Compositions and methods for non-viral delivery of therapeutic compounds

Abstract

The present disclosure provides compositions and methods for non-viral delivery of therapeutic agents and methods of making the compositions.

Description

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 610,979, filed December 15, 2023, which is incorporated herein by reference for all purposes.

[0002] Throughout this application, various publications, patents, and / or patent applications are cited. The disclosures of these publications, patents, and / or patent applications are hereby incorporated in their entirety by reference in order to more fully describe the prior art in the field to which this disclosure pertains. Technical Field

[0003] This disclosure provides compositions and methods for delivering nonviral therapeutic agents, as well as methods for preparing said compositions. Background Technology

[0004] Delivering therapeutic agents to patients can be hampered by the limited ability of such compounds to reach target cells or tissues, or by restrictions on the entry of therapeutic agents into cells or tissues. Typically, the delivery of therapeutic agents is limited by cell membranes. Therefore, to overcome these constraints and limitations on delivery, one approach is to use therapeutic agent concentrations much higher than required for treatment, which leads to an increased risk of adverse drug reactions and side effects.

[0005] Another strategy to address delivery constraints and limitations is to use lipid molecules to improve the transport of compounds to target cells or tissues. Lipids can utilize existing mechanisms for selective cellular entry. For example, cationic lipids can interact with drugs and provide contact with the cell membrane. Lipid molecules can also be organized into liposomes or particles as carriers for drug agents. Liposome drug carriers can protect drug molecules from degradation while improving their uptake by cells. Lipid-containing nanoparticle compositions, liposomes, and lipid complexes have proven effective as transport mediators for the entry of bioactive substances, such as small molecule drugs, proteins, and nucleic acids, into cells and / or intracellular compartments.

[0006] Transfection is a method of delivering nucleic acids into cells. Transfection can be viral or non-viral. Viral vectors generally promote gene transfer to eukaryotic cells very efficiently, but they tend to be highly immunogenic and not suitable for repeated administration. Non-viral vectors tend to be much less efficient in terms of transfection efficiency, but are generally less likely to induce a strong inflammatory response than viral vectors. Among non-viral vectors, cationic lipids and cationic polymers have been extensively studied, and their transfection efficiency has been clearly demonstrated in vitro (Drean M. (2017) Biomacromolecules). Biomacromolecules )》 18:440; Rose V. (2017) Polymer Chemistry ( Polym. Chem.)》 8:353; Pitard B. (2002) Somatic Cell and Molecular Genetics ( Somat Cell Mol. Genet (27:5-15). However, their accumulation in tissue fluid, toxicity, and low in vivo efficiency hinder their clinical application.

[0007] There are many compositions and methods in the art for delivering plasmid DNA (therapeutic agents) into cells. A more efficient transfection method is through electroporation (Sardesai N. et al., (2011) Current Immunological Perspectives (…). Curr. Opin. Immunol. (23:421-429) However, electroporation requires specialized and expensive instruments for injection (e.g., Cellectra). ® Furthermore, it can lead to permanent cell damage. Methods that rely on simple polymer-based formulations to transfect and deliver therapeutic agents into cells (described, for example, in US7,709,452 and US8,367,631) are significantly less efficient.

[0008] However, other transfection methods rely on lipid-based formulations. A commonly used lipid-based transfection reagent is Lipofectamine, sold by Thermo Fisher Scientific. TM and Invivofectamine TM These reagents are easy to use, require no user optimization, and Lipofectamine TM These reagents can be used for different payloads (DNA and RNA). However, they also have many drawbacks. For example, Lipofectamine... TM It can only be used for in vitro transfection, while Invivofectamine TM It can only be used for in vivo transfection. Although Lipofectamine TM It is effective in some cells, but its efficacy is lower in primary cells. Invivofectamine TM It can only be used to transfect siRNA and miRNA, and can only be administered intravenously. In addition, both transfection reagents are extremely expensive.

[0009] Despite the many strategies available to improve in vivo transfection, cost-effective and efficient targeted delivery of bioactive substances, such as small molecule drugs, proteins, and nucleic acids, remains an ongoing medical challenge.

[0010] Therefore, there is a need to develop compositions and methods to improve the transfection and delivery of therapeutic agents (such as nucleic acids, proteins, and / or small molecule drugs) into cells both in vitro and in vivo. Preferably, such compositions are generally applicable for in vitro and in vivo transfection, have high transfection efficiency in any cell type, and are relatively inexpensive. Summary of the Invention

[0011] The compositions described herein are easy to prepare. In embodiments, the same compositions can be used for both in vitro and in vivo transfection, and these compositions exhibit very high efficacy in all cell types (e.g., with Lipofectamine). TM Compared to Lipofectamine, it is suitable for use with a variety of nucleic acids, and the composition can be more effective than Lipofectamine. TM and Invivofectamine TM Cheaper.

[0012] On the one hand, this article provides a composition for transfecting cells in vitro or in vivo, the composition comprising at least one nucleic acid contained in an LPS (lipid shell)-LPX (lipid complex) delivery system, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule.

[0013] On the one hand, this article provides a method for transfecting cells with one or more nucleic acids, the method comprising: contacting the cells in vitro with a composition comprising at least one nucleic acid contained in an LPS-LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule.

[0014] On the other hand, this article provides a method for treating or preventing a disease or alleviating the symptoms of a disease, the method comprising administering to a subject in need a composition for transfecting cells, the composition comprising at least one nucleic acid contained in an LPS-LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digestive agent or a small molecule.

[0015] On the other hand, this article provides a method for preparing a composition for in vitro or in vivo transfection of cells, the method comprising: (a) preparing a first solution comprising an aqueous phase and one or more nucleic acids; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b), thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids; (d) stirring the LPX prepared in step (c); and (e) preparing a composition comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a... (f) preparing LPS by forming liposomes, micelles or other self-assembled lipid nanoparticles from the third solution prepared in step (e), wherein a targeting ligand, imaging ligand, digestive agent or small molecule is optionally added to an aqueous phase, and then the aqueous phase is mixed with the third solution prepared in step (e); (g) mixing the LPX after step (d) with the LPS prepared in step (f), and optionally adding a targeting ligand, imaging ligand, digestive agent or small molecule; and (h) stirring the mixture prepared in step (g), thereby forming a composition for transfecting cells.

[0016] On the other hand, this article provides a method for preparing a composition for transfecting cells in vitro or in vivo, the method comprising: (a) mixing a first solution comprising an aqueous phase and one or more nucleic acids with a second solution comprising an organic phase and one or more lipids, thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids; (b) stirring the LPX prepared in step (a); (c) preparing a third solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule; (d) preparing LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from the third solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule, wherein optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule is added to the aqueous phase, and then the aqueous phase is mixed with the third solution; (e) mixing the LPX after step (b) with the LPS prepared in step (d), and optionally adding a targeting ligand, an imaging ligand, a digestive agent, or a small molecule; and (f) stirring the mixture prepared in step (e), thereby forming a composition for transfecting cells.

[0017] On the other hand, this article provides a method for preparing a composition for in vitro or in vivo transfection of cells, the method comprising: (a) preparing a first solution comprising an aqueous phase; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) to generate liposomes, micelles, or other self-assembled lipid nanoparticles; (d) preparing a third solution comprising an aqueous phase and one or more nucleic acids; (e) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (c) with the third solution prepared in step (d), thereby forming monodisperse or polydisperse LPX; (f) preparing a composition comprising... An organic phase, one or more lipids, and optionally a targeting ligand, imaging ligand, digestive agent, or small molecule in a fourth solution; (g) preparing LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from the fourth solution prepared in step (f), wherein optionally a targeting ligand, imaging ligand, digestive agent, or small molecule is added to an aqueous phase, and then the aqueous phase is mixed with the fourth solution prepared in step (f); (h) mixing the LPX prepared in step (e) with the LPS prepared in step (g), and optionally adding a targeting ligand, imaging ligand, digestive agent, or small molecule; and (i) stirring the mixture prepared in step (h), thereby forming a composition for transfecting cells.

[0018] On the other hand, this article provides a method for preparing a composition for transfecting cells in vitro or in vivo, the method comprising: (a) mixing a first solution comprising an aqueous phase with a second solution comprising an organic phase and one or more lipids to produce liposomes, micelles, or other self-assembled lipid nanoparticles; (b) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (a) with a third solution comprising an aqueous phase and one or more nucleic acids, thereby forming monodisperse or polydisperse LPX; (c) preparing LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from a fourth solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule, wherein optionally the targeting ligand, imaging ligand, digestive agent, or small molecule is added to the aqueous phase, and then the aqueous phase is mixed with the fourth solution; (d) mixing the LPX prepared in step (b) with the LPS prepared in step (c), and optionally adding a targeting ligand, an imaging ligand, a digestive agent, or a small molecule; and (e) stirring the mixture prepared in step (d), thereby forming a composition for transfecting cells. Attached Figure Description

[0019] Figure 1The two top scatter plots show the transfection efficiency of LCF96 (transfected with 0.5 μg or 1.0 μg GFP mRNA) in HEK293 cells. The two bottom scatter plots are positive controls, showing the transfection efficiency of 0.5 μg or 1.0 μg GFP mRNA using Lipofectamine™ MessengerMAX™ (LIPOMAX). The vertical axis shows lateral scatter plots in arbitrary units. The horizontal axis shows GFP fluorescence in relative fluorescence units.

[0020] Figure 2 The scatter plots show the transfection efficiencies of LCF96, LCF107, LCF108, and LCF109 (transfected with 1.0 μg or 3.0 μg GFP mRNA) in Jurkat cells. The two bottom scatter plots are positive controls, showing the transfection efficiencies of 1.0 μg or 3.0 μg GFP mRNA using Lipofectamine™ MessengerMAX™ (LIPOMAX). The vertical axis shows lateral scatter points in arbitrary units. The horizontal axis shows GFP fluorescence in relative fluorescence units.

[0021] Figure 3 The scatter plots show the transfection efficiencies of LCF96 and LCF108 (transfected with 1.0 μg or 3.0 μg GFP mRNA) in human primary T cells. The two bottom scatter plots are positive controls, showing the transfection efficiencies of 1.0 μg or 3.0 μg GFP mRNA using Lipofectamine™ MessengerMAX™ (LIPOMAX). The vertical axis shows lateral scatter points in arbitrary units. The horizontal axis shows GFP fluorescence in relative fluorescence units.

[0022] Figure 4 In vivo imaging of anesthetized mice injected with rsF591, rsF616, or rsF592 (transfected with 1.0 μg Luc mRNA) followed by D-luciferin injection 3 hours later is shown.

[0023] Figure 5 The two top scatter plots show the transfection efficiency of LCF155 and LCF157 (transfected with 1.0 μg pDNA) in HEK293 cells. The two bottom scatter plots are positive controls, showing the transfection efficiency of 1.0 μg pDNA using Lipofectamine™ 2000 and Lipofectamine™ LTX. The vertical axis shows lateral scatter plots in arbitrary units. The horizontal axis shows GFP fluorescence in relative fluorescence units.

[0024] Figure 6The two top scatter plots show the transfection efficiency of LCF155 and LCF157 (transfected with 1.0 μg pDNA) in Jurkat cells. The bottom scatter plot is the positive control, showing the transfection efficiency of 1.0 μg pDNA using Lipofectamine™ 2000. The vertical axis shows lateral scatter plots in arbitrary units. The horizontal axis shows GFP fluorescence in relative fluorescence units.

[0025] Figure 7 In vivo imaging of anesthetized mice injected with rsF615 (transfected with 8.0 μg pDNA) followed by two D-luciferin injections at 3 and 6 hours later is shown.

[0026] Figure 8A Bioluminescent images of mRNA expression in the heart, lungs, liver, spleen, and kidneys of mice injected with rsF598, rsF599, rsF600, or rsF601 (1.0 μg LucmRNA per mouse) followed by D-luciferin injection 6 hours later are shown.

[0027] Figure 8B It shows Figure 8A The relative biodistribution of the heart, lungs, liver, spleen and kidneys of mice as described in the quantitative IVIS images.

[0028] Figure 9A Bioluminescent images of mRNA expression in the heart, lungs, liver, spleen, and kidneys of mice injected with rsF627 or rsF628 (1.0 μg Luc mRNA per mouse) followed by D-luciferin injection 3 hours later are shown.

[0029] Figure 9B It shows Figure 9A The relative biodistribution of the heart, lungs, liver, spleen and kidneys of mice as described in the quantitative IVIS images.

[0030] Figure 9C It shows Figure 9A The level of luciferase expression in the lungs of mice described in the study.

[0031] Figure 10A Bioluminescent images of mRNA expression in the heart, lung, liver, spleen, and kidney of mice injected with rsF620 or rsF621 (1.0 μg Luc mRNA per mouse) followed by D-luciferin injection 3 hours later are shown.

[0032] Figure 10B It shows Figure 10A The relative biodistribution of the heart, lungs, liver, spleen and kidneys of mice as described in the quantitative IVIS images.

[0033] Figure 10C It shows Figure 10A The level of luciferase expression in the lungs of mice described in the study.

[0034] Figure 11 illustrates the fluid / microfluidic process used to prepare LPS-LPX. Figure 11A A process for preparing LPS-LPX is shown, in which the payload (nucleic acid) is inside the LPX. Figure 11A A process for preparing LPS-LPX is shown, in which the payload (nucleic acid) is external to the LPX. Figure 11C The process for preparing LPS-LPX is shown, in which there are two payloads (nucleic acids), one inside the LPX and the other outside the LPX. Detailed Implementation

[0035] definition Unless otherwise defined, the technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art. Generally, terms relating to the techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, transgenic cell production, protein chemistry and nucleic acid chemistry, and hybridization described herein are well-known and commonly used in the art. Unless otherwise stated, the methods and techniques provided herein are generally performed according to routine procedures well-known in the art and as described in the various general and more specific references cited and discussed herein. See, for example, Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), and Ausubel et al., *Current Protocols in Molecular Biology*, Greene Publishing Associates (1992). Many foundational texts describe standard antibody production processes, including Borrebaeck (ed.), *Antibody Engineering*. Antibody Engineering ), 2nd edition, Freeman and Company, NY, 1995; McCafferty et al., Practical Methods for Antibody Engineering ( Antibody Engineering A Practical Approach(Illegible text: "Antibody Engineering Protocols"), Oxford Press, Oxford, England, 1996; and Paul (1995), "Antibody Engineering Protocols" Antibody Engineering Protocols Humana Press, Towata, NJ, 1995; Paul (ed.), Basic Immunology Fundamental Immunology ), Raven Press, New York, 1993; Coligan (1991) *Contemporary Laboratory Immunology*, *Laboratory Guide to Immunology*. Current Protocols in Immunology Wiley / Greene, NY; Harlow and Lane (1989) *Antibodies: A Laboratory Manual* Antibodies: A Laboratory Manual ( ) Cold Spring Harbor Press, NY; Stites et al., (editors) Basic and Clinical Immunology ( Basic and Clinical Immunology "Encoding Monoclonal Antibodies: Principles and Practice" (4th Edition), published by Lange Medical Publications, Los Altos, California, and its cited references; Coding Monoclonal Antibodies: Principles and Practice (2nd edition) Academic Press, New York, NY, 1986, and Kohler and Milstein's *Nature* (2nd edition). Nature (256: 495-497, 1975). All references cited herein are incorporated herein by reference in their entirety. Enzymatic reactions and enrichment / purification techniques are well known and are performed as commonly practiced in the art or as described herein according to the manufacturer's instructions. The terminology, laboratory procedures, and techniques used in conjunction with the analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are well known and commonly used in the art. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical formulation, formulation and delivery, and the treatment of patients.

[0036] The headings provided herein are not intended to limit any aspect of this disclosure, which may be understood by referring to this specification in its entirety.

[0037] Unless the context otherwise requires, singular terms shall include plural forms, and plural terms shall include singular forms. Unless explicitly and definitively limited to a single referent, the singular forms “a / an” and “the” and any other word used in the singular shall include multiple referents.

[0038] It should be understood that the use of alternative forms (such as "or") in this document means one or both of the alternative forms or any combination thereof.

[0039] As used herein, the term “and / or” will be considered to mean that each of the specified features or components is explicitly disclosed with or without the other. For example, the term “and / or” as used in phrases such as “A and / or B” is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0040] As used herein, the terms “comprising,” “including,” “having,” and “containing,” and their grammatical variations, are intended to be non-restrictive, such that one or more items in the list do not exclude other items that may be substituted for or added to the list. It should be understood that whenever an aspect is described herein with the language “comprising,” other similar aspects described as “consisting of” and / or “substantially consisting of” are also provided.

[0041] As used herein, the term “about” means a value or composition within an acceptable range of error for a particular value or composition, as determined by one of ordinary skill in the art, which will depend in part on how the value or composition was measured or determined, i.e., the limitations of the measurement system. For example, “about” or “approximately” may mean within one or more standard deviations according to practice in the art. Alternatively, depending on the limitations of the measurement system, “about” or “approximately” may mean a range of up to 10% (i.e., ± 10%) or more. For example, about 5 mg may include any number between 4.5 mg and 5.5 mg. Furthermore, with specific reference to a biological system or process, the term may mean up to an order of magnitude or up to 5 times the value. When a particular value or composition is provided in this disclosure, unless otherwise stated, the meaning of “about” or “approximately” should be assumed to be within an acceptable range of error for that particular value or composition.

[0042] The term "coronavirus infection" refers to a person or animal having cells that have been infected with a coronavirus. Infection can be established by testing respiratory samples and / or titrating the virus or by measuring circulating coronavirus-specific antibodies in the blood. Individuals infected with coronaviruses are detected using routine diagnostic methods known to those skilled in the art, such as molecular biology (e.g., PCR).

[0043] As used herein, the term "subject" refers to humans and non-human animals, including vertebrates, mammals and non-mammals. In one embodiment, a subject may be a human, a non-human primate, ape, monkey, mouse (e.g., mouse and rat), cow, pig, horse, dog, cat, goat, wolf, frog or fish.

[0044] The term "administering" (or "administered") and its grammatical variations refer to the physical introduction of a therapeutic agent into a subject using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, percutaneous, spinal, or other parenteral administration routes, such as by injection or infusion. As used herein, the phrase "parenteral administration" means a mode of administration other than enteral and local administration (typically by injection), and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrasheath, intralymphatic, intralesional, intracystic, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcystic, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. In one embodiment, the formulation is administered via a non-parenteral route (e.g., orally). Other non-parenteral routes include local, epidermal, or mucosal administration routes, such as intranasal, vaginal, rectal, sublingual, or local administration. Application can also be performed, for example, once, multiple times, and / or over one or more extended periods.

[0045] The terms "treatment" and "treating" refer to combating cancer or coronavirus infection in human or animal subjects. By administering at least one embodiment of the compositions described herein, the viral infection rate (infection titer) in the subject will be reduced, and the virus may be completely eliminated from the subject's body. The terms "treatment" and "treating" also refer to alleviating symptoms associated with viral infection (e.g., respiratory syndrome, kidney failure, fever, and other symptoms associated with viral infection).

[0046] The terms "effective amount," "therapeutic effective amount," or "effective dose," or related terms, may be used interchangeably and refer to an amount of therapeutic agent sufficient, when administered to a subject, to affect a measurable improvement or prevention of a disease or condition associated with cancer or viral infection. For example, an effective dose sufficient to inhibit the proliferation and / or replication of coronaviruses and / or the development of viral infection within a subject. Therapeutic effective amounts of the therapeutic agents provided herein, when used alone or in combination with antiviral agents, will vary according to the relative activity of the therapeutic agent and according to the subject being treated and the condition of the disease, the subject's weight and age and sex, the severity of the subject's condition, the method of administration, etc., which can be readily determined by one of ordinary skill in the art using known techniques.

[0047] As used herein, the terms "therapeutic agent" and "therapeutics" mean an agent (e.g., a compound or composition described herein) that, when administered to a subject, will have a intended preventive or therapeutic effect, such as preventing or delaying the onset (or recurrence) of an injury, disease, pathology, or symptom, or reducing the likelihood of the onset (or recurrence) of such an injury, disease, pathology, or symptom, or the expected therapeutic effect, such as treating or improving an injury, disease, pathology, or symptom, or the expected therapeutic effect, including any objective or subjective parameter of treatment, such as relief; mitigation; reduction of symptoms or making the patient more tolerant of the injury, pathology, or symptom; slowing the rate of deterioration or decline; reducing the degree of debilitating at the end of deterioration; or improving the patient's physical or mental health.

[0048] The terms “peptide,” “polypeptide,” and “protein,” and other related terms used herein, are used interchangeably and refer to polymers of amino acids and are not limited to any particular length. Polypeptides can include both natural and non-natural amino acids. Polypeptides include recombinant or chemically synthesized forms. These terms encompass natural and artificial proteins of protein sequences, protein fragments and polypeptide analogs (such as mutant proteins, variants, chimeric proteins, and fusion proteins), and proteins that are covalently or non-covalently modified post-translationally or otherwise.

[0049] The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide,” as well as other related terms used herein, are used interchangeably and refer to polymers of nucleotides and are not limited to any particular length. Nucleic acids include recombinant and chemically synthesized forms. Nucleic acids include DNA molecules (e.g., cDNA, pDNA, or genomic DNA), RNA molecules (e.g., mRNA, siRNA, miRNA, RNAi, saRNA, taRNA, or shRNA), analogs of DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. Nucleic acid molecules can be single-stranded or double-stranded. In one embodiment, a nucleic acid comprises one type of polynucleotide or a mixture of two or more different types of polynucleotides.

[0050] The term "plasmid DNA" is used to refer to a plasmid comprising a single gene or protein-coding gene, either a promoter (and enhancer) or a light and heavy chain of a monoclonal antibody (or a bispecific antibody or an antigen-binding fragment of a monoclonal or bispecific antibody), such as encoding an antibody fragment, like Fab or scFv, or a single-chain antibody. In embodiments, the protein-coding gene may encode hormones, cytokines, enzymes, immunogenic peptides, etc.

[0051] In some embodiments, the protein-coding gene may encode an antibody fragment. In some embodiments, the protein-coding gene may encode Fab. In some embodiments, the protein-coding gene may encode scFv. In some embodiments, the protein-coding gene may encode a hormone, cytokine, enzyme, immunogenic peptide, etc. In some embodiments, the protein-coding gene may encode a hormone. In some embodiments, the protein-coding gene may encode a cytokine. In some embodiments, the protein-coding gene may encode an enzyme. In some embodiments, the protein-coding gene may encode an immunogenic peptide.

[0052] In implementation, nucleic acids may carry therapeutic genes, regulatory sequences for transcription or replication, modified or unmodified antisense sequences, regions for binding to other cellular components, etc.

[0053] The terms “mutation,” “modification,” or “variation,” or related terms, refer to changes in the nucleic acid or amino acid sequence that differ from a reference nucleic acid or amino acid sequence. Examples of mutations include point mutations, insertions, deletions, amino acid substitutions, inversions, rearrangements, splicing, sequence fusions (e.g., gene fusions or RNA fusions), truncations, translocations, dissimilar mutations, sequence duplications, single nucleotide polymorphisms (SNPs), or other gene rearrangements.

[0054] As used herein, “antibody” and related terms refer to a complete immunoglobulin or its antigen-binding portion (or fragment thereof) that binds specifically to an antigen. Antigen-binding portions (or fragments thereof) can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of a complete antibody. Antigen-binding portions (or fragments thereof) particularly include Fab, Fab', F(ab')2, Fv, domain antibodies (dAb) and complementarity-determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, and polypeptides containing at least a portion sufficient to confer binding to a polypeptide-specific antigen.

[0055] Antibodies comprise both the antibody and the antigen-binding moiety generated recombinantly. Antibodies include non-human, chimeric, humanized, and fully human antibodies. Antibodies include monospecific and multispecific (e.g., bispecific, trispecific, and higher-order specific) antibodies. Antibodies include tetrameric antibodies, light chain monomers, heavy chain monomers, light chain dimers, and heavy chain dimers. Antibodies include F(ab')2 fragments, Fab' fragments, and Fab fragments. Antibodies include single-domain antibodies, monovalent antibodies, single-chain antibodies, single-chain variable fragments (scFv), camelized antibodies, affinity antibodies, disulfide-linked Fv (sdFv), anti-idiotype antibodies (anti-Id), and microantibodies. Antibodies include monoclonal and polyclonal populations.

[0056] "Neutralizing antibody" and related terms refer to antibodies that specifically bind to neutralizing epitopes of their target antigens (e.g., coronavirus spike protein) and substantially inhibit or eliminate the biological activity of the target antigen (e.g., coronavirus spike protein). Neutralizing antibodies can reduce the biological activity of the target antigen by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even higher levels.

[0057] As used herein, the terms “antigen-binding domain,” “antigen-binding region,” “antigen-binding site,” and other related terms refer to a portion of an antigen-binding protein containing amino acid residues (or other parts) that interact with the antigen and contribute to the specificity and affinity of the antigen-binding protein for the antigen. For antibodies that bind specifically to their antigens, the terms will include at least a portion of at least one domain of their CDR domain.

