a higher apparent pKa of 6.26 (compared to 6.09 for ionizable lipid B-3). This suggests that achieving the highest coating efficiency via the PFL method depends on the minimum difference between the apparent pKa of the ionizable lipid and the pH of the PFL incubation buffer.

Formulation Example 6: Head-to-head in vivo comparison. This study was commissioned to compare the properties of LNPs prepared using conventional methods or embodiments of the methods described herein (e.g., Formulation Example 1). All formulations were prepared using an ionizable lipid/DSPC/cholesterol/PEG lipid ratio of 47.5:10:40.7:1.8.

Based on the parameters described below, four formulations were prepared according to conventional methods (e.g., T-mixing, TFF, concentration, etc.): Table 6a: Parameters of formulations prepared according to conventional methods Sample Ionizable lipids pH of the formulation 6a-1 A-15 6 6a-2 B-3 4 6a-3 B-45 5.9 6a-4 C-18 6

Test samples were also prepared using the following steps: LNP components were mixed in an acetate buffer at pH 5.9 using a T-mixer to obtain empty LNPs; the mixture was dialyzed with an acetate buffer at pH 5.9 to remove ethanol; the mixture was then mixed with mRNA at room temperature to obtain coated mRNA; this coated mRNA was diluted with 2×PBS (1:1 ratio) and concentrated by ultrafiltration (Amicon); and then diluted with a PBS/sucrose mixture and frozen. Four formulations were prepared as described below. Table 6b: Parameters of formulations prepared according to the method disclosed herein. Sample Ionizable lipids pH of the formulation 6b-1 A-15 5.9 6b-2 B-3 5.9 6b-3 B-45 5.9 6b-4 C-18 5.9

The test samples were administered intravenously according to the procedure described in Biological Example 2 below. In addition, a comprehensive analysis of all LNPs was performed, including RNA integrity, lipid adduct formation, cryo-electron microscopy, and encapsulation detection by UPLC. The samples prepared according to the parameters detailed in Tables 6a and 6b are described below. Table 6c: Physical characteristics of samples 6a-1 to 6a-4 and 6b-1 to 6b-4 Sample 6a-1 6b-1 6a-2 6b-2 6a-3 6b-3 6a-4 6b-4 Particle size (nm) ^† 71 81 73 74 64 87 75 77 PDI 0.006 0.042 0.023 0.064 0.065 0.046 0.099 0.016 Coating efficiency (%) 97 99 90 97 96 96 98 99 mRNA:lipids (wt:µmol) 0.026 0.026 0.025 0.026 0.025 0.024 0.023 0.025 ^† Strength Average Particle Size

As illustrated in Figures 1 and 2, the two types of formulations exhibited similar activities at low doses for samples 6a-1 and 6b-1, and samples 6a-2 and 6b-2. Sample 6b-3 was more active than sample 6a-3.

In Example 7, lipid adduct evaluation was performed on samples after storage at -80°C. The lipid adduct formation was initially worse for samples prepared using conventional methods (e.g., those shown in Table 6a) compared to samples prepared according to the embodiments described herein (e.g., those shown in Table 6b). The lipid adduct test results are shown in Figure 3 (samples freshly removed from -80°C) and Figure 4 (the same samples after administration and storage at 4°C for 2 weeks).

Example 8: Coating Assessment. This procedure was used to examine RNA coating and confirm that RNA was not only adsorbed onto the outer surface of the LNP. RiboGreen analysis was performed after drug administration (i.e., after storage at 4°C for approximately one week), and UPLC analysis was performed 6 days later, during which time the sample was stored at 4°C.

Based on RG and UPLC analyses, the RNA coating efficiencies of samples prepared using conventional methods and those prepared according to methods described herein (e.g., formulation example 1) were similar. The tested samples are shown in Tables 6a and 6b. A notable exception is that sample 6a-2 appeared to exhibit particularly low coating efficiency when analyzed by UPLC.

In vivo comparison of formulations delivered intramuscularly: When such formulations are used for intramuscular delivery, this procedure is used to compare the characteristics of LNPs prepared using conventional methods or according to the methods of embodiments described herein (e.g., formulation example 1).

Based on the parameters described below, three formulations were prepared according to conventional methods (e.g., T-mixing, TFF, concentration, etc.): Table 9a: Parameters of formulations prepared according to conventional methods Sample Ionizable lipids pH of the formulation 9a-1 B-3 4 9a-2 A-39 5.9 6a-3 D-1 5.9

Test samples were also prepared using the following steps: LNP components were mixed in an acetate buffer at pH 5.9 using a T-mixer to obtain empty LNPs. The LNPs were then dialyzed with an acetate buffer at pH 5.9 to remove ethanol. The LNPs were then mixed with mRNA at room temperature to obtain coated mRNA, diluted with 2×PBS (1:1 ratio), concentrated by ultrafiltration (Amicon), and then diluted with a PBS/sucrose mixture before freezing. Three formulations were prepared as described below. Table 9b: Parameters of formulations prepared according to the method disclosed herein. Sample Ionizable lipids pH of the formulation 9b-1 B-3 5.9 9b-2 A-39 5.9 9b-3 D-1 5.9

All samples were prepared using PR8HA mRNA, and HAI immunogenicity assays were performed in mice using primary/enhanced immunization when administered intramuscularly (IM). Comprehensive analyses (e.g., RNA integrity, lipid adduct assays, etc.) were performed on all LNP formulations.

The analysis of the prepared samples presents the following data: Table 9c: Physical characteristics of samples 9a-1 to 9a-3 and 9b-1 to 9b-3 Sample 9a-1 9b-1 9a-2 9b-2 9a-3 9b-3 Particle size (nm) ^† 84 81 85 95 80 96 PDI 0.044 0.043 0.017 0.091 0.057 0.101 Coating efficiency (%) 81 94 96 97 95 96 mRNA:lipids (wt:µmol) 0.018 0.022 0.020 0.021 0.019 0.021 Lipid adducts (%) 3.1 0.9 2.2 0.5 10.3 1.6 RNA integrity (%) (RNA=78) 66 72 72 70 72 65 ^† Strength Average Particle Size

In summary, the LNP samples prepared according to Tables 9a and 9b all exhibited similar immunogenicity regardless of the preparation method, with a notable exception being sample 9b-3 at a dose of 0.5 µg, which had a lower titer. At low doses (0.2 µg), the activities of samples 9a-1 and 9b-1 were lower than those of samples 9a-2 and 9b-2 (lower by 1.9×) or samples 9a-3 and 9b-3 (lower by 3.2×). At higher doses (0.5 µg), the immunogenicity of all ionizable lipids was similar.

For the samples described in Table 9b, the particle size, PDI, coating efficiency, and lipid adduct formation generally appear to be good. The particle size of these samples is slightly larger than that of the samples prepared according to Table 9a. Within the lipid range, the in vivo activities of the two formulations prepared according to Tables 9a and 9b are similar. Lipid adduct data show RNA integrity and indicate that the lipid adduct levels of samples prepared according to the embodiments of the methods described herein (e.g., formulation example 1; see, for example, Table 9c, Figure 3) are unexpectedly low. UPLC coating analysis confirms good coating in all formulations prepared according to the embodiments of the methods described herein (e.g., formulation example 1). Cryo-electron microscopy data for all preparation methods appear similar.

Example 10: Study on the Coating Efficiency as a Percentage of RNA Mixture. The concentration of RNA during mixing was investigated to determine the coating efficiency of LNPs prepared according to embodiments of the method described herein (e.g., Example 1). Specifically, test samples were prepared by mixing the LNP components in an acetate buffer at pH 5.9 or pH 5.5 using a T-mixer to obtain empty LNPs. The LNPs were then dialyzed against an acetate buffer at pH 5.9 or pH 5.5 to remove ethanol, mixed with mRNA at room temperature to obtain coated mRNA, diluted with 2× PBS (1:1 ratio), concentrated by ultrafiltration (Amicon), and diluted with a PBS/sucrose mixture before freezing. Three formulations were prepared as described below. Table 10: Parameters of formulations prepared according to the method disclosed herein Sample Ionizable lipids pH of the formulation 10-1 A-15 5.9 10-2 B-3 5.5 10-4 C-18 5.9

The samples were then mixed with mRNA, and the coating efficiency was determined for a series of concentrations (i.e., RNA concentrations of 0.15, 0.5, and 1 mg/mL for lipids A-15 and B-3; and RNA concentrations of 0.075, 0.15, 0.5, and 1 mg/mL for lipid C-18). The coating efficiency and LNP particle size relative to the loading concentration are shown in Figures 9 and 10, respectively. In summary, within the scope of this study, empty LNPs can be mixed with RNA, and the resulting LNPs loaded with RNA have similar particle sizes.

