Lipid nanoparticle compositions and methods of use thereof

Abstract

Disclosed herein are nanoparticle compositions comprising a polymeric component and a lipid component. The present disclosure also relates to lipid nanoparticles (LNPs) having the nanoparticle compositions for nucleic acid delivery and methods of making and using the same. The LNPs can be used for rapid, efficient, and safe delivery of nucleic acids (e.g., DNA / RNA).

Description

[0001] Priority claim

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 313,967, filed on February 25, 2022. The entire contents of the foregoing application are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to nanoparticle compositions comprising a polymer component and a lipid component. The present disclosure also relates to lipid nanoparticles (LNPs) having the nanoparticle compositions for nucleic acid delivery and methods of preparing and using the same. Background Art

[0004] The huge success of COVID-19 vaccines based on messenger RNA (mRNA) has made mRNA and plasmid DNA a new class of therapeutic agents for the prevention and treatment of various diseases. In order to work in vivo, mRNA requires a safe, effective and stable delivery system that protects nucleic acids from degradation and allows cellular uptake and mRNA release. Lipid nanoparticles (LNPs) have successfully entered the clinic for delivering mRNA; specifically, lipid nanoparticle-mRNA vaccines are now clinically used to fight the coronavirus disease 2019 (COVID-19), marking a milestone in mRNA therapy. Obviously, LNP plays a vital role in mRNA delivery. For example, the LNP of the COVID-19 vaccine is made of 50mol% ionizable lipids, 10mol% DSPC, 38.5mol% cholesterol and 1.5mol% PEG2000-DMG. Among these components, ionizable lipids play two key roles in LNPs: 1) encapsulating mRNA through electrostatic interactions between their positively charged amino groups and negatively charged phosphate groups in the mRNA molecular backbone; and 2) disrupting the membrane of the endosome / lysosome to allow LNP to escape from the lysosome and deliver mRNA to the cytosol of the cell. DSPC is an auxiliary lipid used to stabilize LNP; cholesterol is a structural lipid that provides rigidity and stability to LNP; and the PEGylated lipid PEG2000-DMG forms a PEG shell at the surface of LNP, thereby preventing particle aggregation. In addition to the lipid-to-lipid ratio between different lipid molecules, another key parameter is the ratio between ionizable lipids and mRNA encapsulated in the particles, which is expressed as the N / P ratio. Here, N is the number of nitrogen atoms of the positively charged ionizable amino groups of the ionizable lipid, and P is the number of negatively charged phosphate groups of the mRNA. The N / P ratio of the COVID-19 vaccine LNP formulation is 6:1, which indicates an excess of ionizable lipid components from the perspective of charge-charge ratio.

[0005] Currently, this composition ratio (mol%) of 50% ionizable (or cationic) lipid, 10% DSPC, 38.5% cholesterol and 1.5% PEG2000-conjugated lipid represents the gold standard for LNP formulation. For example, recent publications reporting innovative ionizable lipids generally use the above composition. See Sabnis, S. et al. "A novel aminolipid series for mRNA delivery: improved endosomal escape and sustained pharmacology and safety in non-human primates." Molecular Therapy 26.6 (2018): 1509-1519; Li, S. et al. "Payload distribution and capacity of mRNA lipid nanoparticles." Nature Communications 13.1 (2022): 5561; Herrera-Barrera, M. et al. "Peptide-guided lipid nanoparticles deliver mRNA to the neural retina of rodents and non-human primates." nonhuman primates). "Science Advances" 9.2(2023):eadd4623; and Chen, J. et al. "Lipid nanoparticle-mediated lymph node-targeting delivery of mRNA cancer vaccine elicits robust CD8+T cell response." Proceedings of the National Academy of Sciences 119.34(2022):e2207841119.

[0006] However, immunogenic ionizable lipids can cause adverse reactions in patients, ranging from mild symptoms to life-threatening symptoms. See Szebeni, J. et al. "Applying lessons learned from nanomedicines to understand rare hypersensitivity reactions to mRNA-based SARS-CoV-2 vaccines." Nature Nanotechnology 17.4 (2022): 337-346; Oster, ME et al. "Myocarditis cases reported after mRNA-based COVID-19 vaccination in the US from December 2020 to August 2021." JAMA 327.4 (2022): 331-340. The toxicity of LNPs severely limits their application in genetic vaccines and genetic therapeutics, especially for therapeutics that require higher doses of LNPs using repeated dosing regimens.

[0007] Therefore, there is a need to develop next-generation LNPs that can improve DNA / RNA delivery efficiency without cytotoxicity. Summary of the invention

[0008] The present disclosure provides compositions and methods involving polymer-advanced lipid nanoparticles (PALNPs) for delivering nucleic acids (eg, DNA or RNA) to cells.

[0009] Compared to currently FDA-approved LNPs, the PALNPs disclosed herein exhibit one or more or all of the following superior features: i) plasmid DNA and mRNA delivery; ii) 10-50-fold reduction in mRNA dose; iii) approximately 100-300-fold reduction in the dose of cationic (ionizable) lipids; iv) more than 95% cell transfection success rate; v) 10-60-fold higher protein expression; vi) rapid cell uptake and protein expression; vii) non-cytotoxicity; and viii) sustained gene proliferation and protein expression. All of the key factors for significant improvements in combined transfection, including low mRNA dose, robust cell transfection rate, and high protein expression, increase the mRNA / plasmid DNA delivery capacity of PALNPs by hundreds of times compared to currently FDA-approved control LNPs.

[0010] In one aspect, the present disclosure relates to nanoparticle compositions comprising a lipid component and a polymer component, wherein in some embodiments, the polymer component comprises:

[0011] (a) Formula I (PEO x -PPO y -PEO z )

[0012]

[0013] In some embodiments, x is a number selected from 1-100, y is a number selected from 10-500, and z is a number selected from 1-100;

[0014] (b) Formula II (PPO x -PEO y -PPO z )

[0015]

[0016] In some embodiments, x is a number selected from 10-500, y is a number selected from 1-100, and z is a number selected from 10-500;

[0017] (c) Formula III (PEO y -PPO z )

[0018]

[0019] In some embodiments, R1 is H or CH3, R2 is OH or OCH3, y is a number selected from 1-100, and z is a number selected from 5-500; and / or

[0020] (d) Formula IV (PPO x )

[0021]

[0022] In some embodiments, x is a number selected from 1-100.

[0023] In some embodiments, the polymer component comprises about 0.1 mol % to about 20 mol % of the nanoparticle composition.

[0024] In some embodiments, the lipid component comprises ionizable and / or permanently charged cationic lipids, auxiliary lipids, structural lipids and / or PEG (polyethylene glycol) lipids. In some embodiments, the ionizable and / or permanently charged cationic lipids account for about 5mol% to about 30mol% of the nanoparticle composition. In some embodiments, the auxiliary lipids are phospholipids. In some embodiments, the auxiliary lipids account for about 10mol% to about 50mol% of the nanoparticle composition. In some embodiments, the structural lipids are cholesterol. In some embodiments, the structural lipids account for about 20mol% to about 50mol% of the nanoparticle composition. In some embodiments, the mean molecular weight of the PEG lipids is about 500-5000 daltons (e.g., about 2000 daltons). In some embodiments, the PEG lipids account for about 0.5mol% to about 5mol% of the nanoparticle composition.

[0025] In some embodiments, the nanoparticle compositions described herein comprise: (a) about 1 mol% to about 20 mol% of the compound of Formula I; (b) about 5 mol% to about 30 mol% of the ionizable and / or permanently charged cationic lipid (e.g., ALC-0315, SM-102, DLin-DMA, DLin-MC3-DMA and / or DLin-KC2-DMA); (c) about 10 mol% to about 50 mol% of the auxiliary lipid (e.g., DSPC and / or DOPE); (d) about 20 mol% to about 50 mol% of the structural lipid (e.g., cholesterol); and (e) about 0.5 mol% to about 5 mol% of the PEG lipid (e.g., PEG2000-DSPE). In some embodiments, the number x and the number z are the same in Formula I. In some embodiments, the auxiliary lipid comprises about 5 mol% to about 30 mol% of DSPC and / or about 5 mol% to about 30 mol% of DOPE. In some embodiments, the average molecular weight of the compound of Formula I is from about 1000 Daltons to about 30,000 Daltons (e.g., from about 1000 Daltons to about 10,000 Daltons). In some embodiments, the number x is selected from 1-15, the number y is selected from 30-80, and the number z is selected from 1-15. In some embodiments, the compound of Formula I is L121, L92, or L81.

[0026] In some embodiments, the nanoparticle compositions described herein comprise: (a) about 1 mol% to about 20 mol% of the compound of formula II; (b) about 5 mol% to about 30 mol% of the ionizable and / or permanently charged cationic lipids (e.g., ALC-0315, SM-102, DLin-DMA, DLin-MC3-DMA and / or DLin-KC2-DMA); (c) about 10 mol% to about 50 mol% of the auxiliary lipids (e.g., DSPC and / or DOPE); (d) about 20 mol% to about 50 mol% of the structural lipids (e.g., cholesterol); and (e) about 0.5 mol% to about 5 mol% of the PEG lipids (e.g., PEG2000-DSPE). In some embodiments, the quantity x and the quantity z are the same in formula II. In some embodiments, the auxiliary lipids comprise about 5 mol% to about 30 mol% of DSPC and / or about 5 mol% to about 30 mol% of DOPE. In some embodiments, the average molecular weight of the compound of formula II is from about 1000 Daltons to about 30,000 Daltons (e.g., from about 1000 Daltons to about 10,000 Daltons). In some embodiments, the number x is selected from 10-50, the number y is selected from 1-30, and the number z is selected from 10-50. In some embodiments, the compound of formula II is L31R1 or L17R4.

[0027] In some embodiments, the nanoparticle compositions described herein comprise: (a) about 1 mol% to about 20 mol% of the compound of formula IV; (b) about 5 mol% to about 30 mol% of the ionizable and / or permanently charged cationic lipid (e.g., ALC-0315, SM-102, DLin-DMA, DLin-MC3-DMA and / or DLin-KC2-DMA); (c) about 10 mol% to about 50 mol% of the auxiliary lipid (e.g., DSPC and / or DOPE); (d) about 20 mol% to about 50 mol% of the structural lipid (e.g., cholesterol); and (e) about 0.5 mol% to about 5 mol% of the PEG lipid (e.g., PEG2000-DSPE). In some embodiments, the auxiliary lipid comprises about 5 mol% to about 30 mol% of DSPC and / or about 5 mol% to about 30 mol% of DOPE. In some embodiments, the compound of Formula IV has an average molecular weight of about 1000 Daltons to about 30,000 Daltons (e.g., about 1000 Daltons to about 10,000 Daltons). In some embodiments, the number x in the compound of Formula IV is about 30 to about 60. In some embodiments, the compound of Formula IV is PPO 2700 .

[0028] In one aspect, the disclosure relates to lipid nanoparticles (LNPs) having nanoparticle compositions as described herein. In some embodiments, the LNPs described herein further comprise a nucleic acid. In some embodiments, the nucleic acid comprises DNA (e.g., double-stranded DNA (dsDNA), plasmid DNA, single-stranded DNA (ssDNA) or its antisense DNA) or RNA (e.g., small interfering RNA (siRNA), microRNA (miRNA), messenger mRNA (mRNA), guide RNA (gRNA), circular RNA (circRNA), self-amplifying RNA (saRNA) or its antisense RNA).

[0029] In some embodiments, the nanoparticle composition has an N / P ratio of about 0.1 to about 10 (for example, about 0.1 to about 5, about 0.1 to about 4, about 0.1 to about 3, about 0.2 to about 2, or about 0.2 to about 1.5).

[0030] In some embodiments, the hydrophilic lipophilic balance (HLB) value of the compound of Formula I, Formula II, Formula III, Formula IV and / or Formula V is 1 to 18. In some embodiments, the average size of the LNP is about 30nm to about 2000nm (e.g., about 30nm to about 1000nm or about 30nm to about 500nm). In some embodiments, the polydispersity index of the LNP is about 0.001 to about 0.5 (e.g., about 0.01 to about 0.3). In some embodiments, the zeta potential of the LNP is about -30mV to about +20mV. In some embodiments, the w / w ratio of the lipid component to the nucleic acid is about 2: 1 to about 50: 1 (e.g., about 2: 1 to about 20: 1). In some embodiments, the encapsulation efficiency of the nucleic acid is at least 50%, at least 60%, at least 70%, at least 80% or at least 90%.

[0031] On the one hand, the present disclosure relates to a method for delivering nucleic acid to mammalian cells, in some embodiments, the method comprises administering to a subject an LNP as described herein, in some embodiments, the administration comprises contacting the mammalian cell with the nanoparticle composition, thereby delivering the nucleic acid to the mammalian cell. In some embodiments, the mammalian cell is located in a mammal. In some embodiments, the LNP is associated with a therapeutic drug, a vaccine (e.g., a prophylactic vaccine or a therapeutic vaccine), gene editing, or a cell-based therapy (e.g., a chimeric antigen receptor (CAR)-T therapy). In some embodiments, the LNP is associated with the treatment of a disease (e.g., an infectious disease, an autoimmune disease, a cancer, or a genetic disorder). In some embodiments, the LNP is delivered by oral administration, nasal administration, skin administration, intravenous administration, topical administration, ocular administration, and / or mucosal administration, intradermal administration, and intramuscular administration.

[0032] In one aspect, the present disclosure relates to a method for enhancing delivery of a nucleic acid to a target tissue, which in some embodiments comprises administering to a subject an LNP as described herein, which in some embodiments comprises contacting the target tissue with the LNP, thereby delivering the nucleic acid to the target tissue.

[0033] In one aspect, the present disclosure relates to a method of producing a polypeptide of interest in a mammalian cell, the method comprising administering to a subject a LNP as described herein, wherein in some embodiments, the nucleic acid encodes the polypeptide of interest, whereby the nucleic acid can be translated in the mammalian cell to produce the polypeptide of interest.

[0034] In one aspect, the disclosure relates to a method for reducing the expression of a target polypeptide (or protein) in a mammalian cell, the method comprising administering to a subject an LNP as described herein. In some embodiments, the LNP is loaded with one or more antisense polynucleotides (e.g., siRNA) that are substantially complementary to a sequence encoding the target polypeptide (or protein).

[0035] In one aspect, the present disclosure relates to a method for preparing an LNP having a nanoparticle composition comprising a lipid component and a polymer component, in some embodiments, the lipid component comprises: ionizable and / or permanently charged cationic lipids, helper lipids, structural lipids, and PEG (polyethylene glycol) lipids, in some embodiments, the polymer component comprises: a compound of Formula I, Formula II, Formula III, and / or Formula IV, in some embodiments, the method comprises: (a) introducing one or more streams of a solution of lipids in a water-miscible organic solvent through a first set of one or more inlet ports connected to a mixing chamber, in some embodiments, the lipid solution comprises the lipid component and the polymer component, (b) introducing one or more streams of an aqueous solution through a second set of one or more inlet ports connected to the mixing chamber, (c) mixing the one or more streams of the lipid solution and the one or more streams of the aqueous solution in a mixing chamber to produce the LNP, and (d) recovering the LNP through one or more outlet ports connected to the mixing chamber. In some embodiments, the aqueous solution comprises nucleic acids. In some embodiments, the angle between any inlet port in the first set of one or more inlet ports and any inlet port in the second set of one or more inlet ports is 0-180 degrees.

[0036] As used herein, the term "approximately" and "about" as applied to one or more values ​​of interest refer to values ​​similar to the stated reference values. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​falling within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater than or less than) of the stated reference value, unless otherwise stated or otherwise apparent from the context (except for the case where this quantity will exceed 100% of the possible value). For example, when used in the context of the amount of a given compound in the lipid component of a nanoparticle composition, "approximately" can mean + / -10% of the listed value. For example, a nanoparticle composition including a lipid component with about 40% of a given compound can include 30-50% of the compound.

[0037] As used herein, the term "delivery" means providing an entity to a destination. For example, delivering mRNA to a subject may involve administering a nanoparticle composition comprising the mRNA to the subject (e.g., by intravenous, intramuscular, intradermal, or subcutaneous routes). Administering a nanoparticle composition to a mammal or mammalian cell may involve contacting one or more cells with the nanoparticle composition.

[0038] As used herein, a "lipid component" is a component of a nanoparticle composition that includes one or more lipids. For example, the lipid component can include one or more ionizable and / or permanently charged cationic lipids, helper lipids, structural lipids, PEG lipids or other lipids. As used herein, a "polymer component" is a component of a nanoparticle composition that includes one or more polymers.

[0039] The term "alkyl" used alone or in combination with other terms refers to a saturated hydrocarbon group which may be straight or branched. n-m"Alkyl" refers to an alkyl group having n to m carbon atoms. Alkyl formally corresponds to an alkane in which one CH bond is replaced by the point of attachment of the alkyl group to the rest of the compound. The alkyl group may be unsubstituted or substituted. The alkyl group may be substituted by one or more substituents (e.g., one or more halo as defined herein; haloalkyl as defined herein; alkoxy as defined herein; hydroxyalkyl as defined herein; amino as defined herein; amide as defined herein; cyano (e.g., -CN group); nitro (e.g., -NO2 group); hydroxyl (e.g., , -OH group); carboxyl (e.g., -CO2H group); oxo (e.g., =O group); carbaldehyde (e.g., -C(O)H group); etc., and combinations thereof. In some embodiments, the unsubstituted alkyl group contains 1 to 6, 1 to 4, 1 to 3, or 1 to 2 carbon atoms. Examples of alkyl groups include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, higher homologs (e.g., such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl), etc.

[0040] As used herein, the term "alkylene" refers to a polyvalent (e.g., divalent) form of an alkyl group, as described herein. Exemplary alkylene groups include methylene, ethylene, propylene, butylene, and the like. In some embodiments, the alkylene group is C 1-3 , C 1-6 , C 1-12 , C 1-16 , C 1-18 , C 1-20 , C 1-24 , C 2-3 , C 2-6 , C 2-12 , C 2-16 , C 2-18 , C 2-20 or C 2-24 Alkylene. Alkylene can be branched or unbranched. Alkylene can also be substituted or unsubstituted. For example, alkylene can be substituted with one or more substituents, as described herein for alkyl.

[0041] The term "alkenyl," used alone or in combination with other terms, refers to a straight or branched hydrocarbon group corresponding to an alkyl group having one or more carbon-carbon double bonds. Alkenyl formally corresponds to an alkene in which one C-H bond is replaced by the point of attachment of the alkenyl group to the rest of the compound. Alkenyl groups may be unsubstituted or substituted (e.g., substituted with one or more substituents, as described herein for alkyl groups). The term "C- n-m"Alkenyl" refers to an alkenyl group having n to m carbons. In some embodiments, the unsubstituted alkenyl group contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms. Examples of alkenyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl, and the like.

[0042] The term "alkynyl," used alone or in combination with other terms, refers to a straight or branched hydrocarbon group corresponding to an alkyl group having one or more carbon-carbon triple bonds. Alkynyl formally corresponds to an alkyne in which one C-H bond is replaced by an alkyl group at the point of attachment to the rest of the compound. Alkynyl groups may be unsubstituted or substituted (e.g., substituted with one or more substituents, as described herein for alkyl groups). The term "C- n-m "Alkynyl" refers to an alkynyl group having n to m carbon atoms. In some embodiments, the unsubstituted alkynyl group contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl, and the like.

[0043] The term "alkoxy," used alone or in combination with other terms, refers to a radical of the formula -O-alkyl, wherein the alkyl radical is as defined above. The alkoxy radical may be unsubstituted or substituted (e.g., substituted with one or more substituents, as described herein for alkyl). n-m "Alkoxy" refers to an alkoxy group whose alkyl group has n to m carbons. Examples of alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), tert-butoxy, and the like. In some embodiments, the alkyl group of the unsubstituted alkoxy group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0044] As used herein, the term "amide" refers to a group of the formula -C(O)NR N1 R N2 A group in which R N1 and R N2 Each of R is independently H or optionally substituted alkyl, or R N1 and R N2 Together with the respective nitrogen atom to which they are attached, they form a heterocyclic group.

[0045] As used herein, the term "amino" refers to a group of the formula -NR N1 R N2 A group in which R N1 and R N2 Each of R is independently H or optionally substituted alkyl, or R N1 and R N2 Together with the respective nitrogen atom to which they are attached, they form a heterocyclic group.

[0046] The term "halo" or "halogen" used alone or in combination with other terms refers to fluorine, chlorine, bromine and / or iodine. In some embodiments, "halo" refers to a halogen atom selected from F, Cl or Br.

[0047] As used herein, the term "haloalkyl" refers to an alkyl group in which one or more of the hydrogen atoms have been replaced by a halogen atom. n-m "Haloalkyl" refers to a C group having n to m carbon atoms and at least one to {2(n to m)+1} halogen atoms. n-m In some embodiments, the halogenated alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0048] Atom. Examples of haloalkyl groups include CF3, C2F5, CHF2, CCl3, CHCl2, C2Cl5, and the like.

[0049] The term "haloalkoxy" used alone or in combination with other terms refers to a radical of the formula -O-haloalkyl, wherein the haloalkyl is as defined above. n-m "Haloalkoxy" refers to a haloalkoxy group whose haloalkyl group has n to m carbon atoms. Examples of haloalkoxy groups include trifluoromethoxy, difluoromethoxy, and the like. In some embodiments, the haloalkoxy group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. As used herein, the term "hydroxyalkyl" refers to an alkyl group in which one or more of the hydrogen atoms have been replaced by a hydroxyl group (e.g., -OH group). The term "C n-m "Hydroxyalkyl" refers to a C 2- to C 3-alkyl group having n to m carbon atoms and at least one to {2 (n to m) + 1} hydroxyl group. n-m In some embodiments, the hydroxyalkyl group includes one to three hydroxyl groups. In some embodiments, the hydroxyalkyl group has 1 to 6, or 1 to 4, or 1 to 3 carbon atoms. Examples of hydroxyalkyl groups include hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, etc.

[0050] The term "salt" means an ionic form of a compound or structure (e.g., any formula, compound or composition described herein), which includes a cationic or anionic compound to form an electrically neutral compound or structure. Salts (e.g., simple salts, double salts, triple salts, etc. having binary compounds) are well known in the art. Salts are described, for example, in Berge SM et al., "Pharmaceutical salts," J. Pharm. Sci. Jan. 1977; 66(1): 1 19; International Union of Pure and Applied Chemistry. "Nomenclature of Inorganic Chemistry," Butterworth & Co. (Publishers) Ltd., London, England, 1971 (2nd ed.); and "Handbook of Pharmaceutical Salts: Properties, Selection, and Use," Wiley-VCH, April 2011 (2nd revised ed., PH Stahl and CG Wermuth, eds.). The salts can be prepared in situ during the final isolation and purification of the compounds, or separately by reacting the free base group with a suitable organic acid (thereby producing anionic salts) or by reacting the acid group with a suitable metal or organic salt (thereby producing cationic salts).Representative anionic salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, camphorate, camphorsulfonate, chloride, citrate, cyclopentanepropionate, digluconate, dihydrochloride, diphosphate, dodecylsulfate, edetate, ethanesulfonate, fumarate, glucoheptonate, gluconate, glutamate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, isethionate, hydroxynaphthoate, iodide, lactate, lactobionate, laurate, lauryl sulfate , malate, maleate, malonate, mandelate, methanesulfonate, mesylate, methyl bromide, methyl nitrate, methyl sulfate, mucate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, acetate, succinate, sulfate, tannate, tartrate, theophylline, thiocyanate, triethiodide, toluenesulfonate, undecanoate, valerate, etc. Representative cationic salts include metal salts, such as alkali metal salts or alkaline earth metal salts, for example, barium, calcium (e.g., calcium acetate), lithium, magnesium, potassium, sodium, etc.; other metal salts, such as aluminum, bismuth, iron, and zinc; and non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, pyridine, etc. Other cationic salts include organic salts, such as chloroprocaine, choline, dibenzylethylenediamine, diethanolamine, ethylenediamine, methylglucamine, and procaine.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

[0052] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1GFP fluorescence images of HEK293 cells are shown. Upper row: cells transfected with 0.6 μg GFP mRNA delivered by control LNPs (N / P=6) of the indicated cationic lipids (cationic lipid 50%, DSPC 10%, cholesterol 38.5%, PEG2000-DSPE 1.5%). Lower row: 0.1 μg GFP mRNA delivered by PALNPs (N / P=6).

[0054] Figure 2 Shown are flow cytometry results of cells transfected with CPT108 containing 0.05 μg of GFP mRNA and cells transfected with CPT107 ("ACL-0315") containing 0.6 μg of GFP mRNA (control).

[0055] Figure 3 The mean fluorescence intensity and percentage of GFP-positive cells determined by FACS are shown. Cells were transfected with CPT108 loaded with 50 ng, 100 ng, 200 ng or 400 ng GFP mRNA. Cells transfected with CPT107 ("ACL-0315") loaded with 600 ng GFP mRNA were used as controls.

[0056] Figure 4 mRNA delivery enhancement curves are shown. The overall enhancement was calculated as the product of the ratios (mRNA dose, cell transfection efficiency, and fluorescence intensity) between PALNPs and corresponding control LNPs.

[0057] Figures 5A-5C GFP fluorescence images of HEK293 cells transfected with CPT147 LNPs ( FIG. 5A : 0.01 μg mRNA, N / P=1; FIG. 5B : 0.3 μg mRNA, N / P=1) or CPT107 LNPs ( FIG. 5C : 0.3 μg mRNA, N / P=6) are shown.

[0058] Figure 6A shows a set of fluorescent images of cells transfected with a fixed amount of CPT147 LNPs loaded with GFP mRNA. The mRNA was 0.025-0.4 μg, and the corresponding N / P ratio ranged from 0.25 to 4.

[0059] Figure 6B shows a set of fluorescent images of cells transfected with CPT147 LNPs loaded with GFP mRNA. mRNA was fixed at 0.05 μg, and the N / P ratio ranged from 0.25 to 4.

[0060] Figure 6C shows a set of fluorescent images of cells transfected with CPT147-38 LNPs loaded with GFP mRNA. mRNA was fixed at 0.25 μg, and the N / P ratio ranged from 0.25 to 2.

[0061] Figure 6D shows a set of fluorescent images of cells transfected with CPT163-21-18 LNPs loaded with GFP mRNA. mRNA was fixed at 0.25 μg, and the N / P ratio ranged from 0.25 to 2.

[0062] Figure 7 A set of fluorescent images of cells transfected with CPT147 (N / P=1) for 1 hour, 2 hours or 4 hours are shown. The red fluorescence emitted by the cells originates from the LNPs labeled with rhodamine-DSPC (Rhd-LNPs), and the green fluorescence originates from the expressed GFP protein (GFP).

[0063] Fig. 8A Shown are fluorescent images of cells transfected with CPT147 labeled with rhodamine-DSPC 2 hours after transfection.

[0064] Figure 8B Shown are fluorescent images of cells transfected with control LNPs (CPT107) labeled with rhodamine-DSPC 2 hours after transfection.

[0065] Fig. 9A Fluorescence images of cells transfected with CPT147 LNPs loaded with GFP mRNA for 1 hour and then cultured in fresh medium for 3 hours are shown. CPT147 LNPs were labeled with Rhodamine-DSPC.

[0066] Fig. 9B Fluorescence images of cells transfected with CPT147 LNPs loaded with GFP mRNA for 4 hours are shown. CPT147 LNPs were labeled with Rhodamine-DSPC.

[0067] Fig. 9C Shows Fig. 9A (Group A) and Fig. 9B (Panel B) Schematic diagram of mRNA transfection.

[0068] Figures 10A-10B Shown are GFP fluorescence images and bright field images of HEK293 cells 96 hours after transfection with CPT147 LNPs (mRNA 0.05 μg, N / P=1).

[0069] Figures 11A-11B show the results of transfection of CPT147 cells loaded with 0.05 μg GFP plasmid DNA for 48 hours ( Fig.12 A) and 120 hours ( Fig.12 B) Fluorescence image of cells.

[0070] FIG11C shows the overlapped GFP fluorescence image. Fig.12 B is a magnified bright field image of the cells in the rectangle.

[0071] Figures 11D-11E show fluorescent images of cells that were split from the cells in Figure 11B and cultured in fresh medium for an additional 24 hours (Figure 11D) or 144 hours (Figure 11E). Cell colonies that emit green fluorescent signals are marked with circles.

[0072] Figures 11F-11G show fluorescent images of cells that were split from the cells in Figure 11E and cultured in fresh medium for an additional 24 hours (Figure 11F) or 120 hours (Figure 11G). Cell colonies that emit green fluorescent signals are marked by circles.

[0073] Fig.12 A set of fluorescent images of cells transfected with CPT147E LNPs loaded with 0.05 μg GFP mRNA is shown (N / P=1; transfection time 24 hours). LNPs were stored at 25°C for 0 hours ("T0"), 4 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 120 hours, or 168 hours before transfection.

[0074] Fig.13 Shown is a set of fluorescent images of cells transfected with CPT147 ELNPs loaded with 0.05 μg, 0.01 μg, 0.005 μg or 0.0025 μg GFP mRNA. The LNPs were stored at 25°C for 0 h, 48 h, 72 h, 120 h or 168 h before transfection.

[0075] 14A-14F show the use of CPT147E-05 (FIG. 14A), CPT147E 对照 (Figure 14B), CPT149E-01 (Figure 14C), CPT149E 对照 ( FIG. 14D ), CPT162E-01 ( FIG. 14E ), or CPT162E 对照 (FIG. 14F) A set of fluorescent images of transfected cells. LNPs were loaded with 0.2 μg, 0.1 μg, 0.05 μg or 0.025 μg GFP mRNA.

[0076] Fig.15 The results show that the ionizable lipids and PEO x -PPO y -PEO x A set of fluorescent images of HEK-293 cells transfected with LNPs made of triblock copolymers. The LNPs were loaded with 0.2 μg, 0.1 μg, or 0.05 μg of GFP mRNA.

[0077] Fig.16Fluorescence images of HEK-293 cells transfected with CPT153E LNPs are shown. The amount of GFP mRNA (μg / well) is specified on top of the images.

[0078] Fig.17 Fluorescence images of HEK-293 cells transfected with CPT189 LNPs (top row) and CPT202 LNPs (bottom row) are shown. The amount of GFP mRNA (μg / well) is specified on top of the images.

[0079] Figures 18A-18B Fluorescence images of HEK-293 cells transfected with base LNPs plus copolymers L81 or L92 at different molar ratios (mol%) are shown. The molar ratios of the polymers are specified at the top of the image. 0.25 μg of GFP mRNA was added to each well. Images were taken 24 hours after LNPs were added to the cells.

[0080] Figures 19A-19D show bright field images of cells transfected with CPT147P (Figure 19A), CPT107 (Figure 19B), CPT149E (Figure 19C), or CPT109 (Figure 19D) containing 0.2 μg of GFP mRNA for 24 hours.

[0081] Figures 20A-20B Shown are body images of BALB / c mice injected with LNPs carrying luciferase reporter mRNA for 4, 8, 12 or 24 hours. PALNP CPT147E-10 and the corresponding reference LNP CPT107 containing 10 μg of luciferase mRNA were injected via the tail vein of mice.

[0082] Fig.21 Shown are body images of BALB / c mice injected with LNPs carrying luciferase reporter mRNA for 6, 12 or 24 hours. PALNP CPT147E-10 and the corresponding reference LNP CPT107 containing 1 μg of luciferase mRNA were injected via the tail vein of mice.

[0083] Fig. 22 Shown are body images of BALB / c mice injected with LNPs carrying luciferase reporter mRNA for 4, 8, 12, 24, 48 or 72 hours. PALNP CPT147E-10 and the corresponding reference LNP CPT107 containing 1 μg of luciferase mRNA were injected via the tail vein of mice.

[0084] Fig.23 Liver and spleen targeted distribution of mRNA delivered by LNP CPT147E-10 is shown.

[0085] Fig.24 Shown are body images of BALB / c mice injected with LNP CPT147 by intramuscular injection. Images were taken at 4 hours, 8 hours, 12 hours, 24 hours, 36 hours, or 48 hours after injection.

[0086] Fig.25 Shown is the T-mixing setup used to produce LNPs.

[0087] Fig.26 Particle size distribution (size / polydispersity) of LNPs is shown. Blank LNPs (128.3 nm / 0.220); mRNA LNPs prepared from blank LNPs and mRNA (180.1 nm / 0.125); and mRNA LNPs prepared by direct mixing of lipids and mRNA (164.7 nm / 0.075) are marked with arrows.

[0088] Fig. 27 Fluorescence images of HEK-293 cells transfected with CPT201 LNPs are shown. The amount of GFP mRNA (μg / well) is specified on top of the images. DETAILED DESCRIPTION

[0089] The disclosure provides a safe and efficient LNP (e.g., with up to 95%) deduction of ionizable lipid uses compared to standard LNP. However, since the development of the first LNP, the molar ratio of cation / ionizable lipid has been maintained at about 50mol%, and the effective N / P ratio in the siRNA therapeutic agent and mRNA vaccine approved by the FDA is 3-6. This is because the second effect of ionizable lipids destroying the lysosomal membrane to allow mRNA to escape requires a large amount of ionizable lipids. Simply reducing ionizable lipids can endanger the function of LNP. Therefore, the disclosure is intended to identify non-immunogenic, non-toxic materials for LNP confirmed clinically to enhance its cellular uptake and promote mRNA delivery.

[0090] Poloxamers, also known as Pluronics, are a class of nonionic surfactants widely used in pharmaceuticals, cosmetics, and various industrial applications. The poloxamer is a copolymer composed of polyoxyethylene (PEO) and polyoxypropylene (PPO) blocks arranged in a specific structure. The ratio of these blocks and the length of the polyoxyethylene and polyoxypropylene chains determine the properties of the poloxamer. The general formula of poloxamer (Pluronic) is:

[0091] HO-(CH2-CH2-O) n -(CH2-CH(CH3)-O) m -(CH2-CH2-O) n -H

[0092] Where n and m represent the number of repeating units of the polyoxyethylene and polyoxypropylene blocks, respectively. The values ​​of n and m may vary depending on the specific poloxamer product.

