Lipid-polymer compounds, compositions and uses thereof

Abstract

Described herein are compounds comprising lipids attached to a polymer backbone having functional groups. Also described herein are uses of such agents for delivering nucleic acids to cells.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 379,031, filed October 11, 2022, which is incorporated herein by reference in its entirety. Background Art

[0003] The control of life processes can be mediated by nucleic acids. Nucleic acids encode proteins that act as enzymes, hormones, and other regulatory factors, carrying out the processes that enable an organism to function. Nucleic acids also encode regulatory sequences that control protein expression. Because of their central role in organisms, nucleic acids are ideal therapeutic targets.

[0004] A factor that can limit the therapy based on nucleic acid is the ability of nucleic acid to be delivered to the appropriate compartment of the cell. Nucleic acid can be a fragile molecule, which can be highly negatively charged (one negative charge per phosphate group), and can be easily cleaved by nucleases present in both the extracellular fluid and the intracellular compartment. Although some attempts to encapsulate or otherwise stabilize nucleic acids with proteins, peptides, polymers, lipids, liposomes and lipid nanoparticles have shown some prospects, it is still necessary to determine a safe, non-toxic method to stabilize the nucleic acid molecules in the biological system, for example, to improve the therapeutic efficacy of this therapy. A strategy for achieving this goal also has development lipid-polymer conjugates, which can, for example, interact with nucleic acid molecules (such as stabilization), form liposomes or lipid nanoparticles, and / or penetrate into lipid membranes, for example, as a reporter or functional handle in the cell membrane. The other compositions and methods related to lipid-polymer compounds are described herein. Summary of the Invention

[0005] The present disclosure provides lipid-polymer compounds (or, "lipid-polymer conjugates") that can be used in various applications, such as delivering biologically active compounds (e.g., nucleic acid molecules) to cells in biological systems, for example, in in vitro cell transfection studies. The lipid-polymer conjugates can comprise one or more lipid compounds conjugated to a polymer backbone. The present disclosure also provides methods for preparing such compounds, which can have various applications, such as treating disease or for gene therapy applications.

[0006] In one aspect, described herein are compounds comprising a lipid; and a stimuli-responsive unit. Further examples of lipid and stimuli-responsive unit elements are described below.

[0007] In one aspect, described herein are compounds comprising a lipid; a linker comprising a stimulus responsive unit; and a polymer, wherein the linker connects the lipid to the backbone of the polymer. In some embodiments, the polymer may comprise, for example, at least 3 monomeric units, wherein at least 3 monomeric units comprise C 1-20In some embodiments, the stimuli-responsive unit is a temperature-responsive unit, a pH-responsive unit, a light-responsive unit, or a chemical-responsive unit. In some embodiments, the temperature-responsive unit comprises a lower crystallization solution temperature (LCST) of about 27°C to about 35°C. In some embodiments, the temperature-responsive unit comprises a lower crystallization solution temperature (LCST) of about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, or about 35°C. In some embodiments, the temperature-responsive unit comprises poly(N-isopropylacrylamide), poly(N-n-propylacrylamide), poly(N-methyl-N-n-propylacrylamide), poly(N,N-diethylacrylamide), poly(N-isobutylacrylamide), poly(N-sec-butylacrylamide), poly(N-n-butylacrylamide), poly(N-isobutylacrylamide), hydroxypropylcellulose, poly(N-vinylcaprolactam), poly-2-isopropyl-2-oxazoline, or polyvinyl methyl ether, or a combination thereof. In some embodiments, the temperature-responsive unit comprises 2-250 monomer units. More examples of lipids and polymers are described below.

[0008] In one aspect, described herein are compounds comprising a lipid attached to a polymer backbone. The polymer may comprise, for example, at least 3 monomeric units, wherein at least 3 monomeric units comprise C 1-20 Heteroalkyl side chains. In some cases, the polymer comprises 4 or more (e.g., 10 or more, 50 or more) monomeric units. The polymer may comprise about 400 or less (e.g., about 300 or less) monomeric units. In some cases, the polymer comprises about 10-about 200 monomeric units. In certain instances, the polymer comprises about 50-about 150 monomeric units. The polymer may comprise polyacrylate or polyacrylamide. The polymer may comprise one or more acrylate side chains and / or one or more acrylamide side chains. In some embodiments, each monomer comprises acrylamide or acrylate. The polymer may be a peptide or a non-peptide. As used herein, the polymer is typically a non-peptide. For example, the polymer may not contain amino acids. In some embodiments, each monomeric unit is not an amino acid. The polymer may comprise a copolymer. For example, the polymer may comprise a block copolymer. In some cases, the block copolymer comprises a cationic or cationic monomeric unit. The block copolymer may be a random block copolymer. In some cases, the polymer is positively charged in a neutral aqueous solution. The polymer may comprise a pK of about 2 to about 12 (e.g., about 4 to about 11). b Further examples of lipids and polymers are described below.

[0009] In another aspect, provided herein is a compound according to Formula I or a pharmaceutically acceptable salt thereof:

[0010] X—Y—Z

[0011] Formula I;

[0012] in:

[0013] X is a lipid;

[0014] Y is a polymer comprising 3 or more monomer units, wherein each monomer unit comprises C 1-20 heteroalkyl side chains; and

[0015] Z is an unsubstituted or substituted functional group;

[0016] The lipids are covalently bonded to the polymer via the polymer backbone.

[0017] In some embodiments, the lipid comprises a steroid or a fatty acid. In some embodiments, the steroid comprises a sterol or a stanol. In some embodiments, the steroid comprises a sterol. In some embodiments, the sterol comprises cholesterol. In some embodiments, the fatty acid comprises a saturated fatty acid, a monounsaturated fatty acid, a polyunsaturated fatty acid, or a combination thereof. In some embodiments, the fatty acid comprises oleic acid or an ester thereof. In some embodiments, the lipid is hydrophobic. In some embodiments, the lipid is amphiphilic. In some embodiments, the lipid has an octanol:water coefficient (log(K)) of about 2 or greater. OW In some embodiments, provided herein is a compound (eg, a compound of Formula I), wherein lipid (X) has a structure of Formula XA, Formula XB, or Formula XC:

[0018]

[0019]

[0020] In some embodiments, provided herein is a compound (e.g., of Formula I), wherein polymer (Y) has the structure of Formula YA, Formula YB, Formula YC, or Formula YD, or a pharmaceutically acceptable salt thereof:

[0021]

[0022] in:

[0023] A 1 、B 1 、C 1 and D 1 Each of is independently hydrogen or methyl;

[0024] A 2 、B 2 、C 2 and D 2Each of which is independently unsubstituted or substituted C 1-20 heteroalkyl;

[0025] Each of a, b, c, d, e and f is independently an integer from 0 to 200, provided that the total number of monomer units is 3 or more;

[0026] Each substituted C 1-20 The heteroalkyl group is independently substituted with a ring that is unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclyl, unsubstituted or substituted aryl, or unsubstituted or substituted heteroaryl.

[0027] In some embodiments, the polymer comprises from about 4 to about 400 monomer units. In some embodiments, the polymer comprises from about 10 to about 200 monomer units. In some embodiments, the polymer comprises from about 50 to about 150 monomer units. In some embodiments, the polymer comprises a polyacrylate or a polyacrylamide. In some embodiments, A 2 、B 2 、C 2 and D 2 Each of the monomer units independently comprises an acrylate or an acrylamide. In some embodiments, each of the monomer units has a structure of the following formula or one of its pharmaceutically acceptable salts:

[0028]

[0029] in:

[0030] R 1 and R 3 Each of is independently hydrogen or methyl;

[0031] Each R 2 For hydrogen, C 1-6 Alkyl, C 7-20 Aralkyl, C 1-20 Heteroalkyl, or polyethylene glycol chain containing 1-100 ethylene glycol monomers; wherein each C 1-6 Alkyl, C 7-20 Aralkyl and C 1-20 Each of the heteroalkyl groups is unsubstituted or substituted with one or more groups, wherein each of the one or more groups is independently -COOH, -CONH2, -NH2, -NH3 + 、-NHC(NH2 + )NH2, -NHCH3, -N(CH3)2, -N(CH3)3 + , -OH, -OCH3, -SH, -S(O)CH3, -S(O)2CH3 or -S(O)2OH;

[0032] R4 and R 5 Each of which is independently hydrogen, C 1-6 Alkyl, C 7-20 Aralkyl or C 1-20 heteroalkyl; wherein each C 1-6 Alkyl, C 7-20 Aralkyl and C 1-20 Heteroalkyl is unsubstituted or substituted with one or more groups, wherein each of the one or more groups is independently -COOH, -CONH2, -NH2, -NH3 + 、-NHC(NH2 + )NH2, -NHCH3, -N(CH3)2, -N(CH3)3 + , -OH, -OCH3, -SH, -S(O)CH3, -S(O)2CH3 or -S(O)2OH; or a pharmaceutically acceptable salt thereof.

[0033] In some embodiments, each of the monomeric units independently comprises:

[0034]

[0035] In some embodiments, the functional group is a thiol or a sulfide. In some embodiments, the functional group is a thiol. In some embodiments, the functional group is a sulfide. In some embodiments, the sulfide is SR 6 , and where R 6 is a group consisting of 1 to about 200 atoms selected from hydrogen, halogen, C, N, O, and S. In some embodiments, the sulfide is SR 6 , and where R 6 In some embodiments, the functional group comprises a reactive group, a charged group, a detectable group, a peptide group, a capping group, or a combination thereof. In some embodiments, the reactive group comprises an azide or an alkyne. In some embodiments, the charged group comprises one or more cationic groups. In some embodiments, the one or more cationic groups comprise a cyclic amine, a primary amine, a guanidine, or a combination thereof. In some embodiments, the detectable group comprises a fluorophore, a dye, a FRET donor, or an acceptor. In some embodiments, the capping group is an inert group. In some embodiments, the functional group is selected from:

[0036] -SH,

[0037] The functional group is bonded to the polymer via the sulfur atom.

[0038] In some embodiments, the compound is configured to encapsulate nucleic acid or compound with nucleic acid in aqueous solution. In some embodiments, the compound is substantially non-toxic. In some embodiments, the compound is biodegradable. In some embodiments, the compound comprises a molecular weight of about 1 kilodalton (kDa)-about 100kDa.

[0039] In another aspect, described herein are nanoparticles comprising a compound disclosed herein or PLip, wherein the nanoparticles are configured for encapsulation or compounding of nucleic acids. In some embodiments, the nanoparticles are configured for encapsulation or compounding of nucleic acids at a ratio of 0.3:1-100:1 (weight:weight or w / w). In some embodiments, the encapsulation or compounding of nucleic acids increases the half-life of the nucleic acids by at least 2-fold under aqueous or physiological conditions. In some embodiments, nuclease digestion of nucleic acids is inhibited by the encapsulation or compounding. In some embodiments, the encapsulation or compounding of nucleic acids produces a transfection reagent having an average size of about 20 nm to about 2000 nm. In some embodiments, the compounding comprises adsorption of at least a subset of nucleic acids to the surface of the nanoparticles. In some embodiments, the encapsulation or compounding of nucleic acids produces a transfection reagent configured for cellular uptake. In some embodiments, cellular uptake comprises endocytosis.

[0040] In another aspect, provided herein is a transfection reagent comprising nanoparticles described herein (e.g., compounds disclosed herein) having genetic information (e.g., one or more nucleic acid molecules) encapsulated therein. In some embodiments, the nucleic acid comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), or any combination thereof. In some embodiments, the nucleic acid comprises about 1 kilobase pair (kb)-about 100kb. In some embodiments, the nucleic acid comprises about 2kb-about 20kb. In some embodiments, the nucleic acid comprises about 5kb-about 15kb. In some embodiments, the nucleic acid comprises about 8kb-about 12kb. In some embodiments, the nucleic acid comprises about 10kb. In some embodiments, provided herein is a transfection reagent comprising a water solubility of at least 5μg / mL. In some embodiments, provided herein is a transfection reagent comprising a water solubility of about 5μg-about 5mg / mL. In some embodiments, provided herein is a transfection reagent comprising a water solubility of about 10μg / mL-about 50μg / mL.

[0041] In another aspect, provided herein is a method for transfecting cells, the method comprising: (a) providing a transfection reagent comprising a compound disclosed herein and a nucleic acid, and (b) contacting the cell with the transfection reagent, wherein the contact is carried out under conditions suitable for the nucleic acid to enter the cell. In some embodiments, step (a) includes contacting the compound with the nucleic acid under conditions sufficient to form a transfection complex. In some embodiments, the conditions sufficient to form a transfection complex include conditions sufficient to perform an ion gel method. In some embodiments, the nucleic acid includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), locked nucleic acid (LNA), peptide nucleic acid (PNA) or any combination thereof. In some embodiments, the transfection complex includes a positive charge under conditions suitable for the nucleic acid to enter the cell. In some embodiments, the contact is less than 24 hours. In some embodiments, the cell includes an animal cell, a plant cell, a fungal cell, a bacterial cell or any combination thereof.

[0042] In another aspect, provided herein is a pharmaceutical composition comprising nanoparticles disclosed herein (e.g., comprising compounds disclosed herein (e.g., Formula I)) and bioactive molecules. In some embodiments, the nanoparticles are lipid nanoparticles. In some embodiments, the nanoparticles are covalently bonded to the bioactive molecules. In some embodiments, the nanoparticles are ionically bonded to the bioactive molecules. In some embodiments, the nanoparticles encapsulate the bioactive molecules. In some embodiments, the bioactive molecules include nucleic acid molecules. In some embodiments, the nucleic acid molecules include RNA or DNA. In some embodiments, the nucleic acid molecules include mRNA, siRNA, or tRNA. In some embodiments, the bioactive molecules include therapeutic agents. In some embodiments, the therapeutic agent is a chemotherapeutic agent, a radiotherapeutic agent, an oligonucleotide, or an oligopeptide. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0043] In yet another aspect, provided herein is a method of treating a condition or disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a transfection reagent or pharmaceutical composition disclosed herein. In some embodiments, the transfection reagent or pharmaceutical composition is administered to the subject by injection.

[0044] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be appreciated, the present disclosure is capable of other and different embodiments, and its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not restrictive.

[0045] Incorporate by reference

[0046] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. If a publication, patent, or patent application incorporated by reference contradicts the disclosure contained in this specification, the specification is intended to supersede and / or take precedence over any such contradictory material. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The novel features of the present invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention may be obtained by reference to the following detailed description which sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "Figures" and "FIG."), in which:

[0048] Figure 1 The CPCPA-diol of Example S1 is shown 1 H NMR spectroscopy.

[0049] Figure 2 The CPCPA-dilinoleoyl RAFT agent of Example S2 is shown 1 H NMR spectroscopy.

[0050] Figure 3 The CPCPA-cholesterol RAFT agent of Example S3 is shown 1 H NMR spectroscopy.

[0051] Figure 4 The cationic diacyl PLip of Example S8(a) is shown 1 H NMR spectroscopy.

[0052] Figure 5 The P(HPMA)PLip of Example S9 is shown 1 H NMR spectroscopy.

[0053] Figure 6 Shown are the DNA encapsulation percentages of lipid nanoparticles formulated with the cationic PLip of Example B1(a).

[0054] Figure 7 Shown is the effect on LNP binding resulting from incorporation of 0.5 mol% PLip 7 into the LNP formulation of Example B2(b).

[0055] Figure 8 Dynamic light scattering intensity distributions of LNPs containing PLip 6 and 8 are shown.

[0056] Figure 9Anti-Spike protein IgG in mice at day 14 and day 35 after LNP-encapsulated mRNA delivery is shown, where each bar represents an individual mouse.

[0057] Figure 10 Shown is the dose response priming of Jurkat cells with PLip 12 insertion.

[0058] Figure 11 Shown are fluorescence measurements at different temperatures after the fluorescent PLip 13 was inserted into the cytoplasmic membrane of 293F cells.

[0059] Figure 12 Shown are the radius and normalized fluorescence of LNPs 14-19 after mixing with azidefluor-488.

[0060] Figure 13 Shown is a comparison of 293F cells transfected with cationic PLip 20-22 alone.

[0061] Figure 14 Shown in the presence and absence of cationic PLip 20-22 by Comparison with POLY1-transfected 293F cells.

[0062] Figure 15 Shown are the effects of temperature cycling on the LNP radius for two LNPs containing 0.6 and 1.2 mol% of temperature-sensitive PLip 23 and one control LNP without PLip 23.

[0063] Figure 16 Shown are the effects of temperature cycling on normalized intensity data (number of particles) from dynamic light scattering analysis.

[0064] Figure 17A Shown are the percentage of intact capsids (following transfection) over time in the presence or absence of stabilized PLip when complex formation was assessed. Figure 17B Shown are genome counts over time (after transfection) in the presence or absence of stabilized PLip when assessing complex formation.

[0065] Figure 18A Shown are the concentrations of complex formation in the absence of stabilized PLip measured by dynamic light scattering (DLS). Figure 18B Shown are the concentrations of complex formation in the presence of stabilized PLip measured by DLS.

[0066] Figure 19AShown are the concentrations of complex formation over time in the presence of stabilized PLip as measured by genome titer and percentage of intact capsids. Figure 19B Another example of the concentration of complex formation over time as measured by genome titer and percentage of intact capsids is shown in the presence of stabilized PLip.

[0067] The novel features of the present disclosure are set forth with particularity in the appended claims.A better understanding of the features and advantages of the present disclosure may be obtained by reference to the following detailed description which sets forth illustrative embodiments. Summary of the Invention

[0068] The control of life processes is mediated by nucleic acids. Nucleic acids encode proteins, which act as enzymes, hormones, and other regulatory factors and carry out the processes that enable an organism to function. Nucleic acids also encode regulatory sequences that control protein expression. It is believed that many diseases can be controlled by manipulating nucleic acids in organisms.

[0069] One factor that limits the therapeutics based on nucleic acid manipulation is the ability to deliver nucleic acids to the appropriate compartments of the cell. Nucleic acids are fragile molecules that are highly negatively charged (one negative charge per phosphate group) and are easily cleaved by nucleases present in both the extracellular fluid and the intracellular compartment. As a highly charged molecule, it will not pass through the lipid membrane surrounding the cell, nor can it easily escape from the endosomal compartment involved in the uptake of macromolecules into the cell. Even RNA interference (RNAi) molecules, despite their small molecular weight, face significant problems in stability and uptake.

[0070] By using various vesicles, attempts have been made to effectively deliver bioactive compounds to the intracellular space of cells. Liposomes are microvesicles comprising amphiphilic molecules, which contain both hydrophobic and hydrophilic regions. In addition, bioactive compounds can be delivered to the intracellular space of cells through liposome-like structures (e.g., lipid nanoparticles (LNPs)). LNPs can effectively embed multiple bioactive compounds, such as nucleic acids (e.g., mRNA, microRNA, siRNA, etc.). Liposomes and LNPs can also be used for delivering multiple bioactive compounds, such as nucleic acids, to the intracellular space of cells. This paper describes the development, synthesis, and characterization of lipid-polymer compounds, as well as their various uses in the treatment and / or detection of biological systems.

[0071] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many changes, modifications, and substitutions may occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0072] The present invention relates to lipid-polymer compounds comprising one or more lipid compounds conjugated to a polymer backbone (or "lipid-polymer conjugates," or simply "compounds") that can be used to deliver nucleic acids to cells in biological systems, such as in in vitro cell transfection studies. The invention also relates to methods of preparing such compounds and, potentially, to gene therapy using such compounds.

[0073] The present invention also provides a compound that promotes the transfer of genetic material (e.g., nucleic acid molecules) into animal cells via a complex comprising a nucleic acid and a polymer containing an ionic or nonionic side chain moiety. The lipid-polymer compounds described herein can be used, for example, as nucleic acid transfection agents. In some cases, the lipid-polymer conjugates or lipid polymer compounds described herein are used in conjunction with endosomally soluble lipids to transfect nucleic acids into cells.

[0074] Some of the methods described herein can be used for the purpose of altering the expression of one or more genes in one or more cells.

[0075] In some cases, the present invention also provides compositions and compounds that can promote nucleic acid delivery to animal cells (or cells) in vitro and / or in vivo. The nucleic acid can comprise a double-stranded structure, and its nucleotide sequence is substantially identical to the target nucleic acid portion expressed in the cell. Further, the use of a lipid connected to a polymer backbone as provided herein can significantly improve nucleic acid transfer efficiency. The nucleic acid can then change the expression of a selected endogenous nucleic acid.

[0076] Lipids attached to polymer backbones as described herein can be used to facilitate transfection of DNA, RNA, mRNA or RNAi into cells. The nucleic acids can then alter the natural processes of the cell.

[0077] definition

[0078] The use of absolute or ordinal terms, such as "shall," "shall not," "should," "should not," "must not," "must not," "first," "initially," "next," "subsequently," "before," "after," "last," and "ultimately," are not meant to limit the scope of the present embodiments disclosed herein, but are intended to be exemplary.

[0079] As used herein, the singular forms "a," "an," and "the" are generally intended to include the plural forms as well, unless the context clearly indicates otherwise. In addition, to the extent that the terms "including," "includes," "having," "has," "with," or variations thereof are used in the detailed description and / or claims, these terms are intended to be inclusive in a manner similar to the term "comprising." Similarly, the terms "between," "from," and "to" when referring to a range are intended to include the entire range. For example, "between 0 and 5" is intended to include both 0 and 5, as well as integers (or non-integers, as appropriate) in between (e.g., 1-4). Similarly, the range "from 0 to 5" is intended to include 0, 1, 2, 3, 4, and 5.

[0080] Whenever the term "at least," "greater than," or "greater than or equal to" precedes the first value in a series of two or more values, the term "at least," "greater than," or "greater than or equal to" applies to each value in the series. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0081] Whenever the term "not more than," "less than," or "less than or equal to" precedes the first value in a series of two or more values, the term "not more than," "less than," or "less than or equal to" applies to every value in the series. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0082] As used herein, "or" can mean "and," "or," or "and / or," and can be used both singly and inclusively. For example, the term "A or B" can mean "A or B," "A but not B," "B but not A," and "A and B." In some cases, the context may dictate a specific meaning.

[0083] When referring to a number or a numerical range, the term "about" generally means that the number or numerical range referred to is an approximate value within the experimental variability (or within the statistical experimental error), and the number or numerical range may, for example, vary from 1% to 15% of the number or numerical range. As used herein, the term "about" generally refers to ±10% of the number or value. For smaller numbers (e.g., 1-10), the term "about" may refer to ±0.5 of the number. For example, if the value of a variable is "about 2," it should be understood that "about 2" refers to a range of 1.5-2.5 (inclusive). Similarly, if the value of a variable is "about 4.5," it should be understood that the term refers to a range from 4 to 5.