Preparation Example 11: Buffer Species Study To determine which buffer systems are ideal for preparing the LNP system according to this disclosure, several buffers were tested. Specifically, the following buffer systems were tested: - Citrate buffer: 5 mM, 50 mM pH 6.0 / 5.9 - Acetate buffer: 25 mM pH 5.9, pH 5.5 - Phosphate buffer: 5 mM, 10 mM, 20 mM and 50 mM pH 5.8, pH 5.5

Generally, acetate and phosphate buffer systems perform well. Citrate buffer showed less than 90% encapsulation efficiency and was more challenging than other test buffers when neutralized with phosphate-buffered saline (PBS). Table 11a: Physical results of the citrate buffer system using LNP (pre-diluted with PBS) prepared with A-15 and 2.5% polyethylene glycol-modified lipids. buffer solution Coverage efficiency (%) at 20 minutes Coverage efficiency (%) at 60 minutes Particle size (nm) after being cultured with RNA PDI 50 mM citrate (pH 4) 89 93 127 (87%) 63 (13%) 0.10 50 mM citrate (pH 6) 29 twenty one 121 0.11 Table 11b: Physical results using a citrate buffer system containing LNP (diluted with PBS) prepared with A-15 and 2.5% polyethylene glycol lipids. buffer solution Coating efficiency (%) pH Particle size (nm) after dilution with PBS PDI 50 mM citrate (pH 4) 55 5.43 122 (90%) 56 (10%) 0.11 50 mM citrate (pH 6) twenty one 6.15 114.8 0.11

Two different pH values (5.5 and 5.9) were tested for the acetate buffer system. This buffer system was tested at different RNA loading concentrations, and particle size (Figures 11 and 12) and encapsulation efficiency (Figure 13) were measured. Compared to the pH 5.5 buffer, the pH 5.9 acetate buffer system generally resulted in smaller particle size and lower polydispersity.

Preparation Example 12: Buffer Concentration (Acetate) Study. Based on the embodiments disclosed herein, several buffer concentrations were tested for particle size, PDI, and coating efficiency. RNA-LNPs were prepared by adding RNA, buffer, and then empty LNPs. The RNA loading concentration was 1.0 mg/mL. Compound A-15 was used as the cationic lipid, along with other components and concentrations described in Preparation Example 1, to prepare the formulation. Empty LNPs were prepared and then stored at 4°C for two weeks before mixing. The results are shown in Figure 14.

Example 13: RNA Loading and PEG Content. The study investigated the content of PEGylated lipids in empty LNPs to determine whether PEG content affected RNA loading. LNPs were prepared using the ionizable lipid C-18 with a range of different PEG concentrations. Concentrations and physical characteristics measured by RG and DLS are shown below. Table 13a: PEG Concentration and Related Data. PEG concentration (mol%) Z-mean (nm) PDI Coating efficiency (%) 5 71 0.134 50.8 2.5 73 0.087 98.2 1.8 78 0.092 98.6 0.5 88 0.074 98.4

LNP loading was measured 10 minutes after mixing, yielding the data in Table 13a above. Additionally, the time series for 5 mol% samples was tested as described below. Table 13b: Time series for 5 mol% PEG time Z-mean (nm) PDI Coating efficiency (%) 10 minutes 78 0.159 57.7 30 minutes 81 0.182 68.6 4 hours 88 0.197 89.2 8 hours 88 0.207 94.9 24 hours 89 0.216 96.6

As demonstrated by the above data, LNPs with a higher percentage of PEG in their composition require a longer time to carry RNA, but they do achieve a higher level of coverage over time.

This phenomenon was further investigated by testing a range of PEG contents (from 1.8% to 8% PEG) using the same ionizable lipid (C-18). The data are shown in Figure 15, illustrating the RNA loading of empty LNPs with PEG contents of 1.8%, 4.0%, 6.0%, and 8.0% over 24 hours. In the samples studied, all LNPs achieved RNA loading rates exceeding 90%, except for the 8% PEG version, which reached approximately 70% RNA loading after 24 hours of incubation. This confirms the effect of higher PEG% on RNA loading in the PFL method.

Example 14: RNA Loading of Empty LNPs at Different pH Values. The loading of empty LNPs (A-15, B3, D-1, C-18) at different buffer pH values was studied to evaluate the particle size and coating efficiency of the LNPs loaded with the RNA. Figures 20, 22, 24, and 26 show the particle size (Z-mean) and PDI of LNPs loaded with RNA prepared using ionizable lipids A-15, B-3, D-1, and C-18 with buffers of different pH values, respectively. In these figures, the bars represent the Z-mean particle size of LNPs at a series of pH values, corresponding to the left scale; the lines/dots represent the PDI values of LNPs, corresponding to the right scale.

Figures 21, 23, 25 and 27 show a comparison of the encapsulation efficiency of LNPs prepared using the same range of ionizable lipids (A-15, B-3, D-1 and C-18, respectively) at different pH values.

Example 15 illustrates the UPLC coating data shown in Figures 28 and 29. UPLC can be used to evaluate the degree of coating during PFL loading. Figure 28 shows the coating efficiency over time of LNPs prepared using 1.8 mol% PEG lipid 1 and ionizable lipid A-15, as measured by UPLC. This LNP almost immediately coated RNA with 97%.

Figure 29 is a graph showing the encapsulation efficiency of LNP prepared using 5 mol% PEG lipid 1 and ionizable lipid A-15 over time, as measured by UPLC. The encapsulation efficiency increased from 20% to 67% over 10 hours.

Example 16: RNase Treatment. The purpose of this study was to determine the amount of free RNA and to determine, using AGE ^, whether free RNA could be degraded by RNases. RNases included: RNase T1: cleaves after G, inhibited by metal ions, optimal at 37°C; Benzosinase: cleaves at all sites, requires ^Mg²⁺ , optimal at 37°C; RNase A: cleaves at pyrimidine C and U, extremely stable, optimal at 37°C; RNase I: cleaves at all sites, requires NaCl, optimal at 37°C; S1 nuclease: cleaves at all sites, but only at single strands, optimal at 37°C.

If any uncoated RNA is present, RNase T1 leaves a visible band in the middle of the gel. This was confirmed by studying 6% PEG LNPs (see, for example, formulation example 13) before they were fully loaded (after approximately 1 hour of incubation). As more RNA is loaded into the LNP and thus protected from RNase degradation, the intensity of this band gradually decreases over time.

In another experiment, empty LNPs containing 1.8% PEG were tested only 10 minutes after loading and incubation. No bands corresponding to unloaded RNA were observed in the gel. This further supports the notion that RNA is encapsulated within the LNP and not merely adhered to the outside.