[0093] Generally, the typical structure of poloxamer is a triblock copolymer, PEO n -PPO m -PEO n or PPO m -PEO n -PPO m At the same time, PEO n -PPO m Diblock copolymers of are also classified as poloxamers. See Alvarez-Lorenzo, C., A. Sosnik and A. Concheiro. "PEO-PPO block copolymers for passive micellar targeting and overcoming multidrug resistance in cancer therapy." "Current Drug Targets" 12.8 (2011): 1112-1130. As an extreme condition, when m or n is equal to 0, the polymer PEO or PPO can also be called poloxamer.

[0094] In addition, poloxamer is non-toxic and non-irritating, making it an attractive material for biomedical applications. In addition, poloxamer has a proven record of FDA approval and is listed in the U.S. and European Pharmacopoeias as stabilizers, emulsifiers, solubilizers, and for topical, parenteral and oral administration. See Russo, E et al., "Poloxamer hydrogels for biomedical applications." Pharmaceutics 11.12 (2019): 671.

[0095] In the present disclosure, poloxamer is incorporated into lipid nanoparticles to form a new class of poloxamer-advanced lipid nanoparticles (PALNPs). In PALNPs, the N / P ratio is reduced from the standard 6 to a range of about 1-0.25 (about 6-24 times deduction), and the weight percentage of mRNA in total lipids is increased from the standard 4% to about 10-20%. In the absence of observed cytotoxicity, the efficiency of gene delivery in vitro and in vivo is significantly increased.

[0096] In certain embodiments, the disclosure relates to nanoparticle compositions including lipid components and polymer components and methods of use thereof. In certain embodiments, the disclosure provides methods for producing a polypeptide of interest in a cell, the method involving contacting an LNP (e.g., any LNP in the LNP described herein) of a load nucleic acid with a mammalian cell, whereby the nucleic acid can be translated to produce a polypeptide of interest. The disclosure further includes methods for delivering nucleic acids to mammalian cells, the method involving applying an LNP (e.g., any LNP in the LNP described herein) of a load nucleic acid to a subject, whereby the application involves contacting a cell with the LNP, whereby the nucleic acid is delivered to the cell.

[0097] In some embodiments, the N / P ratio of a LNP (e.g., PALNP) or a LNP having a nanoparticle composition described herein is less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% compared to a reference LNP that does not include a polymer component (e.g., any of the polymer components described herein), except for polyethylene glycol-conjugated lipids (e.g., LNPs for use as a COVID-10 vaccine described herein).

[0098] In some embodiments, the LNP (e.g., PALNP) or LNP with a nanoparticle composition described herein is more stable than a reference LNP (e.g., LNP used as a COVID-10 vaccine described herein) that does not include a polymer component (e.g., any of the polymer components described herein). For example, the LNP (e.g., PALNP) can be stored at 25° C. for at least one week without substantially affecting its transfection efficiency in cells.

[0099] In some embodiments, LNP (e.g., PALNP) or LNP with nanoparticle composition as described herein is effective for both DNA transfection and RNA transfection. In some embodiments, compared with a reference LNP (e.g., LNP used as a COVID-10 vaccine as described herein) loaded with the same amount of cargo molecules (e.g., any nucleic acid in nucleic acids as described herein) but not including a polymer component (e.g., any polymer component in polymer components as described herein), the cell transfection efficiency of LNP (e.g., PALNP) increases by at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 55 times, 60 times, 65 times, 70 times, 75 times, 80 times, 85 times, 90 times, 95 times or 100 times. In some embodiments, the cell transfection efficiency is determined by measuring the fluorescence emitted from transfected cells.

[0100] In certain embodiments, LNP (for example, PALNP) or LNP with nanoparticle composition as described herein can promote rapid cellular uptake and / or protein expression.For example, after LNP (for example, PALNP) transfection with nucleic acid encoding protein of interest using load, transfected cells can express protein of interest within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours or 24 hours.In certain embodiments, transfection can be less than 2 hours, less than 1.5 hours, less than 1 hour, less than 55 minutes, less than 50 minutes, less than 45 minutes, less than 40 minutes, less than 35 minutes, less than 30 minutes, less than 25 minutes, less than 20 minutes, less than 15 minutes, less than 10 minutes or less than 5 minutes.In certain embodiments, LNP (for example, PALNP) or LNP with nanoparticle composition as described herein can be applied to experimenter (for example, by intramuscular administration), and do not enter experimenter's systemic delivery.

[0101] In some embodiments, a LNP (e.g., PALNP) or a LNP with a nanoparticle composition described herein can increase the protein expression level of a cargo molecule (e.g., any of the nucleic acids described herein) from the same dose of a nucleic acid by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold as compared to a reference LNP (e.g., a LNP that does not include a polymer component (e.g., any of the polymer components described herein), such as a LNP for use as a COVID-10 vaccine described herein).

[0102] In some embodiments, provided herein are methods of delivering cargo molecules (e.g., any nucleic acid in a nucleic acid described herein) by LNPs described herein or LNPs having nanoparticle compositions described herein. When reference is made to an LNP (e.g., an LNP that does not include a polymer component (e.g., any polymer component in a polymer component described herein), such as an LNP used as a COVID-10 vaccine described herein), the method can achieve comparable transfection efficiency using less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% dose of cargo molecules (e.g., any nucleic acid in a nucleic acid described herein).

[0103] In some embodiments, the success rate of cell transfection using LNPs (e.g., PALNPs) or LNPs with nanoparticle compositions described herein is at least 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, or 99% when less than 1000 ng, 500 ng, 200 ng, 100 ng, 50 ng, 40 ng, 30 ng, 20 ng, 10 ng, 5 ng, or 1 ng is used.

[0104] Polymer-Advanced Lipid Nanoparticles (PALNPs)

[0105] In some embodiments, the nanoparticle compositions described herein are PALNPs. In some embodiments, the nanoparticle compositions described herein include a lipid component (e.g., any lipid component of the lipid components described herein), a polymer component (e.g., any polymer component of the polymer components described herein). In some embodiments, the nanoparticle compositions further include a cargo molecule (e.g., any nucleic acid of the nucleic acids described herein).

[0106] Polymer components

[0107] In some embodiments, the polymer components described herein include any compound or combination of compounds described herein. In some embodiments, the compounds described herein are derived from Formula I, Formula II, Formula III, or Formula IV.

[0108] Formula I (triblock copolymer)

[0109] In some embodiments, the polymer components described herein include triblock copolymers. In some embodiments, the triblock copolymer is Pluronic TM Surfactant. In some embodiments, the triblock copolymer is a poloxamer. Specifically, a poloxamer is a nonionic triblock copolymer comprising a central hydrophobic polyoxypropylene (poly(propylene oxide)) chain flanked by two polyoxyethylene (poly(ethylene oxide)) hydrophilic chains.

[0110] In some embodiments, the triblock copolymer is derived from a compound of Formula I having the general structure shown below:

[0111]

[0112] In some embodiments, Formula I is also depicted as PEO x -PPO y -PEO z , [PEO] x -[PPO] y -[PEO] z, PEO-PPO-PEO, poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide), PEG-PPG-PEG, poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol), or H[OCH2CH2] x [OCH(CH3)CH2] y [OCH2CH2] z OH. In some embodiments, x, y and z in Formula I or their equivalents are numbers. For example, x is a number selected from 1-100, y is a number selected from 10-500, and z is a number selected from 1-100. In some embodiments, the numbers x and z are the same. In some embodiments, the number y is at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times or 20 times the number x and / or z.

[0113] In some embodiments, the triblock copolymer is derived from a compound of Formula I' having the general structure shown below:

[0114] or a salt thereof,

[0115] in:

[0116] Each R3, R4, R5, R 5a , R6, R7 and R8 are independently selected from the group consisting of hydrogen (H), C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Haloalkoxy, C 1-6 Hydroxyalkyl, halo, cyano (-CN), nitro (-NO2), hydroxyl (-OH), carboxyl (-COOH), formaldehyde (-C(O)H), amide (e.g., -C(O)NH2, -C(O)NHC 1-4 Alkyl or -C(O)N(C 1-4 Alkyl)2) and amino (e.g., -NH2, -NHC 1-4 Alkyl or -N(C 1-4 alkyl)2; and

[0117] R A Selected from the group consisting of: hydrogen (H), C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl and C 1-6 Hydroxyalkyl.

[0118] In some embodiments, each of R3, R4, R5, R 5a , R6, R7, R8 and R A It's H.

[0119] In some embodiments, each of R3, R4, R5, R 5a , R6, R7 are R8 is H; and R A It's methyl.

[0120] In some embodiments, each R3 is H. In some embodiments, at least one R3 is selected from C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 1-6 In some embodiments, at least one R3 is selected from methyl, ethyl, methoxy, ethoxy, hydroxymethyl, halo and hydroxy. In some embodiments, at least one R3 is methyl. In some embodiments, at least one R3 is ethyl. In some embodiments, at least one R3 is methoxy. In some embodiments, at least one R3 is ethoxy. In some embodiments, at least one R3 is hydroxymethyl. In some embodiments, at least one R3 is halo. In some embodiments, at least one R3 is hydroxy.

[0121] In some embodiments, each R4 is H. In some embodiments, at least one R4 is selected from C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 1-6 In some embodiments, at least one R4 is selected from methyl, ethyl, methoxy, ethoxy, hydroxymethyl, halo and hydroxy. In some embodiments, at least one R4 is methyl. In some embodiments, at least one R4 is ethyl. In some embodiments, at least one R4 is methoxy. In some embodiments, at least one R4 is ethoxy. In some embodiments, at least one R4 is hydroxymethyl. In some embodiments, at least one R4 is halo. In some embodiments, at least one R4 is hydroxy.

[0122] In some embodiments, R5 is H. In some embodiments, R5 is selected from C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 1-6In some embodiments, R5 is selected from methyl, ethyl, methoxy, ethoxy, halo and hydroxy. In some embodiments, R5 is methyl. In some embodiments, R5 is ethyl. In some embodiments, R5 is methoxy. In some embodiments, R5 is ethoxy. In some embodiments, R5 is hydroxymethyl. In some embodiments, R5 is halo. In some embodiments, R5 is hydroxy.

[0123] In some embodiments, R 5a is H. In some embodiments, R 5a Selected from C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 1-6 In some embodiments, R 5a is selected from the group consisting of methyl, ethyl, methoxy, ethoxy, halo and hydroxy. 5a In some embodiments, R 5a In some embodiments, R 5a In some embodiments, R 5a In some embodiments, R 5a In some embodiments, R 5a In some embodiments, R 5a It's a hydroxyl group.

[0124] In some embodiments, each R6 is H. In some embodiments, at least one R6 is selected from C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 1-6 In some embodiments, at least one R6 is selected from methyl, ethyl, methoxy, ethoxy, halo and hydroxy. In some embodiments, at least one R6 is methyl. In some embodiments, at least one R6 is ethyl. In some embodiments, at least one R6 is methoxy. In some embodiments, at least one R6 is ethoxy. In some embodiments, at least one R6 is hydroxymethyl. In some embodiments, at least one R6 is halo. In some embodiments, at least one R6 is hydroxy.

[0125] In some embodiments, each R7 is H. In some embodiments, at least one R7 is selected from C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6Halogenated alkoxy, C 1-6 In some embodiments, at least one R7 is selected from methyl, ethyl, methoxy, ethoxy, halo and hydroxy. In some embodiments, at least one R7 is methyl. In some embodiments, at least one R7 is ethyl. In some embodiments, at least one R7 is methoxy. In some embodiments, at least one R7 is ethoxy. In some embodiments, at least one R7 is hydroxymethyl. In some embodiments, at least one R7 is halo. In some embodiments, at least one R7 is hydroxy.

[0126] In some embodiments, each R8 is H. In some embodiments, at least one R8 is selected from C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 1-6 In some embodiments, at least one R8 is selected from methyl, ethyl, methoxy, ethoxy, halo and hydroxy. In some embodiments, at least one R8 is methyl. In some embodiments, at least one R8 is ethyl. In some embodiments, at least one R8 is methoxy. In some embodiments, at least one R8 is ethoxy. In some embodiments, at least one R8 is hydroxymethyl. In some embodiments, at least one R8 is halo.

[0127] In some embodiments, R A is H. In some embodiments, R A Selected from C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl and C 1-6 In some embodiments, R A In some embodiments, R A In some embodiments,

[0128] R A In some embodiments, R A It's hydroxymethyl.

[0129] In some embodiments, at least one R8 is hydroxyl. In some embodiments, the triblock copolymer is derived from a compound of formula I" having the general structure shown below:

[0130] or a salt thereof,

[0131] in:

[0132] R1 is hydrogen (H), optionally substituted alkyl (e.g., C1-6 , C 1-4 , C 1-3 or C 1-2 alkyl) or optionally substituted hydroxyalkyl (e.g., C 1-6 , C 1-4 , C 1-3 or C 1-2 Hydroxyalkyl);

[0133] R2 is hydroxy (-OH), optionally substituted alkoxy (e.g., C 1-6 , C 1-4 , C 1-3 or C 1-2 alkoxy) or optionally substituted hydroxyalkyl (e.g., C 1-6 , C 1-4 , C 1-3 or C 1-2 Hydroxyalkyl);

[0134] Each of Ak1, Ak2 and Ak3 is independently an optionally substituted alkylene group (e.g., an optionally substituted C 1-6 , C 1-4 , C 1-3 or C 1-2 Alkylene);

[0135] Each of Ak1, Ak2, and Ak3 is different; and

[0136] X, y, and z can be any x, y, and z described herein (eg, as described herein for Formula I).

[0137] In some embodiments, R1 is hydrogen or C 1-6 In some embodiments, R2 is hydroxyl or C 1-6 In some embodiments, R1 is hydrogen and R2 is hydroxy. In some embodiments, R1 is hydrogen and R2 is C 1-6 In some embodiments, R1 is C 1-6 In some embodiments, R1 is C 1-6 alkyl, and R2 is C 1-6 Alkoxy.

[0138] In some embodiments, Ak1 and Ak3 are the same, but Ak2 is different.

[0139] In some embodiments, the optionally substituted alkylene is substituted with one or more substituents. Non-limiting examples of substituents include one or more of halo, haloalkyl, alkoxy, hydroxyalkyl, amino, amide, cyano, nitro, hydroxyl, carboxyl, oxo, formaldehyde, or a combination thereof.

[0140] In some embodiments, at least one of Ak1, Ak2, and Ak3 is independently an optionally substituted C 1-6 , C 1-4 , C 1-3 or C 1-2 In some embodiments, each of Ak1, Ak2, and Ak3 is independently an optionally substituted C 1-6 , C 1-4 , C 1-3 or C 1-2 Alkylene.

[0141] In some embodiments, at least one of Ak1, Ak2, and Ak3 is independently unsubstituted C 1-6 , C 1-4 , C 1-3 or C 1-2 In some embodiments, each of Ak1, Ak2, and Ak3 is independently an unsubstituted C 1-6 , C 1-4 , C 1-3 or C 1-2 Alkylene.

[0142] In some embodiments, at least one of Ak1, Ak2, and Ak3 is independently an unbranched alkylene group (e.g., an unbranched C 1-6 , C 1-4 , C 1-3 or C 1-2 In some embodiments, at least two of Ak1, Ak2, and Ak3 are independently unbranched alkylene. In some embodiments, each of Ak2 and Ak3 is independently unbranched alkylene.

[0143] In some embodiments, at least one of Ak1, Ak2, and Ak3 is a branched alkylene group (e.g., a branched C 2-6 , C 2-4 , C 3-6 or C 3-4 In some embodiments, Ak2 is a branched alkylene group.

[0144] In some embodiments, the number x is about 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-100, 40-90, 40-80, 40-70, 40-60, 40- 50, 50-100, 50-90, 50-80, 50-70, 50-60, 60-100, 60-90, 60-80, 60-70, 70-100, 70-90, 70-80, 80-100, 80-90 or 90-100; the number y is about 10-500, 10-450, 10-400, 10-350, 10-300, 10-250, 10-200, 10-150, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-500, 20-450, 20-400, 20-350, 20-300, 20-250, 20-200, 20-150, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-500, 30-450, 30-400, 30-350, 30-300, 30-250, 30-200, 30-15 0, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-500, 40-450, 40-400, 40-350, 40-300, 40-250, 40-200, 40-150, 40-100, 40-90, 40-80, 40-70 , 40-60, 40-50, 50-500, 50-450, 50-400, 50-350, 50-300, 50-250, 50-200, 50-150, 50-100, 50-90, 50-80, 50-70, 50-60, 60-500, 60-450, 60-400, 60-3 50, 60-300, 60-250, 60-200, 60-150, 60-100, 60-90, 60-80, 60-70, 70-500, 70-450, 70-400, 70-350, 70-300, 70-250, 70-200, 70-150, 70-100, 70-90,70-80, 80-500, 80-450, 80-400, 80-350, 80-300, 80-250, 80-200, 80-150, 80-100, 80-90, 90-500, 90-450, 90-400, 90-350, 90-300, 90-250, 90-200, 90-150, 90-100, 100-500, 100-450, 100-400, 100-350, 100-300, 100-250, 100-2 00, 100-150, 150-500, 150-450, 150-400, 150-350, 150-300, 150-250, 150-200, 200-500, 200-450, 200-400, 200-350, 200-300, 200-250, 250-500, 250-450, 250-400, 250-350, 250-300, 300-500, 300-450, 300-400, 300-350, 350-5 00, 350-450, 350-400, 400-500, 400-450 or 450-500; and the number z is about 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-100, 20-90, 20-80, 20-70, 20-60, 2 In some embodiments, the number x is selected from 1-15, the number y is selected from 30-80, and the number z is selected from 1-15.

[0145] In some embodiments, the number x is about 1, 2, 3, 4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 7, 8, 9, or 10 (e.g., about 5 or about 6) and the number y is about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 , 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 (e.g., about 67 or about 68), and the number z is about 1, 2, 3, 4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 7, 8, 9 or 10 (e.g., about 5 or about 6). In some embodiments, the number x is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 (e.g., about 10), the number y is about 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 (e.g., about 47), and the number z is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 (e.g., about 10).In some embodiments, the number x is about 1, 2, 3, 4, 5, 6, 7, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 10, 11, 12, 13, 14, or 15 (e.g., about 8.2) and the number y is about 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 49.5, 49.6, 49.7, 49.8, 49.9, 50, 50.1, 50.2, 50.3, 50.4, .4, 50.5, 50.6, 50.7, 50.8, 50.9, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 (e.g., about 50 or about 50.3), and the number z is 1, 2, 3, 4, 5, 6, 7, 7.5, 7.6, 7.7, 7.8, 79, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 89, 9, 10, 11, 12, 13, 14 or 15 (e.g., about 8.2). In some embodiments, the number x is about 1, 2, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, or 10 (e.g., about 3 or about 3.1), and the number y is about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 42.1, 42.2, 42.3, 42.4, 42.5, 42.6, In some embodiments, the numbers described herein (e.g., x, y, and z) are averages.

[0146] In some embodiments, the weight percentage of PEO (PEO% (w / w)) is less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of Formula I. In some embodiments, the weight percentage of PPO (PPO% (w / w)) is greater than 80%, greater than 81%, greater than 82%, greater than 83%, greater than 84%, greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%.

[0147] In some embodiments, the weight percentage of PEO is between about 1% to about 20%, between about 1% to about 15%, between about 1% to about 10%, between about 1% to about 5%, between about 5% to about 20%, between about 5% to about 15%, between about 5% to about 10%, between about 10% to about 20%, between about 10% to about 15%, or between about 15% to about 20%.

[0148] In some embodiments, the average molecular weight of Formula I is from about 1000 Daltons to about 30,000 Daltons. In some embodiments, the average molecular weight of Formula I is from about 1000 Daltons to about 20,000 Daltons, from about 1000 Daltons to about 10,000 Daltons, from about 1000 Daltons to about 9,000 Daltons, from about 1000 Daltons to about 8,000 Daltons, from about 1000 Daltons to about 7,000 Daltons, from about 1000 Daltons to about 6,000 Daltons, from about 1000 Daltons to about 5,000 Daltons, from about 1000 Daltons to about 4,000 Daltons, from about 1000 Daltons to about 3,000 Daltons, from about 1000 Daltons to about 2,000 Daltons, from about 2,000 Daltons to about 1,000 Daltons. 0 Daltons, about 2000 Daltons to about 9000 Daltons, about 2000 Daltons to about 8000 Daltons, about 2000 Daltons to about 7000 Daltons, about 2000 Daltons to about 6000 Daltons, about 2000 Daltons to about 5000 Daltons, about 2000 Daltons to about 4000 Daltons, about 2000 Daltons to about 3000 Daltons, about 3000 Daltons to about 10000 Daltons, about 3000 Daltons to about 9000 Daltons, about 3000 Daltons to about 8000 Daltons, about 3000 Daltons to about 7000 Daltons, about 3000 Daltons to about 6000 Daltons, about 3000 Daltons to about 5000 Daltons, about 3000 Daltons to about 4000 Daltons, about 4000 Daltons to about 10000 Daltons, about 4000 Daltons to about 9000 Daltons, about 4000 Daltons to about 8000 Daltons, about 4000 Daltons to about 7000 Daltons, about 4000 Daltons to about 6000 Daltons, about 4000 Daltons to about 5000 Daltons, about 5000 Daltons to about 10000 Daltons, about 5000 Daltons to about 9000 Daltons, about 5000 Daltons to about 8000 Daltons, about 5000 Daltons to about In some embodiments, the present invention relates to a molecule of the present invention to a molecule of the present invention that is at least 500 daltons to about 7000 daltons, about 5000 daltons to about 6000 daltons, about 6000 daltons to about 10000 daltons, about 6000 daltons to about 9000 daltons, about 6000 daltons to about 8000 daltons, about 6000 daltons to about 7000 daltons, about 7000 daltons to about 10000 daltons, about 7000 daltons to about 9000 daltons, about 7000 daltons to about 8000 daltons, about 8000 daltons to about 10000 daltons, about 8000 daltons to about 9000 daltons or about 9000 daltons to about 10000 daltons.In some embodiments, the average molecular weight of Formula I is about 2000 Daltons, about 2500 Daltons, about 2600 Daltons, about 2700 Daltons, about 2750 Daltons, about 2800 Daltons, about 2900 Daltons, about 3000 Daltons, about 3100 Daltons, about 3200 Daltons, about 3300 Daltons, about 3400 Daltons, about 3500 Daltons, about 3600 Daltons, about 3700 Daltons, about 3800 Daltons, about 3900 Daltons, about 4000 Daltons, about 4100 Daltons, about 4200 Daltons, about 4300 Daltons, about 4400 Daltons, about 4500 Daltons, about 4600 Daltons, about 4700 Daltons, about 4800 Daltons, about 4900 Daltons, about 5000 Daltons, about 5100 Daltons, about 5200 Daltons, about 5300 Daltons, about 5400 Daltons, about 5500 Daltons, about 5600 Daltons, about 5700 Daltons, about 5800 Daltons, about 5900 Daltons, about 6000 Daltons, about 6100 Daltons, about 6200 Daltons, about 6300 Daltons, about 6400 Daltons, about 6500 Daltons, about 6600 Daltons, about 6700 Daltons, about 6800 Daltons, about 6900 Daltons, about 7100 Daltons, about 7200 Daltons, about 7300 Daltons About 3650 Daltons, about 3700 Daltons, about 3750 Daltons, about 3800 Daltons, about 3900 Daltons, about 4000 Daltons, about 4100 Daltons, about 4200 Daltons, about 4300 Daltons, about 4400 Daltons, about 4500 Daltons, about 4600 Daltons, about 4700 Daltons, about 4800 Daltons, about 4900 Daltons or about 5000 Daltons.

[0149] In some embodiments, when the number x and the number z are the same, Formula I can also be depicted as:

[0150]

[0151] In some embodiments, the above formula is also depicted as PEO x -PPO y -PEO x , [PEO] x -[PPO] y -[PEO] x or H[OCH2CH2] x [OCH(CH3)CH2] y [OCH2CH2] x OH.

[0152] Formula II (triblock copolymer)

[0153] In some embodiments, the triblock copolymer of formula I can be reversed, that is, the PEO block is located in the center of the triblock and the two PPO blocks are located on either side of the PEO block. In some embodiments, the triblock copolymer is derived from a compound of formula II having the general structure shown below:

[0154]

[0155] In some embodiments, Formula II is also depicted as PPO x -PEO y -PPO z , [PPO] x -[PEO] y -[PPO] z, PPO-PEO-PPO, poly(propylene oxide)-poly(ethylene oxide)-poly(propylene oxide), PPG-PEG-PPG, H[OCH(CH3)CH2] x [OCH2CH2] y [OCH(CH3)CH2] z OH. In some embodiments, x, y and z in Formula I or their equivalents are numbers. For example, x is a number selected from 10-500, y is a number selected from 1-100, and z is a number selected from 10-500. In some embodiments, the numbers x and z are the same. In some embodiments, the number x and / or the number z is at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times or 20 times the number y.

[0156] In some embodiments, the triblock copolymer is derived from a compound of Formula II' having the general structure shown below:

[0157] or a salt thereof,

[0158] in:

[0159] Each R9, R 9a , R 10 , R 11 , R 12 , R 13 , R 13a and R 14 independently selected from the group consisting of: hydrogen (H), C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl, halo, cyano (-CN), nitro (-NO2), hydroxyl (OH), carboxyl (-COOH), formaldehyde (-C(O)H), amide (e.g., -C(O)NH2, -C(O)NHC 1-4 Alkyl or -C(O)N(C 1-4 Alkyl)2) and amino (e.g., -NH2, -NHC 1-4 Alkyl and -N(C 1-4 alkyl)2); and

[0160] R B Selected from the group consisting of: hydrogen (H), C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6Alkynyl, C 1-6 Haloalkyl and C 1-6 Hydroxyalkyl.

[0161] In some embodiments, each R9, R 9a , R 10 , R 11 , R 12 , R 13 , R 13a , R 14 and R B It's H.

[0162] In some embodiments, each R9, R 9a , R 10 , R 11 , R 12 , R 13 , R 13a and R 14 is H; and R B It's methyl.

[0163] In some embodiments, R9 is H. In some embodiments, R9 is selected from C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 1-6 In some embodiments, R9 is selected from methyl, ethyl, methoxy, ethoxy, hydroxymethyl, halo and hydroxy. In some embodiments, R9 is methyl. In some embodiments, R9 is ethyl. In some embodiments, R9 is methoxy. In some embodiments, R9 is ethoxy. In some embodiments, R9 is hydroxymethyl. In some embodiments, R9 is halo. In some embodiments, R9 is hydroxy.

[0164] In some embodiments, R 9a is H. In some embodiments, R 9a Selected from C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 1-6 In some embodiments, R 9a is selected from the group consisting of methyl, ethyl, methoxy, ethoxy, hydroxymethyl, halo and hydroxy. 9a In some embodiments, R 9a In some embodiments, R 9a In some embodiments, R 9a In some embodiments, R9a In some embodiments, R 9a In some embodiments, R 9a It's a hydroxyl group.

[0165] In some embodiments, each R 10 is H. In some embodiments, at least one R 10 Selected from C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl, halo, CN and OH. In some embodiments, at least one R 10 is selected from the group consisting of methyl, ethyl, methoxy, ethoxy, hydroxymethyl, halo and hydroxy. 10 is methyl. In some embodiments, at least one R 10 In some embodiments, at least one R 10 is methoxy. In some embodiments, at least one R 10 In some embodiments, at least one R 10 is hydroxymethyl. In some embodiments, at least one R 10 In some embodiments, at least one R 10 It's a hydroxyl group.

[0166] In some embodiments, each R 11 is H. In some embodiments, at least one R 11 Selected from C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl, halo, CN and OH. In some embodiments, at least one R 11 is selected from the group consisting of methyl, ethyl, methoxy, ethoxy, hydroxymethyl, halo and hydroxy. 11 is methyl. In some embodiments, at least one R 11 In some embodiments, at least one R 11 is methoxy. In some embodiments, at least one R 11 In some embodiments, at least one R 11 is hydroxymethyl. In some embodiments, at least one R 11 In some embodiments, at least one R 11 It's a hydroxyl group.

[0167] In some embodiments, each R12 is H. In some embodiments, at least one R 12 Selected from C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl, halo, CN and OH. In some embodiments, at least one R 12 is selected from the group consisting of methyl, ethyl, methoxy, ethoxy, hydroxymethyl, halo and hydroxy. 12 is methyl. In some embodiments, at least one R 12 In some embodiments, at least one R 12 is methoxy. In some embodiments, at least one R 12 In some embodiments, at least one R 12 is hydroxymethyl. In some embodiments, at least one R 12 In some embodiments, at least one R 12 It's a hydroxyl group.

[0168] In some embodiments, R 13 is H. In some embodiments, R 13 Selected from C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 1-6 In some embodiments, R 13 is selected from the group consisting of methyl, ethyl, methoxy, ethoxy, hydroxymethyl, halo and hydroxy. 13 In some embodiments, R 13 In some embodiments, R 13 In some embodiments, R 13 In some embodiments, R 13 In some embodiments, R 13 In some embodiments, R 13 It's a hydroxyl group.

[0169] In some embodiments, R 13a is H. In some embodiments, R 13a Selected from C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 1-6In some embodiments, R 13a is selected from the group consisting of methyl, ethyl, methoxy, ethoxy, hydroxymethyl, halo and hydroxy. 13a In some embodiments, R 13a In some embodiments, R 13a In some embodiments, R 13a In some embodiments, R 13a In some embodiments, R 13a In some embodiments, R 13a It's a hydroxyl group.

[0170] In some embodiments, each R 14 is H. In some embodiments, at least one R 14 Selected from C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Haloalkoxy, C 1-6 Hydroxyalkyl, halo, CN and OH. In some embodiments, at least one R 14 is selected from the group consisting of methyl, ethyl, methoxy, ethoxy, hydroxymethyl, halo and hydroxy. 14 In some embodiments, at least one R 14 In some embodiments, at least one R 14 is methoxy. In some embodiments, at least one R 14 In some embodiments, at least one R 14 is hydroxymethyl. In some embodiments, at least one R 14 In some embodiments, at least one R 14 It's a hydroxyl group.

[0171] In some embodiments, R B is H. In some embodiments, R B Selected from C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl and C 1-6 In some embodiments, R B In some embodiments, R B In some embodiments, R B In some embodiments, R B It's hydroxymethyl.

[0172] In some embodiments, the triblock copolymer is derived from a compound of Formula II" having the general structure shown below:

[0173] or a salt thereof,

[0174] in:

[0175] R1 is hydrogen (H), optionally substituted alkyl (e.g., C 1-6 , C 1-4 , C 1-3 or C 1-2 alkyl) or optionally substituted hydroxyalkyl (e.g., C 1-6 , C 1-4 , C 1-3 or C 1-2 Hydroxyalkyl);

[0176] R2 is hydroxy (-OH), optionally substituted alkoxy (e.g., C 1-6 , C 1-4 , C 1-3 or C 1-2 alkoxy) or optionally substituted hydroxyalkyl (e.g., C 1-6 , C 1-4 , C 1-3 or C 1-2 Hydroxyalkyl);

[0177] Each of Ak1, Ak2 and Ak3 is independently an optionally substituted alkylene group (e.g., an optionally substituted C 1-6 , C 1-4 , C 1-3 or C 1-2 Alkylene);

[0178] Each of Ak1, Ak2, and Ak3 is different; and

[0179] X, y, and z can be any x, y, and z described herein (eg, as described herein for Formula II).

[0180] In some embodiments, R1, R2, Ak1, Ak2, and Ak3 can be any of R1, R2, Ak1, Ak2, and Ak3 described herein (e.g., for Formula I").

[0181] In some embodiments, at least one of Ak1, Ak2, and Ak3 is independently an unbranched alkylene group (e.g., an unbranched C 1-6 , C 1-4 , C 1-3 or C 1-2 In some embodiments, Ak2 is an unbranched alkylene group.

[0182] In some embodiments, at least one of Ak1, Ak2, and Ak3 is a branched alkylene group (e.g., a branched C 2-6 , C 2-4 , C 3-6 or C 3-4 In some embodiments, each of Ak1 and Ak3 is independently a branched alkylene group.

[0183] In some embodiments, the number x is about 10-500, 10-450, 10-400, 10-350, 10-300, 10-250, 10-200, 10-150, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-500, 20-450, 20-400, 20-350, 20-300, 20-250, 20-200, 20-150, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-500, 30-450, 30-400 , 30-350, 30-300, 30-250, 30-200, 30-150, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-500, 40-450, 40-400, 40-350, 40-300, 40-250, 40- 200, 40-150, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-500, 50-450, 50-400, 50-350, 50-300, 50-250, 50-200, 50-150, 50-100, 50-90, 50-80, 50 -70, 50-60, 60-500, 60-450, 60-400, 60-350, 60-300, 60-250, 60-200, 60-150, 60-100, 60-90, 60-80, 60-70, 70-500, 70-450, 70-400, 70-350, 70-30 0, 70-250, 70-200, 70-150, 70-100, 70-90, 70-80, 80-500, 80-450, 80-400, 80-350, 80-300, 80-250, 80-200, 80-150, 80-100, 80-90, 90-500, 90-450, 90-400, 90-350, 90-300, 90-250, 90-200, 90-150, 90-100, 100-500, 100-450, 100-400, 100-350, 100-300, 100-250, 100-200, 100-150, 150-500, 150 -450, 150-400, 150-350, 150-300, 150-250, 150-200, 200-500, 200-450, 200-400, 200-350, 200-300, 200-250, 250-500, 250-450, 250-400, 250-350,250-300, 300-500, 300-450, 300-400, 300-350, 350-500, 350-450, 350-400, 400-500, 400-450 or 450-500; the number y is about 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20- 30, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-100, 50-90, 50-80, 50-70, 50-60, 60-100, 60-9 0, 60-80, 60-70, 70-100, 70-90, 70-80, 80-100, 80-90 or 90-100; and the number z is about 10-500, 10-450, 10-400, 10-350, 10-300, 10-250, 10-200, 10-150, 10-100, 10 -90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-500, 20-450, 20-400, 20-350, 20-300, 20-250, 20-200, 20-150, 20-100, 20-90, 20-80, 20- 70, 20-60, 20-50, 20-40, 20-30, 30-500, 30-450, 30-400, 30-350, 30-300, 30-250, 30-200, 30-150, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40 , 40-500, 40-450, 40-400, 40-350, 40-300, 40-250, 40-200, 40-150, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-500, 50-450, 50-400, 50-350, 50- 300, 50-250, 50-200, 50-150, 50-100, 50-90, 50-80, 50-70, 50-60, 60-500, 60-450, 60-400, 60-350, 60-300, 60-250, 60-200, 60-150, 60-100, 60-90,60-80, 60-70, 70-500, 70-450, 70-400, 70-350, 70-300, 70-250, 70-200, 70-150, 70-100, 70-90, 70-80, 80-500, 80-450, 80-400, 80-350, 80-300, 80-2 50, 80-200, 80-150, 80-100, 80-90, 90-500, 90-450, 90-400, 90-350, 90-300, 90-250, 90-200, 90-150, 90-100, 100-500, 100-450, 100-400, 100-350, 10 0-300, 100-250, 100-200, 100-150, 150-500, 150-450, 150-400, 150-350, 150-300, 150-250, 150-200, 200-500, 200-450, 200-400, 200-350, 200-300, 2 In some embodiments, the number x is selected from 10-50, the number y is selected from 1-30, and the number z is selected from 10-50.