[0058] As used herein in the context of antibodies or antigen-binding proteins or antibody fragments, the terms "specific binding," "specifically binds," or "specifically binding" and other related terms refer to non-covalent or covalently preferably binding to an antigen relative to other molecules or portions (e.g., an antibody specifically binds to a particular antigen relative to other available antigens). In one embodiment, if the antibody is at 10 -5 M or smaller, or 10 -6 M or smaller, or 10 -7 M or smaller, or 10 -8 M or smaller, or 10 -9 M or smaller, or 10 -10 M or a smaller dissociation constant K D When an antibody binds to an antigen, it specifically binds to the target antigen.

[0059] As used herein, the terms “antibody fragment,” “antibody moiety,” “antigen-binding fragment of an antibody,” or “antigen-binding portion of an antibody,” and other related terms, refer to molecules other than the intact antibody that include the portion of the intact antibody that binds to the antigen. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; Fd; and Fv fragments, as well as dAb; bifunctional antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and polypeptides containing at least a portion sufficient to confer antigen-specific binding to the antibody. Antigen-binding portions of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody. Antigen-binding portions particularly include Fab, Fab', F(ab')2, Fv, domain-specific antibodies (dAb) and complementarity-determining region (CDR) fragments, chimeric antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, and polypeptides containing at least a portion of an immunoglobulin sufficient to confer antigen-binding properties to the antibody fragment.

[0060] The terms "Fab," "Fab fragment," and other related terms refer to components including the variable light chain region (V... L ), constant light chain region (C L ), variable heavy chain region (V H ) and the first constant region (C H1 The Fd fragment is a monovalent fragment consisting of two Fab fragments linked by a disulfide bridge in the hinge region. F(Ab')2 is capable of antigen binding. The Fd fragment includes V... H District and C H1 The Fv segment includes V. L District and V HThe region. Fv can bind to the antigen. The dAb fragment has V. H Structural domain, V L Domain or V H or V L Antigen-binding fragments of the domain (US Patents 6,846,634 and 6,696,245; US Publications 2002 / 02512, 2004 / 0202995, 2004 / 0038291, 2004 / 0009507, 2003 / 0039958; and Ward et al., Nature 341:544-546, 1989).

[0061] Single-chain antibody (scFv) is V L and V H Regions bind to antibodies by linkers (e.g., synthetic sequences of amino acid residues) to form continuous protein chains. Preferably, the linkers are long enough to allow the protein chains to fold themselves and form monovalent antigen-binding sites (see, for example, Bird et al., 1988, Science 242:423-26 and Huston et al., 1988, Proceedings of the National Academy of Sciences of the United States of America 85:5879-83).

[0062] Bifunctional antibodies are bivalent antibodies comprising two polypeptide chains, each of which includes V-linked structures linked by linkers that are too short to pair between two domains on the same chain. H and V L The two polypeptide chains are identical, thus allowing each domain to pair with a complementary domain on the other polypeptide chain (see, for example, Holliger et al., 1993, Proceedings of the National Academy of Sciences 90:6444-48 and Poljak et al., 1994, Structure 2:1121-23). ​​If the two polypeptide chains of a bifunctional antibody are identical, then the bifunctional antibody resulting from their pairing will have two identical antigen-binding sites. Polypeptide chains with different sequences can be used to prepare bifunctional antibodies with two different antigen-binding sites. Similarly, trifunctional and tetrafunctional antibodies are antibodies comprising three and four polypeptide chains, respectively, forming three and four antigen-binding sites, which may be identical or different.

[0063] The term "human antibody" refers to an antibody having one or more variable and constant regions derived from a human immunoglobulin sequence. In one embodiment, all variable and constant domains are derived from a human immunoglobulin sequence (e.g., a fully human antibody). These antibodies can be prepared in various ways, examples of which are described below, including by recombinant methods or by immunization with a mouse antigen of interest, the antigen of interest being genetically modified to express an antibody derived from genes encoding human heavy and / or light chains.

[0064] As used herein, the term "variant" polypeptide and "variant" of a polypeptide refers to a polypeptide having an amino acid sequence having one or more amino acid residues inserted into, deleted from, and / or substituted into an amino acid sequence relative to a reference polypeptide sequence. Polypeptide variants include fusion proteins. In the same manner, variant polynucleotides include nucleotide sequences having one or more nucleotides inserted into, deleted from, and / or substituted into a nucleotide sequence relative to another polynucleotide sequence. Polynucleotide variants include fusion polynucleotides.

[0065] As used herein, the term "derivative" of a polypeptide is a polypeptide (e.g., an antibody) that has been chemically modified, for example, by conjugation, phosphorylation, and glycosylation with another chemical moiety such as, for example, polyethylene glycol, albumin (e.g., human serum albumin). Unless otherwise indicated, the term "antibody" includes its derivatives, variants, fragments, and mutant proteins, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, examples of which are described below.

[0066] The term "hinge" refers to an amino acid segment that is typically present between two domains of a protein and allows for the flexibility of one or both domains to be assembled and moved relative to each other. Structurally, a hinge region comprises about 10 to about 100 amino acids, for example, about 15 to about 75 amino acids, about 20 to about 50 amino acids, or about 30 to about 60 amino acids. In embodiments, the length of the hinge region is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids. The hinge region can be derived from the hinge region of a naturally occurring protein (such as the CD8 hinge region or a fragment thereof, the CD8α hinge region or a fragment thereof), the hinge region of an antibody (e.g., IgG, IgA, IgM, IgE, or IgD antibody), or a hinge region connecting the constant structural domains CH1 and CH2 of an antibody. The hinge region can be derived from an antibody and may or may not include one or more constant regions of the antibody, or the hinge region may include the hinge region of the antibody and the CH3 constant region of the antibody, or the hinge region may include the hinge region of the antibody and the CH2 and CH3 constant regions of the antibody, or the hinge region is a non-naturally occurring peptide, or the hinge region is located between the C-terminus of the scFv and the N-terminus of the transmembrane structural domain. In embodiments, the hinge region includes any region or any combination of two or more regions comprising any one of the upper hinge sequence, core hinge sequence, or lower hinge sequence from an IgG1, IgG2, IgG3, or IgG4 immunoglobulin molecule. In embodiments, the hinge region includes the IgG1 upper hinge sequence EPKSCDKTHT. In this implementation, the hinge region includes the IgG1 core hinge sequence CP. X CP, among which X The sequence is P, R, or S. In one embodiment, the hinge region includes the lower hinge sequence APELLGGP. In one embodiment, the hinge is connected to the Fc region (CH2) having the amino acid sequence SVFLFPPKPKDT. In one embodiment, the hinge region includes the amino acid sequence of the upper hinge, core hinge, or lower hinge and includes EPKSCDKTHTCPPCPAP-ELLGGP. In one embodiment, the hinge region includes one, two, three, or more cysteine ​​residues that can form at least one, two, three, or more interchain disulfide bonds.

[0067] As used herein, the term "Fc" or "Fc region" refers to a portion of the constant region of an antibody heavy chain that begins in or after the hinge region and ends at the C-terminus of the heavy chain. The Fc region includes at least a portion of the CH2 and CH3 regions and may or may not include a portion of the hinge region. Two polypeptide chains, each carrying a half-Fc region, may dimerize to form a complete Fc domain. The Fc domain can bind Fc cell surface receptors as well as some proteins of the immune complement system. The Fc region can bind complement component C1q. The Fc domain exhibits effector functions, including any one or any combination of two or more activities such as complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADP), opsonization, and / or cell binding. The Fc domain can bind Fc receptors, including FcγRI (e.g., CD64), FcγRII (e.g., CD32), and / or FcγRIII (e.g., CD16a). In implementations, the Fc region may include mutations or several mutations that increase or decrease any one or any combination of these functions. In implementations, the Fc domain includes LALA mutations that decrease effector function (e.g., corresponding to L234A, L235A according to Kabat numbering) (see, for example, Hezareh et al., (2001) *Journal of Virology*). J Virol (See 12161-12168). In an embodiment, the Fc domain includes LALA-PG mutations that reduce effector function (e.g., corresponding to L234A, L235A, P329G according to Kabat numbering). In an embodiment, the Fc domain mediates the serum half-life of the protein complex, and mutations in the Fc domain can increase or decrease the serum half-life of the protein complex. In an embodiment, the Fc domain affects the thermal stability of the protein complex, and mutations in the Fc domain can increase or decrease the thermal stability of the protein complex.

[0068] As used herein, the term "labeled antibody" or related terms refer to an unlabeled antibody or antibody bound to a detectable label or portion for detection, wherein the detectable label or portion is radioactive, colorimetric, antigenic, enzymatic, detectable beads (such as magnetic or electron-dense (e.g., gold) beads), biotin, streptavidin, or protein A. A variety of labels may be used, including but not limited to radionuclides, fluorescent agents, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, and ligands (e.g., biotin, haptens).

[0069] As used herein, the terms "percentage of identity" or "percentage of homology" and related terms refer to a quantitative measurement of the similarity between two polypeptide sequences or two polynucleotide sequences. The percentage of identity between two polypeptide sequences is a function of the number of common amino acids shared by the two polypeptide sequences at the aligned position, taking into account the number of vacancies and the length of each vacancy that may need to be introduced to optimize the alignment of the two polypeptide sequences. Similarly, the percentage of identity between two polynucleotide sequences is a function of the number of common nucleotides shared by the two polynucleotide sequences at the aligned position, taking into account the number of vacancies and the length of each vacancy that may need to be introduced to optimize the alignment of the two polynucleotide sequences. Sequence comparisons and the determination of the percentage of identity between two polypeptide sequences or two polynucleotide sequences can be performed using mathematical algorithms. For example, the “percentage of identity” or “percentage of homology” of two polypeptides or two polynucleotide sequences can be determined by comparing sequences using the GAP computer program (GCGWisconsin Package), version 10.3 (Accelrys, San Diego, California) with its default parameters.

[0070] As used herein, “vector” and related terms refer to nucleic acid molecules (e.g., DNA or RNA) that can be operatively linked to foreign genetic material (e.g., nucleic acid transgenes). Vectors can be used as mediators to introduce foreign genetic material into cells (e.g., host cells). Vectors may include at least one restriction endonuclease recognition sequence to insert transgenes into the vector. Vectors may include at least one gene sequence conferring antibiotic resistance or selectable properties to aid in the selection of host cells carrying the vector-transgene construct. Vectors can be single-stranded or double-stranded nucleic acid molecules. Vectors can be linear or circular nucleic acid molecules. One type of vector is a “plasmid,” which refers to a linear or circular double-stranded extrachromosomal DNA molecule that can be linked to a transgene and is capable of replicating and transcribing and / or translating the transgene in a host cell. Viral vectors typically contain a viral RNA or DNA backbone sequence that can be linked to a transgene. The viral backbone sequence can be modified to stop infection but retain the viral backbone and co-linked transgenes for insertion into the host cell genome. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated vectors, baculovirus vectors, multivaccinia virus vectors, vaccinia virus vectors, herpes simplex virus vectors, and Epstein-Barr viral vectors. Some vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors including bacterial origins of replication and free-living mammalian vectors). Other vectors (e.g., non-free-living mammalian vectors) are integrated into the host cell's genome upon introduction and thereby replicate along with the host genome.

[0071] "Expression vector" is a type of vector that may contain one or more regulatory sequences, such as inducible and / or constitutive promoters and enhancers. Expression vectors may include ribosome binding sites and / or polyadenylation sites. Regulatory sequences direct transcription or transcription and translation of transgenes (such as DNA or RNA transgenes) linked to the expression vector transduced into a host cell. Regulatory sequences can control the expression level, timing, and / or location of the transgene. Regulatory sequences can exert their effects on the transgene, for example, directly or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and / or nucleic acids). Regulatory sequences may be part of the vector. Further examples of regulatory sequences are described below: for example, Goeddel, 1990, *Gene Expression Technology: Methods in Enzymology*, 185, Academic Press, San Diego, California; and Baron et al., 1995, *Nucleic Acids Res.*, 23:3605-3606.

[0072] When a link exists between a transgene and a vector to allow the transgene sequence contained in the vector to function or be expressed, the transgene and the vector are “operably linked.” In one implementation, the transgene and the regulatory sequence are “operably linked” when the regulatory sequence affects the expression of the transgene (e.g., the level, timing, or location of expression).

[0073] As used herein, the terms “transfected,” “transformed,” “transduced,” or other related terms refer to the process of transferring or introducing a foreign nucleic acid (e.g., a transgenic) into a host cell. A “transfected,” “transformed,” or “transduced” host cell is a host cell that has been transfected, transformed, or transduced with a foreign nucleic acid (transgenic). Host cells include primary subject cells and their progeny. A foreign nucleic acid encoding at least a portion of any anti-spike protein antibody described herein may be introduced into a host cell. An expression vector including at least a portion of any anti-spike protein antibody described herein may be introduced into a host cell, and the host cell may express a polypeptide including at least a portion of the anti-spike protein antibody.

[0074] The term "transfection" is generally used herein to refer to methods for delivering and introducing biologically functional nucleic acids into cells (e.g., eukaryotic cells) in a manner that preserves the function of the nucleic acids within the cells. As is known in the art, such methods may involve the use of poly(lactide-co-glycolic acid) (PLGA), ISCOM, liposomes, vesicles, virions, block copolymers, Prönkel block copolymers, chitosan and other biodegradable polymers, microparticles, microspheres, calcium phosphate nanoparticles, nanoparticles, nanocapsules, nanospheres, poloxamer nanospheres, electroporation, nuclear transfection, piezoelectric permeabilization, acoustic permeabilization, iontophoresis, ultrasound, SQZ high-speed cell deformation-mediated membrane rupture, corona plasma, plasma-enhanced delivery, tissue-tolerant plasma, laser microperforation, shock wave energy, magnetic fields, non-contact magnetic permeabilization, gene guns, microneedles, microdermabrasion, hydrodynamic delivery, high-pressure tail vein injection, etc.

[0075] The transfection method of the present invention can be applied to cells in vitro or in vivo. The term transfection has a more specific meaning of delivering and introducing an expressible nucleic acid into cells so that the cells are able to express said nucleic acid. The term expressive means any manifestation of the functional presence of the nucleic acid within the cell, including both transient and stable expression. Nucleic acids include both DNA and RNA from any source containing natural and non-natural bases, without size limitations. Nucleic acids can have a variety of biological functions. They can carry therapeutic genes, regulatory sequences for transcription or replication, modified or unmodified antisense sequences, regions for binding to other cellular components, etc. They can direct the synthesis of polypeptides specific to infectious agents or can be able to remedy genetic or acquired defects. They can encode proteins, including regulatory regions, act as inhibitors of gene or RNA expression (e.g., antisense DNA or RNA), act as inhibitors of proteins, inhibit cell growth or kill cells, catalyze reactions, or play a role in diagnostic or other analytical assays.

[0076] As used herein, the term "transfection agent" refers to the compound used herein for transfection. In embodiments, the transfection agent is a polymer, lipid, surfactant, or any combination thereof.

[0077] The term "therapeutic gene" is intended to specifically refer to any gene encoding a protein product that has a therapeutic effect. Such encoded protein products can be proteins, peptides, etc. In embodiments, nucleic acids may also contain one or more genes encoding antigenic peptides capable of generating an immune response in humans or animals. In embodiments, nucleic acids can be used for cell therapy. In embodiments, nucleic acids enable the production of vaccines or immunotherapeutic treatments. In embodiments, vaccines or immunotherapeutic treatments can be used to prevent or treat cancer. In embodiments, such vaccines or immunotherapeutic treatments can be used to treat viral infections. In embodiments, the viral infection is a coronavirus.

[0078] A host cell can be a cultured cell that can be transformed or transfected with a nucleic acid encoded by a polypeptide, which can then be expressed in the host cell. The phrases “transgenic host cell” or “recombinant host cell” can be used to indicate a host cell that has been introduced (e.g., transduced, transformed, or transfected) with a nucleic acid that is expressed or not expressed. A host cell can also be a cell that includes a nucleic acid but does not express it at the desired level until a regulatory sequence is introduced into the host cell such that the regulatory sequence is operatively linked to the nucleic acid. It should be understood that the term host cell refers not only to a specific subject cell but also to the offspring or potential offspring of such cells. Because certain modifications may be present in subsequent passages due to, for example, mutations or environmental influences, such offspring may actually be different from the parent cells but are still included within the scope of the terminology used herein.

[0079] Therefore, as used herein, the terms "host cell" or "host cell population" or related terms may refer to cells (or populations or multiple host cells) used to produce antibodies or fragments thereof, which are one or more cells to which external (exogenous or transgenic) nucleic acids have been introduced to, for example, guide the production of anti-spike protein antibodies by producing host cells. The exogenous nucleic acid may include an expression vector operatively linked to a transgene, and the host cell may be used to express nucleic acids and / or polypeptides encoded by the exogenous nucleic acid (transgene). The host cell (or population thereof) may be a cultured cell, may be extracted from a subject, or may be a cell of an organism including a human subject. Without regard to the number of generations or passages, the host cell (or population of host cells) includes primary subject cells and their progeny. The host cell (or population thereof) includes immortalized cell lines. Progeny cells may or may not carry the same genetic material as parental cells. In one embodiment, producing host cells describe any cell (including its progeny) that has been modified, transfected, transduced, transformed, and / or manipulated in any way to express antibodies as disclosed herein. In one instance, host cells (or populations thereof) can be transfected or transduced using an expression vector operatively linked to a nucleic acid encoding a desired antibody or its antigen-binding moiety, as described herein. The producing host cells and populations thereof may carry an expression vector stably integrated into the host's genome, or they may carry an extrachromosomal expression vector. In one embodiment, the host cells and populations thereof may carry an extrachromosomal vector that exists after several cell divisions or exists transiently and is lost after several cell divisions.

[0080] In other contexts, this disclosure may use the term "host cell" to refer to one or more cells infected by a virus (such as a coronavirus), cells capable of being infected by a virus (e.g., lung cells of a subject), or cells used in assays or experiments testing their ability to be infected by a virus. As a non-limiting example, other terms for virus-infected cells, cells capable of being infected by a virus, or cells used in assays including virus infection procedures may include "target cell," "susceptible cell," "test cell," "virus-transmitting cell," "infected cell," etc.

[0081] The term "lipid" or "lipid moiety" is used according to its common meaning in chemistry and refers to a hydrophobic molecule typically characterized by an aliphatic hydrocarbon chain. In embodiments, a lipid moiety comprises a carbon chain having 3 to 100 carbons. In embodiments, a lipid moiety comprises a carbon chain having 5 to 50 carbons. In embodiments, a lipid moiety comprises a carbon chain having 5 to 25 carbons. In embodiments, a lipid moiety comprises a carbon chain having 8 to 525 carbons. A lipid moiety may comprise saturated or unsaturated carbon chains and may optionally be substituted. In embodiments, a lipid moiety is optionally substituted at the terminal with a charged portion. In embodiments, a lipid moiety is an alkyl or heteroalkyl group optionally substituted at the terminal with a carboxylic acid portion. Lipids are also a group of organic compounds, including but not limited to fatty acid esters, and characterized by being insoluble in water but soluble in many organic solvents. Lipids are generally classified into at least three categories: (1) "simple lipids," which include fats and oils as well as waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0082] As used herein, “LPS-LPX delivery system” refers to the transfection reagent described herein, which comprises a lipid complex (LPX) encapsulated within a lipid shell (LPS), and optionally a targeting ligand, imaging ligand, digestant, or small molecule on the lipid shell (LPS). The lipid complex is a complex of one or more lipids with cargo (e.g., nucleic acid); thus, the cargo (e.g., nucleic acid) can be contained within the LPS-LPX delivery system. In embodiments, the LPX comprises one or more ionizable cationic lipids and optionally one or more auxiliary lipids. In embodiments, the LPS is a lipid micelle, liposome, or self-assembled lipid nanoparticle. In embodiments, the LPS comprises one or more ionizable cationic lipids and / or one or more auxiliary lipids capable of forming lipid micelles, liposomes, or self-assembled lipid nanoparticles. In embodiments, the ionizable cationic lipid can be any of the lipids listed in Table 1. In embodiments, the auxiliary lipid is a phospholipid, PEGylated lipid, cholesterol, or a cholesterol derivative. As described herein, nucleic acids can be incorporated into the LPX prior to their encapsulation within the LPS. In one embodiment, the targeting ligand, imaging ligand, digesting agent, or small molecule may contact the LPS. In another embodiment, the imaging ligand, digesting agent, or small molecule may be inside the LPX. In yet another embodiment, the imaging ligand, digesting agent, or small molecule may be between the surface of the LPS and the LPX.

[0083] As used herein, the term "targeting ligand in contact with LPS" means a targeting ligand on the surface of LPS; or a targeting ligand with one end in a micelle or liposome bilayer and the other end protruding to the outside of the lipid shell.

[0084] The terms “cationic lipid” or “ionizable cationic lipid” are used interchangeably herein and refer to lipids that are protonated (e.g., >50% protonation) at low pH (e.g., pH 4), which makes the lipid positively charged, but which can remain neutral at physiological pH (e.g., pH 7.4). In embodiments, ionizable cationic lipids are any lipids listed in Table 1.

[0085] The term "helper lipid" refers to lipids that improve the stability, flowability, blood compatibility, oligonucleotide delivery efficiency, and transfection activity of nanoparticles. In embodiments, helper lipids include, but are not limited to, certain phospholipids, DOPE, DOPC, ALC-0159, DEPE, DLOPE, POPE, DSPC, cholesterol, cholesterol-based lipids, and polyethylene glycol-modified lipids. In embodiments, the helper lipid can be any lipid listed in Table 2.

[0086] As used herein, the term "organic phase" is used according to its ordinary meaning in chemistry and refers to a solution containing a solvent (organic solvent) that contains carbon. Non-limiting examples of organic solvents include acetic acid, acetone, acetonitrile, benzene, 1-butanol, 2-butanol, 2-butanone, tert-butanol, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, 1,2-dichloroethane, diethylene glycol, diethyl ether, diethylene glycol (dimethyl ether), 1,2-dimethoxyethane (dimethyl glycol dimethyl ether, DME), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,4-dioxane, ethanol, ethyl acetate, ethylene glycol, glycerol, heptane, hexamethylphosphoramide (HMPA), hexamethylphosphotriamide (HMPT), hexane, methanol, methyl tert-butyl ether (MTBE), methylene chloride, N-methyl-2-pyrrolidone (NMP), nitromethane, pentane, petroleum ether (volatile oil), 1-propanol, 2-propanol, pyridine, tetrahydrofuran (THF), toluene, triethylamine, o-xylene, m-xylene, or p-xylene. In the embodiments, the organic solvent is or includes chloroform, dichloromethane, methanol, ethanol, tetrahydrofuran, or dioxane.

[0087] As used herein, the term "aqueous phase" is used according to its common meaning in chemistry and refers to a solution in which the solvent is water.

[0088] As used herein, the term "stirring" refers to any type of stirring or mixing. Some non-limiting examples of stirring include vortexing, sonication, pipetting, mixing, oscillation, etc.

[0089] This disclosure provides methods for treating subjects who test positive for infection or who have cancer. This disclosure also provides methods for treating subjects suspected of being infected with a virus or at risk of viral infection.

[0090] Composition for transfection On one hand, this document provides a composition for transfecting cells in vitro or in vivo, the composition comprising at least one nucleic acid contained in an LPS-LPX delivery system, and optionally a targeting ligand, imaging ligand, digestive agent, or small molecule. In one embodiment, the composition for transfecting cells comprises DNA contained in an LPS-LPX delivery system, and optionally a targeting ligand, imaging ligand, digestive agent, or small molecule. In another embodiment, the composition for transfecting cells comprises RNA contained in an LPS-LPX delivery system, and optionally a targeting ligand, imaging ligand, digestive agent, or small molecule. In yet another embodiment, the composition for transfecting cells comprises DNA and RNA contained in an LPS-LPX delivery system, and optionally a targeting ligand, imaging ligand, digestive agent, or small molecule.

[0091] In one embodiment, the targeting ligand contacts the LPS. In another embodiment, the imaging ligand contacts the LPS, is inside the LPX, or is located on the surface of the LPS between the LPS and the LPX. In another embodiment, the imaging ligand contacts the LPS. In another embodiment, the imaging ligand is inside the LPX. In another embodiment, the imaging ligand is located on the surface of the LPS between the LPS and the LPX. In another embodiment, the digestive agent contacts the LPS, is inside the LPX, or is located on the surface of the LPS between the LPS and the LPX. In another embodiment, the digestive agent contacts the LPS. In another embodiment, the digestive agent is inside the LPX. In another embodiment, the digestive agent is located on the surface of the LPS between the LPS and the LPX. In another embodiment, the small molecule contacts the LPS, is inside the LPX, or is located on the surface of the LPS between the LPS and the LPX. In another embodiment, the small molecule contacts the LPS. In another embodiment, the small molecule is inside the LPX. In another embodiment, the small molecule is located on the surface of the LPS between the LPS and the LPX.