[0084] As used herein, C1-C x Can include C1-C2, C1-C3, ..., C1-C x By way of example only, a group designated as "C1-C4" indicates that there are 1 to 4 carbon atoms in the moiety, i.e., a group containing 1 carbon atom, 2 carbon atoms, 3 carbon atoms, or 4 carbon atoms. Thus, by way of example only, "C1-C4 alkyl" indicates that there are 1 to 4 carbon atoms in the alkyl group, i.e., the alkyl group is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0085] "Alkyl" generally refers to an aliphatic hydrocarbon group. An alkyl group may be branched or straight chain. An "alkyl" group may contain 1 to 10 carbon atoms, i.e., C1-C 10 Alkyl. Whenever it appears in this document, a numerical range such as "1-10" refers to each integer in the given range; for example, "1-10 carbon atoms" means that the alkyl group consists of 1 carbon atom, 2 carbon atoms, and 3 carbon atoms, etc., up to and including 10 carbon atoms, although this definition also covers the occurrence of the term "alkyl" in which no numerical range is specified. In some embodiments, the alkyl group is a C1-C6 alkyl group. In one aspect, the alkyl group is a methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl group. Typical alkyl groups include, but are by no means limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, or hexyl. The alkyl group can have any size. In some instances, the alkyl group has a carbon chain length of about 3 to about 36 carbons, wherein one or more (e.g., 1, 2, 3, 4, 5, or 6) bonds are double bonds. The alkyl group can be a fatty acid (e.g., C6-C6). 30 alkyl or alkenyl chain).

[0086] "Alkoxy" may refer to an (alkyl) O- group, wherein alkyl is as defined herein. In some embodiments, "alkoxy" refers to methoxy (-OCH3), ethoxy (-COH2CH3), and the like.

[0087] "Hydroxyalkyl" refers to an alkyl group in which at least one hydrogen atom is replaced by a hydroxy group. In some embodiments, the hydroxyalkyl group is a C1-C4 hydroxyalkyl group. Typical hydroxyalkyl groups include, but are not limited to, -CH2OH, -CH2CH2OH, -CH2CH2CH2OH, -CH2CH2CH2CH2OH, and the like.

[0088] "Aminoalkyl" may refer to a group in which at least one hydrogen atom is replaced by an amine (-NH2, -NHR, -NR2 or -NR3 +) substituted alkyl. In some embodiments, the aminoalkyl group is a C1-C6 aminoalkyl group. Typical aminoalkyl groups include, but are not limited to, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2NH2, and the like.

[0089] The term "alkenyl" can refer to an alkyl type in which at least one carbon-carbon double bond is present. In one embodiment, the alkenyl group has the formula -C(H)=CR2, wherein R refers to the remainder of the alkenyl group, which may be the same or different. In some embodiments, R is H or an alkyl group. In some embodiments, the alkenyl group is selected from ethenyl (i.e., vinyl), propenyl (i.e., allyl), butenyl, pentenyl, pentadienyl, etc. Non-limiting examples of alkenyl groups include -CH=CH-, -CH=CH2, -C(CH3)=CH2, -CH=CHCH3, -C(CH3)=CHCH3, and -CH2CH=CH2. Alkenes may be cis (or "Z" configuration) or trans (or "E" configuration). Alkenes may also contain multiple double bonds, each of which is independently E or Z. Cis-olefins or cis-polyolefins are preferably selected. For example, cis-olefins may be oleyl (e.g., oleic acid or esters).

[0090] The term "alkynyl" generally refers to a type of alkyl group in which at least one carbon-carbon triple bond is present. In one embodiment, the alkynyl group has the formula -C≡CR, where R refers to the remainder of the alkynyl group. In some embodiments, R is H or an alkyl group. In some embodiments, the alkynyl group is selected from ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Non-limiting examples of alkynyl groups include -C≡C-, -C≡CH, -C≡CCH3, -C≡CCH2CH3, or -CH2C≡CH.

[0091] The term "heteroalkyl" generally refers to an alkyl group in which one or more of the backbone atoms of the alkyl group is selected from atoms other than carbon, such as oxygen, nitrogen (e.g., -NH-, -N(alkyl)-), sulfur, or a combination thereof. The heteroalkyl group is attached to the remainder of the molecule at a carbon atom of the heteroalkyl group. In one aspect, the heteroalkyl group is a C1-C6 heteroalkyl group. In one aspect, the heteroalkyl group is a C6-C 30 Assorted alkyl. Assorted alkyl can include nitrile, amide, ester, ether, amine, thioether, thioester, carbamate, carbonate, polyether, polyamine etc. Assorted alkyl can include alkyl ether (such as polyether), alkyl ester (such as polyester), alkylamine (such as polyamine), alkylamide (such as polyamide) or any combination thereof. Assorted alkyl can include acrylate or acrylamide. In some instances, assorted alkyl includes ether or polyethylene glycol (PEG) group, wherein the PEG group includes 2-100 monomer units. By way of non-limiting example, each of A1-A12 can be considered to have assorted alkyl side chains.

[0092] The term "aromatic" generally refers to a planar ring having a delocalized π-electron system containing 4n+2 π electrons, where n is an integer. The term "aromatic" includes both carbocyclic aromatic ("aryl," e.g., phenyl) and heterocyclic aromatic (or "heteroaryl" or "heteroaromatic") groups (e.g., pyridine). The term includes monocyclic or fused-ring polycyclic (i.e., rings that share adjacent pairs of carbon atoms) groups.

[0093] The term "carbocyclic" or "carbocycle" generally refers to a ring or ring system in which the atoms forming the ring backbone are all carbon atoms. Thus, the term distinguishes carbocycle from a "heterocyclic" ring or "heterocycle," in which the ring backbone contains at least one atom other than carbon. In some embodiments, at least one of the two rings of a bicyclic carbocycle is aromatic. In some embodiments, both rings of a bicyclic carbocycle are aromatic. Carbocycles include aryl and cycloalkyl groups.

[0094] As used herein, the term "aryl" generally refers to an aromatic ring in which each atom forming the ring is a carbon atom. In one aspect, aryl is phenyl or naphthyl. In some embodiments, aryl is phenyl. In some embodiments, aryl is phenyl, naphthyl, indanyl, indenyl, or tetrahydronaphthyl. In some embodiments, aryl is C6-C 24 Aryl. In some embodiments, "aryl" refers to a polycyclic aromatic carbocyclic ring having two or more (e.g., 2, 3, 4, 5, 6, or 7) conjugated aromatic rings. Examples include, but are not limited to, phenanthrene, anthracene, pyrene, benzopyrene, coronene, and the like. Depending on the structure, an aryl group can be a monovalent radical (i.e., an aryl group) or a divalent radical (i.e., an arylene group).

[0095] The term "cycloalkyl" may refer to a monocyclic or polycyclic aliphatic, non-aromatic radical, wherein each atom (i.e., skeleton atom) forming the ring is a carbon atom. In some embodiments, cycloalkyl is a spirocyclic or bridged compound. In some embodiments, cycloalkyl is optionally fused to an aromatic ring, and the point of attachment is located at a carbon that is not an aromatic ring carbon atom. Cycloalkyl includes a group with 3-10 ring atoms. In some embodiments, cycloalkyl is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, spiro [2.2] pentyl, norbornyl, and bicyclo [1.1.1] pentyl. In some embodiments, cycloalkyl is C3-C6 cycloalkyl. In some embodiments, cycloalkyl is C3-C4 cycloalkyl.

[0096] The term "halo" or "halogen" or "halide" generally refers to fluorine, chlorine, bromine or iodine. In some cases, halo can refer to fluorine, chlorine or bromine. As used herein, the term "halo" is not intended to be numerically restrictive. For example, a haloalkyl group can contain 1, 2, 3 or more halogen groups. A haloalkyl group can be, for example, -CHF2, -CH2F or -CF3.

[0097] The term "fluoroalkyl" generally refers to an alkyl group in which one or more hydrogen atoms are replaced by a fluorine atom. In one aspect, a fluoroalkyl group is a C1-C6 fluoroalkyl group. Examples of fluoroalkyl groups include, but are not limited to, -CH2F, -CHF2, -CF3, -CH2CH2F, -CH2CF3, -CF2CF3, etc.

[0098] The term "heterocycle" or "heterocyclic" generally refers to heteroaromatic rings (also known as heteroaryl) and heterocycloalkyl rings containing 1-4 heteroatoms in the ring, wherein each heteroatom in the ring is selected from O, S and N, wherein each heterocyclic group has 3-10 atoms in its ring system, and with the proviso that any ring does not contain two adjacent O or S atoms. Non-aromatic heterocyclic groups (also known as heterocycloalkyl) include rings having 3-10 atoms in their ring system, and aromatic heterocyclic groups include rings having 5-10 atoms in their ring system. Heterocyclic groups include benzo-fused ring systems. Examples of non-aromatic heterocyclic groups are pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, oxazolidinone, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, thioxanyl, piperazinyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepanyl, oxazepinyl, diazepine diazepinyl, thiazolinyl thiazepinyl, 1,2,3,6-tetrahydropyridinyl, pyrrolin-2-yl, pyrrolin-3-yl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothiophenyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl inyl), imidazolidinyl, 3-azabicyclo[3.1.0]hexyl, 3-azabicyclo[4.1.0]heptyl, 3H-indolyl, indolin-2-onyl, isoindolin-1-onyl, isoindolin-1,3-dionyl, 3,4-dihydroisoquinolin-1(2H)-onyl, 3,4-dihydroquinolin-2(1H)-onyl, isoindolin-1,3-dithionyl, benzo[d]oxazol-2(3H)-onyl, 1H-benzo[d]imidazol-2(3H)-onyl, benzo[d]thiazol-2(3H)-onyl, and quinolizinyl. Examples of aromatic heterocyclic groups are pyridyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. Where possible, the above groups are either C-attached (or C-linked) or N-attached. For example, groups derived from pyrrole include both pyrrole-1-yl (N-attached) or pyrrole-3-yl (C-attached). Further, groups derived from imidazole include imidazol-1-yl or imidazol-3-yl (both N-attached) or imidazol-2-yl, imidazol-4-yl or imidazol-5-yl (all C-attached). Non-aromatic heterocycles are optionally substituted with one or two oxo (=O) moieties, such as pyrrolidin-2-one. In some embodiments, at least one of the two rings of the bicyclic heterocycle is aromatic. In some embodiments, both rings of the bicyclic heterocycle are aromatic.

[0099] The term "heteroaryl" or "heteroaromatic" generally refers to an aryl group that includes one or more ring heteroatoms selected from nitrogen, oxygen, and sulfur. Illustrative examples of heteroaryl groups include, but are not limited to, monocyclic heteroaryl groups and bicyclic heteroaryl groups. Monocyclic heteroaryl groups include pyridyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, and furazanyl. Monocyclic heteroaryl groups include indolizine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, and pteridine. In some embodiments, the heteroaryl group contains 0-4 nitrogen atoms in the ring. In some embodiments, the heteroaryl group contains 1-4 nitrogen atoms in the ring. In some embodiments, the heteroaryl group contains 0-4 N atoms, 0-1 O atoms, and 0-1 S atoms in the ring. In some embodiments, the heteroaryl group contains 1-4 N atoms, 0-1 O atoms, and 0-1 S atoms in the ring. In some embodiments, the heteroaryl group is a C1-C9 heteroaryl group. In some embodiments, the monocyclic heteroaryl group is a C1-C5 heteroaryl group. In some embodiments, the monocyclic heteroaryl group is a 5-membered or 6-membered heteroaryl group.

[0100] "Heterocycloalkyl" generally refers to a cycloalkyl group that includes at least one heteroatom selected from nitrogen, oxygen, and sulfur. In some embodiments, the heterocycloalkyl group is fused to an aryl or heteroaryl group. In some embodiments, the heterocycloalkyl group is oxazolidinone, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, piperidin-2-one, pyrrolidine-2,5-dithione, pyrrolidine-2,5-diketone, pyrrolidinone, imidazolidinyl, imidazolidin-2-one, or thiazolidin-2-one. In one aspect, the heterocycloalkyl group is C2-C 10 In some embodiments, the heterocycloalkyl group is C4-C 10 Heterocycloalkyl. In some embodiments, the heterocycloalkyl is monocyclic or bicyclic. In some embodiments, the heterocycloalkyl is monocyclic and is a 3, 4, 5, 6, 7, or 8-membered ring. In some embodiments, the heterocycloalkyl is monocyclic and is a 3, 4, 5, or 6-membered ring. In some embodiments, the heterocycloalkyl is monocyclic and is a 3 or 4-membered ring. In some embodiments, the heterocycloalkyl contains 0-2 N atoms in the ring. In some embodiments, the heterocycloalkyl contains 0-2 N atoms, 0-2 O atoms, and 0-1 S atoms in the ring.

[0101] The term "bond" or "single bond" generally refers to a chemical bond between two atoms or moieties when the atoms connected by the bond are considered part of a larger substructure. In one aspect, when a group described herein is a bond, the referenced group is absent, thereby allowing a bond to form between the remaining identified groups.

[0102] The term "moiety" generally refers to a specific segment or functional group of a molecule. A chemical moiety is often a recognized chemical entity embedded in or appended to a molecule.

[0103] The term "substituted" generally refers to a group in which one or more hydrogen atoms are replaced by a substituent, for example, a substituent selected from the group consisting of halogen, -CN, -NH2, -NH(alkyl), -N(alkyl)2, -OH, -CO2H, -CO2alkyl, -C(=O)NH2, -C(=O)NH(alkyl), -C(=O)N(alkyl)2, -S(=O)2NH2, -S(=O)2NH(alkyl), -S(=O)2N(alkyl)2, alkyl, cycloalkyl, fluoroalkyl, heteroalkyl, alkoxy, fluoroalkoxy, heterocycloalkyl, aryl, heteroaryl, aryloxy, alkylthio, arylthio, alkyl sulfoxide, aryl sulfoxide, alkyl sulfone and aryl sulfone. In some other embodiments, the optional substituents are independently selected from halogen, -CN, -NH2, -NH(CH3), -N(CH3)2, -OH, -CO2H, -CO2(C1-C4 alkyl), -C(=O)NH2, -C(=O)NH(C1-C4 alkyl), -C(=O)N(C1-C4 alkyl)2, -S(=O)2NH2, -S(=O)2NH(C1-C4 alkyl), -S(=O)2N(C1-C4 alkyl)2, C1-C4 alkyl, C3-C6 cycloalkyl, C1-C4 fluoroalkyl, C1-C4 heteroalkyl, C1-C4 alkoxy, C1-C4 fluoroalkoxy, -SC1-C4 alkyl, -S(=O)C1-C4 alkyl and -S(=O)2C1-C4 alkyl. In some embodiments, optional substituents are independently selected from halogen, -CN, -NH2, -OH, -NH(CH3), -N(CH3)2, -CH3, -CH2CH3, -CHF2, -CF3, -OCH3, -OCHF2, and -OCF3. In some embodiments, substituted groups are substituted by one or two of the aforementioned groups. In some embodiments, optional substituents on aliphatic carbon atoms (acyclic or cyclic) include oxo (=O). A group may also be "optionally substituted," meaning that the group may be unsubstituted or substituted as described above.

[0104] The term "acrylate" generally refers to a salt, ester, or conjugate base of acrylic acid. These acrylates (CH2=CHCO2R or CH2=CHCO2H) contain vinyl groups that are easily polymerized, while the carboxylate group has a variety of functionalities. Modified acrylates include, but are not limited to, methacrylates (CH2=C(CH3)CO2R or CH2=C(CH3)CO2H) and cyanoacrylates (CH2=C(CN)CO2R or CH2=C(CN)CO2H). As used herein, the term "acrylate" includes such modified acrylates, including "methacrylates." As used herein, the term "acrylate" includes "methacrylates."

[0105] The term "acrylamide" generally refers to a vinyl-substituted primary, secondary or tertiary amide (CH2=CHC(O)NH2, CH2=CHC(O)NHR or CH2=CHC(O)NR 1 R 2 Modified acrylamides include, but are not limited to, methacrylamide (CH2=C(CH3)C(O)NH2, CH2=C(CH3)C(O)NHR or CH2=C(CH3)C(O)NR 1 R 2 ) and cyanoacrylamide (CH2=C(CN)C(O)NH2, CH2=C(CN)C(O)NHR or CH2=C(CN)C(O)NR 1 R 2 ). As used herein, the term "acrylamide" includes such modified acrylamides, including "methacrylamide". As used herein, the term "acrylamide" includes "methacrylamide".

[0106] " polymer " generally refers to the molecule formed by repeating bonding together of smaller units (referred to as monomers). As used herein, the term " polymer " can include oligomers (which can have 2-about 80 monomers) and polymers with more than 80 monomers. Polymer can include, for example, 4 or more monomeric units (such as 5 or more, 10 or more, 20 or more, 50 or more, 75 or more, 100 or more, 200 or more, 300 or more or 400 or more monomeric units or monomers). Polymer can include, for example, no more than about 1000 monomeric units or monomers (such as no more than about 500, no more than about 400, no more than about 300, no more than about 200, no more than about 150, no more than 100 or no more than about 50 monomeric units or monomers). In some instances, the polymer has about 10 to about 200 monomer units (e.g., about 20 to about 200, about 50 to about 200, about 75 to about 200, or about 100 to about 200 monomers). The polymer can be a linear, branched network, star, comb, or ladder-shaped polymer type. The polymer can be a homopolymer in which a single monomer is used, or a copolymer in which two or more monomers are used. Types of copolymers include alternating, random, block, and grafted. The "main chain" of a polymer or the "backbone chain" of a polymer can refer to the longest series of covalently bonded atoms that together produce a continuous chain of a given molecule. The main chain of a polymer or the backbone of a polymer can be composed of atoms whose bonds are required for the extension of the polymer length in step-growth or chain-growth polymerization. The side chains of a polymer can be composed of atoms whose bonds are not required for the extension of the polymer length. For those skilled in the art of polymerization, there are several types of polymerization processes that can be utilized in the described process. In some embodiments, the polymers described herein have heteroalkyl side chains (e.g., acrylate or acrylamide side chains). The polymer may include an alkyl backbone. The polymer may include an alkenyl backbone. The polymer may include a heteroalkyl backbone. Examples of heteroalkyl backbones include, but are not limited to, polyethers (e.g., polyethylene glycol or PEG) and polyamines (e.g., polyethyleneimine or PEI).

[0107] "Monomer" generally refers to a building block from which a polymer is constructed. As used herein, a "monomer" may be a divalent chemical unit in which each valency of the unit is completed by bonding (e.g., by polymerization) to an adjacent monomer (thereby producing a polymer) or to a terminal group (e.g., a lipid or functional group as described herein). The monomers described herein may be linked to adjacent monomers or terminal groups via a backbone. Monomers may be linked to adjacent monomers or terminal groups via bonds or via linkers (e.g., C 1-20An alkyl linker, which is optionally substituted with an amine, amide, ether, ester, carbonyl, carbamate, carbonate, etc.) is bonded to an adjacent group (e.g., a second monomer, a lipid, or a functional group). For example, a polymer can be attached to a lipid or a functional group via a linker, wherein the linker is C 1-20 Alkyl esters (e.g. C 1-6 alkyl esters).

[0108] "Side chains" or "side-chains" generally refer to groups of atoms within a monomer that are bonded to the main chain, that do not contribute to chain extension, and that may optionally have additional functionality. For example, side chains having amino groups (e.g., linear, branched, cyclic, or primary, secondary, tertiary, or quaternary amines) that can form cations. In some embodiments, side chains having basic or cationic properties interact with the acidic or anionic phosphate backbone of a nucleic acid molecule, thereby enhancing the ability of the monomer (or a compound comprising such a monomer) to bind nucleic acids. In some embodiments, cationic or basic side chains interact with the anionic phosphate groups of nucleic acid molecules, thereby forming a net neutral (or more neutral relative to unbound nucleic acid) molecule that can then pass through the cell membrane. In some embodiments, side chains are specifically designed to enhance the transport of charged molecules (e.g., nucleic acid molecules) into and out of cells. In some embodiments, the side chains are not cationic. In some embodiments, the side chain is a heteroalkyl group that modulates one or more properties of the lipid-polymer conjugate (e.g., toxicity, solubility, stability, nonspecific (e.g., protein plasma) binding, specific (e.g., nucleic acid) binding, polarity, detectability, etc.). A monomer may lack a side chain (e.g., a PEG or PEI group). In some embodiments, the polymer comprises repeating units of two or more monomers, resulting in an ABABAB pattern of side chains, where A and B represent the same or different side chains.

[0109] "Biologically active compounds" or "bioactive compounds" generally refer to compounds with known or suspected biological activity in mammals. As used in this context, a compound can be any atom or molecule that has biological activity (e.g., therapeutic activity) in a mammal (e.g., a human). Examples of biologically active compounds include atoms, small molecules, macrocycles, peptides, proteins, antibodies, antigen-binding fragments of antibodies, or nucleic acid molecules (e.g., DNA or RNA). "Biologically active molecules" include all cations, anions, salts, oxides, solvates, stereoisomers, and isotopes with known or suspected biological activity. Some examples of biologically active compounds include nucleic acid molecules (e.g., mRNA), radiotherapeutic agents (e.g., radioisotopes or chelate complexes comprising radioisotopes), drugs (e.g., antiproliferative agents, antitumor agents, cytotoxic or cytostatic agents, targeted therapeutics, anti-inflammatory drugs, nucleic acid molecules, peptides, etc.). As disclosed herein, some examples of biologically active molecules for use in the present invention include nucleic acid molecules. Examples of nucleic acid molecules include mRNA, tRNA, miRNA, siRNA, ssDNA, dsDNA, cDNA, genomic DNA, or fragments thereof. As used herein, biologically active compounds or biologically active compounds may refer to a payload (e.g., a therapeutic payload) disclosed herein.

[0110] As used herein, "payload" generally refers to a molecule with practicality inside the target cell. Payload can be useful because it can be detected or manipulated within the cell. Payload can also be a biologically active molecule or a bioactive molecule. In this case, the payload can exert a biological effect (e.g., a disease-mitigating effect) within the target cell. Nucleic acid molecules such as plasmid DNA, mRNA, and siRNA can be particularly used as payloads. Lipid nanoparticles comprising compounds disclosed herein and as payloads of nucleic acid molecules can be particularly used as transfection reagents, i.e., for introducing nucleic acids into eukaryotic cells. Payload can also include a variety of different types of molecules. For example, payload can refer to a combination of nucleic acid molecules and one or more other non-nucleic acid molecules (e.g., small molecule therapeutics, chelating agents, binding agents, etc.). Payload can also include detectable agents or detectable groups (e.g., metals, radioactive tracers, dyes, etc.), therapeutic agents (e.g., immunomodulators, anticancer drugs, antivirals, etc.), oligonucleotides (e.g., siRNA, mRNA), etc.

[0111] "Therapeutic agent" or "therapeutic" or "therapeutic payload" generally refers to any disease-modifying or pathogen-directing agent. Therapeutic agents include any drug clinically approved for the treatment of a disease. Therapeutic agents also include small molecules, antibodies, peptides, proteins, radionuclides, radiopharmaceuticals, and the like. Therapeutic agents are also intended to include oligonucleotides (e.g., siRNA or antisense oligonucleotides). Therapeutic oligonucleotides such as fomivirsen, pegaptanib, mipomersen, defibrotide, eteplirsen, nusinersen, inotesen, patisiran, volanesorsen, givosiran, golodirsen, viltolarsen, lumasiran, inclisiran, and casimersen are examples of therapeutic agents that can be used in combination with the immediately disclosed LNPs and PLips.