[0184] In some embodiments, the number x is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 24.5, 25, 25.1, 25.2, 25.3, 25.4, 25.5, 25.6, 25.7, 25.8, 25.9, 26, 26.1, 26.2, 26.3, 26.4, 26.5, 27, 28, 29, or 30 (e.g., about 25.6) and the number y is about 1, 2, 3, 4, 5, 6, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.10, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, .6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 9, 10, 11, 12, 13, 14, or 15 (e.g., about 7.5), and the number z is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 24.5, 25, 25.1, 25.2, 25.3, 25.4, 25.5, 25.6, 25.7, 25.8, 25.9, 26, 26.1, 26.2, 26.3, 26.4, 26.5, 27, 28, 29, or 30 (e.g., about 25.6). In some embodiments, the number x is about 10, 11, 12, 13, 13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 (e.g., about 14) and the number y is about 20, 21, 22, 23, 24, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7, 24.8, 24.9, 25.10, 25.20, 25.30, 25.40, 25.50, 25.60, 25.70, 25.80 In some embodiments, the numbers described herein (e.g., x, y, and z) are averages.

[0185] In some embodiments, the weight percent of PEO (PEO % (w / w)) is less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of Formula I. In some embodiments, the weight percentage of PPO (PPO% (w / w)) is greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 81%, greater than 82%, greater than 83%, greater than 84%, greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%. In some embodiments, the weight percentage of PEO is between about 1% to about 20%, between about 1% to about 15%, between about 1% to about 10%, between about 1% to about 5%, between about 5% to about 20%, between about 5% to about 15%, between about 5% to about 10%, between about 10% to about 20%, between about 10% to about 15%, or between about 15% to about 20%.

[0186] In some embodiments, the average molecular weight of Formula II is from about 1000 Daltons to about 30,000 Daltons. In some embodiments, the average molecular weight of Formula II is from about 1000 Daltons to about 20,000 Daltons, from about 1000 Daltons to about 10,000 Daltons, from about 1000 Daltons to about 9,000 Daltons, from about 1000 Daltons to about 8,000 Daltons, from about 1000 Daltons to about 7,000 Daltons, from about 1000 Daltons to about 6,000 Daltons, from about 1000 Daltons to about 5,000 Daltons, from about 1000 Daltons to about 4,000 Daltons, from about 1000 Daltons to about 3,000 Daltons, from about 1000 Daltons to about 2,000 Daltons, from about 2,000 Daltons to about 1,000 Daltons. 0 Daltons, about 2000 Daltons to about 9000 Daltons, about 2000 Daltons to about 8000 Daltons, about 2000 Daltons to about 7000 Daltons, about 2000 Daltons to about 6000 Daltons, about 2000 Daltons to about 5000 Daltons, about 2000 Daltons to about 4000 Daltons, about 2000 Daltons to about 3000 Daltons, about 3000 Daltons to about 10000 Daltons, about 3000 Daltons to about 9000 Daltons, about 3000 Daltons to about 8000 Daltons, about 3000 Daltons to about 7000 Daltons, about 3000 Daltons to about 6000 Daltons, about 3000 Daltons to about 5000 Daltons, about 3000 Daltons to about 4000 Daltons, about 4000 Daltons to about 10000 Daltons, about 4000 Daltons to about 9000 Daltons, about 4000 Daltons to about 8000 Daltons, about 4000 Daltons to about 7000 Daltons, about 4000 Daltons to about 6000 Daltons, about 4000 Daltons to about 5000 Daltons, about 5000 Daltons to about 10000 Daltons, about 5000 Daltons to about 9000 Daltons, about 5000 Daltons to about 8000 Daltons, about 5000 Daltons to about In some embodiments, the present invention relates to a molecule of the present invention to a molecule of the present invention that is at least 500 daltons to about 7000 daltons, about 5000 daltons to about 6000 daltons, about 6000 daltons to about 10000 daltons, about 6000 daltons to about 9000 daltons, about 6000 daltons to about 8000 daltons, about 6000 daltons to about 7000 daltons, about 7000 daltons to about 10000 daltons, about 7000 daltons to about 9000 daltons, about 7000 daltons to about 8000 daltons, about 8000 daltons to about 10000 daltons, about 8000 daltons to about 9000 daltons or about 9000 daltons to about 10000 daltons.In some embodiments, the average molecular weight of Formula II is about 2000 Daltons, about 2100 Daltons, about 2200 Daltons, about 2300 Daltons, about 2400 Daltons, about 2500 Daltons, about 2600 Daltons, about 2700 Daltons, about 2800 Daltons, about 2900 Daltons, about 3000 Daltons, about 3100 Daltons, about 3200 Daltons, about 3300 Daltons, about 3400 Daltons, about 3500 Daltons, about 3600 Daltons, about 3700 Daltons, about 3800 Daltons, about 3900 Daltons, about 4000 Daltons, about 4100 Daltons, about 4200 Daltons, about 4300 Daltons, about 4400 Daltons, about 4500 Daltons, about 4600 Daltons, about 4700 Daltons, about 4800 Daltons, about 4900 Daltons, about 5000 Daltons, about 5100 Daltons, about 5200 Daltons, about 5300 Daltons, about 5400 Daltons, about 5500 Daltons, about 5600 Daltons, about 5700 Daltons, about 5800 Daltons, about 5900 Daltons, about 6000 Daltons, about 6100 Daltons, about 6200 Daltons, about 6300 Daltons, about 6400 Daltons, about 6500 Daltons, about 6600 Daltons, about 6700 Daltons, about 6800 Daltons, about 6900 Daltons About 3500 Daltons, about 3600 Daltons, about 3700 Daltons, about 3750 Daltons, about 3800 Daltons, about 3900 Daltons, about 4000 Daltons, about 4100 Daltons, about 4200 Daltons, about 4300 Daltons, about 4400 Daltons, about 4500 Daltons, about 4600 Daltons, about 4700 Daltons, about 4800 Daltons, about 4900 Daltons or about 5000 Daltons.

[0187] In some embodiments, when the number x and the number z are the same, Formula II can also be depicted as:

[0188]

[0189] In some embodiments, the above formula is also depicted as PPO x -PEO y -PPO z , [PPO] x -[PEO] y -[PPO] x 、H[OCH(CH3)CH2] x [OCH2CH2] y [OCH(CH3)CH2] x OH.

[0190] Formula III (diblock copolymer)

[0191] In some embodiments, the polymeric components described herein include diblock copolymers.

[0192] In some embodiments, the triblock copolymer is derived from a compound of Formula I having the general structure shown below:

[0193]

[0194] In some embodiments, R1 is H or CH3, and R2 is OH or OCH3.

[0195] In some embodiments, Formula III is also depicted as PEO y -PPO z , PEO] y -[PPO] z, PEO-PPO, poly(ethylene oxide)-poly(propylene oxide), PEG-PPG or R1[OCH2CH2] y [OCH(CH3)CH2] z R2. In some embodiments, y and z in Formula III or their equivalents are numbers. For example, y is a number selected from 1-100, and z is a number selected from 5-500. In some embodiments, the numbers x and z are the same. In some embodiments, the numbers x and z are not the same. In some embodiments, the number z is at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, or 20 times the number y.

[0196] In some embodiments, the triblock copolymer is derived from a compound of Formula III' having the general structure shown below:

[0197] or a salt thereof,

[0198] in:

[0199] R1 is hydrogen (H) or an alkyl group (e.g., C 1-6 Alkyl, such as CH3);

[0200] R2 is hydroxyl (-OH) or alkoxy (e.g., C 1-6 Alkoxy, such as -OCH3); and

[0201] Each R 15 , R 16 , R 17 , R 17a and R 18 independently selected from the group consisting of: hydrogen (H), C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl, halo, cyano (-CN), nitro (-NO2), hydroxyl (-OH), carboxyl (-COOH), formaldehyde (-C(O)H), amide (e.g., -C(O)NH2, -C(O)NHC 1-4 Alkyl or -C(O)N(C 1-4 Alkyl)2) and amino (e.g., -NH2, -NHC 1-4 Alkyl and -N(C 1-4 Alkyl)2).

[0202] In some embodiments, R1 is H. In some embodiments, R1 is CH3.

[0203] In some embodiments, R2 is OH. In some embodiments, R2 is OCH3.

[0204] In some embodiments, R1 is H and R2 is OH. In some embodiments, R1 is H and R2 is OCH3. In some embodiments, R1 is CH3 and R2 is OH. In some embodiments, R1 is CH3 and R2 is OCH3.

[0205] In some embodiments, each R 15 , R 16 , R 17 , R 17a and R 18 It's H.

[0206] In some embodiments, each R 15 is H. In some embodiments, at least one R 15 Selected from C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl, halo, CN and OH. In some embodiments, at least one R 15 is selected from the group consisting of methyl, ethyl, methoxy, ethoxy, hydroxymethyl, halo and hydroxy. 15 is methyl. In some embodiments, at least one R 15 In some embodiments, at least one R 15 is methoxy. In some embodiments, at least one R 15 In some embodiments, at least one R 15 is hydroxymethyl. In some embodiments, at least one R 15 In some embodiments, at least one R 15 It's a hydroxyl group.

[0207] In some embodiments, each R 16 is H. In some embodiments, at least one R 16 Selected from C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl, halo, CN and OH. In some embodiments, at least one R 16is selected from the group consisting of methyl, ethyl, methoxy, ethoxy, hydroxymethyl, halo and hydroxy. 16 is methyl. In some embodiments, at least one R 16 In some embodiments, at least one R 16 is methoxy. In some embodiments, at least one R 16 In some embodiments, at least one R 16 is hydroxymethyl. In some embodiments, at least one R 16 In some embodiments, at least one R 16 It's a hydroxyl group.

[0208] In some embodiments, R 17 is H. In some embodiments, R 17 Selected from C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 1-6 In some embodiments, R 17 is selected from the group consisting of methyl, ethyl, methoxy, ethoxy, hydroxymethyl, halo and hydroxy. 17 In some embodiments, R 17 In some embodiments, R 17 In some embodiments, R 17 In some embodiments, R 17 In some embodiments, R 17 In some embodiments, R 17 It's a hydroxyl group.

[0209] In some embodiments, R 17a is H. In some embodiments, R 17a Selected from C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 1-6 In some embodiments, R 17a is selected from the group consisting of methyl, ethyl, methoxy, ethoxy, hydroxymethyl, halo and hydroxy. 17a In some embodiments, R 17a In some embodiments, R 17a In some embodiments, R 17a In some embodiments, R 17aIn some embodiments, R 17a In some embodiments, R 17a It's a hydroxyl group.

[0210] In some embodiments, each R 18 is H. In some embodiments, at least one R 18 Selected from C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl, halo, CN and OH. In some embodiments, at least one R 18 is selected from the group consisting of methyl, ethyl, methoxy, ethoxy, hydroxymethyl, halo and hydroxy. 18 is methyl. In some embodiments, at least one R 18 In some embodiments, at least one R 18 is methoxy. In some embodiments, at least one R 18 In some embodiments, at least one R 18 is hydroxymethyl. In some embodiments, at least one R 18 In some embodiments, at least one R 18 It's a hydroxyl group.

[0211] In some embodiments, the triblock copolymer is derived from a compound of Formula III" having the general structure shown below:

[0212] or a salt thereof,

[0213] in:

[0214] R1 is hydrogen (H), optionally substituted alkyl (e.g., C 1-6 , C 1-4 , C 1-3 or C 1-2 alkyl) or optionally substituted hydroxyalkyl (e.g., C 1-6 , C 1-4 , C 1-3 or C 1-2 Hydroxyalkyl);

[0215] R2 is hydroxy (-OH), optionally substituted alkoxy (e.g., C 1-6 , C 1-4 , C 1-3 or C 1-2 alkoxy) or optionally substituted hydroxyalkyl (e.g., C 1-6 , C 1-4 , C 1-3 or C1-2 Hydroxyalkyl);

[0216] Each of Ak1 and Ak2 is independently an optionally substituted alkylene group (e.g., an optionally substituted C 1-6 , C 1-4 , C 1-3 or C 1-2 Alkylene);

[0217] Each of Ak1 and Ak2 is different; and

[0218] Y and z can be any y and z described herein (eg, as described herein for Formula III).

[0219] In some embodiments, R1 is hydrogen or C 1-6 In some embodiments, R2 is hydroxyl or C 1-6 In some embodiments, R1 is hydrogen and R2 is hydroxy. In some embodiments, R1 is hydrogen and R2 is C 1-6 In some embodiments, R1 is C 1-6 In some embodiments, R1 is C 1-6 alkyl, and R2 is C 1-6 Alkoxy.

[0220] In some embodiments, the optionally substituted alkylene is substituted with one or more substituents. Non-limiting examples of substituents include one or more of halo, haloalkyl, alkoxy, hydroxyalkyl, amino, amide, cyano, nitro, hydroxyl, carboxyl, oxo, formaldehyde, or a combination thereof.

[0221] In some embodiments, at least one of Ak1 and Ak2 is independently an optionally substituted C 1-6 , C 1-4 , C 1-3 or C 1-2 In some embodiments, each of Ak1 and Ak2 is independently an optionally substituted C 1-6 , C 1-4 , C 1-3 or C 1-2 Alkylene.

[0222] In some embodiments, at least one of Ak1 and Ak2 is independently unsubstituted C 1-6 , C 1-4 , C 1-3 or C 1-2 In some embodiments, each of Ak1 and Ak2 is independently an unsubstituted C 1-6 , C 1-4 , C 1-3or C 1-2 Alkylene.

[0223] In some embodiments, at least one of Ak1 and Ak2 is independently an unbranched alkylene group (e.g., an unbranched C 1-6 , C 1-4 , C 1-3 or C 1-2 In some embodiments, Ak1 is an unbranched alkylene group.

[0224] In some embodiments, at least one of Ak1 and Ak2 is a branched alkylene group (e.g., a branched C 2-6 , C 2-4 , C 3-6 or C 3-4 In some embodiments, Ak2 is a branched alkylene group.

[0225] In some embodiments, the number y is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 and the number z is about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500. In some embodiments, the number y is about 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50 , 30-40, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-100, 50-90, 50-80, 50-70, 50-60, 60-100, 60-90, 60-80, 60-70, 70-100, 70-90, 70-80, 80-100, 80-90 or 90-100; and the number z is about 1-500, 1-450, 1-400, 1-350, 1-300, 1-250, 1-200, 1-150, 1-100, 1-90, 1-80 , 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 10-500, 10-450, 10-400, 10-350, 10-300, 10-250, 10-200, 10-150, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-500, 20-450, 20-400, 20-350, 20-300, 20-250, 20-200, 20-150, 20 -100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-500, 30-450, 30-400, 30-350, 30-300, 30-250, 30-200, 30-150, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-500, 40-450, 40-400, 40-350, 40-300, 40-250, 40-200, 40-150, 40-100,40-90, 40-80, 40-70, 40-60, 40-50, 50-500, 50-450, 50-400, 50-350, 50-300, 50-250, 50-200, 50-150, 50-100, 50-90, 50-80, 50-70, 50-60, 60-500, 60-450, 60-400, 60-350, 60-300, 60-250, 60-200, 60-150, 60-100, 60-90, 60-80, 60-70, 70-500, 70-450, 70-400, 70-350, 70-300, 70-250, 70-200, 70-150, 70-100, 70-90, 70-80, 80-500, 80-450, 80-400, 80-350, 80-300, 80-250, 80-200, 80-150, 80-100, 80-90, 90-5 00, 90-450, 90-400, 90-350, 90-300, 90-250, 90-200, 90-150, 90-100, 100-500, 100-450, 100-400, 100-350, 100-300, 100-250, 100-200, 100-150, 150-500, 150-450, 150-400, 150-350, 150-300, 150-250, 15 0-200, 200-500, 200-450, 200-400, 200-350, 200-300, 200-250, 250-500, 250-450, 250-400, 250-350, 250-300, 300-500, 300-450, 300-400, 300-350, 350-500, 350-450, 350-400, 400-500, 400-450 or 450-500.

[0226] In some embodiments, the weight percent of PEO (PEO % (w / w)) is less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of Formula I. In some embodiments, the weight percentage of PPO (PPO% (w / w)) is greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 81%, greater than 82%, greater than 83%, greater than 84%, greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%. In some embodiments, the weight percentage of PEO is between about 1% to about 20%, between about 1% to about 15%, between about 1% to about 10%, between about 1% to about 5%, between about 5% to about 20%, between about 5% to about 15%, between about 5% to about 10%, between about 10% to about 20%, between about 10% to about 15%, or between about 15% to about 20%.

[0227] In some embodiments, the average molecular weight of Formula II is from about 1000 Daltons to about 30,000 Daltons. In some embodiments, the average molecular weight of Formula II is from about 1000 Daltons to about 20,000 Daltons, from about 1000 Daltons to about 10,000 Daltons, from about 1000 Daltons to about 9,000 Daltons, from about 1000 Daltons to about 8,000 Daltons, from about 1000 Daltons to about 7,000 Daltons, from about 1000 Daltons to about 6,000 Daltons, from about 1000 Daltons to about 5,000 Daltons, from about 1000 Daltons to about 4,000 Daltons, from about 1000 Daltons to about 3,000 Daltons, from about 1000 Daltons to about 2,000 Daltons, from about 2,000 Daltons to about 1,000 Daltons. 0 Daltons, about 2000 Daltons to about 9000 Daltons, about 2000 Daltons to about 8000 Daltons, about 2000 Daltons to about 7000 Daltons, about 2000 Daltons to about 6000 Daltons, about 2000 Daltons to about 5000 Daltons, about 2000 Daltons to about 4000 Daltons, about 2000 Daltons to about 3000 Daltons, about 3000 Daltons to about 10000 Daltons, about 3000 Daltons to about 9000 Daltons, about 3000 Daltons to about 8000 Daltons, about 3000 Daltons to about 7000 Daltons, about 3000 Daltons to about 6000 Daltons, about 3000 Daltons to about 5000 Daltons, about 3000 Daltons to about 4000 Daltons, about 4000 Daltons to about 10000 Daltons, about 4000 Daltons to about 9000 Daltons, about 4000 Daltons to about 8000 Daltons, about 4000 Daltons to about 7000 Daltons, about 4000 Daltons to about 6000 Daltons, about 4000 Daltons to about 5000 Daltons, about 5000 Daltons to about 10000 Daltons, about 5000 Daltons to about 9000 Daltons, about 5000 Daltons to about 8000 Daltons, about 5000 Daltons to about In some embodiments, the present invention relates to a molecule of the present invention to a molecule of the present invention that is at least 500 daltons to about 7000 daltons, about 5000 daltons to about 6000 daltons, about 6000 daltons to about 10000 daltons, about 6000 daltons to about 9000 daltons, about 6000 daltons to about 8000 daltons, about 6000 daltons to about 7000 daltons, about 7000 daltons to about 10000 daltons, about 7000 daltons to about 9000 daltons, about 7000 daltons to about 8000 daltons, about 8000 daltons to about 10000 daltons, about 8000 daltons to about 9000 daltons or about 9000 daltons to about 10000 daltons.In some embodiments, the average molecular weight of Formula II is about 2000 Daltons, about 2100 Daltons, about 2200 Daltons, about 2300 Daltons, about 2400 Daltons, about 2500 Daltons, about 2600 Daltons, about 2700 Daltons, about 2750 Daltons, about 2800 Daltons, about 2900 Daltons, about 3000 Daltons, about 3100 Daltons, about 3200 Daltons, about 3300 Daltons, about 3400 Daltons, about 3500 Daltons, about 3600 Daltons, about 3700 Daltons, about 3800 Daltons, about 3900 Daltons, about 4000 Daltons, about 4100 Daltons, about 4200 Daltons, about 4300 Daltons, about 4400 Daltons, about 4500 Daltons, about 4600 Daltons, about 4700 Daltons, about 4800 Daltons, about 4900 Daltons, about 5000 Daltons, about 5100 Daltons, about 5200 Daltons, about 5300 Daltons, about 5400 Daltons, about 5500 Daltons, about 5600 Daltons, about 5700 Daltons, about 5800 Daltons, about 5900 Daltons, about 6100 Daltons, about 6200 Daltons, about 6300 Daltons, about 6400 Daltons, about 6500 Daltons, about 6600 Daltons, about 6700 Daltons, about 6800 Daltons, about 6900 Daltons 0 Daltons, about 3500 Daltons, about 3600 Daltons, about 3700 Daltons, about 3750 Daltons, about 3800 Daltons, about 3900 Daltons, about 4000 Daltons, about 4100 Daltons, about 4200 Daltons, about 4300 Daltons, about 4400 Daltons, about 4500 Daltons, about 4600 Daltons, about 4700 Daltons, about 4800 Daltons, about 4900 Daltons, or about 5000 Daltons.

[0228] Formula IV (polymer)

[0229] In some embodiments, the polymer components described herein include polymers containing poly(polypropylene oxide) or poly(ethylene glycol). In some embodiments, the polymer is derived from a compound of Formula IV having the general structure shown below:

[0230]

[0231] In some embodiments, Formula IV is also depicted as [PPO] x , PPG, PPO, poly(propylene oxide) or H[OCH(CH3)CH2] x OH.

[0232] In some embodiments, the triblock copolymer is derived from a compound of Formula IV' having the general structure shown below:

[0233] or a salt thereof,

[0234] in:

[0235] Each R 19 , R 19a and R 20 independently selected from the group consisting of: hydrogen (H), C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 1-6Hydroxyalkyl, halo, cyano (-CN), nitro (-NO2), hydroxyl (-OH), carboxyl (-COOH), formaldehyde (-C(O)H), amide (e.g., -C(O)NH2, -C(O)NHC 1-4 Alkyl or -C(O)N(C 1-4 Alkyl)2) and amino (e.g., -NH2, -NHC 1-4 Alkyl and -N(C 1-4 alkyl)2); and

[0236] R C Selected from the group consisting of: hydrogen (H), C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl and C 1-6 Hydroxyalkyl.

[0237] In some embodiments, each R 19 , R 19a , R 20 and R C It's H.

[0238] In some embodiments, each R 19 , R 19a and R 20 is H; and R C It's methyl.

[0239] In some embodiments, R 19 is H. In some embodiments, R 19 Selected from C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 1-6 In some embodiments, R 19 is selected from the group consisting of methyl, ethyl, methoxy, ethoxy, hydroxymethyl, halo and hydroxy. 19 In some embodiments, R 19 In some embodiments, R 19 In some embodiments, R 19 In some embodiments, R 19 In some embodiments, R 19 In some embodiments, R 19 It's a hydroxyl group.

[0240] In some embodiments, R 19a is H. In some embodiments, R 19aSelected from C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Haloalkoxy, C 1-6 In some embodiments, R 19a is selected from the group consisting of methyl, ethyl, methoxy, ethoxy, hydroxymethyl, halo and hydroxy. 19a In some embodiments, R 19a In some embodiments, R 19a In some embodiments, R 19a In some embodiments, R 19a In some embodiments, R 19a In some embodiments, R 19a It's a hydroxyl group.

[0241] In some embodiments, each R 20 is H. In some embodiments, at least one R 20 Selected from C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Haloalkoxy, C 1-6 Hydroxyalkyl, halo, CN and OH. In some embodiments, at least one R 20 is selected from the group consisting of methyl, ethyl, methoxy, ethoxy, hydroxymethyl, halo and hydroxy. 20 In some embodiments, at least one R 20 In some embodiments, at least one R 20 is methoxy. In some embodiments, at least one R 20 In some embodiments, at least one R 20 is hydroxymethyl. In some embodiments, at least one R 20 In some embodiments, at least one R 20 It's a hydroxyl group.

[0242] In some embodiments, R C is H. In some embodiments, R C Selected from C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl and C 1-6 In some embodiments, R C In some embodiments, R CIn some embodiments, R C In some embodiments, R C It's hydroxymethyl.

[0243] In some embodiments, the triblock copolymer is derived from a compound of Formula IV" having the general structure shown below:

[0244] or a salt thereof,

[0245] in:

[0246] R1 is hydrogen (H), optionally substituted alkyl (e.g., C 1-6 , C 1-4 , C 1-3 or C 1-2 alkyl) or optionally substituted hydroxyalkyl (e.g., C 1-6 , C 1-4 , C 1-3 or C 1-2 Hydroxyalkyl);

[0247] R2 is hydroxy (-OH), optionally substituted alkoxy (e.g., C 1-6 , C 1-4 , C 1-3 or C 1-2 alkoxy) or optionally substituted hydroxyalkyl (e.g., C 1-6 , C 1-4 , C 1-3 or C 1-2 Hydroxyalkyl);

[0248] Ak1 is an optionally substituted alkylene group (e.g., an optionally substituted C 1-6 , C 1-4 , C 1-3 or C 1-2 alkylene); and

[0249] X can be any x described herein (eg, as described herein for Formula IV).

[0250] In some embodiments, R1 is hydrogen or C 1-6 In some embodiments, R2 is hydroxyl or C 1-6 In some embodiments, R1 is hydrogen and R2 is hydroxy. In some embodiments, R1 is hydrogen and R2 is C 1-6 In some embodiments, R1 is C 1-6 In some embodiments, R1 is C 1-6 alkyl, and R2 is C 1-6 Alkoxy.

[0251] In some embodiments, the optionally substituted alkylene is substituted with one or more substituents. Non-limiting examples of substituents include one or more of halo, haloalkyl, alkoxy, hydroxyalkyl, amino, amide, cyano, nitro, hydroxyl, carboxyl, oxo, formaldehyde, or a combination thereof.

[0252] In some embodiments, Ak1 is an optionally substituted C 1-6 , C 1-4 , C 1-3 or C 1-2 In some embodiments, Ak1 is an unsubstituted C 1-6 , C 1-4 , C 1-3 or C 1-2 Alkylene.

[0253] In some embodiments, Ak1 is an unbranched alkylene group (e.g., an unbranched C 1-6 , C 1-4 , C 1-3 or C 1-2 In some embodiments, Ak1 is a branched alkylene group (e.g., a branched C 2-6 , C 2-4 , C 3-6 or C 3-4 alkylene).

[0254] In some embodiments, x in Formula IV or its equivalent is a number. For example, x is a number selected from the group consisting of 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30- 70, 70-100, 70-90, 70-80, 80-100, 80-90, 90-100, 10 ... In some embodiments, the number x is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 0, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100. In some embodiments, the number x is about 45.1, 45.2, 45.3, 45.4, 45.5, 45.6, 45.7, 45.8, 45.9, 46, 46.1, 46.2, 46.3, 46.4, 46.5, 46.6, 46.7, 46.8, 46.9, 47, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, or 48 (e.g., about 46.5). In some embodiments, the numbers described herein (e.g., x) are averages.

[0255] In some embodiments, the average molecular weight of Formula IV is from about 1000 Daltons to about 30,000 Daltons. In some embodiments, the average molecular weight of Formula II is from about 1000 Daltons to about 20,000 Daltons, from about 1000 Daltons to about 10,000 Daltons, from about 1000 Daltons to about 9,000 Daltons, from about 1000 Daltons to about 8,000 Daltons, from about 1000 Daltons to about 7,000 Daltons, from about 1000 Daltons to about 6,000 Daltons, from about 1000 Daltons to about 5,000 Daltons, from about 1000 Daltons to about 4,000 Daltons, from about 1000 Daltons to about 3,000 Daltons, from about 1000 Daltons to about 2,000 Daltons, from about 2,000 Daltons to about 1,000 Daltons. 0 Daltons, about 2000 Daltons to about 9000 Daltons, about 2000 Daltons to about 8000 Daltons, about 2000 Daltons to about 7000 Daltons, about 2000 Daltons to about 6000 Daltons, about 2000 Daltons to about 5000 Daltons, about 2000 Daltons to about 4000 Daltons, about 2000 Daltons to about 3000 Daltons, about 3000 Daltons to about 10000 Daltons, about 3000 Daltons to about 9000 Daltons, about 3000 Daltons to about 8000 Daltons, about 3000 Daltons to about 7000 Daltons, about 3000 Daltons to about 6000 Daltons, about 3000 Daltons to about 5000 Daltons, about 3000 Daltons to about 4000 Daltons, about 4000 Daltons to about 10000 Daltons, about 4000 Daltons to about 9000 Daltons, about 4000 Daltons to about 8000 Daltons, about 4000 Daltons to about 7000 Daltons, about 4000 Daltons to about 6000 Daltons, about 4000 Daltons to about 5000 Daltons, about 5000 Daltons to about 10000 Daltons, about 5000 Daltons to about 9000 Daltons, about 5000 Daltons to about 8000 Daltons, about 5000 Daltons to about In some embodiments, the present invention relates to a molecule of the present invention to a molecule of the present invention that is at least 500 daltons to about 7000 daltons, about 5000 daltons to about 6000 daltons, about 6000 daltons to about 10000 daltons, about 6000 daltons to about 9000 daltons, about 6000 daltons to about 8000 daltons, about 6000 daltons to about 7000 daltons, about 7000 daltons to about 10000 daltons, about 7000 daltons to about 9000 daltons, about 7000 daltons to about 8000 daltons, about 8000 daltons to about 10000 daltons, about 8000 daltons to about 9000 daltons or about 9000 daltons to about 10000 daltons.In some embodiments, the average molecular weight of Formula II is about 2000 Daltons, about 2100 Daltons, about 2200 Daltons, about 2300 Daltons, about 2400 Daltons, about 2500 Daltons, about 2600 Daltons, about 2700 Daltons, about 2750 Daltons, about 2800 Daltons, about 2900 Daltons, about 3000 Daltons, about 3100 Daltons, about 3200 Daltons, about 3300 Daltons, about 3400 Daltons, about 3500 Daltons, about 3600 Daltons, about 3700 Daltons, about 3800 Daltons, about 3900 Daltons, about 4000 Daltons, about 4100 Daltons, about 4200 Daltons, about 4300 Daltons, about 4400 Daltons, about 4500 Daltons, about 4600 Daltons, about 4700 Daltons, about 4800 Daltons, about 4900 Daltons, about 5000 Daltons, about 5100 Daltons, about 5200 Daltons, about 5300 Daltons, about 5400 Daltons, about 5500 Daltons, about 5600 Daltons, about 5700 Daltons, about 5800 Daltons, about 5900 Daltons, about 6100 Daltons, about 6200 Daltons, about 6300 Daltons, about 6400 Daltons, about 6500 Daltons, about 6600 Daltons, about 6700 Daltons, about 6800 Daltons, about 6900 Daltons 0 Daltons, about 3500 Daltons, about 3600 Daltons, about 3700 Daltons, about 3750 Daltons, about 3800 Daltons, about 3900 Daltons, about 4000 Daltons, about 4100 Daltons, about 4200 Daltons, about 4300 Daltons, about 4400 Daltons, about 4500 Daltons, about 4600 Daltons, about 4700 Daltons, about 4800 Daltons, about 4900 Daltons, or about 5000 Daltons.

[0256] Other

[0257] In some embodiments, the polymer component described herein includes other polymers, such as the PEO-PPO block polymers described in the following document: Alvarez-Lorenzo, C. et al. "PEO-PPO block copolymers for passive micelle targeting and overcoming multidrug resistance in cancer therapy." Contemporary Drug Targets 12.8 (2011): 1112-1130, such as poloxamine, which is incorporated herein by reference in its entirety.

[0258] In some embodiments, the polymer components described herein include a polymer derived from a compound of Formula A having the general structure shown below:

[0259]

[0260] In some embodiments, the polymer components described herein include a polymer derived from a compound of Formula B having the general structure shown below:

[0261]

[0262] In some embodiments, a in Formula A and Formula B or its equivalent is an integer. For example, a is an integer selected from the group consisting of 1-200, 1-190, 1-180, 1-170, 1-160, 1-150, 1-140, 1-130, 1-120, 1-110, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 2-200, 2-190, 2-180, 2-170, 2-160, 2-150, 2-140, 2-130, 2-120, 2-110, 2-100, 2-90, 2-80, 2-70, 2-60, 2-50, 2-40, 2-30, 2-20, 2-10, 5-200, 5- 190, 5-180, 5-170, 5-160, 5-150, 5-140, 5-130, 5-120, 5-110, 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30, 5-20, 5-10, 10-200, 10-190, 10-180, 10-170, 10-160, 10-150, 10-140, 10-130, 10-120, 10-110, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-200, 20-190, 20 20-180, 20-170, 20-160, 20-150, 20-140, 20-130, 20-120, 20-110, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-200, 30-190, 30-180, 30-170, 30-160, 30-150, 30-140, 30-130, 30-120, 30-110, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-200, 40-190, 40-180, 40-170 , 40-160, 40-150, 40-140, 40-130, 40-120, 40-110, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-200, 50-190, 50-180, 50-170, 50-160, 50-150, 50-140, 50-130, 50-120, 50-110, 50-100, 50-90, 50-80, 50-70, 50-60, 60-200, 60-190, 60-180, 60-170, 60-160, 60-150, 60-140, 60-130, 60-120,60-110, 60-100, 60-90, 60-80, 60-70, 70-200, 70-190, 70-180, 70-170, 70-160, 70-150, 70-140, 70-130, 70-120, 70-110, 70-100, 70-90, 70-80, 80-200, 80-190, 80-180 0, 80-170, 80-160, 80-150, 80-140, 80-130, 80-120, 80-110, 80-100, 80-90, 90-200, 90-190, 90-180, 90-170, 90-160, 90-150, 90-140, 90-130, 90-120, 90-110 or 90-100. 45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200.