Example 17: A study was conducted with low PEG lipid content to determine the effect of PEG lipid concentration on LNP formulations. Empty LNPs were prepared using different concentrations of PEG lipid 1 and ionizable lipids. The physical characteristics of empty LNPs and RNA-loaded LNPs after incubation with fLuc RNA for 10 minutes were tested. Results for ionizable lipid C-18 are shown in Tables 17a-d; results for ionizable lipid B-3 are shown in Tables 17e-g; and results for ionizable lipid D-1 are shown in Tables 17h-j. Table 17a: Showing the Z-mean and polydispersity index values of empty LNPs prepared using different concentrations of PEG lipid 1 and ionizable lipid C-18. PEG lipid concentration (mol%) Z-mean (nm) PDI 1.8 56 0.107 1 66 0.079 0.75 71 0.066 0.5 78 0.072 Table 17b: Showing the Z-mean and polydispersity index of LNPs of RNA-loaded RNA prepared using different concentrations of PEG-1 and ionizable lipid C-18 after 10 minutes of co-culture with RNA. PEG lipid concentration (mol%) Z-mean (nm) PDI Coating efficiency (%) 1.8 66 0.038 99 1 76 0.018 99 0.75 82 0.052 99 0.5 87 0.071 99 Table 17c: Showing the Z-mean, PDI, and coating efficiency values of RNA-loaded LNPs prepared using different concentrations of PEG lipid 1 and ionizable lipid C-18 after storage at 2 to 8°C for 4 weeks and incubation for 10 minutes. PEG lipid concentration (mol%) Z-mean (nm) PDI Coating efficiency (%) 1.8 67 0.039 99 1.0 76 0.011 99 0.75 81 0.028 99 0.5 87 0.026 99 Table 17d: Showing the Z-mean, PDI, and coating efficiency values of RNA-loaded LNPs prepared using different concentrations of PEG lipid 1 and ionizable lipid C-18 after storage at 2 to 8°C for 8 weeks and incubation with RNA for 10 minutes. PEG lipid concentration (mol%) Z-mean (nm) PDI Coating efficiency (%) 1.8 69 0.072 98 1.0 78 0.005 99 0.75 83 0.051 99 0.5 91 0.082 99 Table 17e: Showing the Z-mean and polydispersity index values of empty LNPs prepared using different concentrations of PEG lipid 1 and ionizable lipid B-3. PEG lipid concentration (mol%) Z-mean (nm) PDI 1.0 80 0.243 0.5 87 0.130 Table 17f: Showing the Z-mean and polydispersity index of LNPs of RNA-loaded RNA prepared using different concentrations of PEG lipid 1 and ionizable lipid B-3 after 10 minutes of co-culture with RNA. PEG lipid concentration (mol%) Z-mean (nm) PDI Coating efficiency (%) 1.0 90 0.094 99 0.5 106 0.155 99 Table 17g: Showing the Z-mean and polydispersity index of RNA-loaded LNPs after precursor empty LNPs prepared using different concentrations of PEG lipid 1 and ionizable lipid B-3 were stored at 2 to 8°C for 4 weeks and incubated with RNA for 10 minutes. PEG lipid concentration (mol%) Z-mean (nm) PDI Coating efficiency (%) 1.0 92 0.082 97 0.5 102 0.118 97 Table 17h: Showing the Z-mean and polydispersity index values of empty LNPs prepared using different concentrations of PEG lipid 1 and ionizable lipid D-1. PEG lipid concentration (mol%) Z-mean (nm) PDI 1.0 69 0.119 0.5 82 0.120 Table 17i: Showing the Z-mean and polydispersity index of LNPs of RNA-loaded RNA prepared using different concentrations of PEG-1 and ionizable lipid D-1 after 10 minutes of co-culture with RNA. PEG lipid concentration (mol%) Z-mean (nm) PDI Coating efficiency (%) 1.0 79 0.136 98 0.5 94 0.059 99 Table 17j: Showing the Z-mean and polydispersity index of RNA-loaded LNPs after precursor empty LNPs prepared using different concentrations of PEG lipid 1 and ionizable lipid D-1 were stored at 2 to 8°C for 4 weeks and incubated with RNA for 10 minutes. PEG lipid concentration (mol%) Z-mean (nm) PDI Coating efficiency (%) 1.0 87 0.112 97 0.5 102 0.068 99

Empty LNPs can be prepared using low-concentration PEG lipids. The prepared samples can be stored at 4°C for at least 4 weeks with minimal aggregation. Empty LNPs with low PEG lipid concentrations can rapidly load RNA. After storing empty LNPs at 2 to 8°C for one month or longer, the physical properties of RNA-loaded LNPs remain essentially unchanged. These studies indicate that, in point-of-care settings, this LNP loading technique allows the use of LNPs with lower PEG lipid content than known LNP systems (e.g., for healthcare professionals to load RNA into empty LNPs just before drug administration).

Example 18: On-site Care Study. This study evaluated the efficacy of formulations prepared using the PFL method in "on-site care" settings (e.g., mixing RNA with empty LNPs immediately or shortly before injection/administration). The results are presented graphically in Figure 30.

LNPs were prepared using ionizable lipid C-18 and the following sample parameters were used. Table 18a: Sample parameters of the prepared and tested "site care" samples. Sample describe mRNA addition A T-type mixture prepared as a control sample Before the scene B PFL LNP was prepared and frozen using the same empty stock solution as samples C to E. Before the scene C ⁺ mRNA-LNP in acidic buffer (pH 5.9) was diluted with physiological saline to match the dilution of samples D and E. On-site D ⁺ Dilute the mRNA-LNP to the administered volume using PBS (pH 7.4). On-site E ⁺ mRNA-LNP (pH 7.4) prepared by different operators and diluted with PBS. On-site ⁺ Store empty LNPs at 4°C to prepare RNA-LNPs just before drug administration.

Mice were subsequently administered the sample (0.5 mg/kg) (n=5 mice for each formulation), and serum IgG concentrations were detected. The results are shown in Figure 30 and the table below. Table 18b: Results of the samples prepared according to Table 21a. Sample Serum IgG concentration (µg/mL) Compared to sample A (multiple change) Compared to sample B (multiple change) %CV A 76.0 1.0 0.7 17 B 106.6 1.4 1.0 19 C 78.8 1.0 0.7 17 D 112.8 1.5 1.1 13 E 81.6 1.1 0.8 8

Overall, this data successfully demonstrates that the targeted care LNP formulation is comparable to conventional LNP formulations (e.g., sample A).

Example 19 Stability Study - Empty LNPs: The stability of empty LNPs and subsequent loading were tested to determine the efficacy of this type of sample. Empty LNPs were stored at 2–8°C and -80°C, and samples were tested at time points of 48 hours, 7 days, 1 month, 3 months, 6 months, and beyond. LNP particle size and PDI data were collected. As a preliminary study, ionizable lipid A-15 was used. The results for empty LNPs are shown in Figures 31–32, and the results for LNPs loaded with RNA after storing empty LNPs are shown in Figures 33–35. As shown in Figure 35, at all time points, even after 6 months of storage, all encapsulation efficiencies were greater than 95%. In summary, samples loaded with RNA after storage in empty LNPs exhibit good physical properties. This is also true for samples stored at 2 to 8°C and samples stored at -80°C.

In summary, empty LNPs stored well at 2 to 8°C and -80°C. After storage, all empty LNPs were successfully loaded with RNA to produce loaded LNPs with good granular characteristics.

Preparation Example 20: Stability Study - Empty LNPs were loaded using the PFL method described herein and the conventional T-mix method. The LNPs were loaded with 0.15 mg/mL fLuc RNA and stored at -80°C at an RNA concentration of 1 mg/mL. The cationic lipids used are described below. Table 20: Comparison of physical characteristics at time point 0 between samples prepared using the PFL method and samples prepared using the conventional method (i.e., T-mix). PFL Sample T-shaped mixed samples Cationic lipids Z-mean (nm) PDI Coating efficiency (%) Z-mean (nm) PDI Coating efficiency (%) A-15 79 0.028 97 79 0.03 89 B-3 85 0.095 96 69 0.08 84 B-45 86 0.066 95 61 0.003 84 D-1 96 0.114 97 76 0.028 96 C-18 80 0.064 98 75 0.029 97

Every two months, various physical properties of the samples (such as particle size, PDI, RNA content, coating efficiency, and RNA integrity) are tested.

The T-mix samples are shown in Figures 36A to 36C (z-mean, PDI, and total lipids, respectively) and Figures 38A to 38C (RNA content, coating efficiency, and integrity, respectively). Data for samples prepared using the PFL method are shown in Figures 37A to 37C (z-mean, PDI, and total lipids, respectively) and Figures 39A to 39C (RNA content, coating efficiency, and integrity, respectively). In summary, over a 6-month period, the sample stability of PFL RNA-LNP samples was similar to that of RNA-LNP samples prepared using the T-mix technique.

Example 21: Particle Size and Subsequent Loading Efficiency of Empty LNPs Empty LNPs of the same composition, prepared using one of the ionizable lipids A-15, B-3, or B-45, were each formulated to have unique particle sizes. These empty LNPs were then loaded with 0.075 mg/mL fLuc RNA using the PFL method described herein. Figures 16, 18, and 42A illustrate the coating efficiency of the RNA-loaded particles using each of the ionizable lipids (B-45, A-15, and B-3, respectively). Figures 17, 19 and 42B show the Z-mean particle size of the loaded particles using each of the ionizable lipids (B-45, A-15 and B-3, respectively), as well as the magnitude of the particle size variation between loaded LNPs and empty LNPs.

Example 22: Preparation of Different Types of Nucleic Acids To evaluate whether the PFL method can be used to successfully encapsulate a range of different nucleic acids, empty LNPs were prepared using ionizable lipid B-3 or ionizable lipid C-18 and subsequently loaded with the following nucleic acids: siRNA (Invitrogen silencing negative control), a 1:1 mixture of Cas9 mRNA/gRNA (mg/mg), Cas9 gRNA; or saRNA: encoding the Covid-19 pre-fusion spike protein.