[0092] In one embodiment, this document provides a composition for transfecting cells, the composition comprising one nucleic acid contained in an LPS-LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule. In another embodiment, this document provides a composition for transfecting cells, the composition comprising two nucleic acids contained in an LPS-LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule. In yet another embodiment, this document provides a composition for transfecting cells, the composition comprising three nucleic acids contained in an LPS-LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule. In yet another embodiment, this document provides a composition for transfecting cells, the composition comprising four nucleic acids contained in an LPS-LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule. In the presence of multiple nucleic acids, they may or may not be present in the same individual LPS-LPX structure (e.g., one LPS-LPX may contain a first nucleic acid, and another LPS-LPX may contain a second nucleic acid; or one LPS-LPX may contain both the first and second nucleic acids).

[0093] In one embodiment, the composition for transfecting cells comprises at least one nucleic acid contained in the LPX-LPS delivery system. In another embodiment, the nucleic acid is DNA, RNA, or a combination of DNA and RNA. In yet another embodiment, the nucleic acid is DNA. In yet another embodiment, the nucleic acid is RNA. In yet another embodiment, the nucleic acid is both DNA and RNA. In yet another embodiment, the nucleic acid comprises a therapeutic gene.

[0094] In this embodiment, the RNA is mRNA, siRNA, miRNA, RNAi, saRNA, taRNA, or shRNA. In this embodiment, the RNA is mRNA. In this embodiment, the RNA is siRNA. In this embodiment, the RNA is miRNA. In this embodiment, the RNA is RNAi. In this embodiment, the RNA is saRNA. In this embodiment, the RNA is taRNA. In this embodiment, the RNA is shRNA.

[0095] In this embodiment, the DNA is plasmid DNA. In this embodiment, the plasmid DNA is nanoplasmid DNA. In this embodiment, the nanoplasmid DNA encodes a monoclonal antibody.

[0096] In some embodiments, the protein-coding gene may encode an antibody fragment. In some embodiments, the protein-coding gene may encode Fab. In some embodiments, the protein-coding gene may encode scFv. In some embodiments, the protein-coding gene may encode a hormone, cytokine, enzyme, immunogenic peptide, etc. In some embodiments, the protein-coding gene may encode a hormone. In some embodiments, the protein-coding gene may encode a cytokine. In some embodiments, the protein-coding gene may encode an enzyme. In some embodiments, the protein-coding gene may encode an immunogenic peptide.

[0097] In implementation, nucleic acids may include one or more therapeutic genes, regulatory sequences for transcription or replication, modified or unmodified antisense sequences, regions for binding to other cellular components, etc.

[0098] In some embodiments, the nucleic acid may contain a therapeutic gene. In some embodiments, the nucleic acid may contain a therapeutic gene. In some embodiments, the nucleic acid may contain a regulatory sequence for transcription. In some embodiments, the nucleic acid may contain a regulatory sequence for transcription. In some embodiments, the nucleic acid may contain a regulatory sequence for replication. In some embodiments, the nucleic acid may contain a modified antisense sequence. In some embodiments, the nucleic acid may contain a modified antisense sequence. In some embodiments, the nucleic acid may contain an unmodified antisense sequence. In some embodiments, the nucleic acid may contain an unmodified antisense sequence. In some embodiments, the nucleic acid may contain a region for binding to other cellular components. In some embodiments, the nucleic acid may contain a region for binding to other cellular components.

[0099] The term "therapeutic gene" is intended to refer to any gene that encodes a protein product (e.g., a peptide) having a therapeutic effect. In embodiments, nucleic acids may also include one or more genes encoding antigenic peptides capable of evoking an immune response in humans or animals. In embodiments, nucleic acids can be used for cell therapy. In embodiments, nucleic acids enable the production of vaccines or immunotherapeutic treatments. In embodiments, vaccines or immunotherapeutic treatments can be used to prevent or treat cancer. In embodiments, such vaccines or immunotherapeutic treatments can be used to treat viral infections. In embodiments, the viral infection is a coronavirus infection.

[0100] In one implementation, the LPS-LPX delivery system includes an LPS that encapsulates LPX.

[0101] In some embodiments, LPX comprises one or more ionizable cationic lipids and optionally one or more auxiliary lipids. In some embodiments, LPX comprises one ionizable cationic lipid. In some embodiments, LPX comprises two ionizable cationic lipids. In some embodiments, LPX comprises three ionizable cationic lipids. In some embodiments, LPX comprises four ionizable cationic lipids. In some embodiments, LPX comprises five ionizable cationic lipids.

[0102] In one embodiment, LPX comprises one ionizable cationic lipid and one auxiliary lipid. In another embodiment, LPX comprises one ionizable cationic lipid and two auxiliary lipids. In yet another embodiment, LPX comprises one ionizable cationic lipid and three auxiliary lipids. In yet another embodiment, LPX comprises one ionizable cationic lipid and four auxiliary lipids. In yet another embodiment, LPX comprises one ionizable cationic lipid and five auxiliary lipids.

[0103] In one embodiment, LPX comprises two ionizable cationic lipids and one auxiliary lipid. In another embodiment, LPX comprises two ionizable cationic lipids and two auxiliary lipids. In yet another embodiment, LPX comprises two ionizable cationic lipids and three auxiliary lipids. In yet another embodiment, LPX comprises two ionizable cationic lipids and four auxiliary lipids. In yet another embodiment, LPX comprises two ionizable cationic lipids and five auxiliary lipids.

[0104] In one embodiment, LPX comprises three ionizable cationic lipids and one auxiliary lipid. In another embodiment, LPX comprises three ionizable cationic lipids and two auxiliary lipids. In yet another embodiment, LPX comprises three ionizable cationic lipids and three auxiliary lipids. In yet another embodiment, LPX comprises three ionizable cationic lipids and four auxiliary lipids. In yet another embodiment, LPX comprises three ionizable cationic lipids and five auxiliary lipids.

[0105] In one embodiment, LPX comprises four ionizable cationic lipids and one auxiliary lipid. In another embodiment, LPX comprises four ionizable cationic lipids and two auxiliary lipids. In another embodiment, LPX comprises four ionizable cationic lipids and three auxiliary lipids. In another embodiment, LPX comprises four ionizable cationic lipids and four auxiliary lipids. In yet another embodiment, LPX comprises four ionizable cationic lipids and five auxiliary lipids.

[0106] In one embodiment, LPX comprises five ionizable cationic lipids and one auxiliary lipid. In another embodiment, LPX comprises five ionizable cationic lipids and two auxiliary lipids. In yet another embodiment, LPX comprises five ionizable cationic lipids and three auxiliary lipids. In yet another embodiment, LPX comprises five ionizable cationic lipids and four auxiliary lipids. In yet another embodiment, LPX comprises five ionizable cationic lipids and five auxiliary lipids.

[0107] In some embodiments, LPS is a lipid micelle, a liposome, or a self-assembled lipid nanoparticle. In some embodiments, LPS is a lipid micelle. In some embodiments, LPS is a liposome. In some embodiments, LPS is a self-assembled lipid nanoparticle.

[0108] In some embodiments, LPS comprises one or more ionizable cationic lipids and / or one or more auxiliary lipids. In some embodiments, LPS comprises one ionizable cationic lipid. In some embodiments, LPS comprises two ionizable cationic lipids. In some embodiments, LPS comprises three ionizable cationic lipids. In some embodiments, LPS comprises four ionizable cationic lipids. In some embodiments, LPS comprises five ionizable cationic lipids. In some embodiments, LPS comprises one auxiliary lipid. In some embodiments, LPS comprises two auxiliary lipids. In some embodiments, LPS comprises three auxiliary lipids. In some embodiments, LPS comprises four auxiliary lipids. In some embodiments, LPS comprises five auxiliary lipids.

[0109] In one embodiment, LPS comprises one ionizable cationic lipid and one auxiliary lipid. In another embodiment, LPS comprises one ionizable cationic lipid and two auxiliary lipids. In another embodiment, LPS comprises one ionizable cationic lipid and three auxiliary lipids. In another embodiment, LPS comprises one ionizable cationic lipid and four auxiliary lipids. In another embodiment, LPS comprises one ionizable cationic lipid and five auxiliary lipids. In yet another embodiment, LPS comprises one ionizable cationic lipid and two auxiliary lipids. In yet another embodiment, LPS comprises one ionizable cationic lipid and three auxiliary lipids. In yet another embodiment, LPS comprises one ionizable cationic lipid and four auxiliary lipids. In yet another embodiment, LPS comprises one ionizable cationic lipid and five auxiliary lipids.

[0110] In one embodiment, LPS comprises two ionizable cationic lipids and one auxiliary lipid. In another embodiment, LPS comprises two ionizable cationic lipids and two auxiliary lipids. In yet another embodiment, LPS comprises two ionizable cationic lipids and three auxiliary lipids. In yet another embodiment, LPS comprises two ionizable cationic lipids and four auxiliary lipids. In yet another embodiment, LPS comprises two ionizable cationic lipids and five auxiliary lipids.

[0111] In one embodiment, LPS comprises three ionizable cationic lipids and one auxiliary lipid. In another embodiment, LPS comprises three ionizable cationic lipids and two auxiliary lipids. In yet another embodiment, LPS comprises three ionizable cationic lipids and three auxiliary lipids. In yet another embodiment, LPS comprises three ionizable cationic lipids and four auxiliary lipids. In yet another embodiment, LPS comprises three ionizable cationic lipids and five auxiliary lipids.

[0112] In one embodiment, LPS comprises four ionizable cationic lipids and one auxiliary lipid. In another embodiment, LPS comprises four ionizable cationic lipids and two auxiliary lipids. In yet another embodiment, LPS comprises four ionizable cationic lipids and three auxiliary lipids. In yet another embodiment, LPS comprises four ionizable cationic lipids and four auxiliary lipids. In yet another embodiment, LPS comprises four ionizable cationic lipids and five auxiliary lipids.

[0113] In one embodiment, LPS comprises five ionizable cationic lipids and one auxiliary lipid. In another embodiment, LPS comprises five ionizable cationic lipids and two auxiliary lipids. In yet another embodiment, LPS comprises five ionizable cationic lipids and three auxiliary lipids. In yet another embodiment, LPS comprises five ionizable cationic lipids and four auxiliary lipids. In yet another embodiment, LPS comprises five ionizable cationic lipids and five auxiliary lipids.

[0114] Some non-limiting examples of ionizable cationic lipids (e.g., ionizable cationic lipids of LPS or LPX) include the lipids provided in Table 1 below.

[0115] Table 1: In embodiments, the ionizable cationic lipid (e.g., an ionizable cationic lipid of LPS or LPX) is selected from any of the following U.S. patents or U.S. patent publications: US20150376115A1, US20160376224A1, US20170119904A1, US20180185516A1, and US201900222. No. 47A1, No. US20200046838A1, No. US20200172472A1, No. US20200283372A1, No. US20210122703 No. A1, No. US20210128488A1, No. US20210395188A1, No. US20220081392A1, No. US20220106257A1 US No. 20220218622A1, US No. 20220218622A1, US No. 10077232B2, US No. 20190240339A1, US No. 10561732B2, US No. 10653780B2, US No. 20200282060A1, US No. 20220133636A1, US No. 20150141 U.S. Patent Nos. 678A1, 20150239926A1, 20170190661A1, 20180170866A1, 20180222863A1, 8034376B2, 20090163705A1, and 20110097720A1, all of which are incorporated herein by reference in their entirety.

[0116] In embodiments, the ionizable cationic lipids (e.g., ionizable cationic lipids of LPS or LPX) are SM-102, ALC-0315, DODMA, DOTMA, DOTAP, DC-cholesterol, JK-0315-CA, C12-200, JK-102-CA, ALC-BAE-0315, Me2N+HexDecA, KT-001, TU-C3-HAD, TU-B3-HAD, TU-D3-HAD, or SSEC-ALC-0315.

[0117] In an embodiment, the ionizable cationic lipid (e.g., the ionizable cationic lipid of LPS or LPX) is DOTMA, DOTAP, DC-cholesterol, or KT-001.

[0118] In implementation, the auxiliary lipid (e.g., the auxiliary lipid of LPS or LPX) is a phospholipid, a polyethylene glycol-modified lipid, cholesterol, or a cholesterol derivative.

[0119] In one embodiment, the cofactor lipid (e.g., cofactor lipid of LPS or LPX) is a phospholipid. In another embodiment, the cofactor lipid (e.g., cofactor lipid of LPS or LPX) is a polyethylene glycol-modified lipid. In yet another embodiment, the cofactor lipid (e.g., cofactor lipid of LPS or LPX) is cholesterol. In yet another embodiment, the cofactor lipid (e.g., cofactor lipid of LPS or LPX) is a cholesterol derivative.

[0120] Some non-limiting examples of cofactor lipids (e.g., cofactor lipids of LPS or LPX) include the lipids provided in Table 2 below. All lipids in Table 2 are available from Avanti Polar Lipids.

[0121] Table 2: In some embodiments, the cofactor lipid (e.g., cofactor lipid of LPS or LPX) is DSPC, cholesterol, DMG-PEG2000, or DOPE. In some embodiments, the cofactor lipid (e.g., cofactor lipid of LPS or LPX) is DSPC. In some embodiments, the cofactor lipid (e.g., cofactor lipid of LPS or LPX) is cholesterol. In some embodiments, the cofactor lipid (e.g., cofactor lipid of LPS or LPX) is DMG-PEG2000. In some embodiments, the cofactor lipid (e.g., cofactor lipid of LPS or LPX) is DOPE.

[0122] In this embodiment, LPX comprises KT-001, DSPC, cholesterol, DOTMA, DC-cholesterol, DOPE, or any combination thereof.

[0123] In one embodiment, LPX comprises KT-001 and DSPC. In another embodiment, LPX comprises KT-001, DSPC, and cholesterol. In yet another embodiment, LPX comprises KT-001, DOTMA, DSPC, and cholesterol. In yet another embodiment, LPX comprises KT-001, DSPC, cholesterol, and DMG-PEG2000. In yet another embodiment, LPX comprises DC-cholesterol and DOPE.

[0124] In this embodiment, LPS comprises KT-001, DSPC, cholesterol, DMG-PEG2000, DOTMA, DOTAP, or any combination thereof.

[0125] In one embodiment, LPS comprises DC-cholesterol and DOTAP. In another embodiment, LPS comprises DC-cholesterol and DOPE. In yet another embodiment, LPS comprises KT-001, DSPC, cholesterol, and DMG-PEG2000. In yet another embodiment, LPS comprises KT-001, DOTMA, DSPC, cholesterol, and DMG-PEG2000.

[0126] In embodiments, the molar ratio of KT-001 to DSPC (e.g., in LPX) is in the range of about 1:3 to about 10:1, for example, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 10:1, or about 15:1. In embodiments, the KT-001:DSPC ratio is 4.5:1 to 5.5:1, 4.7:1 to 5.3:1, 4.8:1 to 5.2:1, or 4.9:1 to 5.1:1. In embodiments, the KT-001:DSPC ratio is about 5:1. In embodiments, the KT-001:DSPC ratio is 5:1.

[0127] In embodiments, the molar ratio of KT-001 to DSPC to cholesterol (e.g., in LPX) can be between approximately 20-80:2-30:10-60. In embodiments, the molar ratio of KT-001:DSPC:cholesterol is 40:10:40 to 60:10:40, 40:20:80 to 60:20:50, 45:10:45 to 55:10:45, or 45:10:30 to 55:10:45. In embodiments, the molar ratio of KT-001:DSPC:cholesterol is approximately 50:10:38.5. In embodiments, the molar ratio of KT-001:DSPC:cholesterol is 50:10:38.5.

[0128] In embodiments, the molar ratio of KT-001 to DOTMA to DSPC to cholesterol (e.g., in LPX) can be between approximately 20-80:1-20:2-30:10-60. In embodiments, the molar ratio of KT-001:DOTMA:DSPC:cholesterol is 35:5:10:40 to 55:5:10:40, 35:10:20:80 to 55:10:20:50, 30:5:10:45 to 50:5:10:45, 40:2:4:30 to 50:10:20:45, or 40:5:10:30 to 50:5:10:45. In embodiments, the molar ratio of KT-001:DOTMA:DSPC:cholesterol is approximately 45:5:10:38.5. In this implementation, the molar ratio of KT-001:DOTMA:DSPC:cholesterol is 45:5:10:38.5.

[0129] In embodiments, the molar ratio of KT-001 to DSPC to cholesterol to DMG-PEG2000 (e.g., in LPX or LPS) can be between approximately 20-80:2-30:10-60:0.5-8. In embodiments, the molar ratio of KT-001:DSPC:cholesterol:DMG-PEG2000 is 40:10:40:2 to 60:10:40:2, 40:20:80:1.5 to 60:20:50:1.5, 45:10:45:2 to 55:10:45:2, 45:10:30:1 to 55:10:45:1, or 45:5:30:0.5 to 55:25:45:2.5. In this embodiment, the molar ratio of KT-001:DSPC:cholesterol:DMG-PEG2000 is approximately 50:10:38.5:1.5.

[0130] In embodiments, the molar ratio of DC-cholesterol to DOPE (e.g., in LPX or LPS) is in the range of about 1:3 to about 10:1, for example, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, or about 10:1. In embodiments, the DC-cholesterol:DOPE ratio is 1.5:1 to 2.5:1, 1.7:1 to 2.3:1, 1.8:1 to 2.2:1, or 1.9:1 to 2.1:1. In embodiments, the DC-cholesterol:DOPE ratio is about 2:1. In embodiments, the DC-cholesterol:DOPE ratio is 2:1.

[0131] In embodiments, the molar ratio of DC-cholesterol to DOPE (e.g., in LPS) is in the range of about 3:1 to about 10:1, for example, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, or about 1:5. In embodiments, the DC-cholesterol:DOPE ratio is 1:1.5 to 1:2.5, 1:1.7 to 1:2.3, 1:1.8 to 1:2.2, or 1:1.9 to 1:2.1. In embodiments, the DC-cholesterol:DOPE ratio is about 1:2. In embodiments, the DC-cholesterol:DOPE ratio is 1:2.

[0132] In embodiments, the molar ratio of DC-cholesterol to DOTAP (e.g., in LPS) is in the range of about 1:3 to about 10:1, for example, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, or about 10:1. In embodiments, the DC-cholesterol:DOTAP ratio is 1.5:1 to 2.5:1, 1.7:1 to 2.3:1, 1.8:1 to 2.2:1, or 1.9:1 to 2.1:1. In embodiments, the DC-cholesterol:DOTAP ratio is about 2:1. In embodiments, the DC-cholesterol:DOTAP ratio is 2:1.

[0133] In embodiments, the molar ratio of DC-cholesterol to DOTAP (e.g., in LPS) is in the range of about 3:1 to about 10:1, for example, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, or about 1:5. In embodiments, the DC-cholesterol:DOTAP ratio is 1:1.5 to 1:2.5, 1:1.7 to 1:2.3, 1:1.8 to 1:2.2, or 1:1.9 to 1:2.1. In embodiments, the DC-cholesterol:DOTAP ratio is about 1:2. In embodiments, the DC-cholesterol:DOTAP ratio is 1:2.

[0134] In embodiments, the molar ratio of KT-001 to DOTMA to DSPC to cholesterol to DMG-PEG2000 (e.g., in LPS) can be between approximately 20-80:1-15:2-30:10-60:0.5-8. In embodiments, the molar ratio of KT-001:DOTMA:DSPC:cholesterol:DMG-PEG2000 is 40:5:10:40:2 to 60:5:10:40:2, 35:10:20:80:1.5 to 55:10:20:50:1.5, 40:5:10:45:2 to 50:5:10:45:2, 40:5:10:30:1 to 50:5:10:45:1, or 40:1:5:30:0.5 to 50:15:25:45:2.5. In this embodiment, the molar ratio of KT-001:DOTMA:DSPC:cholesterol:DMG-PEG2000 is approximately 45 / 5 / 10 / 38.5 / 1.5.

[0135] In some embodiments, the composition for transfecting cells comprises a targeting ligand, an imaging ligand, a digestive agent, or a small molecule. In some embodiments, the composition for transfecting cells comprises a targeting ligand. In some embodiments, the composition for transfecting cells comprises an imaging ligand. In some embodiments, the composition for transfecting cells comprises a digestive agent. In some embodiments, the composition for transfecting cells comprises a small molecule.

[0136] In this implementation, the targeting ligand is in contact with LPS. As used herein, the term "in contact with LPS" means a targeting ligand on the surface of LPS; or a targeting ligand with one end in a micelle or liposome bilayer and the other end protruding to the outside of the lipid shell.

[0137] In some embodiments, the targeting ligand is DUPA, folic acid, a peptide, or an antibody or its antigen-binding fragment. In some embodiments, the targeting ligand is DUPA. In some embodiments, the targeting ligand is folic acid. In some embodiments, the targeting ligand is a peptide. In some embodiments, the targeting ligand is an antibody or its antigen-binding fragment. In some embodiments, the targeting ligand is an antibody. In some embodiments, the targeting ligand is Fab. In some embodiments, the targeting ligand is scFv.

[0138] Non-limiting examples of antibodies that can be used as targeting ligands include anti-FAP antibodies, anti-CD2 antibodies, anti-CD25 antibodies, anti-CD3 antibodies, anti-CD33 antibodies, anti-CD30 antibodies, anti-HER2 antibodies, anti-CD22 antibodies, anti-PSMA antibodies, anti-TM4SF antibodies, and anti-ICAM1 antibodies. In embodiments, some non-limiting examples of targeting ligands may be Fabs of the antibodies listed above.

[0139] In this implementation, the targeting ligand is an anti-FAP antibody, an anti-CD2 antibody, an anti-CD25 antibody, an anti-CD3 antibody, an anti-CD33 antibody, an anti-CD30 antibody, an anti-HER2 antibody, an anti-CD22 antibody, an anti-PSMA antibody, an anti-TM4SF antibody, or an anti-ICAM1 antibody.

[0140] In the implementation, the targeted ligand is anti-FAP Fab, anti-CD2 Fab, anti-CD25 Fab, anti-CD3 Fab, anti-CD33 Fab, anti-CD30 Fab, anti-HER2 Fab, anti-CD22 Fab, anti-PSMA Fab, anti-TM4SF Fab, or anti-ICAM1 Fab.

[0141] Non-limiting examples of peptides that can be used as targeting ligands include RGD4C, PL1, angiopep-1, PEN-221, Ty3-octreotide, etc. In embodiments, the targeting ligand is RGD4C, PL1, angiopep-1, PEN-221, or Ty3-octreotide.

[0142] In one embodiment, the imaging ligand is in contact with the LPS, inside the LPX, or between the surface of the LPS and the LPX. In another embodiment, the imaging ligand is in contact with the LPS. In yet another embodiment, the imaging ligand is inside the LPX. In still another embodiment, the imaging ligand is located between the surface of the LPS and the LPX.

[0143] Non-limiting examples of imaging ligands that can be used in compositions for transfecting cells include 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol-IR825 (DSPE-PEG-IR825) and diethylenetriaminepentaacetic acid (DTPA)-Gd. In embodiments, the imaging ligand is 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol-IR825 (DSPE-PEG-IR825). In embodiments, the imaging ligand is diethylenetriaminepentaacetic acid (DTPA)-Gd.

[0144] In one embodiment, the digestant is in contact with the LPS, inside the LPX, or between the surface of the LPS and the LPX. In another embodiment, the digestant is in contact with the LPS. In yet another embodiment, the digestant is inside the LPX. Also in another embodiment, the digestant is between the surface of the LPS and the LPX.

[0145] Non-limiting examples of digestive agents that can be used in compositions for transfecting cells include hyaluronidase and collagenase. In one embodiment, the digestive agent is hyaluronidase. In another embodiment, the digestive agent is collagenase. In yet another embodiment, the digestive agent is a degrading enzyme. In still another embodiment, the digestive agent is an enzyme that cleaves oligomeric or polymeric substrates.

[0146] In one embodiment, the small molecule is in contact with LPS, inside LPX, or between the surface of LPS and LPX. In another embodiment, the small molecule is in contact with LPS. In yet another embodiment, the small molecule is inside LPX. Also in another embodiment, the small molecule is between the surface of LPS and LPX.

[0147] Non-limiting examples of small molecules that can be used in compositions for transfecting cells include the molecules in Table 3.

[0148] Table 3. Other non-limiting examples of small molecules that can be used in compositions for transfecting cells include anti-inflammatory small molecules and toxins. In embodiments, the small molecule is an NSAID or a toxin. In embodiments, the small molecule is a vitamin, sugar, steroid, or chemotherapeutic agent. Other non-limiting examples of small molecules that can be used in compositions for transfecting cells include folic acid, glucose, galactose, and N-acetylgalactosamine (GalNAc). In embodiments, the small molecule is folic acid, glucose, galactose, N-acetylgalactosamine (GalNAc), dexamethasone, paclitaxel, or doxorubicin.

[0149] Pharmaceutical Composition On the one hand, this article provides pharmaceutical compositions comprising compositions for in vitro and in vivo cell transfection as described herein (including embodiments) and pharmaceutically acceptable excipients.