[0263] In some embodiments, b in Formula A and Formula B or its equivalent is an integer. For example, b is an integer selected from the group consisting of 1-300, 1-290, 1-280, 1-270, 1-260, 1-250, 1-240, 1-230, 1-220, 1-210, 1-200, 1-190, 1-180, 1-170, 1-160, 1-150, 1-140, 1-130, 1-120, 1-110, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 2-300, 2-290, 2-280, 2-270, 2-260, 2-250, 2-240, 2-230, 2-220, 2-210, 2-200, 2-190, 2-180, 2-170, 2-160, 2-150, 2-140, 2-130, 2-120, 2-110, 2-100, 2-90, 2-80, 2-70, 2-60, 2-50, 2-40, 2-30, 2-20, 2-10, 4-300, 4-290, 4-280, 4-270, 4-260, 4-250, 4-240, 4-230, 4-220, 4-210, 4-200, 4-190, 4-180, 4-170, 4-160, 4-150, 4-140, 4-130, 4-120, 4-110, 4-100, 4-90, 4- 80, 4-70, 4-60, 4-50, 4-40, 4-30, 4-20, 4-10, 5-300, 5-290, 5-280, 5-270, 5-260, 5-250, 5-240, 5-230, 5-220, 5-210, 5-200, 5-190, 5-180, 5-170, 5-160, 5-150, 5-140, 5-130, 5-120, 5-110, 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30, 5-20, 5-10, 10-300, 10-290, 10-280, 10-270, 10-260, 10-250, 10-240, 10-230, 10-220, 10-210, 10-200, 10-190, 10-180, 10-170, 10-160, 10-150, 10-140, 10-130, 10-120, 10-110, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-300, 20-290, 20-280, 20-270, 20-260, 20-250, 20-240, 20-230, 20-220, 20-210, 20-200, 20-190,20-180、20-170、20-160、20-150、20-140、20-130、20-120、20-110、20-100、20-90、20-80、20-70、20-60、20-50、20-40、20-30、30-300、30-290、30-280、30-270、30-260、30-250、30-240、30-230、30-220、30-210、30-200、30-190、30-180、30-170、30-160、30-150、30-140、30-130、30-120、30-110、30-100、30-90、30-80、30-70、30-60、30-50、30-40、40-300、40-290、40-280、40-270、40-260、40-250、40-240、40-230、40-220、40-210、40-200、40-190、40-180、40-170、40-160、40-150、40-140、40-130、40-120、40-110、40-100、40-90、40-80、40-70、40-60、40-50、50-300、50-290、50-280、50-270、50-260、50-250、50-240、50-230、50-220、50-210、50-200、50-190、50-180、50-170、50-160、50-150、50-140、50-130、50-120、50-110、50-100、50-90、50-80、50-70、50-60、60-300、60-290、60-280、60-270、60-260、60-250、60-240、60-230、60-220、60-210、60-200、60-190、60-180、60-170、60-160、60-150、60-140、60-130、60-120、60-110、60-100、60-90、60-80、60-70、70-300、70-290、70-280、70-270、70-260、70-250、70-240、70-230、70-220、70-210、70-200、70-190、70-180、70-170、70-160、70-150、70-140、70-130、70-120、70-110、70-100、70-90、70-80、80-300、80-290、80-280、80-270、80-260、80-250, 80-240, 80-230, 80-220, 80-210, 80-200, 80-190, 80-180, 80-170, 80-160, 80-150, 80-140, 80-130, 80-120, 80-110, 80-100, 80-90, 90-300, 90-290, 90-280, 90-270, 90-260, 90-250, 90-240, 90-230, 90-220, 90-210, 90-200, 90-190, 90-180, 90-170, 90-160, 90-150, 90-140, 90-130, 90-120, 90-110 or 90-100. In some embodiments, the integer b is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 , 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300. ,

[0264] Lipid components

[0265] In some embodiments, the lipid component described herein includes an ionizable and / or permanently charged cationic lipid (e.g., any of the ionizable and / or permanently charged cationic lipids described herein), a helper lipid (e.g., any of the helper lipids described herein), a structural lipid (e.g., any of the structural lipids described herein), and / or a PEG lipid (e.g., any of the PEG lipids described herein).

[0266] Ionizable and / or permanently charged cationic lipids

[0267] Ionizable lipids are positively charged at acidic pH to condense RNA into LNPs, but are neutral at physiological pH to minimize toxicity. Described ionizable lipids can be protonated in acidic endosomes after cellular uptake, and interact with anionic endosomal phospholipids to form cone-shaped ion pairs that are incompatible with double layers. These cation-anion lipid pairs drive the transition from double-layer structure to inverted hexagonal Hu phase, which promotes membrane fusion / rupture, endosomal escape and cargo release into the cytosol.

[0268] In some embodiments, the ionizable lipids described herein are unsaturated ionizable lipids (e.g., DLin-MC3-DMA (MC3) or A18-Iso5-2DC18), multi-tailed ionizable lipids (e.g., 98N 12 -5, C12-200, cKK-E12 or 9A1P9), ionizable polymer-lipids (e.g., 7C1 or G0-C14), biodegradable ionizable lipids (e.g., MC3, L319, 3040 13 , C12-200, OF-Deg-Lin, OF-02, 306-O12B) or ionizable lipids with branched tails (e.g., 306O i10 Or FTT5). Detailed information on ionizable lipids can be found, for example, in the following documents: Han, X. et al. "An ionizable lipid toolbox for RNA delivery." Nature Communications 12.1 (2021): 7233; and Hou, X. et al. "Lipid nanoparticles for mRNA delivery." Nature Reviews Materials 6.12 (2021): 1078-1094; each of which is incorporated herein by reference in its entirety.

[0269] In some embodiments, the ionizable and / or permanently charged cationic lipids described herein are ionizable lipids (e.g., within the physiological pH range). For example, the ionizable cationic lipids can be selected from: [(4-hydroxybutyl) azadiyl] di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315); 9-heptadecanyl 8-{(2-hydroxyethyl) [6-oxo-6-(undecyloxy) hexyl] amino} caprylate (SM-102); 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA); 4-(dimethylamino) butyric acid heptaheptacontane-6,9,28,31-tetraene -19-yl ester (DLin-MC3-DMA); 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA); 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA); N1-[2-(docosylamino)ethyl]-N1,N4,N4-triadecyl-1,4-piperazinediyldiethylamine (KL22), 14,25-tridecyl -15,18,21,24-tetraaza-octatridecane (KL25); l,2-dioleyloxy-N,N-dimethylaminopropane (DODMA); 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA); (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine -({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2R)); and / or (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)).

[0270] In certain embodiments, ionizable and / or permanently charged cationic lipids as described herein are permanently charged cationic lipids (e.g., within the physiological pH range). In certain embodiments, such lipids are also referred to as fixed cationic lipids. Fixed cationic lipids are permanently positively charged, regardless of the pH of their biological environment. Cationic lipids are similar to other natural lipids, except that typical head groups are substituted with cationic head groups. Fixed cationic lipids have been shown to be good transfection agents in cationic liposomes (CL) and LNP. It is generally considered to be nontoxic at lower concentrations, but due to the tetrasubstituted ammonium moieties, these lipids do have some toxicity issues when used at higher concentrations.

[0271] Fixed cationic lipids are generally cheaper and more readily available alternatives to ionizable lipids. Historically, fixed cationic lipids have been more extensively studied, and therefore a large amount of data has been generated to support their use. The fixed cationic lipids are often used to deliver DNA, and in cases where a smaller payload is required, siRNA and saRNA.

[0272] In some embodiments, the permanently charged cationic lipid can be dioleoyl-3-trimethylammonium propane (DOTAP); 1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA); dimethyldioctadecyl ammonium bromide (DDAB); 2,3-dioleyloxy-N-[2-(sperminamido)ethyl]-N,N-dimethyl-1-propylammonium trifluoroacetate (DOSPA); 2-(((((3S,8S,9S,10R , 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylheptane-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)carbonyl)amino)-N,N-bis(2-hydroxyethyl)-N-methylethane-1-ammonium bromide (BHEM-cholesterol); and / or ethylphosphatidylcholine (ePC). Detailed information on such cationic lipids can be found, for example, in the following literature: Hou, X. et al. "Lipid nanoparticles for mRNA delivery." "Nature Review Materials" 6.12(2021):1078-1094, which is incorporated herein by reference in its entirety.

[0273] In some embodiments, the ionizable and / or permanently charged cationic lipid described herein is N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC"); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride ("DOTMA"); N-(2,3-dioleyloxy)propyl)-N,N-dimethylammonium chloride ("DODMA"); N-(2,3-dioleyloxy)propyl)-N,N-dimethylammonium chloride ("DODAP"); 3-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol ("DC-Chol"); N-(1,2-dimyristoyloxyprop-3-yl)-N,N- Dimethyl-N-hydroxyethylammonium bromide ("DMRIE"); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA); 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA); 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA); or 2-{4-[(3b)-cholest-5-en-3-yloxy]butoxy}-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-amine (CLinDMA), 3β-[N-(dimethylaminoethane)-carbamoyl]-cholesterol (DC-Chol).

[0274] Helper lipids

[0275] Generally include helper lipid as LNP component, to provide particle stability, blood compatibility and enhance cargo delivery efficiency.Have the helper lipid (such as DOPE) of the cone geometry that is conducive to forming hexagonal II phase can promote the endosome release of cargo molecules.Meanwhile, cylindrical lipid phosphatidylcholine can provide larger double layer stability, which is important for the in vivo application of LNP.Detailed information can be found in the following documents, for example: Cheng, X. et al. "The role of helper lipids in lipidnanoparticles (LNPs) designed for oligonucleotide delivery in lipid nanoparticles (LNPs) designed for oligonucleotide delivery." "Advanced Drug Delivery Reviews" 99 (2016): 129-137, the document is incorporated herein by reference in its entirety.

[0276] In some embodiments, the helper lipid described herein is a phospholipid. In some embodiments, the phospholipid comprises a portion selected from the group consisting of: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine and sphingomyelin. In some embodiments, the phospholipid comprises one or more fatty acid moieties selected from the following: lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid and docosahexaenoic acid.

[0277] In some embodiments, the phospholipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC); 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC); 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC); 1,2-heneicosanoyl-sn-glycero-phosphocholine (DUPC); 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (1 8:0 diether PC); 1-oleoyl-2-cholesterol hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC); 1-hexadecyl-sn-glycero-3-phosphocholine (C16Lyso PC); 1,2-dilinoleyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine; 1,2-di(docosahexaenyl)-sn-glycero-3-phosphocholine; 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine; 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine; 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine; 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine; 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphoethanolamine; and / or 1,2-dioleoyl-sn-glycero-3-phospho-racem-(1-glycerol) sodium salt (DOPG) and sphingomyelin. In some embodiments, the lipid component includes both DOPE and DSPC.

[0278] In some embodiments, the helper lipid described herein is a non-cationic lipid. In some embodiments, the helper lipid is a saturated lipid, an unsaturated lipid, or a combination of a saturated lipid and an unsaturated lipid.

[0279] In some embodiments, the helper lipid described herein is an anionic lipid, such as diacylglycerophosphoglycerol (1,2-distearoyl-sn-glycero-3-phosphate (DSPA); 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA); 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA); 1,2-dilauroyl-sn-glycero-3-phosphate (DLPA); 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA)), diacylglycerophosphoglycerol (1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG); 1,2 -dipalmitoyl-sn-glycerol-3-phospho-(1'-racemic-glycerol) (DPPG); 1,2-dimyristoyl-sn-glycerol-3-phospho-(1'-racemic-glycerol) (DMPG); 1,2-dilauroyl-sn-glycerol-3-phospho-(1'-racemic-glycerol) (DLPG); 1,2-dioleoyl-sn-glycerol-3-phospho-(1'-racemic-glycerol) (DOPG)), phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol and other anionic modifying groups attached to neutral lipids. The mixture of lipids can also include neutral lipids. The neutral lipid may be, but is not limited to, diacylglycerophosphocholine (L-α-phosphatidylcholine, hydrogenated (Soy) (HSPC); 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC); 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC); 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPPC); Phosphocholine (DOPC), diacylglycerophosphoethanolamines (1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE); 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE); 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE); 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE); 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and phosphatidylserine.

[0280] Structural lipids

[0281] The lipid component of the nanoparticle composition can include one or more structural lipids. In some embodiments, the structural lipids described herein are selected from cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid and alpha-tocopherol. In some embodiments, the structural lipid is cholesterol.

[0282] The role of cholesterol in the membrane depends largely on the environment. m ) phospholipids, cholesterol contributes to the formation of a liquid ordered phase characterized by reduced membrane fluidity and increased bilayer thickness. m Lipids undergo "condensation" whereby the cross-sectional area of ​​lipid and cholesterol is less than the sum of the individual cross-sectional areas. However, when compared with high T m When lipids combine, cholesterol increases membrane fluidity and narrows the bilayer. In both cases, cholesterol pulls the lipids toward the liquid, ordered phase.

[0283] In LNP formulations containing nucleic acids, the incorporation of cholesterol is largely based on two major findings obtained with liposome formulations of small molecule therapeutics: 1) cholesterol is an exchangeable molecule and can accumulate within liposomes during circulation, and 2) cholesterol significantly reduces the amount of surface-bound proteins and improves circulation half-life. Therefore, relative to endogenous membranes, equimolar amounts of cholesterol are included in LNP formulations; this prevents net efflux or net influx and maintains membrane integrity. In addition, it was found to be essential for particle stability and subsequently appeared for the first time in stable antisense lipid particles (SALPs). Structural lipids such as cholesterol are also necessary for encapsulating nucleic acids. Because cholesterol increases membrane rigidity, it is used to reduce drug leakage from the liposome core. However, this effect may be considered unimportant for large cargoes such as nucleic acids. Detailed information can be found, for example, in Albertsen, CH et al. "The role of lipid components in lipid nanoparticles for vaccines and gene therapy." Advanced Drug Delivery Reviews (2022): 114416, which is incorporated herein by reference in its entirety.

[0284] PEGylated lipids

[0285] The lipid component of the nanoparticle composition of the present disclosure may include one or more PEG or PEG-modified lipids. Such substances may alternatively be referred to as PEGylated lipids. PEG lipids are lipids modified with polyethylene glycol.

[0286] The lipid component of the nanoparticle composition can include one or more PEG lipids. Although PEG lipids constitute the minimum molar percentage of the lipid component in LNP (usually about 1.5mol%), they will affect several key properties: population size and dispersibility; LNP aggregation prevention; and particle stability during both preparation and storage. In addition, PEG lipids also affect factors such as nucleic acid encapsulation efficiency; circulation half-life; in vivo distribution; transfection efficiency; and immune response. All of these characteristics are related to the molar ratio of PEG lipids and the structure and length of PEG chains and lipid tails (alkyl / dialkyl chains) to some extent.

[0287] In some embodiments, the PEG lipid described herein is selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. In some embodiments, the PEG lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol) -2000] (PEG2000-DSPE) or 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DMG).

[0288] In some embodiments, the PEG lipid can be, but is not limited to, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (mPEG-2000-DSPE); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (mPEG-2000-DOPE); 1,2-dipalmitoyl-sn-glycero -3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](mPEG-2000-DPPE); 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](mPEG-2000-DMPE); 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](mPEG-2 000-DLPE); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000](mPEG-5000-DSPE); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000](mPEG-5000-DOPE); 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N -[methoxy(polyethylene glycol)-5000](mPEG-5000-DPPE); 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000](mPEG-5000-DMPE); 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000](mPEG-5000-DLPE).

[0289] In some embodiments, the average molecular weight of the PEG lipid is about 500-5000 Daltons, about 500-4500 Daltons, about 500-4000 Daltons, about 500-3500 Daltons, about 500-3000 Daltons, about 500-2500 Daltons, about 500-2000 Daltons, about 500-1500 Daltons, about 500-1000 Daltons, about 1000-5000 Daltons, about 1000-45 ...3500 Daltons, about 500-3000 Daltons, about 500-2500 Daltons, about 500-2000 Daltons, about 500-1500 Daltons, about 500-1000 Daltons, about 1000-5000 Daltons, about 1000-4500 Daltons, about 1000- 0 Daltons, about 1000-4000 Daltons, about 1000-3500 Daltons, about 1000-3000 Daltons, about 1000-2500 Daltons, about 1000-2000 Daltons, about 1000-1500 Daltons, about 1500-5000 Daltons, about 1500-4500 Daltons, about 1500-4000 Daltons, about 1500-3500 Daltons, about 1500-3000 Daltons, about 1500-2500 Daltons, about 1500-2000 Daltons, about 2000-5000 Daltons, about 2000-4500 Daltons, about 2000-4000 Daltons, about 2000-3500 Daltons, about 2000-3000 Daltons, about 2000-2500 Daltons, about 2500-5000 Daltons, about 2500-4500 Daltons, about 2500-4000 Daltons, about 2500-3 In some embodiments, the present invention relates to an aqueous solution of at least about 200 daltons, about 500 daltons, about 2500-3000 daltons, about 3000-5000 daltons, about 3000-4500 daltons, about 3000-4000 daltons, about 3000-3500 daltons, about 3500-5000 daltons, about 3500-4500 daltons, about 3500-4000 daltons, about 4000-5000 daltons, about 4000-4500 daltons or about 4500-5000 daltons.

[0290] Cargo molecules

[0291] Also provided herein are cargo molecules of the LNPs described herein. In some embodiments, the cargo molecule is a nucleic acid.

[0292] In some embodiments, the nucleic acid described herein is RNA, such as small interfering RNA (siRNA), microRNA (miRNA), messenger mRNA (mRNA), guide RNA (gRNA), circular RNA (circRNA), self-amplifying RNA (saRNA) or its antisense RNA. In some embodiments, the nucleic acid described herein is DNA, such as double-stranded DNA (dsDNA), plasmid DNA, single-stranded DNA (ssDNA) or its antisense DNA.

[0293] Examples of nucleic acids include DNA, such as genomic DNA, methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, in situ synthesized PCR products, and RNA / DNA hybrids.

[0294] Examples of nucleic acids also include RNA, such as various types of coding and non-coding RNA. Examples of different types of RNA include messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), micro RNA (miRNA) and viral RNA. RNA can be a transcript (for example, present in a tissue section). RNA can be very small (for example, less than 200 nucleic acid bases in length) or very large (for example, RNA length is greater than 200 nucleic acid bases). Small RNA mainly includes 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), micro RNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), Piwi interacting RNA (piRNA), tRNA-derived small RNA (tsRNA) and small rDNA derived RNA (srRNA). RNA can be double-stranded RNA or single-stranded RNA. RNA can be circular RNA. RNA can be bacterial rRNA (for example, 16s rRNA or 23s rRNA).

[0295] In some embodiments, the nucleic acid is capable of serving as a component of a gene editing reaction, such as clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing.

[0296] Other components

[0297] In addition to the components described in the aforementioned sections, the nanoparticle composition can also include one or more components. For example, the nanoparticle composition can include one or more small hydrophobic molecules, such as vitamins (e.g., vitamin A or vitamin E) or sterols. The nanoparticle composition can also include one or more permeability enhancer molecules, carbohydrates, polymers, therapeutic agents, surface modifiers or other components. The permeability enhancer molecule can be, for example, the molecule described in U.S. Patent Application Publication No. 2005 / 0222064. Carbohydrates can include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives thereof and the like).

[0298] Polymers can be included in the nanoparticle composition and / or used to encapsulate or partially encapsulate the nanoparticle composition. Polymers can be biodegradable and / or biocompatible. Polymers can be selected from, but are not limited to, polyamines, polyethers, polyamides, polyesters, polyurethanes, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylene, polyethylene, polyethylene imine, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitrile and polyarylates. For example, the polymer may include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-caprolactone-co-glycolide). -co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylates, polyurethanes, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(esteramides), polyamides, poly(etheresters), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as poly( terephthalate), polyvinyl alcohol (PVA), polyvinyl ethers, polyvinyl esters such as polyvinyl acetate, polyvinyl halides such as polyvinyl chloride (PVC), polyvinyl pyrrolidone, polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocellulose, hydroxypropyl cellulose and carboxymethyl cellulose), acrylic polymers such as poly(methyl (meth)acrylate) (PMMA), poly(ethyl (meth)acrylate), poly(butyl (meth)acrylate), poly(isobutyl (meth)acrylate). esters), poly(hexyl (meth)acrylate), poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and copolymers and mixtures thereof, polydioxanone and copolymers thereof, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamer, polyamine oxide, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone) and trimethylene carbonate, polyvinyl pyrrolidone.

[0299] Therapeutic agents may include, but are not limited to, cytotoxic agents, chemotherapeutic agents, and other therapeutic agents. Cytotoxic agents can include, for example, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, rachelmycin, and analogs thereof. Radioactive ions can also be used as therapeutic agents, and can include, for example, radioactive iodine, strontium, phosphorus, palladium, cesium, iridium, cobalt, yttrium, samarium, and praseodymium. Other therapeutic agents can include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil and decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, razithromycin, melphalan, carmustine, lomustine, cyclophosphamide, The invention relates to antimicrobial agents such as phosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) and cisplatin), anthracyclines (e.g., daunorubicin and doxorubicin), antibiotics (e.g., dactinomycin, bleomycin, mithramycin and anthramycin), and antimitotic agents (e.g., vincristine, vinblastine, paclitaxel and maytansine alkaloids).

[0300] Surface-altering agents can include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytics (e.g., acetylcysteine, mugwort, bromelain, papain, zearalenone, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin beta 4, dornase alfa, neltenexine, and erdosteine), and DNA enzymes (e.g., rhDNA enzyme). The surface-altering agent can be disposed within the nanoparticle and / or on the surface of the nanoparticle composition (eg, by coating, adsorption, covalent attachment or other methods).

[0301] In addition to these components, the nanoparticle compositions of the present disclosure may include any substance useful in a pharmaceutical composition. For example, the nanoparticle compositions may include one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as but not limited to one or more solvents, dispersion media, diluents, dispersing aids, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid vehicles, adhesives, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives and other substances. Excipients such as waxes, butters, colorants, coating agents, flavoring agents and aromatics may also be included. Pharmaceutically acceptable excipients are well known in the art (see, for example, Remington's The Science and Practice of Pharmacy, 21st edition, ARGennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006).

[0302] Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and / or combinations thereof. Granulating agents and dispersants may be selected from a non-limiting list consisting of potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponges, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (cross-linked carboxymethyl cellulose), methyl cellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate Sodium lauryl sulfate, quaternary ammonium compounds and / or combinations thereof.

[0303] Surfactants and / or emulsifiers may include, but are not limited to, natural emulsifiers (e.g., gum arabic, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, waxes, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and [magnesium aluminum silicate]), long-chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [ 20], polyoxyethylene sorbitan[ 60], polyoxyethylene sorbitan monooleate[ 80], Sorbitan monopalmitate[ 40], Sorbitan monostearate[ 60], Sorbitan tristearate[ 65], monoolein, sorbitan monooleate[ 80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [ 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate and ), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., ), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [ 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, F68, 188. Cetrimide bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium and / or a combination thereof.

[0304] Binders can be starches (e.g., corn starch and starch paste); gelatin; sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic gums (e.g., gum arabic, sodium alginate, extracts of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate and larch arabinogalactan); alginate; polyethylene oxide; polyethylene glycol; inorganic calcium salt; silicic acid; polymethacrylate; wax; water; alcohol; and combinations thereof or any other suitable binder.

[0305] The example of preservative may include but is not limited to antioxidant, chelating agent, antimicrobial preservative, antifungal preservative, alcohol preservative, acidic preservative and / or other preservative. The example of antioxidant includes but is not limited to alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite and / or sodium sulfite. The example of chelating agent includes ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid and / or trisodium edetate. Examples of antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethanol, glycerol, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Examples of antifungal preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoates, and / or phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid and / or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroximemesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT Methylparaben, 1 15, II. NEOLONE TM 、KATHON TM and / or

[0306] Examples of buffers include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glucuronate, calcium glucoheptonate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propionic acid, calcium ketovalerate, valeric acid, calcium hydrogen phosphate, phosphoric acid, tricalcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dipotassium hydrogen phosphate, monopotassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, monosodium phosphate, sodium phosphate mixtures, tromethamine, sulfamate buffer (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethanol, and / or combinations thereof. The lubricant may be selected from the non-limiting group consisting of magnesium stearate, calcium stearate, stearic acid, silicon dioxide, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

[0307] Examples of oils include, but are not limited to, almond oil, apricot kernel oil, avocado oil, babassu oil, bergamot oil, black currant seed oil, borage oil, juniper oil, chamomile oil, rapeseed oil, caraway oil, Brazilian palm oil, castor oil, cinnamon oil, cocoa butter oil, coconut oil, cod liver oil, coffee oil, corn oil, cottonseed oil, emu oil, eucalyptus oil, evening primrose oil, fish oil, linseed oil, geraniol oil, gourd oil, grape seed oil, hazelnut oil, hyssop oil, isopropyl myristate, jojoba oil, macadamia oil, lavender oil, lavender oil, lemon oil, litsea cubeba oil, macadamia nut oil, mallow oil, mango seed oil, white meadowfoam seed oil, mink oil, nutmeg oil, olive oil, sweet orange oil, deep sea fish oil (orange roughy oil), palm oil, palm kernel oil, peach kernel oil, peanut oil, poppy seed oil, pumpkin seed oil, rapeseed oil, rice bran oil, rosemary oil, safflower oil, sandalwood oil, sasquana oil, savoury oil, sea buckthorn oil, sesame oil, shea butter, ketone oil, soybean oil, sunflower oil, tea tree oil, thistle oil, tsubaki oil, vetiver oil, walnut oil and wheat germ oil, and butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, simethicone, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil and / or combinations thereof.

[0308] Composition of PALNP

[0309] In some embodiments, the nanoparticle composition described herein includes: (a) about 0.1 mol% to about 20 mol% of a compound of Formula I, Formula II, Formula III, Formula IV, or a derivative thereof; (b) about 5 mol% to about 30 mol% of an ionizable and / or permanently charged cationic lipid (e.g., ALC-0315, SM-102, DLin-DMA, DLin-MC3-DMA, and / or DLin-KC2-DMA); (c) about 10 mol% to about 50 mol% of an auxiliary lipid (e.g., DSPC and / or DOPE); (d) about 20 mol% to about 50 mol% of a structural lipid (e.g., cholesterol); and (e) about 0.5 mol% to about 5 mol% of a PEG lipid (e.g., PEG2000-DSPE).

[0310] As used herein, the term "mol%" represents the molar ratio of one or more compounds relative to the total molar amount of a nanoparticle composition described herein (e.g., a nanoparticle composition comprising a lipid component and a polymer component as described herein). In some embodiments, the "mol%" of a particular compound is calculated without taking into account the molar amount of nucleic acids encapsulated in the lipid nanoparticles.

[0311] In some embodiments, the compound of Formula I, Formula II, Formula III, or Formula IV comprises about 1 mol% to about 10 mol% of the nanoparticle composition (e.g., about 1 mol% to about 9 mol%, about 1 mol% to about 8 mol%, about 1 mol% to about 7 mol%, about 1 mol% to about 6 mol%, about 1 mol% to about 5 mol%, about 1 mol% to about 4 mol%, about 1 mol% to about 3 mol%, about 1 mol% to about 2 mol%, about 2 mol% to about 10 mol%, about 2 mol% to about 9 mol%, about 2 mol% to about 8 mol%, about 2 mol% to about 7 mol%, about 2 mol% to about 6 mol%, about 2 mol% to about 5 mol%, about 2 mol% to about 4 mol%, about 2 mol% to about 3 mol%, about 1 mol% to about 2 mol%, about 2 mol% to about 10 mol%, about 2 mol% to about 9 mol%, about 2 mol% to about 8 mol%, about 2 mol% to about 7 mol%, about 3 mol% to about 6 mol%, about 2 mol% to about 5 mol%, about 2 mol% to about 4 mol%, about 2 mol% to about 3 mol%, about 3 mol% to about 10 mol%, about 3 mol% to about 9 mol%, about 3 mol% to about 8 mol%, about 3 mol% to about 7 mol%, about 3 % to about 9mol%, about 4mol% to about 8mol%, about 4mol% to about 7mol%, about 4mol% to about 6mol%, about 4mol% to about 5mol%, about 5mol% to about 10mol%, about 5mol% to about 9mol%, about 5mol% to about 8mol%, about 5mol% to about 7mol%, about 5mol% to about 6mol%, about 6mol% to about 10mol%, about 6mol% to about 9mol%, about 6mol% to about 8mol%, about 6mol% to about 7mol%, about 7mol% to about 10mol%, about 7mol% to about 9mol%, about 7mol% to about 8mol%, about 8mol% to about 10mol%, about 8mol% to about 9mol%, or about 9mol% to about 10mol%). In some embodiments, Formula I is L121, which comprises about 5 mol% to about 7 mol% of the nanoparticle composition (e.g., about 5 mol%, about 5.1 mol%, about 5.2 mol%, about 5.3 mol%, about 5.4 mol%, about 5.5 mol%, about 5.6 mol%, about 5.7 mol%, about 5.8 mol%, about 5.9 mol%, about 6 mol%, about 6.1 mol%, about 6.2 mol%, about 6.3 mol%, about 6.4 mol%, about 6.5 mol%, about 6.6 mol%, about 6.7 mol%, about 6.8 mol%, about 6.9 mol%, or about 7 mol%).In some embodiments, the compound of Formula I is L92, which comprises about 4 mol% to about 5 mol% of the nanoparticle composition (e.g., about 4 mol%, about 4.1 mol%, about 4.2 mol%, about 4.3 mol%, about 4.4 mol%, about 4.5 mol%, about 4.6 mol%, about 4.7 mol%, about 4.8 mol%, about 4.9 mol%, or about 5 mol%). In some embodiments, the compound of Formula I is L81, which comprises about 2 mol% to about 4 mol% of the nanoparticle composition (e.g., about 2, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, or about 4).

[0312] In some embodiments, the ionizable and / or permanently charged cationic lipids described herein comprise about 5 mol% to about 30 mol%, about 5 mol% to about 25 mol%, about 5 mol% to about 20 mol%, about 5 mol% to about 15 mol%, about 5 mol% to about 10 mol%, about 10 mol% to about 30 mol%, about 10 mol% to about 25 mol%, about 10 mol% to about 20 mol%, about 10 mol% to about 15 mol%, about 15 mol% to about 30 mol%, about 15 mol% to about 25 mol%, about 15 mol% to about 20 mol%, about 15 mol% to about 20 mol%, about 20 mol% to about 30 mol%, about 20 mol% to about 25 mol%, or about 25 mol% to about 30 mol% of the nanoparticle composition. In some embodiments, the ionizable and / or permanently charged cationic lipids described herein comprise about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30% of the nanoparticle composition.

[0313] In some embodiments, the ionizable and / or permanently charged cationic lipid within the LNP (e.g., PALNP) or LNP having a nanoparticle composition described herein is less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% of the ionizable and / or permanently charged cationic lipid for a reference LNP (e.g., a LNP that does not include a polymer component (e.g., any of the polymer components described herein), such as a LNP used as a COVID-10 vaccine described herein).

[0314] In some embodiments, the helper lipids described herein comprise about 10 mol % to about 50 mol %, about 10 mol % to about 45 mol %, about 10 mol % to about 40 mol %, about 10 mol % to about 35 mol %, about 10 mol % to about 30 mol %, about 10 mol % to about 25 mol %, about 10 mol % to about 20 mol %, about 10 mol % to about 15 mol %, about 15 mol % to about 50 mol %, about 15 mol % to about 45 mol %, about 15 mol % to about 40 mol %, about 15 mol % to about 35 mol %, about 15 mol % to about 30 mol %, about 15 mol % to about 25 mol %, about 15 mol % to about 20 mol %, about 20 mol % to about 50 mol %, about 20 ...20 mol% to about 40mol%, about 20mol% to about 35mol%, about 20mol% to about 30mol%, about 20mol% to about 25mol%, about 25mol% to about 50mol%, about 25mol% to about 45mol%, about 25mol% to about 40mol%, about 25mol% to about 35mol%, about 25mol% to about 30mol%, about 30mol% to about 50mol%, about 30mol% to about 45mol%, about 30mol% to about 40mol%, about 30mol% to about 35mol%, about 35mol% to about 50mol%, about 35mol% to about 45mol%, about 35mol% to about 40mol%, about 40mol% to about 50mol%, about 40mol% to about 45mol%, or about 45mol% to about 50mol%.

[0315] In some embodiments, the helper lipids described herein include about 5 mol % to about 30 mol % DSPC or HSPC and about 5 mol % to about 30 mol % DOPE.