These nucleic acids were loaded into empty LNPs to achieve a final nucleic acid concentration of 0.075 mg/mL and incubated at room temperature for 10 minutes before neutralization with 2× DPBS. The particle size and PDI of the RNA-loaded LNPs for ionizable lipids B-3 and C-18 are shown in Figures 43A and 43B, respectively. The coating efficiency of the two lipid compositions is shown in Figure 43C. Regardless of the lipid species used in the formulation, all nucleic acids were coated with >90% efficiency. Particle size depends to some extent on the type of nucleic acid used; LNPs loaded with saRNA and siRNA were larger than those formulated with a Cas9 mRNA/gRNA mixture. Regardless of the lipid type, LNPs carrying siRNA exhibited lower polydispersity (approximately 0.01), while LNPs carrying saRNA tended to have higher polydispersity (0.098 for ionizable lipid B-3 and 0.11 for ionizable lipid C-18).

Example 23: Effect of NaCl Concentration A study was conducted to determine the effect of sodium chloride concentration on RNA-LNPs prepared via the PFL method. Empty LNPs were prepared using ionizable lipids B-3 or C-18 in 25 mM acetate at pH 5.5 or pH 5.9, respectively. These acetate buffers contained the following amounts of sodium chloride: 0 mM, 50 mM, 137 mM, and 500 mM. The particle size (Z-mean) of the empty LNP preparations is shown in Figure 44A. When prepared in the presence of 50 mM NaCl, the particle size showed a slight decrease; however, the particle size increased with further increases in NaCl concentration. Empty LNPs were then loaded with 0.075 mg/mL fLuc RNA using the PFL method described herein. Figures 44B and 44C show the particle size (z-mean) and coating efficiency of the loaded LNPs, respectively. The loaded particle size was nearly identical between LNPs prepared with 50 mM NaCl and in the absence of NaCl. However, a significant increase in particle size was observed when prepared with 137 mM NaCl. Particles with the same size as the empty LNPs were obtained when prepared with 500 mM NaCl. The coating efficiency of the two tested ionizable lipids dropped to zero when prepared with 500 mM NaCl. For the other concentrations tested, the coating efficiency generally remained high, with only a slight decrease observed in ionizable lipid C-18 LNPs prepared with 137 mM NaCl. Overall, all LNPs tested could be prepared with a maximum NaCl concentration of 137 mM.

Example 24: Effect of N:P on PFL Loading. To investigate the effect of the N:P ratio on the PFL loading method, ionizable lipid C-18 was used with a series of different N:P ratios from 9 to 3 (9, 7.5, 6, 4.5, 3). Figure 40 shows the z-mean particle size and PDI of LNP as a function of N:P ratio, and Figure 41 shows the coating efficiency of the same LNP as a function of N:P ratio.

As the N:P ratio decreases, the particle size of the LNPs carrying the RNA increases, exhibiting a clear opposite trend. Within this range, the coating efficiency does not appear to be affected by the N:P ratio, demonstrating near-quantitative loading within the scope of this study.

In summary, LNPs prepared using the PFL method exhibit characteristics comparable to those prepared using conventional techniques (e.g., T-mixing). The PFL method operates with high success rates across a wide range of mixing concentrations (i.e., empty LNP and RNA concentrations). In some embodiments, pH is a key factor for successful loading and may depend on the pKa of the cationized lipid. In some embodiments, the concentration of PEG (e.g., polyethylene glycol-modified lipids) in the LNP shell affects the rate of RNA loading.

Biological Example 1: Efficacy evaluation used the following protocol to determine the efficacy of nucleic acid molecules encapsulated in lipid nanoparticle formulations according to this disclosure, using a rodent in vivo luciferase mRNA expression model.

Lipid nanoparticles encapsulating nucleic acids were prepared based on the example described above. The study was conducted in 6- to 8-week-old female C57BL/6 mice (Charles River) and 8- to 10-week-old CD-1 (Harlan) mice (Charles River), following guidelines established by the Animal Care Committee (ACC) and the Canadian Council on Animal Care (CCAC). Different doses of mRNA-lipid nanoparticles were administered systemically via tail intravenous injection, and the animals were euthanized at specific time points after administration (e.g., 4 hours). Liver and spleen were collected in pre-weighed tubes, weighed, immediately flash-frozen in liquid nitrogen, and stored at -80°C until processing and analysis.

For the liver, approximately 50 mg was cut and analyzed in 2 mL FastPrep tubes (MP Biomedicals, Solon OH). Add ¼" ceramic balls (MP Biomedicals) to each test tube, and add 500 µL of Glo lysis buffer-GLB (Promega, Madison WI) equilibrated to room temperature to the liver tissue. Homogenize the liver tissue for 15 seconds using a FastPrep24 instrument (MP Biomedicals) at 2 × 6.0 m/s. Incubate the homogenate at room temperature for 5 minutes, then dilute the homogenate 1:4 in GLB and evaluate using the SteadyGlo luciferase assay system (Promega). Specifically, react 50 µL of diluted tissue homogenate with 50 µL of SteadyGlo, shake for 10 seconds, incubate for 5 minutes, and then use a CentroXS³ LB 960 spectrophotometer (Berthold). Quantification was performed using BCA protein analysis kits (Pierce, Rockford, IL, Germany). The amount of the analyzed protein was determined using a BCA protein analysis kit. The relative luminescence units (RLU) were then standardized relative to the total micrograms of the analyzed protein. To convert RLU to luciferase, a standard curve was generated using QuantiLum recombinant luciferase (Promega).

Fluc mRNA (L-6107) from Trilink Biotechnologies expresses a luciferase protein, originally isolated from the firefly (Photinus pyralis ) . Fluc is commonly used in mammalian cell culture to measure gene expression and cell viability. It emits biofluorescence in the presence of chromogenic luciferin. This capped and polyadenylated mRNA is completely replaced by 5-methylcytidine and pseudouridine.

Activity was determined by measuring luciferase activity in the liver 4 hours after administration via tail vein injection. Activities were compared at doses of 1.0, 0.3, or 0.1 mg mRNA/kg and expressed as nacquet luciferase/gram of liver 4 hours after administration.

Biological Example 2: In vivo evaluation of immunoglobulin G (IgG) mRNA using nanoparticle compositions. This study was conducted in 6- to 8-week-old CD-1/ICR mice (Envigo) following guidelines developed by the Institutional Animal Care Committee (ACC) and the Canadian Council for Animal Care (CCAC). Different doses of mRNA-lipid nanoparticles were administered systemically via tail intravenous injection, and the animals were euthanized at specific time points after administration (e.g., 24 hours). Whole blood was collected, and serum was separated by centrifuging the tubes containing whole blood at 2000 × g for 10 minutes at 4°C and stored at -80°C until analysis.

For the immunoglobulin G (IgG) ELISA (Life Diagnostics, Human IgG ELISA kit), serum samples were diluted 100 to 15000 times with 1× diluent. Two 100 µL aliquots of the diluted serum were applied to 96-well plates coated with anti-human IgG, along with a human IgG standard, and incubated at 25°C and 150 rpm in a disc shaker for 45 minutes. The wells were washed 5 times with 1× washing solution (400 µL/well) using a washer. 100 µL of HRP conjugate was added to each well, and the wells were incubated in a disc shaker under the same conditions as above. The wells were washed again 5 times with 1× washing solution (400 µL/well) using a washer. Add 100 µL of TMB reagent to each well and incubate in a disc oscillator under the same conditions as described above. Stop the reaction by adding 100 µL of stop solution to each well. Read the absorbance at 450 nm (A450) using a microdisc reader. Determine the amount of human IgG in mouse serum by plotting the A450 value of the analytical standard versus the concentration of human IgG.

Biological Example 3: Determination _{of pKa} of Blended Lipids The _pKa of each lipid in lipid nanoparticles was determined using an analysis based on 2-(p-toluidine)-6-naphthalenesulfonic acid (TNS) fluorescence. Lipid nanoparticles containing ionizable lipid/DSPC/cholesterol/PEG lipid 1 (50/10/38.5/1.5 or 47.5:10:40.7:1.8 mol%) present in PBS at a total lipid concentration of 0.4 mM were prepared as a 100 μM stock solution of TNS in distilled water. The vesicles were diluted to a lipid concentration of 24 μM in 2 mL buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl, with a pH range of 2.5 to 11. Aliquots of TNS solution were added to bring the final concentration to 1 µM. After vortex mixing, the fluorescence intensity was measured at room temperature using an SLM Aminco Series 2 luminescence spectrophotometer at an excitation wavelength of 321 nm and an emission wavelength of 445 nm. S-shaped best fit analysis was applied to the fluorescence data, and the pH at which half-maximum fluorescence intensity was achieved was measured as _pKa .