[0150] In embodiments, the compositions for transfecting cells described herein can be formulated into pharmaceutical compositions. Pharmaceutical compositions may include one or more LPS-LPX delivery systems, each containing at least one nucleic acid contained in each LPS-LPX delivery system, and optionally a targeting ligand, imaging ligand, digestive agent, or small molecule. Pharmaceutical compositions may further contain one or more pharmaceutically acceptable excipients or adjuvants, as described herein. General guidance on the formulation and manufacture of pharmaceutical compositions and agents may be available, for example, in Remington's The Science and Practice of Pharmacy, 21st Edition, AR Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006.

[0151] The relative amounts of the one or more compositions for transfecting cells, the one or more pharmaceutically acceptable excipients, and / or any other ingredients in the pharmaceutical compositions according to this disclosure will vary depending on the identity, size, and / or condition of the subject being treated and, further, on the route of administration of the composition. For example, the pharmaceutical composition may contain one or more compositions for transfecting cells in amounts between 0.1% and 100% (wt / wt).

[0152] Non-limiting examples of pharmaceutically acceptable excipients include water for injection (WFI), NaCl, PBS, physiological saline solution, lactated Ringer's solution, standard sucrose, standard glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, saline solutions (such as Ringer's solution), alcohols, fixed oils, polyethylene glycol, glycerol, propylene glycol, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, and pigments.

[0153] Pharmaceutically acceptable excipients are typically added after the composition for transfection of cells has been formed. Therefore, after the composition for transfection of cells has been formed, it can be diluted with a pharmaceutically acceptable excipient, such as normal buffered saline. Pharmaceutical compositions typically contain conventional pharmaceutical excipients and may additionally contain other agents, carriers, adjuvants, additives, etc.

[0154] Pharmaceutical compositions can be prepared in a variety of forms suitable for various routes and methods of administration. For example, the pharmaceutical compositions of the present invention can be prepared as liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable forms, solid dosage forms (e.g., capsules, tablets, pills, powders, and granules), dosage forms for topical and / or transdermal application (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalers, and patches), suspensions, powders, and other forms. Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to the active ingredient, liquid dosage forms may include inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (specifically, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions may contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and / or aromatizers. In some embodiments for parenteral administration, the composition is mixed with a solubilizer (such as Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof).

[0155] Pharmaceutical formulations containing compositions for transfecting cells can be in liquid, solid, semi-solid, or lyophilized powder form, such as solutions, suspensions, emulsions, sustained-release formulations, tablets, capsules, powders, suppositories, creams, ointments, lotions, aerosols, patches, etc., for example, in unit dosage forms suitable for simple administration of precise doses.

[0156] For internal administration, it can be administered in any manner known in the art, such as by injection, oral administration, inhalation (e.g., intranasal or intratracheal), transdermal (topical), transmucosal, or rectal administration.

[0157] In some embodiments, the pharmaceutical composition may be administered parenterally, such as intra-articular, intravenous, intradermal, intrathecal, intraperitoneal, subcutaneous, or intramuscularly. In others, the pharmaceutical composition may be administered intravenously or intraperitoneally via bolus injection. In still others, the parenteral preparation may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0158] Injectable compositions intended for parenteral administration (e.g., intravenous, intramuscular, or intrathecal) typically contain the composition in a suitable IV solution (such as sterile saline solution). The composition may also be formulated as a suspension in an aqueous emulsion.

[0159] Method for preparing compositions for transfection On one hand, this article provides a method for preparing a composition for in vitro or in vivo transfection of cells, the method comprising: (a) preparing a first solution comprising an aqueous phase and one or more nucleic acids; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b), thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids; (d) stirring the LPX prepared in step (c); and (e) preparing a composition comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a... (f) preparing LPS by forming liposomes, micelles or other self-assembled lipid nanoparticles from the third solution prepared in step (e), wherein a targeting ligand, imaging ligand, digestive agent or small molecule is optionally added to an aqueous phase, and then the aqueous phase is mixed with the third solution prepared in step (e); (g) mixing the LPX after step (d) with the LPS prepared in step (f), and optionally adding a targeting ligand, imaging ligand, digestive agent or small molecule; and (h) stirring the mixture prepared in step (g), thereby forming a composition for transfecting cells.

[0160] On one hand, this article provides a method for preparing a composition for transfecting cells in vitro or in vivo, the method comprising: (a) mixing a first solution comprising an aqueous phase and one or more nucleic acids with a second solution comprising an organic phase and one or more lipids, thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids; (b) stirring the LPX prepared in step (a); (c) preparing a third solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule; (d) preparing LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from the third solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule, wherein optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule is added to the aqueous phase, and then the aqueous phase is mixed with the third solution; (e) mixing the LPX after step (b) with the LPS prepared in step (d), and optionally adding a targeting ligand, an imaging ligand, a digestive agent, or a small molecule; and (f) stirring the mixture prepared in step (e), thereby forming a composition for transfecting cells.

[0161] On one hand, this article provides a method for preparing a composition for transfecting cells in vitro or in vivo, the method comprising: (a) preparing a first solution comprising an aqueous phase and one or more nucleic acids; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b), thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids; (d) stirring the LPX prepared in step (c); (e) preparing a third solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule; (f) preparing LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from the aqueous phase, and then mixing the aqueous phase with the third solution prepared in step (e); (g) mixing the LPX after step (d) with the LPS prepared in step (f); and (h) stirring the mixture prepared in step (g), thereby forming a composition for transfecting cells.

[0162] On one hand, this article provides a method for preparing a composition for transfecting cells in vitro or in vivo, the method comprising: (a) preparing a first solution comprising an aqueous phase and one or more nucleic acids; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b), thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids; (d) stirring the LPX prepared in step (c); (e) preparing a third solution comprising an organic phase and one or more lipids; (f) preparing LPS by forming liposomes, micelles or other self-assembled lipid nanoparticles from the third solution prepared in step (e), wherein optionally a targeting ligand, imaging ligand, digestive agent or small molecule is added to the aqueous phase, and then the aqueous phase is mixed with the third solution prepared in step (e); (g) mixing the LPX after step (d) with the LPS prepared in step (f); and (h) stirring the mixture prepared in step (g), thereby forming a composition for transfecting cells.

[0163] On one hand, this article provides a method for preparing a composition for transfecting cells in vitro or in vivo, the method comprising: (a) preparing a first solution comprising an aqueous phase and one or more nucleic acids; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b), thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids; (d) stirring the LPX prepared in step (c); (e) preparing a third solution comprising an organic phase and one or more lipids; (f) preparing LPS by forming liposomes, micelles or other self-assembled lipid nanoparticles from the aqueous phase, and then mixing the aqueous phase with the third solution prepared in step (e); (g) mixing the LPX after step (d) with the LPS prepared in step (f), and optionally adding a targeting ligand, an imaging ligand, a digestive agent or a small molecule; and (h) stirring the mixture prepared in step (g), thereby forming a composition for transfecting cells.

[0164] On the other hand, this article provides a method for preparing a composition for in vitro or in vivo transfection of cells, the method comprising: (a) preparing a first solution comprising an aqueous phase; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) to generate liposomes, micelles, or other self-assembled lipid nanoparticles; (d) preparing a third solution comprising an aqueous phase and one or more nucleic acids; (e) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (c) with the third solution prepared in step (d), thereby forming monodisperse or polydisperse LPX; (f) preparing a composition comprising... An organic phase, one or more lipids, and optionally a targeting ligand, imaging ligand, digestive agent, or small molecule in a fourth solution; (g) preparing LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from the fourth solution prepared in step (f), wherein optionally a targeting ligand, imaging ligand, digestive agent, or small molecule is added to an aqueous phase, and then the aqueous phase is mixed with the fourth solution prepared in step (f); (h) mixing the LPX prepared in step (e) with the LPS prepared in step (g), and optionally adding a targeting ligand, imaging ligand, digestive agent, or small molecule; and (i) stirring the mixture prepared in step (h), thereby forming a composition for transfecting cells.

[0165] On the other hand, this article provides a method for preparing a composition for transfecting cells in vitro or in vivo, the method comprising: (a) mixing a first solution comprising an aqueous phase with a second solution comprising an organic phase and one or more lipids to produce liposomes, micelles, or other self-assembled lipid nanoparticles; (b) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (a) with a third solution comprising an aqueous phase and one or more nucleic acids, thereby forming monodisperse or polydisperse LPX; (c) preparing LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from a fourth solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule, wherein optionally the targeting ligand, imaging ligand, digestive agent, or small molecule is added to the aqueous phase, and then the aqueous phase is mixed with the fourth solution; (d) mixing the LPX prepared in step (b) with the LPS prepared in step (c), and optionally adding a targeting ligand, an imaging ligand, a digestive agent, or a small molecule; and (e) stirring the mixture prepared in step (d), thereby forming a composition for transfecting cells.

[0166] On the other hand, this article provides a method for preparing a composition for in vitro or in vivo cell transfection, the method comprising: (a) preparing a first solution comprising an aqueous phase; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) to generate liposomes, micelles, or other self-assembled lipid nanoparticles; (d) preparing a third solution comprising an aqueous phase and one or more nucleic acids; (e) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (c) with the solution prepared in step (d) The prepared third solution is mixed to form a monodisperse or polydisperse LPX; (f) a fourth solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule is prepared; (g) LPS is prepared by forming liposomes, micelles, or other self-assembled lipid nanoparticles from an aqueous phase, and then the aqueous phase is mixed with the fourth solution prepared in step (f); (h) the LPX prepared in step (e) is mixed with the LPS prepared in step (g); and (i) the mixture prepared in step (h) is stirred to form a composition for transfecting cells.

[0167] On the other hand, this article provides a method for preparing a composition for in vitro or in vivo transfection of cells, the method comprising: (a) preparing a first solution comprising an aqueous phase; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) to generate liposomes, micelles, or other self-assembled lipid nanoparticles; (d) preparing a third solution comprising an aqueous phase and one or more nucleic acids; and (e) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (c) with the third solution prepared in step (d). (f) preparing a fourth solution comprising an organic phase and one or more lipids; (g) preparing LPS by forming liposomes, micelles or other self-assembled lipid nanoparticles from the fourth solution prepared in step (f), wherein optionally a targeting ligand, imaging ligand, digestive agent or small molecule is added to the aqueous phase, and then the aqueous phase is mixed with the fourth solution prepared in step (f); (h) mixing the LPX prepared in step (e) with the LPS prepared in step (g); and (i) stirring the mixture prepared in step (h), thereby forming a composition for transfecting cells.

[0168] On the other hand, this article provides a method for preparing a composition for in vitro or in vivo transfection of cells, the method comprising: (a) preparing a first solution comprising an aqueous phase; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) to generate liposomes, micelles, or other self-assembled lipid nanoparticles; (d) preparing a third solution comprising an aqueous phase and one or more nucleic acids; and (e) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (c) with the composition prepared in step (d) (f) Mix the third solution prepared in step (e) to form a monodisperse or polydisperse LPX; (g) Prepare a fourth solution comprising an organic phase and one or more lipids; (g) Prepare LPS by forming liposomes, micelles or other self-assembled lipid nanoparticles from an aqueous phase, and then mix the aqueous phase with the fourth solution prepared in step (f); (h) Mix the LPX prepared in step (e) with the LPS prepared in step (g), and optionally add a targeting ligand, an imaging ligand, a digestive agent or a small molecule; and (i) Stir the mixture prepared in step (h) to form a composition for transfecting cells.

[0169] On the other hand, this article provides a method for preparing compositions for in vitro or in vivo cell transfection, the method comprising: (a) preparing a first solution comprising an aqueous phase and one or more nucleic acids; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) to generate liposomes, micelles, or other self-assembled lipid nanoparticles; (d) preparing a third solution comprising an aqueous phase and one or more nucleic acids; (e) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (c) with the third solution prepared in step (d), thereby forming monodisperse or polydisperse LPX; (f) A fourth solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule is prepared; (g) LPS is prepared by forming liposomes, micelles, or other self-assembled lipid nanoparticles from the fourth solution prepared in step (f), wherein optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule is added to an aqueous phase, and then the aqueous phase is mixed with the fourth solution prepared in step (f); (h) the LPX prepared in step (e) is mixed with the LPS prepared in step (g), and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule is added; and (i) the mixture prepared in step (h) is stirred, thereby forming a composition for transfecting cells.

[0170] On the other hand, this article provides a method for preparing compositions for in vitro or in vivo cell transfection, the method comprising: (a) mixing a first solution comprising an aqueous phase and one or more nucleic acids with a second solution comprising an organic phase and one or more lipids to produce liposomes, micelles, or other self-assembled lipid nanoparticles; (b) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (a) with a third solution comprising an aqueous phase and one or more nucleic acids, thereby forming monodisperse or polydisperse LPX; (c) by mixing the liposomes, micelles, or other self-assembled lipid nanoparticles comprising an organic phase and one or more lipids with a third solution comprising an aqueous phase and one or more nucleic acids. LPS is prepared by forming liposomes, micelles, or other self-assembled lipid nanoparticles from a fourth solution of optionally targeting ligands, imaging ligands, digestive agents, or small molecules, wherein optionally targeting ligands, imaging ligands, digestive agents, or small molecules are added to an aqueous phase, and then the aqueous phase is mixed with the fourth solution; (d) the LPX prepared in step (b) is mixed with the LPS prepared in step (c), and optionally targeting ligands, imaging ligands, digestive agents, or small molecules are added; and (e) the mixture prepared in step (d) is stirred, thereby forming a composition for transfecting cells.

[0171] On the other hand, this article provides a method for preparing a composition for in vitro or in vivo cell transfection, the method comprising: (a) preparing a first solution comprising an aqueous phase and one or more nucleic acids; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) to generate liposomes, micelles, or other self-assembled lipid nanoparticles; (d) preparing a third solution comprising an aqueous phase and one or more nucleic acids; (e) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (c) with the first solution prepared in step (b) to generate liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (c) with the second solution prepared in step (b) to generate liposomes, micelles, or other self-assembled lipid nanoparticles. (d) The third solution prepared in step (d) is mixed to form a monodisperse or polydisperse LPX; (f) A fourth solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule is prepared; (g) LPS is prepared by forming liposomes, micelles, or other self-assembled lipid nanoparticles from an aqueous phase, and then the aqueous phase is mixed with the fourth solution prepared in step (f); (h) The LPX prepared in step (e) is mixed with the LPS prepared in step (g); and (i) The mixture prepared in step (h) is stirred to form a composition for transfecting cells.

[0172] On the other hand, this article provides a method for preparing a composition for in vitro or in vivo transfection of cells, the method comprising: (a) preparing a first solution comprising an aqueous phase and one or more nucleic acids; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) to generate liposomes, micelles, or other self-assembled lipid nanoparticles; (d) preparing a third solution comprising an aqueous phase and one or more nucleic acids; and (e) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (c) with the third solution prepared in step (d). (f) Mixing solutions to form monodisperse or polydisperse LPX; (g) Preparing a fourth solution comprising an organic phase and one or more lipids; (f) Preparing LPS by forming liposomes, micelles or other self-assembled lipid nanoparticles from the fourth solution prepared in step (f), wherein optionally a targeting ligand, imaging ligand, digestive agent or small molecule is added to the aqueous phase, and then the aqueous phase is mixed with the fourth solution prepared in step (f); (h) Mixing the LPX prepared in step (e) with the LPS prepared in step (g); and (i) Stirring the mixture prepared in step (h) to form a composition for transfecting cells.

[0173] On the other hand, this article provides a method for preparing a composition for in vitro or in vivo transfection of cells, the method comprising: (a) preparing a first solution comprising an aqueous phase and one or more nucleic acids; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) to generate liposomes, micelles, or other self-assembled lipid nanoparticles; (d) preparing a third solution comprising an aqueous phase and one or more nucleic acids; (e) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (c) with the second solution prepared in step (b) to generate liposomes, micelles, or other self-assembled lipid nanoparticles. (d) The third solution prepared in step (d) is mixed to form a monodisperse or polydisperse LPX; (f) A fourth solution comprising an organic phase and one or more lipids is prepared; (g) LPS is prepared by forming liposomes, micelles or other self-assembled lipid nanoparticles from an aqueous phase, and then the aqueous phase is mixed with the fourth solution prepared in step (f); (h) The LPX prepared in step (e) is mixed with the LPS prepared in step (g), and optionally a targeting ligand, an imaging ligand, a digestive agent or a small molecule is added; and (i) The mixture prepared in step (h) is stirred to form a composition for transfecting cells.

[0174] On one hand, this article provides a method for preparing a composition for in vitro or in vivo transfection of cells, the method comprising: (a) preparing a first solution comprising an aqueous phase and one or more nucleic acids; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b), thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids; (d) stirring the LPX prepared in step (c); (e) preparing a third solution comprising an aqueous phase and one or more nucleic acids; (f) mixing the LPX prepared in step (d) with the third solution prepared in step (e); (g) preparing a package... A fourth solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule; (h) preparing LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from the fourth solution prepared in step (g), wherein optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule is added to an aqueous phase, and then the aqueous phase is mixed with the fourth solution prepared in step (g); (i) mixing the LPX from step (f) with the LPS prepared in step (h), and optionally adding a targeting ligand, an imaging ligand, a digestive agent, or a small molecule; and (j) stirring the mixture prepared in step (i), thereby forming a composition for transfecting cells.

[0175] A method for preparing a composition for transfecting cells in vitro or in vivo, the method comprising: (a) mixing a first solution comprising an aqueous phase and one or more nucleic acids with a second solution comprising an organic phase and one or more lipids, thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids; (b) stirring the LPX prepared in step (a); (c) mixing the LPX prepared in step (a) with a third solution comprising an aqueous phase and one or more nucleic acids; (d) preparing LPS by forming liposomes, micelles or other self-assembled lipid nanoparticles from a fourth solution comprising an organic phase, one or more lipids and optionally a targeting ligand, an imaging ligand, a digestive agent or a small molecule, wherein optionally the targeting ligand, imaging ligand, digestive agent or small molecule is added to the aqueous phase and then the aqueous phase is mixed with the fourth solution; (e) mixing the LPX prepared in step (c) with the LPS prepared in step (d), and optionally adding a targeting ligand, an imaging ligand, a digestive agent or a small molecule; and (f) stirring the mixture prepared in step (e), thereby forming a composition for transfecting cells.

[0176] On one hand, this article provides a method for preparing a composition for in vitro or in vivo transfection of cells, the method comprising: (a) preparing a first solution comprising an aqueous phase and one or more nucleic acids; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b), thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids; (d) stirring the LPX prepared in step (c); (e) preparing a third solution comprising an aqueous phase and one or more nucleic acids; (f) mixing the first solution prepared in step (d) with the second solution prepared in step (b) to form a monodisperse or polydisperse LPX. The prepared LPX is mixed with the third solution prepared in step (e); (g) a fourth solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule is prepared; (h) LPS is prepared by forming liposomes, micelles, or other self-assembled lipid nanoparticles from an aqueous phase, and then the aqueous phase is mixed with the fourth solution prepared in step (g); (i) the LPX after step (f) is mixed with the LPS prepared in step (h); and (j) the mixture prepared in step (i) is stirred, thereby forming a composition for transfecting cells.

[0177] On one hand, this article provides a method for preparing a composition for in vitro or in vivo transfection of cells, the method comprising: (a) preparing a first solution comprising an aqueous phase and one or more nucleic acids; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b), thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids; (d) stirring the LPX prepared in step (c); (e) preparing a third solution comprising an aqueous phase and one or more nucleic acids; (f) mixing the LPX prepared in step (d) with the second solution prepared in step (b) in step (c). (e) The third solution prepared in step (e) is mixed; (g) a fourth solution comprising an organic phase and one or more lipids is prepared; (h) LPS is prepared by forming liposomes, micelles or other self-assembled lipid nanoparticles from the fourth solution prepared in step (g), wherein optionally a targeting ligand, imaging ligand, digestive agent or small molecule is added to the aqueous phase, and then the aqueous phase is mixed with the fourth solution prepared in step (g); (i) the LPX after step (f) is mixed with the LPS prepared in step (h); and (j) the mixture prepared in step (i) is stirred, thereby forming a composition for transfecting cells.

[0178] On one hand, this article provides a method for preparing a composition for in vitro or in vivo transfection of cells, the method comprising: (a) preparing a first solution comprising an aqueous phase and one or more nucleic acids; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b), thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids; (d) stirring the LPX prepared in step (c); (e) preparing a third solution comprising an aqueous phase and one or more nucleic acids; (f) mixing the first solution prepared in step (d) with the second solution prepared in step (b). The LPX prepared in step (e) is mixed with the third solution prepared in step (e); (g) a fourth solution comprising an organic phase and one or more lipids is prepared; (h) LPS is prepared by forming liposomes, micelles or other self-assembled lipid nanoparticles from an aqueous phase, and then the aqueous phase is mixed with the fourth solution prepared in step (g); (i) the LPX after step (f) is mixed with the LPS prepared in step (h), and optionally a targeting ligand, an imaging ligand, a digestive agent or a small molecule is added; and (j) the mixture prepared in step (i) is stirred, thereby forming a composition for transfecting cells.

[0179] In embodiments, methods for preparing compositions for in vitro or in vivo cell transfection can be combined with the use of fluidic instruments. In embodiments, methods for preparing compositions for in vitro or in vivo cell transfection can be combined with the use of microfluidic instruments. Schematic illustrations of these processes are depicted in... Figure 11A , Figure 11B and Figure 11C middle.

[0180] In this embodiment, all steps involving the mixing of the aqueous and organic phases can be combined using a fluidic instrument. In this embodiment, all steps involving the mixing of the aqueous and organic phases can be combined using a microfluidic instrument.

[0181] In embodiments, a method for preparing a composition for in vitro or in vivo cell transfection includes a first process for preparing LPX, a second process for preparing LPS, and a third process for mixing LPX with LPS. In embodiments, LPX encapsulates one or more nucleic acids. In embodiments, one or more nucleic acids are on the surface of LPX (e.g., in contact with LPX). In embodiments, LPX encapsulates one or more nucleic acids, and another one or more nucleic acids are on the surface of LPX. In embodiments, a targeting ligand, imaging ligand, digestive agent, or small molecule is in contact with LPS. In embodiments, the targeting ligand, imaging ligand, digestive agent, or small molecule is inside LPX. In embodiments, the targeting ligand, imaging ligand, digestive agent, or small molecule is between the surface of LPS and LPX.

[0182] In one embodiment, the first process involves a first step of preparing a first solution comprising an aqueous phase. In another embodiment, the first process involves a first step of preparing a first solution comprising an aqueous phase and optionally one or more nucleic acids. In yet another embodiment, the first process involves a first step of preparing a first solution comprising an aqueous phase and one or more nucleic acids. In one embodiment, the aqueous phase is an aqueous buffer. In another embodiment, the aqueous buffer is an acetate buffer, citrate buffer, Tyrode buffer, TBT buffer, Tris buffer, TBS buffer, or Tris-sucrose buffer. In another embodiment, the aqueous buffer is an acetate buffer. In another embodiment, the aqueous buffer is a citrate buffer. In another embodiment, the aqueous buffer is a Tyrode buffer. In another embodiment, the aqueous buffer is a TBT buffer. In another embodiment, the aqueous buffer is a Tris buffer. In another embodiment, the aqueous buffer is a TBS buffer. In another embodiment, the aqueous buffer is a Tris-sucrose buffer.

[0183] In one embodiment, the pH range of the aqueous buffer solution is from about pH 2 to about pH 6. In another embodiment, the pH range of the aqueous buffer solution is from about pH 2 to about pH 5.5. In another embodiment, the pH range of the aqueous buffer solution is from about pH 2 to about pH 5. In another embodiment, the pH range of the aqueous buffer solution is from about pH 2 to about pH 4.5. In another embodiment, the pH range of the aqueous buffer solution is from about pH 2.5 to about pH 5.5. In another embodiment, the pH range of the aqueous buffer solution is from about pH 2.5 to about pH 5. In another embodiment, the pH range of the aqueous buffer solution is from about pH 3 to about pH 5.5. In another embodiment, the pH range of the aqueous buffer solution is from about pH 3 to about pH 5.

[0184] In one embodiment, the aqueous buffer solution is approximately pH 2. In one embodiment, the aqueous buffer solution is approximately pH 2.5. In one embodiment, the aqueous buffer solution is approximately pH 3. In one embodiment, the aqueous buffer solution is approximately pH 3.5. In one embodiment, the aqueous buffer solution is approximately pH 4. In one embodiment, the aqueous buffer solution is approximately pH 4.5. In one embodiment, the aqueous buffer solution is approximately pH 5. In one embodiment, the aqueous buffer solution is approximately pH 5.5. In one embodiment, the aqueous buffer solution is approximately pH 6. In one embodiment, the aqueous buffer solution is approximately pH 6.5. In one embodiment, the aqueous buffer solution is approximately pH 7. In one embodiment, the aqueous buffer solution is approximately pH 7.5. In one embodiment, the aqueous buffer solution is approximately pH 8.

[0185] In one embodiment, the pH range of the aqueous buffer solution is from about pH 7.0 to about pH 8.0. In another embodiment, the aqueous buffer solution is about pH 7.0. In another embodiment, the aqueous buffer solution is about pH 7.2. In another embodiment, the aqueous buffer solution is about pH 7.4. In another embodiment, the aqueous buffer solution is about pH 7.6. In another embodiment, the aqueous buffer solution is about pH 7.8. In another embodiment, the aqueous buffer solution is about pH 8.0.