[0112] As used herein, "nucleic acid" or "nucleic acid molecule" generally refers to any biopolymer comprising nucleotides (e.g., cytosine, guanine, adenine, uracil, or thymine). In some embodiments, the nucleic acid comprises natural nucleotides. The nucleic acid may comprise non-natural nucleotides. In some embodiments, the nucleic acid comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), or any combination thereof. In some embodiments, the nucleic acid comprises deoxyribonucleic acid (DNA). In some embodiments, the nucleic acid comprises ribonucleic acid (RNA). In some embodiments, the nucleic acid comprises locked nucleic acid (LNA). In some embodiments, the nucleic acid comprises peptide nucleic acid (PNA). In some embodiments, the DNA is single-stranded DNA (ssDNA). In some embodiments, the DNA is double-stranded DNA (dsDNA). In some embodiments, the DNA is circular DNA (cDNA), such as plasmid DNA. In some embodiments, the DNA is recombinant DNA (rDNA). In some embodiments, the DNA is genomic DNA. In some embodiments, it is synthetic DNA, modified DNA, or non-natural DNA. In some embodiments, the RNA is messenger RNA (mRNA). In some embodiments, the RNA is a transfer RNA (tRNA). In some embodiments, the RNA is a ribosomal RNA (rRNA). In some embodiments, the RNA is a small nuclear RNA (snRNA). In some embodiments, the RNA is a microRNA (miRNA). In some embodiments, the RNA is a silencing RNA (siRNA). In some embodiments, the RNA is naked RNA (e.g., non-enveloped RNA that is not complexed with lipids, proteins, or other stabilizing or protective molecules). In some embodiments, the RNA is a complexed RNA.

[0113] As used herein, an "effective amount" or "therapeutically effective amount" generally refers to a sufficient amount of an agent or compound (e.g., a payload or biologically active molecule) administered to alleviate, to some extent, one or more symptoms of the disease or condition being treated. Results include reduction or alleviation of signs, symptoms, or causes of the disease, or any other desired change in a biological system. For example, an "effective amount" for therapeutic use is the amount of a composition comprising a compound as disclosed herein required to provide a clinically significant alleviation of the symptoms of the disease. In any individual case, an appropriate "effective" amount is optionally determined using techniques such as dose escalation studies.

[0114] “Dispersity” or “polydispersity index” is usually a measure of the breadth of the molecular weight distribution of a polymer, defined as M w / M n , where M w is the weight average molecular weight (or “mass average molar mass”), M nis the number average molecular weight (or, "number average molar mass"). This value corresponds to the heterogeneity of the molecular sizes in the mixture. In a mixture where all molecules have the same size, the dispersity is 1. A lower dispersity can indicate a higher homogeneity within the sample.

[0115] "Steric stabilizers" generally refer to long-chain hydrophilic groups that prevent aggregation of the final polymer by sterically hindering particle-particle electrostatic interactions. Examples include, but are not limited to, alkyl groups, PEG chains, polysaccharides, and alkylamines. Electrostatic interactions are the non-covalent association of two or more substances due to the attractive force between positive and negative charges.

[0116] "Reactive group" generally refers to a chemical moiety that can form an ionic or covalent bond with another compound, particularly in a biological environment (e.g., at about neutral pH and about 37°C). The portion of a reactive compound that can form a covalent bond can be referred to as a reactive functional group. Reactive groups include coupling partners for specific reactions (e.g., alkynes or azides for "click" type chemistry or reactions), as well as common reactive species (e.g., nucleophiles or electrophiles). In some embodiments, the reactive group is maleimide or succinimide. In some embodiments, the reactive group is a chemical group that can undergo a bioorthogonal reaction. In some embodiments, the biorthogonal reaction is a copper-free click reaction (CuAAC). In some embodiments, the reactive group is an azide. In some embodiments, the reactive group is an alkyne. In some embodiments, the alkyne is a strained alkyne (e.g., a cycloalkyne). In some embodiments, the cycloalkyne is monofluorinated cyclooctyne (MOFO), difluorinated cyclooctyne (DIFO), dibenzocyclooctyne (DIBO), dibenzoazepine octyne (DIBAC), diarylazacyclooctyne (BARAC), dimethoxyazacyclooctyne (DIMAC), and the like.

[0117] "Detectable group" generally refers to a compound or chemical moiety that can be detected by any method known in the art. In some embodiments, the detectable group is capable of bioorthogonal detection. In some embodiments, the detectable group includes a conjugated aromatic or heteroaromatic system that can emit light in response to a stimulus (e.g., excitation). In some embodiments, the detectable group includes a fluorescent group (e.g., a fluorescein group or a derivative thereof, a rhodamine group or a derivative thereof, or a coumarin group or a derivative thereof). In some embodiments, the detectable group includes a pyrene group, a benzopyrene group or a derivative thereof. Fluorescent groups, or "fluorophores", are well known in the art, and any known fluorophore is considered to be within the scope of the present invention. In some embodiments, the fluorophore is fluorescein, rhodamine, coumarin, anthocyanin or xanthene or a derivative thereof. In some embodiments, the fluorophore is an Alexa Fluor or DyLight Fluor probe, or a derivative thereof.

[0118] "Steroid" or "steroid derivative" generally refers to a sterol or stanol, or a steroid hormone or analog thereof, in which the hydroxyl moiety has been modified (e.g., acylated). Modifications may include spacers, linkers, or reactive groups. As used herein, a steroid may be any naturally occurring or non-natural steroid (e.g., cholesterol) or a derivative thereof known in the art.

[0119] As used herein, "cell" generally refers to a biological cell. In some embodiments, the cell is an animal cell (e.g., a human cell) or a plant cell. In some embodiments, the cell is a specific type of cell, such as an immune cell, a blood cell, a cancer cell, a healthy cell, etc.

[0120] As used herein, the term "in vivo" can be used to describe an event that occurs in an organism, such as a subject's body. In some embodiments, in vivo refers to an event that occurs in a non-human subject, such as a mouse or rat. In some embodiments, in vivo refers to an event that occurs in a human.

[0121] As used herein, the term "in vitro" may be used to describe an event that occurs in a container that is contained within a laboratory reagent, such that it is separated from the living biological source organism from which the material was obtained. In vitro assays may include cell-based assays, in which living or dead cells are employed. In vitro assays may also include cell-free assays, in which intact cells are not employed.

[0122] Lipid-polymer compound ("PLip" or "compound")

[0123] In one aspect, disclosed herein are polymer-lipid compounds comprising a lipid, a polymer, and a functional group. The lipid can be bonded to the polymer via a backbone. The lipid can also be bonded to the polymer backbone via (non-side chain linkers). The polymer can comprise a backbone and side chains (e.g., C 1-20 In some embodiments, the polymer may have no side chains. In some cases, the polymer comprising a heteroalkyl side chain interacts with one or more nucleic acid molecules to stabilize or encapsulate the nucleic acid molecules. Compared to unsubstituted (e.g., PEG or PEI) polymers, polymers comprising a heteroalkyl side chain may provide advantageous biological or physicochemical properties.

[0124] In another aspect, disclosed herein is a compound having a structure represented by Formula I or a pharmaceutically acceptable salt thereof:

[0125] X—Y—Z

[0126] in:

[0127] X is a lipid;

[0128] Y is a polymer comprising at least 3 monomer units, each of which comprises C 1-20 heteroalkyl side chains; and

[0129] Z is an unsubstituted or substituted functional group;

[0130] wherein the lipid is covalently bonded to the polymer via the backbone.

[0131] Lipid ("X")

[0132] The "lipid" component of the polymer-lipid compound described herein generally refers to an organic compound (or its free radical, in the case of a polymer-lipid compound) that is or contains a lipophilic and / or hydrophobic portion. In many cases, lipids have poor water solubility but are soluble in non-polar solvents. The term lipid as used herein can refer to the lipophilic group itself or its derivatives. The lipid may further comprise a linker or spacer. For example, the lipid may comprise a hydrophobic portion having an optionally substituted alkyl or optionally substituted heteroalkyl linker that connects the hydrophobic portion to the polymer (Y). In some cases, the lipid is a fatty acid or comprises a fatty acid. In some cases, the lipid comprises one or more fatty acids or derivatives thereof and a glyceride group. The lipid may comprise, for example, a monoglyceride, a diglyceride, or a triglyceride. These may alternatively be referred to as (single) acyl, diacyl, or triacylglycerol. As described herein, the lipid may be (or may comprise) an acyl or diacylglycerol bonded to the polymer backbone.

[0133] Lipids can also refer to non-polar groups that do not contain fatty acids. For example, sterols or phospholipids or their derivatives can be referred to as lipids. The lipids described herein can include a linker that connects monoglycerides, diglycerides, sterols, etc. to the polymer backbone. The linker can be C 1-6 alkyl or 1- to 6-membered heteroalkyl, each optionally further substituted. In some cases, the linker is a substituted or unsubstituted C 1-6 An alkyl group that forms an ester bond with the hydroxyl group of the glycerol or sterol. If substituted, the linker group may be substituted, for example, with one or more methyl or cyano groups. In some cases, the linker may have geminal dimethyl substitution or geminal methyl / cyano substitution. In some examples, the linker is a substituted or unsubstituted C 1-6 Alkyl ester. The linker can also be selected from C 1-6 Ether, C 1-6 Amide, C 1-6 Carbamate, C 1-6 Phosphate, C 1-6 Alternatively, longer linkers can be employed, comprising from about 7 to about 20 carbon atoms, conjugated via an ester bond and optionally substituted as described above.

[0134] As used herein, lipid can refer to any steroid (e.g., sterol). For example, in some instances, lipid comprises sterol or stanol. The example of sterol includes but is not limited to cholesterol, campesterol, stigmasterol, rapeseed sterol, avenosterol, sitosterol, and ergosterol.

[0135] The lipid may comprise one or more fatty acids. For example, the lipid may be a diacylglycerol, which comprises one or more saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, or combinations thereof. The fatty acids typically contain an acid group and a hydrocarbon chain (or a fatty acid tail). The fatty acid tail may range in length from about 10 to about 30 carbons and may have a degree of unsaturation from about 0 to about 6. As used herein, the fatty acid tail may be C 10-30 Alkyl, C 10-30 Alkenyl or C 10-30 The fatty acid tail can be substituted or unsubstituted. In some cases, the fatty acid tail is an unsubstituted C 10-30 Alkyl or C 10-30 In some cases, the fatty acid tail is a linear or unbranched C 10-30 Alkyl or C 10-30 Alkenyl.

[0136] In one example, the lipid comprises a monoacylglycerol (MAG) or diacylglycerol (DAG) group with one or more linoleic or oleic acid tails. The lipid may further comprise a geminal disubstituted C 1-6 An alkyl ester linker connects the MAG or DAG to the polymer backbone. In another example, the lipid comprises a cholesterol group. The cholesterol lipid may further comprise a geminal disubstituted C 1-6 An alkyl ester linker that attaches the sterol to the polymer backbone.

[0137] As an example, the lipid may have a structure represented by any of the following formulae: Wherein each L is substituted or unsubstituted C 1-6 Alkyl, S 1 is a sterol group, and FA 1 , FA 2 , FA 3 and FA 4 Each of L is a fatty acid tail having 10-30 carbon atoms and 0-3 double bonds per fatty acid tail. In some embodiments, each L is a substituted or unsubstituted C 1-12 alkyl, or substituted or unsubstituted 2- to 12-membered heteroalkyl. As described herein, L may contain geminal substitution (e.g., dimethyl or methyl / cyano). In other cases, the linker L is monosubstituted. In still other cases, the linker L is unsubstituted. Examples of linkers include, but are not limited to:

[0138] In further examples, the lipid can have a structure represented by any of the following formulae:

[0139]

[0140]

[0141] As used herein, alkenyl groups may include dienes and trienes, and further include both cis (or E) and trans (or Z) isomers. Unless otherwise indicated, all forms of stereoisomers (e.g., enantiomers, diastereomers, etc.) are also considered within the scope of the above formula and throughout this disclosure.

[0142] The lipids may be hydrophobic. In some instances, the lipids are amphiphilic. In some embodiments, the lipids comprise an octanol:water coefficient (log(K)) of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10. OW )). In some cases, the lipid comprises a log(K OW In further instances, the lipid comprises a log(K) range of about 2 to about 3, about 2 to about 4, about 2 to about 5, about 2 to about 6, about 2 to about 7, about 2 to about 8, about 2 to about 9, about 2 to about 10, about 3 to about 4, about 3 to about 5, about 3 to about 6, about 3 to about 7, about 3 to about 8, about 3 to about 9, about 3 to about 10, about 4 to about 5, about 4 to about 6, about 4 to about 7, about 4 to about 8, about 4 to about 9, about 4 to about 10, about 5 to about 6, about 5 to about 7, about 5 to about 8, about 5 to about 9, about 5 to about 10, about 6 to about 7, about 6 to about 8, about 6 to about 9, about 6 to about 10, about 7 to about 8, about 7 to about 9, about 7 to about 10, about 8 to about 9, about 8 to about 10, or about 9 to about 10. OW ) range. The lipid may have a log(K OW ) range. The lipid may have a log(K OW The lipid may have a log(K of at most about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10. OW ).

[0143] The compounds disclosed herein may have a lipid of the formula:

[0144] Among them FA 1 and FA 2 Each of is independently an ester side chain derived from one of the following fatty acids:

[0145] Lauric acid;

[0146] Myristic acid;

[0147] Palmitic acid;

[0148] stearic acid;

[0149] Oleic acid;

[0150] Elaidic acid;

[0151] Vinegar;

[0152] Linoleic acid;

[0153] trans-linoleic acid;

[0154] α-linolenic acid;

[0155] Gamma-linolenic acid; and linolenelaidic acid.

[0156] In further examples, the compounds disclosed herein may have a lipid of the formula: Among them FA 1 and FA 2 Each of which is independently selected from C 1-30 Alkyl, C 1-30 Alkenyl, C 1-30 Alkynyl or 1-30 membered heteroalkyl. Some examples of 1- to 30

[0157]

[0158] The compounds disclosed herein may have a lipid of the formula:

[0159]

[0160] Polymer ("Y")

[0161] In the literature, PEG-DMG is the stabilizing lipid generally utilized in LNP. Several studies and clinical reports show that moderate to severe immunogenic reactions can occur after systemic PEG is used. The therapeutic efficiency reduction of acute hypersensitivity and PEGylated drugs can be due to complement system activation and / or anti-PEG antibody production. Various stabilization PLips have been synthesized, and compared with the LNP stabilized by PEG-DMG, the stabilization LNPs of similar size are shown. Stabilization PLip can contain any variation (cholesterol, diacyl, acyl group etc.) of lipid tail, and the polymer side chain can comprise hydrophilic stabilizing groups. Stabilization polymer side chain can be called assorted alkyl side chain, stabilization side chain, non-cationic side chain or solubilizing side chain. Some examples of stabilized PLip side chains include repeating units of 2-hydroxypropyl methacrylamide (HPMA), (2-(methylsulfinyl)ethyl methacrylate (MSEMA), hydroxyethyl acrylate (HEA), and oligo(ethylene glycol) methacrylate. The size and stability of the LNP can depend on the nature and length of the polymer side chains. In addition, the size and stability of the LNP can be determined or influenced by the identity of the lipid tail present in the stabilized PLip. In some cases, stabilized PLip is used as a substitute for PEG stabilizers (e.g., PEG-DMG). Alternatively, PL PLip can have both stabilizing and cationic properties. For example, PLip can comprise multiple polymer blocks (sequentially, alternatingly, statistically, randomly), wherein one block (or multiple identical monomer units) contains a cation or a group that forms a cation, and a second block (or multiple identical monomer units) contains a stabilizing side chain. Stabilized PLip can comprise a polymer having an alkoxy or hydroxyl side chain. In some cases, stabilized PLip is hydrophilic. Stabilized PLip can contain between about 3 and about 200 stabilizing monomer units. The cationic monomer unit can have a structure of one of the following formulas: Each R group is C 1-6 Alkyl, C 1-6 Alkoxyalkyl, C 1-6 Hydroxyalkyl, or wherein R is a polyethylene glycol (PEG) group containing 1-100 PEG units; and each R' group is hydrogen or methyl.

[0162] Some examples of stabilizing monomeric units include, but are not limited to, the following:

[0163] Each R' thereof is hydrogen or methyl. More specifically, the stabilizing monomer unit may have a structure of one of the following formulae:

[0164] Cationic PLip can contain any variation of lipid tail (cholesterol, diacyl, acyl group etc.), and polymer side chain can comprise cation or the group (such as amine) that forms cation.For example, cationic PLip can comprise cationic polymer, wherein at least one (such as at least 3) monomeric unit of polymer comprises cation or the side chain that forms cation.As used herein, cation is intended to include not only cation, but also include the uncharged group that becomes cation in biological environment.For example, cationic side chain can comprise amide or ester group, wherein amide or ester is replaced by alkylamine or alkylammonium side chain.Cationic PLip can contain cationic monomeric unit between about 3-about 200.Cationic monomeric unit can have the structure of one of following formula: wherein each R group is an alkylamine or alkylammonium group, and wherein each R' group is hydrogen or methyl. More specifically, the cationic monomer unit may have a structure of one of the following formulae or a salt thereof: wherein each R' is independently hydrogen or methyl.

[0165] Some examples of cationic monomer units include, but are not limited to, the following: In each of the aforementioned monomeric unit examples, the alkyl chain can be between 2 and 6 carbons in length (e.g., 2, 3, or 4 carbons in length). For example, in any of the aforementioned monomeric units, the alkyl chain can be an ethyl chain, a propyl chain, or a butyl chain. In a specific embodiment, the cationic monomer unit is or a salt thereof, or a free base thereof.

[0166] In some examples, a polymer (e.g., a polymer having a structure represented by Formula YA, Formula YB, Formula YC, or Formula YD) comprises at least a first plurality of monomeric units represented by one of the following formulae:

[0167] wherein a is an integer from 3 to 400, and wherein n is an integer from 1 to 100.

[0168] More specifically, n can be an integer from 1 to about 50. In more specific embodiments, n can be an integer from 1 to 40, 1 to 30, 1 to 20, 1 to 10, 2 to 10, or 2 to 8. In certain examples disclosed herein, n has an average value between 2 and 10, between 3 and 6, or between 4 and 5 (e.g., n is about 4.5). N can be an integer having a value (average) of about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10.

[0169] In another aspect, provided herein are compounds comprising a lipid attached to a polymer backbone, wherein the polymer comprises 3 monomeric units or more. In some cases, the monomeric units comprise C 1-20 Heteroalkyl side chains.

[0170] The polymer may comprise about 5 monomer units or more, about 10 monomer units or more, about 15 monomer units or more, about 20 monomer units or more, about 30 monomer units or more, about 50 monomer units or more, about 100 monomer units or more, about 200 monomer units or more, about 300 monomer units or more, or about 400 monomer units or more. Each monomer unit may comprise C 1-20 Heteroalkyl side chains.

[0171] In some cases, the polymer may comprise from about 3 monomer units to about 400 monomer units. For example, the polymer may comprise from about 3 monomer units to about 4 monomer units, from about 3 monomer units to about 10 monomer units, from about 3 monomer units to about 30 monomer units, from about 3 monomer units to about 50 monomer units, from about 3 monomer units to about 80 monomer units, from about 3 monomer units to about 100 monomer units, from about 3 monomer units to about 120 monomer units, from about 3 monomer units to about 150 monomer units, from about 3 monomer units to about 200 monomer units, from about 3 monomer units to about 400 monomer units, from about 4 monomer units to about 10 monomer units, from about 4 monomer units to about 30 monomer units, from about 4 monomer units to about 50 monomer units, from about 4 monomer units to about 100 monomer units. about 80 monomer units, about 4 monomer units - about 100 monomer units, about 4 monomer units - about 120 monomer units, about 4 monomer units - about 150 monomer units, about 4 monomer units - about 200 monomer units, about 4 monomer units - about 400 monomer units, about 10 monomer units - about 30 monomer units, about 10 monomer units - about 50 monomer units, about 10 monomer units - about 80 monomer units, about 10 monomer units - about 100 monomer units, about 10 monomer units - about 120 monomer units, about 10 monomer units - about 150 monomer units, about 10 monomer units - about 200 monomer units, about 10 monomer units - about 400 monomer units, about 30 monomer units to about 50 monomer units, about 30 monomer units to about 80 monomer units, about 30 monomer units to about 100 monomer units, about 30 monomer units to about 120 monomer units, about 30 monomer units to about 150 monomer units, about 30 monomer units to about 200 monomer units, about 30 monomer units to about 400 monomer units, about 50 monomer units to about 80 monomer units, about 50 monomer units to about 100 monomer units, about 50 monomer units to about 120 monomer units, about 50 monomer units to about 150 monomer units, about 50 monomer units to about 200 monomer units, about 50 monomer units to about 4 00 monomer units, about 80 monomer units to about 100 monomer units, about 80 monomer units to about 120 monomer units, about 80 monomer units to about 150 monomer units, about 80 monomer units to about 200 monomer units, about 80 monomer units to about 400 monomer units, about 100 monomer units to about 120 monomer units, about 100 monomer units to about 150 monomer units, about 100 monomer units to about 200 monomer units, about 100 monomer units to about 400 monomer units, about 120 monomer units to about 150 monomer units, about 120 monomer units to about 200 monomer units, about 120 monomer units to about 400 monomer units,In some embodiments, the polymer comprises at least about 3 monomeric units, about 4 monomeric units, about 10 monomeric units, about 30 monomeric units, about 50 monomeric units, about 80 monomeric units, about 100 monomeric units, about 120 monomeric units, about 150 monomeric units, about 200 monomeric units, or about 400 monomeric units. In some embodiments, the polymer comprises at least about 3 monomeric units, about 4 monomeric units, about 10 monomeric units, about 30 monomeric units, about 50 monomeric units, about 80 monomeric units, about 100 monomeric units, about 120 monomeric units, about 150 monomeric units, or about 200 monomeric units. In some embodiments, the polymer comprises up to about 4 monomeric units, about 10 monomeric units, about 30 monomeric units, about 50 monomeric units, about 80 monomeric units, about 100 monomeric units, about 120 monomeric units, about 150 monomeric units, about 200 monomeric units, or about 400 monomeric units. Each monomeric unit comprises C, 1-20 Heteroalkyl side chains.

[0172] The polymer can be a polyacrylate, also known as poly(acrylate) or poly(alkyl acrylate). Polyacrylate polymers have one or more monomeric units comprising acrylate, also known as acrylic ester or alkyl acrylate. Polyacrylate polymers can have about 10 or more monomeric units comprising acrylate. In some cases, the polymer can have up to about 400 monomeric units comprising acrylate. For example, the polymer can have from about 10 to about 400 monomeric units comprising acrylate. In other examples, the polymer can have from about 20 to about 300, from about 50 to about 200, or from about 100 to about 150.