Biological Example 4: Immunogenicity of LNP after Intramuscular ( I.M. ) Administration . 30 μL of an LNP-mRNA modulator encoding influenza A/Puerto Rico/8/1934 hemagglutinin ( HA ) was injected intramuscularly into the quadriceps femoris muscle of BALB/c mice at doses of 0.2 or 0.5 µg mRNA on days 0 and 14. Blood samples were collected on days -1 and 13, and final blood was collected on day 28, and serum was obtained. The immunogenicity of the LNP modulator was determined by hemagglutination inhibition (HAI) assay.

Hemagglutination inhibition ( HAI ) assay involves diluting the sample with receptor-degrading enzyme II (RDE) at a 1:4 ratio to inactivate nonspecific hemagglutination inhibitors present in serum. The sample is incubated at 37°C for 18 hours, followed by a further incubation at 56°C for 30 minutes to inactivate the enzyme. The RDE-treated sample is then diluted 1:10 in 0.85% NaCl. A 1% turkey red blood cell (TRBC) suspension is prepared in PBS. Nonspecific agglutinins in the sample are detected by incubating 25 µL of RDE-treated serum, 25 µL of PBS, and 50 µL of the 1% TRBC suspension at room temperature for 30 minutes. If the RBCs completely precipitate in the wells containing serum, the serum sample is acceptable for HAI assay.

Validated RDE-treated serum samples were serially diluted twice in PBS, a total of 12 dilutions ranging from 20 to 40960. Inactivated IFV-A/PR/8/34 (Charles River Laboratories, #10100782) was prepared to a concentration equivalent to 4 hemagglutinin (HA) units and incubated with serum at a 1:1 ratio at room temperature for 30 minutes. The serum/antigen mixture was incubated with 1% TRBC suspension at a 1:1 ratio at room temperature for 30 minutes. Controls containing only TRBC and only antigen were included.

The culture trays were then analyzed and evaluated. Hemagglutination was examined in each well. Inhibition of hemagglutination indicated the presence of HA-specific antibodies. For each serum sample, the highest dilution that completely inhibited hemagglutination was recorded as the HA titer.

Other embodiments can be provided by combining the various embodiments described above. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referenced in this specification are incorporated herein by reference in their entirety. Where necessary, the embodiments may be modified to adopt the concepts of various patents, applications, and publications to provide other embodiments.

In light of the detailed description above, such and other changes may be made to the embodiments. Generally, the terms used in the following patent claims should not be construed as limiting the scope of the patent claims to the specific embodiments disclosed in the specification and patent claims, but rather as including the entire scope of all possible embodiments and equivalents that such patent claims may assert. Therefore, the scope of the patent claims is not limited by this disclosure.