[0186] In one embodiment, the aqueous buffer is an acetate buffer. In another embodiment, the aqueous buffer is a citrate buffer. In another embodiment, the aqueous buffer is an acetate buffer adjusted to approximately pH 5. In another embodiment, the aqueous buffer is an acetate buffer adjusted to approximately pH 4. In another embodiment, the aqueous buffer is a citrate buffer adjusted to approximately pH 3. In another embodiment, the aqueous buffer is a Tris-sucrose buffer. In another embodiment, the aqueous buffer is a Tris-sucrose buffer adjusted to approximately pH 7.4.

[0187] In one embodiment, a first solution is prepared by dissolving one or more nucleic acids in an aqueous phase. In another embodiment, a first solution is prepared by dissolving one type of nucleic acid in an aqueous phase. In yet another embodiment, a first solution is prepared by dissolving two types of nucleic acids in an aqueous phase. In yet another embodiment, a first solution is prepared by dissolving three types of nucleic acids in an aqueous phase.

[0188] In one embodiment, a first solution is prepared by dissolving a nucleic acid in an aqueous buffer. In another embodiment, a first solution is prepared by dissolving a nucleic acid in an acetate buffer. In yet another embodiment, a first solution is prepared by dissolving a nucleic acid in a citrate buffer. In yet another embodiment, a first solution is prepared by dissolving a nucleic acid in a Tris-sucrose buffer.

[0189] In one embodiment, a first solution is prepared by dissolving the two nucleic acids in an aqueous buffer. In another embodiment, a first solution is prepared by dissolving the two nucleic acids in an acetate buffer. In yet another embodiment, a first solution is prepared by dissolving the two nucleic acids in a citrate buffer. In yet another embodiment, a first solution is prepared by dissolving the two nucleic acids in a Tris-sucrose buffer.

[0190] In one embodiment, the first process involves a second step of preparing a second solution comprising an organic phase and one or more lipids. In another embodiment, the organic phase comprises a water-miscible organic solvent. In another embodiment, the water-miscible organic solvent is ethanol, methanol, isopropanol, DMSO, acetone, acetonitrile, DMF, THF, ethylamine, glycerol, or dioxane. In yet another embodiment, the water-miscible organic solvent is ethanol, methanol, isopropanol, DMSO, acetone, acetonitrile, DMF, or THF. In yet another embodiment, the water-miscible organic solvent is ethanol.

[0191] In one embodiment, the second solution is prepared by dissolving one or more lipids in an organic phase. In another embodiment, the second solution is prepared by dissolving one or more lipids in a water-miscible organic solvent. In another embodiment, the second solution is prepared by dissolving one lipid in a water-miscible organic solvent. In another embodiment, the second solution is prepared by dissolving two lipids in a water-miscible organic solvent. In another embodiment, the second solution is prepared by dissolving three lipids in a water-miscible organic solvent. In another embodiment, the second solution is prepared by dissolving four lipids in a water-miscible organic solvent. In yet another embodiment, the second solution is prepared by dissolving five lipids in a water-miscible organic solvent.

[0192] In one embodiment, the second solution is prepared by dissolving one or more lipids in ethanol. In another embodiment, the second solution is prepared by dissolving one lipid in ethanol. In another embodiment, the second solution is prepared by dissolving two lipids in ethanol. In another embodiment, the second solution is prepared by dissolving three lipids in ethanol. In another embodiment, the second solution is prepared by dissolving four lipids in ethanol. In yet another embodiment, the second solution is prepared by dissolving five lipids in ethanol.

[0193] In embodiments, where the first solution does not contain nucleic acids, the first and second solutions are combined using any method known in the art to form liposomes, micelles, or other self-assembled lipid nanoparticles. In embodiments, liposomes, micelles, or other self-assembled lipid nanoparticles can be formed by pipetting, vortexing, or sonicating the first and second solutions together. In embodiments, the first and second solutions can be mixed using a fluid or microfluidic mixture. In embodiments, the second solution can be dried and then rehydrated with the first solution. In embodiments, the second solution (organic phase) can be injected into the first solution. In embodiments, the first and second solutions can be emulsified, i.e., the first solution can be added to the second solution.

[0194] In one embodiment, the first solution and the second solution are mixed to form a monodisperse or polydisperse LPX containing one or more nucleic acids. In another embodiment, the first solution and the second solution are mixed by pipetting to form a monodisperse or polydisperse LPX containing one or more nucleic acids.

[0195] In one embodiment, the first solution and the second solution are mixed. In another embodiment, the first solution and the second solution are mixed by pipetting. In another embodiment, the first solution and the second solution are mixed, followed by stirring. In another embodiment, the first solution and the second solution are mixed, followed by vortexing or sonication. In another embodiment, the first solution and the second solution are mixed by pipetting, followed by vortexing or sonication. In another embodiment, the first solution and the second solution are mixed, followed by vortexing. In another embodiment, the first solution and the second solution are mixed by pipetting, followed by vortexing. In another embodiment, the first solution and the second solution are mixed, followed by sonication. In another embodiment, the first solution and the second solution are mixed by pipetting, followed by sonication. In another embodiment, the first solution and the second solution are mixed using a fluid or microfluidic mixture, such as... Figure 11A -C is shown.

[0196] In one embodiment, where the first solution does not contain nucleic acids, the first process involves a third step of preparing a third solution comprising an aqueous phase and one or more nucleic acids.

[0197] In one embodiment, the third solution is prepared by dissolving one or more nucleic acids in an aqueous phase. In another embodiment, the third solution is prepared by dissolving one nucleic acid in an aqueous phase. In yet another embodiment, the third solution is prepared by dissolving two nucleic acids in an aqueous phase. In yet another embodiment, the third solution is prepared by dissolving three nucleic acids in an aqueous phase.

[0198] In one embodiment, the third solution is prepared by dissolving a nucleic acid in an aqueous buffer. In another embodiment, the third solution is prepared by dissolving a nucleic acid in an acetate buffer. In yet another embodiment, the third solution is prepared by dissolving a nucleic acid in a citrate buffer. In yet another embodiment, the third solution is prepared by dissolving a nucleic acid in a Tris-sucrose buffer.

[0199] In one embodiment, the third solution is prepared by dissolving the two nucleic acids in an aqueous buffer. In another embodiment, the third solution is prepared by dissolving the two nucleic acids in an acetate buffer. In yet another embodiment, the third solution is prepared by dissolving the two nucleic acids in a citrate buffer. In yet another embodiment, the third solution is prepared by dissolving the two nucleic acids in a Tris-sucrose buffer.

[0200] In one embodiment, where the first solution does not contain nucleic acids, the first and second solutions are combined to form liposomes, micelles, or other self-assembled lipid nanoparticles. In another embodiment, the liposomes, micelles, or other self-assembled lipid nanoparticles are mixed with a third aqueous solution containing one or more nucleic acids, thereby forming monodisperse or polydisperse LPX.

[0201] In one embodiment, the second process involves a first step of preparing a third solution comprising an organic phase and one or more lipids. In another embodiment, the second process involves a first step of preparing a third solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule.

[0202] In this embodiment, the organic phase comprises a water-miscible organic solvent. In this embodiment, the water-miscible organic solvent is ethanol, methanol, isopropanol, DMSO, acetone, acetonitrile, DMF, THF, ethylamine, glycerol, or dioxane. In this embodiment, the water-miscible organic solvent is ethanol.

[0203] In one embodiment, the third solution is prepared by dissolving one or more lipids in an organic phase. In another embodiment, the third solution is prepared by dissolving one or more lipids in a water-miscible organic solvent. In another embodiment, the third solution is prepared by dissolving one lipid in a water-miscible organic solvent. In another embodiment, the third solution is prepared by dissolving two lipids in a water-miscible organic solvent. In another embodiment, the third solution is prepared by dissolving three lipids in a water-miscible organic solvent. In another embodiment, the third solution is prepared by dissolving four lipids in a water-miscible organic solvent. In yet another embodiment, the third solution is prepared by dissolving five lipids in a water-miscible organic solvent.

[0204] In one embodiment, the third solution is prepared by dissolving one or more lipids in ethanol. In another embodiment, the third solution is prepared by dissolving one lipid in ethanol. In another embodiment, the third solution is prepared by dissolving two lipids in ethanol. In another embodiment, the third solution is prepared by dissolving three lipids in ethanol. In another embodiment, the third solution is prepared by dissolving four lipids in ethanol. In yet another embodiment, the third solution is prepared by dissolving five lipids in ethanol.

[0205] In one embodiment, a third solution is prepared by dissolving one or more lipids in an organic phase and a targeting ligand, imaging ligand, digestive agent, or small molecule. In another embodiment, a third solution is prepared by dissolving one or more lipids in a water-miscible organic solvent and a targeting ligand, imaging ligand, digestive agent, or small molecule. In another embodiment, a third solution is prepared by dissolving one lipid in a water-miscible organic solvent and a targeting ligand, imaging ligand, digestive agent, or small molecule. In another embodiment, a third solution is prepared by dissolving two lipids in a water-miscible organic solvent and a targeting ligand, imaging ligand, digestive agent, or small molecule. In another embodiment, a third solution is prepared by dissolving three lipids in a water-miscible organic solvent and a targeting ligand, imaging ligand, digestive agent, or small molecule. In another embodiment, a third solution is prepared by dissolving four lipids in a water-miscible organic solvent and a targeting ligand, imaging ligand, digestive agent, or small molecule. In yet another embodiment, a third solution is prepared by dissolving five lipids in a water-miscible organic solvent and a targeting ligand, imaging ligand, digestive agent, or small molecule.

[0206] In one embodiment, a third solution is prepared by dissolving one or more lipids and a targeting ligand in an organic phase. In another embodiment, a third solution is prepared by dissolving one or more lipids and a targeting ligand in a water-miscible organic solvent. In another embodiment, a third solution is prepared by dissolving one lipid and a targeting ligand in a water-miscible organic solvent. In another embodiment, a third solution is prepared by dissolving two lipids and a targeting ligand in a water-miscible organic solvent. In another embodiment, a third solution is prepared by dissolving three lipids and a targeting ligand in a water-miscible organic solvent. In another embodiment, a third solution is prepared by dissolving four lipids and a targeting ligand in a water-miscible organic solvent. In yet another embodiment, a third solution is prepared by dissolving five lipids and a targeting ligand in a water-miscible organic solvent.

[0207] In one embodiment, a third solution is prepared by dissolving one or more lipids and imaging ligands in an organic phase. In another embodiment, a third solution is prepared by dissolving one or more lipids and imaging ligands in a water-miscible organic solvent. In another embodiment, a third solution is prepared by dissolving one lipid and imaging ligand in a water-miscible organic solvent. In another embodiment, a third solution is prepared by dissolving two lipids and imaging ligands in a water-miscible organic solvent. In another embodiment, a third solution is prepared by dissolving three lipids and imaging ligands in a water-miscible organic solvent. In another embodiment, a third solution is prepared by dissolving four lipids and imaging ligands in a water-miscible organic solvent. In yet another embodiment, a third solution is prepared by dissolving five lipids and imaging ligands in a water-miscible organic solvent.

[0208] In one embodiment, a third solution is prepared by dissolving one or more lipids and a digestive agent in an organic phase. In another embodiment, a third solution is prepared by dissolving one or more lipids and a digestive agent in a water-miscible organic solvent. In another embodiment, a third solution is prepared by dissolving one lipid and a digestive agent in a water-miscible organic solvent. In another embodiment, a third solution is prepared by dissolving two lipids and a digestive agent in a water-miscible organic solvent. In another embodiment, a third solution is prepared by dissolving three lipids and a digestive agent in a water-miscible organic solvent. In another embodiment, a third solution is prepared by dissolving four lipids and a digestive agent in a water-miscible organic solvent. In yet another embodiment, a third solution is prepared by dissolving five lipids and a digestive agent in a water-miscible organic solvent.

[0209] In one embodiment, the third solution is prepared by dissolving one or more lipids and small molecules in an organic phase. In another embodiment, the third solution is prepared by dissolving one or more lipids and small molecules in a water-miscible organic solvent. In another embodiment, the third solution is prepared by dissolving one lipid and small molecule in a water-miscible organic solvent. In another embodiment, the third solution is prepared by dissolving two lipids and small molecules in a water-miscible organic solvent. In another embodiment, the third solution is prepared by dissolving three lipids and small molecules in a water-miscible organic solvent. In another embodiment, the third solution is prepared by dissolving four lipids and small molecules in a water-miscible organic solvent. In yet another embodiment, the third solution is prepared by dissolving five lipids and small molecules in a water-miscible organic solvent.

[0210] In one embodiment, without nucleic acids in the first solution, a fourth solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule is prepared. The fourth solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule is prepared in the same manner as the third solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule.

[0211] In one embodiment, the second process involves a second step in preparing LPS using a third solution and an aqueous phase for preparing liposomes, micelles, or other self-assembled lipid nanoparticles by any method known in the art. In another embodiment, the second process involves a second step in preparing LPS using a third solution and an aqueous phase for preparing liposomes, micelles, or other self-assembled lipid nanoparticles by any method known in the art, wherein optionally a targeting ligand, imaging ligand, digestive agent, or small molecule is added to the aqueous phase.

[0212] In one embodiment, where the first solution does not contain nucleic acids, the second process involves a second step of preparing LPS using a fourth solution and an aqueous phase for preparing liposomes, micelles, or other self-assembled lipid nanoparticles by any method known in the art. In another embodiment, the second process involves a second step of preparing LPS using a fourth solution and an aqueous phase for preparing liposomes, micelles, or other self-assembled lipid nanoparticles by any method known in the art, wherein optionally a targeting ligand, imaging ligand, digestive agent, or small molecule is added to the aqueous phase.

[0213] In one embodiment, the second process involves a second step of preparing LPS using a third solution and mixing it with an aqueous phase by pipetting. In another embodiment, the second process involves a second step of preparing LPS using a third solution and mixing it with an aqueous phase by stirring. In another embodiment, the second process involves a second step of preparing LPS using a third solution and mixing it with an aqueous phase by vortexing. In another embodiment, the second process involves a second step of preparing LPS using a third solution and mixing it with an aqueous phase by ultrasonic treatment. In another embodiment, the second process involves a second step of preparing LPS using a third solution and mixing it with an aqueous phase using a microfluidic system. In another embodiment, the second process involves a second step of preparing LPS using a third solution, drying it, and then rehydrating it with an aqueous phase (thin-film method). In another embodiment, the second process involves a second step of preparing LPS using a third solution and mixing it with an aqueous phase by injecting the third solution (organic phase) into the aqueous phase. In another embodiment, the second process involves a second step of preparing LPS using a third solution and mixing it with an aqueous phase by emulsification, i.e., slowly adding the aqueous phase to the third solution (organic phase).

[0214] In one embodiment, the targeting ligand, imaging ligand, digestive agent, or small molecule is dissolved in the aqueous phase before mixing with the third solution. In another embodiment, the targeting ligand is dissolved in the aqueous phase before mixing with the third solution. In yet another embodiment, the imaging ligand is dissolved in the aqueous phase before mixing with the third solution. In yet another embodiment, the digestive agent is dissolved in the aqueous phase before mixing with the third solution. In yet another embodiment, the small molecule is dissolved in the aqueous phase before mixing with the third solution.

[0215] In one embodiment, the second process involves a second step of preparing LPS using a fourth solution and mixing it with an aqueous phase by pipetting. In another embodiment, the second process involves a second step of preparing LPS using a fourth solution and mixing it with an aqueous phase by stirring. In another embodiment, the second process involves a second step of preparing LPS using a fourth solution and mixing it with an aqueous phase by vortexing. In another embodiment, the second process involves a second step of preparing LPS using a fourth solution and mixing it with an aqueous phase by ultrasonic treatment. In another embodiment, the second process involves a second step of preparing LPS using a fourth solution and mixing it with an aqueous phase using a microfluidic system. In another embodiment, the second process involves a second step of preparing LPS using a fourth solution, drying it, and then rehydrating it with an aqueous phase (thin-film method). In another embodiment, the second process involves a second step of preparing LPS using a fourth solution and mixing it with an aqueous phase by injecting the fourth solution (organic phase) into the aqueous phase. In another embodiment, the second process involves a second step of preparing LPS using a fourth solution and mixing it with an aqueous phase by emulsification, i.e., slowly adding the aqueous phase to the fourth solution (organic phase).

[0216] In one embodiment, the targeting ligand, imaging ligand, digestive agent, or small molecule is dissolved in the aqueous phase before mixing with the fourth solution. In another embodiment, the targeting ligand is dissolved in the aqueous phase before mixing with the fourth solution. In yet another embodiment, the imaging ligand is dissolved in the aqueous phase before mixing with the fourth solution. In yet another embodiment, the digestive agent is dissolved in the aqueous phase before mixing with the fourth solution. In yet another embodiment, the small molecule is dissolved in the aqueous phase before mixing with the fourth solution.

[0217] In one embodiment, LPX is mixed with LPS. In another embodiment, a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is added when LPX and LPS are mixed. In yet another embodiment, a targeting ligand is added when LPX and LPS are mixed. In yet another embodiment, an imaging ligand is added when LPX and LPS are mixed. In yet another embodiment, a digesting agent is added when LPX and LPS are mixed. In yet another embodiment, a small molecule is added when LPX and LPS are mixed.

[0218] In the embodiments, both the first solution and the third solution contain an aqueous phase and one or more nucleic acids.

[0219] Methods for treating or preventing diseases and for in vitro transfection The transfection method of the present invention can be applied to cells in vitro or in vivo. This disclosure provides a method for transfecting cells in vitro. In one aspect, this document provides a method for transfecting cells with one or more nucleic acids, the method comprising: contacting the cells in vitro with a composition comprising at least one nucleic acid contained in an LPS-LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule. In an embodiment, this document provides the method for transfecting cells with one nucleic acid, the method comprising: contacting the cells in vitro with a composition comprising one nucleic acid contained in an LPS-LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule. In an embodiment, this document provides the method for transfecting cells with two nucleic acids, the method comprising: contacting the cells in vitro with a composition comprising two nucleic acids contained in an LPS-LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule. In an embodiment, this document provides a method for transfecting cells with three nucleic acids, the method comprising: contacting the cells in vitro with a composition comprising three nucleic acids contained in an LPS-LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule.

[0220] In this embodiment, the cell is a eukaryotic cell.

[0221] This disclosure provides a method for the prophylactic treatment of a subject who has not yet been infected with a viral infection or may be in the early stages of infection but has not yet shown symptoms of viral infection. The method includes administering to the subject a composition for cell transfection as described herein, included in the embodiments.

[0222] This disclosure provides a method for the preventive treatment of subjects who have not yet been infected with coronavirus or may be in the early stages of infection but have not yet shown symptoms of coronavirus infection.

[0223] In one implementation, prophylactic treatment may be administered to asymptomatic subjects approximately 1–24 hours, approximately 24–48 hours, or approximately 48 hours to 3 days after exposure to, or potential exposure to, or suspected exposure to a viral infection. In another implementation, prophylactic treatment may be administered to asymptomatic subjects approximately 3–5 days, or approximately 5–10 days, or approximately 10–14 days, or approximately 14–21 days, or approximately 21–30 days or longer after exposure to a viral infection.

[0224] In one implementation, prophylactic treatment may be administered to asymptomatic subjects approximately 1–24 hours, approximately 24–48 hours, or approximately 48 hours to 3 days after exposure to, or potential exposure to, or suspected exposure to the coronavirus. In another implementation, prophylactic treatment may be administered to asymptomatic subjects approximately 3–5 days, or approximately 5–10 days, or approximately 10–14 days, or approximately 14–21 days, or approximately 21–30 days or longer after exposure to the coronavirus.

[0225] This disclosure provides a method for treating a subject, the method comprising administering to a subject in need a composition for cell transfection as described herein, included in embodiments.

[0226] This disclosure provides a method for alleviating symptoms of a disease in a subject, the method comprising administering to a subject in need a composition for cell transfection as described herein, included in embodiments. In embodiments, this disclosure provides a method for treating a subject in need of cell therapy.

[0227] In one embodiment, the disease is a viral infection, diabetes, an autoimmune disease, or cancer. In another embodiment, the disease is any disease that can be treated with cell therapy. In one embodiment, the disease is cancer. In another embodiment, the disease is diabetes. In another embodiment, the disease is an autoimmune disease. In another embodiment, the disease is a viral infection. In another embodiment, the viral infection is a coronavirus.

[0228] The methods disclosed herein can be used to generate transfected cells expressing useful gene products. These methods can be used as steps in any therapeutic process requiring the introduction of nucleic acids into cells, including gene therapy and viral suppression methods, as well as for introducing antisense or antigene nucleic acids, ribozymes, or RNA regulatory sequences or associated repressive or regulatory nucleic acids into cells. Specifically, these methods can be used in cancer treatment, treatment of infectious diseases, in vivo and in vitro gene therapy, and diagnostic methods.

[0229] The dosage of a cell transfection composition administered to a patient will depend on many factors, including the method and site of administration, the patient's age, weight, and condition. A person skilled in the art can readily adjust the dosage for a given administration type, a given patient, and a given therapeutic application.

[0230] In one implementation, the subject is a mammal. In another implementation, the subject is a human.

[0231] Application method The compositions described herein can be formulated as solutions or suspensions. In some embodiments, the compositions described herein can be formulated as solutions. In some embodiments, the compositions described herein can be formulated as suspensions.

[0232] In embodiments, the compositions described herein and formulated as solutions or suspensions may be administered to a subject by injection. In embodiments, the compositions described herein and formulated as solutions or suspensions may be administered to a subject by intramuscular, subcutaneous, or intradermal injection.

[0233] In one embodiment, the composition described herein and formulated as a suspension may be administered to the subject via intramuscular injection. In another embodiment, the composition described herein and formulated as a suspension may be administered to the subject via subcutaneous injection. In yet another embodiment, the composition described herein and formulated as a suspension may be administered to the subject via intradermal injection.

[0234] The compositions described herein can be administered in single or multiple doses. In one embodiment, the composition is administered in a single dose. In another embodiment, the composition is administered in two doses. In yet another embodiment, the composition is administered in three doses. In a third embodiment, the composition is administered in four doses. In yet another embodiment, the composition is administered in five doses.

[0235] The compositions described herein can be used to treat viral infections, diabetes, autoimmune diseases, or cancer. The compositions described herein can also be used to prevent viral infections.

[0236] In one embodiment, when treating a subject with a viral infection, the injection may be delivered intramuscularly, subcutaneously, or intradermally. In another embodiment, when treating a subject with a viral infection, the injection may be delivered intramuscularly. In yet another embodiment, when treating a subject with a viral infection, the injection may be delivered subcutaneously. In yet another embodiment, when treating a subject with a viral infection, the injection may be delivered intradermally.

[0237] In one embodiment, when a subject is vaccinated to prevent viral infection, the injection may be delivered intramuscularly, subcutaneously, or intradermally. In another embodiment, when a subject is vaccinated to prevent viral infection, the injection may be delivered intramuscularly. In yet another embodiment, when a subject is vaccinated to prevent viral infection, the injection may be delivered subcutaneously. In yet another embodiment, when a subject is vaccinated to prevent viral infection, the injection may be delivered intradermally.

[0238] In one implementation, in the case of treating a subject's cancer, the injection can be delivered intratumorally.

[0239] In one embodiment, the vaccine or treatment is delivered directly to the lymphatic system. In another embodiment, the vaccine or treatment is administered subcutaneously to the lymphatic system. In yet another embodiment, the vaccine or treatment is administered intramuscularly to the lymphatic system. In a third embodiment, the vaccine or treatment is administered intranodally to the lymphatic system. In a fourth embodiment, the vaccine or treatment is administered percutaneously to the lymphatic system.

[0240] In one embodiment, the vaccine or treatment is administered to the lymphatic system via a patch. In another embodiment, the patch comprises a polymer. In yet another embodiment, the polymer is an absorbable polymer.

[0241] In one implementation, a vaccine or treatment is administered to the lymphatic system via a medical device that includes a microneedle array.

[0242] Medical devices including the microneedle arrays applicable to this article are known in the art. Specific exemplary structures and devices, including means for the controllable delivery of vaccines or treatments to subjects, are described in International Patent Application Publications Nos. WO2014 / 188343, WO 2014 / 132239, WO 2014 / 132240, WO 2013 / 061208, WO2012 / 046149, WO 2011 / 135531, WO 2011 / 135530, WO 2011 / 135533, WO2014 / 132240, and WO 2015 / 16821, and International Patent Applications PCT / US2015 / 028154 (published as WO 2015 / 168214 A1) and PCT / US2015 / 028150 (published as WO 2015 / 168214 A1). 2015 / 168210 A1), PCT / US2015 / 028158 (published as WO 2015 / 168215 A1), PCT / US2015 / 028162 (published as WO 2015 / 168217 A1), PCT / US2015 / 028164 (published as WO 2015 / 168219 A1), PCT / US2015 / 038231 (published as WO 2016 / 003856 A1), PCT / US2015 / 038232 (published as WO 2016 / 003857 A1), PCT / US2016 / 043623 (published as WO 2017 / 019526 A1), PCT / US2016 / 043656 (published as WO 2017 / 019535 A1), PCT / US2017 / 027879 (published as WO 2017 / 189258 A1), PCT / US2017 / 027891 (published as WO 2017 / 189259 A1), PCT / US2017 / 064604 (published as WO 2018 / 111607 A1), PCT / US2017 / 064609 (published as WO 2018 / 111609 A1), PCT / US2017 / 064614 (published as WO 2018 / 111611 A1), PCT / US2017 / 064642 (published as WO 2018 / 111616 A1), PCT / US2017 / 064657 (published as WO The entire international patent applications, including PCT / US2017 / 064668 (published as WO 2018 / 111621 A1), are incorporated herein by reference in their entirety.