[0173] In some embodiments, the polymer is polyacrylamide. In some embodiments, the polymer has monomeric units comprising acrylamide, also known as acrylic amide. In some embodiments, the polymer comprises a combination of acrylate and acrylamide.

[0174] In some embodiments, the polymer comprises a series of repeating units, wherein the repeating units comprise two monomers, three monomers, or four monomers. In some embodiments, the repeating units comprise both acrylamide and acrylate. In some embodiments, the repeating units comprise two or more acrylamides. In some embodiments, the repeating units comprise two or more acrylates.

[0175] In some embodiments, the polymer is a homopolymer comprising an acrylate. In some embodiments, the homopolymer comprises a cationic monomer unit. In some embodiments, the homopolymer is positively charged in a neutral aqueous solution.

[0176] In some embodiments, the polymer is a homopolymer comprising acrylamide. In some embodiments, the homopolymer comprises cationic monomer units. In some embodiments, the homopolymer is positively charged in neutral aqueous solution.

[0177] In some embodiments, the polymer is a copolymer comprising a block copolymer, an alternating copolymer, a random or statistical copolymer, or a gradient copolymer. In some embodiments, the copolymer comprises cationic monomer units. In some embodiments, the polymer is positively charged in a neutral aqueous solution.

[0178] In some embodiments, the polymer is a cationic polymer, e.g., comprising a plurality of cations or cation-forming groups (e.g., primary amines). In some embodiments, the cationic polymer comprises a plurality of monomeric units, wherein each monomeric unit comprises an aminoalkyl side chain (e.g., CH2CH2NH2, CH2CH2CH2NH2, or a cation thereof). In some embodiments, the cationic polymer comprises a plurality of monomeric units, wherein each monomeric unit comprises an alkylammonium side chain (e.g., CH2CH2N(CH3)3 + 、CH2CH2CH2N(CH3)3 + or a salt thereof). In some embodiments, the cationic PLip comprises at least a first plurality of monomeric units, wherein each of said monomeric units in said first plurality of monomeric units comprises an aminoalkyl (or alkylamino) side chain (e.g., comprising -NH2 or -N(CH3)3 + In some embodiments, the cationic PLip comprises a second plurality of monomeric units, wherein each of said monomeric units in said second plurality of monomeric units comprises a hydroxyalkyl or alkoxyalkyl side chain.

[0179] In some embodiments, the polymer comprises a pK in the range of about 2 to about 12. b In some embodiments, the polymer comprises a pK in the range b: about 2 to about 3, about 2 to about 4, about 2 to about 5, about 2 to about 6, about 2 to about 7, about 2 to about 8, about 2 to about 9, about 2 to about 10, about 2 to about 11, about 2 to about 12, about 3 to about 4, about 3 to about 5, about 3 to about 6, about 3 to about 7, about 3 to about 8, about 3 to about 9, about 3 to about 10, about 3 to about 11, about 3 to about 12, about 4 to about 5, about 4 to about 6, about 4 to about 7, about 4 to about 8, about 4 to about 9, about 4 to about 10, about 4 to about 11, about 4 to about 12, about 5 to about 6, about 5 to about 5 In some embodiments, the polymer comprises a pK in the range of about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12. b In some embodiments, the polymer comprises a pK in the range of at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or about 11. b In some embodiments, the polymer comprises a pK in the range of at most about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12. b .

[0180] The polymer may have a structure represented by Formula YA, Formula YB, Formula YC, or Formula YD, or a pharmaceutically acceptable salt thereof:

[0181]

[0182] in:

[0183] A 1 、B 1 、C 1 and D 1 is independently selected at each occurrence from hydrogen and methyl;

[0184] A 2 、B 2 、C 2 and D 2 In each case, independently selected from C 1-20 heteroalkyl;

[0185] a, b, c, d, e and f are each independently an integer selected from 0-400 (e.g., 0-200 (e.g., 40-120)), with the proviso that the total number of monomer units is 3 or more;

[0186] If C 1-20 When the heteroalkyl group is substituted, it is substituted by unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclyl, unsubstituted or substituted aryl, or unsubstituted or substituted heteroaryl.

[0187] In some embodiments, the cycloalkyl group is C 3-24 In some embodiments, the cycloalkyl group is C 3-15 In some embodiments, the cycloalkyl group is C 3-10 In some embodiments, the cycloalkyl group is C 3-6 In some embodiments, the heterocyclyl group is a 3- to 24-membered heterocyclic ring. In some embodiments, the heterocyclyl group is a 3- to 15-membered heterocyclic ring. In some embodiments, the heterocyclyl group is a 3- to 10-membered heterocyclic ring. In some embodiments, the heterocyclyl group is a 3- to 6-membered heterocyclic ring. In some embodiments, the heterocyclyl group is oxirane, oxetane, tetrahydrofuran, tetrahydropyran, aziridine, azetidine, pyrrolidine, piperidine, piperazine, morpholine, or dioxane. In some embodiments, the heterocyclyl group is a saturated or partially saturated heterocyclic ring composed of atoms selected from hydrogen, C, N, O, and S.

[0188] In some embodiments, aryl is C 6-24 In some embodiments, the aryl group is C 6-16 In some embodiments, the aryl group is C 6-10 In some embodiments, aryl is phenyl, naphthyl, phenanthrenyl, anthracenyl, fluoroanthenyl, pyrenyl, In some embodiments, the heteroaryl group is phenyl, naphthyl, or pyrenyl. In some embodiments, the heteroaryl group is a 3- to 24-membered aromatic heterocyclic ring. In some embodiments, the heteroaryl group is a 3- to 15-membered aromatic heterocyclic ring. In some embodiments, the heteroaryl group is a 3- to 10-membered aromatic heterocyclic ring. In some embodiments, the heteroaryl group is a 3- to 6-membered aromatic heterocyclic ring. In some embodiments, the heteroaryl group is an aromatic heterocyclic ring composed of atoms selected from hydrogen, C, N, O, and S.

[0189] In some embodiments, the substituted cycloalkyl, substituted heterocyclyl, substituted aryl, or substituted heteroaryl is substituted with one or more groups selected from the group consisting of: oxo, -COOH, -CONH2, -NH2, -NH3 + 、-NHC(NH2+ )NH2, -NHCH3, -N(CH3)2, -N(CH3)3 + , -OH, -OCH3, -SH, -S(O)CH3, -S(O)2CH3 and -S(O)2OH.

[0190] In some embodiments, the polymer has the structure of Formula YA or a pharmaceutically acceptable salt thereof:

[0191]

[0192] in:

[0193] A 1 is hydrogen or methyl;

[0194] A 2 Selected from unsubstituted or substituted C 1-20 heteroalkyl; and

[0195] a is an integer of 3-400 (eg, 3-200 (eg, 40-120)).

[0196] In some embodiments, each A 2 are identical (i.e., the polymer comprises a single repeating monomer). In some embodiments, each A 2 are not identical (i.e., the polymer comprises multiple different monomers). In some embodiments, A 1 In some embodiments, A 1 In some embodiments, A 2 is an acid, an ester, a carboxamide, or a substituted amide. In some embodiments, the polymer comprises 3-400 monomers (e.g., A1-A12) selected from the following:

[0197]

[0198] Wherein a is an integer from 0 to 100.

[0199] In some embodiments, a is an integer from 3 to 400. In some embodiments, a is an integer from 10 to 200. In some embodiments, a is an integer from about 10 to about 30. In some embodiments, a is an integer from about 30 to about 60. In some embodiments, a is an integer from about 60 to about 90. In some embodiments, a is an integer from about 90 to about 120. In some embodiments, a is an integer from about 120 to about 150. In some embodiments, a is an integer from about 40 to about 120. In some embodiments, a is an integer from about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 200, or more. In some embodiments, a is about 20. In some embodiments, a is about 30. In some embodiments, a is about 40. In some embodiments, a is about 50. In some embodiments, a is about 75. In some embodiments, a is about 95. In some embodiments, a is about 100. In some embodiments, a is about 110. In some embodiments, a is about 115.

[0200] In some embodiments, the polymer has the structure of Formula YB or a pharmaceutically acceptable salt thereof:

[0201]

[0202] in:

[0203] A 1 and B 1 is independently selected at each occurrence from hydrogen and methyl;

[0204] A 2 and B 2 In each case, independently selected from C 1-20 heteroalkyl; and

[0205] a and b are each independently an integer from 0 to 400 (eg, 0 to 200 (eg, 40 to 120)), with the proviso that the total number of monomer units is 3 or more.

[0206] In some embodiments, each A 2 are the same, and each B 2 In some embodiments, the polymer of formula YB is a polymer comprising two different monomers. In some embodiments, the polymer of formula YB is a polymer comprising two adjacent sequences of repeating monomers (i.e., a block copolymer), wherein each A 2 is the same in each case, and where each B 2 is the same in each case, but each B 2With each A 2 In some embodiments, each A 2 and B 2 C 1-10 In some embodiments, each A 2 is an amide, and each B 2 In some embodiments, each A 2 is an ester, and each B 2 In some embodiments, each A 2 and B 2 having the formula -C(O)OR 2 or -C(O)NR 4 R 5 C 1-20 heteroalkyl, wherein each R 2 、R 4 and R 5 As defined herein. In some embodiments, each A 2 and B 2 having the formula -C(O)OR 12 or -C(O)NR 14 R 15 C 1-20 heteroalkyl, wherein each R 12 、R 14 and R 15 As defined herein.

[0207] In some embodiments, the polymer of formula YB comprises monomers:

[0208]

[0209] In some embodiments, the polymer has the structure of Formula Y-AB or a pharmaceutically acceptable salt thereof:

[0210]

[0211] in:

[0212] A 1 and B 1 is independently selected at each occurrence from hydrogen and methyl;

[0213] A 2 and B 2 In each case, independently selected from C 1-20 heteroalkyl;

[0214] a, b and e are each independently an integer from 0 to 400 (eg, 0 to 200 (eg, 40 to 120)), with the proviso that the total number of monomer units is 3 or more.

[0215] In some embodiments, the polymer of formula Y-AB is a polymer comprising two adjacent monomers forming a repeating unit, wherein each A 2 is the same in each case, and where each B 2 is the same in each case, but each B 2 With each A 2 In some embodiments, a and b are each 1, and the polymer of formula Y-AB is an "alternating" copolymer. In some embodiments, a, b, and e are each an integer selected from 0 to 400 (e.g., a random copolymer). In some embodiments, each A 2 and B 2 C 1-10 In some embodiments, each A 2 is an amide, and each B 2 In some embodiments, each A 2 is an ester, and each B 2 In some embodiments, each A 2 and B 2 having the formula -C(O)OR 2 or -C(O)NR 4 R 5 C 1-20 heteroalkyl, wherein each R 2 、R 4 and R 5 As defined herein. In some embodiments, each A 2 and B 2 having the formula -C(O)OR 12 or -C(O)NR 14 R 15 C 1-20 heteroalkyl, wherein each R 12 、R 14 and R 15 As defined herein.

[0216] In some embodiments, the polymer has the structure of Formula YC or a pharmaceutically acceptable salt thereof:

[0217]

[0218] in:

[0219] A 1 、B 1 and C 1 is independently selected at each occurrence from hydrogen and methyl;

[0220] A 2 、B 2 and C2 In each case, independently selected from C 1-20 heteroalkyl;

[0221] a, b, c and e are each independently an integer selected from 0-400 (eg 0-200 (eg 40-120)), with the proviso that the total number of monomer units is 3 or more.

[0222] In some embodiments, the polymer of formula YC is a polymer comprising two adjacent monomers forming a repeating unit, wherein each A 2 is the same in each case, and where each B 2 is the same in each case, but each B 2 With each A 2 Different; and each C 2 To be able to 2 Same as B 2 The same or different monomer side chains.

[0223] In some embodiments, each A 2 、B 2 and C 2 C 1-10 In some embodiments, each A 2 For amide, each B 2 is an ester, and each C 2 In some embodiments, each A 2 For amide, each B 2 is an ester, and each C 2 In some embodiments, each A 2 For esters, each B 2 is an amide, and each C 2 In some embodiments, each A 2 For esters, each B 2 is an amide, and each C 2 In some embodiments, each A 2 、B 2 and C 2 having the formula -C(O)OR 2 or -C(O)NR 4 R 5 C 1-20 heteroalkyl, wherein each R 2 、R 4 and R 5 As defined herein. In some embodiments, each A 2 、B 2 and C 2 having the formula -C(O)OR12 or -C(O)NR 14 R 15 C 1-20 heteroalkyl, wherein each R 12 、R 14 and R 15 As defined herein.

[0224] In some embodiments, the polymer of formula YC is:

[0225]

[0226] Similar to Formula Y-AB, in some embodiments, the polymer has a structure of Formula Y-ABC or a pharmaceutically acceptable salt thereof:

[0227]

[0228] in:

[0229] A 1 、B 1 and C 1 is independently selected at each occurrence from hydrogen and methyl;

[0230] A 2 、B 2 and C 2 In each case, independently selected from C 1-20 heteroalkyl; and

[0231] a, b, c and f are each independently an integer selected from 0-400 (eg 0-200 (eg 40-120)), with the proviso that the total number of monomer units is 3 or more.

[0232] In some embodiments, a, b, and c are each 1, and the polymer of formula Y-ABC is an "alternating" copolymer. In some embodiments, the polymer of formula Y-ABC contains trimer repeat units (wherein A, B, and C represent adjacent monomer units in a given repeat unit). In some embodiments, the polymer of formula Y-ABC has f repeat units, where f is an integer between 0-400 (e.g., 0-200 (e.g., 40-120)), provided that the total number of monomer units is 3 or more. In some embodiments, each A 2 、B 2 and C 2 C 1-10 In some embodiments, each A 2 、B 2 and C 2 In some embodiments, each A 2 、B 2 and C2 In some embodiments, the polymer has a ratio of amide side chains to ester side chains of about 2:1.

[0233] In some embodiments, the polymer has the structure of Formula YD or a pharmaceutically acceptable salt thereof:

[0234]

[0235] in:

[0236] A 1 、B 1 、C 1 and D 1 is independently selected at each occurrence from hydrogen and methyl;

[0237] A 2 、B 2 、C 2 and D 2 In each case, independently selected from C 1-20 heteroalkyl;

[0238] a, b, c, d, e and f are each independently an integer selected from 0-400 (eg 0-200 (eg 40-120)), with the proviso that the total number of monomer units is 3 or more.

[0239] In some embodiments, a, b, and c are each 1, and the polymer of formula Y-ABC is a mixed copolymer comprising a first block having trimer repeat units and a second block having monomer repeat units. In other embodiments, f is 1, and the polymer is a block copolymer having 1, 2, 3, or 4 monomer repeat units (with side chains A 2 、B 2 、C 2 、D 2 ). In some embodiments, each A 2 、B 2 、C 2 and D 2 In some embodiments, each A 2 、B 2 、C 2 and D 2 For esters.

[0240] In some embodiments, the polymer of formula YD is:

[0241]

[0242] In some embodiments, the ratio of amide side chains to ester side chains is about 1: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 20: 1, 30: 1, 50: 1, 100: 1 or more. In some embodiments, the ratio of ester side chains to amide side chains is about 1: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 20: 1, 30: 1, 50: 1, 100: 1 or more. In some embodiments, the polymer comprises about 0%, about 1%, about 5%, about 10%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 90%, or about 95% acrylate monomer in a given polymer chain. In some embodiments, the polymer comprises about 0%, about 1%, about 5%, about 10%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 90%, or about 95% acrylamide monomers in a given polymer chain.

[0243] In some embodiments, the polymer comprises 3 monomeric units or more. In some embodiments, the polymer comprises about 5 monomeric units or more, about 10 monomeric units or more, about 15 monomeric units or more, about 20 monomeric units or more, about 30 monomeric units or more, about 50 monomeric units or more, about 100 monomeric units or more, about 200 monomeric units or more, about 300 monomeric units or more, or about 400 monomeric units or more.

[0244] In some embodiments, the polymer comprises from about 3 monomer units to about 400 monomer units. In some embodiments, the polymer comprises from about 3 monomer units to about 4 monomer units, from about 3 monomer units to about 10 monomer units, from about 3 monomer units to about 30 monomer units, from about 3 monomer units to about 50 monomer units, from about 3 monomer units to about 80 monomer units, from about 3 monomer units to about 100 monomer units, from about 3 monomer units to about 120 monomer units, from about 3 monomer units to about 150 monomer units, from about 3 monomer units to about 200 monomer units, from about 3 monomer units to about 400 monomer units, from about 4 monomer units to about 10 monomer units, from about 4 monomer units to about 30 monomer units, from about 4 monomer units to about 50 monomer units, 1 monomer unit, about 4 monomer units to about 80 monomer units, about 4 monomer units to about 100 monomer units, about 4 monomer units to about 120 monomer units, about 4 monomer units to about 150 monomer units, about 4 monomer units to about 200 monomer units, about 4 monomer units to about 400 monomer units, about 10 monomer units to about 30 monomer units, about 10 monomer units to about 50 monomer units, about 10 monomer units to about 80 monomer units, about 10 monomer units to about 100 monomer units, about 10 monomer units to about 120 monomer units, about 10 monomer units to about 150 monomer units, about 10 monomer units to about 200 monomer units, about 10 monomer units to about 400 monomer units, about 30 monomer units to about 50 monomer units, about 30 monomer units to about 80 monomer units, about 30 monomer units to about 100 monomer units, about 30 monomer units to about 120 monomer units, about 30 monomer units to about 150 monomer units, about 30 monomer units to about 200 monomer units, about 30 monomer units to about 400 monomer units, about 50 monomer units to about 80 monomer units, about 50 monomer units to about 100 monomer units, about 50 monomer units to about 120 monomer units, about 50 monomer units to about 150 monomer units, about 50 monomer units to about 200 monomer units, about 50 monomer units to about 400 monomer units, about 80 monomer units to about 100 monomer units, about 80 monomer units to about 120 monomer units, about 80 monomer units to about 150 monomer units, about 80 monomer units to about 200 monomer units, about 80 monomer units to about 400 monomer units, about 100 monomer units to about 120 monomer units, about 100 monomer units to about 150 monomer units, about 100 monomer units to about 200 monomer units, about 100 monomer units to about 400 monomer units, about 120 monomer units to about 150 monomer units, about 120 monomer units to about 200 monomer units,In some embodiments, the polymer comprises at least about 3 monomeric units, about 4 monomeric units, about 10 monomeric units, about 30 monomeric units, about 50 monomeric units, about 80 monomeric units, about 100 monomeric units, about 120 monomeric units, about 150 monomeric units, about 200 monomeric units, or about 400 monomeric units. In some embodiments, the polymer comprises at least about 3 monomeric units, about 4 monomeric units, about 10 monomeric units, about 30 monomeric units, about 50 monomeric units, about 80 monomeric units, about 100 monomeric units, about 120 monomeric units, about 150 monomeric units, or about 200 monomeric units. In some embodiments, the polymer comprises up to about 4 monomer units, about 10 monomer units, about 30 monomer units, about 50 monomer units, about 80 monomer units, about 100 monomer units, about 120 monomer units, about 150 monomer units, about 200 monomer units, or about 400 monomer units. Each monomer unit comprises C, 1-20 Heteroalkyl side chains.

[0245] In some embodiments, the polymer is a polyacrylate, also known as poly(acrylate) or poly(alkyl acrylate). In some embodiments, the polymer has monomeric units comprising acrylate (also known as acrylic ester or alkyl acrylate). In some embodiments, the polymer is a polyacrylamide. In some embodiments, the polymer has monomeric units comprising acrylamide (also known as acrylic amide). In some embodiments, each A 2 、B 2 、C 2 and D 2 Contains acrylates or acrylamides.

[0246] In some embodiments, the monomer unit has a structure represented by the following formula or a pharmaceutically acceptable salt thereof:

[0247]

[0248] in:

[0249] R 1 and R 3 is independently selected at each occurrence from hydrogen and methyl; and

[0250] Each R 2 For hydrogen, C 1-6 Alkyl, C 7-20Aralkyl, C 1-20 Heteroalkyl, or polyethylene glycol chain containing 1-100 ethylene glycol monomers; wherein each of said C 1-6 Alkyl, C 7-20 Aralkyl and C 1-20 Heteroalkyl is unsubstituted or substituted with one or more groups, wherein each of the one or more groups is independently -COOH, -CONH2, -NH2, -NH3 + 、-NHC(NH2 + )NH2, -NHCH3, -N(CH3)2, -N(CH3)3 + , -OH, -OCH3, -SH, -S(O)CH3, -S(O)2CH3 or -S(O)2OH; and

[0251] R 4 and R 5 Each of which is independently hydrogen, C 1-6 Alkyl, C 7-20 Aralkyl or C 1-20 heteroalkyl; wherein each of said C 1-6 Alkyl, C 7-20 Aralkyl and C 1-20 Heteroalkyl is unsubstituted or substituted with one or more groups, wherein each of the one or more groups is independently -COOH, -CONH2, -NH2, -NH3 + 、-NHC(NH2 + )NH2, -NHCH3, -N(CH3)2, -N(CH3)3 + , -OH, -OCH3, -SH, -S(O)CH3, -S(O)2CH3 or -S(O)2OH.

[0252] In some embodiments, R 4 In some embodiments, R 2 and R 5 is independently selected at each occurrence from substituted or unsubstituted C 1-6 In some embodiments, R 2 and R 5 Each independently selected from substituted C 1-6 Alkyl, wherein the alkyl group is composed of one or more selected from -NH2, -NH3 + 、-NHC(NH2 + )NH2, -NHCH3, -N(CH3)2, -N(CH3)3 + , -OH, -OCH3, -S(O)CH3, -S(O)2CH3 and -S(O)2OH or a pharmaceutically acceptable salt thereof. In some embodiments, each R 2and R 5 is independently selected from aminoalkyl, hydroxyalkyl, carboxyalkyl, alkoxy, haloalkyl, or any combination thereof. 2 In some embodiments, R 4 In some embodiments, R 5 is substituted or unsubstituted C 1-6 aminoalkyl, wherein if the aminoalkyl group is substituted, it is composed of one or more groups selected from -NH2, -NH3 + 、-NHC(NH2 + )NH2, -NHCH3, -N(CH3)2, -N(CH3)3 + , -OH, -OCH3, -S(O)CH3, -S(O)2CH3 and -S(O)2OH groups or pharmaceutically acceptable salts thereof.

[0253] In some embodiments, the monomer unit has a structure represented by the following formula or a pharmaceutically acceptable salt thereof:

[0254]

[0255] in:

[0256] R 11 and R 13 is independently selected at each occurrence from hydrogen and methyl; and

[0257] R 12 、R 14 and R 15 In each case, independently C 1-6 Alkyl, C 1-6 Heteroalkyl, C 3-10 Cycloalkyl or 3- to 10-membered heterocycloalkyl; wherein each alkyl, heteroalkyl, cycloalkyl and heterocycloalkyl is optionally substituted by one or more groups, each group independently selected from cycloalkyl, heterocycloalkyl, aryl, heteroaryl, oxo, -COOH, -CONH2, -NH2, -NH3 + 、-NHC(NH2 + )NH2, -NHCH3, -N(CH3)2, -N(CH3)3 + , -OH, -OCH3, -SH, -S(O)CH3, -S(O)2CH3 and -S(O)2OH or pharmaceutically acceptable salts thereof;

[0258] or R 14 and R 15 Together they form a substituted or unsubstituted heterocyclic ring.