[0316] In some embodiments, the helper lipids described herein include about 10 mol% to about 30 mol% (e.g., about 10 mol% to about 28 mol%, about 10 mol% to about 26 mol%, about 10 mol% to about 24 mol%, about 10 mol% to about 22 mol%, about 10 mol% to about 20 mol%, about 10 mol% to about 18 mol%, about 10 mol% to about 16 mol%, about 10 mol% to about 14 mol%, about 10 mol% to about 12 mol%, about 12 mol% to about 30 mol%, about 12 mol% to about 28 mol%, about 12 mol% to about 16 mol%, about 12 mol% to about mol% to about 24mol%, about 12mol% to about 22mol%, about 12mol% to about 20mol%, about 12mol% to about 18mol%, about 12mol% to about 16mol%, about 12mol% to about 14mol%, about 14mol% to about 30mol%, about 14mol% to about 28mol%, about 14mol% to about 26mol%, about 14mol% to about 24mol%, about 14mol% to about 22mol%, about 14mol% to about 20mol%, about 14mol% to about 18mol%, about 14mol% to about 16mol%, about 16mol% to about 30mol%, about 14mol% to about 28mol%, about 14mol% to about 26mol%, about 14mol% to about 24mol%, about 14mol% to about 22mol%, about 14mol% to about 20mol%, about 14mol% to about 18mol%, about 14mol% to about 16mol%, about 16mol% to about 30mol%. mol%, about 16 mol% to about 28 mol%, about 16 mol% to about 26 mol%, about 16 mol% to about 24 mol%, about 16 mol% to about 22 mol%, about 16 mol% to about 20 mol%, about 16 mol% to about 18 mol%, about 18 mol% to about 30 mol%, about 18 mol% to about 28 mol%, about 18 mol% to about 26 mol%, about 18 mol% to about 24 mol%, about 18 mol% to about 22 mol%, about 18 mol% to about 20 mol%, about 20 mol% to about 30 mol%, about 20 mol% to about 28 mol%, about 18 mol% to about 26 mol%, about 18 mol% to about 24 mol%, about 18 mol% to about 22 mol%, about 18 mol% to about 20 mol%, about 20 mol% to about 30 mol%, about 20 mol% to about 28 mol%, about 20 mol% to about % to about 26 mol%, about 20 mol% to about 24 mol%, about 20 mol% to about 22 mol%, about 22 mol% to about 30 mol%, about 22 mol% to about 28 mol%, about 22 mol% to about 26 mol%, about 22 mol% to about 24 mol%, about 24 mol% to about 30 mol%, about 24 mol% to about 28 mol%, about 24 mol% to about 26 mol%, about 26 mol% to about 30 mol%, about 26 mol% to about 28 mol%, about 28 mol% to about 30 mol%) of DSPC or a derivative thereof and about 10 mol% to about 30 mol% (e.g.,about 10 mol% to about 28 mol%, about 10 mol% to about 26 mol%, about 10 mol% to about 24 mol%, about 10 mol% to about 22 mol%, about 10 mol% to about 20 mol%, about 10 mol% to about 18 mol%, about 10 mol% to about 16 mol%, about 10 mol% to about 14 mol%, about 10 mol% to about 12 mol%, about 12 mol% to about 30 mol%, about 12 mol% to about 28 mol%, about 12 mol% to about 16 mol%, about 12 mol% to about 24 mol%, about 12 mol% to about 2 %, about 12 mol% to about 20 mol%, about 12 mol% to about 18 mol%, about 12 mol% to about 16 mol%, about 12 mol% to about 14 mol%, about 14 mol% to about 30 mol%, about 14 mol% to about 28 mol%, about 14 mol% to about 26 mol%, about 14 mol% to about 24 mol%, about 14 mol% to about 22 mol%, about 14 mol% to about 20 mol%, about 14 mol% to about 18 mol%, about 14 mol% to about 16 mol%, about 16 mol% to about 30 mol%, about 16 mol% to about %, about 16 mol% to about 26 mol%, about 16 mol% to about 24 mol%, about 16 mol% to about 22 mol%, about 16 mol% to about 20 mol%, about 16 mol% to about 18 mol%, about 18 mol% to about 30 mol%, about 18 mol% to about 28 mol%, about 18 mol% to about 26 mol%, about 18 mol% to about 24 mol%, about 18 mol% to about 22 mol%, about 18 mol% to about 20 mol%, about 20 mol% to about 30 mol%, about 20 mol% to about 28 mol%, % to about 26 mol%, about 20 mol% to about 24 mol%, about 20 mol% to about 22 mol%, about 22 mol% to about 30 mol%, about 22 mol% to about 28 mol%, about 22 mol% to about 26 mol%, about 22 mol% to about 24 mol%, about 24 mol% to about 30 mol%, about 24 mol% to about 28 mol%, about 24 mol% to about 26 mol%, about 26 mol% to about 30 mol%, about 26 mol% to about 28 mol%, or about 28 mol% to about 30 mol%) of DOPE or a derivative thereof.

[0317] In some embodiments, the structural lipids described herein comprise about 20 mol% to about 50 mol%, about 20 mol% to about 45 mol%, about 20 mol% to about 40 mol%, about 20 mol% to about 35 mol%, about 20 mol% to about 30 mol%, about 20 mol% to about 25 mol%, about 25 mol% to about 50 mol%, about 25 mol% to about 45 mol%, about 25 mol% to about 40 mol%, about 25 mol% to about 35 mol%, about 30 mol% to about 50 mol%, about 30 mol% to about 45 mol%, about 30 mol% to about 40 mol%, about 30 mol% to about 45 mol%, about 35 mol% to about 50 mol%, about 35 mol% to about 45 mol%, about 35 mol% to about 40 mol%, about 40 mol% to about 50 mol%, about 40 mol% to about 45 mol%, or about 45 mol% to about 50 mol% of the nanoparticle composition.

[0318] In some embodiments, the PEG lipids described herein comprise about 0.5 mol% to about 5 mol%, about 0.5 mol% to about 4.5 mol%, about 0.5 mol% to about 4 mol%, about 0.5 mol% to about 3.5 mol%, about 0.5 mol% to about 3 mol%, about 0.5 mol% to about 2.5 mol%, about 0.5 mol% to about 2 mol%, about 0.5 mol% to about 1.5 mol%, about 0.5 mol% to about 1 mol%, about 1 mol% to about 5 mol%, about 1 mol% to about 4.5 mol%, about 1 mol% to about 4 mol%, about 1 mol% to about 3.5 mol%, about 1 mol% to about 3 mol%, about 1 mol% to about 2.5 mol%, about 1 mol% to about 2 mol%, about 1 mol% to about 1.5 mol%, about 1 mol% to about 1.5 mol%, about 1 mol% to about 5 mol%, about 1 mol% to about 4.5 mol%, about 1 mol% to about 4 mol%, about 1 mol% to about 3.5 mol%, about 1 mol% to about 3 mol%, about 1 mol% to about 2.5 mol%, about 1 mol% to about 2 % to about 5mol%, about 1.5mol% to about 4.5mol%, about 1.5mol% to about 4mol%, about 1.5mol% to about 3.5mol%, about 1.5mol% to about 3mol%, about 1.5mol% to about 2.5mol%, about 1.5mol% to about 2mol%, about 2mol% to about 5mol%, about 2mol% to about 4.5mol%, about 2mol% to about 4mol%, about 2mol% to about 3.5mol%, about 2mol% to about 3mol%, about 2mol% to about 2.5mol%, about 2.5mol% to about 5mol%, about 2.5mol% to about 4.5mol%, about 2.5mol% to about 4mol%, about 2.5mol% to about 3.5mol%, about 2.5mol% to about 3mol%, about 3mol% to about 5mol%, about 3mol% to about 4 5mol%, about 3mol% to about 4mol%, about 3mol% to about 3.5mol%, about 3.5mol% to about 5mol%, about 3.5mol% to about 4.5mol%, about 3.5mol% to about 4mol%, about 4mol% to about 5mol%, about 4mol% to about 4.5mol% or about 4.5mol% to about 5mol%.

[0319] In some embodiments, provided herein is CPT067 or an LNP having a formulation substantially similar to CPT067, wherein the CPT067 or LNP comprises: (a) about 0.1 mol % to about 10 mol % (e.g., about 5 mol %) of a compound of the copolymer described herein (e.g., L121); (b) about 30 mol % to about 50 mol % (e.g., about 41.5 mol %) of DLin-DMA or a derivative thereof; (c) about 5 mol % to about 20 mol % (e.g., about 10 mol %) of DSPC or a derivative thereof; (d) about 20 mol % to about 50 mol % (e.g., about 38.5 mol %) of cholesterol or a derivative thereof; and (e) about 0.5 mol % to about 5 mol % (e.g., about 1.5 mol %) of PEG2000-DSPE or a derivative thereof.

[0320] In some embodiments, provided herein is CPT069 or an LNP having a formulation substantially similar to CPT069, comprising: (a) about 0.1 mol % to about 10 mol % (e.g., about 5 mol %) of a compound of the copolymer described herein (e.g., L121); (b) about 30 mol % to about 50 mol % (e.g., about 41.5 mol %) of DLin-KC2-DMA or a derivative thereof; (c) about 5 mol % to about 20 mol % (e.g., about 10 mol %) of DSPC or a derivative thereof; (d) about 20 mol % to about 50 mol % (e.g., about 41.5 mol %) of cholesterol or a derivative thereof; and (e) about 0.5 mol % to about 5 mol % (e.g., about 2 mol %) of PEG2000-DSPE or a derivative thereof.

[0321] In some embodiments, provided herein is CPT104 or an LNP having a formulation substantially similar to CPT104, wherein the CPT104 or LNP comprises: (a) about 0.1 mol % to about 10 mol % (e.g., about 5 mol %) of a compound of the copolymer described herein (e.g., L121); (b) about 30 mol % to about 60 mol % (e.g., about 45 mol %) of DLin-KC2-DMA or a derivative thereof; (c) about 5 mol % to about 20 mol % (e.g., about 10 mol %) of DSPC or a derivative thereof; (d) about 20 mol % to about 50 mol % (e.g., about 38.5 mol %) of cholesterol or a derivative thereof; and (e) about 0.5 mol % to about 5 mol % (e.g., about 1.5 mol %) of PEG2000-DSPE or a derivative thereof.

[0322] In some embodiments, provided herein is CPT106 or an LNP having a formulation substantially similar to CPT106, wherein the CPT106 or LNP comprises: (a) about 0.1 mol % to about 10 mol % (e.g., about 5 mol %) of a compound of the copolymer described herein (e.g., L121); (b) about 30 mol % to about 60 mol % (e.g., about 45 mol %) of DLin-MC3-DMA or a derivative thereof; (c) about 5 mol % to about 20 mol % (e.g., about 10 mol %) of DSPC or a derivative thereof; (d) about 20 mol % to about 50 mol % (e.g., about 38.5 mol %) of cholesterol or a derivative thereof; and (e) about 0.5 mol % to about 5 mol % (e.g., about 1.5 mol %) of PEG2000-DSPE or a derivative thereof.

[0323] In some embodiments, provided herein is CPT108 or an LNP having a formulation substantially similar to CPT108, wherein the CPT108 or LNP comprises: (a) about 0.1 mol % to about 10 mol % (e.g., about 5 mol %) of a compound of the copolymer described herein (e.g., L121); (b) about 30 mol % to about 60 mol % (e.g., about 45 mol %) of ALC-0315 or a derivative thereof; (c) about 5 mol % to about 20 mol % (e.g., about 10 mol %) of DSPC or a derivative thereof; (d) about 20 mol % to about 50 mol % (e.g., about 38.5 mol %) of cholesterol or a derivative thereof; and (e) about 0.5 mol % to about 5 mol % (e.g., about 1.5 mol %) of PEG2000-DSPE or a derivative thereof.

[0324] In some embodiments, provided herein is CPT147 or an LNP having a formulation substantially similar to CPT147, comprising: (a) about 0.1 mol % to about 10 mol % (e.g., about 5 mol %) of a compound of the copolymer described herein (e.g., L121); (b) about 1 mol % to about 20 mol % (e.g., about 10 mol %) of ALC-0315 or a derivative thereof; (c) about 10 mol % to about 30 mol % (e.g., about 22.5 mol %) of DSPC or a derivative thereof and about 10 mol % to about 30 mol % (e.g., about 22.5 mol %) of DOPE or a derivative thereof; (d) about 20 mol % to about 50 mol % (e.g., about 38.5 mol %) of cholesterol or a derivative thereof; and (e) about 0.5 mol % to about 5 mol % (e.g., about 1.5 mol %) of PEG2000-DSPE or a derivative thereof.

[0325] In some embodiments, provided herein is CPT147-38 or an LNP having a formulation substantially similar to CPT147-38, wherein the CPT147-38 or LNP comprises: (a) about 0.1 mol% to about 10 mol% (e.g., about 6.8 mol%) of a compound of the copolymer described herein (e.g., L121); (b) about 10 mol% to about 30 mol% (e.g., about 18 mol%) of ALC-0315 or a derivative thereof; (c) about 10 mol% to about 30 mol% (e.g., about 20 mol%) of DSPC or a derivative thereof and about 10 mol% to about 30 mol% (e.g., about 20 mol%) of DOPE or a derivative thereof; (d) about 20 mol% to about 50 mol% (e.g., about 34 mol%) of cholesterol or a derivative thereof; and (e) about 0.5 mol% to about 5 mol% (e.g., about 1.5 mol%) of PEG2000-DSPE or a derivative thereof.

[0326] In some embodiments, provided herein is CPT163-21-18 or an LNP having a formulation substantially similar to CPT163-21-18, wherein the CPT163-21-18 or LNP comprises: (a) about 0.1 mol% to about 10 mol% (e.g., about 1.8 mol%) of a compound of the copolymer described herein (e.g., L81); (b) about 10 mol% to about 30 mol% (e.g., about 19 mol%) of ALC-0315 or a derivative thereof; (c) about 10 mol% to about 30 mol% (e.g., about 21 mol%) of DSPC or a derivative thereof and about 10 mol% to about 30 mol% (e.g., about 21 mol%) of DOPE or a derivative thereof; (d) about 20 mol% to about 50 mol% (e.g., about 36 mol%) of cholesterol or a derivative thereof; and (e) about 0.5 mol% to about 5 mol% (e.g., about 1.4 mol%) of PEG2000-DSPE or a derivative thereof.

[0327] In some embodiments, provided herein is CPT147E or an LNP having a formulation substantially similar to CPT147E, comprising: (a) about 0.1 mol % to about 10 mol % (e.g., about 6.8 mol %) of a compound of the copolymer described herein (e.g., L121); (b) about 10 mol % to about 30 mol % (e.g., about 18 mol %) of ALC-0315 or a derivative thereof; (c) about 10 mol % to about 30 mol % (e.g., about 19.8 mol %) of DSPC or a derivative thereof and about 10 mol % to about 30 mol % (e.g., about 19.8 mol %) of DOPE or a derivative thereof; (d) about 20 mol % to about 50 mol % (e.g., about 34.2 mol %) of cholesterol or a derivative thereof; and (e) about 0.5 mol % to about 5 mol % (e.g., about 1.4 mol %) of PEG2000-DSPE or a derivative thereof.

[0328] In some embodiments, provided herein is CPT147E-05 or an LNP having a formulation substantially similar to CPT147E-05, wherein the CPT147E-05 or LNP comprises: (a) about 0.1 mol% to about 10 mol% (e.g., about 6.8 mol%) of a compound of the copolymer described herein (e.g., L121); (b) about 10 mol% to about 30 mol% (e.g., about 18 mol%) of ALC-0315 or a derivative thereof; (c) about 10 mol% to about 30 mol% (e.g., about 19.8 mol%) of DSPC or a derivative thereof and about 10 mol% to about 30 mol% (e.g., about 19.8 mol%) of DOPE or a derivative thereof; (d) about 20 mol% to about 50 mol% (e.g., about 34.2 mol%) of cholesterol or a derivative thereof; and (e) about 0.5 mol% to about 5 mol% (e.g., about 1.4 mol%) of PEG2000-DSPE or a derivative thereof.

[0329] In some embodiments, provided herein is CPT149E-01 or an LNP having a formulation substantially similar to CPT149E-01, wherein the CPT149E-01 or LNP comprises: (a) about 0.1 mol% to about 10 mol% (e.g., about 6.8 mol%) of a compound of the copolymer described herein (e.g., L121); (b) about 10 mol% to about 30 mol% (e.g., about 18 mol%) of SM-102 or a derivative thereof; (c) about 10 mol% to about 30 mol% (e.g., about 19.8 mol%) of DSPC or a derivative thereof and about 10 mol% to about 30 mol% (e.g., about 19.8 mol%) of DOPE or a derivative thereof; (d) about 20 mol% to about 50 mol% (e.g., about 34.2 mol%) of cholesterol or a derivative thereof; and (e) about 0.5 mol% to about 5 mol% (e.g., about 1.5 mol%) of PEG2000-DSPE or a derivative thereof.

[0330] In some embodiments, provided herein is CPT162E-01 or an LNP having a formulation substantially similar to CPT162E-01, wherein the CPT162E-01 or LNP comprises: (a) about 0.1 mol% to about 10 mol% (e.g., about 6.8 mol%) of a compound of the copolymer described herein (e.g., L121); (b) about 10 mol% to about 30 mol% (e.g., about 18 mol%) of DLin-KC2-DMA or a derivative thereof; (c) about 10 mol% to about 30 mol% (e.g., about 19.8 mol%) of DSPC or a derivative thereof and about 10 mol% to about 30 mol% (e.g., about 19.8 mol%) of DOPE or a derivative thereof; (d) about 20 mol% to about 50 mol% (e.g., about 34.2 mol%) of cholesterol or a derivative thereof; and (e) about 0.5 mol% to about 5 mol% (e.g., about 1.5 mol%) of PEG2000-DSPE or a derivative thereof.

[0331] In some embodiments, provided herein is CPT147E-04 or an LNP having a formulation substantially similar to CPT147E-04, wherein the CPT147E-04 or LNP comprises: (a) about 0.1 mol% to about 10 mol% (e.g., about 5 mol%) of a compound of the copolymer described herein (e.g., L121); (b) about 1 mol% to about 20 mol% (e.g., about 10 mol%) of ALC-0315 or a derivative thereof; (c) about 10 mol% to about 30 mol% (e.g., about 22.5 mol%) of DSPC or a derivative thereof and about 10 mol% to about 30 mol% (e.g., about 22.5 mol%) of DOPE or a derivative thereof; (d) about 20 mol% to about 50 mol% (e.g., about 38.5 mol%) of cholesterol or a derivative thereof; and (e) about 0.5 mol% to about 5 mol% (e.g., about 1.5 mol%) of PEG2000-DSPE or a derivative thereof.

[0332] In some embodiments, provided herein is CPT163-10 or an LNP having a formulation substantially similar to CPT163-10, wherein the CPT163-10 or LNP comprises: (a) about 0.1 mol% to about 10 mol% (e.g., about 1.9 mol%) of a compound of the copolymer described herein (e.g., L81); (b) about 1 mol% to about 30 mol% (e.g., about 19 mol%) of ALC-0315 or a derivative thereof; (c) about 10 mol% to about 30 mol% (e.g., about 20.9 mol%) of DSPC or a derivative thereof and about 10 mol% to about 30 mol% (e.g., about 20.9 mol%) of DOPE or a derivative thereof; (d) about 20 mol% to about 50 mol% (e.g., about 36 mol%) of cholesterol or a derivative thereof; and (e) about 0.5 mol% to about 5 mol% (e.g., about 1.4 mol%) of PEG2000-DSPE or a derivative thereof.

[0333] In some embodiments, provided herein is CPT161E or an LNP having a formulation substantially similar to CPT161E, comprising: (a) about 0.1 mol % to about 10 mol % (e.g., about 6.8 mol %) of a compound of the copolymer described herein (e.g., L121); (b) about 1 mol % to about 30 mol % (e.g., about 18 mol %) of DLin-DMA or a derivative thereof; (c) about 10 mol % to about 30 mol % (e.g., about 19.8 mol %) of DSPC or a derivative thereof and about 10 mol % to about 30 mol % (e.g., about 19.8 mol %) of DOPE or a derivative thereof; (d) about 20 mol % to about 50 mol % (e.g., about 34.2 mol %) of cholesterol or a derivative thereof; and (e) about 0.5 mol % to about 5 mol % (e.g., about 1.5 mol %) of PEG2000-DSPE or a derivative thereof.

[0334] In some embodiments, provided herein is CPT164E or an LNP having a formulation substantially similar to CPT164E, comprising: (a) about 0.1 mol % to about 10 mol % (e.g., about 4.6 mol %) of a compound of the copolymer described herein (e.g., L92); (b) about 1 mol % to about 30 mol % (e.g., about 18.3 mol %) of DLin-KC2-DMA or a derivative thereof; (c) about 10 mol % to about 30 mol % (e.g., about 20.3 mol %) of DSPC or a derivative thereof and about 10 mol % to about 30 mol % (e.g., about 20.3 mol %) of DOPE or a derivative thereof; (d) about 20 mol % to about 50 mol % (e.g., about 35.1 mol %) of cholesterol or a derivative thereof; and (e) about 0.5 mol % to about 5 mol % (e.g., about 1.4 mol %) of PEG2000-DSPE or a derivative thereof.

[0335] In some embodiments, provided herein is CPT149-01 or an LNP having a formulation substantially similar to CPT149-01, wherein the CPT149-01 or LNP comprises: (a) about 0.1 mol% to about 10 mol% (e.g., about 6.8 mol%) of a compound of the copolymer described herein (e.g., L121); (b) about 1 mol% to about 30 mol% (e.g., about 18 mol%) of SM-102 or a derivative thereof; (c) about 10 mol% to about 30 mol% (e.g., about 19.8 mol%) of DSPC or a derivative thereof and about 10 mol% to about 30 mol% (e.g., about 19.8 mol%) of DOPE or a derivative thereof; (d) about 20 mol% to about 50 mol% (e.g., about 34.2 mol%) of cholesterol or a derivative thereof; and (e) about 0.5 mol% to about 5 mol% (e.g., about 1.5 mol%) of PEG2000-DSPE or a derivative thereof.

[0336] In some embodiments, provided herein is CPT200-07 or an LNP having a formulation substantially similar to CPT200-07, wherein the CPT200-07 or LNP comprises: (a) about 0.1 mol% to about 10 mol% (e.g., about 2.2 mol%) of a compound of the copolymer described herein (e.g., L81); (b) about 1 mol% to about 30 mol% (e.g., about 22.3 mol%) of SM-102 or a derivative thereof; (c) about 10 mol% to about 30 mol% (e.g., about 15.6 mol%) of DSPC or a derivative thereof and about 10 mol% to about 30 mol% (e.g., about 15.6 mol%) of DOPE or a derivative thereof; (d) about 30 mol% to about 60 mol% (e.g., about 43 mol%) of cholesterol or a derivative thereof; and (e) about 0.5 mol% to about 5 mol% (e.g., about 1.1 mol%) of PEG2000-DSPE or a derivative thereof.

[0337] In some embodiments, provided herein is CPT153E or an LNP having a formulation substantially similar to CPT153E, comprising: (a) about 0.1 mol % to about 10 mol % (e.g., about 6.8 mol %) of a compound of the copolymer described herein (e.g., L121); (b) about 1 mol % to about 30 mol % (e.g., about 17.9 mol %) of DOTAP or a derivative thereof; (c) about 10 mol % to about 30 mol % (e.g., about 19.9 mol %) of DSPC or a derivative thereof and about 10 mol % to about 30 mol % (e.g., about 19.9 mol %) of DOPE or a derivative thereof; (d) about 30 mol % to about 60 mol % (e.g., about 34.1 mol %) of cholesterol or a derivative thereof; and (e) about 0.5 mol % to about 5 mol % (e.g., about 1.4 mol %) of PEG2000-DSPE or a derivative thereof.

[0338] In some embodiments, provided herein is CPT189 or an LNP having a formulation substantially similar to CPT189, comprising: (a) about 0.1 mol % to about 10 mol % (e.g., about 2.4 mol %) of a compound of the copolymer described herein (e.g., L31R1); (b) about 1 mol % to about 30 mol % (e.g., about 21.7 mol %) of ALC-0315 or a derivative thereof; (c) about 10 mol % to about 30 mol % (e.g., about 16.9 mol %) of DSPC or a derivative thereof and about 10 mol % to about 30 mol % (e.g., about 16.9 mol %) of DOPE or a derivative thereof; (d) about 30 mol % to about 60 mol % (e.g., about 41 mol %) of cholesterol or a derivative thereof; and (e) about 0.5 mol % to about 5 mol % (e.g., about 1.2 mol %) of PEG2000-DSPE or a derivative thereof.

[0339] In some embodiments, provided herein is CPT202 or an LNP having a formulation substantially similar to CPT202, wherein the CPT202 or LNP comprises: (a) about 0.1 mol% to about 10 mol% (e.g., about 2.4 mol%) of a compound of the copolymer described herein (e.g., L17R4); (b) about 1 mol% to about 30 mol% (e.g., about 21.7 mol%) of ALC-0315 or a derivative thereof; (c) about 10 mol% to about 30 mol% (e.g., about 16.9 mol%) of DSPC or a derivative thereof and about 10 mol% to about 30 mol% (e.g., about 16.9 mol%) of DOPE or a derivative thereof; (d) about 30 mol% to about 60 mol% (e.g., about 41 mol%) of cholesterol or a derivative thereof; and (e) about 0.5 mol% to about 5 mol% (e.g., about 1.2 mol%) of PEG2000-DSPE or a derivative thereof.

[0340] In some embodiments, provided herein is CPT147P or an LNP having a formulation substantially similar to CPT147P, comprising: (a) about 0.1 mol % to about 10 mol % (e.g., about 5 mol %) of a compound of the copolymer described herein (e.g., L121); (b) about 1 mol % to about 30 mol % (e.g., about 18.1 mol %) of ALC-0315 or a derivative thereof; (c) about 10 mol % to about 30 mol % (e.g., about 18.1 mol %) of DSPC or a derivative thereof and about 10 mol % to about 30 mol % (e.g., about 18.1 mol %) of DOPE or a derivative thereof; (d) about 30 mol % to about 60 mol % (e.g., about 39.2 mol %) of cholesterol or a derivative thereof; and (e) about 0.5 mol % to about 5 mol % (e.g., about 1.5 mol %) of PEG2000-DSPE or a derivative thereof.

[0341] In some embodiments, provided herein is CPT149E or an LNP having a formulation substantially similar to CPT149E, comprising: (a) about 0.1 mol % to about 10 mol % (e.g., about 6.8 mol %) of a compound of the copolymer described herein (e.g., L121); (b) about 1 mol % to about 30 mol % (e.g., about 18 mol %) of SM-102 or a derivative thereof; (c) about 10 mol % to about 30 mol % (e.g., about 19.8 mol %) of DSPC or a derivative thereof and about 10 mol % to about 30 mol % (e.g., about 19.8 mol %) of DOPE or a derivative thereof; (d) about 30 mol % to about 60 mol % (e.g., about 34.1 mol %) of cholesterol or a derivative thereof; and (e) about 0.5 mol % to about 5 mol % (e.g., about 1.5 mol %) of PEG2000-DSPE or a derivative thereof.

[0342] In some embodiments, provided herein is CPT201 or an LNP having a formulation substantially similar to CPT201, comprising: (a) about 0.1 mol % to about 10 mol % (e.g., about 2.2 mol %) of a compound of a copolymer described herein (e.g., PPO 2700 ); (b) about 1 mol % to about 30 mol % (e.g., about 22.5 mol %) of ALC-0315 or a derivative thereof; (c) about 10 mol % to about 30 mol % (e.g., about 15.7 mol %) of DSPC or a derivative thereof and about 10 mol % to about 30 mol % (e.g., about 15.7 mol %) of DOPE or a derivative thereof; (d) about 30 mol % to about 60 mol % (e.g., about 42.7 mol %) of cholesterol or a derivative thereof; and (e) about 0.5 mol % to about 5 mol % (e.g., about 1.1 mol %) of PEG2000-DSPE or a derivative thereof.

[0343] Physical properties

[0344] The properties of a nanoparticle composition can depend on its components. For example, a nanoparticle composition comprising cholesterol as a structural lipid can have different properties than a nanoparticle composition comprising a different structural lipid. Similarly, the properties of a nanoparticle composition can depend on the absolute or relative amounts of its components. For example, a nanoparticle composition comprising a higher mole fraction of phospholipids can have different properties than a nanoparticle composition comprising a lower mole fraction of phospholipids. The properties can also vary depending on the method and conditions of preparation of the nanoparticle composition.

[0345] Nanoparticle compositions can be characterized by a variety of methods. For example, a microscope (e.g., a transmission electron microscope or a scanning electron microscope) can be used to examine the morphology and size distribution of the nanoparticle composition. Dynamic light scattering or potentiometric methods (e.g., potentiometric titration) can be used to measure the zeta potential. Dynamic light scattering can also be used to determine particle size. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure multiple characteristics of the nanoparticle composition, such as particle size, polydispersity index, and zeta potential.

[0346] In some embodiments, the N / P ratio of the LNP or LNP having a nanoparticle composition described herein is about 0.1 to about 10, for example, about 0.1 to about 9, about 0.1 to about 8, about 0.1 to about 7, about 0.1 to about 6, about 0.1 to about 5, about 0.1 to about 4, about 0.1 to about 3, about 0.1 to about 2, about 0.1 to about 1, about 0.1 to about 0.9, about 0.1 to about 0.8, about 0.1 to about 0.7, about 0.1 to about 0.6, about 0.1 to about 0.5, about 0.1 to about 0.4, about 0.1 to about 0.3, about 0.1 to about 0.2, about 0.2 to about 10, about 0.2 to about 9, about 0.2 to about 8, about 0.2 to about 7, about 0.2 to about 6, about 0.2 to about 5, about 0.1 to about 4, about 0.1 to about 3, about 0.1 to about 2, about 0.1 to about 1, about 0.1 to about 0.9, about 0.1 to about 0.8, about 0.1 to about 0.7, about 0.1 to about 0.6, about 0.1 to about 0.5, about 0.1 to about 0.4, about 0.1 to about 0.3 about 0.2 to about 4, about 0.2 to about 3, about 0.2 to about 2, about 0.2 to about 1, about 0.2 to about 0.9, about 0.2 to about 0.8, about 0.2 to about 0.7, about 0.2 to about 0.6, about 0.2 to about 0.5, about 0.2 to about 0.4, about 0.2 to about 0.3, about 0.3 to about 10, about 0.3 to about 9, about 0.3 to about 8, about 0.3 to about 7, about 0.3 to about 6, about 0.3 to about 5, about 0.3 to about 4, about 0.3 to about 3, about 0.3 to about 2, about 0.3 to about 1, about 0.3 to about 0.9, about 0.3 to about 0.8, about 0.3 to about 0.7, about 0.3 to about 0.6, about 0.3 to about 0.5, about 0.3 to about 0.4, about 0.4 to about 1 0, about 0.4 to about 9, about 0.4 to about 8, about 0.4 to about 7, about 0.4 to about 6, about 0.4 to about 5, about 0.4 to about 4, about 0.4 to about 3, about 0.4 to about 2, about 0.4 to about 1, about 0.4 to about 0.9, about 0.4 to about 0.8, about 0.4 to about 0.7, about 0.4 to about 0.6, about 0.4 to about 0.5, about 0.5 to about 10, about 0.5 to about 9, about 0.5 to about 8, about 0.5 to about 7, about 0.5 to about 6, about 0.5 to about 5, about 0.5 to about 4, about 0.5 to about 3, about 0.5 to about 2, about 0.5 to about 1, about 0.5 to about 0.9, about 0.5 to about 0.8, about 0.5 to about 0.7, about 0.5 to about 0.6 about 10, about 0.6 to about 9, about 0.6 to about 8, about 0.6 to about 7, about 0.6 to about 6, about 0.6 to about 5, about 0.6 to about 4, about 0.6 to about 3, about 0.6 to about 2, about 0.6 to about 1, about 0.6 to about 0.9, about 0.6 to about 0.8, about 0.6 to about 0.7, about 0.7 to about 10, about 0.7 to about 9, about 0.7 to about 8, about 0.7 to about 7, about 0.7 to about 6, about 0.7 to about 5, about 0.7 to about 4, about 0.7 to about 3, about 0.7 to about 2, about 0.7 to about 1, about 0.7 to about 0.9, about 0.7 to about 0.8, about 0.8 to about 10, about 0.7 to about 9, about 0.7 to about 8, about 0.7 to about 7, about 0.7 to about 6, about 0.7 to about 5, about 0.7 to about 4, about 0.7 to about 3, about 0.7 to about 2,8 to about 5, about 0.8 to about 4, about 0.8 to about 3, about 0.8 to about 2, about 0.8 to about 1, about 0.8 to about 0.9, about 0.9 to about 10, about 0.9 to about 9, about 0.9 to about 8, about 0.9 to about 7, about 0.9 to about 6, about 0.9 to about 5, about 0.9 to about 4, about 0.9 to about 3, about 0.9 to about 2, about 0.9 to about 1, about 1 to about 10, about 1 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, about 1 to about 2, about 2 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, about 2 to about 3, about 3 to about 10, about 3 to about 9, about 3 to about 8, about 3 to about 7, about 3 to about 6, about 3 to about 5, about 3 to about 4, about 4 to about 10, about 4 to about 9, about 4 to about 8, about 4 to about 7, about 4 to about 6, about 4 to about 5, about 5 to about 10, about 5 to about 9, about 5 to about 8, about 5 to about 7, about 5 to about 6, about 6 to about 10, about 6 to about 9, about 6 to about 8, about 6 to about 7, about 7 to about 10, about 7 to about 9, about 7 to about 8, about 8 to about 10, about 8 to about 9, or about 9 to 10. .

[0347] In some embodiments, the N / P ratio of the LNP or LNP having a nanoparticle composition described herein is about 0.25 to about 1, for example, about 0.26, about 0.27, about 0.28, about 0.29, about 0.3, about 0.31, about 0.32, about 0.33, about 0.34, about 0.35, about 0.36, about 0.37, about 0.38, about 0.39, about 0.4, about 0.41, about 0.42, about 0.43, about 0.44, about 0.45, about 0.46, about 0.47, about 0.48, about 0.49, about 0.5, about 0.51, about 0.52, about 0.53, about 0.54, about 0.55, about 0.56, about 0.57, about 0.58, about 0.59, about 0.60, about 0.61, about 0.62, about 0.63, about 0.64, about 0.65 .6, about 0.61, about 0.62, about 0.63, about 0.64, about 0.65, about 0.66, about 0.67, about 0.68, about 0.69, about 0.7, about 0.71, about 0.72, about 0.73, about 0.74, about 0.75, about 0.76, about 0.77, about 0.78, about 0.79, about 0.8, about 0.81 , about 0.82, about 0.83, about 0.84, about 0.85, about 0.86, about 0.87, about 0.88, about 0.89, about 0.9, about 0.91, about 0.92, about 0.93, about 0.94, about 0.95, about 0.96, about 0.97, about 0.98, about 0.99, about 1, or any range between two of the above values.