[0243] In one embodiment, a vaccine or treatment is administered by applying one or more medical devices to one or more sites on the skin of a subject. In another embodiment, the vaccine or treatment is delivered directly to the lymphatic system. A non-limiting example of a medical device comprising multiple microneedles suitable for use with all the methods disclosed herein is the Sofusa™ drug delivery platform, available from Sorrento Therapeutics, Inc. See, for example, U.S. Patent No. 10,737,082; International Patent Application PCT / US2019 / 034736 (published as WO 2019 / 232265), which are incorporated herein by reference in their entirety.

[0244] In one embodiment, the medical device is positioned in direct contact with the subject's skin. In another embodiment, an intermediate layer or structure exists between the subject's skin and the medical device. For example, surgical tape or gauze may be used to reduce potential skin irritation between the medical device and the subject's skin. As the microneedles extend from the device, they will contact and, in some cases, penetrate the subject's epidermis or dermis to deliver a vaccine or treatment to the subject. Delivery of the vaccine or treatment may be to the circulatory system, lymphatic system, interstitium, subcutaneous, intramuscular, intradermal, or a combination thereof. In one embodiment, the vaccine or treatment is delivered directly to the subject's lymphatic system. In another embodiment, the vaccine or treatment is delivered to superficial blood vessels of the lymphatic system.

[0245] In some embodiments, the target of vaccination or treatment is a lymph node, lymphatic vessel, organ that is part of the lymphatic system, or a combination thereof. In some embodiments, the target of vaccination or treatment is a lymph node. In some embodiments, the target of vaccination or treatment is a specific lymph node as described elsewhere herein.

[0246] In one embodiment, the medical device may include an array of needles in the form of a patch. In another embodiment, the needle array is capable of penetrating the outermost layer of the stratum corneum and delivering a vaccine or treatment, as described herein, to at least a portion or all of the inactive epidermis, at least a portion or all of the active epidermis, and / or at least a portion of the active dermis of a subject, and subsequently to the subject's lymphatic system. These needles may further include nanomorphic patterns on the surface of the needles in a random or organized pattern. In another embodiment, the nanomorphic pattern may exhibit fractal geometry.

[0247] Abbreviations Example Example 1. In HEK293 cells, mRNA was transfected using the LPS-LPX delivery system compared to Lipofectamine™ MessengerMAX™.

[0248] Of all formulations containing mRNA, mRNA is primarily located within the lipid complex (LPX).

[0249] The LCF96 formulation was prepared as follows: 1) Nuclear (lipid complex, LPX)-green fluorescent protein (GFP) mRNA (20 µL, 1 mg / mL in water) and sodium acetate buffer (80 µL, 25 mM, pH 5) were added to a 1 mL Eppendorf tube to prepare a 100 µL mRNA solution. KT-001 (14.6 µL, 20 mg / mL in EtOH), DSPC (3.01 µL, 20 mg / mL in EtOH), and cholesterol (5.68 µL, 20 mg / mL in EtOH) (lipid:KT-001 / DSPC / cholesterol molar ratio = 50 / 10 / 38.5) were added to a 2 mL serum vial to prepare a 23.28 µL lipid solution. The lipid solution was then rapidly pipetted into the mRNA solution (with several up-and-down pipetting to mix), followed by vortexing at the highest speed setting for 5–10 seconds.

[0250] Sodium acetate buffer 25 mM, pH 5, is prepared as follows: 1 M commercially available pH 5.0 acetate buffer (Thermo Scientific, catalog number J60964-AK) is diluted 40 times with water for injection.

[0251] 2) Add lipid shell (LPS)-KT-001 (729.8 µL, 20 mg / mL in EtOH), DSPC (150.5 µL, 20 mg / mL in EtOH), cholesterol (283.8 µL, 20 mg / mL in EtOH), and DMG-PEG2000 (74.3 µL, 20 mg / mL in EtOH) to a 5 mL Eppendorf tube to prepare a 1238.4 µL lipid solution (molar ratio of lipid:KT-001 / DSPC / cholesterol / DMG-PEG2000 = 50 / 10 / 38.5 / 1.5). Aspirate the lipid solution into a 3 mL BD syringe. Remove air bubbles by gently tapping the syringe. Then, load the syringe onto the Ignite NanoAssemblr cartridge. Aspirate 3715.11 µL of Tris-sucrose buffer into a 10 mL BD syringe. Remove air bubbles by gently tapping the syringe. Next, the syringe was loaded onto the Ignite NanoAssemblr cartridge. The flow rate was set to 12 mL / min, with a ratio of Tris-sucrose buffer / liposome solution = 3 / 1, to generate liposomes. The liposomes were then collected into a dialysis bag (100 KD) and dialyzed with Tris-sucrose buffer. The buffer was changed 3 times every 2 hours for a total of 6 hours.

[0252] Tris-sucrose buffer, pH approximately 7.4, was prepared as follows: Tris base (9.688 g, 0.08 mol), sucrose (320.00 g), and water for injection (WFI 3.8 L) were added to a 6 L beaker. The mixture was stirred at room temperature for 2 hours or until all materials were dissolved. The pH of the solution was adjusted to 7.3 to 7.4 with HCl (1 N). The total volume of the solution was brought to 4 L, and the solution was aseptically filtered using a 0.22 µM filter.

[0253] 3) mRNA-LPS-LPX was prepared as follows: 100 µL of lipid shell (LPS) was pipetted into 123.28 µL of lipid complex (LPX), and then pipetted up and down several times to mix.

[0254] Transfection in HEK293 cells: Day 1 - Gently pipette cells to mix thoroughly. Using an automated cell counter, perform trypan blue exclusion cell counting with 10 μL aliquots of cell suspension to determine cell concentration and viability. Calculate the total cell count. Transfer cells to 50 ml centrifuge tubes, centrifuge at 1200 rpm or 250 g for 5 minutes, discard the old medium, and resuspend the cells in fresh HEK293 complete medium (DMEM containing 10% FBS) to adjust the cell concentration to 0.4 × 10⁻⁶ cells / mL. 6 Cells / ml. Then, load 0.5 ml of cell suspension (0.2 × 10⁻⁶ cells / ml) into each well of a 24-well plate. 6 The plate was incubated overnight at 37°C and 5% CO2. On day 2, the medium was replaced with fresh complete cell culture medium before transfection. 0.5 μg or 1 μg mRNA-LPS-LPX (prepared as above) was added to each well, and the plate was incubated at 37°C and 5% CO2 for 24 hours. 0.5 μg or 1 μg / well of mRNA + Lipofectamine™ MessengerMAX™ (from Thermo Fisher Scientific) was used as a positive control. HEK293 cells were used as a negative control.

[0255] After 24 hours, transfection efficiency was determined by flow cytometry, and transfected cell viability was determined by trypan blue exclusion cell counting using an automated cell counter.

[0256] Figure 1 The scatter plot illustrates the transfection efficiency of LCF96 and Lipofectamine™ MessengerMAX™ (0.5 μg and 1.0 μg GFP mRNA) in HEK293 cells. In easily transfected cells, such as HEK293, the LPS-LPX delivery system improved transfection efficiency from 90.4% to 99.4% (for 0.5 μg) and from 96.6% to 99.3% (for 1.0 μg) compared to Lipofectamine™ MessengerMAX™. Cell viability also improved from 82% to 94% compared to Lipofectamine™ MessengerMAX™, as shown in Table 4.

[0257] Table 4: Example 2. In Jurkat cells, mRNA was transfected using the LPS-LPX delivery system compared to Lipofectamine™ MessengerMAX™.

[0258] The formulation LCF96 was prepared as described in Example 1.

[0259] The formulation LCF107 was prepared as follows: 1) Nucleolipin complex (LPX)-luciferase (Luc) mRNA (20 µL, 1 mg / mL in water) and sodium acetate buffer (80 µL, 25 mM, pH 5) were added to a 1 mL Eppendorf tube to prepare a 100 µL mRNA solution. KT-001 (13.14 µL, 20 mg / mL in EtOH), DOTMA (1.28 µL, 20 mg / mL in EtOH), DSPC (3.01 µL, 20 mg / mL in EtOH) and cholesterol (5.68 µL, 20 mg / mL in EtOH) (molar ratio of lipids: KT-001 / DOTMA / DSPC / cholesterol = 45 / 5 / 10 / 38.5) were added to a 2 mL serum vial to prepare a 23.1 µL lipid solution. Then, the lipid solution was rapidly pipetted into the mRNA solution (and pipetted up and down several times to mix), followed by vortexing at the highest speed setting for 5-10 seconds.

[0260] 2) Add lipid shell (LPS)-KT-001 (729.8 µL, 20 mg / mL in EtOH), DSPC (150.5 µL, 20 mg / mL in EtOH), cholesterol (283.8 µL, 20 mg / mL in EtOH), and DMG-PEG2000 (74.3 µL, 20 mg / mL in EtOH) to a 5 mL Eppendorf tube to prepare a 1238.4 µL lipid solution (molar ratio of lipid:KT-001 / DSPC / cholesterol / DMG-PEG2000 = 50 / 10 / 38.5 / 1.5). Aspirate the lipid solution into a 3 mL BD syringe. Remove air bubbles by gently tapping the syringe. Then, load the syringe onto the Ignite NanoAssemblr cartridge. Aspirate 3715.11 µL of Tris-sucrose buffer into a 10 mL BD syringe. Remove air bubbles by gently tapping the syringe. Next, the syringe was loaded onto the Ignite NanoAssemblr cartridge. The flow rate was set to 12 mL / min, with a ratio of Tris-sucrose buffer / liposome solution = 3 / 1, to generate liposomes. The liposomes were then collected into a dialysis bag (100 KD) and dialyzed with Tris-sucrose buffer. The buffer was changed 3 times every 2 hours for a total of 6 hours.

[0261] 3) mRNA-LPS-LPX was prepared as follows: 100 µL of lipid shell (LPS) was pipetted into 123.1 µL of lipid complex (LPX), and then pipetted up and down several times to mix.

[0262] The LCF108 formulation was prepared as follows: 1) Nuclear (lipid complex, LPX)-green fluorescent protein (GFP) mRNA (20 µL, 1 mg / mL in water) and sodium acetate buffer (80 µL, 25 mM, pH 5) were added to a 1 mL Eppendorf tube to prepare a 100 µL mRNA solution. KT-001 (14.6 µL, 20 mg / mL in EtOH), DSPC (3.01 µL, 20 mg / mL in EtOH), and cholesterol (5.68 µL, 20 mg / mL in EtOH) (lipid:KT-001 / DSPC / cholesterol molar ratio = 50 / 10 / 38.5) were added to a 2 mL serum vial to prepare a 23.28 µL lipid solution. The lipid solution was then rapidly pipetted into the mRNA solution (and pipetted up and down several times to mix), followed by vortexing at the highest speed setting for 5–10 seconds.

[0263] 2) Lipid shell (LPS)-KT-001 (656.8 µL, 20 mg / mL in EtOH), DOTMA (63.9 µL, 20 mg / mL in EtOH), DSPC (150.5 µL, 20 mg / mL in EtOH), cholesterol (283.8 µL, 20 mg / mL in EtOH), and DMG-PEG2000 (74.3 µL, 20 mg / mL in EtOH) were added to a 5 mL Eppendorf tube to prepare a 1229.3 µL lipid solution (molar ratio of lipid:KT-001 / DOTMA / DSPC / cholesterol / DMG-PEG2000 = 45 / 5 / 10 / 38.5 / 1.5). The lipid solution was aspirated into a 3 mL BD syringe. Air bubbles were removed by gently tapping the syringe. The syringe was then loaded onto the Ignite NanoAssemblr cartridge. Draw 3687.9 µL of Tris-sucrose buffer into a 10 mL BD syringe. Remove air bubbles by gently tapping the syringe. Then, load the syringe onto the Ignite NanoAssemblr cartridge. Set the flow rate to 12 mL / min, with a ratio of Tris-sucrose buffer / liposome solution = 3 / 1, to generate liposomes. The liposomes are then collected in a dialysis bag (100 KD) and dialyzed with Tris-sucrose buffer. Change the buffer 3 times every 2 hours for a total of 6 hours.

[0264] 3) mRNA-LPS-LPX was prepared as follows: 100 µL of lipid shell (LPS) was pipetted into 123.28 µL of lipid complex (LPX), and then pipetted up and down several times to mix.

[0265] The formulation LCF109 was prepared as follows: 1) Nucleolipin complex (LPX)-luciferase (Luc) mRNA (20 µL, 1 mg / mL in water) and sodium acetate buffer (80 µL, 25 mM, pH 5) were added to a 1 mL Eppendorf tube to prepare a 100 µL mRNA solution. KT-001 (13.14 µL, 20 mg / mL in EtOH), DOTMA (1.28 µL, 20 mg / mL in EtOH), DSPC (3.01 µL, 20 mg / mL in EtOH) and cholesterol (5.68 µL, 20 mg / mL in EtOH) (molar ratio of lipids: KT-001 / DOTMA / DSPC / cholesterol = 45 / 5 / 10 / 38.5) were added to a 2 mL serum vial to prepare a 23.1 µL lipid solution. Then, the lipid solution was rapidly pipetted into the mRNA solution (and pipetted up and down several times to mix), followed by vortexing at the highest speed setting for 5-10 seconds.

[0266] 2) Lipid shell (LPS)-KT-001 (656.8 µL, 20 mg / mL in EtOH), DOTMA (63.9 µL, 20 mg / mL in EtOH), DSPC (150.5 µL, 20 mg / mL in EtOH), cholesterol (283.8 µL, 20 mg / mL in EtOH), and DMG-PEG2000 (74.3 µL, 20 mg / mL in EtOH) were added to a 5 mL Eppendorf tube to prepare a 1229.3 µL lipid solution (molar ratio of lipid:KT-001 / DOTMA:DSPC / cholesterol / DMG-PEG2000 = 45 / 5 / 10 / 38.5 / 1.5). The lipid solution was aspirated into a 3 mL BD syringe. Air bubbles were removed by gently tapping the syringe. The syringe was then loaded onto an Ignite NanoAssemblr cartridge. Draw 3687.9 µL of Tris-sucrose buffer into a 10 mL BD syringe. Remove air bubbles by gently tapping the syringe. Then, load the syringe onto the Ignite NanoAssemblr cartridge. Set the flow rate to 12 mL / min, with a ratio of Tris-sucrose buffer / liposome solution = 3 / 1, to generate liposomes. The liposomes are then collected in a dialysis bag (100 KD) and dialyzed with Tris-sucrose buffer. Change the buffer 3 times every 2 hours for a total of 6 hours.

[0267] 3) mRNA-LPS-LPX was prepared as follows: 100 µL of lipid shell (LPS) was pipetted into 123.1 µL of lipid complex (LPX), and then pipetted up and down several times to mix.

[0268] Transfection in Jurkat cells: Gently pipette cells to mix thoroughly. Using an automated cell counter, count cells by trypan blue exclusion with 10 μL aliquots of the cell suspension to determine cell concentration and viability. Calculate the total cell count. Transfer cells to 50 ml centrifuge tubes, centrifuge at 1200 rpm or 250 g for 5 minutes, discard the old medium, and resuspend the cells in fresh RPMI serum-reduced medium (SRM) to adjust the cell concentration to 1.0 × 10⁻⁶ cells / mL. 6 Cells / ml. Then, load 0.5 ml of cell suspension (0.5 × 10⁶ cells / ml) into each well of a 24-well plate. 6 Add 1.0 μg or 3.0 μg mRNA-LPS-LPX (prepared in Examples 1 and 2) to each well and incubate the plate at 37°C and 5% CO2 for 4 hours. 1 μg / well or 3 μg / well of mRNA + Lipofectamine™ MessengerMAX™ was used as a positive control. Jurkat cells were used as a negative control.

[0269] Four hours later, the cells were centrifuged at 250 g for 5 minutes and the old medium was discarded. 500 μL of Jurkat complete medium (RPMI 1640 with 10% FBS) was added to each well and the plate was incubated at 37°C and 5% CO2 for 24 hours.

[0270] After 24 hours, transfection efficiency was determined by flow cytometry, and transfected cell viability was determined by trypan blue exclusion cell counting using an automated cell counter.

[0271] Figure 2 The scatter plot illustrates the transfection efficiency of LCF96, LCF107, LCF108, LCF109, and Lipofectamine™ MessengerMAX™ (1.0 μg and 3.0 μg) in Jurkat cells. In difficult-to-transfect cells, such as Jurkat, the LPS-LPX delivery system (LCF107, LCF108, and LCF109) significantly improved transfection efficiency from 14.1% to 99.8% (for 1.0 μg) and from 23.4% to 99.9% (for 3.0 μg) compared to Lipofectamine™ MessengerMAX™. Cell viability was slightly lower compared to Lipofectamine™ MessengerMAX™, as shown in Table 5.

[0272] Table 5: Example 3. In human primary T cells, mRNA was transfected using the LPS-LPX delivery system compared to Lipofectamine™ MessengerMAX™.

[0273] Compound LCF96 was prepared as described in Example 1. Compound LCF108 was prepared as described in Example 2.

[0274] Transfection in primary human T cells: Day 1 – Rapidly thaw frozen vials of 3-day activated T cells in a bead bath. Wipe the exterior of the vials with 70% ethanol. Transfer the cell suspension to a 50 mL tube containing 10 mL of complete T cell culture medium. Centrifuge the cells at 200 g for 10 min, then resuspend the cell pellet in 10 mL of complete T cell culture medium. Using an automated cell counter, perform trypan blue exclusion cell counting with 10 μL aliquots of the cell suspension to determine cell concentration and viability. Adjust the T cell concentration to 1 × 10⁻⁶ cells / mL in a T75 flask with complete T cell culture medium. 6 Achieve a cell concentration of [number] cells / ml. Incubate the flasks overnight at 37°C and 5% CO2. Day 2 – Replace the medium with fresh medium before transfection. Use an automated cell counter for trypan blue exclusion cell counting to obtain cell concentration and viability. Adjust the cell concentration to 1 × 10⁻⁶ cells / ml with complete T-cell medium. 6 Cells / ml. 1 ml of cells was dispensed at 1 × 10⁻⁶. 6 Cells / ml were added to 24-well plates. 1.0 μg or 3.0 μg mRNA-LPS-LPX (prepared in Examples 1 and 2) was added to each well, and the plates were incubated at 37°C and 5% CO2 for 24 hours. 1.0 μg / well or 3.0 μg / well of mRNA + Lipofectamine™ MessengerMAX™ (from Thermo Fisher Scientific) was used as a positive control. Human primary T cells were used as a negative control.

[0275] After 24 hours, transfection efficiency was determined by flow cytometry, and transfected cell viability was determined by trypan blue exclusion cell counting using an automated cell counter.

[0276] Figure 3The scatter plot illustrates the transfection efficiencies of LCF96, LCF108, and Lipofectamine™ MessengerMAX™ (1.0 μg and 3.0 μg) in human primary T cells. In difficult-to-transfect cells, such as human primary T cells, the LPS-LPX delivery system (LCF108) significantly improved transfection efficiency from 3.6% to 93.1% (for 1.0 μg) and from 9.1% to 99.5% (for 3.0 μg) compared to Lipofectamine™ MessengerMAX™. Cell viability was slightly lower compared to Lipofectamine™ MessengerMAX™, as shown in Table 6.

[0277] Table 6: Example 4. In vivo, mRNA was transfected using the LPS-LPX delivery system compared to Lipofectamine™ MessengerMAX™.

[0278] Formula rsF591 was prepared as described above for formula LCF96 in Example 1, with the only difference being that the mRNA used in formula rsF591 is luciferase mRNA instead of green fluorescent protein (GFP) mRNA used in formula LCF96. Formula rsF616 was prepared as described above for formula LCF108 in Example 2, with the only difference being that the mRNA used in formula rsF616 is luciferase mRNA instead of green fluorescent protein (GFP) mRNA used in formula LCF108.

[0279] Formula rsF592 was used as a control. Formula rsF592 has the same LPX (core) as formulas rsF591 and rsF616 (the core was prepared in the same manner), but it does not have a lipid shell (LPS).

[0280] In vivo transfection: The formulations rsF591, rsF592, or rsF616 were reconstituted in sterile PBS, and each formulation was administered intravenously to four healthy mice at a dose of 1 µg / mouse. Three hours post-injection, D-fluorescein was administered intraperitoneally to the mice at a dose of 150 mg / kg body weight. Mice were anesthetized and placed ventrally on the imaging stage of an IVIS imaging system. Images were acquired 10 minutes post-D-fluorescein injection using an IVIS imaging system (Xenogen, Alameda, CA). Whole-body photon emission was quantified using Living Image software (Xenogen).

[0281] Mice administered rsF591 or rsF616 exhibited high levels of mRNA expression (i.e., luciferase activity), including 1 × 10⁻⁶ mRNAs spanning most or all of the trunk width. 7 p / sec / cm 2 / sr or a larger luminescent region. Mice administered rsF592 showed significantly lower mRNA expression (i.e., luciferase activity) and no luminescence exceeding 0.3 × 10⁻⁶. 7 p / sec / cm 2 / sr indicates that LPX alone is insufficient for transfection (shown in...). Figure 4 middle).

[0282] Example 5. In HEK293 cells, pDNA was transfected using the LPS-LPX delivery system compared to Lipofectamine™ LTX or Lipofectamine™ 2000.

[0283] In formulations containing pDNA, the pDNA is primarily located on the exterior of the lipid complex (LPX).

[0284] The formulation LCF155 was prepared as follows: 1) Lipid shell-DC-cholesterol (500 µL, 20 mg / mL in EtOH) and DOPE (346 µL, 20 mg / mL in EtOH) were added to a 5 mL Eppendorf tube to prepare an 846 µL lipid solution (lipid:DC-cholesterol / DOPE molar ratio = 2 / 1). The lipid solution was aspirated into a 3 mL BD syringe. Air bubbles were removed by gently tapping the syringe. The syringe was then loaded onto an Ignite NanoAssemblr cartridge. 4230 µL of Tris-sucrose buffer was aspirated into a 10 mL BD syringe. Air bubbles were removed by gently tapping the syringe. The syringe was then loaded onto an Ignite NanoAssemblr cartridge. The flow rate was set to 12 mL / min, with a ratio of Tris-sucrose buffer / lipid solution = 5 / 1, to generate liposomes. The liposomes were then collected into a dialysis bag (100 KD) and dialyzed with Tris-sucrose buffer. The buffer solution was changed 3 times every 2 hours, for a total of 6 hours.

[0285] 2) Rapidly pipette the core (lipid complex, LPX)-green fluorescent protein (GFP) pDNA (5 µL, 4 mg / mL in water) into a DC-cholesterol / DOPE lipid shell (8.47 µL) to obtain 13.47 µL of lipid complex (pDNA / DC-cholesterol = 1 / 1 by weight). Bring this solution to a final volume of 100 µL with Tris-sucrose buffer at pH 7.4.

[0286] 3) Add lipid shell (LPS)-KT-001 (656.8 µL, 20 mg / mL in EtOH), DOTMA (63.9 µL, 20 mg / mL in EtOH), DSPC (150.5 µL, 20 mg / mL in EtOH), cholesterol (283.8 µL, 20 mg / mL in EtOH), and DMG-PEG2000 (74.3 µL, 20 mg / mL in EtOH) to a 5 mL Eppendorf tube to prepare a 1229.3 µL lipid solution (molar ratio of lipid:KT-001 / DOTMA / DSPC / cholesterol / DMG-PEG2000 = 45 / 5 / 10 / 38.5 / 1.5). Aspirate the lipid solution into a 3 mL BD syringe. Remove air bubbles by gently tapping the syringe. Then, load the syringe onto the Ignite NanoAssemblr cartridge. Draw 3687.9 µL of Tris-sucrose buffer into a 10 mL BD syringe. Remove air bubbles by gently tapping the syringe. Then, load the syringe onto the Ignite NanoAssemblr cartridge. Set the flow rate to 12 mL / min, with a ratio of Tris-sucrose buffer / liposome solution = 3 / 1, to generate liposomes. The liposomes are then collected in a dialysis bag (100 KD) and dialyzed with Tris-sucrose buffer. Change the buffer 3 times every 2 hours for a total of 6 hours.

[0287] 4) pDNA-LPS-LPX is prepared as follows: 100 µL of lipid shell (LPS) is pipetted into 100 µL of lipid complex (LPX), and then pipetted up and down several times to mix.