[0259] In some embodiments, R 14In some embodiments, R 14 and R 15 Together they form a substituted or unsubstituted heterocyclic ring (e.g., a 3- to 10-membered heterocycloalkyl ring). 14 and R 15 Together they form an unsubstituted or substituted aziridine, an unsubstituted or substituted azetidine, an unsubstituted or substituted pyrrolidine, an unsubstituted or substituted piperidine, an unsubstituted or substituted piperazine, an unsubstituted or substituted morpholine, an unsubstituted or substituted azepane, an unsubstituted or substituted azocane, and the like. In some embodiments, R 14 is hydrogen, and R 15 C 1-6 Alkyl, where C 1-6 The alkyl group is substituted with a heterocycle which is unsubstituted or substituted aziridine, unsubstituted or substituted azetidine, unsubstituted or substituted pyrrolidine, unsubstituted or substituted piperidine, unsubstituted or substituted piperazine, unsubstituted or substituted morpholine, unsubstituted or substituted azepane, unsubstituted or substituted azacyclooctane, and the like.

[0260] In some embodiments, the polymer has the structure of Formula YB:

[0261]

[0262] Each monomer unit (i.e. each ) has a structure represented by one of the following formulae:

[0263]

[0264] Each R 1 、R 2 、R 3 、R 4 、R 5 、R 11 、R 12 、R 13 、R 14 and R 15 As defined above.

[0265] In some embodiments, the polymer has a structure of Formula Y-B1 or Formula Y-B2:

[0266]

[0267] In some embodiments, the monomeric unit is selected from:

[0268]

[0269] Functional group ("Z")

[0270] The compounds disclosed herein comprise a functional group "Z" that is bonded to a polymer "Y" either via a bond or optionally separated by a linker. The functional group can be any functional group known in the art. Examples of functional groups include amines, amides, alcohols, acids, esters, thiols, sulfides, sulfoxides, halogens, nitriles, carbocycles, and heterocycles. As used herein, the functional groups disclosed herein typically comprise a sulfur group (e.g., -SR 6 In some embodiments, the functional group comprises a reactive group, a charged group, a detectable group, a blocking group, or a combination thereof.

[0271] The functional group can be or include a reactive group, a charged group, a detectable group, a capping group, a binding group, a peptide group, a therapeutic group, a chelating group, a temperature-sensitive group, a photosensitive group, a radioactive group, a cytotoxic group, or a combination thereof.

[0272] In some embodiments, the functional group is a thiol or mercapto group, such as -SH. In some embodiments, the functional group is a sulfide, such as -SR 6 In some embodiments, the functional group is a hydroxyl group, such as -OH. In some embodiments, the functional group is an ether group, such as -OR 6 In some embodiments, the functional group is a sulfoxide, such as -S(O)R 6 , or sulfone, such as -S(O)2R 6 .

[0273] In some embodiments, R 6 is a group consisting of 1 to about 200 atoms selected from hydrogen, halogen, C, N, O, and S. In some embodiments, R 6 In some embodiments, R 6 C 1-100 In some embodiments, R 6 C 1-20 In some embodiments, R 6 The invention also includes sulfur atoms having attached thereto an alkyl or heteroalkyl group, each consisting of 1 to about 200 atoms, wherein the non-carbon atoms of the heteroalkyl group are selected from hydrogen, halogen, nitrogen, oxygen, and sulfur. Salts of these groups, such as sodium, lithium, potassium, magnesium, calcium, chloride, nitrate, phosphate, and the like, are considered within the scope of the invention.

[0274] In some embodiments, R 6 Contains a reactive group, a charged group, a detectable group, a peptide group, a blocking group, or a combination thereof.

[0275] In some embodiments, R 6The reactive group of comprises an azide or an alkyne. In some embodiments, R is reacted via "click" chemistry or "click" reaction, particularly [3+2] cycloaddition, such as Huisgen 1,3-dipolar cycloaddition, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAc), strain-promoted azide-alkyne cycloaddition (SPAAC), strain-promoted alkyne-nitrone cycloaddition (SPANC). 6 The reactive group of is capable of reacting with another molecule containing an alkyne or an azide. In some embodiments, the alkyne is a cyclooctyne, such as dibenzylcyclooctyne, biarylazacyclooctynone and fluorinated cyclooctyne, or bicyclononyne.

[0276] In some embodiments, R 6 The charged groups of include one or more cationic groups. In some embodiments, R 6 The one or more cationic groups of R include cyclic amines, primary amines, guanidines, or combinations thereof. 6 The charged groups include multiple cationic groups (e.g., 3 or more cationic groups). Cationic groups as described herein may include amines or other organic groups that can stably maintain a positive charge or a partial positive charge at physiologically relevant pH. For example, primary amines, secondary amines, tertiary amines, or quaternary amines may all be considered cationic groups. In particular, quaternary amines have a positive (cationic) charge, regardless of the protonation state, and can be used to prepare the compounds described herein.

[0277] In some embodiments, R 6 The detectable group comprises a fluorophore, a dye or Resonance Energy Transfer (FRET) donors or acceptors. In some embodiments, fluorophores (also known as fluorescent dyes, chromophores, or fluorescent probes), dyes, or FRET donors or acceptors are fluorescent compounds that can re-emit light upon excitation. Examples of fluorophores, dyes, FRET donors or acceptors include, but are not limited to, organic dyes (e.g., fluorescein, rhodamine, coumarin, and their derivatives), biological fluorophores (e.g., green fluorescent protein, phycoerythrin, allophycocyanin), and quantum dots. In some embodiments, R 6 The detectable group comprises a fluorescein compound. In some embodiments, R 6 The detectable group comprises a rhodamine compound. In some embodiments, R 6The detectable group comprises a coumarin compound. In some embodiments, the detectable group comprises fluorescein, rhodamine, or a derivative of a coumarin compound. In some embodiments, the detectable group comprises or is derived from a fluorescent azide (e.g., azidefluor-488).

[0278] In some embodiments, the detectable group comprises a near-infrared fluorescent probe, which is a molecule or portion of a molecule that emits a signal in response to light in the near-infrared portion of the spectrum. Other detectable groups considered within the scope of the present invention include molecules or portions of molecules that emit a signal in response to an excitation source comprising infrared, near-infrared, visible, or ultraviolet light.

[0279] In some embodiments, R 6 The end-capping group is an inert group. In some embodiments, the inert group is a chemically inert group or a chemically non-reactive group. In some embodiments, the end-capping group is a hydrocarbon (e.g., C 1-20 In some embodiments, the capping group is a thiol.

[0280] Functional groups can include binding groups. For example, binding groups can be small molecules that can bind to other atoms or molecules. Binding groups can include chelating agents that are configured to bind atoms such as metals. In a specific example, the compound can include a functional group that is a chelating agent known in the art (e.g., DOTA, DOTA-TATE, DOTATOC), optionally chelated to a metal atom. The metal atom can be an ion or an oxide. Specifically, the metal atom can be a lanthanide or an actinide. In some embodiments, the metal atom is an alpha emitter, a beta emitter, or a gamma emitter. In some embodiments, the metal atom is actinium, yttrium, gadolinium, actinium, lutetium, or an isotope thereof.

[0281] The functional group may also comprise a peptide group. In some cases, the functional group comprises a targeting group (e.g., wherein the peptide group is the targeting group). The targeting group may be a peptide. The targeting group may be a cyclic peptide. In one embodiment, the functional group comprises a targeting integrin receptor (e.g., α v3-integrin receptor). For example, the functional group may comprise an RGD peptide. Radiolabeled cyclic peptides containing the (Arg-Gly-Asp)RGD sequence have been reported for use in positron emission tomography (PET) imaging, single photon emission computed tomography (SPECT) imaging, and targeted radionuclide therapy of cancer. Any RGD peptide known in the art can be attached as a functional group as disclosed herein. In one embodiment, a functional group comprising a cyclic (-Arg-Gly-Asp-D-Phe-Lys) ("cRGDfK") group was synthesized. Radiolabeled analogs, salts, or radioisotope derivatives thereof are also contemplated within the scope of the present invention. In examples having a functional group comprising a cRGDfK group, the cRGDfK group can be linked to the polymer (Y), for example, via a lysine nitrogen atom, and optionally via a linker. As used herein, the term "linker" includes any alkyl, heteroalkyl, cyclyl or heterocyclyl group or any combination thereof, which contains 1-100 atoms selected from C, H, N, O and S. The linker can include, for example, maleimide, ethylene units, propylene units, amides, amines, esters, ethers, thioethers, polyethylene glycol chains, alkyl chains, click reagents, peptides or any combination thereof. In some embodiments, the functional group (optionally comprising a linker) is any one of the groups (Z) shown in Table 1.

[0282] Functional groups can include therapeutic groups. For example, functional groups can include drugs connected through cleavable linkers. As mentioned above, therapeutic groups can also be binding agents, or radiotherapy groups comprising chelating agents and radioisotopes. In some embodiments, functional groups include chelating groups. Chelating groups can be coordinated with metal ions. Chelating groups can be used as therapeutic agents or as detectable agents (for example, in PET or SPECT imaging). Chelating groups can be cyclic or non-cyclic and often include two or more basic amines or acidic carboxylates. An example of a chelating functional group is an amino polycarboxylic acid group, nitrilotriacetic acid (NTA). Other amino polycarboxylic acid chelating agents include NTA, EDTA, DTPA, EGTA, BAPTA, NOTA, DOTA and derivatives thereof.

[0283] In some embodiments, the functional group (Z) is selected from:

[0284] -SH,

[0285] wherein the functional group is bonded to the polymer via the left sulfur atom of each of the aforementioned structures.

[0286] In another aspect, provided herein is a compound of Formula I or a pharmaceutically acceptable salt thereof:

[0287] X—Y—Z;

[0288] in:

[0289] X is a lipid selected from XA, XB and XC:

[0290] XA:

[0291] XB:

[0292] XC:

[0293] Y is a polymer having a structure as described below by formula YA, YB, YC or YD; and

[0294] Z is a functional group as described below.

[0295] Specific embodiments of the present invention are disclosed in Table 1 below.

[0296] Table 1.

[0297]

[0298]

[0299]

[0300]

[0301] In some embodiments, the compound is configured to encapsulate or complex with a nucleic acid in an aqueous solution. In some embodiments, the compound is substantially non-toxic.

[0302] In some embodiments, the compound is biodegradable. In some embodiments, the compound comprises a molecular weight of about 1 kDa to about 100 kDa. In some embodiments, the compound comprises the following molecular weights: about 1 kDa to about 10 kDa, about 1 kDa to about 20 kDa, about 1 kDa to about 50 kDa, about 1 kDa to about 70 kDa, about 1 kDa to about 90 kDa, about 1 kDa to about 100 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 50 kDa, about 10 kDa to about 70 kDa, about 10 kDa to about 90 kDa, about 1 In some embodiments, the compound comprises a molecular weight of about 1 kDa, about 10 kDa, about 20 kDa, about 50 kDa, about 70 kDa, about 90 kDa, about 20 kDa to about 100 kDa, about 50 kDa to about 70 kDa, about 50 kDa to about 90 kDa, about 50 kDa to about 100 kDa, about 70 kDa to about 90 kDa, about 70 kDa to about 100 kDa, or about 90 kDa to about 100 kDa. In some embodiments, the compound comprises a molecular weight of at least about 1 kDa, about 10 kDa, about 20 kDa, about 50 kDa, about 70 kDa, about 90 kDa, or about 100 kDa. In some embodiments, the compound comprises a molecular weight of at most about 10 kDa, about 20 kDa, about 50 kDa, about 70 kDa, about 90 kDa, or about 100 kDa.

[0303] Liposomes and nanoparticles

[0304] The lipid-polymer compounds disclosed herein can be used to prepare liposomes and / or lipid nanoparticles. For example, the PLip disclosed herein can be used for various purposes. For example, the PLip disclosed herein can increase the stability or other practicality of lipid nanoparticles (LNPs). Lipid nanoparticles are generally spherical vesicles made of ionizable lipids that can be positively charged at low pH (allowing RNA to complex) and neutral at physiological pH (reducing potential toxic effects compared to positively charged lipids such as liposomes). Due to their size and properties, lipid nanoparticles can be taken up by cells via endocytosis, and the ionization of lipids at low pH can promote endosomal escape, thereby allowing the payload to be released into the cytoplasm of target cells. The PLip disclosed herein can be used to prepare lipid nanoparticles (LNPs). Therefore, the use of the PLip disclosed herein for preparing lipid nanoparticles, as well as any lipid nanoparticles comprising the PLip disclosed herein (or "compound"), are within the scope of the present invention. The PLip disclosed herein can contribute to the stability of LNPs (e.g., stabilized PLip). In many cases, PLip can have more than one practicality. For example, PLip can be both stabilized and reactive PLip, which means that PLip has a stabilized polymer block and the functional group comprises a reactive portion (e.g., strained cyclooctyne or azide). Similarly, PLip can be cationic and comprise a reactive portion. In some cases, the reactive portion reacts with a dye to form fluorescently labeled LNPs. Stabilized PLip can be used as an alternative to PEG, which has shown toxicity and problematic side effects in some biological systems. Stabilized PLip can comprise a PEG side chain, or a miscellaneous alkyl acrylate or acrylamide side chain.

[0305] Cationic PLip may comprise a plurality of cationic groups. In some cases, cationic PLip comprises cationic monomer units between about 3 and about 20. In some embodiments, cationic PLip may replace the cationic lipids used in LNP formulations. Cationic PLip may be used as a transfection reagent (e.g., wherein the cationic amine (N) non-covalently interacts with the phosphate backbone (P) of the nucleic acid). In some cases, the N:P ratio is about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 8:1, about 10:1, about 16:1, about 20:1, about 32:1, about 50:1, about 64:1, about 100:1, about 128:1, about 150:1, about 200:1 or more. In a specific example, the N:P ratio is between about 1:4 and about 128:1. In a more specific example, the N:P ratio is about 4:1 to about 16:1. Cationic PLip may have a positive charge. In some instances, the cationic PLip has a positive charge of 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 or more. The positive charge can be directly related to the polymer length of the polymer comprising the cationic side chain. For example, a cationic PLip having a polymer comprising 19 cationic monomer units can have a +19 charge. Similarly, a cationic PLip having a polymer comprising 13 cationic monomer units can have a +13 charge. In other instances, the polymer can comprise a block of two repeating monomer units, wherein the first block comprises a stabilizing side chain, and wherein the second block comprises a cationic side chain. In this case, the stabilizing polymer block can exceed 2 times, 3 times, 4 times, 5 times, 8 times, 10 times, 16 times, 20 times, 32 times, 50 times, 64 times or 100 times or more of the cationic block length. For example, in some cases, the polymer comprises a stabilizing block comprising about 100 to about 200 (e.g., about 170 to about 190) monomer units, and a second cationic block comprising monomer units comprising about 3 to about 30 cationic monomer units (e.g., about 3 to about 10 monomer units). In alternative embodiments, the polymer comprises blocks of alternating monomer units, wherein the first monomer unit has a stabilizing (uncharged) side chain, and wherein the second monomer unit has a cationic side chain.

[0306] Lipid-polymer compounds disclosed herein (e.g., cationic PLip) can have practicality such as preparing nanoparticles and / or transfection reagents. As used herein, "nanoparticles" generally refer to aggregates of compounds that can form vesicles. As described herein, nanoparticles can be configured to encapsulate one or more nucleic acid molecules, thereby producing transfection reagents, i.e., reagents configured to transfer genetic information to host cells. For example, nanoparticles can be configured to encapsulate nucleic acids (e.g., DNA, RNA, etc.) or form complexes with nucleic acids. Nanoparticles prepared as described herein can be configured for the compound of the encapsulation or the nucleic acid at a ratio of 0.3: 1-100: 1 (weight: weight). For example, the transfection reagent encapsulates one or more nucleic acids or is complexed with one or more nucleic acids at a ratio of 0.5:1-100:1, 1:1-100:1, 5:1-100:1, 10:1-100:1, 20:1-100:1, 30:1-100:1, 40:1-100:1, 50:1-100:1, 60:1-100:1, 70:1-100:1, 80:1-100:1, 90:1-100:1 or 95:1-100:1 (weight:weight). Generally, a transfection reagent refers to a combination of nanoparticles and genetic material, while a nanoparticle refers to the vesicle itself. Additionally, the nanoparticles can comprise a variety of compounds or PLips disclosed herein. The advantages of nanoparticles and transfection reagents comprising compounds or PLips disclosed herein are apparent throughout the examples.

[0307] In some embodiments, encapsulation or complexation of nucleic acids increases the half-life of the nucleic acids under aqueous or physiological conditions by at least 2-fold. In some embodiments, encapsulation or complexation of nucleic acids increases the half-life of the nucleic acids under aqueous or physiological conditions by at least 1.1-fold, at least 1.3-fold, at least 1.5-fold, at least 1.7-fold, at least 2-fold, at least 2.5-fold, or at least 3-fold.

[0308] In some embodiments, the encapsulation or complexation inhibits nuclease digestion of the nucleic acid. In some embodiments, the encapsulation or complexation of the nucleic acid produces a transfection complex having an average size of 20-2000 nm. In some embodiments, the encapsulation or complexation of the nucleic acid produces a transfection complex having an average size of about 20 nm to about 2000 nm. In some embodiments, the encapsulation or complexation of the nucleic acid produces a transfection complex having an average size of about 20 nm to about 30 nm, about 20 nm to about 50 nm, about 20 nm to about 100 nm, about 20 nm to about 200 nm, about 20 nm to about 300 nm, about 20 nm to about 400 nm, about 20 nm to about 500 nm, about 20 nm to about 1,000 nm, about 20 nm to about 1,500 nm, about 20 nm to about 2,000 nm, about 30 nm to about 50 nm, about 30 nm to about 100 nm, about 30 nm to about 200 nm, about 30 nm to about 400 nm, about 20 nm to about 500 nm, about 20 nm to about 1,000 nm, about 20 nm to about 1,500 nm, about 20 nm to about 2,000 nm, about 30 nm to about 50 nm, about 30 nm to about 100 nm, about 30 nm to about 200 nm, about 30 nm to about 300 nm, about 30 nm to about 400 nm, about 30 nm to about 500 nm, about 30 nm to about 1,000 nm, about 30 nm to about 1,500 nm, about 30 nm to about 2,000 nm, about 50 nm to about 100 nm, about 50 nm to about 200 nm, about 50 nm to about 300 nm, about 50 nm to about 400 nm, about 50 nm to about 500 nm, about 50 nm to about 1,000 nm, about 50 nm to about 1,500 nm, about 50 nm to about 2,000 nm, about 100 nm to about 200 nm, about 10 0 nm to about 300 nm, about 100 nm to about 400 nm, about 100 nm to about 500 nm, about 100 nm to about 1,000 nm, about 100 nm to about 1,500 nm, about 100 nm to about 2,000 nm, about 200 nm to about 300 nm, about 200 nm to about 400 nm, about 200 nm to about 500 nm, about 200 nm to about 1,000 nm, about 200 nm to about 1,500 nm, about 200 nm to about 2,000 nm, about 300 nm to about 400 nm, about 300 nm to about 500 nm, about 30 In some embodiments, the present invention relates to a nanostructured carbonyl group comprising: a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group,In some embodiments, the encapsulation or complexation of nucleic acids produces transfection complexes having an average size of about 20 nm, about 30 nm, about 50 nm, about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 1,000 nm, about 1,500 nm, or about 2,000 nm. In some embodiments, the encapsulation or complexation of nucleic acids produces transfection complexes having an average size of at least about 20 nm, about 30 nm, about 50 nm, about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 1,000 nm, or about 1,500 nm. In some embodiments, the encapsulation or complexation of nucleic acids produces transfection complexes having an average size of at most about 30 nm, about 50 nm, about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 1,000 nm, about 1,500 nm, or about 2,000 nm. In some embodiments, the encapsulation or complexation of nucleic acids produces transfection complexes having an average radius of about 100 nm to 300 nm (e.g., about 100 to about 200 nm). In some embodiments, the LNPs have an average radius of about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, or about 200 nm. In some embodiments, temperature-sensitive LNPs can induce temperature-dependent expansion of the LNPs, for example, from a radius of about 180 nm to a radius of about 300 nm. In some examples, temperature-sensitive LNP expansion can be a reversible process (e.g., wherein less than about 10%, less than about 5%, less than about 3%, less than about 2%, or less than about 1% of the LNP particles degrade or aggregate after heating (e.g., to about 45° C.)). The process can be completely or nearly completely reversible (i.e., less than about 2% loss of LNP per thermal cycle).

[0309] In some embodiments, encapsulation or complexation comprises adsorption of at least a subset of the nucleic acids to the surface of the transfection reagent. In some embodiments, encapsulation or complexation of the nucleic acids produces a transfection complex configured for cellular uptake. In some embodiments, cellular uptake comprises endocytosis.

[0310] In some embodiments, encapsulation or complexation of nucleic acids produces a transfection complex comprising a water solubility of at least 5 μg / mL. In some embodiments, encapsulation or complexation of nucleic acids produces a transfection complex comprising a water solubility of at least 1 μg / mL, at least 5 μg / mL, at least 10 μg / mL, at least 50 μg / mL, at least 100 μg / mL, at least 200 μg / mL, at least 500 μg / mL, at least 1000 μg / mL, at least 1500 μg / mL, at least 2000 μg / mL, at least 2500 μg / mL, at least 3000 μg / mL, at least 3500 μg / mL, at least 4000 μg / mL, at least 4500 μg / mL, or at least 5000 μg / mL.