In the diagram, similar elements are indicated by the same symbol. The size and relative position of the elements in the diagram are not necessarily drawn to scale, and some of these elements have been enlarged and positioned to improve the readability of the diagram. In addition, the shapes of the drawn elements are not intended to convey any information about the actual shape of the elements and are chosen solely for ease of identification of the diagram. Figure 1 illustrates IgG performance tested using the procedure described in Biological Example 2. Samples 6a-1, 6b-1, 6a-2, 6b-2, 6a-3, 6b-3, 6a-4, and 6b-4 are shown from left to right. In the figure, # indicates that sample 6b-3 is p < 0.01 compared to sample 6a-3 (one-tailed test), and * indicates p < 0.05. Samples were tested at a dose of 0.3 mg/kg. Figure 2 shows the IgG performance tested using the procedure described in Biological Example 2. Samples 6a-2 and 6b-2 are shown from left to right. In the figure, p < 0.01 compared to sample 6a-2. Samples were tested at a dose of 1 mg/kg. Figure 3 shows a diagram of the lipadsorption formation test performed on the samples after storage at -80°C. Samples were prepared and tested as described in Tables 6a and 6b; in the figure, samples 6a-1, 6b-1, 6a-2, 6b-2, 6a-3, 6b-3, 6a-4, and 6b-4 are shown from left to right. Figure 4 is a diagram of the lipadsorption formation test performed on the samples after post-administration storage (i.e., storage at 4°C for 2 weeks). Samples were prepared and tested as described in Tables 6a and 6b; in the figure, samples 6a-1, 6b-1, 6a-2, 6b-2, 6a-3, 6b-3, 6a-4, and 6b-4 are shown from left to right. Figure 5 is a graph showing the coating percentage of the samples prepared according to Table 6a. The samples shown include (from left to right) sample 6a-1 analyzed by UPLC, sample 6a-1 analyzed by RG, sample 6a-2 analyzed by UPLC, sample 6a-2 analyzed by RG, sample 6a-3 analyzed by UPLC, sample 6a-3 analyzed by RG, sample 6a-4 analyzed by UPLC, and sample 6a-4 analyzed by RG. Figure 6 is a graph showing the coating percentage of the samples prepared according to Table 6a. The samples shown include (from left to right): sample 6b-1 analyzed by UPLC, sample 6b-1 analyzed by RG, sample 6b-2 analyzed by UPLC, sample 6b-2 analyzed by RG, sample 6b-3 analyzed by UPLC, sample 6b-3 analyzed by RG, sample 6b-4 analyzed by UPLC, and sample 6b-4 analyzed by RG. Figure 7 shows the HAI units measured for samples prepared according to Tables 9a and 9b at a dosage of 0.2 µg. This figure shows the data for samples 9a-1, 9b-1, 9a-2, 9b-2, 9a-3, and 9b-3 (from left to right). Figure 8 shows the HAI units measured for samples prepared according to Tables 9a and 9b at a dosage of 0.5 µg. This figure shows data for samples 9a-1, 9b-1, 9a-2, 9b-2, 9a-3, and 9b-3 (from left to right). Figure 9 shows the percentage of coverage relative to the load concentration at the 10-minute time point. For each concentration (i.e., RNA concentrations of 0.075, 0.15, 0.5, and 1 mg/mL), the percentage of coverage for samples 10⁻¹, 10⁻², and 10⁻⁴ is shown (from left to right). Samples 10⁻¹ and 10⁻² were not tested at a concentration of 0.075. Figure 10 shows the Z-mean particle size (nm) of the samples prepared according to Table 10 (squares indicate sample 10⁻¹; diamonds indicate sample 10⁻²; circles indicate sample 10⁻⁴). Samples were detected at the 10-minute time point. Figure 11 shows the physical characteristics of LNPs loaded in 25 mM acetate buffer at pH 5.5 at different RNA concentrations. Figure 12 shows the physical characteristics of LNPs loaded in 25 mM acetate buffer at pH 5.9 at different RNA concentrations. Figure 13 shows the encapsulation efficiency of LNPs loaded in 25 mM acetate buffer at pH 5.5 and pH 5.9 at different RNA concentrations. Figure 14 shows the effect of acetate concentration on PFL LNPs at pH 5.9; the unit of acetate concentration at pH 5.9 is [mM]. Figure 15 shows the RNA encapsulation efficiency of LNPs prepared with different mol% PEG lipids over time. Figure 16 is a graph showing the coating efficiency of LNPs of different sizes prepared using the PFL method with ionizable lipid B-45 as a function of incubation time. Figure 17 shows the change in particle size (Z-mean) after 90 minutes of incubation. In the figure, the bars represent the particle size change of LNPs after formation (i.e., the PFL method), and the particle size change corresponds to the left scale; the lines/dots represent the LNP particle size after RNA loading, corresponding to the right scale. Figure 18 shows a comparison of coating efficiency related to particle size during incubation. Figure 19 shows the change in particle size (Z-mean) after 90 minutes of incubation. In the figure, the bars show the particle size changes of LNPs after loading (i.e., the PFL method), with the particle size changes corresponding to the left-hand scale; the lines/dots show the particle size of LNPs after RNA loading, corresponding to the right-hand scale. Figure 20 shows the particle size (Z-mean) and PDI of LNPs prepared using buffers with different pH values and ionizable lipid A-15. In the figure, the bars show the Z-mean particle size of LNPs at a series of pH values, corresponding to the left-hand scale; the lines/dots show the LNP PDI value, corresponding to the right-hand scale. Figure 21 shows a comparison of the encapsulation efficiency of LNPs prepared using ionizable lipid A-15 at different pH values. Figure 22 shows the particle size (Z-mean) and PDI of LNPs prepared using buffers with different pH values and ionizable lipid B-3. In the figure, the bars show the particle size changes of LNPs at a series of pH values, corresponding to the left scale; the lines/dots show the LNP PDI values, corresponding to the right scale. Figure 23 shows a comparison of the encapsulation efficiency of LNPs prepared using ionizable lipid B-3 at different pH values. Figure 24 shows the particle size (Z-mean) and PDI of LNPs prepared using buffers with different pH values and ionizable lipid D-1. In the figure, the bars show the particle size changes of LNPs at a series of pH values, corresponding to the left scale; the lines/dots show the LNP PDI values, corresponding to the right scale. Figure 25 shows a comparison of the encapsulation efficiency of LNPs prepared using ionizable lipid D-1 at different pH values. Figure 26 shows the particle size (Z-mean) and PDI of LNPs prepared using buffers with different pH values and ionizable lipid C-18. In the figures, the bars show the particle size changes of LNPs at a series of pH values, corresponding to the left scale; the lines/dots show the LNP PDI values, corresponding to the right scale. Figure 27 shows a comparison of the encapsulation efficiency of LNPs prepared using ionizable lipid C-18 at different pH values. Figure 28 is a curve showing the encapsulation efficiency over time of LNPs prepared using 1.8 mol% PEG lipid 1 and ionizable lipid A-15, measured by UPLC. This LNP encapsulated RNA almost immediately at 97%. Figure 29 is a graph showing the coating efficiency of LNP prepared using 5 mol% PEG lipid 1 and ionizable lipid A-15 over time, measured by UPLC. The coating efficiency increased from 20% to 67% over 10 hours. Figure 30 shows serum IgG performance at a dose of 0.5 mg/kg. Samples were prepared according to the description in Formulation Example 18. From left to right, Figure 30 shows the results for samples A, B, C, D, and E. In Figure 30, * indicates p < 0.05 compared to the T-type control (sample A), and ** indicates p < 0.01 between samples D and E. Figure 31 shows the particle size (top set of lines) and PDI (bottom set of lines) measurements of empty LNP samples at different time points after storage at 2 to 8°C. Figure 32 shows the particle size (top line) and PDI (bottom line) measurements of empty LNP samples at different time points after storage at -80°C. Figure 33 shows the particle size (top line) and PDI (bottom line) measurements of samples after storage at 2 to 8°C followed by RNA loading at different time points. Figure 34 shows the particle size (top line) and PDI (bottom line) measurements of samples after storage at -80°C followed by RNA loading at different time points. Figure 35 shows the RNA loading efficiency of stored empty LNPs as a measure of coating efficiency. The results at various time points (i.e., 1 month, 3 months, and 6 months) are shown for concentrations of 0.5 mg/mL at 2–8°C, 1.0 mg/mL at 2–8°C, 2.0 mg/mL at 2–8°C, 3.1 mg/mL at 2–8°C, 0.1 mg/mL at -80°C, 0.5 mg/mL at -80°C, 1.0 mg/mL at -80°C, and 2.0 mg/mL at -80°C. Figure 36A shows the z-mean particle size of the sample prepared using the T-mix technique as described in detail in Formulation Example 19. Figure 36B shows the PDI of the sample prepared using the T-mix technique as described in detail in Formulation Example 19. Figure 36C shows the total lipid content (mg/mL) of the sample prepared using the T-mix technique as described in detail in Formulation Example 19. Figure 37A shows the z-mean particle size of the sample prepared using the PFL method as described in detail in Formulation Example 19. Figure 37B shows the PDI of the sample prepared using the PFL method as described in detail in Formulation Example 19. Figure 37C shows the total lipid content (mg/mL) of the sample prepared using the PFL method as described in detail in Formulation Example 19. Figure 38A shows the RNA content of the sample prepared using the T-mix technique as described in detail in Formulation Example 19. Figure 38B shows the encapsulation efficiency of the sample prepared using the T-mix technique as described in detail in Formulation Example 19. Figure 38C shows the RNA integrity of the sample prepared using the T-mix technique as described in detail in Preparation Example 19. Figure 39A shows the RNA content of the sample prepared using the PFL method as described in detail in Preparation Example 19. Figure 39B shows the encapsulation efficiency of the sample prepared using the PFL method as described in detail in Preparation Example 19. Figure 39C shows the LNP integrity of the sample prepared using the PFL method as described in detail in Preparation Example 19. Figure 40 shows the LNP z-mean particle size (shown as bars, corresponding to the left scale) and PDI (shown as dots/lines, corresponding to the right scale) as a function of the N:P ratio. Figure 41 shows the encapsulation efficiency of LNPs as a function of the N:P ratio (N:P ratio from 9 to 3). Figure 42A shows the RNA coating efficiency of empty LNPs of different sizes prepared using ionizable lipid B-3 after incubation with RNA at ambient temperature for 10 minutes. Figure 42B shows the particle size change (Z-mean) after 10 minutes of incubation. In the figures, the bars represent the particle size change of the loaded ionizable lipid B-3 LNPs (i.e., the PFL method), and the particle size change corresponds to the left scale; the lines/dots represent the LNP particle size after RNA loading, corresponding to the right scale. Figure 43A shows the z-mean size (in bars, corresponding to the left scale) and PDI (in dots/lines, corresponding to the right scale) of ionizable lipid B-3 LNPs loaded with siRNA, saRNA, or mRNA/gRNA mixtures using the PFL method. Figure 43B shows the z-mean size (in bars, corresponding to the left scale) and PDI (in dots/lines, corresponding to the right scale) of ionizable lipid C-18 LNPs loaded with siRNA, saRNA, or mRNA/gRNA mixtures using the PFL method. Figure 43C shows the coating efficiency of LNPs prepared from ionizable lipids B-3 or C-18 (as indicated) loaded with siRNA, saRNA, or mRNA/gRNA mixtures using the PFL method. Figure 44A shows the z-mean size of empty LNPs prepared in the presence of an aqueous buffer containing increasing concentrations of sodium chloride. Figure 44B shows the z-mean particle size of LNPs prepared using the PFL method in the presence of an aqueous buffer containing increasing concentrations of sodium chloride. Figure 44C shows the coating efficiency of LNPs prepared using the PFL method in the presence of increasing concentrations of sodium chloride.

Claims (88)