[0288] The formulation LCF157 was prepared as follows: 1) Lipid shell-DC-cholesterol (500 µL, 20 mg / mL in EtOH) and DOPE (346 µL, 20 mg / mL in EtOH) were added to a 5 mL Eppendorf tube to prepare an 846 µL lipid solution (lipid:DC-cholesterol / DOPE molar ratio = 2 / 1). The lipid solution was aspirated into a 3 mL BD syringe. Air bubbles were removed by gently tapping the syringe. The syringe was then loaded onto an Ignite NanoAssemblr cartridge. 4230 µL of Tris-sucrose buffer was aspirated into a 10 mL BD syringe. Air bubbles were removed by gently tapping the syringe. The syringe was then loaded onto an Ignite NanoAssemblr cartridge. The flow rate was set to 12 mL / min, with a ratio of Tris-sucrose buffer / lipid solution = 5 / 1, to generate liposomes. The liposomes were then collected into a dialysis bag (100 KD) and dialyzed with Tris-sucrose buffer. The buffer solution was changed 3 times every 2 hours, for a total of 6 hours.

[0289] 2) Rapidly pipette the core (lipid complex, LPX)-green fluorescent protein (GFP) pDNA (5 µL, 4 mg / mL in water) into a DC-cholesterol / DOPE lipid shell (8.47 µL) to obtain 13.47 µL of lipid complex (pDNA / DC-cholesterol = 1 / 1 by weight). Bring this solution to a final volume of 100 µL with Tris-sucrose buffer at pH 7.4.

[0290] 3) Prepare the lipid shell (LPS) as described above for LCF155.

[0291] 4) pDNA-LPS-LPX is prepared as follows: 100 µL of lipid shell (LPS) is pipetted into 100 µL of lipid complex (LPX), and then pipetted up and down several times to mix.

[0292] Transfection in HEK293 cells: Day 1 - Gently pipette cells to mix thoroughly. Use an automated cell counter to perform trypan blue exclusion cell counting with 10 μL aliquots of cell suspension to determine cell concentration and viability. Calculate the total cell count. Transfer cells to 50 ml centrifuge tubes, centrifuge at 1200 rpm or 250 g for 5 minutes, discard the old medium, and resuspend the cells in fresh HEK293 complete medium (DMEM containing 10% FBS) to adjust the cell concentration to 0.4 × 10⁻⁶ cells / mL. 6 Cells / ml. Then, load 0.5 ml of cell suspension (0.2 × 10⁶ cells / ml) into each well of a 24-well plate. 6On day 2, replace the medium with fresh complete cell culture medium before transfection. Add 1 μg pDNA-LPS-LPX (prepared as above) to each well and incubate the plate at 37°C and 5% CO2 for 24 hours. 1 μg / well pDNA + Lipofectamine™ LTX or 1 μg / well pDNA + Lipofectamine™ 2000 (from Thermo Fisher Scientific) is used as a positive control. HEK293 cells are used as a negative control.

[0293] After 24 hours, transfection efficiency was determined by flow cytometry, and transfected cell viability was determined by trypan blue exclusion cell counting using an automated cell counter.

[0294] Figure 5 The scatter plot illustrates the transfection efficiency of LCF155, LCF157, Lipofectamine™ 2000, and Lipofectamine™ LTX (1.0 μg pDNA) in HEK293 cells. In easily transfected cells, such as HEK293, the LPS-LPX delivery system significantly increased transfection efficiency from approximately 67% to approximately 89% compared to Lipofectamine™ 2000 or Lipofectamine™ LTX. Cell viability was comparable to that of the control Lipofectamine™ 2000 or Lipofectamine™ LTX, as shown in Table 7.

[0295] Table 7: Example 6. In Jurkat cells, pDNA was transfected using the LPS-LPX delivery system compared to Lipofectamine™ 2000.

[0296] Formulas LCF155 and LCF157 were prepared as described in Example 5.

[0297] Transfection in Jurkat cells: Gently pipette cells to mix thoroughly. Using an automated cell counter, count cells by trypan blue exclusion with 10 μL aliquots of the cell suspension to determine cell concentration and viability. Calculate the total cell count. Transfer cells to 50 ml centrifuge tubes, centrifuge at 1200 rpm or 250 g for 5 minutes, discard the old medium, and resuspend the cells in fresh RPMI serum-reduced medium (SRM) to adjust the cell concentration to 1.0 × 10⁻⁶ cells / mL. 6 Cells / ml. Then, load 0.5 ml of cell suspension (0.5 × 10⁶ cells / ml) into each well of a 24-well plate. 61.0 μg pDNA-LPS-LPX (prepared in Example 5) was added to each well, and the plate was incubated at 37°C and 5% CO2 for 4 hours. 1 μg / well of mRNA + Lipofectamine™ 2000 was used as a positive control. Jurkat cells were used as a negative control.

[0298] Four hours later, the cells were centrifuged at 250 g for 5 minutes and the old medium was discarded. 500 μL of Jurkat complete medium (RPMI 1640 with 10% FBS) was added to each well and the plate was incubated at 37°C and 5% CO2 for 24 hours.

[0299] After 24 hours, transfection efficiency was determined by flow cytometry, and transfected cell viability was determined by trypan blue exclusion cell counting using an automated cell counter.

[0300] Figure 6 The scatter plot illustrates the transfection efficiency of LCF155, LCF157, and Lipofectamine™ 2000 (1.0 μg pDNA) in Jurkat cells. In difficult-to-transfect cells, such as Jurkat, the LPS-LPX delivery system (LCF155 and LCF157) significantly improved transfection efficiency from approximately 2.5% to approximately 36% (for 1.0 μg pDNA) compared to Lipofectamine™ 2000. Cell viability was comparable to that of the control Lipofectamine 2000, as shown in Table 8. Example 7. Transfection of pDNA in vivo using the LPS-LPX delivery system.

[0301] Formula rsF615 was prepared as described above for formula LCF96 in Example 1, with the only difference being that luciferase pDNA (20 µL, 1 mg / mL in water) was used in formula rsF615 instead of green fluorescent protein (GFP) mRNA (pDNA is mainly present in LPX) used in formula LCF96.

[0302] In vivo transfection: The formulation rsF615 was reconstituted in sterile PBS and administered intravenously to two healthy mice at a dose of 8 µg / mouse. Three and six hours post-injection, D-fluorescein was administered intraperitoneally at a dose of 150 mg / kg body weight. Mice were anesthetized and placed ventrally on the imaging stage of an IVIS imaging system. Images were acquired 10 minutes post-D-fluorescein injection using an IVIS imaging system (Xenogen, Alameda, CA). Whole-body photon emission was quantified using Living Image software (Xenogen).

[0303] Following administration of rsF615, bioluminescent images of mice showed high levels of pDNA expression (i.e., Luc expression) (shown in...). Figure 7 (in the middle), where the luminous area spanning most or all of the width of the torso is 2 × 10 6 p / sec / cm 2 / sr or larger.

[0304] Example 8. Manipulating organ specificity of LPS-LPX delivery system by controlling the lipid composition of LPS.

[0305] In this experiment, the composition of LPX remained constant, while the composition of LPS was altered.

[0306] For all four formulations (rsF598, rsF599, rsF600, and rsF601), the nucleus (LPX) was prepared in the same manner. 1) Nucleus (lipid complex, LPX)-luciferase mRNA (20 µL, 1 mg / mL in water) and sodium acetate buffer (80 µL, 25 mM, pH 5) were added to a 1 mL Eppendorf tube to prepare a 100 µL mRNA solution. KT-001 (14.6 µL, 20 mg / mL in EtOH), DSPC (3.01 µL, 20 mg / mL in EtOH), and cholesterol (5.68 µL, 20 mg / mL in EtOH) (lipid:KT-001 / DSPC / cholesterol molar ratio = 50 / 10 / 38.5) were added to a 2 mL serum vial to prepare a 23.28 µL lipid solution. Then, the lipid solution was rapidly pipetted into the mRNA solution (and pipetted up and down several times to mix), followed by vortexing at the highest speed setting for 5-10 seconds.

[0307] rsF598 2) Lipid shell (LPS)-DC-cholesterol (500 µL, 20 mg / mL in EtOH) and DOPE (346 µL, 20 mg / mL in EtOH) were added to a 1 mL Eppendorf tube to prepare an 846 µL lipid solution (lipid:DC-cholesterol / DOPE molar ratio = 2 / 1). The lipid solution was aspirated into a 1 mL BD syringe. Air bubbles were removed by gently tapping the syringe. The syringe was then loaded onto an Ignite NanoAssemblr cartridge. 4.23 mL of Tris-sucrose buffer was aspirated into a 10 mL BD syringe. Air bubbles were removed by gently tapping the syringe. The syringe was then loaded onto an Ignite NanoAssemblr cartridge. The flow rate was set to 12 mL / min, with a ratio of Tris-sucrose buffer / lipid solution = 5 / 1, to produce liposomes. The liposomes were then collected into a dialysis bag (100 KD) and dialyzed with Tris-sucrose buffer. The buffer solution was changed 3 times every 2 hours, for a total of 6 hours.

[0308] rsF599 2) Add DC-cholesterol (250 µL, 20 mg / mL in EtOH) and DOPE (692 µL, 20 mg / mL in EtOH) to a 1 mL Eppendorf tube to prepare a 942 µL lipid solution (lipid:DC-cholesterol / DOPE molar ratio = 1 / 2). Aspirate the lipid solution into a 1 mL BD syringe. Remove air bubbles by gently tapping the syringe. Then, load the syringe onto the Ignite NanoAssemblr cartridge. Aspirate 4.71 mL of Tris-sucrose buffer into a 10 mL BD syringe. Remove air bubbles by gently tapping the syringe. Then, load the syringe onto the Ignite NanoAssemblr cartridge. Set the flow rate to 12 mL / min, where the ratio is Tris-sucrose buffer / lipid solution = 5 / 1, to generate liposomes. The liposomes are then collected into a dialysis bag (100 KD) and dialyzed with Tris-sucrose buffer. The buffer solution was changed 3 times every 2 hours, for a total of 6 hours.

[0309] 2) Add DC-cholesterol (500 µL, 20 mg / mL in EtOH) and DOTAP (325 µL, 20 mg / mL in EtOH) to a 1 mL Eppendorf tube to prepare an 825 µL lipid solution (lipid:DC-cholesterol / DOTAP molar ratio = 2 / 1). Aspirate the lipid solution into a 1 mL BD syringe. Remove air bubbles by gently tapping the syringe. Then, load the syringe onto the Ignite NanoAssemblr cartridge. Aspirate 4.125 mL of Tris-sucrose buffer into a 10 mL BD syringe. Remove air bubbles by gently tapping the syringe. Then, load the syringe onto the Ignite NanoAssemblr cartridge. Set the flow rate to 12 mL / min, where the ratio is Tris-sucrose buffer / lipid solution = 5 / 1, to generate liposomes. The liposomes are then collected into a dialysis bag (100 KD) and dialyzed with Tris-sucrose buffer. The buffer solution was changed 3 times every 2 hours, for a total of 6 hours.

[0310] rsF601 2) Add DC-cholesterol (250 µL, 20 mg / mL in EtOH) and DOTAP (650 µL, 20 mg / mL in EtOH) to a 1 mL Eppendorf tube to prepare a 900 µL lipid solution (lipid:DC-cholesterol / DOTAP molar ratio = 1 / 2). Aspirate the lipid solution into a 1 mL BD syringe. Remove air bubbles by gently tapping the syringe. Then, load the syringe onto the Ignite NanoAssemblr cartridge. Aspirate 4.5 mL of Tris-sucrose buffer into a 10 mL BD syringe. Remove air bubbles by gently tapping the syringe. Then, load the syringe onto the Ignite NanoAssemblr cartridge. Set the flow rate to 12 mL / min, where the ratio is Tris-sucrose buffer / lipid solution = 5 / 1, to generate liposomes. The liposomes are then collected into a dialysis bag (100 KD) and dialyzed with Tris-sucrose buffer. The buffer solution was changed 3 times every 2 hours, for a total of 6 hours.

[0311] 3) For all four formulations (rsF598, rsF599, rsF600 and rsF601), mRNA-LPS-LPX was prepared as follows: 100 µL of lipid shell (LPS) was pipetted into 123.28 µL of lipid complex (LPX), and then pipetted up and down several times to mix.

[0312] In vivo transfection: The formulations rsF598, rsF599, rsF600, or rsF601 were reconstituted in sterile PBS and administered intravenously to two healthy mice at a dose of 1 µg / mouse for each formulation. Six hours post-injection, D-fluorescein was administered intraperitoneally to the mice at a dose of 150 mg / kg body weight. Major organs such as the heart, lungs, liver, spleen, and kidneys were immediately removed and placed on the imaging stage of an IVIS imaging system for imaging. Images were acquired 10 minutes post-D-fluorescein injection using an IVIS imaging system (Xenogen, Alameda, California). Photons emitted by organs and tissues were quantified using Living Image software (Xenogen).

[0313] For the four formulations tested (rsF598, rsF599, rsF600, and rsF601), the composition of the lipid complex (LPX) was identical. The LPS composition contained a common component (DC-cholesterol) and a modified component. Formulations rsF598 and rsF599 contained varying amounts of neutral lipids (DOPE). Formulations rsF600 and rsF601 contained varying amounts of cationic lipids (DOTAP).

[0314] Bioluminescent images of organs (heart, lung, liver, spleen, and kidney) in mice after administration of rsF598, rsF599, rsF600, or rsF601. Figure 8A The results show that rsF598 is significantly better than rsF599 (both < 0.4 × 10⁻⁶). 6 p / sec / cm 2 / sr) has more lung accumulation (> 1 × 10 in one sample) 6 p / sec / cm 2 / sr, and approximately 0.6 × 10 in another sample. 6 p / sec / cm 2 / sr), while rsF599 is lower than rsF598 (both < 0.4 × 10). 6 p / sec / cm 2 / sr) had more spleen accumulation (all > 1 × 10 6 p / sec / cm 2 / sr). Compared to a lower DC-CHOL / DOPE ratio (rsF599), a higher DC-CHOL / DOPE ratio (rsF598) was associated with higher lung accumulation and lower spleen accumulation. rsF600 had the highest liver accumulation (>1 ×10⁻⁶). 6 p / sec / cm 2 / sr), where rsF598 is approximately 0.6-1 × 106 p / sec / cm 2 / sr, and others in approximately 0.1-0.6 × 10 6 p / sec / cm 2 The emission values ​​given here and elsewhere in the embodiments correspond to the peak emission observed anywhere on the surface of the object being analyzed.

[0315] Replacing neutral lipids (DOPE) with cationic lipids (DOTAP) increased liver accumulation and decreased lung accumulation.

[0316] Figure 8B The relative biodistribution of quantitative IVIS images of the heart, lungs, liver, spleen, and kidneys is shown. rsF598 showed a greater lung distribution (30.7%) compared to 7.0% lung distribution in rsF599 (higher DC-CHOL / DOPE ratio). rsF599 showed a greater spleen distribution (48.1%) compared to 6.6% spleen distribution in rsF598 (lower DC-CHOL / DOPE ratio). Both rsF600 and rsF601 showed strong liver accumulation (80.2% and 74.6%, respectively). These results demonstrate that the distribution of LPS-LPX delivery systems can be manipulated by altering LPS composition, enabling passive targeting to specific organs.

[0317] Example 9. Enhancing organ specificity of LPS-LPX delivery system with anti-ICAM Fab.

[0318] The formulation rsF627 was prepared as follows: 1) Nucleolipin complex (LPX)-luciferase (Luc) mRNA (10 µL, 1 mg / mL in water) and sodium acetate buffer (90 µL, 25 mM, pH 5) were added to a 1 mL Eppendorf tube to prepare a 100 µL mRNA solution. KT-001 (14.6 µL, 10 mg / mL in EtOH), DSPC (3.01 µL, 10 mg / mL in EtOH), and cholesterol (5.68 µL, 10 mg / mL in EtOH) (molar ratio of lipid:KT-001 / DSPC / cholesterol = 50 / 10 / 38.5) were added to a 2 mL serum vial to prepare a 23.28 µL lipid solution. The lipid solution was then rapidly pipetted into the mRNA solution (with several up-and-down pipetting to mix), followed by vortexing at the highest speed setting for 5–10 seconds.

[0319] 2) Lipid shell (LPS) _KT-001 (73 µL, 10 mg / mL in EtOH), DSPC (15.1 µL, 10 mg / mL in EtOH), cholesterol (28.4 µL, 10 mg / mL in EtOH), and DMG-PEG2000 (7.4 µL, 10 mg / mL in EtOH) were added to a 1 mL Eppendorf tube to prepare a 123.9 µL lipid solution (molar ratio of lipids: KT-001 / DSPC / cholesterol / DMG-PEG2000 = 50 / 10 / 38.5 / 1.5). The lipid solution was aspirated into a 1 mL BD syringe. Air bubbles were removed by gently tapping the syringe. The syringe was then loaded onto an Ignite NanoAssemblr cartridge. Anti-ICAM Fab (139.9 µL, 664 µg / mL) and 231.8 µL Tris-sucrose buffer were aspirated into a 1 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Next, the syringe was loaded onto the Ignite NanoAssemblr cartridge. The flow rate was set to 12 mL / min, with a ratio of Tris-sucrose buffer / liposome solution = 3 / 1, to generate liposomes. The liposomes were then collected into a dialysis bag (100 KD) and dialyzed with Tris-sucrose buffer. The buffer was changed 3 times every 2 hours for a total of 6 hours.

[0320] 3) mRNA-LPS-LPX was prepared as follows: 100 µL of lipid shell (LPS) was pipetted into 123.28 µL of lipid complex (LPX), and then pipetted up and down several times to mix.

[0321] The formulation rsF628 was prepared as follows: 1) Core (lipid complex, LPX) - prepared as described above for rsF627.

[0322] 2) Lipid shell (LPS) _KT-001 (729.8 µL, 10 mg / mL in EtOH), DSPC (150.5 µL, 10 mg / mL in EtOH), cholesterol (283.8 µL, 10 mg / mL in EtOH), and DMG-PEG2000 (74.3 µL, 10 mg / mL in EtOH) were added to a 5 mL Eppendorf tube to prepare a 1238.4 µL lipid solution (molar ratio of lipids:KT-001 / DSPC / cholesterol / DMG-PEG2000 = 50 / 10 / 38.5 / 1.5). The lipid solution was aspirated into a 3 mL BD syringe. Air bubbles were removed by gently tapping the syringe. The syringe was then loaded onto an Ignite NanoAssemblr cartridge. 3715.2 µL of Tris-sucrose buffer was aspirated into a 10 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Next, the syringe was loaded onto the Ignite NanoAssemblr cartridge. The flow rate was set to 12 mL / min, with a ratio of Tris-sucrose buffer / liposome solution = 3 / 1, to generate liposomes. The liposomes were then collected into a dialysis bag (100 KD) and dialyzed with Tris-sucrose buffer. The buffer was changed 3 times every 2 hours for a total of 6 hours.

[0323] 3) mRNA-LPS-LPX was prepared as follows: 100 µL of lipid shell (LPS) was pipetted into 123.28 µL of lipid complex (LPX), and then pipetted up and down several times to mix.

[0324] Four mice were intravenously injected with lipopolysaccharide (from E. coli O111:B4, Sigma-Aldrich) at a dose of 7.5 mg / kg to establish endotoxemia-induced acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS). One hour after injection, in vivo transfection was initiated using either rsF627 or rsF628.

[0325] In vivo transfection: The formulations rsF627 or rsF628 were reconstituted in sterile PBS, and each formulation was administered intravenously to two ARDS mice at a dose of 1 µg / mouse. Three hours post-injection, D-fluorescein was administered intraperitoneally to the mice at a dose of 150 mg / kg body weight. Major organs such as the heart, lungs, liver, spleen, and kidneys were immediately removed and placed on the imaging stage of an IVIS imaging system for imaging. Images were acquired 10 minutes post-D-fluorescein injection using an IVIS imaging system (Xenogen, Alameda, California). Photons emitted by organs and tissues were quantified using Living Image software (Xenogen).

[0326] For both formulations tested (rsF627 and rsF628), the lipid complex (LPX) composition was identical. The lipid composition and lipid ratio of the LPS in both formulations were the same, but rsF627 additionally contained anti-ICAM Fab on the LPS.

[0327] Bioluminescent images of organs (heart, lung, liver, spleen, and kidney) in ARDS mice after administration of rsF627 or rsF628. Figure 9A The results show that rsF627 is lower than rsF628 (below the detection limit, i.e., < 0.05 × 10⁻⁶). 7 p / sec / cm 2 / sr) has more lung accumulation (luminescence approximately 0.1-0.3 × 10 7 p / sec / cm 2 / sr).

[0328] rsF628 is a liver-targeted modulator in which over 95% of total luciferase is expressed in the liver. LPS modified with anti-ICAMFab to produce the modulator rsF627 exhibits a 14% lung distribution, such as... Figure 9B As shown.

[0329] Figure 9C The results showed that the total luciferase expression level in the lungs with formulation rsF627 was 8 times that with the control formulation rsF628.

[0330] These results demonstrate that active targeting with a targeting ligand (e.g., an antibody or antibody fragment) can enhance organ specificity. By having a targeting ligand (e.g., an antibody or antibody fragment) on the LPS, the distribution of the LPS-LPX delivery system can be manipulated, and active targeting to specific organs can be achieved.

[0331] Example 10. Organ specificity of LPS-LPX delivery system by controlling LPS lipid composition and using anti-ICAM Fab.

[0332] The formulation rsF620 was prepared as follows: 1) Core (lipid complex, LPX) - prepared as described above for rsF627 in Example 9.

[0333] 2) Lipid shell (LPS)-DC-cholesterol (500 µL, 10 mg / mL in EtOH) and DOPE (346 µL, 10 mg / mL in EtOH) were added to a 1 mL Eppendorf tube to prepare an 846 µL lipid solution (lipid:DC-cholesterol / DOPE molar ratio = 2 / 1). The lipid solution was aspirated into a 1 mL BD syringe. Air bubbles were removed by gently tapping the syringe. The syringe was then loaded onto an Ignite NanoAssemblr cartridge. 4.23 mL of Tris-sucrose buffer was aspirated into a 10 mL BD syringe. Air bubbles were removed by gently tapping the syringe. The syringe was then loaded onto an Ignite NanoAssemblr cartridge. The flow rate was set to 12 mL / min, with a ratio of Tris-sucrose buffer / lipid solution = 5 / 1, to produce liposomes. The liposomes were then collected into a dialysis bag (100 KD) and dialyzed with Tris-sucrose buffer. The buffer solution was changed 3 times every 2 hours, for a total of 6 hours.

[0334] 3) mRNA-LPS-LPX was prepared as follows: 100 µL of lipid shell (LPS) was pipetted into 123.28 µL of lipid complex (LPX), and then pipetted up and down several times to mix.

[0335] The formulation rsF621 was prepared as follows: 1) Core (lipid complex, LPX) - prepared as described above for rsF627 in Example 9.

[0336] 2) Add lipid shell (LPS)-DC-cholesterol (41 µL, 10 mg / mL in EtOH) and DOPE (56.6 µL, 10 mg / mL in EtOH) to a 1 mL Eppendorf tube to prepare a 97.6 µL lipid solution (lipid:DC-cholesterol / DOPE molar ratio = 2 / 1). Aspirate the lipid solution into a 1 mL BD syringe. Remove air bubbles by gently tapping the syringe. Then, load the syringe onto the Ignite NanoAssemblr cartridge. Aspirate anti-ICAM Fab (183.72 µL, 664 µg / mL) and 304.28 µL Tris-sucrose buffer into a 1 mL BD syringe. Remove air bubbles by gently tapping the syringe. Then, load the syringe onto the Ignite NanoAssemblr cartridge. Set the flow rate to 12 mL / min, where the ratio is Tris-sucrose buffer / lipid solution = 5 / 1, to generate liposomes. The liposomes were then collected in a dialysis bag (100 KD) and dialyzed with Tris-sucrose buffer. The buffer was changed 3 times every 2 hours for a total of 6 hours.

[0337] 3) mRNA-LPS-LPX was prepared as follows: 100 µL of lipid shell (LPS) was pipetted into 123.28 µL of lipid complex (LPX), and then pipetted up and down several times to mix.

[0338] Four mice were intravenously injected with lipopolysaccharide (from E. coli O111:B4, Sigma-Aldrich) at a dose of 7.5 mg / kg to establish endotoxemia-induced acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS). One hour post-injection, in vivo transfection was initiated using either rsF620 or rsF621.

[0339] In vivo transfection: The formulations rsF620 or rsF621 were reconstituted in sterile PBS, and each formulation was administered intravenously to two ARDS mice at a dose of 1 µg / mouse. Three hours post-injection, D-fluorescein was administered intraperitoneally to the mice at a dose of 150 mg / kg body weight. Major organs such as the heart, lungs, liver, spleen, and kidneys were immediately removed and placed on the imaging stage of an IVIS imaging system for imaging. Images were acquired 10 minutes post-D-fluorescein injection using an IVIS imaging system (Xenogen, Alameda, California). Photons emitted by organs and tissues were quantified using Living Image software (Xenogen).