[0311] In some embodiments, encapsulation or complexation of nucleic acids produces transfection complexes comprising about 10 μg / mL to about 50 μg / mL water solubility. In some embodiments, encapsulation or complexation of nucleic acids produces transfection complexes comprising about 1 μg / mL to about 100 μg / mL water solubility. In some embodiments, encapsulation or complexation of nucleic acids produces a transfection complex comprising a water-soluble mixture of about 1 μg / mL to about 5 μg / mL, about 1 μg / mL to about 10 μg / mL, about 1 μg / mL to about 20 μg / mL, about 1 μg / mL to about 30 μg / mL, about 1 μg / mL to about 40 μg / mL, about 1 μg / mL to about 50 μg / mL, about 1 μg / mL to about 100 μg / mL, about 5 μg / mL to about 10 μg / mL, about 5 μg / mL to about 20 μg / mL, about 5 μg / mL to about 30 μg / mL, about 5 μg / mL to about 40 μg / mL, about 5 μg / mL to about 50 μg / mL, about 5 μg / mL to about 100 μg / mL, about 10 μg / mL to about In some embodiments, the present invention provides an aqueous solution of at least about 20 μg / mL, about 10 μg / mL to about 30 μg / mL, about 10 μg / mL to about 40 μg / mL, about 10 μg / mL to about 50 μg / mL, about 10 μg / mL to about 100 μg / mL, about 20 μg / mL to about 30 μg / mL, about 20 μg / mL to about 40 μg / mL, about 20 μg / mL to about 50 μg / mL, about 20 μg / mL to about 100 μg / mL, about 30 μg / mL to about 40 μg / mL, about 30 μg / mL to about 50 μg / mL, about 30 μg / mL to about 100 μg / mL, about 40 μg / mL to about 50 μg / mL, about 40 μg / mL to about 100 μg / mL, or about 50 μg / mL to about 100 μg / mL. In some embodiments, the encapsulation or complexation of nucleic acids produces a transfection complex comprising a water solubility of about 1 μg / mL, about 5 μg / mL, about 10 μg / mL, about 20 μg / mL, about 30 μg / mL, about 40 μg / mL, about 50 μg / mL, or about 100 μg / mL. In some embodiments, the encapsulation or complexation of nucleic acids produces a transfection complex comprising a water solubility of at least about 1 μg / mL, about 5 μg / mL, about 10 μg / mL, about 20 μg / mL, about 30 μg / mL, about 40 μg / mL, or about 50 μg / mL. In some embodiments, the encapsulation or complexation of nucleic acids produces a transfection complex comprising a water solubility of at most about 5 μg / mL, about 10 μg / mL, about 20 μg / mL, about 30 μg / mL, about 40 μg / mL, about 50 μg / mL, or about 100 μg / mL.

[0312] In some embodiments, encapsulation or complexation of nucleic acids produces a transfection complex comprising a water solubility of about 5 μg / mL to about 5,000 μg / mL. In some embodiments, encapsulation or complexation of nucleic acids produces a transfection complex comprising a water solubility of about 5 μg / mL to about 50 μg / mL, about 5 μg / mL to about 100 μg / mL, about 5 μg / mL to about 500 μg / mL, about 5 μg / mL to about 1,000 μg / mL, about 5 μg / mL to about 2,000 μg / mL, about 5 μg / mL to about 3,000 μg / mL, about 5 μg / mL to about 4,000 μg / mL, about 5 μg / mL to about 5,000 μg / mL, about 50 μg / mL to about 100 μg / mL, about mL, about 50 μg / mL to about 500 μg / mL, about 50 μg / mL to about 1,000 μg / mL, about 50 μg / mL to about 2,000 μg / mL, about 50 μg / mL to about 3,000 μg / mL, about 50 μg / mL to about 4,000 μg / mL, about 50 μg / mL to about 5,000 μg / mL, about 100 μg / mL to about 500 μg / mL, about 100 μg / mL to about 1,000 μg / mL, about 100 μg / mL to about 2,000 μg / mL, about 100 μg / mL to about 3 ,000μg / mL, about 100μg / mL-about 4,000μg / mL, about 100μg / mL-about 5,000μg / mL, about 500μg / mL-about 1,000μg / mL, about 500μg / mL-about 2,000μg / mL, about 500μg / mL-about 3,000μg / mL, about 500μg / mL-about 4,000μg / mL, about 500μg / mL-about 5,000μg / mL, about 1,000μg / mL-about 2,000μg / mL, about 1,000μg / mL-about 3 ,000 μg / mL, about 1,000 μg / mL to about 4,000 μg / mL, about 1,000 μg / mL to about 5,000 μg / mL, about 2,000 μg / mL to about 3,000 μg / mL, about 2,000 μg / mL to about 4,000 μg / mL, about 2,000 μg / mL to about 5,000 μg / mL, about 3,000 μg / mL to about 4,000 μg / mL, about 3,000 μg / mL to about 5,000 μg / mL, or about 4,000 μg / mL to about 5,000 μg / mL. In some embodiments, encapsulation or complexation of nucleic acids produces a transfection complex comprising a water solubility of about 5 μg / mL, about 50 μg / mL, about 100 μg / mL, about 500 μg / mL, about 1,000 μg / mL, about 2,000 μg / mL, about 3,000 μg / mL, about 4,000 μg / mL, or about 5,000 μg / mL.In some embodiments, encapsulation or complexation of nucleic acids produces a transfection complex comprising a water solubility of at least about 5 μg / mL, about 50 μg / mL, about 100 μg / mL, about 500 μg / mL, about 1,000 μg / mL, about 2,000 μg / mL, about 3,000 μg / mL, or about 4,000 μg / mL. In some embodiments, encapsulation or complexation of nucleic acids produces a transfection complex comprising a water solubility of at most about 50 μg / mL, about 100 μg / mL, about 500 μg / mL, about 1,000 μg / mL, about 2,000 μg / mL, about 3,000 μg / mL, about 4,000 μg / mL, or about 5,000 μg / mL.

[0313] In some embodiments, the compound has a dispersivity (i.e., molecular weight distribution) of about 2.0 or less. In some embodiments, the compound has a dispersivity of about 1.5 or less. In some embodiments, the compound has a dispersivity of about 1.3 or less. In some embodiments, the compound has a dispersivity of about 1.2 or less. In some embodiments, the compound has a dispersivity of at least 1.0. In some embodiments, the compound has a dispersivity of at least 1.1. In some embodiments, the compound has a dispersivity of at least 1.2. In some embodiments, the compound has a dispersivity of at least 1.3. In some embodiments, the compound has a dispersivity of at least 1.4. In some embodiments, the compound has a dispersivity of at least 1.5. In some embodiments, the compound has a dispersivity of about 1.1 to about 1.5. In some embodiments, the compound has a dispersivity of about 1.2 to about 1.5. In some embodiments, the compound has a dispersivity of about 1.3 to about 1.5. In some embodiments, the compound has a dispersivity of about 1.2 to about 1.8. In some embodiments, the compound has a dispersibility of about 1.5 to about 1.8. In some embodiments, the compound has a dispersibility of about 1.5 to about 2.0. In some embodiments, the compound has a dispersibility of about 1.8 to about 2.0. In some embodiments, the compound has a dispersibility of about 1.8 to about 2.3. In some embodiments, the compound has a dispersibility of about 2.0 to about 2.3. In some embodiments, the compound has a dispersibility of about 2.0 to about 2.5. In some embodiments, the compound has a dispersibility of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 3.0, or less.

[0314] Transfection reagent

[0315] In one aspect, provided herein is a reagent (e.g., transfection reagent) comprising a compound disclosed herein and a nucleic acid. Transfection reagents can be used as delivery vehicles for payloads (e.g., bioactive compounds (e.g., nucleic acids). Payload can be specifically matched with a given transfection reagent, or a transfection reagent can be capable of delivering various payloads. Transfection reagents can provide a variety of advantageous properties to help payloads be effectively delivered to a target (e.g., a target cell). For example, a transfection reagent can include liposomes or lipid nanoparticles (LNPs). Liposomes and LNPs can be used as microvesicles, have an approximately spherical shape, and contain an interior and exterior. Advantageously, by encapsulating payload in the interior of a liposome or LNP, payloads can be shielded from the effects of biological and chemical processes that would otherwise degrade the payload. Alternatively, the encapsulated payload can be located within a lipid bilayer, rather than within an internal compartment. In still another example, the internal compartment can be customized to stabilize a specific payload.

[0316] In another example, the transfection reagent can form a lipid bilayer around the payload, which, upon contact with the target cell, can fuse with the cell and release the payload inside the cell. The transfection reagent can also physically exclude enzymes or ribozymes, or can isolate the internal payload from external changes in the environment (e.g., pH, ion concentration, etc.).

[0317] In another aspect, the transfection reagent comprising the compound disclosed herein can directly interact with the payload, such as to stabilize or chelate certain groups. For example, the transfection reagent comprising the compound disclosed herein can form polarity, ions or other non-covalent interactions with the nucleic acid payload. Although some vesicles may contain simple lipid shells and aqueous internal compartments, other vesicles (such as LNPs) may have compounds distributed in the internal space (such as interacting with the payload of internalization). In a specific example, the transfection reagent comprising the compound disclosed herein simultaneously (a) produces a vesicle that separates the internal compartment from the external environment, and (b) encapsulates the internal payload with the compound disclosed herein. It is particularly useful that the polymer disclosed herein may include a hydrocarbon backbone capable of forming a double layer, and the polymer side chain may form a stabilization interaction with the nucleic acid payload (such as the phosphate backbone of the nucleic acid payload). The nucleic acid payload may include DNA or RNA (such as siRNA, saRNA, mRNA, microRNA, etc.).

[0318] In still another aspect, the transfection reagent disclosed herein may include a lipid-polymer compound having a functional group used as a reporter portion or a reactive portion. In addition to the above-mentioned stabilization, shielding, and delivering the payload to the intracellular space of the target cell, the transfection reagent comprising the reporter compound or the reactive compound can be fused with the target cell, thereby becoming incorporated into or inserted into the target cell. In a specific example, a transfection reagent comprising a polymer-lipid compound with a fluorescent functional group can deliver the nucleic acid payload to the target cell, and when fused with the target cell, the target cell is labeled with the fluorescent functional group. Similarly, a transfection reagent comprising a reactive functional group (e.g., a click handle) can contact and be inserted into the target cell, thereby providing a reactive handle on the target cell. Other functional groups disclosed herein can be similarly incorporated into the target cell, thereby functionalizing the target cell with the functional group.

[0319] The nucleic acid may include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), or a combination thereof. In some embodiments, the nucleic acid comprises about 1 kb to about 100 kb (e.g., about 1 kb to about 2 kb, about 1 kb to about 5 kb, about 1 kb to about 8 kb, about 1 kb to about 10 kb, about 1 kb to about 12 kb, about 1 kb to about 15 kb, about 1 kb to about 20 kb, about 1 kb to about 50 kb, about 1 kb to about 100 kb, about 2 kb to about 5 kb, about 2kb to about 8kb, about 2kb to about 10kb, about 2kb to about 12kb, about 2kb to about 15kb, about 2kb to about 20kb, about 2kb to about 50kb, about 2kb to about 100kb, about 5kb to about 8kb, about 5kb to about 10kb, about 5kb to about 12kb, about 5kb to about 15kb, about 5kb to about 20kb, about 5kb to about 50kb, about 5kb to about 100kb, about 8kb to about 10kb, about 8kb to about 12kb, about 8kb to about 15kb, about 8kb to about 20kb, about 8kb to about 50kb, about 8kb to about 100kb, about 10kb to about 12kb, about 10kb to about 15kb, about 10kb to about 20kb, about 10kb to about 50kb, about 10 kb to about 100 kb, about 12 kb to about 15 kb, about 12 kb to about 20 kb, about 12 kb to about 50 kb, about 12 kb to about 100 kb, about 15 kb to about 20 kb, about 15 kb to about 50 kb, about 15 kb to about 100 kb, about 20 kb to about 50 kb, about 20 kb to about 100 kb, or about 50 kb to about 100 kb). The nucleic acid can comprise about 1 kb, about 2 kb, about 5 kb, about 8 kb, about 10 kb, about 12 kb, about 15 kb, about 20 kb, about 50 kb, or about 100 kb. In some embodiments, the nucleic acid comprises at least about 1 kb, about 2 kb, about 5 kb, about 8 kb, about 10 kb, about 12 kb, about 15 kb, about 20 kb, or about 50 kb. In some embodiments, the nucleic acid comprises at most about 2 kb, about 5 kb, about 8 kb, about 10 kb, about 12 kb, about 15 kb, about 20 kb, about 50 kb, or about 100 kb. In some cases, the nucleic acid comprises about 2 kb-about 20 kb. (For example, about 5 kb-about 15 kb (for example, about 8 kb-about 12 kb)). In a specific example, the nucleic acid comprises about 10 kb.

[0320] Chemical transfection of nucleic acids can provide a convenient and robust alternative to viral, liposome encapsulation and electroporation delivery. After complexing with nucleic acids, the physical properties of the resulting nucleic acid complex can change over time and can affect the functional performance of the complex. In some cases, a transfection complex with a radius of 200-400 nm can be the best choice for many applications. Complex formation time can be one of the main parameters that can be changed in order to control the size of the transfection complex to achieve nucleic acid delivery. Although the functional performance of the transfection complex may not be entirely due to the size of the complex, dynamic light scattering (DLS) can be used to track changes in the size of the aggregates as they continue to grow over time. Various factors, including the composition, salt and pH of the transfection reagent, can affect the size and functional performance of the resulting complex after the addition of nucleic acids.

[0321] In some embodiments, commercial transfection reagent protocols may specify optimal complex formation times. For example, the recommended The transfection complex formation time is <5min, and The formation time of the transfection complex is 15-30 minutes. However, in some cases, including, for example, large-scale automated transfection, this time frame may not be preferred or practical because the addition and mixing of the transfection complex may require longer time. Therefore, it is desirable to design a method that uses stabilized polymer-lipid hybrids (PLips) in transfection reagent formulations prior to complexing with DNA to control or prolong the time it takes for the transfection complex to form.

[0322] In some embodiments, the optimal concentration for complex formation may be a factor of the commercial transfection reagent protocol. For example, transfection reagent complexes may be formed at an initial concentration of 10x the concentration of the complex after addition to the cells in the culture medium (assuming 10% vol / vol of the complex is added compared to the total volume of culture medium in which the cells are transfected). For a 200 L bioreactor, the volume of transfection complex to be added for a typical transfection reagent is 20 L. Adding this volume in a timely manner and maintaining the functionality of the transfection reagent complex may be difficult, and it may be preferable to add lower volumes of complex (shorter addition time and smaller volumes to process). Therefore, adding a more concentrated complex would be beneficial, unless the complex grows more rapidly and heterogeneously at higher concentrations, thereby creating quality issues (particularly in a GMP environment). Adding stabilized PLip may mitigate the growth of more concentrated transfection complexes.

[0323] Stimulus-sensitive transfection

[0324] In one aspect, the lipopolymer compounds provided herein comprise a stimulus-sensitive component in the linker region between the lipid tail and the polymer head group comprising a stabilizing monomeric unit. These types of stimulus-sensitive, stabilized PLips not only increase the stability of the transfection reagent complex, but also control the time of release of the transfection reagent in the reactor.

[0325] When nucleic acids are combined with transfection reagents (which may include a mixture of polymers and lipids), transfection complexes are formed. Through a combination of electrostatic and other non-covalent interactions, cationic, non-viral, non-liposomal formulations can bind negatively charged nucleic acids to stabilize and concentrate them. For in vitro delivery, the net positive charge of the complex can enhance cell surface binding. When the molar number of available positively charged amines (N) on the polymer head and / or lipid tail is greater than the molar number of available negatively charged phosphates (P) on the nucleic acid, a net positively charged complex can be obtained. The N:P ratio is often calculated to determine whether the complex is net positive, neutral or negative. When incubated with cells, the positively charged complex can electrostatically interact with the negatively charged cell membrane, allowing cellular uptake by endocytosis. Once endocytosed, the transfection complex can become trapped in the endosome, potentially leading to undesirable consequences of lysosomal degradation or cellular export. The PLip disclosed herein not only stabilizes the transfection reagent, but is also stimulus-responsive and, when added to cells maintained at 37°C, imparts a higher net positive charge to the complex.

[0326] In some embodiments, the physical properties of the transfection complex that evolve during the complex formation step can affect the functional performance of the transfection complex. These physical properties, such as the size and charge of the transfection complex, can vary depending on several factors, including but not limited to:

[0327] Chemical composition of transfection reagents

[0328] The time it takes for the complex to form between the transfection reagent and the nucleic acid

[0329] The type and size of the nucleic acid delivered

[0330] Culture medium in which transfection complexes are formed

[0331] The molar charge ratio of the transfection reagent to the nucleic acid (often reported as the amine / phosphate charge ratio, N / P)

[0332] Concentration of reagents and nucleic acids

[0333] The method disclosed herein wherein will be used for adding the optimal window of transfection complex to cells expands from several minutes to several hours (and possibly several days) without changing the functional performance of the reagent. The electrostatic interaction between the transfection complex and the cellular components slows down, making the growth of the transfection complex much slower or "stable" over time. Stabilized PLip is incorporated into the transfection complex (which in most cases contains nucleic acid, cationic polymer and cationic lipid) through the hydrophobic interaction of its lipid tail. Once incorporated into the complex, the polymer head group of PLip provides the "stabilization" part. This polymer head group is a sufficiently long hydrophilic chain that stabilizes the complex by interfering with the electrostatic interaction responsible for the continuous growth of the complex over time.

[0334] Scheme 1 shows an example design of a stabilized PLip and a stimuli-responsive stabilized PLip. The difference between the two designs is the insertion of a stimuli-responsive linker unit between the lipid tail and the stabilizing polymer head group in the stimuli-responsive stabilized PLip. Other designs are possible. For example, multiple stimuli-responsive linker units can be incorporated into the structure of the PLip. For example, one of the monomer units of the stabilizing polymer head group can contain one or more stimuli-responsive linker units. Upon arrival or application of a stimulus, the stimuli-responsive linker unit can change its chemical and / or physical properties, thereby affecting the function of the transfection complex. The stimulus can be heat, light, chemical, pH, etc., to trigger changes in the properties of the PLip. These changes can range from an extended form or coil form to a condensed form or globule aggregate, from positive to neutral or negative charge, from neutral to negative charge, from neutral to positive charge, from negative charge to neutral or positive charge, from stable to unstable, etc.

[0335] Option 1:

[0336]

[0337] Temperature-responsive PLip

[0338] Particularly for in vitro transfection complexes, the zeta potential and overall positive charge of the particles can affect surface delivery to negatively charged cell membrane surfaces. Therefore, the properties and concentration of the stabilized PLip can play a role. For example, on the one hand, adding too much stabilized PLip can prevent electrostatic interactions with other transfection complexes and with cells. On the other hand, adding too little PLip may not have the desired effect of stabilizing the complex and extending the optimal time for optimal size and transfection efficiency. Similarly, PLip with too long or too short head groups can have similar effects.

[0339] In some embodiments, the PLip can be stimulus-responsive. In some embodiments, the temperature-sensitive PLip can contain: (i) at one end, a lipid tail group (for interaction with the transfection complex), (ii) in the middle, a temperature-sensitive linker unit group comprising a polymer moiety (e.g., poly(N-isopropylacrylamide) ("P(NIPAm)")), and (ii) at the other end, a stabilizing polymer head group comprising an additional polymer moiety (e.g., PEG, poly(2-hydroxyethyl acrylate) ("PHEA"), or poly[oligo(ethylene glycol) methyl ether methacrylate]

[0340] ("POEGMA"). The use of temperature-sensitive PLip can take advantage of the fact that transfection complexes can be formed at room temperature (25°C) or lower, while transfection of cells is performed at 37°C. Some polymers, such as P(NIPAm), can have a lower crystallization solution temperature (LCST) of 32°C or lower in water, meaning that they undergo a transition from being soluble (hydrophilic) in water below this temperature to being insoluble ("hydrophobic") above this temperature. Above the LCST, the P(NIPAm) polymer chain may not adopt an extended random coil structure because the protons on the P(NIPAm) side chains no longer form hydrogen bonds with surrounding water molecules, but instead interact with protons from other P(NIPAm) side chains. This transition can cause the P(NIPAm) to adopt a collapsed structure and exclude water molecules. When incorporated into a transfection complex, the net effect can be a complex that is more stable and less cationic at room temperature, but less stable and more cationic at 37°C. Scheme 2 below shows a schematic diagram of the stabilized hydrodynamic volume associated with the transfection complex at 25°C and 37°C.

[0341] Option 2:

[0342]

[0343] When compared to 37° C., the hydrodynamic volume can be larger and the stability of the transfection complex can be higher at 25° C. These properties may be a result of the extended P(NIPAm) linker unit at 25° C. and the presence of the POEGMA unit in the polymer head group. At 37° C., the P(NIPAm) block has collapsed and is no longer hydrophilic, leaving only the outer POEGMA block as a stabilizing moiety.

[0344] In some embodiments, the temperature responsive unit comprises a lower crystallization solution temperature (LCST) of about 27° C. to about 35° C. In some embodiments, the temperature responsive unit comprises a lower crystallization solution temperature (LCST) of about 27° C., about 28° C., about 29° C., about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., or about 35° C. In some embodiments, the temperature responsive unit comprises poly(N-isopropylacrylamide), poly(N-n-propylacrylamide), poly(N-methyl-N-n-propylacrylamide), poly(N,N-diethylacrylamide), poly(N-isobutylacrylamide), poly(N-sec-butylacrylamide), poly(N-n-butylacrylamide), poly(N-isobutylacrylamide), hydroxypropylcellulose, poly(N-vinylcaprolactam), poly-2-isopropyl-2-oxazoline, or polyvinyl methyl ether, or a combination thereof. In some embodiments, the temperature responsive unit comprises 2-250 monomer units.

[0345] Other examples of stimulus responsiveness may be pH-responsive Plip. In some embodiments, pH-responsive Plip can stabilize the transfection complex at pH 7.4, but can become less stable at lower pH, such as the pH encountered in endosomes or other cells. pH-responsive Plip can swell, collapse, or change its structure or shape, depending on the pH of its environment. This pH-dependent behavior can be exhibited due to the presence of certain pH-sensitive functional groups in the polymer chain. Sensitivity can be acidic or alkaline, responding to alkaline or acidic pH conditions as a stimulus. For example, polymers with acidic groups (such as -COOH and -SO3H) and polymers with basic groups (-NH2) can be pH-sensitive polymers. The response mechanisms of the two groups can be similar, except that the stimuli are different.

[0346] The example of polyacid polymer (anionic polymer) comprises acidic functional group, comprises carboxylic acid (-COOH), sulfonic acid (-SO3H), phosphonic acid and boric acid.Therefore, polyacid can accept proton at low pH value.At higher pH value, they can deprotonate and become negatively charged.Negative charge can produce repulsive force, causes polymer expansion.When pH is greater than the pKa of polymer, this swelling behavior can be observed.The example of polyacid polymer can include polymethyl methacrylate polymer and cellulose acetate phthalate.In some cases, poly (methacrylic acid) (PMAAc) can accept proton at low pH, and release proton at neutral and high pH.Other examples of polyacid polymer can include: poly (carboxylic acid), poly (phosphoric acid), poly (sulfonic acid), poly (amino acid) and poly (boric acid).

[0347] The example of polybasic polymer can be the alkaline equivalent of polyacid polymer, and can also be referred to as cationic polymer.They can accept protons at low pH like polyacid polymer, but then they can become positively charged.By contrast, they are neutral at higher pH values.Swelling behavior is visible when pH is lower than the pKa of polymer.Poly [(2-dimethylamino)ethyl methacrylate] (PDMA) can accept protons at low pH, thereby forming positively charged polymer chains.Other examples can include polymers containing tertiary amine groups, morpholino groups, pyrrolidino groups, piperazine groups, pyridine groups, imidazole groups as part of polymer side chains.