[0348] As used herein, "N / P ratio" is the molar ratio of ionizable nitrogen atoms in ionizable lipids or permanently charged nitrogen atoms in permanently charged cationic lipids relative to phosphate groups in the nucleic acid backbone. In some embodiments, the nucleic acid is DNA or RNA (e.g., mRNA).

[0349] In some embodiments, LNPs or LNPs with nanoparticle compositions described herein are stable after storage at a temperature of about 20°C to about 40°C (e.g., about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, or 3 weeks. In some embodiments, the stability of the LNP is determined by testing the transfection efficiency of the LNP after storage.

[0350] In some embodiments, the LNP or LNPs having nanoparticle compositions described herein have a hydrophilic lipophilic balance (HLB) value of about 1 to about 18, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18. HLB defines the affinity of a surfactant for a solvent and ranges from 0 to 20. The greater the HLB, the greater the affinity of the surfactant for water, and conversely, the lower the HLB, the higher the affinity of the surfactant for oil. Detailed information on HLB determination can be found, for example, in Griffin, WC "Calculation of HLB values ​​of non-ionic surfactants", Journal of the Society of Cosmetic Chemists (J. Soc. Cosmet. Chem.) 5 (1954): 249-256, which is incorporated herein by reference in its entirety. For example, the HLB value of L121 is about 1; the HLB value of L81 is about 2, and the HLB value of L92 is about 6.

[0351] In some embodiments, the average size of the LNPs or LNPs having the nanoparticle compositions described herein is from about 30 nm to about 2000 nm, for example, from about 30 nm to about 1800 nm, from about 30 nm to about 1600 nm, from about 30 nm to about 1400 nm, from about 30 nm to about 1200 nm, from about 30 nm to about 1000 nm, from about 30 nm to about 900 nm, from about 30 nm to about 800 nm, from about 30 nm to about 700 nm, from about 30 nm to about 600 nm, from about 30 nm to about 500 nm, from about 30 nm to about 400 nm, from about 30 nm to about 300 nm, from about 30 nm to about 200 nm, from about 30 nm to about 100 nm, from about 30 nm to about 900 nm, nm, about 30 nm to about 80 nm, about 30 nm to about 70 nm, about 30 nm to about 60 nm, about 30 nm to about 50 nm, about 30 nm to about 40 nm, about 40 nm to about 2000 nm, about 40 nm to about 1800 nm, about 40 nm to about 1600 nm, about 40 nm to about 1400 nm, about 40 nm to about 1200 nm, about 40 nm to about 1000 nm, about 40 nm to about 900 nm, about 40 nm to about 800 nm, about 40 nm to about 700 nm, about 40 nm to about 600 nm, about 40 nm to about 500 nm, about 40 nm to about 400 nm, about 40 nm to about 300 nm, about 40 nm to about 200 nm. m, about 40nm to about 100nm, about 40nm to about 90nm, about 40nm to about 80nm, about 40nm to about 70nm, about 40nm to about 60nm, about 40nm to about 50nm, about 50nm to about 2000nm, about 50nm to about 1800nm, about 50nm to about 1600nm, about 50nm to about 1400nm, about 50nm to about 1200nm, about 50nm to about 1000nm, about 50nm to about 900nm, about 50nm to about 800nm, about 50nm to about 700nm, about 50nm to about 600nm, about 50nm to about 500nm, about 50nm to about 400nm, about 50nm to about 300nm , about 50 nm to about 200 nm, about 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, about 50 nm to about 60 nm, about 60 nm to about 2000 nm, about 60 nm to about 1800 nm, about 60 nm to about 1600 nm, about 60 nm to about 1400 nm, about 60 nm to about 1200 nm, about 60 nm to about 1000 nm, about 60 nm to about 900 nm, about 60 nm to about 800 nm, about 60 nm to about 700 nm, about 60 nm to about 600 nm, about 60 nm to about 500 nm, about 60 nm to about 400 nm, about 60 nm to about 300 nm,from about 60 nm to about 200 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 2000 nm, from about 70 nm to about 1800 nm, from about 70 nm to about 1600 nm, from about 70 nm to about 1400 nm, from about 70 nm to about 1200 nm, from about 70 nm to about 1000 nm, from about 70 nm to about 900 nm, from about 70 nm to about 800 nm, from about 70 nm to about 700 nm, from about 70 nm to about 600 nm, from about 70 nm to about 500 nm, from about 70 nm to about 400 nm, from about 70 nm to about 300 nm, from about 70 nm to about 200 nm. m, about 70nm to about 100nm, about 70nm to about 90nm, about 70nm to about 80nm, about 80nm to about 2000nm, about 80nm to about 1800nm, about 80nm to about 1600nm, about 80nm to about 1400nm, about 80nm to about 1200nm, about 80nm to about 1000nm, about 80nm to about 900nm, about 80nm to about 800nm, about 80nm to about 700nm, about 80nm to about 600nm, about 80nm to about 500nm, about 80nm to about 400nm, about 80nm to about 300nm, about 80nm to about 200nm, about 80nm to about 100nm, about 80nm to about 9 From about 90 nm to about 2000 nm, from about 90 nm to about 1800 nm, from about 90 nm to about 1600 nm, from about 90 nm to about 1400 nm, from about 90 nm to about 1200 nm, from about 90 nm to about 1000 nm, from about 90 nm to about 900 nm, from about 90 nm to about 800 nm, from about 90 nm to about 700 nm, from about 90 nm to about 600 nm, from about 90 nm to about 500 nm, from about 90 nm to about 400 nm, from about 90 nm to about 300 nm, from about 90 nm to about 200 nm, from about 90 nm to about 100 nm, from about 100 nm to about 2000 nm, from about 100 nm to about 1800 nm, from about 100 nm to about 1600 nm. m, about 100 nm to about 1400 nm, about 100 nm to about 1200 nm, about 100 nm to about 1000 nm, about 100 nm to about 900 nm, about 100 nm to about 800 nm, about 100 nm to about 700 nm, about 100 nm to about 600 nm, about 100 nm to about 500 nm, about 100 nm to about 400 nm, about 100 nm to about 300 nm, about 100 nm to about 200 nm, about 200 nm to about 2000 nm, about 200 nm to about 1800 nm, about 200 nm to about 1600 nm, about 200 nm to about 1400 nm, about 200 nm to about 1200 nm, about 200 nm to about 1000 nm,about 200 nm to about 900 nm, about 200 nm to about 800 nm, about 200 nm to about 700 nm, about 200 nm to about 600 nm, about 200 nm to about 500 nm, about 200 nm to about 400 nm, about 200 nm to about 300 nm, about 300 nm to about 2000 nm, about 300 nm to about 1800 nm, about 300 nm to about 1600 nm, about 300 nm to about 1400 nm, about 300 nm to about 1200 nm, about 300 nm to about 1000 nm, about 300 nm to about 900 nm, about 300 nm to about 800 nm, about 300 nm to about 700 nm, about 300 nm to about 600 nm, about 300 nm to about nm to about 500 nm, about 300 nm to about 400 nm, about 400 nm to about 2000 nm, about 400 nm to about 1800 nm, about 400 nm to about 1600 nm, about 400 nm to about 1400 nm, about 400 nm to about 1200 nm, about 400 nm to about 1000 nm, about 400 nm to about 900 nm, about 400 nm to about 800 nm, about 400 nm to about 700 nm, about 400 nm to about 600 nm, about 400 nm to about 500 nm, about 500 nm to about 2000 nm, about 500 nm to about 1800 nm, about 500 nm to about 1600 nm, about 500 nm to about 1400 nm, about 500 nm to about nm to about 1200 nm, about 500 nm to about 1000 nm, about 500 nm to about 900 nm, about 500 nm to about 800 nm, about 500 nm to about 700 nm, about 500 nm to about 600 nm, about 600 nm to about 2000 nm, about 600 nm to about 1800 nm, about 600 nm to about 1600 nm, about 600 nm to about 1400 nm, about 600 nm to about 1200 nm, about 600 nm to about 1000 nm, about 600 nm to about 900 nm, about 600 nm to about 800 nm, about 600 nm to about 700 nm, about 700 nm to about 2000 nm, about 700 nm to about 1800 nm, about 700 nm to about nm to about 1600 nm, about 700 nm to about 1400 nm, about 700 nm to about 1200 nm, about 700 nm to about 1000 nm, about 700 nm to about 900 nm, about 700 nm to about 800 nm, about 800 nm to about 2000 nm, about 800 nm to about 1800 nm, about 800 nm to about 1600 nm, about 800 nm to about 1400 nm, about 800 nm to about 1200 nm, about 800 nm to about 1000 nm, about 800 nm to about 900 nm, about 900 nm to about 2000 nm, about 900 nm to about 1800 nm, about 900 nm to about 1600 nm, about 900 nm to about 1400 nm,In some embodiments, the average size of the LNPs is a mass average size, a volume average size, an intensity average size, or a number average size. In some embodiments, the average size of the LNPs is determined by a light scattering-based method.

[0352] LNP or LNP with nanoparticle composition as described herein can be relatively homogeneous.Polydispersity index can be used to indicate the homogeneity of nanoparticle composition, for example, the particle size distribution of nanoparticle composition.Small (e.g., less than 0.5) polydispersity index usually indicates narrow particle size distribution. In some embodiments, the polydispersity index of the LNPs or LNPs having the nanoparticle compositions described herein is about 0.001 to about 0.5, about 0.001 to about 0.4, about 0.001 to about 0.3, about 0.001 to about 0.2, about 0.001 to about 0.1, about 0.001 to about 0.05, about 0.001 to about 0.01, about 0.001 to about 0.005, about 0.005 to about 0.5, about 0.005 to about 0.4, about 0.005 to about 0.3, about 0.005 to about 0.2, about 0.001 to about 0.1, about 0.001 to about 0.05, about 0.001 to about 0.01, about 0.001 to about 0.05, about 0.005 to about 0.5, about 0.005 to about 0.4, about 0.005 to about 0.3, about 0.005 to about 0.2, about 0.005 to about 0.1 , about 0.01 to about 0.5, about 0.01 to about 0.4, about 0.01 to about 0.3, about 0.01 to about 0.2, about 0.01 to about 0.1, about 0.01 to about 0.05, about 0.05 to about 0.5, about 0.05 to about 0.4, about 0.05 to about 0.3, about 0.05 to about 0.2, about 0.05 to about 0.1, about 0.1 to about 0.5, about 0.1 to about 0.4, about 0.1 to about 0.3, about 0.1 to about 0.2, about 0.2 to about 0.5, about 0.2 to about 0.4, about 0.2 to about 0.3, about 0.3 to about 0.5, about 0.3 to about 0.4, or about 0.4 to about 0.5.

[0353] The zeta potential of LNP can be used to indicate the electrokinetic potential of the composition. For example, the zeta potential can describe the surface charge of the LNP. LNPs with relatively low charge (positive or negative) are generally desirable because more highly charged species may interact with cells, tissues, and other elements in the body undesirably. In some embodiments, the zeta potential of the LNP or an LNP having a nanoparticle composition described herein is about -30 mV to about +20 mV, about -30 mV to about +10 mV, about -30 mV to about 0 mV, about -30 mV to about -10 mV, about -30 mV to about -20 mV, -20 mV to about +20 mV, about -20 mV to about +10 mV, about -20 mV to about 0 mV, about -20 mV to about -10 mV, -10 mV to about +20 mV, about -10 mV to about +10 mV, about -10 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +10 mV, about -10 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +10 mV, or about 10 mV to about 20 mV.

[0354] In some embodiments, the w / w ratio of the lipid component to the cargo molecule (e.g., any of the nucleic acids described herein) is about 2:1 to about 50:1, about 2:1 to about 40:1, about 2:1 to about 30:1, about 2:1 to about 20:1, about 2:1 to about 10:1, about 2:1 to about 8:1, about 2:1 to about 6:1, about 2:1 to about 4:1, about 4:1 to about 50:1, about 4:1 to about 40:1, about 4:1 to about 30:1, about 4:1 to about 20:1, about 4:1 to about 10:1, about 4:1 to about 8:1, about 4:1 to about 6:1, about 6:1 to about 50:1, about 6:1 to about 40:1, about 4:1 to about 30:1, about 4:1 to about 20:1, about 4:1 to about 10:1, about 4:1 to about 8:1, about 4:1 to about 6:1, about 6:1 to about 50:1, about 6:1 to about 40 : 1, about 6: 1 to about 30: 1, about 6: 1 to about 20: 1, about 6: 1 to about 10: 1, about 6: 1 to about 8: 1, about 8: 1 to about 50: 1, about 8: 1 to about 40: 1, about 8: 1 to about 30: 1, about 8: 1 to about 20: 1, about 8: 1 to about 10: 1, about 10: 1 to about 50: 1, about 10: 1 to about 40: 1, about 10: 1 to about 30: 1, about 10: 1 to about 20: 1, about 20: 1 to about 50: 1, about 20: 1 to about 40: 1, about 20: 1 to about 30: 1, about 30: 1 to about 50: 1, about 30: 1 to about 40: 1, or about 40: 1 to about 50: 1. The amount of cargo molecule in the nanoparticle composition can be measured, for example, using absorption spectroscopy (e.g., UV-visible spectroscopy).

[0355] In some embodiments, LNPs or LNPs with nanoparticle compositions described herein encapsulate cargo molecules (e.g., any of the nucleic acids described herein). In some embodiments, the encapsulation efficiency is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%.

[0356] Pharmaceutical composition

[0357] In some embodiments, the LNP or LNP having a nanoparticle composition described herein is associated with a therapeutic drug, vaccine, gene editing, or cell-based therapy.

[0358] The nanoparticle compositions disclosed herein can be formulated in whole or in part as pharmaceutical compositions. The pharmaceutical compositions disclosed herein may include one or more nanoparticle compositions. For example, a pharmaceutical composition may include one or more nanoparticle compositions, the one or more nanoparticle compositions comprising one or more different cargo molecules (e.g., DNA or mRNA). The pharmaceutical compositions disclosed herein may further include one or more pharmaceutically acceptable excipients or adjuvants, such as the pharmaceutically acceptable excipients or adjuvants described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and medicaments can be obtained, for example, in Remington's Pharmaceutical Science and Practice, 21st edition, A.R. Gennaro; Lippincott Williams & Wilkins Publishing Company, Baltimore, Maryland, 2006. Conventional excipients and adjuvants can be used in any pharmaceutical composition disclosed herein, unless any conventional excipient or adjuvant may be incompatible with one or more components of the nanoparticle composition disclosed herein. If the excipient or adjuvant is combined with the components of the nanoparticle composition, it may cause any undesirable biological effect or other harmful effect, then the excipient or adjuvant may be incompatible with the component.

[0359] In some embodiments, one or more excipients or auxiliary ingredients may account for more than 50% of the total mass or volume of the pharmaceutical composition including the nanoparticle composition of the present disclosure. For example, the one or more excipients or auxiliary ingredients may constitute 50%, 60%, 70%, 80%, 90% or more of pharmaceutical practice. In some embodiments, the purity of the pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%. In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient is approved by the United States Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia and / or the International Pharmacopoeia.

[0360] The relative amounts of the one or more nanoparticle compositions, the one or more pharmaceutically acceptable excipients and / or any additional ingredients in a pharmaceutical composition according to the present disclosure will vary depending on the identity, size and / or condition of the subject being treated and further depending on the route of administration of the composition. For example, a pharmaceutical composition may comprise between 0.1% and 100% (wt / wt) of one or more nanoparticle compositions.

[0361] Nanoparticle compositions and / or pharmaceutical compositions comprising one or more nanoparticle compositions can be administered to any patient or subject, including those patients or subjects who can benefit from the therapeutic effect provided by delivering DNA or mRNA to one or more specific cells, tissues, organs or systems or groups thereof (such as the renal system). Although the description of nanoparticle compositions and pharmaceutical compositions comprising nanoparticle compositions provided herein mainly relates to compositions suitable for administration to humans, it will be appreciated by those skilled in the art that such compositions are generally suitable for administration to any other mammal. It is well known that compositions suitable for administration to humans are modified in order to make the compositions suitable for administration to various animals, and veterinary pharmacologists of ordinary skill can design and / or perform such modifications using only ordinary (if any) experiments. Subjects contemplated for administration of the composition include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals such as cattle, pigs, hoses, sheep, cats, dogs, mice, and / or rats.

[0362] Pharmaceutical compositions comprising one or more nanoparticle compositions can be prepared by any method known or hereafter developed in the art of pharmacology. Generally, such preparation methods involve associating the active ingredient with an excipient and / or one or more other auxiliary ingredients, and then, if desired or necessary, dividing, shaping and / or packaging the product into desired single or multiple dose units.

[0363] Pharmaceutical compositions according to the present disclosure can be prepared, packaged and / or sold in batches as a single unit dose and / or as multiple single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of active ingredient (e.g., nanoparticle composition). The amount of the active ingredient is generally equal to the dosage of the active ingredient to be administered to the subject and / or a convenient fraction of such dosage, such as half or one-third of such dosage.

[0364] The pharmaceutical compositions disclosed herein can be prepared into various forms suitable for various routes and methods of administration. For example, the pharmaceutical compositions disclosed herein can be prepared into 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 administration (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and patches), suspensions, powders and other forms.

[0365] 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, the liquid dosage form may contain an inert diluent 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-butylene glycol, dimethylformamide, oil (specifically, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan and mixtures thereof. In addition to the inert diluent, the oral composition may include adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavorings and / or aromatics. In certain embodiments of parenteral administration, the composition is mixed with a solubilizer (such as alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers and / or combinations thereof).

[0366] Injectable preparations, such as sterile injectable aqueous or oily suspensions can be formulated using suitable dispersants, wetting agents and / or suspending agents according to known techniques. Sterile injectable preparations can be sterile injectable solutions, suspensions and / or emulsions in nontoxic parenteral acceptable diluents and / or solvents, for example, as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used are water, Ringer's solution, USP and isotonic sodium chloride solution. Sterile fixed oils are generally used as solvents or suspension media. For this purpose, any gentle fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid can be used to prepare injectables.

[0367] The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter and / or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0368] In order to prolong the effect of active ingredients, it is usually desirable to slow down the absorption of active ingredients from subcutaneous or intramuscular injections. This can be achieved by using a liquid suspension of a crystalline or amorphous material with poor water solubility. Then, the absorption rate of the drug depends on its dissolution rate, which in turn can depend on crystal size and crystalline form. Alternatively, the delayed absorption of the drug form for parenteral administration is achieved by dissolving or suspending the drug in an oily vehicle. The injectable reservoir type (depot) form is prepared by forming a microcapsule matrix of the drug in a biodegradable polymer such as polylactide polyglycolide. According to the ratio of the drug to the polymer and the properties of the specific polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Reservoir type injectable formulations are prepared by trapping the drug in a liposome or microemulsion compatible with body tissues.

[0369] Compositions for rectal or vaginal administration are usually suppositories, which can be prepared by mixing the composition with a suitable non-irritating excipient (such as cocoa butter, polyethylene glycol or suppository wax), which is solid at room temperature but liquid at body temperature and can therefore melt in the rectum or vaginal cavity and release the active ingredient. Solid dosage forms for oral administration include capsules, tablets, pills, films, powders and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and / or fillers or extenders (e.g., starch, lactose, sucrose, glucose, mannitol and silicic acid), binders (e.g., carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and gum arabic), humectants (e.g., glycerol), disintegrants (e.g., agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate), solution retardants (e.g., paraffin), absorption promoters (e.g., quaternary ammonium compounds), wetting agents (e.g., cetyl alcohol and glyceryl monostearate), absorbents (e.g., kaolin and bentonite, silicates) and lubricants (e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate) and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may contain a buffer.

[0370] Solid compositions of similar types can be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycol. Solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as other coatings known in the field of enteric coatings and drug formulations. The solid dosage form can optionally contain an emulsifier and can be a composition that the solid dosage form can release the active ingredient only or preferentially in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of similar types can be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycol.

[0371] Dosage forms for topical and / or transdermal administration of the composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and / or patches.

[0372] Typically, the active ingredient is mixed with a pharmaceutically acceptable excipient and / or any desired preservative and / or buffer that may be needed under aseptic conditions. Additionally, the disclosure contemplates the use of transdermal patches, which typically have the added advantage of allowing the compound to be delivered to the body in a controlled manner. Such dosage forms can be prepared, for example, by dissolving the compound and / or being dispersed in an appropriate medium. Alternatively or additionally, the rate can be controlled by providing a rate-controlled membrane and / or by dispersing the compound in a polymer matrix and / or a gel.

[0373] Devices suitable for delivering intradermal pharmaceutical compositions described herein include short needle devices, such as those described in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288; 4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositions can be administered by devices that limit the effective penetration length of a needle into the skin, such as those described in PCT Publication WO 99 / 34850 and functional equivalents thereof. Jet injection devices that deliver liquid compositions to the dermis by means of a liquid jet injector and / or by means of a needle that pierces the stratum corneum and produces a jet that reaches the dermis are suitable. Jet injection devices are described in, for example, U.S. Patents 5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189; 5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335; 5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880; 4,940,460; and PCT Publications WO 97 / 37705 and WO 97 / 13537. Ballistic powder / particle delivery devices that use compressed gas to accelerate the vaccine in powder form through the outer layers of the skin to the dermis are suitable. Alternatively or additionally, conventional syringes may be used in the classical mantoux method of intradermal administration.

[0374] Formulations suitable for topical administration include, but are not limited to, liquid and / or semi-liquid preparations, such as liniments, lotions, oil-in-water and / or water-in-oil emulsions, such as creams, ointments and / or pastes, and / or solutions and / or suspensions. Topically administrable formulations may, for example, contain from about 1% to about 10% (wt / wt) of the active ingredient, but the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent.

[0375] Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

[0376] Pharmaceutical compositions can be prepared, packaged and / or sold in the form of formulations suitable for pulmonary administration through the buccal cavity. Such formulations can include dry particles containing active ingredients and having a diameter in the range of about 0.5 nm to about 7 nm or about 1 nm to about 6 nm. Such compositions are conveniently in the form of dry powders for use with a device containing a dry powder reservoir (to which a propellant stream can be directed to disperse the powder) and / or with a self-propelling solvent / powder dispensing container (such as a device containing an active ingredient dissolved and / or suspended in a low-boiling propellant in a sealed container). Such powders include particles, wherein at least 98% of the particles by weight have a diameter greater than 0.5 nm and at least 95% of the particles by number have a diameter less than 7 nm. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nm and at least 90% of the particles by number have a diameter less than 6 nm. Dry powder compositions can include solid fine powder diluents such as sugars and are conveniently provided in unit dose form.

[0377] Low boiling point propellants typically include liquid propellants with a boiling point below 65°F at atmospheric pressure. Typically, the propellant may constitute 50% to 99.9% (w / w) of the composition, and the active ingredient may constitute 0.1% to 20% (w / w) of the composition. The propellant may further comprise additional ingredients, such as liquid nonionic and / or solid anionic surfactants and / or solid diluents (the solid diluents may have a particle size of the same order of magnitude as the particles comprising the active ingredient).

[0378] The pharmaceutical composition formulated for pulmonary delivery can provide active ingredients in the form of droplets of solution and / or suspension. Such formulations can be prepared, packaged and / or sold in the form of aqueous and / or diluted alcoholic solutions and / or suspensions that are optionally sterile, contain active ingredients, and can be conveniently applied using any atomization and / or atomization device. Such formulations can further include one or more additional ingredients, including but not limited to flavoring agents such as sodium saccharin, volatile oils, buffers, surfactants and / or preservatives such as methyl hydroxybenzoate. The average diameter of the droplets provided by this route of administration can be in the range of about 1nm to about 200nm.

[0379] The formulations described herein that can be used for pulmonary delivery can be used for intranasal delivery of pharmaceutical compositions. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle size of about 0.2 μm to 500 μm. Such formulations are administered in the manner of snuff, i.e., by rapid inhalation through the nasal passages from a powder container held close to the nostrils.

[0380] The formulation suitable for nasal administration may, for example, contain as little as about 0.1% (wt / wt) and as much as 100% (wt / wt) of active ingredient, and may contain one or more additional ingredients of the additional ingredients described herein. The pharmaceutical composition may be prepared, packaged and / or sold in the form of a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets and / or lozenges prepared using conventional methods, and may be, for example, 0.1% to 20% (wt / wt) of active ingredient, with the remainder comprising an oral soluble and / or degradable composition and optionally one or more additional ingredients of the additional ingredients described herein. Alternatively, formulations suitable for buccal administration may include powders and / or aerosolized and / or atomized solutions and / or suspensions including active ingredients. Such powdered, aerosolized and / or aerosolized formulations may have an average particle and / or droplet size when dispersed in the range of about 0.1 nm to about 200 nm, and may further include one or more additional ingredients of any additional ingredients described herein.

[0381] Pharmaceutical compositions can be prepared, packaged and / or sold in the form of formulations suitable for ocular administration. Such formulations can be, for example, in the form of eye drops, including, for example, 0.1 / 1.0% (wt / wt) solutions and / or suspensions of active ingredients in aqueous or oily liquid excipients. Such drops may further include one or more other additional ingredients of a buffer, salt and / or any additional ingredients described herein. Other useful ocularly administrable formulations include formulations comprising active ingredients in microcrystalline form and / or in the form of liposomal formulations. Ear drops and / or eye drops are considered to be within the scope of the present disclosure.

[0382] Method for preparing LNP

[0383] The present disclosure provides methods for preparing LNPs described herein (e.g., any of the PALNPs described herein) or LNPs having nanoparticle compositions described herein (e.g., any of the nanoparticle compositions described herein). nPort disclosed in U.S. Pat. Nos. 11,497,715 B2 and 9,693,958 B2 can be used. TM The invention relates to the production of LNPs using techniques described in US Pat. No. 5,877,679, filed on Oct. 28, 2009, each of which is incorporated herein by reference in its entirety.

[0384] In one aspect, the present disclosure relates to a method for preparing an LNP (e.g., any of the PALNPs described herein or an LNP having a nanoparticle composition described herein (e.g., any of the nanoparticle compositions described herein)), the method comprising: (a) introducing one or more streams of a solution of lipids in a water-miscible organic solvent through a first set of one or more inlet ports connected to a mixing chamber, wherein the lipid solution comprises a lipid component and a polymer component, (b) introducing one or more streams of an aqueous solution through a second set of one or more inlet ports connected to the mixing chamber, (c) mixing the one or more streams of the lipid solution and the one or more streams of the aqueous solution in a mixing chamber to produce the LNP, and (d) recovering the LNP through one or more outlet ports connected to the mixing chamber.

[0385] In certain embodiments, the aqueous solution does not include nucleic acid. For example, after blank LNP is produced, they can be loaded with nucleic acid (for example, any nucleic acid in nucleic acid described herein) by mixing blank LNP with a solution containing nucleic acid at about 30-40 ° C (for example, about 37 ° C) for about 1-30 minutes (for example, about 10 minutes). In certain embodiments, the volume ratio of blank LNP to nucleic acid is about 1: 1, about 2: 1, about 3: 1, about 4: 1, about 5: 1, about 6: 1, about 7: 1 or about 8: 1. In certain embodiments, the relative amount (for example, volume ratio) of blank LNP and nucleic acid determines the N / P ratio of the LNP of load described herein.

[0386] In certain embodiments, the aqueous solution includes nucleic acids (e.g., any of the nucleic acids described herein). For example, after one or more streams of a lipid solution and one or more streams of an aqueous solution are mixed in a mixing chamber, an LNP containing a load of nucleic acids is formed immediately. Therefore, no additional loading step is required to produce an LNP containing a load of nucleic acids.

[0387] The present disclosure provides a device suitable for performing the methods disclosed herein, such as a manifold system described herein. In some embodiments, the present technology provides a device for preparing LNPs encapsulating APIs, the device comprising a manifold, the manifold may have a mixing chamber, at least one lipid solution inlet port connected to the chamber; and a plurality of aqueous solution inlet ports connected to the chamber. In a preferred embodiment, the device may include an LNP solution outlet port connected to the chamber. Preferably, the device may include a lipid solution reservoir connected to the lipid solution inlet port by a lipid solution conduit, and an aqueous solution reservoir connected to the aqueous solution inlet port by an aqueous solution conduit.

[0388] The inlet port of lipid solution and aqueous solution and the outlet port of liposome solution can have the same or different internal diameters. Preferably, the internal diameter of the inlet port and the outlet port is about 0.1mm to about 10mm. More preferably, the internal diameter of the port is about 0.15mm to about 5mm.

[0389] In some embodiments, the mixing chamber is located at the transition point of the conduit, and the mixing chamber itself can be formed by two or more conduits that pass through or intersect each other without any change in the shape of the conduit. For example, the mixing chamber can be formed by drilling two or more through passages that all intersect at the transition point in the solid material. In addition, one or more conduits can be sealed so that fluid is not allowed to pass through the conduit. Such seals can be located immediately before the intersection or away from the intersection.

[0390] In some embodiments, a manifold may contain more than one mixing chamber. For example, one set of inlet ports intersects at one chamber, and another set of inlet ports intersects at another chamber, and the two chambers are connected by conduits to a third mixing chamber, which is connected to an outlet port.

[0391] Preferably, a pump is used to induce a forward flow of the lipid solution and the aqueous solution. The pump may be an inline pump or a syringe pump.

[0392] Typically, the mixing chamber can be connected to 2 to about 20 aqueous solution inlet ports. Preferably, there can be 3 to about 11 such ports, 3 to about 12 such ports. More preferably, there are 3 to about 10 or 3 to about 7 aqueous solution entry inlet ports. The mixing chamber can also be connected to 1 to about 5 lipid solution inlet ports. Preferably, there are 1 to about 3 lipid solution inlet ports. Most preferably, there are 1 or 2 lipid solution inlet ports. In a preferred embodiment, the mixing chamber is connected to at least 1 (for example, 1, 2, 3, 4 or 5) lipid solution port and at least 2 (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) aqueous solution inlet ports.

[0393] The mixing chamber may be further connected to 1 to about 3 outlet ports for the liposome solution to control the particle size, preferably, there is 1 (eg, 1, 2, or 3) outlet ports.

[0394] In some aspects, the angle between the inlet port of the lipid solution and the inlet port of the aqueous solution is about 18 ° to about 180 °. Preferably, the angle can be about 24 ° to about 180 °, more preferably about 30 ° to about 180 °. In some embodiments, the angle between at least one lipid solution inlet port and at least one aqueous solution inlet port is not 180 ° or a substantially similar angle. For example, the angle between at least one lipid solution inlet port and at least one aqueous solution inlet port is about 120 ° or less, about 90 ° or less. The angle between the ports is the angle at which the corresponding solution flow is directed into the mixing chamber.

[0395] The lipid solution and the aqueous solution can have the same flow through the manifold. Alternatively, the solution can have different flow rates. The flow rate of the lipid solution and the aqueous solution can be 1 ml / min to about 6,000 ml / min, for example, about 1 ml / min to about 1,500 ml / min. Preferably, the flow rate can be about 5 ml / min to about 1,000 ml / min, for example, about 5 ml / min to about 400 ml / min. More preferably, the flow rate can be about 20 ml / min to about 600 ml / min or about 10 ml / min to about 300 ml / min. In certain embodiments, the flow rate is adjusted based on the size of the inlet port to obtain desired LNP size, morphology, PDI and manufacturing scale.

[0396] The present disclosure also provides a method for preparing lipid nanoparticles (LNP), the method comprising: a) i) introducing one or more streams of a lipid solution through a first group of one or more inlet ports of a manifold and ii) introducing one or more streams of an aqueous solution through a second group of one or more inlet ports of the manifold, thereby mixing the lipid solution and the aqueous solution to produce an LNP solution; and b) reclaiming the LNP solution through one or more outlet ports of the manifold; wherein the angle between at least one lipid solution inlet port and at least one aqueous solution inlet port is not 180 ° or a substantially similar angle. In some aspects, at least one stream of a lipid solution collides with at least one stream of an aqueous solution at an angle less than about 180 °. Therefore, in some aspects, the method does not include a T-connector.

[0397] In some embodiments, the angle between at least one lipid solution inlet port and at least one aqueous solution inlet port is about 120° or less, e.g., 115° or less, 100° or less, 90° or less, 80° or less, 72° or less, 60° or less, 45° or less, 30° or less, 18° or less,

[0398] In some embodiments, the aqueous solution in step ii) is introduced through at least two (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) inlet ports. In some embodiments, the aqueous solution in step ii) is introduced through at least 3 but no more than 11 (e.g., at least 3 but no more than 7, at least 3 but no more than 5, at least 4 but no more than 11, at least 5 but no more than 11, at least 6 but no more than 11) inlet ports.

[0399] In some embodiments, at least two (e.g., 3, 4, 5, 6, 7, etc.) aqueous solution inlet ports and at least one (e.g., 2, 3, 4, 5, etc.) lipid solution inlet port are located in the same plane.

[0400] In some embodiments, at least one (e.g., 2) outlet port is substantially perpendicular to the plane of the inlet port. In other embodiments, at least one (e.g., 2, 3, 4, 5, etc.) outlet port is not substantially perpendicular to the plane of the inlet port. In some embodiments, at least two (e.g., 3, 4, 5, 6, 7, etc.) aqueous solution inlet ports and at least one (e.g., 2, 3, 4, 5, etc.) lipid solution inlet port are not located in the same plane. In some embodiments, the aqueous solution introduced into at least one of the inlet ports is different from the second aqueous solution introduced into another inlet port.

[0401] In some embodiments, the aqueous solution and / or lipid solution comprises an active pharmaceutical ingredient (API).