[0340] For both formulations tested (rsF620 and rsF621), the lipid complex (LPX) composition was identical. The lipid composition and lipid ratio of the LPS were the same for both formulations, but rsF621 additionally contained anti-ICAM Fab on the LPS. The lipid composition of the LPS was selected to target the lungs.

[0341] Bioluminescent images of organs (heart, lung, liver, spleen, and kidney) in ARDS mice after administration of rsF620 or rsF621. Figure 10A The results show that rsF621 is superior to rsF620 (< 3.5 × 10⁻⁶). 5 p / sec / cm 2 / sr) has more lung accumulation (>5×10 5 p / sec / cm 2 / sr).

[0342] rsF620 is a lung-targeted modulator in which approximately 48% of total luciferase is expressed in the lungs. Modifying rsF620 with LPS containing anti-ICAM Fab yields the modulator rsF621, which exhibits approximately 78% lung distribution, such as... Figure 10B As shown.

[0343] Figure 10C The results showed that the total luciferase expression level in the lungs with formulation rsF621 was 9 times that with the control formulation rsF620.

[0344] These results demonstrate that active targeting with a targeting ligand (e.g., an antibody or antibody fragment) can enhance organ specificity. By having a targeting ligand (e.g., an antibody or antibody fragment) on the LPS, the distribution of the LPS-LPX delivery system can be manipulated, and active targeting to specific organs can be achieved.

[0345] Example 11. Characterization of the LPS-LPX delivery system.

[0346] Various LPS-LPX delivery systems were fabricated, and their size, surface charge, and encapsulation efficiency were determined.

[0347] The size, surface charge, and polydispersity index (PDI) of various LPS were determined using a dynamic light scattering analyzer. The findings are summarized in Tables 9 and 10 below. The PDI values ​​indicate that the LPS are monodisperse.

[0348] Table 9: Positively charged shells Table 10: Neutral Shell Formulations rsF598, rsF599, rsF600, and rsF601 were prepared as described above in Example 8. The size, surface charge, and encapsulation efficiency of the formulations were measured. The results of these measurements are summarized in Table 11.

[0349] Table 11: Formulas LCF102, LCF96, LCF104, LCF105 and LCF106 were prepared as follows: all formulas have the same externality (LPS) and were prepared as described above for LCF96 in Example 1.

[0350] LCF102: Internal (LPX) preparation is as follows: Green fluorescent protein (GFP) mRNA (20 µL, 1 mg / mL in water) and sodium acetate buffer (80 µL, 25 mM, pH 5) are added to a 1 mL Eppendorf tube to prepare a 100 µL mRNA solution. KT-001 (14.6 µL, 20 mg / mL in EtOH) and DSPC (4.3 µL, 20 mg / mL in EtOH) (lipid:KT-001 / DSPC molar ratio = 5 / 1) are added to a 2 mL serum vial to prepare an 18.9 µL lipid solution. The lipid solution is then rapidly pipetted into the mRNA solution (with several up-and-down pipetting to mix), followed by vortexing at the highest speed setting for 5–10 seconds.

[0351] mRNA-LPS-LPX was prepared as follows: 100 µL of lipid shell (LPS) was pipetted into 118.9 µL of lipid complex (LPX), and then pipetted up and down several times to mix.

[0352] LCF104: Internal (LPX) was prepared as follows: Green fluorescent protein (GFP) mRNA (20 µL, 1 mg / mL in water) and sodium citrate buffer (80 µL, 10 mM, pH 3) were added to a 1 mL Eppendorf tube to prepare a 100 µL mRNA solution. KT-001 (14.6 µL, 20 mg / mL in EtOH), DSPC (3.01 µL, 20 mg / mL in EtOH), cholesterol (5.68 µL, 20 mg / mL in EtOH), and DMG-PEG2000 (1.49 µL, 20 mg / mL in EtOH) (lipid: molar ratio of KT-001 / DSPC / cholesterol / DMG-PEG2000 = 50 / 10 / 38.5 / 1.5) were added to a 2 mL serum vial to prepare a 24.77 µL lipid solution. Then, the lipid solution was rapidly pipetted into the mRNA solution (and pipetted up and down several times to mix), followed by vortexing at the highest speed setting for 5-10 seconds.

[0353] Sodium citrate buffer 10 mM, pH 3: Dilute commercially available 0.5 M, pH 3.0 citrate buffer (Thermo Fisher Scientific, catalog number J61391-AK) 50 times with water for injection.

[0354] mRNA-LPS-LPX was prepared as follows: 100 µL of lipid shell (LPS) was pipetted into 124.77 µL of lipid complex (LPX), and then pipetted up and down several times to mix.

[0355] LCF105: Internal (LPX) was prepared as follows: Green fluorescent protein (GFP) mRNA (20 µL, 1 mg / mL in water) and sodium acetate buffer (80 µL, 25 mM, pH 4) were added to a 1 mL Eppendorf tube to prepare a 100 µL mRNA solution. KT-001 (14.6 µL, 20 mg / mL in EtOH), DSPC (3.01 µL, 20 mg / mL in EtOH), cholesterol (5.68 µL, 20 mg / mL in EtOH), and DMG-PEG2000 (1.49 µL, 20 mg / mL in EtOH) (lipid: molar ratio of KT-001 / DSPC / cholesterol / DMG-PEG2000 = 50 / 10 / 38.5 / 1.5) were added to a 2 mL serum vial to prepare a 24.77 µL lipid solution. Then, the lipid solution was rapidly pipetted into the mRNA solution (and pipetted up and down several times to mix), followed by vortexing at the highest speed setting for 5-10 seconds.

[0356] Sodium acetate buffer 25 mM, pH 4: Dilute commercially available 1 M pH 4.0 acetate buffer (Thermo Fisher Scientific, catalog number J60104-AK) 40 times with water for injection.

[0357] mRNA-LPS-LPX was prepared as follows: 100 µL of lipid shell (LPS) was pipetted into 124.77 µL of lipid complex (LPX), and then pipetted up and down several times to mix.

[0358] LCF106: Internal preparation (LPX) was as follows: Green fluorescent protein (GFP) mRNA (20 µL, 1 mg / mL in water) and sodium acetate buffer (80 µL, 25 mM, pH 5) were added to a 1 mL Eppendorf tube to prepare a 100 µL mRNA solution. KT-001 (14.6 µL, 20 mg / mL in EtOH), DSPC (3.01 µL, 20 mg / mL in EtOH), cholesterol (5.68 µL, 20 mg / mL in EtOH), and DMG-PEG2000 (1.49 µL, 20 mg / mL in EtOH) (lipid: molar ratio of KT-001 / DSPC / cholesterol / DMG-PEG2000 = 50 / 10 / 38.5 / 1.5) were added to a 2 mL serum vial to prepare a 24.77 µL lipid solution. Then, the lipid solution was rapidly pipetted into the mRNA solution (and pipetted up and down several times to mix), followed by vortexing at the highest speed setting for 5-10 seconds.

[0359] mRNA-LPS-LPX was prepared as follows: 100 µL of lipid shell (LPS) was pipetted into 124.77 µL of lipid complex (LPX), and then pipetted up and down several times to mix.

[0360] The size, surface charge, and encapsulation efficiency of the formulation were measured. The results of these measurements are summarized in Table 12.

[0361] Table 12: LPS determines the final surface charge / composition of the LPS-LPX delivery system. When LPS is neutral, the LPS-LPX surface is always neutral regardless of the LPX charge. When LPS is positively charged, the LPS-LPX surface is always positively charged.

[0362] All public disclosures cited in this paper are incorporated herein by full reference as if each disclosure were individually and fully described and incorporated herein.

[0363] Various modifications and variations to the embodiments disclosed herein will become apparent to those skilled in the art without departing from the scope and spirit of this disclosure. The illustrative embodiments and examples are provided by way of example only and are not intended to limit the scope of the invention.

Claims

1. A composition for transfecting cells in vitro or in vivo, said composition comprising at least one nucleic acid contained in an LPS (lipid shell)-LPX (lipid complex) delivery system, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule.

2. A method for transfecting cells with one or more nucleic acids, the method comprising: The cells are brought into contact with a composition in vitro, the composition comprising at least one nucleic acid contained in an LPS-LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule.

3. A method for treating or preventing a disease or alleviating symptoms of a disease, the method comprising administering to a subject in need a composition for transfecting cells, the composition comprising at least one nucleic acid contained in an LPS-LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule.

4. The composition or method for transfecting cells according to any one of claims 1 to 3, wherein the LPS-LPX delivery system comprises LPS encapsulating LPX.

5. A method for preparing a composition for in vitro or in vivo transfection of cells, the method comprising: (a) Prepare a first solution comprising an aqueous phase and one or more nucleic acids; (b) Prepare a second solution comprising an organic phase and one or more lipids; (c) The first solution prepared in step (a) is mixed with the second solution prepared in step (b) to form a monodisperse or polydisperse LPX containing one or more nucleic acids; (d) Stirring the LPX prepared in step (c); (e) Prepare a third solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule; (f) LPS is prepared by forming liposomes, micelles or other self-assembled lipid nanoparticles from the third solution prepared in step (e), wherein a targeting ligand, imaging ligand, digestive agent or small molecule is optionally added to the aqueous phase, and then the aqueous phase is mixed with the third solution prepared in step (e); (g) The LPX obtained after step (d) is mixed with the LPS prepared in step (f), and optionally a targeting ligand, imaging ligand, digestive agent, or small molecule is added; and (h) Stir the mixture prepared in step (g) to form a composition for transfecting cells.

6. A method for preparing a composition for in vitro or in vivo transfection of cells, the method comprising: (a) A first solution containing an aqueous phase and one or more nucleic acids is mixed with a second solution containing an organic phase and one or more lipids, thereby forming a monodisperse or polydisperse LPX containing one or more nucleic acids; (b) Stirring the LPX prepared in step (a); (c) Prepare a third solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule; (d) LPS is prepared by forming liposomes, micelles or other self-assembled lipid nanoparticles from a third solution comprising an organic phase, one or more lipids and optionally a targeting ligand, an imaging ligand, a digestive agent or a small molecule, wherein the targeting ligand, imaging ligand, digestive agent or small molecule is optionally added to an aqueous phase and then the aqueous phase is mixed with the third solution; (e) The LPX obtained after step (b) is mixed with the LPS prepared in step (d), and optionally a targeting ligand, imaging ligand, digestive agent, or small molecule is added; and (f) Stir the mixture prepared in step (e) to form a composition for transfecting cells.

7. A composition for transfecting cells in vitro or in vivo, said composition being prepared by a method comprising: (a) Prepare a first solution comprising an aqueous phase and one or more nucleic acids; (b) Prepare a second solution comprising an organic phase and one or more lipids; (c) The first solution prepared in step (a) is mixed with the second solution prepared in step (b) to form a monodisperse or polydisperse LPX containing one or more nucleic acids; (d) Stirring the LPX prepared in step (c); (e) Prepare a third solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule; (f) LPS is prepared by forming liposomes, micelles or other self-assembled lipid nanoparticles from the third solution prepared in step (e), wherein a targeting ligand, imaging ligand, digestive agent or small molecule is optionally added to the aqueous phase, and then the aqueous phase is mixed with the third solution prepared in step (e); (g) The LPX obtained after step (d) is mixed with the LPS prepared in step (f), and optionally a targeting ligand, imaging ligand, digestive agent, or small molecule is added; and (h) Stir the mixture prepared in step (g) to form a composition for transfecting cells.

8. A composition for transfecting cells in vitro or in vivo, said composition being prepared by a method comprising: (a) A first solution containing an aqueous phase and one or more nucleic acids is mixed with a second solution containing an organic phase and one or more lipids, thereby forming a monodisperse or polydisperse LPX containing one or more nucleic acids; (b) Stirring the LPX prepared in step (a); (c) Prepare a third solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule; (d) LPS is prepared by forming liposomes, micelles or other self-assembled lipid nanoparticles from a third solution comprising an organic phase, one or more lipids and optionally a targeting ligand, an imaging ligand, a digestive agent or a small molecule, wherein the targeting ligand, imaging ligand, digestive agent or small molecule is optionally added to an aqueous phase and then the aqueous phase is mixed with the third solution; (e) The LPX obtained after step (b) is mixed with the LPS prepared in step (d), and optionally a targeting ligand, imaging ligand, digestive agent, or small molecule is added; and (f) Stir the mixture prepared in step (e) to form a composition for transfecting cells.

9. A method for preparing a composition for in vitro or in vivo transfection of cells, the method comprising: (a) Prepare a first solution containing an aqueous phase; (b) Prepare a second solution comprising an organic phase and one or more lipids; (c) The first solution prepared in step (a) is mixed with the second solution prepared in step (b) to generate liposomes, micelles or other self-assembled lipid nanoparticles; (d) Prepare a third solution comprising an aqueous phase and one or more nucleic acids; (e) The liposomes, micelles or other self-assembled lipid nanoparticles prepared in step (c) are mixed with the third solution prepared in step (d) to form a monodisperse or polydisperse LPX; (f) Prepare a fourth solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule; (g) LPS is prepared by forming liposomes, micelles or other self-assembled lipid nanoparticles from the fourth solution prepared in step (f), wherein a targeting ligand, imaging ligand, digestive agent or small molecule is optionally added to the aqueous phase, and then the aqueous phase is mixed with the fourth solution prepared in step (f); (h) The LPX prepared in step (e) is mixed with the LPS prepared in step (g), and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule is added; and (i) Stir the mixture prepared in step (h) to form a composition for transfecting cells.

10. A method for preparing a composition for in vitro or in vivo transfection of cells, the method comprising: (a) Mixing a first solution containing an aqueous phase with a second solution containing an organic phase and one or more lipids to produce liposomes, micelles or other self-assembled lipid nanoparticles; (b) The liposomes, micelles or other self-assembled lipid nanoparticles prepared in step (a) are mixed with a third solution containing an aqueous phase and one or more nucleic acids to form monodisperse or polydisperse LPX. (c) LPS is prepared by forming liposomes, micelles or other self-assembled lipid nanoparticles from a fourth solution comprising an organic phase, one or more lipids and optionally a targeting ligand, an imaging ligand, a digestive agent or a small molecule, wherein the targeting ligand, imaging ligand, digestive agent or small molecule is optionally added to an aqueous phase and then the aqueous phase is mixed with the fourth solution; (d) Mix the LPX prepared in step (b) with the LPS prepared in step (c), and optionally add a targeting ligand, an imaging ligand, a digestive agent, or a small molecule; and (e) Stir the mixture prepared in step (d) to form a composition for transfecting cells.

11. A composition for transfecting cells in vitro or in vivo, said composition being prepared by a method comprising: (a) Prepare a first solution containing an aqueous phase; (b) Prepare a second solution comprising an organic phase and one or more lipids; (c) The first solution prepared in step (a) is mixed with the second solution prepared in step (b) to generate liposomes, micelles or other self-assembled lipid nanoparticles; (d) Prepare a third solution comprising an aqueous phase and one or more nucleic acids; (e) The liposomes, micelles or other self-assembled lipid nanoparticles prepared in step (c) are mixed with the third solution prepared in step (d) to form a monodisperse or polydisperse LPX; (f) Prepare a fourth solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule; (g) LPS is prepared by forming liposomes, micelles or other self-assembled lipid nanoparticles from the fourth solution prepared in step (f), wherein a targeting ligand, imaging ligand, digestive agent or small molecule is optionally added to the aqueous phase, and then the aqueous phase is mixed with the fourth solution prepared in step (f); (h) The LPX prepared in step (e) is mixed with the LPS prepared in step (g), and optionally a targeting ligand, an imaging ligand, a digestive agent, or a small molecule is added; and (i) Stir the mixture prepared in step (h) to form a composition for transfecting cells.

12. A composition for transfecting cells in vitro or in vivo, said composition being prepared by a method comprising: (a) Mixing a first solution containing an aqueous phase with a second solution containing an organic phase and one or more lipids to produce liposomes, micelles or other self-assembled lipid nanoparticles; (b) The liposomes, micelles or other self-assembled lipid nanoparticles prepared in step (a) are mixed with a third solution containing an aqueous phase and one or more nucleic acids to form monodisperse or polydisperse LPX. (c) LPS is prepared by forming liposomes, micelles or other self-assembled lipid nanoparticles from a fourth solution comprising an organic phase, one or more lipids and optionally a targeting ligand, an imaging ligand, a digestive agent or a small molecule, wherein the targeting ligand, imaging ligand, digestive agent or small molecule is optionally added to an aqueous phase and then the aqueous phase is mixed with the fourth solution; (d) Mix the LPX prepared in step (b) with the LPS prepared in step (c), and optionally add a targeting ligand, an imaging ligand, a digestive agent, or a small molecule; and (e) Stir the mixture prepared in step (d) to form a composition for transfecting cells.

13. The composition or method for transfecting cells according to any one of claims 5 to 12, wherein the aqueous phase of the first solution and / or the third solution is an aqueous buffer.

14. The composition or method for transfecting cells according to claim 13, wherein the aqueous buffer is a citrate buffer or an acetate buffer.

15. The composition or method for transfecting cells according to claim 13 or 14, wherein the aqueous buffer is in a pH range of about pH 2 to about pH 6.

16. The composition or method for transfecting cells according to claim 13, wherein the aqueous buffer is Tris-sucrose buffer.

17. The composition or method for transfecting cells according to claim 13 or 16, wherein the aqueous buffer is in a pH range of about pH 7 to about pH 8.

18. The composition or method for transfecting cells according to any one of claims 5 to 17, wherein the organic phase comprises a water-miscible organic solvent.

19. The method or composition according to claim 18, wherein the water-miscible organic solvent is ethanol, methanol, isopropanol, DMSO, acetone, acetonitrile, DMF or THF.

20. The composition or method for transfecting cells according to any one of claims 1 to 19, wherein the nucleic acid is DNA, RNA, or DNA and RNA.

21. The composition or method for transfecting cells according to claim 20, wherein the RNA is or includes mRNA, siRNA, miRNA, RNAi, saRNA, taRNA, or shRNA.

22. The composition or method for transfecting cells according to claim 21, wherein the RNA is or includes mRNA.

23. The composition or method for transfecting cells according to claim 20, wherein the DNA is plasmid DNA.

24. The composition or method for transfecting cells according to claim 23, wherein the plasmid DNA is nanoparticle DNA.

25. The composition or method for transfecting cells according to any one of claims 20 to 24, wherein the nucleic acid comprises a therapeutic gene.

26. The composition or method for transfecting cells according to any one of claims 1 to 25, wherein the LPS is a lipid micelle, liposome, or self-assembled lipid nanoparticle.

27. The composition or method for transfecting cells according to claim 26, wherein the LPS comprises one or more ionizable cationic lipids and / or one or more auxiliary lipids.

28. The composition or method for transfecting cells according to any one of claims 1 to 27, wherein the LPX comprises one or more ionizable cationic lipids and optionally one or more auxiliary lipids.

29. The composition or method for transfecting cells according to claim 27 or 28, wherein the ionizable cationic lipid is selected from the lipids shown in Table 1.

30. The composition or method for transfecting cells according to any one of claims 27 to 29, wherein the auxiliary lipid is a phospholipid, a polyethylene glycol-modified lipid, cholesterol, or a cholesterol derivative.

31. The composition or method for transfecting cells according to claim 30, wherein the auxiliary lipid is a phospholipid.

32. The composition or method for transfecting cells according to claim 30, wherein the auxiliary lipid is a polyethylene glycol-modified lipid.

33. The composition or method for transfecting cells according to claim 30, wherein the auxiliary lipid is cholesterol.

34. The composition or method for transfecting cells according to claim 30, wherein the auxiliary lipid is a cholesterol derivative.

35. The method according to any one of claims 3 to 34, wherein the disease is cancer.

36. The composition or method for transfecting cells according to any one of claims 1 to 35, wherein the LPX comprises KT-001, DSPC, cholesterol, DOTMA, DC-cholesterol, DOPE, or any combination thereof.

37. The composition or method for transfecting cells according to any one of claims 1 to 35, wherein the LPS comprises KT-001, DSPC, cholesterol, DMG-PEG2000, DOTMA, DOTAP, or any combination thereof.

38. The composition or method for transfecting cells according to claim 36 or 37, wherein the LPX comprises KT-001 and DSPC.

39. The composition or method for transfecting cells according to claim 38, wherein the molar ratio of KT-001:DSPC in the LPX is 5:

1.

40. The composition or method for transfecting cells according to claim 36 or 37, wherein the LPX comprises KT-001, DSPC, and cholesterol.

41. The composition or method for transfecting cells according to claim 40, wherein the molar ratio of KT-001:DSPC:cholesterol in the LPX is 50:10:38.

5.

42. The composition or method for transfecting cells according to claim 36 or 37, wherein the LPX comprises KT-001, DOTMA, DSPC, and cholesterol.

43. The composition or method for transfecting cells according to claim 42, wherein the molar ratio of KT-001:DOTMA:DSPC:cholesterol in the LPX is 45:5:10:38.

5.

44. The composition or method for transfecting cells according to claim 36 or 37, wherein the LPX comprises KT-001, DSPC, cholesterol, and DMG-PEG2000.

45. The composition or method for transfecting cells according to claim 44, wherein the molar ratio of KT-001:DSPC:cholesterol:DMG-PEG2000 in the LPX is 50:10:38.5:1.

5.

46. ​​The composition or method for transfecting cells according to claim 36 or 37, wherein the LPX comprises DC-cholesterol and DOPE.

47. The composition or method for transfecting cells according to claim 46, wherein the molar ratio of DC-cholesterol to DOPE in the LPX is 2:

1.

48. The composition or method for transfecting cells according to any one of claims 36 to 47, wherein the LPS comprises KT-001, DSPC, cholesterol, and DMG-PEG2000.

49. The composition or method for transfecting cells according to claim 48, wherein the molar ratio of KT-001:DSPC:cholesterol:DMG-PEG2000 in the LPS is 50 / 10 / 38.5 / 1.

5.

50. The composition or method for transfecting cells according to any one of claims 36 to 47, wherein the LPS comprises KT-001, DOTMA, DSPC, cholesterol, and DMG-PEG2000.

51. The composition or method for transfecting cells according to claim 50, wherein the molar ratio of KT-001:DOTMA:DSPC:cholesterol:DMG-PEG2000 in the LPS is 45 / 5 / 10 / 38.5 / 1.

5.

52. The composition or method for transfecting cells according to any one of claims 36 to 47, wherein the LPS comprises DC-cholesterol and DOPE.

53. The composition or method for transfecting cells according to claim 52, wherein the molar ratio of DC-cholesterol to DOPE in the LPS is 2:1 or 1:

2.

54. The composition or method for transfecting cells according to any one of claims 36 to 47, wherein the LPS comprises DC-cholesterol and DOTAP.

55. The composition or method for transfecting cells according to claim 54, wherein the molar ratio of DC-cholesterol to DOTAP in the LPS is 2:1 or 1:

2.

56. The composition or method for transfecting cells according to any one of claims 1 to 55, wherein the composition comprises a targeting ligand.

57. The composition or method for transfecting cells according to claim 56, wherein the targeting ligand is in contact with the LPS.

58. The composition or method for transfecting cells according to claim 56 or 57, wherein the targeting ligand is DUPA, folic acid, peptide, or antibody or an antigen-binding fragment thereof.

59. The composition or method for transfecting cells according to any one of claims 1 to 58, wherein the composition comprises an imaging ligand.

60. The composition or method for transfecting cells according to claim 59, wherein the imaging ligand is in contact with the LPS, inside the LPX, or between the surface of the LPS and the LPX.

61. The composition or method for transfecting cells according to claim 59 or 60, wherein the imaging ligand is 1,2-distearate-sn-glycerol-3-phosphoethanolamine-polyethylene glycol-IR825 (DSPE-PEG-IR825) or diethylenetriaminepentaacetic acid (DTPA)-Gd.

62. The composition or method for transfecting cells according to any one of claims 1 to 61, wherein the composition comprises a digestive agent.

63. The composition or method for transfecting cells according to claim 62, wherein the digestive agent is in contact with the LPS, inside the LPX, or between the surface of the LPS and the LPX.

64. The composition or method for transfecting cells according to claim 62 or 63, wherein the digestive agent is hyaluronidase or collagenase.

65. The composition or method for transfecting cells according to any one of claims 1 to 64, wherein the composition comprises a small molecule.

66. The composition or method for transfecting cells according to claim 65, wherein the small molecule is in contact with the LPS, inside the LPX, or between the surface of the LPS and the LPX.

67. The composition or method for transfecting cells according to claim 65 or 66, wherein the small molecule is an NSAID or a toxin.

68. The composition or method for transfecting cells according to claim 65 or 66, wherein the small molecule is a vitamin, sugar, steroid, or chemotherapeutic agent.

69. The composition or method for transfecting cells according to claim 65 or 66, wherein the small molecule is folic acid, glucose, galactose, N-acetylgalactosamine (GalNAc), dexamethasone, paclitaxel, or doxorubicin.

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