[0348] Other pH sensitive polymers may include alginate, chitosan, carboxymethyl cellulose, carboxymethyl dextran, gelatin A and B, and hyaluronic acid.

[0349] How to use

[0350] Disclosed herein are methods for transfecting cells, wherein the method comprises (a) providing a transfection reagent comprising a compound disclosed herein and a nucleic acid, and (b) contacting the cell with the transfection reagent, wherein the contact is carried out under conditions suitable for the entry of the nucleic acid into the cell. In some embodiments, step (a) comprises contacting the compound with the nucleic acid under conditions sufficient to form the transfection complex. In some embodiments, in steps (a) and (b), the conditions sufficient to form the transfection complex include conditions sufficient to perform an ionotropic gelation method.

[0351] In some embodiments, provided herein are methods for transfecting a cell, wherein the transfection complex comprises a positive charge under conditions suitable for entry of the nucleic acid into the cell.

[0352] In some embodiments, provided herein are methods for transfecting cells, wherein the nucleic acid comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), or any combination thereof.

[0353] The present invention provides methods for transfecting cells, wherein the contacting lasts for less than about 24 hours. In some embodiments, the contacting lasts for less than about 20 hours, less than about 18 hours, less than about 16 hours, less than about 14 hours, less than about 12 hours, less than about 10 hours, less than about 8 hours, less than about 6 hours, less than about 4 hours, less than about 2 hours, or less than about 1 hour. In some embodiments, the cells comprise animal cells, plant cells, fungal cells, bacterial cells, or any combination thereof.

[0354] In some embodiments, provided herein are methods for performing lipid-mediated cell transfection, comprising contacting cells with a transfection reagent disclosed herein (e.g., a composition comprising a compound described herein and a nucleic acid). In some embodiments, provided herein are methods for preparing compounds (e.g., lipid-polymer conjugates) or transfection reagents comprising the same, as described in the following examples.

[0355] abbreviation

[0356] Unless otherwise specified herein, the abbreviations used throughout this disclosure are defined as follows:

[0357] 1 H NMR proton nuclear magnetic resonance

[0358] AIBN 2,2'-azobis(2-methylpropionitrile)

[0359] AF488 azidefluor-488

[0360] C Celsius

[0361] CPCPA 4-Cyano-4-((phenylthiocarbonyl)thio)pentanoic acid

[0362] DBCO dibenzocyclooctyne

[0363] DCM dichloromethane

[0364] DIC diisopropylcarbodiimide

[0365] DLIN(6Z,9Z,28Z,31Z)-heptatriacontane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate or DLin-MC3-DMA

[0366] DLS Dynamic Light Scattering

[0367] DMAP 4-dimethylaminopyridine

[0368] DMF N,N-dimethylformamide

[0369] DNA deoxyribonucleic acid

[0370] DOPE dioleoylphosphatidylethanolamine

[0371] EtOH

[0372] HEA Hydroxyethyl Acrylate

[0373] HPLC high performance liquid chromatography

[0374] HPMA N-(2-Hydroxypropyl)methacrylamide

[0375] LC / MS liquid chromatography / mass spectrometry

[0376] LNP lipid nanoparticles

[0377] MeOH

[0378] MFI mean fluorescence intensity

[0379] MSEMA 2-(methylsulfinyl)ethyl methacrylate

[0380] NI normalized intensity

[0381] NIPAm N-isopropylacrylamide

[0382] PEG-DMG 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol

[0383] P(HPMA) Poly(N-(2-hydroxypropyl)methacrylamide)

[0384] PLIP polymer-lipid compound

[0385] RAFT reversible addition-fragmentation chain transfer polymerization

[0386] RFU relative fluorescence unit

[0387] SMFI-specific mean fluorescence intensity

[0388] TLC thin layer chromatography

[0389] v / v volume / volume ratio Example

[0390] The following illustrative examples represent embodiments of the compounds, compositions, and methods described herein and are not meant to be limiting in any way.

[0391] Synthesis Example

[0392] The compounds disclosed herein can be synthesized according to or in analogy to the following synthesis examples. In addition, the embodiments disclosed herein can be adjusted and modified according to principles known in the art to produce various polymer-lipid compounds, synthetic intermediates (e.g., RAFT agents), and transfection reagents (e.g., LNPs) as disclosed herein.

[0393] Example S1: Synthesis and characterization of dioleyl RAFT agents

[0394]

[0395] i. To a sample of 4-cyano-4-((phenylthiocarbonyl)thio)pentanoic acid) ("CPCPA") (4.00 g, 14.3 mmol) in DMF (20 mL) were added an excess of glycerol (16.0 g, 0.118 mol) and diisopropylcarbodiimide (DIC, 2.80 mL, 17.9 mmol) and stirred at room temperature for 30 min. Next, 4-dimethylaminopyridine (DMAP, 78.0 mg, 0.64 mmol) was added and the mixture was stirred overnight. Ethyl acetate (40 mL) was added and the resulting mixture was extracted with water (40 mL) to remove DMF. The aqueous layer was extracted with ethyl acetate (15 mL) and combined with the organic layers. The organic layer was then re-extracted with water (40 mL). The organic solution was dried on a rotary evaporator and the resulting oil was dissolved in methanol (12.5 mL). The methanol solution was then precipitated by adding dropwise to water (80 mL) twice. Chloroform (40 mL) was added to the precipitate, and the solution was dried over sodium sulfate and then filtered through filter paper. The solution was dried over a rotary evaporator and redissolved in chloroform (10 mL). The chloroform solution was then precipitated twice in hexane (80 mL) and then dried under reduced pressure. The product was analyzed by TLC (chloroform: methanol 90: 10%, Rf = 0.23) and 1 H NMR spectroscopy (see Figure 1 ) Characterization of CPCPA-diol product. Yield: 3.60 g.

[0396]

[0397] ii. The CPCPA-diol product of step 1 (0.500 g, 1.42 mmol) was dissolved in DCM (15 mL). Oleic acid (1.20 g, 4.25 mmol) and DIC (0.665 mL, 4.25 mmol) were added and stirred at room temperature for 30 min. Then, DMAP (26.5 mg. 0.217 mmol) was added and the mixture was stirred for 2 h. The solution was extracted twice with water (15 mL), and the organic layer was dried over sodium sulfate. The solution was filtered through fluted filter paper and dried by rotary evaporation. The resulting mixture was dissolved in chloroform (2 mL) and precipitated into 9:1 MeOH / H2O (v / v) solution (40 mL) twice. The product was dissolved in chloroform (10 mL) and dried over sodium sulfate, then filtered and dried in vacuo. The product was analyzed by TLC (chloroform: methanol 95:5%, Rf = 0.87) and 1 The product was characterized by H NMR spectroscopy.

[0398] Example S2: Synthesis and characterization of dilinoleoyl RAFT agents

[0399]

[0400] The CPCPA-diol product (0.500 g, 1.42 mmol) from step (i) of Example S1 was dissolved in DCM (15 mL). Linoleic acid (1.19 g, 4.25 mmol) and DIC (0.665 mL, 4.25 mmol) were added and stirred at room temperature for 30 min. Then, DMAP (26.5 mg. 0.217 mmol) was added and the mixture was stirred for 2 h. The solution was extracted twice with water (15 mL), and the organic layer was dried over sodium sulfate. The solution was filtered through fluted filter paper and dried by rotary evaporation. The concentrate was dissolved in chloroform (2 mL) and precipitated into 9:1 MeOH / H2O (v / v) solution (40 mL) twice. The precipitate was collected and dissolved in chloroform (10 mL), dried over sodium sulfate, then filtered and dried in vacuo. The product was characterized by TLC (chloroform: methanol 95: 5%, Rf = 0.87) and 1 The products were characterized by H NMR spectroscopy. Figure 2 shown.

[0401] Example S3: Synthesis and characterization of cholesterol RAFT agents

[0402]

[0403] To a sample of CPCPA (1.00 g, 3.58 mmol) in DCM (15 mL) was added cholesterol (1.94 g, 5.01 mmol) and DIC (2.80 mL, 17.9 mmol) and stirred at room temperature for 30 min. Subsequently, DMAP (31.6 mg. 0.259 mmol) was added and the mixture was stirred overnight. The solution was extracted twice with water (30 mL), after which the organic layer was dried over sodium sulfate. The solution was filtered through fluted filter paper and dried by rotary evaporation. The concentrate was dissolved in DCM (3 mL) and precipitated into a 9:1 MeOH / H2O (v / v) solution (45 mL) twice. The mixture was rotated slowly to separate the oil, then dissolved in DCM (3 mL) and precipitated again in MeOH / H2O. The precipitate was collected and dissolved in DCM (10 mL), dried over sodium sulfate, then filtered and dried in vacuo. The product was analyzed by TLC (chloroform: methanol 95: 5%, Rf = 0.62) and 1 The products were characterized by H NMR spectroscopy. Figure 3 shown.

[0404] Example S4: Synthesis of additional diacyl RAFT agents

[0405] The following diacyl RAFT agents were synthesized similarly to Examples S1-S3:

[0406]

[0407]

[0408] Example S5: Synthesis of monoacyl RAFT agent

[0409] The following monoacyl RAFT agents were synthesized similarly to Examples S1-S3:

[0410]

[0411] Example S6: Synthesis of trithiocarbonate RAFT agents

[0412] The following trithiocarbonate RAFT agents were synthesized similarly to Examples S1-S3:

[0413]

[0414] Example S7: Synthesis of Polymer-Lipid Compound ("PLIP")

[0415]

[0416] Polymer-lipid compounds (PLips) can be synthesized using reversible addition-fragmentation chain transfer polymerization (RAFT) using acrylates or acrylamides and any of the RAFT agents disclosed herein (e.g., in Examples S1-S6) in combination with a free radical initiator (e.g., 2,2'-azobis(2-methylpropionitrile), "AIBN"). The monomers and RAFT agent are heated in the presence of an initiator in an inert atmosphere using a suitable solvent such as dioxane. Grafting from the polymerization process results in the growth of a polymer chain with the lipid tail at the α-terminus of the polymer. More than one monomer can be incorporated into the chain to form random / statistical copolymers (monomers added together) or block / gradient copolymers (monomers added sequentially).

[0417] Example S8: Synthesis of Cationic PLIP

[0418] (a): Synthesis of cationic diacyl PLip

[0419]

[0420] i. CPCPA-diacyl RAFT agent starting material was prepared starting from CPCPA-diol of Example S1 according to the general procedures disclosed in Examples S2 and S3.

[0421] ii. Prepare a 10 mg / mL solution of AIBN in dioxane for polymerization. To a 2 mL sealed glass vial with a septum cap was added Boc-aminopropyl acrylate (55.0 mg, 0.241 mmol), the RAFT reagent of Scheme S8 (24.1 mg, 0.0275 mmol), an AIBN solution (0.0677 mL, 0.00413 mmol AIBN) and dioxane (0.37 mL). The flask was sealed and the reaction solution was purged with nitrogen for 20 min, then heated at 80 ° C for 8 h while stirring. After cooling to room temperature, the solution was precipitated into hexane (14 mL) and the solution was transferred using DCM (0.2 mL). The precipitation was repeated again from 0.5 mL DCM into hexane (14 mL). The precipitate was then dried under vacuum and filtered through 1 H NMR and GPC analysis. Mn 7,100 (PDI 1.26). Yield = 35-50 mg (45-65%).

[0422] iii. The dried precipitate (40 mg) from step (ii) was dissolved in 2N HCl in acetic acid solution (1.0 mL) and stirred for 1 h. Deionized water (2 mL) was added to the solution, which was then placed in a dialysis bag (MWCO 1,000) and dialyzed against saline and then deionized water. The solution was then removed from the dialysis bag and lyophilized to dryness. 1 H NMR characterization of cationic PLip( Figure 4 ).

[0423] iv. Prepare a 20 mg / mL solution by dissolving the lyophilized PLip from step (iii) above in a 1:1 mixture of EtOH / H 2 O. Store the solution at 4° C. Yield = 10-15 mg (35-60%).

[0424] (b): Synthesis of cationic sterol PLip

[0425]

[0426] Cationic cholesterol PLip was synthesized according to Example S8(a), using the cholesterol RAFT agent of Example S3 instead of the diacyl RAFT agent. The subsequent isolation, acidification, dialysis and formulation steps were similar to steps (ii-iv) of Example S3.

[0427] Example S9: Synthesis of non-cationic PLIP

[0428]

[0429] i. N-(2-hydroxypropyl)methacrylamide (HPMA) (1.05 g, 7.36 mmol), CPCPA-diacyl RAFT agent (86.7 mg, 0.0981 mmol), AIBN solution (3.2 mg, 0.0196 mmol AIBN) and dioxane (7.36 mL) were combined in a 20 mL glass vial, which was then purged with nitrogen for 40 min and then heated at 80 ° C while stirring. After 8 h, the solution was cooled to room temperature and PLip was precipitated into hexane (90 mL) using ethanol as a transfer solvent (5 mL) in triplicate. The precipitate was dried in vacuo and filtered through a 5% ethanol precipitate. 1 H NMR ( Figure 5 ) and GPC analysis. n 11,200. Yield = 801 mg (70%).

[0430] ii. Prepare a 20 mg / mL solution by dissolving the dried PLip from the previous step (i) in a 1:1 mixture of EtOH / H 2 O. Store the solution at 4°C.

[0431] Example S10: Additional PLIP

[0432] The following PLips were prepared according to the previous examples S1-S9:

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439] Biological Examples

[0440] Example B1: Encapsulation of DNA in LNPs containing cationic PLIP

[0441] (a) Cationic PLip was incorporated into LNP formulation for DNA encapsulation.

[0442] Cationic PLips 1-5 having the structures shown in Table B1(a) were synthesized similarly to Synthesis Examples S1-S9. Cationic PLips were incorporated into lipid nanoparticle (LNP) formulations, and the resulting LNPs were evaluated for DNA encapsulation.

[0443] Table B1(a). Structure of cationic block copolymer PLip incorporated into LNP

[0444]

[0445]

[0446] (b) Formulation of LNPs containing cationic PLip 1-5

[0447] Cationic PLips 1-5 from Table B1(a) were incorporated into corresponding LNP formulations, referred to as LNP Formulations 1-5. Each LNP contained between approximately 0.3% and 1% of a given cationic PLip. For example, the composition of LNP Formulation 1 is shown in Table B1(b). LNP Formulations 2-5 were prepared according to the same specifications, replacing PLip with the corresponding PLip 2-5 in each case.

[0448] Table B1(b). Example Formulation 1 of LNPs containing cationic PLip 1:

[0449]

[0450] (c) DNA encapsulation into LNPs containing cationic PLip

[0451] DNA concentration and encapsulation were determined using a modified RiboQuant assay (Thermofisher). The LNPs successfully encapsulated DNA (encapsulation efficiency 73-88%), indicating that the presence of cationic PLip did not disrupt encapsulation ( Figure 6 ).

[0452] Example B2: PLIP-stabilized LNPs and their effect on DNA encapsulation

[0453] (a) PLip was incorporated into LNP formulations for DNA encapsulation.

[0454] PLip 6-8 having the structures shown in Table B2(a) were synthesized similarly to Synthesis Examples S1-S9. PLip was incorporated into lipid nanoparticle (LNP) formulations, and the resulting LNPs were evaluated for DNA encapsulation.

[0455] Table B2(a). Structures of stabilized PLip 6 and cationic PLip 7

[0456]

[0457]

[0458] (b) Formulation of LNPs containing stabilized PLip 6 and 8 and cationic PLip 7

[0459] LNPs containing PLip 6-8 were prepared with the mol% disclosed in Table B2(b):

[0460] Table B2(b). Example LNP formulations containing PLip 6-8:

[0461]

[0462] DNA encapsulation into LNPs containing cationic PLip

[0463] 500 ng of MFP-488-labeled pDNA encapsulated in LNPs was exposed to cells for 3.5 hours at 37° C. The fluorescence of the cells was evaluated by flow cytometry. Figure 7 The specific mean fluorescence intensity (SMFI) or cell brightness was measured relative to untreated cells. pDNA concentration and encapsulation were determined by a modified RiboQuant assay (Thermofisher). When compared to the same LNP formulation without cationic PLip 7, formulation 7 showed enhanced LNP binding in two of the three cell lines (e.g., Figure 7 The enhanced binding was attributed to the cationic polymer head group on PLip, allowing ionic interactions with the cell surface that would otherwise be shielded by the stabilizing uncharged lipids.

[0464] (d) Size and stability of LNPs stabilized by PLip

[0465] like Figure 7 As shown in Figure 2, stabilized PLip 7 was successfully used to stabilize LNPs in vitro and bind them to cells. Dynamic light scattering (DLS) showed the formation of well-defined stable LNPs (the radius of the LNP stabilized with PLip 6 was 129 nm and the PD was 9.8%). Similarly, PLip 8 - a PLip with poly(MSEMA) side chains - formed a stable LNP with a radius of 120 nm (PD = 17%). The LNP formulation containing PLip 8 is disclosed in Table B2(b) above. The DLS intensity distribution of LNPs containing PLip 6 and 8 is shown in Figure 2. Figure 8 shown.

[0466] Example B3: PLIP-stabilized LNPs and their effects on in vivo mRNA delivery

[0467] (a) Synthesis and structure of stabilized PLip 9-11

[0468] Additional examples of stabilized PLips are provided in Table B3(a) below. PLips 9-11 were prepared and formulated as in the previous examples.

[0469] Table B3(a). Structure of PLip 9-11

[0470]

[0471] (b) Formulations of LNPs containing stabilized PLip 11 vs. PEG-DMG

[0472] According to Table B3(b), stabilized PLip 11 containing a diacyl tail and a P(HPMA) head group was used to prepare LNP formulation 11. Reference LNPs were prepared using PEG-DMG instead of PLip 11.

[0473] Table B3 (b). Example LNP formulation 11 containing PLip 11 and a reference LNP formulation containing PEG-DMG.

[0474]

[0475] (c) In vivo delivery of mRNA via LNPs stabilized with PLip

[0476] On days 1 and 21, 4 μg of LNP-encapsulated mRNA samples were injected into four 20-25 g female Balb-c mice. Serum samples were evaluated for anti-spike IgG by ELISA on days 14 and 35. From day 14 to day 35, LNPs containing PLip 11 showed a significant increase in anti-spike IgG by ELISA, which was comparable to LNPs prepared with PEG-DMG ( Figure 8 ). Each bar represents an individual mouse.

[0477] Example B4: Cell membrane insertion of PLIP

[0478] (a) PLip with fluorescent functional groups is inserted into the cell membrane

[0479] The insertion of functional and / or labeled lipids into cell membranes to "prime" membranes has been investigated for various potential applications, including the incorporation of sugars, functional groups (such as bioorthogonal "click" chemistry), and fluorescent labels on the cell surface. To demonstrate that PLip can be inserted into the membranes of living cells, a fluorescently labeled PLip12 with the following structure was inserted:

[0480]

[0481] Add serum or reduced serum medium ("RSM") (Thermo Fisher Scientific Opti-MEM TMIn Jurkat cells cultured in a reduced serum medium (1% PLip 12), increasing concentrations of fluorescent PLip 12 were incubated with the cells for 1.5 h at 37°C and then for 60 min at room temperature. The cells were washed once before fluorescence evaluation by flow cytometry. The brightness of the cells was recorded as a function of the "pretreatment" concentration of PLip ( Figure 10 ). The presence of serum inhibited cell preconditioning, and under these conditions preconditioning efficiency appeared to plateau at ∼4 μM PLip.

[0482] (b) Temperature-sensitive PLip with fluorescent side groups was inserted into 293F cells

[0483] Fluorescently labeled PLip 13 with the following structure:

[0484]

[0485] It has a random copolymer head group of 1-pyrenemethyl methacrylate and NIPAm and is incorporated into the cytoplasmic membrane of 293F cells. PLip was incubated with cells at 12°C to prevent endocytosis of PLip and then washed three times with phosphate-buffered saline (PBS) to remove free, unincorporated PLip. Fluorescence measurements showed that PLip was incorporated into cells or cell membranes ( Figure 11 After heating the cells from room temperature to 37.5°C, the emission peak associated with the pyrene dimer (approximately 480 nm) decreased compared to the emission peak associated with single monomeric pyrene (approximately 396 nm). This decrease in dimer emission may be due to the local environment of pyrene: for example, if pyrene is located in a hydrophobic region, it becomes less proximal.

[0486] 293F cells (500k cells / ml in untreated 6-well plates) were pre-cooled to 12.5°C for 2 hours. Pyrene PLip was added to the cells and incubated at 12.5°C for 1 hour to allow outer membrane incorporation (in the absence of endocytosis). 1 mL aliquots of each condition were washed three times in PBS. 200 μl of each condition (+ / - washes) were measured on a Tecan fluorescence plate reader at RT and 37°C.

[0487] Example B5: PLIPs with bioorthogonal reactive groups at the termini or along the backbone

[0488] Synthesize PLips that contain bioorthogonal reactive groups that can participate in chemical reactions in biological media or environments (in vivo). One such example of a bioorthogonal reactive group is a "click" chemical group, such as a strained alkyne or azide moiety. An example of a strained alkyne is a dibenzocyclooctyne (DBCO) group.

[0489] (a) General reaction scheme for end group modification of PLip.

[0490]

[0491] Where R = cholesterol, diacyl or acyl lipid tail

[0492] X=O、NH

[0493] R' = side chain

[0494] Following the aforementioned protocol, reactive PLips containing bioorthogonal DBCO functional groups were incorporated into PLips 14-19, which had the following structures:

[0495]

[0496]

[0497] wherein each n is independently 0 to 100. More specifically, n is typically an integer from 1 to about 50. In certain embodiments disclosed herein, the average value of n is between 2 and 10 (eg, n=4.5).

[0498] (b) Formulation of LNPs containing reactive / stabilized PLip 14

[0499] Stabilized PLip 16, containing a cholesterol lipid tail, a PEG methacrylate polymer, and a DBCO functional group, was used to formulate / stabilize LNPs. 1 mol% of the reactive PLip was formulated with a typical LNP mixture. According to Table B5(b), an example of an LNP formulation comprising a reactive PLip (e.g., PLip 16) is LNP formulation 16. This formulation was compared with a reference formulation identical to Example B3(b), which is reproduced below from Table B3(b). The LNPs obtained from formulation 16 and the reference formulation are referred to as PLip 16 LNP and PEG LNP, respectively.

[0500] Table B5 (b). Example LNP formulation 16 containing PLip 16 and reference LNP formulation containing PEG-DMG.

[0501]

[0502]

[0503] (c) Coupling of fluorescent labeling to PLip-stabilized LNPs

[0504] LNPs were prepared from various PLips with reactive (e.g., DBCO) functional groups. LNPs were analyzed by DLS 20 min before the addition of an excess of azide containing a fluorophore (azidefluor-488 (AF488)). Excess AF488 was removed by dialysis. Increased fluorescence of the LNPs (green color) indicated the presence of the DBCO moiety in the LNPs and the ability to undergo a "click" reaction ( Figure 12 ). The reference LNP containing 0.6% PEG2k but without DBCO-PLip did not show any fluorescence, indicating that AF488 was covalently linked to DBCO-LNP.