A method for encapsulating a nucleic acid-containing payload within lipid nanoparticles (LNPs) comprises: i) providing a first aqueous solution containing a plurality of LNPs, the LNPs containing a plurality of ionizable lipids, wherein the first solution is substantially free of any destabilizing agent, and the difference between the pH of the first solution and the pKa of the ionizable lipids is less than 1.0; and ii) mixing the first solution with a dried payload or a payload in a second aqueous solution, wherein the second aqueous solution is substantially free of any destabilizing agent, thereby encapsulating at least a majority of the payload in the LNPs in the resulting solution. A method for encapsulating a nucleic acid-containing payload within lipid nanoparticles (LNPs) comprises: i) providing a first dried composition comprising a plurality of LNPs, the LNPs containing a plurality of ionizable lipids; and ii) mixing the first composition with the payload in a second aqueous solution, wherein the second aqueous solution substantially contains no destabilizing agent, thereby encapsulating at least a majority of the payload in the LNPs in the first solution. A method for encapsulating a nucleic acid-containing payload within lipid nanoparticles (LNPs) comprises: i) providing a first dried composition comprising a plurality of LNPs, the LNPs containing a plurality of ionizable lipids; ii) mixing the first composition with a second composition comprising the dried payload; and iii) mixing the first composition and the second composition with a first aqueous solution, wherein the first solution is substantially free of any destabilizing agent and the difference between the pH of the first solution and the pKa of the ionizable lipids is less than 1.0, thereby encapsulating at least a majority of the payload within the LNPs. The method of any one of claims 1 to 3, wherein the LNPs in the first solution or the first composition are not coated with an effective load. The method of any of claims 1 to 3, wherein the pH of the first solution is less than 7.0. The method of any of claims 1 to 3, wherein the pH of the first solution is less than 6.8. The method of any of claims 1 to 3, wherein the pH of the first solution is less than 6.5. The method of any one of claims 1 to 3, wherein the pH of the first solution is less than 6.4, less than 6.3, less than 6.2, less than 6.1, less than 6.0, less than 5.9, less than 5.8 or less than 5.7. The method of any of claims 1 to 3, wherein the pH of the first solution is less than 5.0. The method of any of claims 1 to 3, wherein the pH of the first solution is less than 5.7. The method of any one of claims 1 to 3, wherein the pH of the first solution is in the range of 4.5 to 7.4, 5.5 to 6.5, or 5.7 to 6.2. The method of any one of claims 1 to 3, wherein the difference between the pH of the first solution and the pKa of the ionizable lipids is in the range of 0.1 to 1.5, 0.1 to 1.0, 0.1 to 0.5, 0.2 to 0.5, 0.5 to 0.5, 0.3 to 0.6, 0.2 to 0.6, or 0.4 to 0.8. The method of any one of claims 1 to 3, wherein the difference between the pH of the first solution and the pKa of the ionizable lipids is less than 1.0. The method of any of claims 1 to 3, wherein the difference between the pH of the first solution and the pKa of the ionizable lipids is less than 0.7. The method of any one of claims 1 to 3, wherein the difference between the pH of the first solution and the pKa of the ionizable lipids is less than 0.5, less than 0.3, or less than 0.1. The method of any one of claims 1 to 3, wherein the difference between the pH of the first solution and the pKa of the ionizable lipids is at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.2 or at least 1.5. The method of any one of claims 1 to 3, wherein the difference between the pH of the first solution and the pKa of the ionizable lipids is in the range of 0.3 to 1.0, 0.2 to 0.9, 0.1 to 1.1, 0.1 to 0.8, or 0.2 to 0.7. The method of any of the requests 1 to 3, wherein the coverage efficiency is at least 70%. The method of any of the requests 1 to 3, wherein the coverage efficiency is at least 80%. The method of any of the requests 1 to 3, wherein the coverage efficiency is at least 85%. The method of any of the requests 1 to 3, wherein the coverage efficiency is at least 88%. The method of any of the requests 1 to 3, wherein the coverage efficiency is at least 90%. The method of any of the requests 1 to 3, wherein the coverage efficiency is at least 95%. The method of claim 1, wherein the pH of the resulting solution is in the range of 6.0 to 7.8. The method of claim 1, wherein the pH of the resulting solution is in the range of 6.5 to 7.8. The method of claim 1, wherein the pH of the resulting solution is in the range of 7.0 to 7.5. The method of any one of claims 1 to 3, wherein the particle size of the LNPs effectively loaded in the first solution or the resulting solution is in the range of 20 to 200 nm, 20 to 120 nm, 30 to 90 nm, 40 to 120 nm, 50 to 120 nm, or 30 to 100 nm. The method of any one of claims 1 to 3, wherein the particle size of the LNPs effectively loaded in the first solution or the resulting solution is in the range of 60 to 100 nm. The method of any one of claims 1 to 3, wherein the particle size of the LNPs effectively loaded in the first solution or the resulting solution is in the range of 70 to 90 nm. The method of any one of claims 1 to 3, wherein the particle size of the LNPs coated with the effective load in the first solution or the resulting solution is less than 90 nm. The method of any one of claims 1 to 3, wherein the particle size of the LNPs coated in the first solution or the resulting solution is less than 80 nm. The method of any one of claims 1 to 3, wherein the particle size of the LNPs coated in the first solution or the resulting solution is less than 70 nm, less than 60 nm or less than 50 nm. The method of any of claims 1 to 3, wherein the destabilizing agent in the first or second solution is an organic solvent. The method of any one of claims 1 to 3, wherein the destabilizing agent in the first or second solution is methanol, ethanol, isopropanol, tetrahydrofuran, dimethylformamide, dimethyl sulfoxide, acetonitrile, sodium dodecyl sulfate, or a combination thereof. The method of any one of claims 1 to 3, wherein the temperature of the first solution prior to mixing is lower than 27°C, lower than 25°C, or lower than 22°C. The method of any one of claims 1 to 3, wherein the temperature of the second solution during mixing is lower than 27°C, lower than 25°C, or lower than 22°C. The method of claim 1, wherein the temperature of the resulting solution immediately after mixing is lower than 27°C, lower than 25°C, or lower than 22°C. The method of any one of claims 1 to 3, wherein the plurality of LNPs in the first solution or the resulting solution contains 20 to 90 mol% or 30 to 90 mol% of the ionizable lipids. The method of any one of claims 1 to 3, wherein the plurality of LNPs in the first solution or the resulting solution comprises 40 to 55 mol% of the ionizable lipids. The method of any one of claims 1 to 3, wherein the plurality of LNPs in the first solution or the resulting solution comprises 46 to 49 mol% of the ionizable lipids. The method of any one of claims 1 to 3, wherein the plurality of LNPs in the first solution or the resulting solution comprises one or more components of lipids selected from neutral lipids, steroids and bound polymers. The method of any one of claims 1 to 3, wherein the plurality of LNPs in the first solution or the resulting solution contain a concentration of neutral lipids in the range of about 5 to about 15 mol% of the lipid nanoparticles. The method of any one of claims 1 to 3, wherein the concentration of the steroid contained in the plurality of LNPs in the first solution or the resulting solution is in the range of about 30 to about 50 mol% of the lipid nanoparticles. The method of any one of claims 1 to 3, wherein the plurality of LNPs in the first solution or the resulting solution contain polyethylene glycol-modified lipids at a concentration of 0.1 to 10 mol%, 0.1 to 5 mol%, 0.1 to 3 mol%, 0.1 to 2 mol%, 0.1 to 1 mol%, 0.5 to 10 mol%, 0.5 to 5 mol%, 0.5 to 3 mol%, 0.5 to 2 mol%, 0.5 to 1 mol%, 1.0 to 10 mol%, 1.0 to 5 mol%, 1.0 to 3 mol%, 1.0 to 2 mol%, 1.5 to 10 mol%, 1.5 to 5 mol%, 1.5 to 3 mol%, 1.5 to 2 mol%, 2.0 to 10 mol%, 2.0 to 5 mol%, 2.0 to 3 mol%, 2.5 to 10 mol%, 2.5 to 5 mol%. mol%, or in the range of 2.5 to 3 mol%. The method of any of claims 1 to 3, wherein the ionizable lipids have the following structure: Or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein: ^L1 and ^L2 are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -SS-, -C(=O) _S- , -SC(=O)-, -NR a C(=O)-, -C(=O)NR ^a- , -NR ^a C(=O)NR ^a- , -OC(=O)NR ^a- , ^{-NR a} C(=O)O-, or a direct bond; ^G1 is a _C1 - _C2 alkylene group, -(C=O)-, -O(C=O)-, -SC(=O)-, -NR ^a C(=O)-, or a direct bond; G ² is -C(=O)-, -(C=O)O-, -C(=O)S-, -C(=O) ^NRa- , or a direct bond; ^G3 is a _C1 - _C6 alkyl group; ^Ra is H or _C1 - _C12 alkyl group; ^R1a and ^R1b are independently: (a) H or _C1 - _C12 alkyl group; or (b) ^R1a is H or _C1 - _C12 alkyl group, and ^R1b together with its bonded carbon atom forms a carbon-carbon double bond with the adjacent ^R1b and its bonded carbon atom; ^R2a and ^R2b are independently: (a) H or _C1 - _C12 alkyl group; or (b) ^R2a is H or _C1 - _C12 alkyl group, and R ^R2b, together with its bonded carbon atom, forms a carbon-carbon double bond with the adjacent ^R2b and its bonded carbon atom; ^R3a and ^R3b, in each occurrence, are independently: (a) H or _C1 - _C12 alkyl; or (b) ^R3a is H or _C1 - _C12 alkyl, and ^R3b , together with its bonded carbon atom, forms a carbon-carbon double bond with the adjacent ^R3b and its bonded carbon atom; ^R4a and ^R4b, in each occurrence, are independently: (a) H or _C1 - _C12 alkyl; or (b) ^R4a is H or _C1 - _C12 alkyl, and ^R4b , together with its bonded carbon atom, forms a carbon-carbon double bond with the adjacent ^R4b and its bonded carbon atom; ^R5 and R ⁶ is independently H or methyl; ^R7 is _C4 - _C20 alkyl; ^R8 and ^R9 are independently _C1 - _C12 alkyl; or ^R8 and ^R9 together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocycle; a, b, c, and d are independently integers from 1 to 24; and x is 0, 1, or 2. The method of any of claims 1 to 3, wherein the ionizable lipids have the following structure: Or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein: one of ^L1 or ^L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) _x- , -SS-, -C(=O)S-, -SC(=O)-, -NR ^a C(=O)-, -C(=O)NR ^a- , NR ^a C(=O)NR ^a- , -OC(=O)NR ^a- , or -NR ^a C(=O)O-, and the other of ^L1 or ^L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) _x- , -SS-, -C(=O)S-, SC(=O)-, -NR ^a C(=O)-, -C(=O)NR ^a- , NR ^a C(=O)NR ^a- , -OC(=O)NR ^a- , or -NR ^a C(=O)O-, or direct bond; ^G1 and ^G2 are each independently unsubstituted _C1 - _C12 alkyl or _C1 - _C12 alkenyl; ^G3 is _C1 - _C24 alkyl, _C1 -C24 alkenyl, _C3 - _C8 cycloalkyl, or _C3 - _C8 cycloalkyl; ^Ra is H or _{C1 -C12 alkyl} ; ^R1 and ^R2 are each independently _C6 - _C24 alkyl or _C6 - _C24 alkenyl; ^R3 is H, ^OR5 , CN, -C(=O) ^OR4 , -OC(=O) ^R4 , or ^-NR5 C(=O) ^R4 ; ^R4 is _C1 - _C12 alkyl; ^R5 is H or C ₁ -C ₆ alkyl; and x is 0, 1 or 2. The method of any of claims 1 to 3, wherein the ionizable lipids have the following structure: Or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein: ^R1 is, where appropriate, a substituted _C1 - _C24 alkyl or a substituted _C2 - _C24 alkenyl; ^R2 and ^R3 are each independently, where appropriate, a substituted _C1 - _C36 alkyl; ^R4 and ^R5 are each independently, where appropriate, a substituted _C1 - _C6 alkyl, or ^R4 and ^R5 are combined with their attached N to form a heterocyclic or heteroaryl group; ^L1 , ^L2 , and ^L3 are each independently, where appropriate, a substituted _C1 - _C18 alkyl; ^G1 is a direct bond, -( _CH2 ) _nO (C=O)-, -( _CH2 ) _n (C=O)O- or -(C=O)-; ^G2 and ^G3 are each independently -(C=O)O- or -O(C=O)-; and n is an integer greater than 0. The method of any of claims 1 to 3, wherein the ionizable lipids have the following structure: Or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein: ^L1a and ^L1b are each independently a substituted _C3 - _C12 alkyl group, as appropriate; ^R1a is -C(=O) ^OR4a or -O(C=O) ^R4a ; ^R1b is -C(=O) ^OR4b or -O(C=O) ^R4b ; ^R2 is ^-NR6 (C=O) ^R5 , -(C=O)N( ^R6 ) ^R5 , or -(C=O) ^OR7 ; ^R3 and ^R6 are each independently hydrogen or a substituted _C1 - _C12 alkyl group, as appropriate; ^R4a , ^R4b , and ^R5 are each independently a substituted alkyl group, as appropriate; ^R7 is a substituted _C1 alkyl group, as appropriate. -C ₆ alkyl or, where applicable, substituted aryl alkyl; n1 is 2, 3, 4, 5 or 6; and X is C ₂ -C ₆ alkyl or C ₄ -C ₂₀ alkylene oxide. The method of any one of claims 1 to 3, wherein the ionizable lipids have one of the structures in Table A or B. The method of any of claims 1 to 3, wherein the ionizable lipids have one of the structures in Table C. The method of any one of claims 1 to 3, wherein the ionizable lipids have one of the structures in Table D. The method of any one of claims 1 to 3, wherein the plurality of LNPs in the first solution or the resulting solution comprises one or more neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. The method of claim 52, wherein the neutral lipid is DSPC. The method of claim 41, wherein the steroid is cholesterol. The method of claim 41, wherein the lipid of the bound polymer is a polyethylene glycol-modified lipid. The method of claim 55, wherein the polyethylene glycol ester is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer or PEG dialkoxypropylcarbamate. The method of claim 55, wherein the polyethylene glycol-modified lipid has the following structure: Or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: R ¹⁰ and R ¹¹ are each independently a straight-chain or branched-chain alkyl, alkenyl or alkynyl group containing 10 to 30 carbon atoms, wherein each alkyl, alkenyl or alkynyl group is, where appropriate, substituted with at least one fluorine; and z is an integer in the range of 30 to 60. The method of claim 57, wherein R ¹⁰ and R ¹¹ are each independently a straight-chain alkyl group containing 12 to 16 carbon atoms. As in the method of request item 57, where z is in the range of 45 to 50. The method of any one of claims 1 to 3, wherein the effective payload is DNA, siRNA, microcircular RNA, PNA, aptamer, guide RNA, PE guide RNA, saRNA, circular RNA, antisense RNA, messenger RNA, Cas9 mRNA, ribonucleoprotein, or a combination thereof. The method of any of requests 1 to 3, wherein the effective payload is mRNA. The method of any of claims 1 to 3, wherein the effective payload contained in the LNP is delivered to the patient in need. Use of a lipid nanoparticle (LNP) prepared as claimed in any of claims 1 to 61 for the manufacture of a medicament for the treatment of diseases or symptoms caused by infectious entities and/or protein deficiency and/or of a genetic basis. For the purpose described in claim 63, the agent is administered within 72 hours of mixing. For the purpose described in claim 63, the agent is administered within 24 hours of mixing. For the purpose described in claim 63, the agent is administered within 12 hours of mixing. For the purpose described in claim 63, the agent is administered within 4 hours of mixing. For any of the uses described in items 63 to 67, the effective load is less than 1.5 mg per kilogram of patient. For any of the uses described in claims 63 to 67, the effective load is less than 1.0 mg per kilogram of patient. For any of the uses described in items 63 to 67, the effective load is less than 0.5 mg per kilogram of patient. For any of the uses in requests 63 to 67, the effective load is less than 150 micrograms. For any of the uses specified in items 63 to 67, the effective load is less than 100 micrograms or less than 75 micrograms. For any of the uses specified in items 63 to 67, the effective load is in the range of 1 to 30 micrograms. A pharmaceutical composition comprising an LNP prepared by any one of claims 1 to 61 and a pharmaceutically acceptable diluent or excipient. A kit comprising: i) a first aqueous solution containing a plurality of LNPs, the LNPs containing a plurality of ionizable lipids, wherein the first solution is substantially free of any destabilizing agent, and the difference between the pH of the first solution and the pKa of the ionizable lipids is less than 1.0; and ii) a dried effective load containing nucleic acids or an effective load containing nucleic acids in a second aqueous solution substantially free of any destabilizing agent. A kit comprising a first aqueous solution containing a plurality of LNPs, the LNPs containing a plurality of ionizable lipids, wherein the first solution substantially contains no destabilizing agent, and the difference between the pH of the first solution and the pKa of the ionizable lipids is less than 1.0. A kit comprising a first dried composition, comprising: i) a plurality of dried LNPs comprising a plurality of ionizable lipids; and ii) a first solution substantially free of any destabilizing agent, wherein the difference between the pH of the first solution and the pKa of the ionizable lipids is less than 1.0. The kit of claim 77 further comprises: a dried effective load containing nucleic acids or an effective load containing nucleic acids in a second aqueous solution that is substantially free of any destabilizing agent. The kit of any of claims 75 to 78, wherein the kit further includes instructions for mixing the first solution with the dried LNP, the dried effective load, the second solution, or a combination thereof. As in any of claims 75 to 78, a set wherein when the first solution is mixed with the dried effective load, the second solution, or a combination thereof, a majority of the effective load is coated by the LNPs. Such as the kit in any of the requests 75 to 78, wherein the kit includes one or more unit doses of an effective payload containing nucleic acids. For example, in the kit requested in item 81, the unit dose is less than 1.5 mg per kilogram of patient. For example, in the kit of request item 81, the unit dose is less than 1.0 mg per kilogram of patient. For example, in the kit requested in item 81, the unit dose is less than 0.5 mg per kilogram of patient. For example, the kit in request item 81, wherein the unit dose is less than 150 micrograms. For example, the kit in request item 81, wherein the unit dose is less than 100 micrograms. For example, the kit in request item 81, wherein the unit dose is less than 75 micrograms. For example, the kit in request item 81, wherein the unit dose is in the range of 1 to 30 micrograms.