[0402] In some embodiments, step a) further comprises iii) introducing one or more streams of a non-aqueous solution through one or more inlet ports of the manifold.

[0403] Methods for producing polypeptides in cells

[0404] The present disclosure provides a method for producing a polypeptide of interest in a mammalian cell. The method for producing a polypeptide involves contacting a cell with a nanoparticle composition comprising a nucleic acid (e.g., mRNA) encoding the polypeptide of interest. When contacting the cell with the nanoparticle composition, the nucleic acid (e.g., mRNA) can be absorbed and translated in the cell to produce the polypeptide of interest.

[0405] Usually, the step of contacting mammalian cells with the nanoparticle composition comprising a nucleic acid (for example, mRNA) encoding a polypeptide of interest can be carried out in vivo, in vitro, in culture or in vitro. The amount of the nanoparticle composition contacted with cells and / or the amount of nucleic acid (for example, mRNA) therein can depend on the type of the cell or tissue contacted, the means of application, the physicochemical properties of the nanoparticle composition and nucleic acid (for example, mRNA) such as size, electric charge and chemical composition therein, and other factors. Usually, the nanoparticle composition of effective dose will allow efficient polypeptide production in cells. The measurement of efficiency can include polypeptide translation (indicated by polypeptide expression), nucleic acid (for example, mRNA) degradation level and immune response index.

[0406] The step of contacting the nanoparticle composition including nucleic acid (e.g., mRNA) with the cell can involve or cause transfection. The phospholipid included in the lipid component of the nanoparticle composition can promote transfection and / or increase transfection efficiency, for example, by interacting and / or fusing with a cell membrane or intracellular membrane. Transfection can allow nucleic acid (e.g., mRNA) to be translated in the cell.

[0407] In some embodiments, nanoparticle compositions described herein can be used as therapeutic agents. For example, nucleic acids (e.g., mRNA) included in nanoparticle compositions can encode therapeutic polypeptides (e.g., in a translatable region) and produce the therapeutic polypeptides when contacting and / or entering (e.g., transfecting) cells. In other embodiments, nucleic acids (e.g., mRNA) included in nanoparticle compositions of the present disclosure can encode polypeptides that can improve or increase the immunity of a subject. For example, nucleic acids (e.g., mRNA) can encode granulocyte colony stimulating factor or trastuzumab.

[0408] In certain embodiments, the nucleic acid (e.g., mRNA) included in the nanoparticle composition of the present disclosure can encode a recombinant polypeptide that can replace one or more polypeptides that may be substantially absent in the cell in contact with the nanoparticle composition. The one or more substantially absent polypeptides can be lacking due to genetic mutations of the encoding gene or its regulatory pathway. Alternatively, the recombinant polypeptide produced by translating the nucleic acid (e.g., mRNA) can antagonize the activity of endogenous proteins present in the cell, on the cell surface, or secreted from the cell. Antagonistic recombinant polypeptides may be desirable to counteract the harmful effects caused by the activity of endogenous proteins (such as the activity or positioning of the changes caused by mutations). In another alternative, the recombinant polypeptide produced by the translation of the nucleic acid (e.g., mRNA) can indirectly or directly antagonize the activity of biological parts present in the cell, on the cell surface, or secreted from the cell. The biological parts of the antagonism can include, but are not limited to, lipids (e.g., cholesterol), lipoproteins (e.g., low-density lipoproteins), nucleic acids, carbohydrates, and small molecule toxins. A recombinant polypeptide produced by translation of a nucleic acid (eg, mRNA) can be engineered to be localized within a cell, such as within a specific compartment (eg, the nucleus), or can be engineered to be secreted from a cell or translocated to the plasma membrane of the cell.

[0409] In certain embodiments, contacting a cell with a nanoparticle composition comprising a nucleic acid (e.g., mRNA) can reduce the innate immune response of the cell to exogenous nucleic acids. The cell can be contacted with a first nanoparticle composition comprising a first amount of a first exogenous nucleic acid (e.g., mRNA), wherein the first exogenous nucleic acid comprises a translatable region, and the innate immune response level of the cell to the first exogenous nucleic acid (e.g., mRNA) can be determined. Subsequently, the cell can be contacted with a second composition comprising a second amount of a first exogenous nucleic acid (e.g., mRNA), wherein the second amount is a lesser amount of the first exogenous nucleic acid (e.g., mRNA) than the first amount. Alternatively, the second composition can include a first amount of a second exogenous nucleic acid (e.g., mRNA) that is different from the first exogenous nucleic acid (e.g., mRNA). The step of contacting the cell with the first composition and the second composition can be repeated once or multiple times.

[0410] Additionally, the efficiency of polypeptide production (eg, translation) in the cell can optionally be determined, and the cell can be repeatedly re-contacted with the first composition and / or the second composition until the target protein production efficiency is achieved.

[0411] Methods for delivering nucleic acids to cells

[0412] The present disclosure provides a method for delivering nucleic acid (e.g., mRNA) to mammalian cells. Delivery of nucleic acid (e.g., mRNA) to cells involves administering to a subject a nanoparticle composition comprising nucleic acid (e.g., mRNA), wherein the administration of the composition involves contacting the cell with the composition. When contacting the cell with the nanoparticle composition, a translatable nucleic acid (e.g., mRNA) can be translated in the cell to produce a polypeptide of interest. However, substantially non-translatable nucleic acids (e.g., mRNA) can also be delivered to cells. Substantially non-translatable nucleic acids (e.g., mRNA) can be used as vaccines and / or can chelate the translation components of cells to reduce the expression of other species in the cell.

[0413] In some embodiments, nanoparticle compositions of the present disclosure can target cells of a particular type or category (e.g., cells of a particular organ or its system). For example, nanoparticle compositions comprising nucleic acids of interest (e.g., mRNA) can be specifically delivered to the kidney of a mammal. Specific delivery to cells, organs or systems of a particular category or its group means that, relative to other destinations, for example, when nanoparticle compositions are applied to mammals, a higher proportion of nanoparticle compositions comprising nucleic acids (e.g., mRNA) are delivered to the destination of interest (e.g., tissue). In some embodiments, compared with another destination (e.g., liver), specific delivery can increase the amount of nucleic acids (e.g., mRNA) delivered to the targeted destination (e.g., tissue of interest, such as kidney) by more than 2 times, 5 times, 10 times, 15 times or 20 times per 1g. In a particular embodiment, tissue of interest is kidney. In certain embodiments, tissue of interest is the vascular endothelium of kidney. In other embodiments, tissue of interest is selected from the group consisting of: vascular endothelium in blood vessels (e.g., in coronary arteries or femurs) and tumor tissue (e.g., by intratumoral injection).

[0414] As another example of targeted or specific delivery, nucleic acid (e.g., mRNA) of protein binding partners (e.g., antibodies or their functional fragments, scaffold proteins or peptides) or receptors on the coding cell surface can be included in the nanoparticle composition. Nucleic acid (e.g., mRNA) can be used to instruct the synthesis and extracellular location of lipid, carbohydrate or other biological parts in addition or alternatively. Alternatively, other elements (e.g., lipid or part) of nanoparticle composition can be selected based on their affinity to specific receptors (e.g., low-density lipoprotein receptors) so that the nanoparticle composition can more easily interact with the target cell colony comprising receptors. For example, ligands can include, but are not limited to, members of specific binding pairs, antibodies, monoclonal antibodies, Fv fragments, single-chain Fv (scFv) fragments, Fab' fragments, F(ab')2 fragments, single domain antibodies, camelized antibodies and fragments thereof, humanized antibodies and fragments thereof, and multivalent versions thereof; multivalent binding agents, including monospecific antibodies or bispecific antibodies, such as disulfide-stabilized Fv fragments, scFv tandems, bifunctional antibodies, trifunctional antibodies or tetrafunctional antibodies; and aptamers, receptors and fusion proteins.

[0415] In some embodiments, the ligand can be a surface-bound antibody, which can allow for the modulation of cell targeting specificity. This is particularly useful because highly specific antibodies can be generated for epitopes of interest at desired targeting sites. In one embodiment, multiple antibodies are expressed on the cell surface, and each antibody can have a different specificity for the desired target. Such methods can increase the affinity and specificity of the targeting interaction.

[0416] The ligand can be selected, for example, by a technician in the biological field based on the location or function of the desired cell. For example, estrogen receptor ligands such as tamoxifen can target cells to estrogen-dependent breast cancer cells with an increased number of estrogen receptors on the cell surface. Other non-limiting examples of ligand / receptor interactions include CCR1 (e.g., for treating inflamed joint tissue or brain in rheumatoid arthritis and / or multiple sclerosis), CCR7, CCR8 (e.g., targeting lymph node tissue), CCR6, CCR9.CCR10 (e.g., targeting intestinal tissue), CCR4, CCR10 (e.g., for targeting skin), CXCR4 (e.g., for general enhanced migration), HCELL (e.g., for treating inflammation and inflammatory conditions, bone marrow), α4β7 (e.g., for intestinal mucosal targeting) and VLA-4NCAM-1 (e.g., targeting endothelium). Generally, any receptor related to targeting (e.g., cancer metastasis) can be utilized for the methods and compositions described herein.

[0417] Target cells can include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, β cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells.

[0418] In certain embodiments, nanoparticle compositions of the present disclosure can target hepatocytes. Apolipoproteins such as apolipoprotein E (apoE) have been shown to associate with neutral or nearly neutral lipid-containing nanoparticle compositions in vivo and are known to associate with receptors such as low-density lipoprotein receptors (LDLR) found on the surface of hepatocytes. Therefore, nanoparticle compositions comprising lipid components having neutral or nearly neutral charges administered to a subject can obtain apoE in the subject, and nucleic acids (e.g., mRNA) can then be delivered to hepatocytes comprising LDLR in a targeted manner.

[0419] Nanoparticle compositions of the present disclosure can be used to treat diseases, disorders or conditions characterized by missing or abnormal protein or polypeptide activity. After nucleic acid (e.g., mRNA) encoding missing or abnormal polypeptide is delivered to cells, translation of the nucleic acid (e.g., mRNA) can produce the polypeptide, thereby reducing or eliminating the problems caused by lacking the polypeptide or the problems caused by abnormal activity caused by the polypeptide. Because translation can occur rapidly, methods and compositions of the present disclosure can be used to treat acute diseases, disorders or conditions, such as sepsis, stroke and myocardial infarction. Nucleic acids (e.g., mRNA) included in nanoparticle compositions of the present disclosure can also be able to change the transcription rate of a given species, thereby affecting gene expression.

[0420] Diseases, disorders and / or conditions characterized by dysfunctional or abnormal protein or polypeptide activity to which the compositions of the present disclosure may be administered include, but are not limited to, cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases and renal vascular diseases, and metabolic diseases. A variety of diseases, disorders and / or conditions may be characterized by the absence (or substantial reduction, such that proper protein function does not occur) of protein activity. Such proteins may not be present, or such proteins may be substantially non-functional. A specific example of a dysfunctional protein is a missense mutation variant of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which produces a dysfunctional protein variant of the CFTR protein that causes cystic fibrosis. The present disclosure provides a method for treating such diseases, disorders and / or conditions of a subject by administering a nanoparticle composition, the nanoparticle composition comprising a nucleic acid (e.g., mRNA) and a lipid component, the lipid component comprising KL10, a phospholipid (optionally unsaturated), a PEG lipid and a structural lipid, wherein the mRNA encodes a polypeptide that antagonizes or otherwise overcomes the abnormal protein activity present in the cells of the subject.

[0421] In some embodiments, nanoparticle compositions can be designed and administered to promote the transient expression of polypeptides in specific organs or their systems. Transient expression can be used alone as a therapeutic treatment or in combination with other therapeutic treatments including drug administration and / or surgical procedures as a therapeutic treatment. Specifically, the transient expression of vascular endothelial growth factor A (VEG FA) can be promoted by administering nanoparticle compositions of the present disclosure. VEG FA plays an important role in angiogenesis (particularly in vascular endothelium). Although the overexpression of VEGF-A is associated with a variety of cancers, the transient expression of VEG FA in, for example, renal vascular structures can reduce arterial stent implantation to treat vascular diseases such as atherosclerotic renal vascular disease (ARVD). Therefore, nanoparticle compositions comprising mRNA encoding VEGF-A and optimized to selectively deliver nucleic acid (e.g., mRNA) to the renal system (e.g., kidney) can be used to treat ARVD and related conditions.

[0422] The present disclosure provides methods involving administration of nanoparticle compositions comprising nucleic acids (eg, mRNA) or pharmaceutical compositions comprising the nanoparticle compositions.

[0423] The compositions of the present disclosure or their imaging compositions, diagnostic compositions or preventive compositions can be administered to a subject using any reasonable amount and any route of administration that is effective for preventing, treating, diagnosing or imaging a disease, disorder and / or condition and / or any other purpose. The specific amount administered to a given subject may vary according to the species, age and general condition of the subject; the purpose of administration; the specific composition; the mode of administration; and the like. The compositions according to the present disclosure can be formulated in dosage unit form for ease of administration and uniformity of dosage. However, it will be understood that the total daily dosage of the compositions of the present disclosure will be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective, preventively effective or otherwise appropriate dosage level (e.g., for imaging) for any particular patient will depend on a variety of factors, including the severity and identification of the disorder (if any) being treated; the one or more nucleic acids (e.g., mRNA) employed, the specific composition employed; the patient's age, weight, general health, sex and diet; the time of administration, route of administration and excretion rate of the specific pharmaceutical composition employed; the duration of treatment; the drugs used in combination or concomitantly with the specific pharmaceutical composition employed and similar factors well known in the medical field.

[0424] Nanoparticle compositions including one or more nucleic acids (e.g., mRNA) can be administered by any route. In some embodiments, the compositions of the present disclosure (including preventive compositions, diagnostic compositions, or imaging compositions comprising one or more nanoparticle compositions of the present disclosure) are administered by one or more of a variety of routes, including oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal or intradermal, interdermal, rectal, intravaginal, intraperitoneal, topical (e.g., by powders, ointments, creams, gels, lotions, and / or drops), mucosal, nasal, buccal, intestinal, vitreous, intratumoral, sublingual, intranasal; by intratracheal instillation, bronchial instillation, and / or inhalation; as oral sprays and / or powders, nasal sprays and / or aerosols, and / or by portal vein catheters. In some embodiments, the compositions can be administered intravenously, intramuscularly, intradermally, intraarterially, intratumorally, subcutaneously, or by inhalation. However, the present disclosure encompasses the delivery of the compositions of the present disclosure by any appropriate route that takes into account possible advances in drug delivery science. Generally, the most appropriate route of administration will depend on a variety of factors, including the properties of the nanoparticle composition comprising one or more mRNAs (e.g., its stability in various bodily environments such as the bloodstream and gastrointestinal tract), the patient's condition (e.g., whether the patient can tolerate a particular route of administration), etc.

[0425] In certain embodiments, the compositions according to the present disclosure may be administered in an amount sufficient to deliver in a given dose from about 0.0001 mg / kg to about 10 mg / kg, from about 0.001 mg / kg to about 10 mg / kg, from about 0.005 mg / kg to about 10 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.05 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 10 mg / kg, from about 2 mg / kg to about 10 mg / kg, from about 5 mg / kg to about 10 mg / kg. g, about 0.0001 mg / kg to about 5 mg / kg, about 0.001 mg / kg to about 5 mg / kg, about 0.005 mg / kg to about 5 mg / kg, about 0.01 mg / kg to about 5 mg / kg, about 0.05 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 1 mg / kg to about 5 mg / kg, about 2 mg / kg to about 5 mg / kg, about 0.0001 mg / kg to about 2.5 mg / kg, about 0.001 mg / kg to about 2.5 mg / kg, about 0.005 mg / kg to about 5 mg / kg g / kg to about 2.5 mg / kg, about 0.01 mg / kg to about 2.5 mg / kg, about 0.05 mg / kg to about 2.5 mg / kg, about 0.1 mg / kg to about 2.5 mg / kg, about 1 mg / kg to about 2.5 mg / kg, about 2 mg / kg to about 2.5 mg / kg, about 0.0001 mg / kg to about 1 mg / kg, about 0.001 mg / kg to about 1 mg / kg, about 0.005 mg / kg to about 1 mg / kg, about 0.01 mg / kg to about 1 mg / kg, about 0.05 mg / kg The nanoparticle composition of the present disclosure may be administered at a dosage level of about 0.001 mg / kg to about 1 mg / kg, about 0.1 mg / kg to about 1 mg / kg, about 0.0001 mg / kg to about 0.25 mg / kg, about 0.001 mg / kg to about 0.25 mg / kg, about 0.005 mg / kg to about 0.25 mg / kg, about 0.01 mg / kg to about 0.25 mg / kg, about 0.05 mg / kg to about 0.25 mg / kg, or about 0.1 mg / kg to about 0.25 mg / kg, wherein a dose of 1 mg / kg provides 1 mg of the composition per 1 kg of subject body weight. In specific embodiments, a nanoparticle composition of the present disclosure may be administered at a dosage of about 0.001 mg / kg to about 10 mg / kg. In other embodiments, a nanoparticle composition of about 0.005 mg / kg to about 2.5 mg / kg may be administered. In certain embodiments, a dosage of about 0.1 mg / kg to about 1 mg / kg may be administered. In other embodiments, a dose of about 0.05 mg / kg to about 0.25 mg / kg may be administered.Dosage can be applied once or multiple times with the same or different amounts every day to obtain the desired level of nucleic acid (e.g., mRNA) expression and / or therapeutic effect, diagnostic effect, preventive effect or imaging effect. The desired dose can be delivered, for example, three times a day, twice a day, once a day, every other day, every three days, every week, every two weeks, every three weeks or every four weeks. In certain embodiments, multiple administrations (e.g., twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times or more) can be used to deliver the desired dose. In certain embodiments, for example, before or after a surgical procedure, or in the case of an acute disease, illness or condition, a single dose can be applied.

[0426] Nanoparticle compositions including one or more nucleic acids (e.g., mRNA) can be used in combination with one or more other therapeutic agents, prophylactic agents, diagnostic agents, or imaging agents. "Combination with..." is not intended to imply that medicaments must be administered simultaneously and / or formulated for delivery together, although these delivery methods are within the scope of the present disclosure. For example, one or more nanoparticle compositions including one or more different mRNAs can be administered in combination. Compositions can be administered simultaneously with one or more other desired therapeutic agents or medical procedures, administered before them, or administered after them. Typically, each medicament will be administered with a dosage and / or schedule determined for the medicament. In certain embodiments, the present disclosure encompasses the delivery of a combination of compositions of the present disclosure or its imaging compositions, diagnostic compositions, or prophylactic compositions with an agent that improves its bioavailability, reduces and / or changes its metabolism, inhibits its excretion, and / or changes its distribution in vivo.

[0427] It should be further understood that the therapeutically active agent, preventive active agent, diagnostic active agent or imaging active agent used in combination can be administered together in a single composition or separately in different compositions. Generally, it is expected that the medicament used in combination will be used at a level that does not exceed the level at which it is used alone. In some embodiments, the level used in combination may be lower than the level used alone.

[0428] The particular combination of therapies (therapeutics or procedures) employed in a combination regimen will take into account compatibility of the desired therapeutics and / or procedures and the desired therapeutic effect to be achieved. It will also be understood that the therapies employed may achieve the desired effect for the same condition (e.g., a composition useful for treating cancer may be administered simultaneously with a chemotherapeutic agent), or the therapies may achieve different effects (e.g., controlling any side effects).

[0429] Examples

[0430] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0431] The experiment begins by adding a polymer (e.g., L121) to a reference LNP formulation to test whether the polymer can improve the delivery of mRNA to cells. Due to significant improvements observed (e.g., as shown in Examples 1-3), additional experiments were designed to test additional formulations containing various types of polymers to optimize the LNP formulations with significantly improved RNA / DNA delivery efficiency combined with significantly reduced ionizable lipid mol% and increased mRNA loading, as indicated by significantly reduced N / P ratios.

[0432] Example 1. GFP cell fluorescence image: Visualization of mRNA delivery improved by adding polymer to reference LNP

[0433] Different LNPs were tested for in vitro transfection of GFP (green fluorescent protein) mRNA. Reference LNPs (formulation codes: CPT101, CPT103, CPT105, and CPT107) were produced to have a molar ratio of mRNA COVID-19 vaccine, for example, 50 mol% cationic (or ionizable) lipids, 10 mol% DSPC (distearoylphosphatidylcholine), 38.5 mol% cholesterol, and 1.5 mol% PEG2000-DSPE. Pluronic TM The surfactant triblock copolymers L121 were prepared to produce the corresponding polymer-advanced lipid nanoparticles or PALNPs (formulation codes: CPT067, CPT069, CPT106 and CPT108), respectively. The molar percentage (mol%) of LNPs is shown in the table below.

[0434] Table 1: Formulations of PALNP and reference LNP (molar ratio)

[0435]

[0436] PALNPs share the same cationic lipids as the corresponding control (reference) LNPs, but also include L121 in the formulation. The cationic lipids selected for this study are ACL-0315, DLin-MC3-DMA, DLin-KC2-DMA, and DLin-DMA. All LNPs were prepared using nPort as disclosed in US 11,497,715 B2. TM Technologically prepared.

[0437] L121 is Pluronic TM A triblock copolymer of a surfactant consisting of a central hydrophobic polypropylene glycol (PPO) block flanked by two hydrophilic polyethylene glycol (PEO) blocks. TM The general formula of surfactant is shown below.

[0438] or

[0439] H[OCH2CH2] x [OCH(CH3)CH2] y [OCH2CH2] z OH; or

[0440] [PEO] x [PPO] y [PEO] z

[0441] For L121, x=5, Y=68, z=5, and the average molecular weight is about 4,400 Daltons.The total weight / weight (w / w) ratio of the two PEO blocks is about 10% of the molecule.

[0442] HEK293, A549 and HepG2 cell lines were used to test the in vitro transfection of GFP mRNA (as a reporter gene) by the above-mentioned LNPs. Specifically, cells were cultured in black 96-well plates with cover glass bottoms. Cell fluorescence images were taken by a Nikon SLR digital camera mounted on a Nikon TE-2000U microscope equipped with a high-resolution plan apochromat (APO) objective. Figure 1 Shown are representative GFP fluorescence images of HEK293 cells (bottom row) transfected with PALNP (respectively CPT108, CPT106, CPT069 and CPT067) containing 0.1 μg mRNA per well and HEK293 cells (upper row) transfected with control LNP (respectively CPT107, CPT105, CPT103 and CPT101) containing 0.6 μg mRNA. The N / P ratio of all LNPs is 6.0. Bright green fluorescence was detected from most of the cells processed by PALNP, wherein among four kinds of control LNPs, only CPT105LNP (containing DLin-MC3-DMA) induced medium fluorescence, and the remaining three kinds were significantly weaker. The result shows that L121 significantly enhances the transfection efficiency of GFP mRNA.

[0443] Example 2. Improved mRNA delivery determined by FACS analysis

[0444] FACS (fluorescence activated cell sorting) was used for quantitative studies of GFP mRNA transfection. Several main PALNPs made of cationic lipids (ALC-0315, DLin-MC3-DMA, DLin-KC2-DMA or DLin-DMA) were analyzed by FACS and compared with their control LNPs. Specifically, 0.05 μg, 0.10 μg, 0.20 μg or 0.4 μg GFP mRNA were loaded into each PALNP, while 0.6 μg GFP mRNA was loaded into the corresponding control LNP. Figure 2 The FACS results of CPT108 containing ALC-0315 as the cationic lipid are shown. Both the fluorescent spots representing cells transfected with CPT108 containing 0.05 μg of mRNA and the fluorescent spots representing cells transfected with control LNP (CPT107) containing 0.6 μg of mRNA are indicated by circles.

[0445] About 11% of cells transfected with CPT107 (control) were very weakly GFP-positive, with an average fluorescence intensity of about 175 (arbitrary units (au)), while 89% of cells transfected with CPT108 (loaded with only one-twelfth of the mRNA) were GFP-positive, with an average GFP fluorescence of about 5 times that of the control. Figure 3 As shown, when the mRNA was increased to 0.4 μg with CPT108, more than 95% of the cells emitted GFP signals, and the fluorescence intensity was about 24 times that of the control.

[0446] Example 3. Improvement of cell transfection efficiency determined by FACS analysis

[0447] The five types of PALNPs in Table 1 were loaded with 0.05 μg GFP mRNA, and the cell transfection efficiency was determined to be about 76-94%, with a fluorescence intensity (average) ranging from 660-3000 (au). When the mRNA dose was increased to 0.4 μg, the cell transfection efficiency was stably maintained at more than 95%, with a fluorescence intensity of 4000-10600 (au). In contrast, when the corresponding reference LNP was loaded with 0.6 μg mRNA, only a transfection efficiency of 10-60% and an intensity of 175-788 (au) were observed, as shown in the fluorescence intensity table below.

[0448] Table 2: Overview of FACS analysis

[0449]

[0450] As shown in the following table and Figure 4As shown, combining all the significantly improved transfection factors, including lower mRNA dose, higher cell transfection rate and much higher protein expression level in the PALNP group, PALNPs exhibited increased overall mRNA delivery efficiency, which was approximately 10-445 times that of control LNPs.

[0451] Table 3: Enhanced mRNA delivery by PALNPs

[0452]

[0453] Example 4. Enhanced mRNA transfection efficiency at low N / P ratio and low mRNA dose

[0454] The above results prove that adding Pluronic TM Polymer significantly improves the transfection efficiency of LNP, indicating the potential of developing LNP with improved characteristics. In reference LNP, RNA / DNA encapsulation and LNP escape from lysosomes require excessive cationic lipids. The severe toxicity of high-dose cationic lipids has become a huge obstacle for gene delivery based on LNP. In order to solve this problem, an efficient LNP formulation has been developed to reduce both the N / P ratio and the dosage of mRNA. For example, in CPT147LNP, the N / P ratio is reduced to 1, which means that 6 times lower cationic lipids can be used in the formulation of the same amount of mRNA / DNA. Compared with the reference or control LNP (CPT107) (Figure 5C) with 0.3μg mRNA and N / P=6, the CPT147LNP with 0.01μg mRNA and N / P=1 induces comparable GFP fluorescence (Figure 5A). The RNA dosage has been reduced by 30 times (0.01μg to 0.3μg), and therefore the cationic lipid ALC-0315 has been reduced by 180 times. When 0.3 μg of mRNA was used, CPT147 LNP induced supersaturated GFP fluorescence in cells ( FIG. 5B ). The formulation of CPT147 LNP is shown in the table below.

[0455] Table 4: Formulation of CPT147LNP (mol%)

[0456] Element ALC-0315 DSPC DOPE cholesterol PEG2000-DSPE L121 N / P Mol% 10 22.5 22.5 38.5 1.5 5 1

[0457] To track the uptake of LNPs by cells, LNPs were labeled with rhodamine-DSPC (Rhd-DSPC), and the punctate red fluorescence in the images of Figures 5B-5C is from rhodamine-DSPC. In Figure 5A, the lipid concentration is too low (1 / 180 of the control) to produce red fluorescence detectable by a microscope.

[0458] Example 5. Determination of the Optimized N / P Ratio for GFP Expression

[0459] Determine the optimized N / P ratio of CPT147LNP from two opposite methods.As shown in Figure 6A, when using different amounts of mRNA, the LNP amount of all holes is fixed (1 μ l), so that various N / P ratios are within the range of 0.25-4.In Figure 6B, mRNA is fixed at 0.05 μg, but different LNP amounts are used for each hole, so that the desired N / P ratio is 0.25 to 4.The brightest fluorescence of the cell appears in the group where the N / P ratio is between 0.5 and 1.When the N / P ratio is increased to 2 or reduced to 0.25, it is observed that GFP expression drops sharply.Therefore, the optimized N / P ratio of CPT147LNP is 0.25-1, and the ratio is about 1 / 24-1 / 6 of the reference LNP.

[0460] In a separate study, the effect of N / P ratio on GFP mRNA transfection was determined using CPT147-38LNP (Fig. 6C) and CPT163-21-18LNP (Fig. 6D). The basis of CPT147-38 and CPT147 is the same, while the ratios of ALC-0315 and L121 are significantly different from each other. Although the lipid ratios of LNP CPT163-21-18 and CPT147-38 are similar, the block polymers are different. Interestingly, despite differences in formulations and / or lipid ratios, these LNPs share a similarly optimized N / P ratio in the range of 0.25 to 1.

[0461] Table 5: Formulations of LNPs tested for N / P ratio optimization (mol%)

[0462] ALC-0315 DSPC DOPE cholesterol PEG2000-DSPE L121 L81 CPT147 10 22.5 22.5 38.5 1.5 5 CPT147-38 18 20 20 34 1.5 6.8 CPT163-21-18 19 21 21 36 1.4 1.8

[0463] Example 6. Rapid cellular uptake and protein expression of PALNP

[0464] The cellular uptake of PALNP was determined. Specifically, red fluorescence from the LNP labeled with rhodamine-DSPC (Rhd-LNP) was observed in the cell plasma membrane a few minutes after CPT147LNP was added to the cells. This rapid uptake by the cells promotes the rapid delivery of mRNA to the cytosol, resulting in rapid expression of GFP protein. Figure 7 As shown, strong GFP fluorescence was observed 4 hours after CPT147 LNP (N / P=1, mRNA 0.2 μg) was added to the cells.

[0465] Also in Figures 5A-5CIn the figure, the molar ratio of ALC-0315 to Rhd-DSPC is the same in both CPT-0147 and control LNP (CPT107). Due to the low N / P ratio, the amount of Rhd-DSPC used in the cell sample of FIG. 5B is only one-sixth of the amount of Rhd-DSPC used in the control sample ( FIG. 5C ). However, the red fluorescence intensity of FIG. 5B is comparable to that of FIG. 5C , indicating that CPT147 is more effective than the control LNP (CPT107) in increasing cellular uptake by about six times.

[0466] In another experiment, cells were treated with 0.25 μg of mRNA labeled with rhodamine-DSPC in CPT147 ( Fig. 8A ) or control LNPs (CPT107) containing cationic lipid ALC-0315 and 0.25 μg mRNA ( Figure 8B ) transfection. Due to the low N / P ratio of CPT147, Fig. 8A The total lipids and rhodamine-DSPC used in the LNPs were Figure 8B The total lipid content of LNPs used in the experiment was one sixth of that of rhodamine-DSPC. Fig. 8A The red fluorescence shown in Figure 8B This indicates that CPT 147 undergoes a much faster cellular uptake rate than the control LNPs.

[0467] Example 7. Transient mRNA transfection by PALNP is sufficient for protein expression

[0468] High mRNA transfection efficiency can be further demonstrated by observing that even transient mRNA transfection is sufficient for substantial GFP expression. In this study, CPT147LNPs were removed from the wells by medium exchange one hour after transfection ( Fig. 9A ) and then cultured in fresh medium for another 3 hours. Alternatively, cells were cultured for 4 hours ( Fig. 9B The images were taken four hours after transfection began ( Fig. 9C ). Fig. 9A The bright fluorescence of the cells in the culture medium demonstrated that within one hour of transfection, CPT147LNPs delivered sufficient mRNA into the cells for protein expression. Fig. 9B The results showed that the longer the exposure to the mRNA, the greater the uptake of LNPs by the cells (as observed by stronger red fluorescence) and the greater the protein expression (as observed by brighter green fluorescence). The results showed that transient exposure of cells (1 hour) was sufficient for protein expression using CPT147 LNPs.

[0469] Example 8. Reduced cytotoxicity of PALNP

[0470] Cationic lipids have strong cytotoxicity. For example, the cationic lipids can damage the cell membrane, thereby causing a large number of cell apoptosis and hemolysis. Therefore, the cytotoxicity of LNPs containing cationic lipids has brought considerable safety issues to clinical applications. In order to test the cytotoxicity of PALNPs, cells were transfected with CPT147LNPs for 96 hours. Figures 10A-10B As shown, both the fluorescence image and the corresponding bright field image of the cell were recorded. The results show that the transfected cells successfully expressed GFP and also maintained normal cell cycle, morphology and growth. Therefore, no significant cytotoxicity of CPT147LNP was observed, as well as a significant increase in gene transfection efficiency.

[0471] Example 9. PALNP is very effective for plasmid DNA transfection

[0472] In biopharmaceuticals, mRNA has great advantages over DNA in terms of delivery and safety. However, due to the shortcomings of mRNA to DNA in commercial production, stability, and sustained protein expression in transfected cells, mRNA is not a substitute for DNA in gene therapy. Therefore, LNPs for plasmid DNA delivery are still in great demand for gene therapy. Due to the greater challenges faced by DNA delivery, after nearly 40 years of efforts, there are still no commercially available DNA-based therapeutics or vaccines in the world. A large number of in vitro tests have been conducted on the main LNPs for GFP plasmid DNA delivery.

[0473] In a head-to-head comparison with a reference LNP (CPT107), CPT147 effectively delivers plasmid DNA to cells for protein expression. Specifically, 48 hours after transfection, no fluorescence was observed from cells transfected with the reference LNP, while cells transfected with CPT147 emitted detectable GFP fluorescence (Figure 11A). During transfection, cells grew normally, for example, without cell aggregation, necrosis or apoptosis. Figures 11B-11C show that after 120 hours of transfection, 1) the confluence of cells with normal morphology on the plate reached 100%, indicating healthy growth during transfection; and 2) nearly 100% of the cells emitted bright green fluorescence, indicating PALNP-mediated plasmid DNA transfection and protein expression. In addition, the cells in Figure 11B were rinsed, digested with trypsin, and re-seeded to grow in fresh culture medium for another 24 hours (Figure 11D) or 144 hours (Figure 11E). The cells maintained healthy growth conditions and formed colonies (circled cells) that emitted green fluorescence. The cells in Figure 11E were further washed, trypsinized, and re-seeded to grow in fresh medium for another 24 hours (Figure 11F) or 120 hours (Figure 11G). The cells maintained healthy growth conditions and once again formed colonies (circled cells) that emitted green fluorescent signals.

[0474] In general, the above results demonstrate that PALNP can not only deliver mRNA, but also effectively deliver plasmid DNA into cells. No obvious cytotoxicity was observed when cells were transfected with PALNP. In addition, the results show that the delivered plasmid DNA genes can be passed from parent cells to daughter cells for many generations and achieve sustainable protein expression.