[0505] Example B6: Transfection Reagent In Vitro Cationic PLIP Delivered Alone or Added to a Formulation

[0506] (a) Effects of N:P ratio and cationic polymer length on transfection

[0507] 293F cells were transfected with DNA (pCILuc) complexed with various cationic PLips containing a cholesterol tail and a poly(propylaminoacrylate) head group of 5, 13, and 19 units in length at various PLip / DNA ratios. The structures are shown below.

[0508]

[0509] The N:P ratio is calculated from the stoichiometry of the number of cationic amines (N) on the PLip and the number of phosphate groups (P) on the DNA. (Mirus Bio) was used as a comparison. Cells were transfected with CMV-driven firefly luciferase pDNA constructs at various PLip:DNA (N:P) ratios for 48 hours. 48 hours after transfection, cells were harvested by lysing the entire well with 1% triton-X 100 at 4°C for 30 minutes. The lysates were evaluated for luciferase in a Veritas luminometer using standard conditions. In contrast, some higher N:P ratio cationic PLip enhanced the delivery of pCILuc. In general, transfection increased at higher N:P ratios until PLip became too toxic ( Figure 13 ).

[0510] (b) Effects of N:P ratio and cationic polymer length on polymer / DNA complex delivery

[0511] In another experiment, 293F cells were transfected with various DNAs complexed with the proprietary polymer POLY1 (pCILuc) in the presence and absence of cationic PLip 20-22. (2:1 polymer:DNA by weight) as a comparison. Cells were transfected with a CMV-driven firefly luciferase pDNA construct at various compound:DNA mass ratios (also denoted as N:P ratio) for 48 hours. 48 hours after transfection, cells were harvested by lysing the entire well with 1% triton-X 100 for 30 minutes at 4°C. The lysates were evaluated for luciferase in a Veritas luminometer using standard conditions. PLip 20 did not enhance the delivery of POLY1:DNA complexes. However, cationic PLips with longer polymer head group chain lengths (PLip 21 and PLip 22) showed significant improvements in transfection ( Figure 16 ).

[0512] Example B7: Temperature-sensitive PLIP for (de)stabilization of LNPs

[0513] (a) Preparation of temperature-sensitive PLip for stabilizing LNPs

[0514] Poly(N-isopropylacrylamide) ("P(NIPAm)") is a polymer with a low crystalline solution temperature (LCST) of 32°C. Below the LCST, the polymer is completely soluble in water (in this case, P(NIPAm) is considered hydrophilic). Above the LCST, the polymer becomes insoluble due to intramolecular hydrogen bonding interactions and aggregates / precipitates (essentially, P(NIPAm) is considered hydrophobic). A temperature-sensitive dioleyl diglyceride PLip (PLip 23) with P(NIPAm) side chains and 55 monomer units was prepared according to the previous examples and has the following structure:

[0515]

[0516] (b) PLip 18 incorporated into LNPs

[0517] As disclosed in Table B6(b), three LNP formulations were prepared with varying amounts of PLip 23. Formulation 23(a) was a control and had 0 mol% PLip, while LNP formulations 23(b) and 23(c) had 0.6 mol% and 1.2 mol%, respectively.

[0518] Table B7 (b). Example LNP formulations containing PLip 23 at 0, 0.6, and 1.2 mol%.

[0519]

[0520] The LNPs were analyzed by DLS to determine the effect of temperature on LNP size during cyclic temperature scans. At room temperature (below the LCST of P(NIPAm)), LNPs with 0.6 and 1.2 mol% PLip23 were stable at 180 nm radius ( Figure 15 ). PLip-free LNPs with a radius of 120 nm were used as a control. When the temperature exceeded 32°C, the LNPs with PLip began to grow. This increase in radius was due to physical changes in the P(NIPAm) chains, leading to lipid rearrangement. When the temperature was increased to 45°C, the LNPs containing PLip23 grew to a radius of 300 nm. When the temperature was cycled back below the LCST, the LNPs shrank back to their initial size.

[0521] This process appears to be reversible. Figure 16 As shown, there was no change in the normalized intensity data related to the number of particle counts. These data indicate that the particles did not aggregate due to the transition. The control LNPs showed no change in size or normalized intensity.

[0522] Example C1: Synthesis of Additional Temperature-Sensitive "Stabilized" PLIP

[0523]

[0524] i) Preparation of Block A PLip: N-(isopropylacrylamide) (800 mg, 7.08 mmol), CPCPA-cholesterol (69.3 mg, 0.107 mmol), AIBN solution (2.62 mg, 0.0160 mmol AIBN), and dioxane (4.20 mL) were added to a 20 mL glass vial with a septum cap and a stir bar. The flask was sealed with a cap and the solution was bubbled with nitrogen for 30 minutes using a long needle immersed in the solution and a second needle above the solution as an outlet. The syringe was removed without removing the cap and the vial was immersed in an oil bath set at 80°C for 8 hours while stirring. The solution was allowed to cool to room temperature and precipitated into hexane (40 mL). It was reprecipitated into hexane twice from 2.5 mL of chloroform (40 mL each). The collected precipitate was dried under reduced pressure. A small sample of the polymer was removed for NMR and GPC analysis to determine the length of the NIPAm chain compared to the RAFT (aromatic) end group. NIPAm units = 115 (m = 115). Yield = 566 mg (65%).

[0525] ii) Addition of Block B (PHEA): HEA (28.0 mg, 0.241 mmol), the above-obtained Block A PLip (40.0 mg, 0.00294 mmol), AIBN solution (0.0980 mg, 0.000598 mmol AIBN), and dioxane (0.32 mL) were added to a 2 mL glass vial with a septum cap and a stir bar. The flask was sealed with a cap and the solution was bubbled with nitrogen for 20 minutes using a long needle immersed in the solution and a second needle above the solution as an outlet. The syringe was removed without removing the cap, and the vial was immersed in an oil bath set at 80°C for 8 hours while stirring. The solution was allowed to cool to room temperature and precipitated into hexane (15 mL). It was reprecipitated into hexane twice from 1 mL of ethanol (15 mL each). The collected precipitate was dried under reduced pressure. A small sample of the polymer was removed for NMR and GPC analysis to determine the length of the HEA chains compared to that of Block A. HEA units = 92 (n = 92). Yield = 58 mg (85%).

[0526] Example D1: Complex stable over time

[0527] Analysis of the effect of stabilized PLip (MP64240) on transfection complexes (e.g. ) Impact of functional performance: Percentage of intact capsid ( Figure 17A ) and genome ( Figure 17B Immediately before transfection, 293-VP 2.0 cells were seeded at 3 million cells / mL in virus production medium (VPM). Complexes were generated and complex formation was measured at 30 minutes and 3.5 hours. Plasmids: pMIR699, pMIR 732 AAV8, and pMIR701 AAV harvested 72 hours after transfection. Genome counts / mL culture were measured by digital PCR (dPCR). AAV capsid immunoassay measures AAV8 capsids. Figure 17B It shows that in the presence of temperature-sensitive PLip within 3.5 h For stabilization of genomic titers, add it to the transfection reagent in ethanol before mixing with plasmid DNA.

[0528] Example D2: Stabilization of concentrated transfection complexes

[0529] Dynamic light scattering (DLS) was used to track the aggregation behavior of transfection complexes over time. The stabilizing effect of temperature-sensitive PLip (MP64240) added to concentrates (1-5 times the normal (recommended) concentration of VG and DNA in PBS) is shown in Figure 3. Figure 18A and Figure 18B 2× and 5× indicate The concentrations of DNA were 2 and 5 times that of the recommended protocol. The presence of temperature-sensitive PLip stabilized transfection complexes that originally exceeded 1 μm in diameter after only a few minutes (without PLip) to less than 1 μm in diameter for >2 hours (with PLip).

[0530] Determine the 2× and Functional properties of the composite. Figure 19A and Figure 19B Highlighted are the changes in total genome titer and intact capsid percentage for "normal" unstabilized and stabilized complexes at 30 min and 3.5 h. At the 30 min time point, the 2× complex showed similar results to the standard VirusGEN (1×) complex in terms of titer and intact percentage. The 5× complex showed lower titer and similar intact percentage, which may be due to the larger size of the complex at this time. At 3.5 h, both the unstabilized 2× and 5× complexes showed very little functional efficacy. In contrast, when the 2× and 5× complexes were stabilized with temperature-sensitive PLip, functional performance was maintained over a longer period of time and for the more concentrated complexes (2× and 5×).

[0531] ***

[0532] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided in the specification. Although the present invention has been described with reference to the above description, the description and illustration of the embodiments herein are not intended to be interpreted in a limiting sense. Many variations, changes, and substitutions will now occur to those skilled in the art without departing from the present invention. In addition, it will be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions depending on various conditions and variables set forth herein. It will be understood that various alternatives to the embodiments of the present invention described herein may be adopted when practicing the present invention. Therefore, it is contemplated that the present invention also encompasses any such alternatives, modifications, variations, or equivalents. The appended claims are intended to define the scope of the present invention, and thus encompass methods and structures within the scope of these claims and their equivalents.

Claims

1. A compound comprising: (a) lipids; and (b) Stimulus-response unit.

2. A compound comprising: (a) lipids; (b) a linker comprising a stimuli-responsive unit; and (c) polymers, wherein the linker connects the lipid to the backbone of the polymer.

3. The compound of claim 2, wherein the polymer comprises at least 3 monomeric units, wherein the at least 3 monomeric units comprise C 1-20 Heteroalkyl side chains.

4. The compound according to any one of claims 1 to 3, wherein the stimulus-responsive unit is a temperature-responsive unit, a pH-responsive unit, a light-responsive unit, or a chemical-responsive unit.

5. The compound of claim 4, wherein the temperature-responsive unit comprises a lower crystallization solution temperature (LCST) of about 27°C to about 35°C.

6. The compound of claim 4, wherein the temperature-responsive unit comprises a lower crystallization solution temperature (LCST) of about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, or about 35°C.

7. The compound of any one of claims 4 to 6, wherein the temperature-responsive unit comprises poly(N-isopropylacrylamide), poly(N-n-propylacrylamide), poly(N-methyl-N-n-propylacrylamide), poly(N,N-diethylacrylamide), poly(N-isobutylacrylamide), poly(N-sec-butylacrylamide), poly(N-n-butylacrylamide), poly(N-isobutylacrylamide), hydroxypropyl cellulose, poly(N-vinylcaprolactam), poly-2-isopropyl-2-oxazoline, or polyvinyl methyl ether, or a combination thereof.

8. The compound of claim 7, wherein the temperature-responsive unit comprises 2-250 monomer units.

9. A compound comprising a lipid attached to a polymer backbone, said polymer comprising at least 3 monomeric units, wherein said at least 3 monomeric units comprise C 1-20 Heteroalkyl side chains.

10. The compound according to any one of claims 1 to 9, wherein the polymer comprises 4 or more of the monomer units.

11. The compound of any one of claims 10, wherein the polymer comprises about 10 or more of the monomer units.

12. The compound of claim 10, wherein the polymer comprises about 50 or more of the monomer units.

13. The compound of claim 10, wherein the polymer comprises about 400 or fewer of the monomer units.

14. The compound of claim 10, wherein the polymer comprises about 300 or fewer of the monomer units.

15. The compound of claim 10, wherein the polymer comprises from about 10 to about 200 of the monomer units.

16. The compound of claim 10, wherein the polymer comprises about 50 to about 150 of the monomer units.

17. The compound of any one of claims 1 to 16, wherein the polymer comprises a polyacrylate or a polyacrylamide.

18. The compound of any one of claims 1 to 17, wherein each of the monomer units comprises acrylamide or acrylate.

19. The compound of any one of claims 1 to 18, wherein the polymer is not a peptide.

20. The compound of any one of claims 1-19, wherein each of the monomeric units is not an amino acid.

21. The compound of any one of claims 1-20, wherein the polymer is a copolymer.

22. The compound of claim 21, wherein the copolymer is a block copolymer.

23. The compound of claim 22, wherein the block copolymer comprises a block comprising a cationic monomer unit.

24. The compound of claim 22, wherein the block copolymer is a random block copolymer.

25. The compound of any one of claims 1-24, wherein the polymer is positively charged in neutral aqueous solution.

26. The compound of any one of claims 1-25, wherein the polymer comprises a pK of about 2 to about 12. b .

27. The compound of any one of claims 1-26, wherein the polymer comprises a pK of about 4 to about 11. b .

28. The compound of any one of claims 1 to 24, wherein the polymer comprises: in: R 10 is hydrogen or C1-C6 alkyl, and x is an integer from 1 to 20.

29. A compound according to formula I or a pharmaceutically acceptable salt thereof: X—Y—Z Formula I; in: X is a lipid; Y is a polymer comprising 3 or more monomeric units, wherein each of said monomeric units comprises C 1-20 heteroalkyl side chains; and Z is an unsubstituted or substituted functional group; wherein the lipid is covalently bonded to the polymer via the backbone of the polymer.

30. The compound of any one of claims 1-29, wherein the lipid comprises a steroid or a fatty acid.

31. The compound of claim 30, wherein the steroid comprises a sterol or a stanol.

32. The compound of claim 31 , wherein the steroid comprises the sterol.

33. The compound of any one of claims 31-32, wherein the sterol comprises cholesterol.

34. The compound of claim 30, wherein the fatty acid comprises a saturated fatty acid, a monounsaturated fatty acid, a polyunsaturated fatty acid, or a combination thereof.

35. A compound according to claim 30 or claim 34, wherein the fatty acid comprises oleic acid or an ester thereof.

36. The compound of any one of claims 1-35, wherein the lipid is hydrophobic.

37. The compound of any one of claims 1-36, wherein the lipid is amphiphilic.

38. The compound of any one of claims 1-37, wherein the lipid has an octanol:water coefficient (log(K)) of about 2 or greater. OW )).

39. The compound of any one of claims 1-38, wherein the lipid has a structure of Formula XA, Formula XB, or Formula XC:

40. The compound of any one of claims 1-39, wherein the polymer has a structure of Formula YA, Formula YB, Formula YC, Formula YD, or a pharmaceutically acceptable salt thereof: in: A 1 、B 1 、C 1 and D 1 Each of is independently hydrogen or methyl; A 2 、B 2 、C 2 and D 2 Each of which is independently unsubstituted or substituted C 1-20 heteroalkyl; Each of a, b, c, d, e and f is independently an integer from 0 to 200, provided that the total number of said monomer units is 3 or more; wherein each of the substituted C 1-20 The heteroalkyl group is independently substituted with a ring that is unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclyl, unsubstituted or substituted aryl, or unsubstituted or substituted heteroaryl.

41. The compound of any one of claims 29-40, wherein the polymer comprises from about 4 to about 400 of the monomer units.

42. The compound of any one of claims 29-41, wherein the polymer comprises from about 10 to about 200 of the monomer units.

43. The compound of any one of claims 29-42, wherein the polymer comprises from about 50 to about 150 of the monomer units.

44. The compound of any one of claims 29-43, wherein the polymer comprises a polyacrylate or a polyacrylamide.

45. according to the compound described in any one of claim 29-44, wherein said A 2 、B 2 、C 2 and D 2 Each of which independently comprises acrylate or acrylamide.

46. The compound according to any one of claims 3 to 45, wherein each of the monomeric units has a structure of the following formula or one of its pharmaceutically acceptable salts: in: R 1 and R 3 Each of is independently hydrogen or methyl; Each R 2 For hydrogen, C 1-6 Alkyl, C 7-20 Aralkyl, C 1-20 heteroalkyl, or a polyethylene glycol chain containing 1-100 ethylene glycol monomers; wherein the C 1-6 Alkyl, C 7-20 Aralkyl and C 1-20 Each of the heteroalkyl groups is unsubstituted or substituted with one or more groups, wherein each of the one or more groups is independently -COOH, -CONH2, -NH2, -NH3 + 、-NHC(NH2 + )NH2, -NHCH3, -N(CH3)2, -N(CH3)3 + , -OH, -OCH3, -SH, -S(O)CH3, -S(O)2CH3 or -S(O)2OH; R 4 and R 5 Each of which is independently hydrogen, C 1-6 Alkyl, C 7-20 Aralkyl or C 1-20 heteroalkyl; wherein said C 1-6 Alkyl, C 7-20 Aralkyl and C 1-20 Each of the heteroalkyl groups is unsubstituted or substituted with one or more groups, wherein each of the one or more groups is independently -COOH, -CONH2, -NH2, -NH3 + 、-NHC(NH2 + )NH2, -NHCH3, -N(CH3)2, -N(CH3)3 + , -OH, -OCH3, -SH, -S(O)CH3, -S(O)2CH3 or -S(O)2OH; or a pharmaceutically acceptable salt thereof.

47. A compound according to any one of claims 3 to 46, wherein each of the monomeric units independently comprises:

48. The compound of any one of claims 29-47, wherein the functional group is a thiol or a sulfide.

49. The compound according to any one of claims 29-48, wherein the functional group is the thiol.

50. The compound of any one of claims 29-49, wherein the functional group is the sulfide.

51. The compound according to claim 50, wherein the sulfide is SR 6 , and where R 6 is a group consisting of 1 to about 200 atoms selected from hydrogen, halogen, C, N, O and S.

52. The compound according to claim 50, wherein the sulfide is SR 6 , and where R 6 These include reactive groups, charged groups, detectable groups, peptide groups, capping groups, or combinations thereof.

53. The compound of claim 52, wherein the reactive group comprises an azide or an alkyne.

54. The compound of claim 52, wherein the charged group comprises one or more cationic groups.

55. The compound of claim 54, wherein the one or more cationic groups comprise a cyclic amine, a primary amine, a guanidine, or a combination thereof.

56. The compound of claim 52, wherein the detectable group comprises a fluorophore, a dye, a FRET donor, or an acceptor.

57. The compound of claim 52, wherein the capping group is an inert group.

58. The compound of any one of claims 28-56, wherein the functional group is selected from: -SH、 wherein the functional group is bonded to the polymer via a sulfur atom.

59. The compound of any one of claims 1-58, wherein the compound is configured to encapsulate or complex with a nucleic acid in an aqueous solution.

60. The compound of any one of claims 1-59, wherein the compound is substantially non-toxic.

61. The compound of any one of claims 1-60, wherein the compound is biodegradable.

62. The compound of any one of claims 1-61, wherein the compound comprises a molecular weight of about 1 kilodalton (kDa) to about 100 kDa.

63. A nanoparticle comprising a compound according to any one of claims 1-62, wherein the nanoparticle is configured for encapsulation or complexation of nucleic acids.

64. The nanoparticle of claim 63, wherein the nanoparticle is configured for the encapsulation or the complexing of the nucleic acid at a ratio of 0.3:1-100:1 (weight:weight).

65. The nanoparticle of any one of claims 63-64, wherein the encapsulation or the complexing of the nucleic acid increases the half-life of the nucleic acid by at least 2-fold under aqueous or physiological conditions.

66. The nanoparticle of any one of claims 63-65, wherein nuclease digestion of the nucleic acid is inhibited by the encapsulation or complexation.

67. The nanoparticle of any one of claims 63-66, wherein the encapsulation or the complexing of the nucleic acid produces a transfection reagent having an average size of about 20 nm to about 2000 nm.

68. The nanoparticle of any one of claims 63-67, wherein the complexing comprises adsorption of at least a subset of the nucleic acids to the surface of the nanoparticle.

69. The nanoparticle of any one of claims 63-68, wherein the encapsulation or the complexing of the nucleic acid produces a transfection reagent configured for cellular uptake.

70. The nanoparticle of claim 69, wherein the cellular uptake comprises endocytosis.

71. A transfection reagent comprising the nanoparticles according to claim 63 and a nucleic acid encapsulated therein.

72. The transfection reagent of claim 71, wherein the nucleic acid comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), or any combination thereof.

73. The transfection reagent of any one of claims 71-72, wherein the nucleic acid comprises about 1 kilobase pair (kb) to about 100 kb.

74. The transfection reagent of any one of claims 71-73, wherein the nucleic acid comprises about 2 kb to about 20 kb.

75. The transfection reagent of any one of claims 71-74, wherein the nucleic acid comprises about 5 kb to about 15 kb.

76. The transfection reagent of any one of claims 71-75, wherein the nucleic acid comprises about 8 kb to about 12 kb.

77. The transfection reagent of any one of claims 71-76, wherein the nucleic acid comprises about 10 kb.

78. The transfection reagent of any one of claims 71-77, comprising a water solubility of at least 5 μg / mL.

79. The transfection reagent of any one of claims 71-78, comprising about 5 μg to about 5 mg / mL of water solubility.

80. The transfection reagent of any one of claims 71-79, comprising a water solubility of about 10 μg / mL to about 50 μg / mL.

81. A method for transfecting a cell, the method comprising: (a) providing a transfection reagent comprising a compound according to any one of claims 1 to 53 and a nucleic acid, and (b) contacting the cell with the transfection reagent, wherein the contacting is performed under conditions suitable for entry of the nucleic acid into the cell.

82. The method of claim 81, wherein (a) comprises contacting the compound with the nucleic acid under conditions sufficient to form the transfection complex.

83. The method of any one of claims 81-82, wherein the conditions sufficient to form the transfection complex comprise conditions sufficient to perform ionic gelation.

84. The method of any one of claims 81-83, wherein the nucleic acid comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), or any combination thereof.

85. The method of any one of claims 81-84, wherein the transfection complex comprises a positive charge under the conditions suitable for entry of the nucleic acid into the cell.

86. The method of any one of claims 81-85, wherein the contacting is less than 24 hours.

87. The method of any one of claims 81-86, wherein the cell comprises an animal cell, a plant cell, a fungal cell, a bacterial cell, or any combination thereof.

88. A pharmaceutical composition comprising the nanoparticles of claim 63 and a biologically active molecule.

89. The pharmaceutical composition of claim 88, wherein the nanoparticles are covalently bonded to the biologically active molecule.

90. The pharmaceutical composition of claim 88, wherein the nanoparticles are ionically bonded to the biologically active molecule.

91. The pharmaceutical composition of claim 88, wherein the nanoparticles encapsulate the biologically active molecule.

92. The pharmaceutical composition of claim 88, wherein the biologically active molecule comprises a nucleic acid molecule.

93. The pharmaceutical composition of claim 92, wherein the nucleic acid molecule comprises RNA or DNA.

94. The pharmaceutical composition of claim 93, wherein the nucleic acid molecule comprises mRNA, siRNA, or tRNA.

95. The pharmaceutical composition of claim 88, wherein the biologically active molecule comprises a therapeutic agent.

96. The pharmaceutical composition of claim 95, wherein the therapeutic agent is a chemotherapeutic agent, a radiotherapeutic agent, an oligonucleotide, or an oligopeptide.

97. The pharmaceutical composition of any one of claims 88-96, further comprising a pharmaceutically acceptable excipient.

98. A method of treating a condition or disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 88.

99. The method of claim 98, wherein the pharmaceutical composition is administered to the subject by injection.

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