[0475] Example 10. Accelerated stability assessment

[0476] mRNA is rapidly degraded in solution, and the presence of trace amounts of RNA enzymes can greatly promote mRNA degradation. Therefore, the stability of mRNA in LNP is one of the key monitoring characteristics of LNP. Unfortunately, mRNA shows poor stability in the LNP of mRNA LNP vaccine for COVID-19 of both Pfizer and Moderna. For example, the former is only stable for up to 2 hours at room temperature, and the latter is only stable for up to 8 hours. The poor stability is attributed to the formation of the bubble of the reference LNP assembled with the formulation, and a part of the mRNA molecule encapsulated in the LNP is released into the aqueous phase in the bubble. Detailed information can be found, for example, in the following literature: Mark LB et al., Encapsulation state of messenger RNA inside lipid nanoparticles, Biophysical Journal 120, 2766-2770, July 20, 2021.

[0477] To evaluate the stability of PALNPs, CPT147E LNPs were prepared and stored at 25° C. for one week. The formulation of CPT147E is shown in the table below.

[0478] Table 6: Formulation of CPT147E LNP

[0479] Element ALC-0315 DSPC DOPE cholesterol PEG2000-DSPE L121 N / P Mol% 18.0 19.8 19.8 34.2 1.4 6.8 1

[0480] First, the GFP expression levels of LNPs at different time points were compared. Fig.12 As shown, no significant differences were observed in both the cell transfection percentage and cell fluorescence intensity of samples containing 0.05 μg mRNA stored for different times (from time 0 (T0) to 168 hours). Second, in order to avoid false stability results induced by mRNA saturation, cells were transfected with serial dilutions of mRNA ( Fig.13 ). It was observed that when the amount of mRNA decreased, the cell transfection efficiency decreased, indicating that the cells were not saturated with mRNA. Fig.13 As shown, the overall comparable cell transfection efficiency at the same mRNA concentration demonstrates that PALNPs are stable for at least 7 days at 25°C. This is a significant improvement compared to the COVID-19 mRNA vaccines, which are only stable for 2-8 hours at room temperature.

[0481] Example 11. Block copolymers play an important role in mRNA transfection

[0482] To demonstrate the critical role of the coblock polymer in LNPs for mRNA transfection, the in vitro GFP mRNA transfection efficiency of LNP groups made of different ionizable lipids was compared. For example, in the presence of triblock polymer L121, CPT147E-05, CPT149E-01, and CPT162E-01 contained ALC-0315, SM-102, and Dlin-KC2-DMA, respectively. 对照 、CPT149E 对照 and CPT162E 对照 Contains the same ionizable lipids but without L121.

[0483] Table 7: LNP formulations in the presence and absence of triblock polymers (mol%)

[0484]

[0485] As shown in Figures 14A-14F, all LNPs containing L121 exhibited mRNA transfection efficiencies at least 10-fold higher than the mRNA transfection efficiencies of corresponding control LNPs containing the same lipid composition but without L121.

[0486] Example 12. Production of PALNPs using different block copolymers with a wide range of molecular weights

[0487] Other triblock polymers, such as L92 and L81, were tested to replace L121 in PALNP. The molecular structures of the tested triblock polymers are shown in the table below.

[0488] Table 8: Molecular structures of different triblock polymers tested

[0489]

[0490] LNPs were produced using the methods described herein, with formulations listed in the table below.

[0491] Table 9: Formulations of PALNPs containing different block copolymers

[0492]

[0493] HEK293 cells were transfected with these LNPs loaded with 0.05-0.2 μg GFP mRNA, and fluorescence images were recorded after 24 hours. Fig.15 As shown, all transfected cells emitted bright green fluorescence signals of GFP, indicating that not only L121, but also other block copolymers with a wide range of molecular weights can be used to produce PALNPs.

[0494] It was also found to contain three types of Pluronic TM The mRNA delivery efficiency of the copolymer LNPs was in the order of L121-L81 > L92. The results suggest that 1) the hydrophobicity of the polymer plays a key role in mRNA delivery, and a lower weight percentage of PEO blocks is associated with higher mRNA delivery efficiency; and 2) molecular weight plays a minor role here, as the efficiency of L81 is slightly lower than that of L121, while its molecular weight is significantly lower than L121.

[0495] Example 13. Production of PALNPs using permanently charged cationic lipids

[0496] Dioleoyl-3-trimethylaminopropane (DOTAP) is an atypical cationic lipid with a permanent positive charge. It is widely used in gene delivery studies and exhibits potent cytotoxicity in a concentration-dependent manner.

[0497]

[0498] CPT153E LNPs were produced using the methods described herein and their formulations are listed in the table below.

[0499] Table 10: Formulation of CPT153E LNP (Mol%)

[0500] DOTAP DSPC DOPE cholesterol PEG2000-DSPE L121 N / P CPT153E 17.9 19.9 19.9 34.1 1.4 6.8 1

[0501] HEK293 cells were transfected with CPT153E LNPs loaded with 0.0125-0.2 μg GFP mRNA, and fluorescence images were recorded after 24 hours. Fig.16 As shown, all transfected cells emitted bright green fluorescence signals of GFP, indicating that not only transiently charged cationic lipids but also permanently charged cationic lipids can be used to produce PALNPs.

[0502] Example 14. PPO with reverse triblock copolymer x -PEO y -PPO x LNP

[0503] The block structure of the copolymer can also be reversed, that is, one PEO block can be located in the middle of the triblock, and two PPO blocks can be located on the sides of the PEO block. An exemplary structure of a reverse copolymer is shown below.

[0504] or

[0505] H[OCH(CH3)CH2] x [OCH2CH2] y [OCH(CH3)CH2]x OH; or

[0506] {PPO] x [PFO] y [PPO] x

[0507] Specifically, the average molecular weight of L31R1 (x=25.6, y=7.5) is about 3300 Daltons, of which the weight percentage of PEO is about 10%; the average molecular weight of L17R4 (x=14, y=24.5) is about 2700 Daltons, of which the weight percentage of PEO is about 40%.

[0508] CPT189 LNPs and CPT202 LNPs were produced using the methods described herein and their formulations are listed in the table below.

[0509] Table 11: Formulations of LNP CPT189 and CPT202

[0510] ALC-0315 DSPC DOPE cholesterol PEG2000-DSPE L31R1 L17R4 N / P CPT189 21.7 16.9 16.9 41.0 1.2 2.4 1 CPT202 21.7 16.9 16.9 41.0 1.2 2.4 1

[0511] HEK293 cells were transfected with CPT189 LNPs loaded with 0.0125-1 μg GFP mRNA, and fluorescence images were recorded after 24 hours. Fig.17 As shown, all transfected cells emit bright green fluorescence of GFP, indicating that copolymers with reverse structures can also be used to produce PALNPs. The results also show that the transfection efficiency of CPT202 is significantly weaker than that of CPT189, although the molecular weights of the two polymers are very close. The only difference between the two PALNPs is the PEO:PPO ratio. Specifically, the weight percentage of PEO in CPT189 is 10%, compared to 40% in L17R4. The results show that lower PEO content is beneficial for mRNA transfection.

[0512] Example 15. Optimization of the molar ratio of triblock copolymers

[0513] In vitro testing was used to identify the optimal molar ratio of the triblock copolymer. Figures 18A-18B As shown, the basic LNP formula is fixed to contain 18% ALC-0315, 20% DSPC, 20% DOPE, 34% cholesterol, 1.5% PEG2000-DEPE and less than 10% (e.g., 1%, 2%, 4%, 5% or 6%) of the triblock copolymer L81 or L92. The results show that the optimized molar ratio of L81 is about 2-4%, the optimized molar ratio of L92 is about 4-5%, and the optimized molar ratio of L121 is about 5-7%.

[0514] Example 16. PALNP is non-cytotoxic

[0515] Cationic or ionizable lipids, RNA and DNA are all immunostimulants. For example, low doses of cationic lipids and / or oligonucleotides can be used as adjuvants in vaccines to stimulate innate immune responses. Therefore, the combination of cationic lipids and RNA / DNA in nanoparticles can induce a strong innate immune response and cause serious adverse reactions to patients.

[0516] In fact, cationic and ionizable lipids have been reported to stimulate the secretion of proinflammatory cytokines and reactive oxygen species, although the molecular mechanisms of the immunogenicity of these lipids are not fully understood. The cytotoxicity of lipid materials is also a safety issue, which depends on the dose, lipid properties, and cell type. It has been reported that the in vivo application of lipid nanoparticles can induce liver and lung damage in rodents, which can be attributed to the cytotoxicity of the materials and the induction of proinflammatory factors. The toxicity of LNPs severely limits their application in gene vaccines and gene therapeutic agents.

[0517] As shown in the results of PALNP above, when the ratio of ionizable (cationic) lipid to mRNA (DNA) or N / P ratio is reduced by 6-24 times, the mRNA / DNA transfection efficiency of LNP is significantly increased compared with reference LNP. Combined with these two significant improvements of PALNP, when the mRNA dose is reduced by one half and the dose of ionizable lipid is reduced by 6-24 times, the ionizable (cationic) lipid used can be reduced by hundreds of times. Therefore, PALNP can eliminate or minimize the toxicity of LNP and promote its application in genetic vaccines, genetic therapeutic agents and gene editing.

[0518] Cationic lipids can also destroy cell membranes and the endosomes and lysosomes of cells. In fact, the destruction of endosomal / lysosomal membranes by cationic lipids plays a key role in LNP escape from lysosomes for mRNA / DNA delivery. However, lysosomal destruction is a double-edged sword. For example, the method can also release cytotoxic enzymes into the cytosol, such as proteases, nucleases, phosphatases, sulfatases, and lipid degrading enzymes. These enzymes can destroy normal organelles, thereby causing acute or apoptotic cell death and further causing systemic adverse reactions. Detailed information can be found, for example, in the following literature: Forster III, J. et al. "MRNA-carrying lipid nanoparticles that induce lysosomal rupture activate NLRP3 inflammasomes and reduce mRNA transfection efficiency." Biomaterials Science 10.19(2022):5566-5582.

[0519] In addition, since toxicity is cationic lipid dose-dependent, the PALNPs disclosed herein with very low doses of cationic lipids and enhanced gene transfection efficiency can solve the persistent toxicity problem of LNPs. To demonstrate this extraordinary potential of PALNPs, HEK293 cells were transfected with CPT147P LNPs and corresponding reference LNPs CPT107. Similarly, CPT149E LNPs and corresponding reference LNPs CPT109 were used for cell transfection. The formulations of these LNPs are listed in the table below.

[0520] Table 12: Formulations (mol %) of LNPs used in cytotoxicity studies.

[0521] ALC-0315 SM-102 DSPC DOPE cholesterol PEG2000-DSPE L121 N / P CPT147P 18.1 18.1 18.1 39.2 1.5 5 1 CPT107 50 10 38.5 1.5 6 CPT149E 18 19.8 19.8 34.1 1.5 6.8 1 CPT109 50 10 38.5 1.5 6

[0522] All LNPs were added to cells in wells of a 96-well plate containing 0.2 μg of GFP mRNA. As shown in Figures 19A-19D, bright field images of cells were taken 24 hours after the LNPs were added to the cells.

[0523] The images showed that cells exposed to PALNPs (CPT147P and CPT149E) exhibited healthy morphology. In contrast, many cells exposed to reference LNPs (CPT107 and CPT109) aggregated or detached from the bottom of the well, indicating that a large number of cells were undergoing apoptosis. The results prove that PALNPs can reduce or eliminate cytotoxicity and have great potential for application in the development of safe and effective genetic vaccines and genetic therapeutics.

[0524] Example 17. In vivo distribution and improved expression of mRNA delivered by PALNPs

[0525] Several animal studies were conducted to evaluate mRNA transfection function in vivo. Specifically, LNP CPT147E-10 and corresponding reference LNP CPT107, which share the same formula as CPT147E-05 in Table 7, were administered to BALB / c mice. LNPs carrying luciferase reporter gene mRNA were administered by tail vein injection or intramuscular injection. At selected time points (4-24 hours after injection), mice were anesthetized with isoflurane and administered with substrate luciferin. Body images were taken by measuring the photon intensity of bioluminescence, which indicated the expression level of luc mRNA in mice and the distribution of mRNA in organs.

[0526] like Figures 20A-20B As shown in Figure 1, when LNPs were loaded with 10 μg of mRNA, CPT147E-10 LNPs significantly enhanced mRNA expression in vivo compared to CPT107. For example, throughout the experimental period, mice injected with CPT147 showed brighter photon intensity compared to mice injected with CPT107. In another experiment, when LNPs were loaded with 1 μg of mRNA, mice injected with CPT147E-10 also showed brighter photon intensity compared to mice injected with CPT107 ( Fig.21 ).

[0527] In different experiments, PALNP CPT147E-10 and the corresponding reference LNPCPT107 were injected into mice via the tail vein, and body images of the mice were taken within 4-72 hours. Fig. 22 As shown, throughout the experimental period, mice injected with CPT147 showed brighter photon intensity than mice injected with CPT107. The results also showed that the mRNA delivered by CPT147E-10 maintained persistent protein expression, for example, for at least 72 hours. In addition, mice were sacrificed 6 hours after being injected with CPT147E-10 containing 10 μg of mRNA, and bioluminescent images of organs including the heart, kidney, spleen, and liver were isolated. Fig.23As shown, strong bioluminescence was observed from the liver and spleen. No detectable luminescence was observed from the heart and kidney.

[0528] Example 18. PALNPs prevent unwanted systemic delivery of mRNA via intramuscular administration

[0529] The intramuscular route of administration is used for the injection of COVID-19 mRNA vaccines. Although the targeted distribution organ of mRNA is the muscle cells at the injection site, the actual distribution in the human body is currently unknown. Various adverse reactions include liver damage caused by COVID-19 mRNA vaccines. In addition, renal delivery of mRNA vaccines has been attributed to life-threatening myocarditis. Detailed information can be found, for example, in Mann, R. et al. "Drug-induced liver injury after COVID-19 vaccine." Cureus 13.7 (2021); and Cadegiani, FA, "Catecholamines Are the Key Trigger of COVID-19 mRNA Vaccine-Induced Myocarditis: A Compelling Hypothesis Supported by Epidemiological, Anatomical Pathology, Molecular, and Physiological Findings." Cureus 14.8 (2022). In fact, it is very common that after injection of mRNA LNP, a large amount of mRNA expression is observed in the liver and other organs in addition to the local injection site. Preventing intramuscularly administered mRNA from entering systemic delivery is clinically critical and technically challenging. Surprisingly, CPT147LNP provides a solution to this challenging problem.

[0530] Fig.24 Results are shown for BALB / c mice administered PALNP CPT147 by intramuscular injection. Specifically, mice were injected with 10 μg luc mRNA encapsulated in CPT147. Strong protein expression was detected only from the injection site, and no bioluminescence was observed from the supine position. It is thought that the reason for the limited local delivery under ideal conditions is that the LNP is rapidly taken up by muscle cells at the injection site in a manner similar to the rapid cellular uptake clearly observed in the above in vitro studies, which makes it almost impossible to deliver systemically.

[0531] Example 19. Preparation of mRNA / DNA lipid nanoparticles

[0532] Currently, LNP formulations for pharmaceutical research and commercial products use T-type or Y-type mixing setups, such as Fig.25 As shown. In this process, a solution of lipids in ethanol can be fed into a T-joint from one arm of a T-mixer by a pump, and an mRNA aqueous solution can be fed into the opposite arm of the T-mixer by another pump. The volume ratio of aqueous stream to organic stream is in the range of 3-10. The mixed solution can flow out of the T-joint from the T-mixer through a channel, and LNPs are formed in the mixed solution by self-assembly. The quality of LNP depends on the sufficient degree of mixing of the two solutions. For example, when the assembly process occurs in a homogeneous solution, nanoparticles with homogeneous particle size and morphology can be formed. However, when the assembly process occurs in a partially mixed solution, metastable LNPs with heterogeneous composition, particle size and morphology can be formed. The foaming problem of LNPs prepared by a T-mixer, which is usually observed, may be associated with feed imbalance, which may make the lipid solution and the aqueous solution insufficiently mixed during the T-mixing process.

[0533] All LNPs described herein are manufactured using nPort disclosed in U.S. Pat. No. 11,497,715 B2 and U.S. Pat. No. 9,693,958 B2. TM The invention discloses a method for preparing LNPs by using a LNP self-assembly method. In the process, a stainless steel mixing device with 5-6 ports is used to mix lipid / ethanol solution with an aqueous solution in the presence or absence of mRNA / DNA. An organic stream is pumped into a mixing chamber through an inlet port, and the aqueous solution is uniformly fed through multiple inlet ports. The flowing stream is quickly and uniformly mixed in the mixing chamber with a time scale of about sub-milliseconds. The mixed solution flows out through the outlet port and is collected. LNP self-assembly is carried out in the solution. The size of the inlet, outlet, mixing chamber and the flow rate of the solution are all reasonably designed to ensure that the solution is fully mixed under the preparations of various scales. In some preparations, two or more aqueous solutions are used to meet the chemical and physicochemical characteristics of LNP components. Residual ethanol is removed from the product, and buffer exchange is carried out by dialysis (for samples with small volumes) or by tangential flow filtration (TFF). In a fume hood, ethanol is also removed from some small samples by vacuum.

[0534] When mRNA / DNA is included in aqueous buffer, the LNP obtained by the above method is complete LNP, and when residual ethanol is removed from the mixture by evaporation or dialysis or tangential flow filtration, LNP is complete. When mRNA / DNA is not included in aqueous buffer, the LNP obtained by the above method is blank LNP. Blank LNP suspension and mRNA solution are mixed to prepare the LNP of load mRNA by vortex or stirring. The exemplary method for preparing LNP CPT163-012 is as follows.

[0535] Preparation of CPT163-012

[0536] 1) Lipid / ethanol solution: lipid / dehydratase ethanol solution containing 6.00 mg ALC-0315, 6.75 mg DSPC, 6.41 mg DOPE, 5.76 mg cholesterol.2 15 mg L81 and 1.48 mg PEG2000-DSPE.

[0537] 2) Aqueous buffer: 5 mM citrate, 75 mM NaCl, pH

[0538] 3) Mixing process. 1mL lipid / ethanol solution is loaded into a 1ml syringe driven by a syringe pump; and 4.4mL citrate buffer is evenly distributed into 3 syringes driven by another syringe pump. The syringe is connected to the inlet port of the 5-port device. The port size (diameter) and the mixing chamber diameter are 0.25mm; each syringe is connected to the inlet port by a tube. The diameter and length of the outlet tube installed to the outlet port are 0.17mm and 60mm respectively. The lipid and aqueous buffer loaded in the syringe are pumped into the mixing chamber by a syringe pump at a total flow rate of 8 ml / min. The LNP solution is discharged through the outlet tube and collected in a glass vial. After removing residual ethanol from the solution by vacuum, blank LNP (without mRNA / DNA) is obtained. The lipid concentration is determined by HPLC.

[0539] 4) mRNA loading. The final mRNA LNP is obtained by simple mixing of the mRNA solution with the blank LNP. For example, 20.5 μl of black LNP containing 55.4 μg lipid (including L81 and 11.71 μg ALC-0315) is placed in a 0.5 mL Eppendorf tube, and 5 μl of mRNA solution containing 5 μg green fluorescent protein mRNA is added by pipetting, followed by gentle vortexing. The mixture is then incubated at 37°C for 10 minutes. The final mRNA LNP is obtained. The N / P ratio of the preparation is 1. The w / w ratio of total lipid:mRNA is 11:1, and the w / w ratio of ionizable ALC-0315 to mRNA is 2.322:1. Similarly, LNPs with N / P = 0.5 can be prepared by mixing 20.5 μl of black LNPs with 10 μg of mRNA; and LNPs with N / P = 2 can be prepared by mixing 20.5 μl of black LNPs with 2.5 μg of mRNA.

[0540] 5) Characterization of LNP. The encapsulation percentage of mRNA / DNA was measured by the Ribogreen method. Detailed information can be found, for example, in the following literature: Naderi SA et al. "Development of an mrna-lnp vaccine against sars-Cov-2: Evaluation of immune response in mouse and rhesus macaque." Vaccines 9.9 (2021): 1007. The encapsulation percentage was in the range of 75-95%. The particle size of blank LNP and mRNA-loaded LNP was determined by dynamic light scattering method (NanoZetasizer, Malvern). Fig.26 The particle size distribution of CPT163-12 is shown in Figure 3. The particle size and polydispersity of blank LNP are 128.3nm and 0.220, and the particle size and polydispersity of mRNA-loaded LNP are 180.1nm and 0.125. The significant change in particle size between blank LNP and mRNA-loaded LNP and the high homogeneity of mRNA-loaded LNP indicate the reorganization of LNP after blank LNP is mixed with mRNA, rather than the disordered aggregation of LNP. In addition, excellent mRNA encapsulation percentage and high mRNA transfection efficiency prove that mRNA is encapsulated in the reorganized LNP interior rather than being adsorbed to the surface of blank LNP.

[0541] 6) Direct mixing of mRNA with lipid / ethanol solution. LNPs loaded with mRNA can be prepared by directly mixing lipid / ethanol solution with aqueous solution of mRNA. For example, 0.5 mL of lipid / ethanol solution can be loaded into a 1 mL gas-tight glass syringe containing the following: 0.5 mg ALC-0315 ionizable lipid, 0.563 mg DSPC, 0.533 mg DOPE, 0.480 mg cholesterol, 0.179 mg L81 and 0.124 mg PEG2000-DSPE. 2.2 mL of mRNA solution (5 mM citrate, 75 mM NaCl, pH 5.5) containing 0.215 mg mRNA is evenly distributed into 3 syringes driven by another syringe pump. The syringe is connected to the inlet port of the 5-port device. The port size (diameter) and mixing chamber diameter are 0.25 mm; each syringe is connected to the inlet port by a tube. The diameter and length of the outlet tube installed to the outlet port are 0.17 mm and 60 mm, respectively. The lipid and aqueous buffer loaded in the syringe are pumped into the mixing chamber at a total flow rate of 8 ml / min by a syringe pump. The LNP solution is discharged through an outlet tube and collected in a glass vial. Residual ethanol is removed from the solution by vacuum. The N / P ratio of LNP is 1. The w / w ratio of total lipid:mRNA is 11:1. The particle diameter is 164.7nm, and the polydispersity is 0.075. Fig.26 The particle size distribution curve is shown in .

[0542] Example 20. LNPs with polymer propylene glycol (PPO)

[0543] The copolymer used to produce PALNPs was also replaced with polypropylene glycol (PPO). An exemplary structure of polyPPO is shown below

[0544]

[0545] Specifically, the average molecular weight of poly PPO (x=46.5) is about 2700 Daltons (PPO 2700 CPT201 LNPs were produced using the methods described herein and the formulations are listed in the table below.

[0546] Table 13: Formulation of LNP CPT201

[0547] ALC-0315 DSPC DOPE cholesterol PEG2000-DSPE <![CDATA[PPO 2700 ]]> N / P CPT201 22.5 15.7 15.7 42.7 1.1 2.2 1

[0548] HEK293 cells were transfected with CPT201LNPs loaded with 0.0125-0.2 μg GFP mRNA, and fluorescence images were recorded after 24 hours. Fig. 27As shown, all transfected cells emitted bright green fluorescence signals of GFP, indicating that poly-PPO can be used to produce PALNPs.

[0549] Other embodiments

[0550] It should be understood that although the invention has been described in conjunction with the detailed description of the invention, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

Claims

1. A nanoparticle composition comprising lipid components, and Polymer components, Wherein the polymer component comprises: (a) Formula I (PEO x -PPO y -PEO z ) wherein x is a number selected from 1-100, y is a number selected from 10-500, and z is a number selected from 1-100; (b) Formula II (PPO x -PEO y -PPO z ) wherein x is a number selected from 10-500, y is a number selected from 1-100, and z is a number selected from 10-500; (c) Formula III (PEO y -PPO z ) wherein R1 is H or CH3, R2 is OH or OCH3, y is a number selected from 1-100, and z is a number selected from 5-500; and / or (d) Formula IV (PPO x ) Where x is a number selected from 1-100.

2. The nanoparticle composition of claim 1, wherein the polymer component comprises about 0.1 mol% to about 20 mol% of the nanoparticle composition.

3. The nanoparticle composition according to claim 1 or 2, wherein the lipid component comprises ionizable and / or permanently charged cationic lipids, Helper lipids, Structured lipids, and / or PEG (polyethylene glycol) lipids.

4. The nanoparticle composition of claim 3, wherein the ionizable and / or permanently charged cationic lipid comprises about 5 mol% to about 30 mol% of the nanoparticle composition.

5. The nanoparticle composition of claim 3 or 4, wherein the helper lipid is a phospholipid.

6. The nanoparticle composition of any one of claims 3 to 5, wherein the helper lipid comprises about 10 mol% to about 50 mol% of the nanoparticle composition.

7. The nanoparticle composition of any one of claims 3 to 6, wherein the structural lipid is cholesterol.

8. The nanoparticle composition of any one of claims 3 to 7, wherein the structural lipids comprise about 20 mol% to about 50 mol% of the nanoparticle composition.

9. The nanoparticle composition according to any one of claims 3 to 8, wherein the average The molecular weight is about 500-5000 Daltons (eg, about 2000 Daltons).

10. The nanoparticle composition of any one of claims 3 to 9, wherein the PEG lipid comprises about 0.5 mol% to about 5 mol% of the nanoparticle composition.

11. The nanoparticle composition according to any one of claims 1 to 10, comprising: (a) about 1 mol % to about 20 mol % of the compound of formula I; (b) about 5 mol % to about 30 mol % of said ionizable and / or permanently charged cationic lipid (e.g., ALC-0315, SM-102, DLin-DMA, DLin-MC3-DMA and / or DLin-KC2-DMA); (c) about 10 mol % to about 50 mol % of the helper lipid (e.g., DSPC and / or DOPE); (d) about 20 mol % to about 50 mol % of said structural lipid (e.g., cholesterol); and (e) about 0.5 mol % to about 5 mol % of the PEG lipid (e.g., PEG2000-DSPE).

12. The nanoparticle composition of claim 11, wherein the number x and the number z in Formula I are the same.

13. The nanoparticle composition of claim 11 or 12, wherein the helper lipid comprises about 5 mol% to about 30 mol% DSPC and / or about 5 mol% to about 30 mol% DOPE.

14. The nanoparticle composition of any one of claims 11 to 13, wherein the compound of Formula I has an average molecular weight of about 1000 Daltons to about 30,000 Daltons (e.g., about 1000 Daltons to about 10,000 Daltons).

15. The nanoparticle composition of any one of claims 11 to 14, wherein the number x is selected from 1-15, the number y is selected from 30-80, and the number z is selected from 1-15.

16. The nanoparticle composition of any one of claims 11 to 15, wherein the compound of formula I is L121, L92 or L81.

17. The nanoparticle composition according to any one of claims 1 to 10, comprising: (a) about 1 mol % to about 20 mol % of the compound of formula II; (b) about 5 mol % to about 30 mol % of said ionizable and / or permanently charged cationic lipid (e.g., ALC-0315, SM-102, DLin-DMA, DLin-MC3-DMA and / or DLin-KC2-DMA); (c) about 10 mol % to about 50 mol % of the helper lipid (e.g., DSPC and / or DOPE); (d) about 20 mol % to about 50 mol % of said structural lipid (e.g., cholesterol); and (e) about 0.5 mol % to about 5 mol % of the PEG lipid (e.g., PEG2000-DSPE).

18. The nanoparticle composition of claim 17, wherein the number x and the number z in Formula II are the same.

19. The nanoparticle composition of claim 17 or 18, wherein the helper lipid comprises about 5 mol% to about 30 mol% DSPC and / or about 5 mol% to about 30 mol% DOPE.

20. The nanoparticle composition of any one of claims 17 to 19, wherein the compound of Formula II has an average molecular weight of about 1000 Daltons to about 30,000 Daltons (e.g., about 1000 Daltons to about 10,000 Daltons).

21. The nanoparticle composition of any one of claims 17 to 20, wherein the number x is selected from 10-50, the number y is selected from 1-30, and the number z is selected from 10-50.

22. The nanoparticle composition of any one of claims 17 to 21, wherein the compound of Formula II is L31R1 or L17R4.

23. The nanoparticle composition according to any one of claims 1 to 10, comprising: (a) about 1 mol % to about 20 mol % of the compound of formula IV; (b) about 5 mol % to about 30 mol % of said ionizable and / or permanently charged cationic lipid (e.g., ALC-0315, SM-102, DLin-DMA, DLin-MC3-DMA and / or DLin-KC2-DMA); (c) about 10 mol % to about 50 mol % of the helper lipid (e.g., DSPC and / or DOPE); (d) about 20 mol % to about 50 mol % of said structural lipid (e.g., cholesterol); and (e) about 0.5 mol % to about 5 mol % of the PEG lipid (e.g., PEG2000-DSPE).

24. The nanoparticle composition of claim 23, wherein the helper lipid comprises about 5 mol% to about 30 mol% DSPC and / or about 5 mol% to about 30 mol% DOPE.

25. The nanoparticle composition of claim 23 or 24, wherein the compound of Formula IV has an average molecular weight of about 1000 Daltons to about 30,000 Daltons (e.g., about 1000 Daltons to about 10,000 Daltons).

26. The nanoparticle composition of any one of claims 23 to 25, wherein the number x in the compound of formula IV is from about 30 to about 60.

27. The nanoparticle composition of any one of claims 23 to 26, wherein the compound of formula IV is PPO 2700 .

28. A lipid nanoparticle (LNP) having a nanoparticle composition according to any one of claims 1 to 27.

29. The LNP of claim 28, further comprising a nucleic acid, wherein the nucleic acid comprises DNA (e.g., double-stranded DNA (dsDNA), plasmid DNA, single-stranded DNA (ssDNA), or antisense DNA thereof) or RNA (e.g., small interfering RNA (siRNA), micro RNA (miRNA), messenger mRNA (mRNA), guide RNA (gRNA), circular RNA (circRNA), self-amplifying RNA (saRNA), or antisense RNA thereof).

30. The LNP of claim 28 or 29, wherein the nanoparticle composition has an N / P ratio of about 0.1 to about 10 (e.g., about 0.1 to about 5, about 0.1 to about 4, about 0.1 to about 3, about 0.2 to about 2, or about 0.2 to about 1.5).

31. The LNP of any one of claims 28 to 30, wherein the compound of Formula I, Formula II, Formula III, Formula IV and / or Formula V has a hydrophile-lipophile balance (HLB) value of 1 to 18.

32. The LNP of any one of claims 28 to 31, wherein the average size of the LNP is from about 30 nm to about 2000 nm (e.g., from about 30 nm to about 1000 nm or from about 30 nm to about 500 nm).

33. The LNP of any one of claims 28 to 32, wherein the LNP has a polydispersity index of about 0.001 to about 0.5 (e.g., about 0.01 to about 0.3).

34. The LNP of any one of claims 28 to 33, wherein the LNP has a zeta potential of about -30 mV to about +20 mV.

35. The LNP of any one of claims 29 to 34, wherein the w / w ratio of the lipid component to the nucleic acid is from about 2:1 to about 50:1 (e.g., from about 2:1 to about 20:1).

36. The LNP of any one of claims 29 to 35, wherein the nucleic acid has an encapsulation efficiency of at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

37. A method of delivering a nucleic acid to a mammalian cell, wherein the method comprises administering to a subject the LNP of any one of claims 28 to 36, wherein the administration comprises contacting the mammalian cell with the nanoparticle composition, thereby delivering the nucleic acid to the mammalian cell.

38. The method of claim 37, wherein the mammalian cell is located in a mammal.

39. The method of claim 37 or 38, wherein the LNP is associated with a therapeutic drug, a vaccine (e.g., a prophylactic vaccine or a therapeutic vaccine), gene editing, or a cell-based therapy (e.g., a chimeric antigen receptor (CAR)-T therapy).

40. The method of claim 37 or 38, wherein the LNP is associated with the treatment of a disease (eg, an infectious disease, an autoimmune disease, cancer, or a genetic disorder).

41. The method of any one of claims 37 to 40, wherein the LNP is delivered by oral administration, nasal administration, cutaneous administration, intravenous administration, topical administration, ocular administration and / or mucosal administration, intradermal administration, and intramuscular administration.

42. A method for enhancing the delivery of a nucleic acid to a target tissue, wherein the method comprises Administering the LNP of any one of claims 28 to 36 to a subject, wherein the administering comprises contacting the target tissue with the LNP, thereby delivering the nucleic acid to the target tissue.

43. A method for producing a polypeptide of interest in a mammalian cell, the method comprising The LNP of any one of claims 29 to 36 is administered to a subject, wherein the nucleic acid encodes the polypeptide of interest, whereby the nucleic acid can be translated in the mammalian cell to produce the polypeptide of interest.

44. A method for preparing LNPs having a nanoparticle composition, the nanoparticle composition comprising a lipid component and a polymer component, wherein the lipid component comprises: ionizable and / or permanently charged cationic lipids, helper lipids, structural lipids and PEG (polyethylene glycol) lipids, wherein the polymer component comprises: a compound of Formula I, Formula II, Formula III and / or Formula IV, The method comprises: (a) introducing one or more streams of a solution of lipids in a water-miscible organic solvent through a first set of one or more inlet ports connected to a mixing chamber, wherein the lipid solution comprises the lipid component and the polymer component, (b) introducing one or more streams of an aqueous solution through a second set of one or more inlet ports connected to the mixing chamber, (c) mixing the one or more streams of the lipid solution and the one or more streams of the aqueous solution in a mixing chamber to produce the LNPs, and (d) recovering the LNPs via one or more outlet ports connected to the mixing chamber.

45. The method of claim 44, wherein the aqueous solution comprises nucleic acids.

46. ​​The method of claim 44 or 45, wherein the angle between any inlet port in the first set of one or more inlet ports and any inlet port in the second set of one or more inlet ports is 0-180 degrees.

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